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Human Genetics and Childhood Diseases

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Human Genetics and Childhood Diseases

Curator: Larry H. Bernstein, MD, FCAP

Publication Roundup: HGMD

HGMD^®, the Human Gene Mutation Database is used by scientists around the world to find information on reported genetic mutations. The papers below use the database to advance our understanding of disease, DNA dynamics, and more.

https://www.qiagenbioinformatics.com/blog/translational/publication-roundup-hgmd

Local DNA dynamics shape mutational patterns of mononucleotide repeats in human genomes
First author: Albino Bacolla

Scientists in the US and UK published results in Nucleic Acids Research of a detailed analysis of single-base substitutions and indels in the human genome. Their findings show that certain base positions are more susceptible to mutagenesis than others. They used HGMD Professional to find mutations in specific genomic regions for analysis; the paper includes charts showing mutation patterns, germline SNPs, and more from HGMD data.

High prevalence of CDH23 mutations in patients with congenital high-frequency sporadic or recessively inherited hearing loss
First author: Kunio Mizutari

This Orphanet Journal of Rare Diseases paper from scientists in Japan sequenced 72 patients with unexplained hearing loss, finding several CDH23 mutations, some of which were novel. Mutations in the gene have been linked to Usher syndrome and other forms of hereditary hearing loss. The scientists used HGMD to find all known CDH23 mutations within nearly 70 coding regions.

Mutation analyses and prenatal diagnosis in families of X-linked severe combined immunodeficiency caused by IL2Rγ gene novel mutation
First author: Q.L. Bai

In Genetics and Molecular Research, scientists report the utility of mutation analysis of the interleukin-2 receptor gamma gene to assess carrier status and perform prenatal diagnosis for X-linked severe combined immunodeficiency. They studied two high-risk families, along with 100 controls, to evaluate the approach. Sequence variation was determined using HGMD Professional and an X-SCID database, and a new mutation was discovered in the project.

Impact of glucocerebrosidase mutations on motor and nonmotor complications in Parkinson’s disease
First author: Tomoko Oeda

Researchers from three hospitals in Japan published this Neurobiology of Aging report that may help stratify Parkinson’s disease patients by prognosis. They sequenced mutations in the GBA gene in 215 patients, finding that those who had mutations associated with Gaucher disease suffered dementia and psychosis much earlier than those who didn’t. The team found previously reported GBA mutations using HGMD Professional.

Comprehensive Genetic Characterization of a Spanish Brugada Syndrome Cohort
First author: Elisabet Selga

In this PLoS One publication, scientists from a number of institutions in Spain examined genetic variation among patients with Brugada syndrome, a rare genetic cardiac arrhythmia. They sequenced 14 genes in 55 patients, identifying 61 variants and finding the subset that appear pathogenic. Variants were filtered against a number of databases, including HGMD.

Local DNA dynamics shape mutational patterns of mononucleotide repeats in human genomes

Albino Bacolla¹, Xiao Zhu², Hanning Chen³, Katy Howells⁴, David N. Cooper⁴ and Karen M. Vasquez¹

Nucl. Acids Res. (26 May 2015) 43(10): 5065-5080. http://dx.doi.org:/10.1093/nar/gkv364

Single base substitutions (SBSs) and insertions/deletions are critical for generating population diversity and can lead both to inherited disease and cancer. Whereas on a genome-wide scale SBSs are influenced by cellular factors, on a fine scale SBSs are influenced by the local DNA sequence-context, although the role of flanking sequence is often unclear. Herein, we used bioinformatics, molecular dynamics and hybrid quantum mechanics/molecular mechanics to analyze sequence context-dependent mutagenesis at mononucleotide repeats (A-tracts and G-tracts) in human population variation and in cancer genomes. SBSs and insertions/deletions occur predominantly at the first and last base-pairs of A-tracts, whereas they are concentrated at the second and third base-pairs in G-tracts. These positions correspond to the most flexible sites along A-tracts, and to sites where a ‘hole’, generated by the loss of an electron through oxidation, is most likely to be localized in G-tracts. For A-tracts, most SBSs occur in the direction of the base-pair flanking the tracts. We conclude that intrinsic features of local DNA structure, i.e. base-pair flexibility and charge transfer, render specific nucleotides along mononucleotide runs susceptible to base modification, which then yields mutations. Thus, local DNA dynamics contributes to phenotypic variation and disease in the human population.

INTRODUCTION

Changes in human genomic DNA in the form of base substitutions and insertions/deletions (indels) are essential to ensure population diversity, adaptation to the environment, defense from pathogens and self-recognition; they are also a critical source of human inherited disease and cancer. On a genome-wide scale, base substitutions result from the combined action of several factors, including replication fidelity, lagging versus leading strand DNA synthesis, repair, recombination, replication timing, transcription, nucleosome occupancy, etc., both in the germline and in cancer (1–4). On a much finer scale [(over a few base pairs (bp)], rates of base substitutions may be strongly influenced by interrelationships between base–protein and base–base interactions. For example, the mutator role of activation-induced deaminase (AID) in B-cells during class-switch recombination and somatic hypermutation (5) targets preferentially cytosines within WRC (W: A|T; R: A|G) sequences (6), whereas apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) overexpression displays a preference for base substitutions at cytosines in TCW contexts (7). Other examples, such as the induction of C→T transitions at CG:CG dinucleotides by cytosine-5-methylation and the role of UV light in promoting base substitutions at pyrimidine dimers have been well documented (reviewed in (4,8)). More recently, complex patterns of base substitution at guanosines in cancer genomes have been found to correlate with changes in guanosine ionization potentials as a result of electronic interactions with flanking bases (9), suggesting a role for electron transfer and oxidation reactions in sequence-dependent mutagenesis. However, despite these advances, the increasing number of sequence-dependent patterns of mutation noted in genome-wide sequencing studies has met with a lack of understanding of most of the underlying mechanisms (10). Thus, a picture is emerging in which mutations are often heavily dependent on sequence-context, but for which our comprehension is limited.

Mononucleotide repeats comprise blocks of identical base pairs (A|T or C|G; hereafter referred to as A-tracts and G-tracts) and display distinct features: they are abundant in vertebrate genomes; mutations within the tracts occur more frequently than the genome-wide average; mutations generally increase with increasing tract length; length instability is a hallmark of mismatch repair-deficiency in cancers; and sequence polymorphism within the general population has been linked to phenotypic diversity (11–15). Thus, mononucleotide repeats appear ideal for addressing the question of sequence-dependent mutagenesis since base pairs within the tracts are flanked by identical neighbors. Both historic and recent investigations concur with the conclusion that a major source of mononucleotide repeat polymorphism is the occurrence of slippage (i.e. repeat misalignment) during semiconservative DNA replication, which gives rise to the addition or deletion of repeat units (11,12). An additional and equally important source of mutation has recently been suggested to arise from errors in DNA replication by translesion synthesis DNA polymerases, such as pol η and pol κ (13), also on slipped intermediates, leading to single base substitutions.

A key question that remains unanswered in these studies and which is relevant to the issue of sequence context-dependent mutagenesis is whether all base pairs within mononucleotide repeats display identical susceptibility to single base changes and whether indels (which are consequent to DNA breakage) occur randomly within the tracts.

Herein, we combine bioinformatics analyses on mononucleotide repeat variants from the 1000 Genomes Project and cancer genomes with molecular dynamics simulations and hybrid quantum mechanics/molecular mechanics calculations to address the question of sequence-dependent mutagenesis within these tracts. We show that mutations along both A-tracts and G-tracts are highly non-uniform. Specifically, both base substitutions and indels occur preferentially at the first and last bp of A-tracts, whereas they are concentrated between the second and third G:C base pairs in G-tracts. These positions coincide with the most flexible base pairs for A-tracts and with the preferential localization of a ‘hole’ that results when one electron is lost due to an oxidation reaction anywhere along G-tracts. Thus, despite the uniformity of sequence composition, mutations occur in a sequence-dependent context at homopolymeric runs according to a hierarchy that is imposed by both local DNA structural features and long-range base–base interactions. We also show that the repair processes leading to base substitution must differ between A- and G-tracts, since in the former, but not in the latter, base substitutions occur predominantly in the direction of the base immediately flanking the tracts. Additional sequence-dependent patterns of mutation are likely to arise from studies of more heterogeneous sequence combinations, possibly involving other aspects intrinsic to the structure of DNA.

RESULTS

Mononucleotide repeat variation is defined by tract length and flanking base composition

We define mononucleotide repeats in the GRCh37/hg19 (hg19) human genome assembly as uninterrupted runs of A:T and G:C base pairs (hereafter referred to as A-tracts and G-tracts, respectively) from 4 to 13 base pairs in length (Figure 1A). We retrieved a total of 48,767,945 A-tracts and 13,633,781 G-tracts, both of which displayed a biphasic distribution with an inflection point between tract lengths of 8 and 9 (bp) and with the number of runs declining with length more dramatically for G-tracts than for A-tracts (Figure 1B), as noted previously (29). Both the number of short tracts and the extent of decline varied with flanking base composition, TA[n]T runs being two- to three-fold more abundant than CA[n]Cs (Supplementary Figure S1A) and AG[n]As declining the most rapidly (Supplementary Figure S1B). Thus, mononucleotide runs exist as a collection of separate pools of sequences in extant human genomes, each maintained at distinctive rates of sequence stability, as determined by factors such as bp composition (A:T versus G:C), tract length and flanking sequence composition.

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Figure 1.

Mononucleotide repeat variation, evolutionary conservation and association with transcription. (A) The search algorithm was designed to retrieve runs of As or Ts (A-tracts) and Gs or Cs (G-tracts) length n (n = 4 to 13), along with their 5′ (n = 0) and 3′ (n = n + 1) nearest neighbors from hg19. Tract bases were numbered 5′ to 3′ with respect to the purine-rich sequence. The panel exemplifies the nomenclature for A- and G-tracts of length 4. (B) Logarithmic plot of the number of A-tracts (closed circles) and G-tracts (open circles) in hg19 as a function of length. (C) Normalized fractions of polymorphic tracts (F SNV) (number of SNVs divided by both hg19 number of tracts and n) from the 1KGP for A-tracts (closed circles) and G-tracts (open circles). (D) Radial plot of SNVs in the 1KGP at the 5′ and 3′ nearest neighbors of A-tracts. Periphery, tract length; horizontal axis, scale for the fraction of SNVs (F SNV). (E) Radial plot of SNVs in the 1KGP at the 5′ and 3′ nearest neighbors of G-tracts. (F) Percent difference in the numbers of A-tracts (closed circles) and G-tracts (open circles) between syntenic regions of hg19 and HN genomes. (G) The exponents of Benjamini-corrected P-values for A-tract-containing genes enriched in transcription-factor binding sites plotted as a function of A-tract length (triangles); each value represents the median of the top 11 USCS_TFBS terms. The percent A-tracts (closed circles) and G-tracts (open circles) intersecting genomic regions pulled-down by chromatin immunoprecipitation using antibodies against transcription factors are plotted as a function of tract length. (H) List of gene enrichment terms with a Benjamini-corrected P-value of <0.05 in common between genes containing A- and G-tracts of lengths 4–13, excluding the UCSC_TFBS terms.

We examined the extent of sequence variation in the human population by mapping 38,878,546 single nucleotide variants (SNVs) from 1092 haplotype-resolved genomes (the 1000 Genomes Project, 1KGP) (30) to the hg19 A- and G-tracts. The normalized fractions of polymorphic tracts (F SNV) were greater for G-tracts than A-tracts and both displayed Gaussian-type distributions, with maxima of 0.067 for G-tracts of length 8 and 0.017 for A-tracts of length 9 (Figure 1C). CA[n]C and AG[n]A runs displayed the highest F SNV values for A- and G-tracts, respectively (Supplementary Figure S1C and D), with F SNV values for AG[n]As attaining ∼0.10 at length 8. We conclude that flanking base composition influences the rates of SNV within mononucleotide runs and, as a consequence, their representation in the reference human genome.

F SNV values at the flanking 5′ and 3′ bp were similar between A- and G-tracts, except for minor differences for the least represented (i.e. longest) tracts and did not exceed 0.02 (Supplementary Figure S1E). These fractions are expected to be greater than at more distant positions from the tracts, based on previous data (29). SNVs at G-tracts, but not at A-tracts, were more frequent than at flanking base pairs. F SNVs for base pairs flanking short (≤8 bp) tracts were at least twice as high as those flanking long tracts; F SNVs also displayed distinct sequence preference with most (∼0.1) variants occurring at Ts 3′ of G-tracts (Figure 1D and E). In summary, SNVs at mononucleotide runs do not increase monotonically with length but peak at 8–9 bp. This behavior mirrors the genomic distributions, both with respect to the total number of tracts (Figure 1B) and the subsets flanked by specific-sequence combinations (Supplementary Figure S1A–D). Variation at flanking base pairs also displayed a biphasic pattern centered at a length of 8–9 bp, with a greater chance of variation adjacent to G- than A-tracts and with characteristic sequence preferences.

Long tracts are evolutionarily conserved and associated with high transcription

To assess whether more variable monosatellite runs (Figure 1C) might have undergone a greater reduction in number in extant humans relative to extinct hominids, we compared the number of A- and G-tracts between syntenic regions of five individuals comprising hg19 and three Neanderthal (HN) specimens (31). The difference between hg19 and HN was very small (<±2%) for the short tracts, but it displayed more negative values in hg19 with increasing tract length, which reached a maximum of −11.8 and −32.7% for A- and G-tracts, respectively, of length 9. Beyond this threshold, the numbers of tracts converged for A-tracts, whereas they were more abundant in hg19 for G-tracts >11 bp (Figure 1F). In summary, the largest difference in the number of mononucleotide runs between hg19 and HN sequences was centered at 9 bp for both A- and G-tracts, suggesting that the length distributions (Figure 1A and Supplementary Figure S1A and B) reflect distinct rates of evolutionary gains and losses due to differential sequence mutability (Figure 1C) as a function of length and flanking sequence composition (12).

The fact that long (>9 bp) mononucleotide runs display low variability in the human population (Figure 1C) and sequence conservation during evolutionary divergence (Figure 1F) raises the possibility that they might serve functional roles. Through gene enrichment analyses, we found that genes containing A- and G-tracts were enriched for genes associated with the term ‘UCSC_TFBS’, which pertains to transcripts harboring frequent transcription factor binding sites (32,33). For A-tract-containing genes, the median P-values for the top 11 UCSC_TFBS terms decreased from 2.95E-26 for tracts of length 4 to 5.22E-241 for tracts of length 13 (Figure 1G). The percent of A-tracts intersecting genomic fragments amplified from chromatin immunoprecipitation using transcription-factor binding antibodies (32,33) also increased from 8.7 to 9.9 from length 6 to 13, whereas it was constant (mean ± SD, 22.4 ± 1.1) for G-tracts (Figure1G). For gene classes excluding ‘UCSC_TFBS’, a search for categories enriched at P < 0.05 and common to all A- and G-tract-containing genes returned a set of 25 terms, 22 of which were associated with high levels of tissue-specific gene expression (Figure 1H). In summary, these analyses extend prior work (14) supporting a role for mononucleotide tracts in enhancing gene expression, a function that for A-tracts appears to increase with increasing tract length.

Repeat variability is highly skewed

Next we addressed whether bp along A- and G-tracts display equal probability and type of variation. In the 1KGP dataset, the number of SNVs at each position along both A- and G-tracts of length 4 was within a two-fold difference (144,000–240,000); for both types of sequence, transitions (i.e. A→G and G→A) were the predominant (51–78%) type of base substitution (Supplementary Figure S2A and B). However, with increasing length, the number of SNVs decreased up to 30-fold more drastically for G-tracts than for A-tracts, with increasing numbers of transversions (A→T and G→C|T) being predominant. Normalizing the data for the number of tracts genome-wide revealed that the extent of SNV varied by up to 10-fold, depending upon tract length and bp position. Specifically, the highest degree of variation was observed at the first and last A within the A-tracts (i.e. A₁ and A_n), which underwent up to 61% A→T and 43% A→C transversions, respectively, at length 9 (Figure 2A). Likewise, for G-tracts, the most polymorphic sites were G₃, followed by G₂, for mid-size tracts of 8–10 bp, with 44% G→C transversions at G₃ for tracts of length 8 (Figure2B). Thus, the extent of SNV at mononucleotide runs is grossly skewed in human genomes, both along the sequence itself and across tract length, which must account for the bell-shape behavior in F SNV for the tracts as a whole (Figure 1C).

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Figure 2.

Population variation spectra. (A) Variation spectra of A-tracts. Percent (number of SNVs at each position divided by the number of tracts in hg19 × 100) of A→T (black), A→C (red) and A→G (green) SNVs in the 1KGP dataset (left). Percent SNVs at A₁ as a function of tract length (right). (B) Variation spectra of G-tracts. As in panel A with G→T (black), G→C (red) and G→A (cyan) (left). Percent SNVs at G₃ as a function of tract length (right). (C) Percent A→T, A→C and A→G transitions at each position along A-tracts (stars) preceded and followed by a T (TA[n]T, left), C (CA[n]C), center) and G (GA[n]G, right) as a function of tract length. (D) Percent G→T, G→C and G→A transitions at each position along G-tracts (stars) preceded and followed by a T (TG[n]T, left), C (CG[n]C), center) and A (AG[n]A, right) as a function of tract length. (E) Percent transitions at base pairs (stars) preceding or following A-tracts (left) and G-tracts (right) as a function of tract length (n). *, mutated position.

We assessed whether SNV hypervariability was associated with specific combinations of nearest neighbors. For A-tracts flanked 5′ by a T, C or G, the highest percentage of SNVs was observed at A₁ when preceded by a T, which reached 7.9% for TA[n] tracts of length 9 (Supplementary Figure S2C). By contrast, for 3′ T, C or G, the greatest effect was elicited by a C, with the highest percentage (7.1%) of SNVs at A_n for A[n]C tracts of length 9 (Supplementary Figure S2D). Therefore, flanking base pairs play a critical role both in the spectra and frequencies of SNVs at A-tracts. More detailed plots along A-tracts either preceded (Supplementary Figure S2E), followed (Supplementary Figure S2F) or preceded and followed (Figure 2C) by a T, C or G revealed the dramatic and long-range (up to 9–10 bp for the longest tracts, higher than the value of 4 bp predicted by mathematical models of slippage (11)) influence of flanking base pairs on variation spectra, in which up to 95% of the changes were in the direction of the base flanking the tract. Because the number of A-tracts preceded or followed by a specific base varies by up to three-fold (Supplementary Figure S2G), we conclude that for A-tracts, the overall mutation fractions and spectra are the result of at least three variables; length, position along the tract, and base composition of the 5′ and 3′ nearest-neighbors.

For G-tracts flanked 5′ by a T, C or A, high percentages (10–12%) of SNVs were observed at G₁ for tracts preceded by a C, an effect that decreased with increasing tract length (Supplementary Figure S3A). This result, together with an exceedingly low number of G→A transitions at G₁ for tracts not preceded by a C (Supplementary Figure S3C) relative to all tracts (Supplementary Figure S2B), is consistent with the known high mutability of CG:CG dinucleotides as a result of cytosine-5 methylation (9). The hypermutability at G₂ was observed preferentially for tracts preceded by an A, and to a lesser extent T, whereas that at G₃ was insensitive to flanking sequence composition. Likewise, G-tracts flanked 3′ by a T, C or A did not display marked sequence-dependent effects (Supplementary Figure S3B). Detailed plots of the SNV spectra along G-tracts either preceded (Supplementary Figure S3D), followed (Supplementary Figure S3E), or preceded and followed (Figure 2D) by a T, C or A revealed a noticeable effect only for 5′ T in association with G→T substitutions at G₁for tracts of length ≥8. Thus, despite a consistent over-representation of G-tracts flanked 5′ by a T (Supplementary Figures S3F and S1B), which must account for the high absolute number of SNVs at G₁ for TG[n] relative to AG[n] and CG[n] (Supplementary Figure S3G), nearest-neighbor base composition seems to play a lesser role in SNV spectra at G-tracts than at A-tracts.

With respect to SNVs at the flanking 5′ and 3′ nearest positions, no B→A or H→G substitutions (Figure 1A) were found above a length threshold of 9 for A-tracts and 8 for G-tracts (Figure 2E, gray shading) out of 5969 SNVs, implying that tract expansion by recruiting flanking base pairs is disfavored at these lengths. In summary, base substitution along mononucleotide repeats is strongly skewed towards the edges of A-tracts and within the 5′ half of G-tracts, with frequencies that peak at midsize lengths (8–9 bp). For A-tracts ≥7 bp, base substitution occurred almost exclusively in the direction of the flanking nearest-neighbors. Finally, base substitution at flanking bases did not contribute to tract expansion for mononucleotide runs longer than 8–9 bp.

Insertions and deletions display length and positional preference

In addition to SNVs, mononucleotide runs are polymorphic in length as a result of indels. Herein, we consider separately two types of indels: one in which tract length changes by ±1 and flanking bp composition is not altered (slippage); the other comprising all other cases involving the addition or removal of 1–200 bp (indels). Slippage is a widely accepted mutational mechanism (11–12,34), whereby DNA replication errors at reiterated DNA motifs cause changes in the number of motifs (most often +/−1). The normalized fractions of slippage in the 1KGP dataset peaked at lengths of 8 bp for A-tracts and 9 bp for G-tracts (Figure 3A), generating bell-shaped curves similar to those observed for SNVs (Figure1C) and with no differences in the highest fraction of ‘slipped’ tracts, which peaked at ∼0.02. By contrast, +1 slippage occurred more frequently than −1 slippage at A-tracts (Figure 3B). These results support recent studies on microsatellite repeats (12) and contrast with previous conclusions that slippage increases monotonically with tract length, and that the extent of slippage differs between A- and G-tracts (35,36).

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Figure 3.

Population insertions and deletions. (A) Normalized fractions of A-tracts (closed circles) and G-tracts (open circles) displaying +/−1 bp slippage in the 1KGP dataset as a function of tract length. Data were obtained by dividing the number of events by both the number of hg19 tracts and tract length (n). (B) Ratio of the number of +1 to −1 slippage for A-tracts (closed circles) and G-tracts (open circles). (C) Indels at A-tracts. For positions along the tracts (‘Tract’), ‘F Indel’ is the ratio between the number of indels and the number of tracts in hg19 multiplied by tract length. For the positions immediately flanking the tracts genomic coordinates (‘Before tract’ and ‘After tract’), ‘F Indel’ is the ratio between the number of indels and the number of tracts in hg19. (D) Indels at G-tracts, calculated as described in panel C. (E) Heatmap representation of insertions along A-tracts. The percent insertions (i.e. the number of insertions at each position divided by the number of tracts in hg19) (y-axis) plotted as a function of location (x-axis) from position 0 (insertion between the bp 5′ to the tract and the first bp of the tract) to position n + 1 (insertion between the bp 3′ to the last bp of the tract and the following bp) (see Figure 1A) and as a function of tract length (z-axis). (F) Heatmap representation of insertions along G-tracts.

With respect to indels, the normalized fractions were low (<1 × 10⁻³) along short (4–6 bp) A- and G-tracts, but rose to a plateau for longer tracts as reported earlier (11); this plateau was 10-fold higher for G-tracts (∼0.03) than for A-tracts (∼0.003) (Figure 3C and D). Indels also occurred more frequently (up to six-fold for A-tracts of length 11) at nearest-neighboring base pairs (‘Before tract’ and ‘After tract’ in Figure 3C and D) than along the tracts. Thus, contrary to SNVs and slippage, indels increased to a plateau with mononucleotide tract length.

We analyzed in detail the locations of insertions along the tracts and the flanking positions with respect to the 5′ to 3′ orientation of the tracts (Figure 1A). The normalized fractions demonstrated that insertions peaked at the 3′, and to a lesser extent 5′, ends of the longest A-tracts (Figure 3E), but remained low. For G-tracts, insertions occurred most efficiently at two locations (G_2–3 and G₅) (Figure 3F), they increased with tract length (up to ∼0.04), and attained ∼10-fold higher values than for A-tracts. In conclusion, insertion sites at A- and G-tracts followed the patterns observed for SNVs (Figure 2A and B), suggesting that factors associated with local DNA dynamics sensitize specific bases along the tracts to genetic alteration, inducing both SBS and indels.

Base pair flexibility and charge localization map to sites of sequence changes

To elucidate elements of intrinsic DNA dynamics that may be responsible for the biases in SNV and insertion sites, we performed molecular dynamics (MD) and hybrid quantum mechanics/molecular mechanics (QM/MM) simulations on model A[6], A[9], G[6] and G[9] duplex DNA fragments. We focused on water bridge coordination (Figure 4A), bp step flexibility, and for the G[6] and G[9], charge localization, as these properties are known to impact the susceptibility of DNA to base damage, repair and mutation. The fractions of one water coordination increased along the A[9] and A[6] structures in a 5′ to 3′ direction, irrespective of flanking sequence composition, in concert with a decrease in minor groove width (Figure 4B and Supplementary Figure S4A) as predicted (37). V_step, a measure of bp structural fluctuation, displayed a prominent peak of ∼40 Å³deg³ at the 5′-TA-3′ step for both structures (Figure 4C and Supplementary Figure S4B), which together with low water occupancy points to 5′-TA-3′ being a preferred location for base modification and mutation. In the G[9] and G[6] structures water coordination involved mostly two-water bridges due to wide (∼14 Å) minor grooves (Figure 4Dand Supplementary Figure S4C), whereas flexibility was modest (∼20–22 Å³deg³, Figure 4E and Supplementary Figure S4D). Thus, bp dynamics are likely to impact mutations at A-tracts to a greater extent than at G-tracts. Guanine has the lowest ionization potential (IP) of all four bases and IP further decreases at guanine runs, rendering them targets for electron loss, charge localization, oxidation and eventually mutation (4,38). Because after electron loss the ensuing charge (hole) can migrate along the DNA double-helix and relocalize at specific guanines, we addressed whether the preferred sites of mutation along G-tracts, i.e. G_2–3 and G₅, would also be preferred sites for charge localization. The QM/MM determinations indicated that whereas for the short G[6] fragment the difference in the density-derived atomic partial charges (DDAPC) (i.e. the hole) localized most often (∼50%) to the first position (Figure 4F), for the long G[9] fragment charge localization shifted downstream (mostly to the second, but also to positions 6–7, Figure 4G). Importantly, the charge was found exclusively around the guanine rings (Figure 4H). Thus, the two main sites of sequence change along G-tracts, i.e. G_2–3 and G₅, coincide with positions where charge localization and hence one-electron oxidation reactions is predicted to occur most frequently. In summary, bp flexibility at A-tracts and charge transfer at G-tracts likely represent intrinsic DNA features underlying the bias in SNV and insertions at mononucleotide runs in human genomes.

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Figure 4.

MD and QM/MM simulations. (A) Molecular modeling of one (left) and two (right) minor groove water bridge coordination. (B) Fraction of one-water bridge occupancy (left axis) at A[9] DNA sequences flanked 5′ and 3′ by a T (black circles), C (red circles) or G (green circles). Minor groove widths (right axis), as determined from intrastrand phosphate-to-phosphate distances. (C) V_step for A[9] DNA sequences, determined as the product of the square root of the eigenvalues (λ_i) described by the six bp step parameters shift, slide, rise, tilt, roll and twist; i.e. Vstep=∏6i=1λi−−√. (D) Fraction of one- (black circles) and two-water (red circles) bridge occupancy (left axis) at G[9] DNA sequences. Minor groove widths (right axis), as assessed from intrastrand phosphate-to-phosphate distances. (E) V_step for G9 DNA sequences. (F) Average charge redistribution (open circles and right axis) for G[6] DNA structures upon vertical ionization, examined by calculating the difference on the density-derived atomic partial charges (DDAPC) for the neutral and negatively charged states. Histogram of the number of instances (left axis) in which the largest charge redistribution occurred at a specific position along the G[6] structures. (G) DDAPC for G[9] DNA structures (open circles and right axis) and histogram of the number of instances (left axis) in which the largest charge redistribution occurred at a specific position. (H) VMD rendering of a G[9] DNA structure displaying hole localization at G₂. Capped base pairs were removed for clarity.

Position and orientation along nucleosome core particles modulate sequence variation

DNA wrapped around histones in nucleosomes is subject to local deformation (39), which may impact mutation. Thus, we analyzed the 1KGP SNVs at A- and G-tracts predicted to overlap with well-positioned nucleosome core particles (NCPs) (16). In hg19, the percentage of tracts that overlap with NCPs decreased moderately from ∼90% at length of 4 to 81% and 71% for A- and G-tracts of length 13, respectively (Figure 5A), suggesting that mononucleotide runs are not depleted in NCPs in human genomes as previously proposed (40). A-tracts of lengths 4–8 base pairs displayed distinctive peaks along the NCP surface in phase with the helical repeat of DNA (10.5 bp) and with minor grooves facing toward the inner protein core (lengths 4–5) (16) (Figure 5B and Supplementary Figure S5A). A-tracts of length of 9–13 bp exhibited only half (six) the peaks evident for the shorter tracts. For the G-tracts, only small peaks with no clear minor groove-inward-facing regions were detected (Supplementary Figure S5B).

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Figure 5.

Positioning along nucleosome core particles. (A) Percent of A-tract (open circles) and G-tract (closed circles) base pairs in hg19 overlapping with well-positioned NCP genomic coordinates as a function of tract length. (B) Counts of base pairs in hg19 A-tracts of length 5 overlapping with NCPs genomic regions as a function of distance from the histone octamer dyad axis. Minor groove-inward-facing regions (gray) were derived from the X-ray crystal structure of NCP147 (41). (C) Percent SNVs in the 1KGP dataset (left axis) at every bp along A-tracts of length 5 for tracts centered at maxima (black) and minima (gray) along NCPs (Figure 5B). Percent increase (right axis) of SNVs at minima relative to maxima (green). P-values for paired t-tests: 0.013 (*), 0.002 (**) and 4.7 × 10⁻⁶ (***). (D) Whisker plots of%SNVs (left axis) at A₁ for A-tracts of length 5 centered at maxima and minima (black) along NCPs (Figure 5B). Percent difference (right axis) in the number of A-tracts of length 5 in hg19 preceded by C, T or G (red) between those centered at minima and those centered at maxima (Figure5B). (E) C-containing/G-containing ratios (see text) for G-tracts of length 5 in hg19 as a function of distance from the NCP dyad axis (black) and location of core histones (maroon and green). Peaks correspond to negative iSAT (i.e. tilt parameters multiplied by the corresponding sin θ) values (gray) (39). Ratios of%SNV at G₁ (upshifted by 0.5 for clarity) between C-containing (5′-CCCCCG-3′ sequences on the hg19 forward strand) and G-containing (5′-CGGGGG-3′ sequences on the hg19 forward strand) (Figure 1A) CG[5] tracts mapping NCP Chip-seq genomic intervals (red) fitted by a non-parametric local regression (loess; sampling proportion, 0.100; polynomial degree, 3). (F) VMD rendering (top) of TATTT residues 34–38 (yellow) and the complementary AAATA residues 672–753 (pink) from the 1EQZ pdb nucleosomal crystal structure, corresponding to peak area from −40 to −36 in Figure 5E. The switch in G-tract (lengths of 5 and 7) orientation along NCPs (bottom) serves to position the C-containing strand on the outside (yellow) and, correspondingly, the G-containing strand on the inside (pink).

To assess if tract-positioning along NCPs influences SNVs, we selected A-tracts of lengths 5, 7 and 9 bp and G-tracts of lengths 5 and 7 bp whose central positions coincided with either the maxima or minima (41) (Figure 5B and Supplementary Figure S5A and B) and conducted pair-wiset-tests (330 total) between permutations of ‘categories’, including ‘tracts centered at maxima versus minima’, ‘position along the tracts’, ‘flanking sequence composition’, ‘specific NCP locations’ and ‘tract orientation’. For A-tracts, 79/207 (38%) significant pairs were found, 68 (86%) of which were related to differences between tracts centered at maxima versus minima, with a preponderance (63%) of tests displaying increased %SNVs at minima (Supplementary Figure S5C and E). For example, %SNVs at length 5 bp were greater at minima than at maxima at each position along the A-tracts (Figure 5C). A→C substitutions at A₁ were more abundant at maxima than at minima (mean ± SD, 18.7 ± 0.7% at max and 17.6 ± 0.8% at min; P-value 0.001), whereas A→T substitutions at the same position displayed the opposite trend (mean ± SD, 18.4 ± 0.5% at max and 19.8 ± 1.1% at min; P-value 0.0005) (Figure 5D). A-tracts of length 7 also exhibited a similar pattern at A₇ (Supplementary Figure S5H). The percentages of CA[5] and A[7]C tracts in hg19 centered at maxima were greater than at minima and the reverse was observed for the TA[5] and A[7T] tracts (Figure 5D and Supplementary Figure S5H). Thus, we conclude that positioning along the NCP surface of both the double-helical grooves and junctions with flanking base pairs influence SNVs along A-tracts. However, this influence is complex and for the most part, difficult to predict.

For G-tracts, most pairwise comparisons (18/34, 53%) indicated SNV variation according to sequence orientation (Supplementary Figure S5F and G). In hg19, the ratio of the numbers of G-tracts of lengths 5 and 7 for which the C-containing strand coincided with the forward sequence (downstream example sequence in Figure 1A) to the numbers of G-tracts for which the G-containing strand coincided with the forward sequence (upstream example sequence in Figure 1A) (C-containing/G-containing ratios) displayed a prominent 10.5-bp oscillation in phase with iSAT (Figure 5E), a measure of ‘inside’ and ‘outside’ bases, according to the bp step tilt parameter (39). Analysis of the helical path of a 146-bp DNA fragment wrapped around histones showed that the oscillation in the C-containing/G-containing ratios corresponds to a preference for guanine bases to face the protein core (Figure 5F). We analyzed the subset of G-tracts preceded by a 5′ C (i.e. CG[5]) to assess whether SNVs at G₁, the position known to be mutable due to CpG methylation also oscillated with the C-containing/G-containing ratios. Oscillation in SNV-C-containing/SNV-G-containing values was evident, with peaks aligning to the hg19 troughs (Figure 5E) implying that the cytosines facing the protein surface harbor more variants than those facing away. We conclude that A- and G-tracts display preferential positioning (the former) and orientation (the latter) along NCPs, which in turn modulate the rate of sequence variation.

Mutations associated with human disease

Knowing that the first and last As of long A-tracts and G_2–3 in G-tracts are the major sites of SNV in the human population, we addressed whether these features are also discernible in mutated mononucleotide tracts associated with human genetic disease. We collected 9,450,456 unique SBSs (both SBSs and SNVs refer to single base changes) from sequenced cancer genomes and normalized the percent mutations along A- and G-tracts to enable a direct comparison with the 1KGP dataset. For A-tracts (Figure 6A and Supplementary Figure S6A), SBSs displayed the same trend as the 1KGP data (Figure 2A) with respect to the bell-shape increase in mutations at A₁ and A_n and the mutation spectra, although the susceptibility to mutation as a function of tract length attained greater values (6.36% for length 11 in cancer versus 4.15% for length 9 in the 1KGP datasets at A₁). The first and last 3 bp also harbored more SBSs than in the 1KGP dataset for tracts >7 bp, a feature that we found to be due exclusively to a large cancer dataset (42) containing high-level microsatellite instability (MSI) samples (Supplementary Figure S6B and C), which are known to result from mismatch-repair deficiency (15). Thus, A-tracts display similar patterns of base substitution between the germline and somatic cancer tissues. For G-tracts, mutation spectra were characterized by G→T transversions at tract lengths >7, particularly at G₁, the most frequently mutated position for tracts lengths up to 11 bp (Figure 6B and Supplementary Figure S6D). This trend persisted even when the high rates of methylation-mediated deamination mutations at the CG dinucleotide were removed (Supplementary Figure S6E). Thus, mutation patterns in cancer genomes contrast with those observed in the germline, both with respect to the most mutable position (G₁ versus G_2–3) and the types of base substitution (G→T in cancer genomes versus G→T and G→C in the germline).

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Figure 6.

Mutation patterns in cancer genomes. (A) Mutation spectra for SBSs at A-tracts. Percent values were obtained by dividing the total number of SBSs at each position by the number of tracts in hg19 and then multiplying by 3.2516 to equalize the percentage of A-tracts of length 4 between the cancer genomes and the 1KGP datasets. (B) Mutation spectra for SBSs at G-tracts in cancer genomes. Percent values were obtained as in (A) using a multiplication factor of 3.7419. (C) Normalized fractions of A-tracts (closed circles) and G-tracts (open circles) displaying +/−1 bp slippage, obtained by dividing the number of events by both the number of tracts in hg19 and tract length. (D) Indels at A-tracts, calculated as described in Figure 3C. (E) Indels at G-tracts, calculated as described in Figure3C. (F) Heatmap representation of insertions along G-tracts, as described in Figure 3E.

With respect to slippage, the fractions for A-tracts elicited an excess at lengths 9 and 10 bp relative to the 1KGP dataset, which was also due to the MSI-containing dataset. For G-tracts, the fractions peaked at length 8, as for the 1KGP dataset (Figures 3A and 6C), implying that the propensity to undergo slippage is indistinguishable between the germline and soma. Indels were also more abundant at flanking base pairs than along the tracts (Figure 6D and E), particularly for G-tracts of length >7, similar to the 1KGP dataset (Figure 3C and D). Detailed analyses of insertions revealed that both G₁ and the preceding position were the most significant sites of mutation (F-values up to 0.08 at G₁ for tracts of length 8) (Figure 6F). Thus, the 5′ end of long G-tracts is the most susceptible site for both SBSs and insertions in cancer genomes, in contrast to the germline where these occur within the runs, typically at G_2–3.

We also extracted the mutated A- and G-tracts from the Human Gene Mutation Database (HGMD), a collection of >150,000 germline gene mutations associated with human inherited disease. A total of 1519 genes were mutated at A- or G-tracts out of a total of 3972 (38%); 3480 SBSs and 2866 slippage events were noted within these tracts, 85 and 46% of which were predicted to be disease-causing, respectively (Figure 7A and Supplementary Table S1). Ranking genes by the number of literature reports indicated that among the top 10 entries three were associated with cancer (BRCA1, BRCA2 and APC), two with hemophilia (F8 and F9), four with debilitating lesions of the skin (COL71A), muscle (DMD), lung (CFTR) and kidney (PKD1), with one causing hypercholesterolemia (LDLR) (Figure 7B). Thus, mutations within A- and G-tracts carry a high social burden by contributing to some of the most common human pathological conditions.

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Figure 7.

Mutation patterns in HGMD and model for sequence context-dependent changes. (A) Number of germline SBSs and slippage events (Slip.) at A- and G-tracts in HGMD. Gene alterations were classified as disease-causing mutation (DM), likely disease-causing mutation (DM?), disease-associated and putatively functional polymorphism (DFP), disease-associated polymorphism with additional supporting functional evidence (DP) and invitro/laboratory orinvivo functional polymorphism (FP). Codon changes (SIFT predictor) were classified as damaging (d), null (n), tolerated (t) and low-confidence prediction (l). (B) The 10 most commonly reported genes in HGMD with mutations at A- and G-tracts. Various mutated tracts were generally reported for the same gene in different reports. (C) Mutation spectra for SBSs at A- (left) and G-tracts (right) in HGMD. Percent values were obtained by dividing the total number of SBSs at each position by the number of tracts in hg19 exons. A|G→T (black), A|G→C (red), A→G (green), G→A (cyan). (D) Normalized fractions of A-tracts (closed circles) and G-tracts (open circles) displaying +/−1 bp slippage, obtained by dividing the total number of events by the number of tracts in hg19 exons and by tract length. (E) Model for sequence context-dependent changes at A-tracts (left) and G-tracts (right). *, site of base modification.

For both A- and G-tracts, SBSs occurred mostly at tract lengths of 4–7, with patterns more similar to those in the 1KGP than in the cancer datasets, both with respect to the location of the most mutable positions (first and last As and first/second Gs) and the types of base substitution (A→T and G→H) (Figure 7C and Supplementary Figure S6F). Likewise, slippage events peaked at tract lengths of 7–9 as observed in the 1KGP dataset (Figure 7D). In summary, the patterns of both SBSs and slippage in the HGMD dataset followed the trend observed in the 1KGP dataset, suggesting that germline variants at mononucleotide repeats leading to either population variation or human inherited disease may have arisen through similar mechanisms.

DISCUSSION

Why are specific A:T and G:C base pairs within A- and G-tracts more susceptible to sequence changes than their identical neighbors? For A-tracts, bp flexibility may play a role. Chemical damage to DNA, such as by hydroxyl radicals has been shown to be proportional to the geometrical solvent-accessible surface of the atomic groups, which increases with DNA flexibility (43). Along A-tracts flexibility is restricted, but it is high at both the 5′ and 3′ junctions. Thus, the fact that the highest rates of mutation coincide with the highest degree of flexibility at the 5′-TA-3′ bp step is consistent with the view that this position may be susceptible to DNA damage as a result of flexibility. Other sources of DNA dynamics are also likely to be relevant, such as sugar flexibility at the junctions, which increases with tract length (44). Chemical modification at these junctions may then lead to base substitution and indels, the latter as a result of strand breaks.

With respect to SNV mutation spectra, these were found mostly in the direction of flanking base composition above a length of 7–8 bp. We interpret this behavior in terms of DNA slippage along A-tracts when attempts are made during translesion synthesis (TLS) to bypass a damaged site (Figure 7Ei). Two scenarios may be considered to account for A→T transitions at A₁. In the first, the last tract-template base would loop out into the polymerase active site permitting base-pairing and strand elongation (Figure 7Eii) using the tract-flanking base as a template (34,45–46). In the second (Figure 7Eiii), slippage would occur behind the polymerase, prompting extension past the newly created A*:T mispair generated by primer/template misalignment. Either pathway would yield a common intermediate (Figure 7Eiv) that contains the base complementary to the junction across from the damaged site upon slippage resolution (34). Following DNA synthesis (S) and/or repair (R) (Figure 7Ev and vi), this mispair will generate a base change that is always identical to the tract-flanking base.

For G-tracts, the high rates of G→T transversions at G₁ in cancer genomes are also consistent with preferred chemical attack at this site due to high flexibility (Figure 7F top). Direct chemical attack at a guanine is known to result in stable products, such as 8-oxo-G and Fapy-G, both of which are known to yield G→T transversions (47–50). Thus, G₁ may be the most susceptible site for such reactions for G-tracts of lengths ≥7 (Figure 7Fright), which in cancer genomes would become a mutation hotspot. In the germline, SNVs peaked inside G-tract base pairs, while mutational spectra were insensitive to flanking base composition; these events are inconsistent with a role for template misalignment and slippage as noted for A-tracts. Rather, the correspondence between hotspot mutations at G_2–3 and G₅ and the QM/MM simulations suggest a role for charge transfer. A large body of work during the past 20 years using computational, theoretical chemistry and biophysical techniques on short oligonucleotides, has shown that guanine is the most easily oxidizable base in DNA and that indeed a guanine radical cation can be generated through long-range hole transfer from an oxidant via one-electron oxidation mechanisms (51–55). GGG triplets were found to act as the most effective traps in hole transfer by both experimental and theoretical work (56–59), demonstrating that the resulting guanine radical cation (or its neutral deprotonated form) became rather delocalized, but it preferentially centered at the first and second G. These well-established patterns of chemical reactivity are consistent with our experimental observation of high mutation frequencies at G₁ for short G-tracts and the results from QM/MM simulations on G6. For longer tracts, the downstream shift in mutation hotspots, i.e., G_2–3 and G₅, also correlate well with the charge localization predicted from QM/MM simulations, which explicitly included solvent effects and structural fluctuations. Thus, in conjunction with the constrained density functional theory (60), both the neutral and oxidized forms of a guanine nucleobase can be reliably constructed to infer the accurate determination of mutational patterns of mononucleotide repeats in human genomic DNA.

The compact organization of the sperm genome (61), and presumably low levels of oxidative stress in the germline, may enable guanine oxidization through one-electron oxidation reactions rather than by direct chemical attack, thereby favoring the formation of radical cations. A charge injected at G₁ by electron loss would then migrate to neighboring guanines and localize at sites of low IP, such as G₂ (Figure 7F left). Guanine radical cations are known to readily undergo further chemical modification leading to products such as 8-oxo-G, oxazolone, imidazolone, guanidinohydantoin, and spiroiminodyhydantoin (62) (M in Figure 7F), to yield G→T, G→C and G→A substitutions (4,63). Our model is in line with recent observations in which mutations at guanines within short G-runs (1–4 bp) correlate with sequence-dependent IPs at the target guanine in cancer genomes (9). Interestingly, these correlations were not observed in the germline (9). We interpret these composite observations as follows. The IP values for G-runs have been shown to decrease asymptotically with tract length, although the absolute values vary according to the methods and assumptions used (we obtained a value of 5.43 eV for both G[6] and G[9]) (64,65). We suggest that short G-runs with high IPs undergo one-electron oxidation reactions in the oxidative environment of cancer cells but would be refractory to such a mechanism in the germline (Figure 7Fright yellow and left white sectors). As length increases and IP values fall, G-runs would be attacked directly by oxidants abundant in tumor cells (Figure 7F orange sector), whereas oxidation will be limited to electron loss in the germline environment (Figure 7F left yellow sector).

These models (template misalignment for A-tracts and charge transfer for G-tracts) suggest a more complex scenario for mechanisms underlying mononucleotide repeat polymorphism in the human population than recently proposed (13), in which nucleotide misincorporation by error-prone polymerases is proposed as a primary source of mutations at both A- and G-tracts. As already stated, the directionality of SNVs toward tract-flanking bases in A-tracts and the hotspot mutations at G_2–3, supports multiple and distinct mechanisms of base substitution at mononucleotide repeats.

Our analyses highlight additional information, including the lack of mutations in the direction of tract-base composition for base pairs flanking long tracts, the association with gene expression and the preference of guanines for the inner NCP surface, and extend prior observations (12) such as the bell-shape character of base substitution and slippage, whose mechanisms remain to be fully clarified. Finally, we document the contribution of mononucleotide mutagenesis to key aspects of human pathology beyond the well-established MSI instability in cancer (15), including hemophilia and tissue degeneration. Our collective work supports the conclusion that as the human genome undergoes evolutionary diversification and along the way suffers disease-associated mutations, oxidation reactions including charge transfer may play a prominent role.

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online.

Mutation analyses and prenatal diagnosis in families of X-linked severe combined immunodeficiency caused by IL2Rγ gene novel mutation

Q.L. Bai, N. Liu, X.D. Kong, X.J. Xu, Z.H. Zhao

Genet. Mol. Res. 14 (2): 6164 – 6172 DOI: 10.4238/2015.June.9.2

Severe combined immunodeficiency diseases (SCIDs) are a group of primary immunodeficiency diseases characterized by a severe lack of T cells (or T cell dysfunction) caused by various gene abnormalities and accompanied by B cell dysfunction (WHO, 1992; Buckley et al., 1997). The incidence rates in infants were 1/75,000-1/10,0000 (WHO, 1992), but no morbidity statistics are available in China. The 2 genetic modes of SCID include X-linked recessive and autosomal recessive genetic inheritance. X-linked severe combined immunodeficiency (X-SCID) is the most common form, accounting for 50-60% of SCID cases (Noguchi et al., 1993). Immune system abnormalities in patients with X-SCID include T-B+NK-, in which T cells (CD3+) and natural killer (NK) cells (CD16+/CD56+) are absent or significantly reduced, and the number of B cells (CD19+) is normal or increased, causing reduced immunoglobulin production and class switching disorder (Buckley, 2004; Fischer et al., 2005). The IL- 2Rg gene mutation has been confirmed to be a major cause of X-SCID (Noguchi et al., 1993). In recent years, great progress has been made in understanding the pathogenesis of primary immunodeficiency disease and its application in clinical treatment, particularly regarding the development of critical care medicine and immune reconstruction technology. With timely control of infection and early bone marrow or stem cell transplantation, X-SCID patients can be treated, prolonging survival time. Therefore, early diagnosis of X-SCID is very important for patient treatment. Gene diagnosis has become a better early diagnosis or differential diagnosis method. In addition, familial X-SCID brings a great psychological burden to the relatives of patients. Ordinary chromosome analysis and immunological evaluation cannot be used for female carrier identification and fetal diagnosis, and gene diagnosis is the most effective method of carrier detection and prenatal diagnosis. In this study, we detected mutations in 2 families with X-SCID and identified 2 novel mutations, confirming the X-SCID pedigrees. Prenatal diagnosis was performed for the pregnant fetus in the mother of one of the probands based on gene diagnosis. Female individuals in this family were subjected to carrier detection.

IL2Rg gene mutation test Direct sequencing of 1-8 exons and the flanking region of the IL2Rg gene by PCR in family 1 showed that the 3rd exon of the proband contained the c.361-363delGAG heterozygous deletion mutation, which led to deletion of the 121st amino acid glutamate (p.E121del) in its coding product. There were no sequence variations in other coding regions or in the shear zone. The proband’s mother carried the same heterozygous mutation, while his father did not carry the mutation site (Figure 2a, b, c). This mutation was not observed in any cases of the control group, and this family was identified as an X-SCID family. The c.510-511insGAACT insertion heterozygous mutation was present in the 4th exon of the proband’s mother in family 2. This mutation was a 5-base repeat of GAACT, resulting in a change in amino acid 173 from tryptophan into a stop codon (p.W173X). While there were no sequence variations in other coding regions or in the shear zone, the patient’s father did not carry the mutation (see Figure 2d, e). We did not find this mutation in the healthy control group. We presumed that the 4th exon of the deceased child in family 2 contained the c.510-511insGAACT insertion mutation, leading to X-SCID symptoms, and thus we speculated that this family was an X-SCID pedigree. Prenatal diagnosis We verified the chorionic villus status of the fetus in family 1 using the PowerPlex 16 HS System kit. The results of prenatal diagnosis showed that the fetal tissue contained no maternal contamination and that this fetus was female. The results of prenatal diagnosis showed that there was no c.361-363delGAG (p.E121del) heterozygous mutation in the female fetus of family 1.

Figure 2. Sequencing graph of IL2Rg gene in 2 pedigrees with X-chain severe combined immunodeficiency. a.-c. Family 1. a. Normal control (rectangle indicates 3 edentulous bases of this patient). b. Proband carrying the c.361- 363delGAG (p.E121del) mutation (arrow indicates deletion of fragment connection sites). c. The proband’s mother contained a c.361-363delGAG (p.E121del) heterozygous mutation (arrow). d.-e. Family 2. d. The proband’s mother carried the c.510-511insGAACT (p.W173X) heterozygous mutation (arrow indicates that the reverse sequencing graph was positive). e. Normal control (rectangular box indicates 2 normal copies of GAACT (the mutation fragment was 3 copies). Carrier detection results For the c.361-363delGAG (p.E121del) site, the gene analysis results of the female individual in family 1 showed that I2 (proband’s grandmother) was a heterozygous carrier and that II3 (proband’s aunt) was a non-carrier and had no mutations.

IL-2 can combine with the IL-2 receptor (IL-2R) of the immune cell membrane. IL-2R is composed of 3 subunits, including the IL-2Ra chain (CD25), IL-2Rb chain (CD122), and IL- 2Rg chain (CD132). IL-2Rg functional units in common with IL-4, IL-7, IL-9, IL-15, IL-21, and other cytokine receptors, and these regions are referred to as the total chain (Li et al., 2000). The IL-2Rg chain can maintain the integrity of the IL-2R complex and is required for the internalization of the IL-2/IL-2R complex; it is also the link that contacts the cell membrane surface factor region and downstream cell signal transduction molecules. Therefore, the integrity of the IL-2Rg chain is vital for the immune function of an organism (Malka et al., 2008; Shi et al., 2009).

Mutations in the IL2Rg gene, which encodes IL-2Rg, were identified to be a major cause of X-SCID in 1993 (Noguchi et al., 1993). The IL2Rg gene is located on chromosome X q21.3-22, is 37.5 kb length, and contains 8 exons, which encode 369 IL-2Rg amino acids. The IL2Rg chain exhibits varying structural regions, such as the signal peptide [amino acids (AA) 1-22], extracellular domain (AA 23-262), transmembrane region (AA 263-283), and intracellular region (AA 284-369). The WSXWS motif is located in the extracellular region (AA 237-241), while Box 1 is located in the intracellular region (AA 286-294).

By the end of 2013, the Human Gene Mutation Database contained a total of 200 mutations in the IL2Rg gene (HGMD Professional 2013.4). The most common mutation types in the IL2Rg gene were the missense or nonsense mutations, which result from single base changes. A total of 100 missense or nonsense mutations have been identified, followed by insertion or deletion mutations in a total of 50 species. The 3rd most common type of mutations includes shear mutations in approximately 30 species. Eight exons contained mutations, and mutations in 3rd or 4th exons were the highest, accounting for a total mutation rate of 43% (86/200). According to the X-SCID gene database (IL2RGbase) (http://research.nhgri. nih.gov/scid/), the gene mutations in IL2Rg mainly occurred in the extracellular region of the IL2Rg chain (Fugmann et al., 1998). Zhang et al. (2013) reported that the IL2Rg gene mutations in 10 patients with X-SCID in China were located in the extracellular region. Two mutations reported in our study were also located in the extracellular region. The mutation of IL2Rg gene in family 1 was a codon mutation in the 3rd exon, resulting in a 3-base deletion. The c.361-363delGAG (p.E121del) mutation was located in the extracellular area of the IL- 2Rg subunit, and we inferred that the 121 glutamate deletion caused by the mutation would lead to changes in the structure of the peptide chain, affecting signal transmission and resulting in serious symptoms. The mutation of family 2 was a GAACT repeat of ILR2g gene; this repeat of 5 bases resulted in 173 codon changes from tryptophan into a stop codon. Generation of the peptide chain with the mutation lacked 196 amino acids compared to the normal chain, including the intracellular, transmembrane, and some extracellular regions, directly affecting the structure and function of receptors and causing disease. No studies have been reported regarding these 2 mutations. We combined with the mutation characteristics and clinical manifestations and diagnosed family 1 as X-SCID pedigrees. Although the patient in family 2 was deceased, it can be speculated that the 2 deceased patients in family 2 were X-SCID pedigrees caused by c.510-511insGAACT (W173X).

Prenatal diagnosis can accurately identify fetal situations and be used to avoid birth defects, which can also ease the anxiety of the pregnant mother. Gene diagnosis for pedigrees of patients based on DNA samples has advanced recently, particularly with the application of high-throughput sequencing technology (Alsina et al., 2013). We can now perform gene analysis for varied clinical infectious diseases for differential diagnosis. However, the effectiveness of prenatal diagnosis for pedigrees in which the proband is dead remains unclear. Because the gene mutations in the proband is unknown in these cases, the patient’s situation was only inferred by his mother’s genotypes. However, we considered that for the deceased, if we can define the mother was a pathogenic gene carrier, even if the proband is not X-SCID, the woman also has a risk of having X-SCID children and this pedigree may be X-linked recessive inheritance. Prenatal diagnosis may provide a choice for preventing the birth of patients in these families in the premise of informed consent.

Gene diagnosis of IL2Rg can also be used for carrier detection of suspected females in the family.

In the present study, we performed carrier detection of the patient’s grandmother and aunt in family 1 and determined that the patient’s pathogenic mutations were from his grandmother. His aunt did not inherit the pathogenic gene, and thus she was a non-carrier and her fertility will not be affected. In this study, we used direct sequencing of PCR products and identified IL2Rg gene mutations in 2 pedigrees with X-SCID. We found 2 unreported mutations in the IL2Rg gene, and prenatal diagnosis and carrier detection were conducted in 1 X-SCID family. Because the incidence rate of X-SCID is extremely low, it is difficult to promote the widespread use and application of genetic diagnosis. However, this study may provide some implications for the diagnosis of infants with immunodeficiency, and gene diagnosis techniques such as conventional or high-throughput sequencing should be used as soon as possible during pregnancy, which can be used to guide treatment. This method can also provide reliable prenatal diagnosis and carrier detection service for these families.

MEF2A gene mutations and susceptibility to coronary artery disease in the Chinese population

J. Li1 , H.-X. Chen2 , J.-G. Yang3 , W. Li3 , R. Du3 and L. Tian3 DOI http://dx.doi.org/10.4238/2014.October.20.15

Coronary artery disease (CAD) has high morbidity and mortality rates worldwide. Thus, the pathogenesis of CAD has long been the focus of medical studies. Myocyte enhancer factor 2A (MEF2A) was first discovered as a CAD-related gene by Wang (2005) and Wang et al. (2003, 2005). Three mutation points in exon 7 of MEF2A were subsequently identified by Bhagavatula et al. (2004); however, Altshuler and Hirschhorn (2005) and Weng et al. (2005) predicted that the MEF2A gene lacked mutations. Zhou et al. (2006a,b) analyzed the mutations and polymorphisms in exons 7 and 11 of the MEF2A gene in the Han population in Beijing, and various rare mutations were found in exon 11 rather than in exon 7. The clinical significance of specific 21-bp deletions in MEF2A was also explored, and previous studies have shown mixed results. In this study, polymerase chain reaction-singlestrand conformation polymorphism (PCR-SSCP) and DNA sequencing were used to detect exon 11 of the MEF2A gene in samples collected from 210 CAD patients and 190 healthy controls and to investigate the function of the MEF2A gene in CAD pathogenesis and their correlation.

CAD, a common disease in China, is induced by multiple factors, such as genetics, the environment, and lifestyle. Thus, a multi-faceted approach is necessary in the study of CAD pathogenesis, particularly in molecular biology research, which is important for developing comprehensive treatment of CAD based on gene therapy. The MEF2A gene was first identified as a CAD-related gene through linkage analysis of a large family with CAD (9 of 13 patients developed MI) in 2003.

In this study, we found the following mutations: 1) codon 451G/T (147191) heterozygous or homozygous mutation; 2) loss of 1 (Q), 2 (QQ), 3 (QQP), 6 (425QQQQQQ430), and 7 (424QQQQQQQ430) amino acids (147108-147131); and 3) codon 435G/A (147143) heterozygous mutation. Among these mutations, the synonymous mutation at locus 147191 was confirmed by reference to the National Center for Biotechnology Information (NCBI) database to be a single nucleotide polymorphism, which was also demonstrated in our study by the extensive presence of this polymorphism in healthy controls. However, the heterozygous mutation at locus 147143 was only found in the genomes of CAD patients, and was therefore identified as a mutation.

Given that MEF2A is a CAD-related gene, the results of various studies are controversial among several countries. Weng et al. (2005) screened gene mutations in exon 11 of the MEF2A gene from 300 CAD patients and 1500 healthy controls. They hypothesized that the changes in 5-12 CAG repeats are genetic polymorphisms and that the 21-base deletion in exon 11 of the MEF2A gene did not induce autosomal dominant genetic CAD. Gonzalez et al. (2006) suggested that the CAG repeat polymorphism was independent of MI susceptibility in Spanish patients. Kajimoto et al. (2005) reported that the CAG repeat sequence was not correlated with MI susceptibility in Japanese patients. Horan et al. (2006) also found that the CAG repeat sequence was not associated with the susceptibility to early-onset familial CAD in an Irish population. Hsu et al. (2010) identified no correlation between the CAG repeat sequence and CAD susceptibility in the Taiwanese population. Dai et al. (2010) found that the structural change in exon 11 was not related to CAD in the Chinese Han population. Lieb et al. (2008) and Guella et al. (2009) hypothesized that MEF2A was independent of CAD. However, Yuan et al. (2006) and Han et al. (2007) suggested that the CAG repeat sequence was correlated with CAD because 9 CAG repeats was an independent predictor of CAD. Elhawari et al. (2010) and Maiolino et al. (2011) suggested that MEF2A is a susceptibility gene for CAD. Dai et al. (2013) showed that mutations in exon 12 are associated with the early onset of CAD in the Chinese population. Liu et al. (2012) failed to demonstrate a correlation between the CAG repeat sequence and CAD through case-control analysis, systematic review, and meta-analysis, but found that the 21- base deletion in exon 11 was strongly associated with CAD, and that genetic variations in MEF2A may be a relatively rare, but specific, pathogenic gene for CAD/MI. Kajimoto et al. (2005) reported 4-15 CAG repeats. However, only 4-11 CAG repeats were observed in our study, possibly because of genetic differences in patients in this study. Eleven CAG repeats were observed in most samples from the control group, and the proportion of 10, 9, and 8 repeats exceeded 1%. The heterozygous mutation at 147143, as well as the 4 and 5 CAG repeats, was only observed in CAD patients. Thus, we speculated that the CAG repeat sequence is correlated with CAD susceptibility, and the presence of 4 or 5 repeats may be a risk factor for CAD, which was inconsistent with the results obtained by Han et al. (2007). The inconsistency in these results may be explained by the differences in subjects and sample sizes among studies.

Impact of glucocerebrosidase mutations on motor and nonmotor complications in Parkinson’s disease

Tomoko Oeda ^a, Atsushi Umemura ^a, Yuko Mori ^a, Satoshi Tomita ^a, Masayuki Kohsaka ^a, Kwiyoung Park ^a, Kimiko Inoue ^b, et al.

Neurobiology of Aging 7 Sept 2015; doi:10.1016/j.neurobiolaging.2015.08.027

Homozygous and compound heterozygous mutations in GBA encoding glucocerebrosidase lead to Gaucher disease (GD). A link between heterozygous GBAmutations and Parkinson’s disease (PD) has been suggested ( Bembi et al., 2003,Goker-Alpan et al., 2004, Halperin et al., 2006, Machaczka et al., 1999, Neudorfer et al., 1996, Tayebi et al., 2001 and Tayebi et al., 2003). In 2009, a 16-center worldwide analysis of GBA revealed that heterozygous GBA mutation carriers have a strong risk of PD ( Sidransky et al., 2009).

In addition, heterozygote GBA mutations not only carry a risk for PD development but also the possibility of some risk burden on the progression of PD clinical course. In cross-sectional analyses of GBA mutations in PD patients, earlier disease onset, increased cognitive impairment, a greater family history of PD, and more frequent pain were reported in patients with mutations, compared with no mutations ( Chahine et al., 2013,Clark et al., 2007, Gan-Or et al., 2008, Kresojevic et al., 2015, Lwin et al., 2004, Malec-Litwinowicz et al., 2014, Mitsui et al., 2009, Neumann et al., 2009, Nichols et al., 2009,Seto-Salvia et al., 2012, Sidransky et al., 2009, Swan and Saunders-Pullman, 2013 and Wang et al., 2012). Recently, a few prospective studies have investigated clinical features of PD with GBA and showed a more rapid progression of motor impairment and cognitive decline in GBA mutation cases than in PD controls ( Beavan et al., 2015, Brockmann et al., 2015 and Winder-Rhodes et al., 2013). However, in terms of motor complications such as wearing-off and dyskinesia, no studies exist in the longitudinal course of PD with GBA mutations.

Here, we conducted a multicenter retrospective cohort analysis, and the data were investigated by survival time analysis to show the impact of GBA mutations on PD clinical course. We also investigated regional cerebral blood flow (rCBF) and cardiac sympathetic nerve degeneration of subjects with GBA mutations, compared with matched PD controls.

3.1. Subjects

Among the 224 eligible PD patients (the subjects were not related to each other), 9 subjects were excluded from the analysis (4 due to multiple system atrophy findings on subsequent brain MRI and 5 because of insufficient clinical information). Therefore, 215 PD patients [female, 52.1%; age, 66.7 ± 10.8 (mean ± standard deviation)] were analyzed. For non-PD healthy controls, 126 patients’ spouses (female, 58.7%; age, 67.3 ± 10.3) without a family history of PD or GD were enrolled.

3.2. GBA mutations and risk ratios for PD

In the PD subjects, we identified 10 nonsynonymous and 2 synonymous GBA variants. Within the nonsynonymous variants, 7 mutations were previously reported in GD [R120W, L444P-A456P-V460 (RecNciI), L444P, D409H, A384D, D380N, and⁴⁴⁴L(1447-1466 del 20, insTG)] as GD-associated mutations. Three nonsynonymous mutations have never been reported in GD patients [I(-20)V, I489V, and there was one novel mutation (Y11H)].

GD-associated GBA mutations were found in 19 of the 215 (8.8%) PD patients but none in the healthy controls. The risk of PD development relative to these GD-associated mutations was estimated as an OR of 25.1 [95% confidence interval (CI), 1.50–420,p = 0.0001] with 0-cell correction. The nonsynonymous mutations that were not reported in GD patients had no association with PD development (p = 0.506; OR, 1.3; 95% CI, 0.7–2.6) ( Table 1). Four subjects had double mutations. For subsequent analyses, 2 subjects with double mutations of I (-20)V and K466K were adopted to the group of mutations unreported in GD, and 2 subjects with double mutations of R120W and I(-20)V, and of R120W and L336L were adopted to the group of GD-associated mutations.

Table 1.Frequency of glucocerebrosidase gene allele in Parkinson’s disease patients and controls

Allele name	PD (n = 215)	Controls (n = 126)	p	Odds ratio (95% CI)
GD-associated mutations
R120W	7^a	0	0.050	9.1 (0.5–160.8)
RecNciI (L444P-A456P-V460)	4	0
L444P	4	0
D409H	1	0
A384D	1	0
D380N	1	0
⁴⁴⁴L(1447-1466 del 20, insTG)	1	0
Subtotal, n (%)	19 (8.8%)	0 (0%)	<0.001	25.1 (1.5–419.8)^b
Nonsynonymous mutations not reported in GD
I(-20)V	27^a	13	0.603	1.3 (0.6–2.5)
I489V	3	0
Y11H^c	0	1
Subtotal, n (%)	30 (14.0%)	14 (11.1%)	0.506	1.3 (0.7–2.6)
Synonymous, n
K466K	2^a	1
L336L	1^a	0

Allele names refer to the processed protein (excluding the 39-residue signal peptide).

Key: CI, confidence interval; GD, Gaucher disease; PD, Parkinson’s disease.

a Four subjects had double mutations; 2 of I(-20)V and K466K, 1 of I(-20)V and R120W, and 1 of R120W and L336L.

b Odds ratio was calculated by adding 0.5 to each value.

c Novel mutation.

Table options

3.3. Clinical features of PD patients by GBA mutation groups

The clinical features of PD patients with GD-associated mutations, those with mutations unreported in GD, and those without mutations are shown in Table 2. In the GD-associated mutation group, females, those with a family history and those with dementia (DSM IV) were significantly more frequent than those in the no-mutation group (p = 0.047, 0.012, and 0.020, respectively). The age of PD onset was lower in patients with GD-associated mutations (55.2 ± 9.9 years ± standard deviation), compared with those without mutations (59.3 ± 11.5), although the statistical difference was not significant. There were no differences in clinical manifestations between subjects with mutations unreported in GD and those without mutations, except for dopamine agonist dosage (p = 0.026) ( Table 2).

Table 2.Epidemiological and clinical features of PD patients with Gaucher disease–associated GBA mutations, those with mutations previously unreported in GD and those without mutations

Variables	Total n = 215	Mutation (-)	GD-associated mutations		Mutations unreported in GD
Variables	Total n = 215	167	19^a	p^b	29^c	p^d
Sex	Female, n (%)	83 (49.7)	14 (73.7)	0.047	15 (51.7)	ns
Age	Mean (SD)	67.0 (10.8)	62.2 (10.7)	0.063^e	67.5 (11.2)	ns^f
Disease duration (y)	Mean (SD)	7.7 (5.5)	6.9 (4.6)	ns^f	7.2 (4.9)	ns^f
Onset age	Mean (SD)	59.3 (11.5)	55.2 (9.9)	ns	60.3 (11.8)	ns
Family history	Yes, n (%)	17 (11.0)^g	6 (31.6)	0.012	0 (0.0)	ns
Dementia (DSM-IV)	Yes, n (%)	29 (17.4)	9 (47.4)	0.020	5 (17.2)	ns
MMSE	Mean (SD)	25.8 (5.4)^h	23.3 (7.7)	ns^f	27.0 (3.4)ⁱ	ns^f
Onset symptom (tremor vs. others)	Tremor, n (%)	78 (46.8)	9 (47.4)	ns	15 (51.7)	ns
Modified H-Y on (<3 vs. ≥3)	≥3, n (%)	82 (49.1)	14 (73.7)	0.042	16 (55.2)	ns
UPDRS part 3	Mean (SD)	23.6 (12.2)^j	28.5 (13.8)	ns^f	21.9 (8.7)	ns^f
Wearing off	Yes, n (%)	70 (41.9)	9 (47.4)	ns	13 (44.8)	ns
Dyskinesia	Yes, n (%)	49 (29.3)	8 (42.1)	ns	8 (27.6)	ns
Mood disorder	Yes, n (%)	43 (25.7)	8 (42.1)	ns	7 (24.1)	ns
Orthostatic hypotension symptom	Yes, n (%)	21 (12.6)	5 (26.3)	ns	7 (24.1)	ns
Psychosis history	Yes, n (%)	59 (35.3)	10 (52.6)	ns	7 (24.1)	ns
ICD history	Yes, n (%)	8 (4.8)	1 (5.3)	ns	1 (3.4)	ns
Stereotactic brain surgery for PD	Yes, n (%)	4 (2.4)	0 (0.0)	ns	0 (0.0)	ns
Agonist LED mg/d	Mean (SD)	92.8 (114.2)	72.1 (137.7)	ns^e	163.7 (155.6)	0.026^e
Levodopa LED mg/d	Mean (SD)	400.7 (184.2)	456.7 (206.9)	ns^f	369.2 (230.3)	ns^e
Total LED mg/d	Mean (SD)	496.4 (233.7)	537.9 (258.9)	ns^f	525.7 (287.4)	ns^f

Categorical data were examined by Fisher’s exact test.

Key: DSM-IV, Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition; GBA, glucocerebrosidase gene; GD, Gaucher disease; H-Y, Hoehn and Yahr; ICD, impulse control disorder; LED, levodopa equivalent dose; ns, not significant; MMSE, Mini-Mental State Examination; PD, Parkinson’s disease; SD, standard deviation; UPDRS, Unified Parkinson’s Disease Rating Scale.

a Including a double-mutation subject (with a mutation unreported in GD).

b GD-associated mutations versus mutation (-).

c Two subjects with double mutation, including GD-associated mutations, were assigned to GD-associated mutation group.

d Other mutations versus mutation (-).

e Examined by Student t test after Levene’s test for equality of variances.

f Examined by Mann-Whitney U-test because of non-Gaussian distribution.

g n = 155 due to 10 missing data.

h n = 164 due to 3 missing data.

i n = 28 due to 1 missing datum.

j n = 165 due to 2 missing data.

Table options

3.4. Survival time analyses to develop dementia, psychosis, dyskinesia, and wearing-off

Time to develop clinical outcomes (dementia, psychosis, dyskinesia, and wearing-off) was compared in 19 subjects with GD-associated mutations, 29 with mutations unreported in GD, and 167 without mutation. The median observation time was 6.0 years. The subjects with GD-associated mutations showed a significantly earlier development of dementia and psychosis, compared with subjects without mutation (p < 0.001 and p = 0.017) ( Supplementary Table e-1, Fig. 1A and B). We rereviewed the clinical record of the subject who showed early dementia (defined by DSM IV) ( Fig. 1A) and made sure it did not satisfy the criteria of DLB ( McKeith et al., 2005).

: Fig. 1.

Kaplan–Meier curves of dementia and psychosis in Parkinson’s disease (PD) patients with Gaucher disease (GD)-associated glucocerebrosidase gene (GBA) mutations and those without mutations. PD patients with GD-associated GBA mutations and those without GBA mutations were compared to investigate the time taken to develop dementia (A) and psychosis (B). Because of insufficient information in several patients, the numbers in each analysis were different. The patients with and without mutations were 17 and 165 (A), 18 and 165 (B) against a total of 19 and 167. DSM IV, Diagnostic and Statistical Manual of Mental Disorders, revised fourth edition. p-Values were calculated by log-rank tests.; Figure options

The associations of GBA mutations and these symptoms were estimated as HRs, adjusting for sex and age at PD onset. HRs were 8.3 for dementia (95% CI, 3.3–20.9; p < 0.001) and 3.1 for psychosis (95% CI, 1.5–6.4; p = 0.002). The time until development of wearing-off and dyskinesia complications was not statistically significant, with HRs of 1.5 (95% CI, 0.8–3.1; p = 0.219) and 1.9 (95% CI, 0.9–4.1; p = 0.086) ( Table 3).

Table 3.Hazard ratios of GBA pathogenic mutations for clinical symptoms

Model	Clinical feature	Hazard ratio	95% CI	p
1	Dementia (DSM-IV)	8.3	3.3–20.9	<0.001
2	Psychosis	3.1	1.5–6.4	0.002
3	Wearing-off	1.5	0.8–3.1	0.219
4	Dyskinesia	1.9	0.9–4.1	0.086

Each model was adjusted for sex and age at onset.

Key: CI, confidence interval; DSM-IV; The Diagnostic and Statistical Manual of Mental Disorders part 1IV; GBA, glucocerebrosidase.

Table options

Subjects with mutations unreported in GD did not show significant differences in time to develop all 4 outcomes, compared with no mutation subjects. Therefore, subjects with GD-unreported mutations were regarded as subjects without GBA mutations in further analyses.

3.5. rCBF on SPECT in patients with GD-associated GBA mutations

We conducted pixel-by-pixel comparisons of rCBF on SPECT between PD subjects with mutations (cases) and sex-, age-, and disease duration-matched PD subjects without any mutations in GBA (controls). Four controls were adopted for each case (except for a 34-year-old female case who was matched to a control), and in total 12 cases (female 50%, age at SPECT mean ± standard error (SE); 58.9 ± 3.3 years, disease duration at SPECT 7.3 ± 1.5 years) and 45 controls (female 64.4%, age at SPECT mean ± SE; 61.0 ± 1.3 years, disease duration at SPECT 7.1 ± 0.7 years) were analyzed. As a result, a significantly lower rCBF was seen in the cases compared to the controls in the bilateral parietal cortex, including the precuneus ( Fig. 2).

: Fig. 2.

Regional cerebral blood flow in the group with GD-associated mutations compared with the matched Parkinson’s disease group without mutations. Regions with lower regional cerebral blood flow in the group with GD-associated mutations displayed on an anatomic reference map. Abbreviation: GD, Gaucher disease.; Figure options

3.6. H/M ratios on MIBG scintigraphy in patients with GD-associated GBA mutations

Cardiac MIBG scintigraphy visualizes catecholaminergic terminals in vivo that are reduced as well as brain dopaminergic neurons in PD patients. We also investigated MIBG scintigraphy between 16 cases (female 68.8%, age at examination mean ± SE; 60.2 ± 2.6 years, disease duration at examination 6.2 ± 1.2 years) and sex-, age- and disease duration-matched 61 controls [(63.8 %, age 62.0 ± 1.1 years, disease duration 5.5 ± 0.6 years) (1:4 except for 1 young 34-year-old female case who was matched to a control)]. In the results, both early and late H/M ratios declined in both groups and did not show any significant differences (p = 0.309 and 0.244) ( Supplementary Table e-2).

4. Discussion

4.1. Contributions of GD-associated GBA mutations to the development of PD

In the analysis of 215 PD patients and 126 non-PD controls, we identified 10 nonsynonymous heterozygous GBA mutations, including 1 novel mutation. Among these mutations, 7 were GD-associated, and the patients carrying these mutations represented 8.8% of the PD cohort. No significant association was found between the GD-unreported mutations and PD development, which suggests that only the GD-associated mutations are a genetic risk for PD. According to a worldwide multicenter analysis of 1883 fully sequenced PD patients, 7% of the GD-associated mutations are found in non-Ashkenazi Jewish PD patients ( Sidransky et al., 2009). Although the mutation frequency in the present study was similar to previous results, the OR of GD-associated heterozygous mutations (25.1) was significantly greater than the OR (5.43) of other ethnic cohorts (Sidransky et al., 2009) and was consistent with an OR of 28.0 from a previous Japanese report ( Mitsui et al., 2009). These results, taken together, suggest the possibility thatGBA mutations are at a distinct risk for PD in the Japanese population. However, a larger Japanese cohort study is required to confirm this.

4.2. Cross-sectional clinical figures of PD with GBA mutations

Before the survival time analyses, we investigated clinical features at enrollment between mutation groups. The lower onset age, more frequent family history and dementia, and worse disease severity of PD in patients with GBA mutations, compared with those without mutations, were consistent with previous cross-sectional case-control reports ( Anheim et al., 2012, Brockmann et al., 2011, Chahine et al., 2013, Lesage et al., 2011, Li et al., 2013, Mitsui et al., 2009, Neumann et al., 2009, Seto-Salvia et al., 2012 and Sidransky et al., 2009). In contrast, female-predominance (73.7%, p = 0.047) in patients with mutations observed in the present study is inconsistent ( Neumann et al., 2009 and Seto-Salvia et al., 2012).

4.3. Impact of GBA mutations on the clinical course of PD

To investigate the impact of GBA mutations on the clinical course of PD, a prospective-designed study over a long period is preferred. Although there has been a few longitudinally designed study to date, follow-up clinical data for a median of 6 years of 121 PD cases from a community-based incident cohort was recently reanalyzed; results demonstrate that progression to dementia defined by DSM IV (HR 5.7) and Hoehn and Yahr stage 3 (HR 3.2) are significantly earlier in 4 GBA mutation-carrier patients compared with 117 patients with wild-type GBA ( Winder-Rhodes et al., 2013). A 2-year follow-up clinical report of 28 heterozygous GBA carriers who were recruited from relatives of GD-patients shows slight but significant deterioration of cognition and smelling, compared to healthy controls ( Beavan et al., 2015). Brockmann et al. (2015)assessed motor and nonmotor symptoms including cognitive and mood disturbances for 3 years in 20 PD patients with GBA mutations and showed a more rapid disease progression of motor impairment and cognitive decline in GBA mutation cases comparing to sporadic PD controls. The current long-term retrospective cohort study up to 12 years reinforced these results. It revealed that dementia and psychosis developed significantly earlier in subjects with GD-associated mutations compared with those without mutation, and the HRs of GBA mutations were estimated at 8.3 for dementia and 23.1 for psychosis, with adjustments for sex and PD onset age. In contrast, the results showed no significant difference in developing wearing-off and dyskinesia.

In this study, we also investigated whether GD-unreported mutations affected the clinical course of PD. In both cross-sectional and survival time analyses, the mutations unreported in GD carried no increased burden on clinical symptoms such as dementia, psychosis, wearing-off, and dyskinesia.

4.4. Reduced rCBF in PD with GBA mutations compared with matched PD controls

We found a significantly decreased rCBF, reflecting decreased synaptic activity, in the bilateral parietal cortex including the precuneus, in subjects with GD-associated mutations compared with matched subjects without mutations. The pattern of reduced rCBF was very similar to the pattern of H₂¹⁵O positron-emission tomography that Goker-Alpan et.al. (2012) reported, showing decreased resting rCBF in the lateral parietal association cortex and the precuneus bilaterally in GD subjects with parkinsonism (7 subjects with homozygous or compound heterozygous GBA mutations), compared with 11 PD without GBA mutations. Results suggest that PD with heterozygous GBAmutations and GD patients presenting parkinsonism had a common reduced pattern of rCBF. Interestingly, in their study, rCBF in the precuneus—but not in the lateral parietal cortex—correlated with IQ, suggesting that the involvement of the precuneus is critical for defining GBA-associated patterns.

4.5. Reduced cardiac MIBG H/M ratios as well as matched PD controls

We also showed that cardiac MIBG H/M ratios in subjects with GD-associated mutations were lower than the cutoff point for PD discrimination (Sawada et al., 2009), suggesting that postganglionic sympathetic nerve terminals to the epicardium were denervated, as well as in PD without mutations.

4.6. Mechanisms of impact on PD clinical course by GD-associated GBA mutations

Experimental studies suggesting a bidirectional pathogenic loop between α-synuclein and glucocerebrosidase have been accumulated (Fishbein et al., 2014, Gegg et al., 2012, Mazzulli et al., 2011, Noelker et al., 2015, Schondorf et al., 2014 and Uemura et al., 2015). Loss of glucocerebrosidase function compromises α-synuclein degradation in lysosome, whereas aggregated α-synuclein inhibits normal lysosomal function of glucocerebrosidase. The pathogenic loop may facilitate neurodegeneration in GD-associated PD brain, resulting in early development of dementia or psychosis as shown in the present study. Several recent researches propose the possibility that the similar mechanism as in PD with GBA mutations exists even in idiopathic PD brain ( Alcalay et al., 2015, Chiasserini et al., 2015, Gegg et al., 2012 and Murphy et al., 2014). On the other hand, the impacts of GD-associated GBA mutations for the development of motor complications such as wearing-off and dyskinesia were not statistically significant, suggesting other pathophysiological mechanisms in the striatal circuit brought out after long-term therapy especially by l-dopa.

4.7. Limitations

Our study has several limitations. In the design of the study, we assumed that the sample size was 215 (PD patients) for survival time analyses and investigated 224 PD patients. We assumed that the mutation prevalence would be 9.4%, and in fact, we found 19 patients with mutations (8.5%) of the 224 patients. Based on these figures, we estimated the risk ratios of heterozygous GBA mutations for the risk of PD development and PD clinical symptoms as ORs in the cross-sectional multivariate analyses, although the 95% CIs were broad. More of subject numbers will be needed to determine robust risk ratios.

Comprehensive Genetic Characterization of a Spanish Brugada Syndrome Cohort

Elisabet Selga, Oscar Campuzano, Mel·lina Pinsach-Abuin, Alexandra Pérez-Serra, Irene Mademont-Soler, Helena Riuró, Ferran Picó, [ … ], Ramon Brugada

Published: July 14, 2015 DOI: http://dx.doi.org:/10.1371/journal.pone.0132888

Brugada syndrome (BrS) was identified as a new clinical entity in 1992 [1]. Six years later, the first genetic basis for the disease was identified, with the discovery of genetic variations inSCN5A [2]. Nowadays, more than 300 pathogenic variations in this first gene are known to be associated with BrS [3]. SCN5A encodes for the α subunit of the cardiac voltage-dependent sodium channel (Na_v1.5), which is responsible for inward sodium current (I_Na), and thus plays an essential role in phase 0 of the cardiac action potential (AP). Genetic variations in this gene can explain around 20–25% of BrS cases [3].

Since BrS was classified as a genetic disease, several other genes have been described to confer BrS-susceptibility [4–7]. Pathogenic variations have been mainly described in: 1) genes encoding proteins that modulate Na_v1.5 function, and 2) other calcium and potassium channels and their regulatory subunits. All these proteins participate, either directly or indirectly, in the development of the cardiac AP. Although the incidence of pathogenic variations in these BrS-associated genes is low [6], it is considered that, among all of them, they could provide a genetic diagnosis for up to an extra 5–10% of BrS cases. Hence, altogether, a genetic diagnosis can be achieved approximately in 35% of clinically diagnosed BrS patients.

Other types of genetic abnormalities have been suggested to explain the remaining percentage of undiagnosed patients. Indeed, multiplex ligation-dependent probe amplification (MLPA) has allowed the detection of large-scale gene rearrangements involving one or several exons ofSCN5A in BrS cases. However, the low proportion of BrS patients carrying large genetic imbalances identified to date suggests that this type of rearrangements will provide a genetic diagnosis for a modest percentage of BrS cases [8–10].

BrS has been associated with an increased risk of sudden cardiac death (SCD), despite the reported variability in disease penetrance and expressivity [11]. The prevalence of BrS is estimated at about 1.34 cases per 100 000 individuals per year, with a higher incidence in Asia than in the United States and Europe [12]. However, the dynamic nature of the typical electrocardiogram (ECG) and the fact that it is often concealed, hinder the diagnosis of BrS. Therefore, an exhaustive genetic testing and subsequent family screening may prove to be crucial in identifying silent carriers. A large percentage of these pathogenic variation carriers are clinically asymptomatic, and may be at risk of SCD, which is, sometimes, the first manifestation of the disease [13].
In the present work, we aimed to determine the spectrum and prevalence of genetic variations in BrS-susceptibility genes in a Spanish cohort diagnosed with BrS, and to identify variation carriers among relatives, which would enable the adoption of preventive measures to avoid SCD in their families.

Results

Study population

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Table 1. Demographics of the 55 Spanish BrS patients included in the study.

The table shows the demographic characteristics of all the patients included in the study. Numbers in parentheses represent the relative percentages for each condition. T1 ECG refers to Type 1 BrS diagnostic electrocardiogram (ECG), obtained either spontaneously, or after drug challenge. The information regarding both the electrophysiological studies (EPS) and the treatment was not available for all the patients. Two of the patients that didn’t receive any treatment died, and were not taken into account for the calculations of percentages (+2 dead). ICD, intracardiac cardioverter defibrillator.

http://dx.doi.org:/10.1371/journal.pone.0132888.t001

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Table 2. Characteristics of the Spanish BrS patients carrying rare genetic variations.

The table shows the clinical characteristics of the probands who carried rare genetic variations in SCN5A, SCN2B, or RANGRF. All of them are potentially pathogenic except that found in RANGRF, which is of unknown significance (see discussion). All the potentially pathogenic variations (PPVs) that had been previously reported, except p.P1725L and p.R1898C, had been identified in BrS patients. p.P1725L had been associated with Long QT Syndrome and p.R1898C was found in Exome Variant Server with a MAF of 0.0079%. No rare variations were identified in the control population. Patient’s age is expressed in years. Bold identifies the patients carrying variations that had not been described previously. M, male; F, female; S, syncope; ICD, intracardiac cardioverter defibrillator; UK, unknown; EPS, electrophysiological studies (+, positive response;-, negative response; N/P, not performed). The two patients who carried two PPVs each are identified by ^a and ^b, respectively.

http://dx.doi.org:/10.1371/journal.pone.0132888.t002

Sequencing of genes associated with BrS

We performed a genetic screening of 14 genes (SCN5A, CACNA1C, CACNB2, GPD1L,SCN1B, SCN2B, SCN3B, SCN4B, KCNE3, RANGRF, HCN4, KCNJ8, KCND3, and KCNE1L), which allowed the identification of 61 genetic variations in our cohort. Of these, 20 were classified as potentially pathogenic variations (PPVs), one variation of unknown significance, and 40 common or synonymous variants considered benign.

The 20 PPVs were found in 18 of the 55 patients (32.7% of the patients, 83.3% males; Table 2). Sixteen patients (88.9%) carried one PPV, and two patients (11.1%) carried two different PPVs each. Nineteen out of the 20 PPVs identified were localized in SCN5A and one in SCN2B.

The vast majority of the PPVs identified were missense (70%). We also detected 2 nonsense variations (10%), 3 insertions or deletions causing frameshifts (15%), and one splicing variation (5%). The three frameshifts (p.R569Pfs*151, p.E625Rfs*95 and p.R1623Efs*7) were identified in SCN5A. These were not found in any of the databases consulted (see Methods), and were thus considered potentially pathogenic (see below). The other 16 rare variations identified inSCN5A had been previously described, and hence were also considered potentially pathogenic. Fourteen of them had been identified in BrS patients. Of these, 6 had also been identified in individuals diagnosed with other cardiac electric diseases (i.e. Sick Sinus Syndrome, Long QT Syndrome, Sudden Unexplained Nocturnal Death Syndrome or Idiopathic Ventricular Fibrillation [2,15,16,20,21,25]). The other 2, p.P1725L and p.R1898C, had only been associated with Long QT Syndrome or found in Exome Variant Server with a MAF of 0.0079%, respectively. Furthermore, we identified a variation in SCN2B (c.632A>G in exon 4 of the gene, resulting in p.D211G) which was considered pathogenic. This patient was included within our cohort, but the functional characterization of channels expressing SCN2B p.D211G was object of a previous study from our group [7]. We also identified a nonsense variation in RANGRFwhich has been formerly reported as rare genetic variation of unknown significance [29].

Additionally, we screened the relatives of those probands carrying a PPV. We analysed a total of 129 relatives, 69 of which (53.5%) were variation carriers. Genotype-phenotype correlations evidenced that 8 of the families displayed complete penetrance (S3 Table). Additionally, no relatives were available for one of the probands carrying a PPV, thus hampering genotype-phenotype correlation assessment. The other 12 families showed incomplete penetrance.

MLPA analysis

The 37 patients with negative results after the genetic screening of the 14 BrS-associated genes underwent MLPA analyses of SCN5A. This technique did not reveal any large exon deletion or duplication in this gene for any of the patients.

SCN5A p.R569Pfs*151 (c.1705dupC), a novel PPV

A 41-year-old asymptomatic male presented a type 3 BrS ECG which was suggestive of BrS. Flecainide challenge unmasked a type 1 BrS ECG (Fig 1A, left), which was also spontaneously observed sometimes during medical follow up. Sequencing of SCN5A revealed a duplication of a cytosine at position 1705 (c.1705dupC; Fig 1A, right), which originated a frameshift that lead to a truncated Na_v1.5 channel (p.R569Pfs*151). The proband’s sister also carried this duplication, but had never presented signs of arrhythmogenesis. The proband’s twin daughters were also variation carriers, displayed normal ECGs and, to date, are asymptomatic (Fig 1A, middle). Thus, p.R569Pfs*151 represents a novel genetic alteration in the Na_v1.5 channel that could potentially lead to BrS, but with incomplete penetrance.

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Fig 1. Characteristics of the probands carrying non-reported potentially pathogenic variations (PPVs) in SCN5A and their families.

Left: Electrocardiograms of the probands: (A) patient carrying the p.R569Pfs*151 variation, showing the ST elevation characteristic of BrS in V1 at the time of the flecainide test; (B) patient carrying the p.E625Rfs*95 variation, showing the spontaneous ST elevation characteristic of BrS in V1 and V2; and (C) patient carrying the p.R1623Efs*7 variation, showing the spontaneous ST elevation characteristic of BrS in V1 and V2. Middle: Family pedigrees. Open symbols designate clinically normal subjects, filled symbols mark clinically affected individuals and question marks identify subjects without an available clinical diagnosis. Plus signs indicate the carriers of the PPVs and minus signs, non-carriers. The crosses mark deceased individuals and arrows identify the proband. Right: Detail of the electropherograms obtained after SCN5Asequence analysis of a control subject (left panels) and of the probands (right panels).

http://dx.doi.org:/10.1371/journal.pone.0132888.g001

SCN5A p.E625Rfs*95 (c.1872dupA), a novel PPV

A 51-year-old asymptomatic male was diagnosed with BrS since he presented a spontaneous ST segment elevation in leads V1 and V2 characteristic of type 1 BrS ECG (Fig 1B, left). The sequencing of SCN5A evidenced an adenine duplication at position 1872 (c.1872dupA, Fig 1B, right). This genetic variation results in a truncated Na_v1.5 channel (p.E625Rfs*95). The genetic analysis of the proband’s relatives proved that only her mother carried the variation (Fig 1B, middle). She was asymptomatic, but a BrS ECG was unmasked upon ajmaline challenge. The proband’s sister was found dead in her crib at 6 months of age, which suggests that her death might be compatible with BrS. Therefore, the p.E625Rfs*95 variation in the Na_v1.5 channel represents a novel genetic alteration potentially causing BrS.

SCN5A p.R1623Efs*7 (c.4867delC), a novel PPV

The proband, a 31-year-old male, was admitted to hospital after suffering a syncope. His baseline 12-lead ECG showed a ST segment elevation in leads V1 and V2 that strongly suggested BrS type 1 (Fig 1C, left). A deletion of the cytosine at position 4867 (c.4867delC) was observed upon SCN5A sequencing (Fig 1C, right). This base deletion leads to a frameshift that originates a truncated Na_v1.5 channel (p.R1623Efs*7). Genetic screening of his parents and sisters evidenced that none of them carried this novel variation (Fig 1C, middle). None of them had presented any signs of arrhythmogenicity, nor had a BrS ECG. Nevertheless, in uterogenetic analysis of one of his daughters proved that she had inherited the variation. She died when she was 1 year of age of non-arrhythmogenic causes. Hence, the p.R1623Efs*7 variation in the Na_v1.5 channel is a novel genetic alteration originated de novo in the proband that could potentially lead to BrS.

Synonymous and common genetic variations portrayal

In our cohort, we identified 40 single nucleotide variations which were common genetic variants and/or synonymous variants (S2 Table). Twenty-nine had a minor allele frequency (MAF) over 1%, and were thus considered common genetic variants.

We also identified 11 variants with MAF less than 1%. Of them, 9 were synonymous variants, what made us assume that they were not disease-causing. Four of these synonymous variants were not found in any of the databases consulted, and thus their MAF was considered to be less than 1%. Each of these synonymous variations was identified in 1 patient of the cohort. A similar proportion of individuals carrying these novel variations was detected upon sequencing of 300 healthy Spanish individuals (600 alleles). The remaining 2 variants were missense, and although they had either a MAF of less than 1% or an unknown MAF according to the Exome Variant Server and dbSNP websites, they were common in our cohort (29.2 and 50%, respectively; S2 Table), and a similar MAF was detected in a Spanish cohort of healthy individuals (26.7% and 48.8%, respectively).

Influence of phenotype and age on PPV discovery

To assess if a connection existed between the probands’ phenotype and the PPV detection yield, we classified the patients in our cohort according to their ECG (spontaneous or induced type 1), the presence of BrS cases within their families, and the presence/absence of symptoms. Even though the overall PPV detection yield was 32.7%, it was even higher for symptomatic patients (Fig 2). Indeed, in this group of patients, having a family history of BrS was identified as a factor for increased PPV discovery yield. In the case of absence of BrS in the family, the variation discovery yield was almost double for those patients having a spontaneous type 1 BrS ECG than for patients with drug-induced type 1 ECG (45.5% vs 25%, respectively). In addition, we identified a PPV in 44.4% of the asymptomatic patients who presented family history of BrS and a spontaneous type 1 BrS ECG. When the patient presented drug-induced type 1 ECG or in the absence of family history of BrS, the PPV discovery yield was of around 15%.

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Fig 2. Influence of the phenotype on PPV discovery yield.

Bar graph comparing the PPV detection yield in 8 different clinical categories (stated below the graph). Each bar shows the total number of patients for each clinical category divided in those with a PPV (black) and those without an identified PPV (white). The number of patients (in brackets) and percentages are given. Pos, positive; Neg, negative; Spont, spontaneous type 1 BrS ECG; Drug, drug-induced type 1 BrS ECG; n, number of patients.

http://dx.doi.org/:10.1371/journal.pone.0132888.g002

We also investigated the role of age on the PPV occurrence. No significant age differences were observed between variation carriers and non-carriers (38.6±10.3 and 43.5±14.4, respectively, p = 0.16). However, the PPV discovery yield was higher for patients with ages between 30 and 50 years: out of the total of patients carrying a PPV, 83.3% of the patients were in this age range, while 11.1% were younger and 5.6% were older patients (Fig 3A, upper panel). The PPV discovery yield was significantly higher for symptomatic than for asymptomatic patients (42.3% vs 24.1%, respectively; Fig 3A, lower panels).

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Fig 3. Influence of the age on PPVs discovery yield.

(A) Pie charts showing the distribution of patients in the overall population as well as in the categories of symptomatic and asymptomatic patients regarding PPV discovery. The percentage and the number of patients (in brackets) are given for each group. The small pie charts correspond to the age distribution of patients with an identified PPV. (B) Bar graphs of the PPV detection yields obtained for each of the age groups (< 30 years, 30–50 years and > 50 years). Numbers inside each bar correspond to the number of patients carrying a PPV for each category and the percentages represent the variation detection yield.

http://dx.doi.org:/10.1371/journal.pone.0132888.g003

Noteworthy, in the 30–50 age range, 52.9% (9/17) of the symptomatic patients and 35.3% (6/17) of asymptomatic patients carried one PPV (Fig 3B, middle). Additionally, 40% (2/5) of the symptomatic young patients (< 30 years) were variation carriers, while no PPVs were identified in asymptomatic patients within this age range.

Overall, 55 unrelated Spanish patients clinically diagnosed with BrS were included in our study.Table 1 shows the demographics of this cohort, and Table 2 and S1 Table show the clinical and genetic characteristics of all the patients included in the study. The mean age at clinical diagnosis was of 41.9±13.3 years. Although the majority of patients were males (74.5%), their age at diagnosis was not different than that of females (41.8±12.1 years and 42.3±16.3 years, respectively; p = 0.92). A type 1 BrS ECG was present spontaneously in 37 patients (67.3%), and drug challenge revealed a type 1 BrS ECG for the remaining 18 patients (32.7%). Almost half of the patients had experienced symptoms, including 2 SCD and 4 aborted SCD. Patients who had not previously experienced any signs of arrhythmogenicity despite having a BrS ECG were considered asymptomatic. Comparison of symptomatic vs asymptomatic patients evidenced a similar percentage of males (73.1% and 75.9%, respectively). However, the mean age at diagnosis was different between the two groups of patients (37.7±14.3 and 45.7±11.4, respectively; p<0.05).

Discussion

To the best of our knowledge, this is the first comprehensive genetic evaluation of 14 BrS-susceptibility genes and MLPA of SCN5A in a Spanish cohort. Well delimited BrS cohorts from Japan, China, Greece and even Spain have been genetically studied [24,30–32]. Additionally, an international compendium of BrS genetic variations identified in more than 2100 unrelated patients from different countries was published in 2010 [3]. However, all these studies screenedSCN5A exclusively. In 2012, Crotti et al. reported the spectrum and prevalence of genetic variations in 12 BrS-susceptibility genes in a BrS cohort [5]. However, this study included patients of different ethnicity. Here, we report the analysis of 14 genes which has been conducted on a well-defined BrS cohort of the same ethnicity.

Our results confirm that SCN5A is still the most prevalent gene associated with BrS. Indeed,SCN5A-mediated BrS in our cohort (30.9%) is higher than the proportion described in other European reports [3,23], where a potentially causative variation is identified in only 20–25% of BrS patients. The reason for this discrepancy is unclear but could point towards a higher prevalence of SCN5A PPVs in the Spanish population or to selection bias. Additionally, we identified a genetic variation in SCN2B (c.632A>G, which results in p.D211G). We have formerly published the comprehensive electrophysiological characterization of this variation, and showed that indeed this variation could be responsible of the phenotype of the patient, thus linking SCN2B with BrS for the first time [7]. Also, we identified a variation in RANGRF. This variation (c.181G>T leading to p.E61X) had been previously reported in a Danish atrial fibrillation cohort [33]. Surprisingly, the authors reported an incidence of 0.4% for this variation in the healthy Danish population, which brought into question its pathogenicity. Our finding of this variation in an asymptomatic patient displaying a type 2 BrS ECG also points toward considering it as a rare genetic variation with a potential modifier effect on the phenotype but not clearly responsible for the disease [29].

No PPVs were identified in the other genes tested. Certainly, it is well accepted that the contribution of these genes to the disease is minor, and thus should only be considered under special circumstances [13,34]. In addition, recent studies have questioned the causality of variations identified in some of these minority genes [35].

We also used the MLPA technique for the detection of large exon duplications and/or deletions in SCN5A in patients without PPVs, and no large rearrangements were identified. This is in accordance with previous reports, which revealed that such imbalances are uncommon [8–10].

Kapplinger et al. [3] reported a predominance of PPVs in transmembrane regions of Na_v1.5. Indeed, it has been proposed that most rare genetic variations in interdomain linkers may be considered as non-pathogenic [36]. In contrast, PPVs identified in this study are mainly located in extracellular loops and cytosolic linker regions of Na_v1.5 (Fig 4). Additionally, 2 of our non-previously reported frameshifts are located in the DI-DII linker. These 2 genetic variations lead to truncated proteins, which would lack around 75% of the protein sequence, and thus are presupposed to be pathogenic.

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Fig 4. Na_v1.5 channel scheme showing the relative position of the SCN5A PPVs identified in our cohort.

Open symbols indicate already described variations and closed symbols locate novel variations reported in this study. DI to DIV designate the 4 domains of the protein, and numbers 1–6 identify the different segments within each domain. Crosses mark the voltage sensor.

http://dx.doi.org:/10.1371/journal.pone.0132888.g004

In our cohort, we have identified 40 synonymous or common genetic variations, 4 of which have not been previously reported. These variations are gradually becoming more and more important in the explanation of certain phenotypes of genetic diseases. Only a few common variations identified here are already published as phenotypic modifiers [37,38]. The effect of these and other common variants identified in our cohort on BrS phenotype should be further studied.

Unexpectedly, almost 40% (7/18) of the PPV carriers did not present signs of arrhythmogenicity. We also performed genotype-phenotype correlations of the PPVs identified in the families (S3 Table). These studies uncovered relatives, most of whom were young individuals, who carried a familial variation but had never exhibited any clinical manifestations of the disease. This is in agreement with Crotti et al. and Priori et al. [5,23], who postulated that a positive genetic testing result is not always associated with the presence of symptoms. Indeed, the existence of asymptomatic patients carrying genetic variations described to cause a severe Na_v1.5 channel dysfunction has been reported [39]. The identification of silent carriers is of paramount importance since it allows the adoption of preventive measures before any lethal episode takes place. Unknown environmental factors, medication and modifier genes have been suggested to influence and/or predispose to arrhythmogenesis [11]. Hence, this group of patients has to be cautiously followed in order to avoid fatal events.

Our studies on the connection between patients’ phenotype and the PPV detection yield highlighted the presence of symptoms as a factor for an increased variation discovery yield. Within the group of symptomatic individuals, a PPV was identified in a higher proportion of patients displaying a spontaneous type 1 BrS ECG than for patients showing a drug-induced ECG. Likewise, within the asymptomatic patients with family history of BrS, those who presented spontaneous type 1 BrS ECG carried a PPV more often than those with a drug-induced ECG (Fig 2). Referring to age, the vast majority (17/20, 85%) of the PPVs were identified in patients around their fourth decade of age (30–50 years). This is in accordance with the accepted mean age of disease manifestation. Moreover, in this age range, more than 50% of the patients who presented symptoms carried a variation that could be pathogenic (Fig 3). Importantly, 35.3% of asymptomatic patients of around 40 years of age also carried one of such variations. These data highlight the importance of performing a genetic test even in the absence of clinical manifestations of the disease, and particularly when in the 30–50 years range, which is in accordance with consensus recommendations [13,34].

In conclusion, we have analysed for the first time 14 BrS-susceptibility genes and performed MLPA of SCN5A in a Spanish BrS cohort. Our cohort showed male prevalence with a mean age of disease manifestation around 40 years. BrS in this cohort was almost exclusivelySCN5A-mediated. The mean PPV discovery yield in our Spanish BrS patients is higher than that described for other BrS cohorts (32.7% vs 20–25%, respectively), and is even higher for patients in the 30–50 years age range (up to 53% for symptomatic patients). All these evidences support the genetic testing, at least of SCN5A, in all clinically well diagnosed BrS patients.

Study Limitations

First of all, drug challenge tests were not performed for all the relatives who were asymptomatic variation carriers. This fact hampered their clinical diagnosis and represents an impediment to definitely assess the link between PPVs and BrS. These patients are nowadays under follow-up.

New PPVs have been identified in our cohort. The clinical information available for the families suggests that these new variations could be pathogenic. Still, in vitro studies of these variations are required in order to evaluate their functional effects and verify their pathogenic role. Additionally, genotyping in an independent cohort would help reduce the likelihood of type I (false positive) error in genetic variant discovery.

We have to acknowledge that the study set is relatively small. Consequently, the classification of patients according to the different clinical categories rendered rather small sub-groups, which may lead to over-interpretation of the results. Future studies will be directed to the genetic screening of additional Spanish BrS patients, which will probably reinforce the significance of the tendencies observed here.

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Supportive Treatments: Hold the Mind Strong During Cancer

Posted in Behavior, CANCER BIOLOGY & Innovations in Cancer Therapy, Cell Biology, Signaling & Cell Circuits, Clinical & Translational, Clinical Diagnostics, Cognition, Neurological Diseases, Neuroscience, tagged Demet Sag, Dr. Demet Sag, Functional TransGenomics on November 3, 2015| Leave a Comment »

Supportive Treatments: Hold the Mind Strong During Cancer

Demet Sag, PhD

Psychiatric treatments

Cancer is described under a general terminology of uncontrolled cell proliferation and changes that results in out of control development. Thus, correcting the cell division and immune control are the two focus areas. Yet, on the other side of the coin like any given terminal diseases there is another big factor that needs to be resolved that is mental health. This is usually not well discussed among many. After all fighting with a disease is a game of strength. I think that is one of the reason we say congrats to many cancer survivors since they won not only with their treatment but also with their psychological strength. However, it is like a balloon after the disease the battle is still on.

Here are the few articles discussing mainly advanced cancer patient’s psychiatric conditions, their clinical treatments, and training of the healthcare givers including oncologists, nurses, social workers, and other ancillary staff.

Last fifty years there is an improvement to cure mental illnesses yet there are many unresolved issues like passing blood brain barrier or specificity etc. Many of these drugs also used for the adjuvant treatment of cancer-related symptoms. Some of these are pain, hot flashes, pruritus, nausea and vomiting, fatigue, and cognitive impairment. However, the condition of cancer patient requires making psychopharmacology to improve quality life of cancer patients.

There new drugs with less side-effects and safer pharmacological profiles, has been a major advance in clinical psycho-oncology.

Since at least 25-30% of patients with cancer and an even higher percentage of patients in an advanced phase of illness meet the criteria for a psychiatric diagnosis, including depression, anxiety, stress-related syndromes, adjustment disorders, sleep disorders and delirium.

About 50% of patients with advanced cancer meet criteria for a psychiatric disorder, the most common being adjustment disorders (11%-35%) and major depression (5%-26%).

At least 30-40% of patients with cancer and even a higher percentage of patients in an advanced phase of illness.

In addition, age is a big issue since the outcomes and treatments changes based on expectations and challenges in their life. It is now possible to diagnose early and treat more means tolerance level to aggressive treatments also increases. In older patients aging and cancer and in younger patient’s career and relationships broken. This is not just a longevity but improving the quality of life of a patient after cancer’s transition from likely death to survival. Therefore, it is equally important to give their life back fully so there is an increased awareness on psychosocial issues and quality of life.

For example, there is a Psycho-oncology group in National Cancer Center. They are now conducting several clinical studies such as biological studies (neuro-imaging studies), studies to establish novel treatment strategy (n-3 poly unsaturated fatty acid), and multi-faceted intervention study (screening and individually tailored psychotherapy and pharmacotherapy). Hope to see more studies combining not only treat the physiological symptoms but psychological factors.

Table 1. Prevalence of Psychiatric Disorders in Advanced Cancer
	Advanced disease	Terminal illness	Caregivers
Adjustment disorder	14%–34.7%	10.6%–16.3%	—
Anxiety disorders
Generalized anxiety	3.2%–5.3%	5.80%	3.50%
Panic disorder	4.20%	5.50%	8.00%
Post-traumatic stress	2.40%	0%	4.00%
Unspecified	—	4.70%	—
Any	6%–8.2%	13.90%	—

References:

PMID: 26012508

Mehta RD¹, Roth AJ². Psychiatric considerations in the oncology setting. CA Cancer J Clin. 2015 Jul-Aug;65(4):300-14. doi: 10.3322/caac.21285. Epub 2015 May 26.

PMID:23949568

Caruso R¹, Grassi L, Nanni MG, Riba M. Psychopharmacology in psycho-oncology. Curr Psychiatry Rep. 2013 Sep;15(9):393. doi: 10.1007/s11920-013-0393-0.

PMID: 24716500

Grassi L¹, Caruso R, Hammelef K, Nanni MG, Riba M. Efficacy and safety of pharmacotherapy in cancer-related psychiatric disorders across the trajectory of cancer care: a review. Int Rev Psychiatry. 2014 Feb;26(1):44-62. doi: 10.3109/09540261.2013.842542.

PMID: 17847017

Miovic M¹, Block S. Psychiatric disorders in advanced cancer. Cancer. 2007 Oct 15;110(8):1665-76.

PMID: 15387268

Akechi T¹, Nakano T, Uchitomi Y. [Scientific background of psycho-oncology]. Seishin Shinkeigaku Zasshi. 2004;106(6):764-71. [Article in Japanese]

PMID: 25417593

Thekdi SM¹, Trinidad A, Roth A. Psychopharmacology in cancer.

Curr Psychiatry Rep. 2015 Jan;17(1):529. doi: 10.1007/s11920-014-0529-x.

References for the table treatment:

Holland JC,Morrow GR,Schmale A, et al. A randomized clinical trial of alprazolam versus progressive muscle relaxation in cancer patients with anxiety and depressive symptoms. J Clin Oncol. 1991; 9: 1004–1011.

Razavi D,Kormoss N,Collard A,Farvacques C,Delvaux N. Comparative study of the efficacy and safety of trazodone versus clorazepate in the treatment of adjustment disorders in cancer patients: a pilot study. J Int Med Research. 1999; 27: 264–272.

Pugliese P,Perrone M,Nisi E, et al. An integrated psychological strategy for advanced colorectal cancer patients. Health Qual Life Outcomes. 2006; 4: 9.

Kornblith AB,Dowell JM,Herndon JE2nd, et al. Telephone monitoring of distress in patients aged 65 years or older with advanced stage cancer: a cancer and leukemia group B study. Cancer. 2006; 107: 2706–2714.

Goodwin PJ,Leszcz M,Ennis M, et al. The effect of group psychosocial support on survival in metastatic breast cancer. N Engl J Med. 2001; 345: 1719–1726. Web of Science® Times Cited: 249

Classen C,Butler LD,Koopman C, et al. Supportive-expressive group therapy and distress in patients with metastatic breast cancer: a randomized clinical intervention trial. Arch Gen Psychiatry. 2001; 58: 494–501. Web of Science® Times Cited: 156

Pirl WF,Siegel GI,Goode MJ,Smith MR. Depression in men receiving androgen deprivation therapy for prostate cancer: a pilot study. Psychooncology. 2002; 11: 518–523. Web of Science® Times Cited: 43

Chochinov HM,Wilson KG,Enns M, et al. Desire for death in the terminally ill. Am J Psychiatry. 1995; 152: 1185–1191. Web of Science® Times Cited: 309

CA Cancer J Clin. 2015 Jul-Aug;65(4):300-14. doi: 10.3322/caac.21285. Epub 2015 May 26.

Hanna A,Sledge G,Mayer ML, et al. A phase II study of methylphenidate for the treatment of fatigue. Support Care Cancer. 2006;14: 210–215. Web of Science® Times Cited: 18

Dalton SO,Johansen C,Mellemkjaer L, et al. Antidepressant medications and risk for cancer. Epidemiology. 2000; 11: 171–176.PubMed,Web of Science® Times Cited: 31

Pitman RK,Lanes DM,Williston SK, et al. Psychophysiologic assessment of posttraumatic stress disorder in breast cancer patients.Psychosomatics. 2001; 42: 133–140. Web of Science® Times Cited: 21

Derogatis LR,Morrow GR,Fetting J, et al. The prevalence of psychiatric disorders among cancer patients. JAMA. 1983; 249:751–757.PubMed, Web of Science® Times Cited: 837

Travado L,Grassi L,Gil F,Ventura C,Martins C;Southern European Psycho-Oncology Study Group. Physician-patient communication among Southern European cancer physicians: the influence of psychosocial orientation and burnout.Psychooncology. 2005; 14: 661–670. Web of Science® Times Cited: 16

“CREATE Trial Providing Valuable Information on Epoetin Treatment for Anemia.” Hematology Week August 25, 2003: 10.

“Doubts Over Epoetin in Cancer.” SCRIP World Pharmaceutical News October 24, 2003: 24.

“Researcher Working on Medical Patch to Deliver Marijuanalike Drug.” Cancer Weekly September 9, 2003: 126.

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Advances in acoustics and in learning

Posted in Behavior, Biological Networks, Cell Biology, Signaling & Cell Circuits, Cerebrovascular and Neurodegenerative Diseases, Clinical & Translational, Curation, Human Sensation and Cellular Transduction: Physiology and Therapeutics, Innovations, Innovations in Neurophysiology & Neuropsychology, Neurodegenerative Diseases, Neurological Diseases, Neuroscience, tagged acoustics, Aging, Cognition, mechanosensation, memory loss on October 31, 2015| Leave a Comment »

Advances in acoustics and in learning

Larry H. Brnstein, MD, FCAP, Curator

LPBI

Controlling acoustic properties with algorithms and computational methods

http://www.kurzweilai.net/controlling-acoustic-properties-with-algorithms-and-computational-methods

October 28, 2015

Computer scientists at Columbia Engineering, Harvard, and MIT have demonstrated that acoustic properties — both sound and vibration — can be controlled by 3D-printing specific shapes.

They designed an optimization algorithm and used computational methods and digital fabrication to alter the shape of 2D and 3D objects, creating what looks to be a simple children’s musical instrument — a xylophone with keys in the shape of zoo animals.

Practical uses

“Our discovery could lead to a wealth of possibilities that go well beyond musical instruments,” says Changxi Zheng, assistant professor of computer science at Columbia Engineering, who led the research team.

“Our algorithm could lead to ways to build less noisy computer fans and bridges that don’t amplify vibrations under stress, and advance the construction of micro-electro-mechanical resonators whose vibration modes are of great importance.”

Zheng, who works in the area of dynamic, physics-based computational sound for immersive environments, wanted to see if he could use computation and digital fabrication to actively control the acoustical property, or vibration, of an object.

Zheng’s team decided to focus on simplifying the slow, complicated, manual process of designing “idiophones” — musical instruments that produce sounds through vibrations in the instrument itself, not through strings or reeds.

The surface vibration and resulting sounds depend on the idiophone’s shape in a complex way, so designing the shapes to obtain desired sound characteristics is not straightforward, and their forms have so far been limited to well-understood designs such as bars that are tuned by careful drilling of dimples on the underside of the instrument.

Optimizing sound properties

To demonstrate their new technique, the team settled on building a “zoolophone,” a metallophone with playful animal shapes (a metallophone is an idiophone made of tuned metal bars that can be struck to make sound, such as a glockenspiel).

What happens in the brain when we learn

http://www.kurzweilai.net/what-happens-in-the-brain-when-we-learn

Findings could enhance teaching methods and lead to treatments for cognitive problems

October 28, 2015

A Johns Hopkins University-led research team has proven a working theory that explains what happens in the brain when we learn, as described in the current issue of the journal Neuron.

More than a century ago, Pavlov figured out that dogs fed after hearing a bell eventually began to salivate when they heard the bell ring. The team looked into the question of how Pavlov’s dogs (in “classical conditioning”) managed to associate an action with a delayed reward to create knowledge. For decades, scientists had a working theory of how it happened, but the team is now the first to prove it.

“If you’re trying to train a dog to sit, the initial neural stimuli, the command, is gone almost instantly — it lasts as long as the word sit,” said neuroscientist Alfredo Kirkwood, a professor with the university’s Zanvyl Krieger Mind/Brain Institute. “Before the reward comes, the dog’s brain has already turned to other things. The mystery was, ‘How does the brain link an action that’s over in a fraction of a second with a reward that doesn’t come until much later?’ ”

Eligibility traces

The working theory — which Kirkwood’s team has now validated experimentally — is that invisible “synaptic eligibility traces” effectively tag the synapses activated by the stimuli so that the learning can be cemented with the arrival of a reward. The reward is a neuromodulator* (neurochemical) that floods the dog’s brain with “good feelings.” Though the brain has long since processed the “sit” command, eligibility traces in the synapse respond to the neuromodulators, prompting a lasting synaptic change, a.k.a. “learning.”

The team was able to prove the eligibility-traces theory by isolating cells in the visual cortex of a mouse. When they stimulated the axon of one cell with an electrical impulse, they sparked a response in another cell. By doing this repeatedly, they mimicked the synaptic response between two cells as they process a stimulus and create an eligibility trace.

When the researchers later flooded the cells with neuromodulators, simulating the arrival of a delayed reward, the response between the cells strengthened (“long-term potentiation”) or weakened (“long-term depression”), showing that the cells had “learned” and were able to do so because of the eligibility trace.

“This is the basis of how we learn things through reward,” Kirkwood said, “a fundamental aspect of learning.”

In addition to a greater understanding of the mechanics of learning, these findings could enhance teaching methods and lead to treatments for cognitive problems, the researchers suggest.

Scientists at the University of Texas at Houston and the University of California, Davis were also involved in the research, which was supported by grants from JHU’s Science of Learning Institute and National Institutes of Health.

* The neuromodulators tested were norepinephrine, serotonin, dopamine, and acetylcholine, all of which have been implicated in cortical plasticity (ability to grow and form new connections to other neurons).

Abstract of Distinct Eligibility Traces for LTP and LTD in Cortical Synapses

In reward-based learning, synaptic modifications depend on a brief stimulus and a temporally delayed reward, which poses the question of how synaptic activity patterns associate with a delayed reward. A theoretical solution to this so-called distal reward problem has been the notion of activity-generated “synaptic eligibility traces,” silent and transient synaptic tags that can be converted into long-term changes in synaptic strength by reward-linked neuromodulators. Here we report the first experimental demonstration of eligibility traces in cortical synapses. We demonstrate the Hebbian induction of distinct traces for LTP and LTD and their subsequent timing-dependent transformation into lasting changes by specific monoaminergic receptors anchored to postsynaptic proteins. Notably, the temporal properties of these transient traces allow stable learning in a recurrent neural network that accurately predicts the timing of the reward, further validating the induction and transformation of eligibility traces for LTP and LTD as a plausible synaptic substrate for reward-based learning.

references:

Kaiwen He, Marco Huertas, Su Z. Hong, XiaoXiu Tie, Johannes W. Hell, Harel Shouval, Alfredo Kirkwood. Distinct Eligibility Traces for LTP and LTD in Cortical Synapses. Neuron (2015); DOI: 10.1016/j.neuron.2015.09.037

Holographic sonic tractor beam lifts and moves objects using soundwaves

Another science-fiction idea realized

October 27, 2015

British researchers have built a working Star-Trek-style “tractor beam” — a device that can attract or repel one object to another from a distance. It uses high-amplitude soundwaves to generate an acoustic hologram that can grasp and move small objects.

The technique, published in an open-access paper in Nature Communications October 27, has a wide range of potential applications, the researchers say. A sonic production line could transport delicate objects and assemble them, all without physical contact. Or a miniature version could grip and transport drug capsules or microsurgical instruments through living tissue.

The device was developed at the Universities of Sussex and Bristol in collaboration with Ultrahaptics.

https://youtu.be/wDzhlW-rKvM
University of Sussex | Levitation using sound waves

The researchers used an array of 64 miniature loudspeakers. The whole system consumes just 9 Watts of power, used to create high-pitched (40Khz), high-intensity sound waves to levitate a spherical bead 4mm in diameter made of expanded polystyrene.

The tractor beam works by surrounding the object with high-intensity sound to create a force field that keeps the objects in place. By carefully controlling the output of the loudspeakers, the object can be held in place, moved, or rotated.

Three different shapes of acoustic force fields work as tractor beams: an acoustic force field that resembles a pair of fingers or tweezers; an acoustic vortex, the objects becoming trapped at the core; and a high-intensity “cage” that surrounds the objects and holds them in place from all directions.

Previous attempts surrounded the object with loudspeakers, which limits the extent of movement and restricts many applications. Last year, the University of Dundee presented the concept of a tractor beam, but no objects were held in the ray.

The team is now designing different variations of this system. A bigger version aims at levitating a soccer ball from 10 meters away and a smaller version aims at manipulating particles inside the human body.

https://youtu.be/g_EM1y4MKSc
Asier Marzo, Matt Sutton, Bruce Drinkwater and Sriram Subramanian | Acoustic holograms are projected from a flat surface and contrary to traditional holograms, they exert considerable forces on the objects contained within. The acoustic holograms can be updated in real time to translate, rotate and combine levitated particles enabling unprecedented contactless manipulators such as tractor beams.

Abstract of Holographic acoustic elements for manipulation of levitated objects

Sound can levitate objects of different sizes and materials through air, water and tissue. This allows us to manipulate cells, liquids, compounds or living things without touching or contaminating them. However, acoustic levitation has required the targets to be enclosed with acoustic elements or had limited maneuverability. Here we optimize the phases used to drive an ultrasonic phased array and show that acoustic levitation can be employed to translate, rotate and manipulate particles using even a single-sided emitter. Furthermore, we introduce the holographic acoustic elements framework that permits the rapid generation of traps and provides a bridge between optical and acoustical trapping. Acoustic structures shaped as tweezers, twisters or bottles emerge as the optimum mechanisms for tractor beams or containerless transportation. Single-beam levitation could manipulate particles inside our body for applications in targeted drug delivery or acoustically controlled micro-machines that do not interfere with magnetic resonance imaging.

references:

Asier Marzo, Sue Ann Seah, Bruce W. Drinkwater, Deepak Ranjan Sahoo, Benjamin Long, Sriram Subramanian. Holographic acoustic elements for manipulation of levitated objects. Nature Communications, 2015; 6: 8661 DOI: 10.1038/ncomms9661 (open access)

A drug-delivery technique to bypass the blood-brain barrier

http://www.kurzweilai.net/a-drug-delivery-technique-to-bypass-the-blood-brain-barrier

Could benefit a large population of patients with neurodegenerative disorders

October 26, 2015

Researchers at Massachusetts Eye and Ear/Harvard Medical School and Boston University have developed a new technique to deliver drugs across the blood-brain barrier and have successfully tested it in a Parkinson’s mouse model (a line of mice that has been genetically modified to express the symptoms and pathological features of Parkinson’s to various extents).

Their findings, published in the journal Neurosurgery, lend hope to patients with neurological conditions that are difficult to treat due to a barrier mechanism that prevents approximately 98 percent of drugs from reaching the brain and central nervous system.

“Although we are currently looking at neurodegenerative disease, there is potential for the technology to be expanded to psychiatric diseases, chronic pain, seizure disorders, and many other conditions affecting the brain and nervous system down the road,” said senior author Benjamin S. Bleier, M.D., of the department of otolaryngology at Mass. Eye and Ear/Harvard Medical School.

The nasal mucosal grafting solution

Researchers delivered glial derived neurotrophic factor (GDNF), a therapeutic protein in testing for treating Parkinson’s disease, to the brains of mice. They showed that their delivery method was equivalent to direct injection of GDNF, which has been shown to delay and even reverse disease progression of Parkinson’s disease in pre-clinical models.

Once they have finished the treatment, they use adjacent nasal lining to rebuild the hole in a permanent and safe way. Nasal mucosal grafting is a technique regularly used in the ENT (ear, nose, and throat) field to reconstruct the barrier around the brain after surgery to the skull base. ENT surgeons commonly use endoscopic approaches to remove brain tumors through the nose by making a window through the blood-brain barrier to access the brain.

The safety and efficacy of these methods have been well established through long-term clinical outcomes studies in the field, with the nasal lining protecting the brain from infection just as the blood brain barrier has done.

By functionally replacing a section of the blood-brain barrier with nasal mucosa, which is more than 1,000 times more permeable than the native barrier, surgeons could create a “screen door” to allow for drug delivery to the brain and central nervous system.

The technique has the potential to benefit a large population of patients with neurodegenerative disorders, where there is still a specific unmet need for blood-brain-penetrating therapeutic delivery strategies.

The study was funded by The Michael J. Fox Foundation for Parkinson’s Research (MJFF).

Abstract of Heterotopic Mucosal Grafting Enables the Delivery of Therapeutic Neuropeptides Across the Blood Brain Barrier

BACKGROUND: The blood-brain barrier represents a fundamental limitation in treating neurological disease because it prevents all neuropeptides from reaching the central nervous system (CNS). Currently, there is no efficient method to permanently bypass the blood-brain barrier.

OBJECTIVE: To test the feasibility of using nasal mucosal graft reconstruction of arachnoid defects to deliver glial-derived neurotrophic factor (GDNF) for the treatment of Parkinson disease in a mouse model.

METHODS: The Institutional Animal Care and Use Committee approved this study in an established murine 6-hydroxydopamine Parkinson disease model. A parietal craniotomy and arachnoid defect was repaired with a heterotopic donor mucosal graft. The therapeutic efficacy of GDNF (2 [mu]g/mL) delivered through the mucosal graft was compared with direct intrastriatal GDNF injection (2 [mu]g/mL) and saline control through the use of 2 behavioral assays (rotarod and apomorphine rotation). An immunohistological analysis was further used to compare the relative preservation of substantia nigra cell bodies between treatment groups.

RESULTS: Transmucosal GDNF was equivalent to direct intrastriatal injection at preserving motor function at week 7 in both the rotarod and apomorphine rotation behavioral assays. Similarly, both transmucosal and intrastriatal GDNF demonstrated an equivalent ratio of preserved substantia nigra cell bodies (0.79 +/- 0.14 and 0.78 +/- 0.09, respectively, P = NS) compared with the contralateral control side, and both were significantly greater than saline control (0.53 +/- 0.21; P = .01 and P = .03, respectively).

CONCLUSION: Transmucosal delivery of GDNF is equivalent to direct intrastriatal injection at ameliorating the behavioral and immunohistological features of Parkinson disease in a murine model. Mucosal grafting of arachnoid defects is a technique commonly used for endoscopic skull base reconstruction and may represent a novel method to permanently bypass the blood-brain barrier.

references:

Bleier, Benjamin S. MD; Kohman, Richie E. PhD; Guerra, Kevin BS; Nocera, Angela L. MS; Ramanlal, Shreshtha BS; Kocharyan, Armine H. MD; Curry, William T. MD; Han, Xue PhD. Heterotopic Mucosal Grafting Enables the Delivery of Therapeutic Neuropeptides Across the Blood Brain Barrier. Neurosurgery, 2015; DOI: 10.1227/NEU.0000000000001016

Creating an artificial sense of touch by electrical stimulation of the brain

http://www.kurzweilai.net/creating-an-artificial-sense-of-touch-by-electrical-stimulation-of-the-brain

DARPA-funded study may lead to building prosthetic limbs for humans using a direct brain-electrode interface to recreate the sense of touch

October 26, 2015

Neuroscientists in a project headed by the University of Chicago have determined some of the specific characteristics of electrical stimuli that should be applied to the brain to produce different sensations in an artificial upper limb intended to restore natural motor control and sensation in amputees.

The research is part of Revolutionizing Prosthetics, a multi-year Defense Advanced Research Projects Agency (DARPA).

For this study, the researchers used monkeys, whose sensory systems closely resemble those of humans. They implanted electrodes into the primary somatosensory cortex, the area of the brain that processes touch information from the hand. The animals were trained to perform two perceptual tasks: one in which they detected the presence of an electrical stimulus, and a second task in which they indicated which of two successive stimuli was more intense.

The sense of touch is made up of a complex and nuanced set of sensations, from contact and pressure to texture, vibration and movement. The goal of the research is to document the range, composition and specific increments of signals that create sensations that feel different from each other.

To achieve that, the researchers manipulated various features of the electrical pulse train, such as its amplitude, frequency, and duration, and noted how the interaction of each of these factors affected the animals’ ability to detect the signal.

Of specific interest were the “just-noticeable differences” (JND),” — the incremental changes needed to produce a sensation that felt different. For instance, at a certain frequency, the signal may be detectable first at a strength of 20 microamps of electricity. If the signal has to be increased to 50 microamps to notice a difference, the JND in that case is 30 microamps.*

“When you grasp an object, for example, you can hold it with different grades of pressure. To recreate a realistic sense of touch, you need to know how many grades of pressure you can convey through electrical stimulation,” said Sliman Bensmaia, PhD, Associate Professor in the Department of Organismal Biology and Anatomy at the University of Chicago and senior author of the study, which was published today (Oct. 26) in the Proceedings of the National Academy of Sciences. “Ideally, you can have the same dynamic range for artificial touch as you do for natural touch.”

“This study gets us to the point where we can actually create real algorithms that work. It gives us the parameters as to what we can achieve with artificial touch, and brings us one step closer to having human-ready algorithms.”

Researchers from the University of Pittsburgh and Johns Hopkins University were also involved in the DARPA-supported study.

* The study also has important scientific implications beyond neuroprosthetics. In natural perception, a principle known as Weber’s Law states that the just-noticeable difference between two stimuli is proportional to the size of the stimulus. For example, with a 100-watt light bulb, you might be able to detect a difference in brightness by increasing its power to 110 watts. The JND in that case is 10 watts. According to Weber’s Law, if you double the power of the light bulb to 200 watts, the JND would also be doubled to 20 watts.

However, Bensmaia’s research shows that with electrical stimulation of the brain, Weber’s Law does not apply — the JND remains nearly constant, no matter the size of the stimulus. This means that the brain responds to electrical stimulation in a much more repeatable, consistent way than through natural stimulation.

“It shows that there is something fundamentally different about the way the brain responds to electrical stimulation than it does to natural stimulation,” Bensmaia said.

Abstract of Behavioral assessment of sensitivity to intracortical microstimulation of primate somatosensory cortex

Intracortical microstimulation (ICMS) is a powerful tool to investigate the functional role of neural circuits and may provide a means to restore sensation for patients for whom peripheral stimulation is not an option. In a series of psychophysical experiments with nonhuman primates, we investigate how stimulation parameters affect behavioral sensitivity to ICMS. Specifically, we deliver ICMS to primary somatosensory cortex through chronically implanted electrode arrays across a wide range of stimulation regimes. First, we investigate how the detectability of ICMS depends on stimulation parameters, including pulse width, frequency, amplitude, and pulse train duration. Then, we characterize the degree to which ICMS pulse trains that differ in amplitude lead to discriminable percepts across the range of perceptible and safe amplitudes. We also investigate how discriminability of pulse amplitude is modulated by other stimulation parameters—namely, frequency and duration. Perceptual judgments obtained across these various conditions will inform the design of stimulation regimes for neuroscience and neuroengineering applications.

references:

Sungshin Kim, Thierri Callier, Gregg A. Tabot, Robert A. Gaunt, Francesco V. Tenore, and Sliman J. Bensmaia. Behavioral assessment of sensitivity to intracortical microstimulation of primate somatosensory cortex. PNAS 2015; doi:10.1073/pnas.1509265112

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Role of Neurotransmitters and other such Neurosignaling Molecules

Posted in Neurohumoral Transmission, Neurological Diseases, Neuroscience, tagged neurotransmitters on October 27, 2015| Leave a Comment »

Role of Neurotransmitters and other such Neurosignaling Molecules

Curator: Larry H Bernstein, MD, FCAP

Circuit Dynamo

Eve Marder’s quest to understand neurotransmitter signaling is more than 40 years old and still going strong.

By Anna Azvolinsky | October 1, 2015

http://www.the-scientist.com//?articles.view/articleNo/44061/title/Circuit-Dynamo/

Eve Marder has her junior-year college roommate to thank for her initial fascination with neuroscience. “She came back from the first day of an abnormal psychology course and said, ‘Eve, you have to take this course! The professor has an English accent, wears a three-piece suit, and has a dueling scar,’” recalls Marder, a professor of biology at Brandeis University in Waltham, Massachusetts. “Of course, I agreed. What could be more romantic than that?” The course focused on schizophrenia, which at the time, in 1967, was thought to stem from a genetic predisposition coupled with competing sensory inputs or stressors that the brain couldn’t turn off. “The professor, in passing, said that some people think there may be a [cellular] basis for schizophrenia, including deficient inhibition of electrochemical signals in the brain. I thought, ‘What does that mean, inhibition in the brain?’” says Marder. To find out, she read everything she could about the role of inhibitory neurotransmitters—and, in the process, decided she would become a neuroscientist.

Marder began her graduate studies in biology at the University of California, San Diego (UCSD) in 1969. “I was a molecular biologist at heart because I was intrigued by molecules and cells rather than the large systems many were studying,” she says. That same year, a new assistant professor, Allen Selverston, joined the department. “He was the only real neurobiologist in the biology department, so I decided to work with him.” In the summer of 1970, Selverston introduced Marder to the lobster stomatogastric ganglion (STG), a then-new and relatively simple model for studying neuronal connectivity that she has studied ever since.

“Every time we were really stuck—not trivially, but stuck on a deep intellectual level—that has driven us to rethink, go sideways, or turn the problem inside out and come up with something new.”

Here, Marder explains why her choice to become a scientist during the 1960s counterculture was more conservative than what her friends and peers were up to, how her penchant for beautifully written neuropharmacology papers led her to a postdoc in Paris, and why we are not training too many PhDs.

Marder’s Momentum

A political slant. Growing up in the 1960s in Westchester County, New York, at the height of the civil rights movement, Marder was involved in a local youth civil rights group. In 1965, when she entered Brandeis University as a freshman, Marder thought she wanted to be a civil rights lawyer. But during her sophomore year, a post–World War II European history course that required memorization of every country’s political parties, “all alphabet soup and boring, with an odious black textbook with double columns of text,” changed her mind. After taking the final, Marder walked out of the lecture hall and pressed her mental delete button. “I forgot all of it on purpose and changed my major to biology,” she says.

Perfect sense. “I remember in high school when we learned about respiration and photosynthesis and the other molecular machinery hiding inside things that on the surface looked solid. I realized all of these molecular dynamics were inside cells, and that is what really fascinated me—these mechanisms of biological systems. Biology just made complete sense to me.”

The road less travelled. Before graduating and heading off to grad school, Marder presented an honors thesis on muscle biochemistry. At the time, most students who did an honors science thesis were men headed to medical school. “I remember having a funny conversation with one of them my senior year. He asked if I was also applying to medical school, and after I said no, said, ‘But why not? You’d get in.’ I said, ‘But why would I want to go to medical school if I don’t want to be a doctor?’ And he just kept saying, ‘But you would get in,’ and ‘Why wouldn’t you want to be a doctor?’”

A new model. When Marder joined Selverston’s lab as his first graduate student, he had just learned how to make in vitro preparations of the STG. “It was brand new and a simple example of a pattern-generating circuit,” says Marder. The STG, just 30 neurons, controls the rhythmic movement of the lobster’s stomach muscles during digestion, similar to the circuitry that controls breathing or walking. The STG cellular circuitry continues to generate motor patterns in vitro that resemble in vivo action potentials, but without the need for external stimuli. “This was something not possible then with vertebrate neuronal preparations.”

Thinking outside the circuit. For her graduate thesis, Marder set out to identify the neurotransmitters of the STG. “It was already clear that there were a bunch of different molecules used as transmitters, but no one had any idea why there were so many. There were researchers studying GABA, dopamine, serotonin, and other neurotransmitters separately, but no one was asking why there were so many transmitters in the brain and what their functional organization was. I wanted to understand the whole circuitry,” she says. Marder was the first to describe neurotransmitters in the STG. She discovered that acetylcholine functions as both an excitatory and inhibitory transmitter in some of the STG’s neurons. “This turned into a lifelong chase into transmitters and modulators in functional circuits.”

neurohormones and neuromodulators

Marder’s Merits

A singular vision. Marder learned a lot in graduate school, but her desire to understand the molecular underpinnings of neuronal communication remained strong. “At the time, the field was interested in working out wiring diagrams, because the scientists, who mostly came from electrical engineering, thought the neuron circuits worked like electrical circuits. My advisor echoed what many said at the time. ‘What you are doing is just pharmacology, it doesn’t really matter.’ In their minds, it only mattered whether the signal was inhibitory or excitatory; the actual signaling molecule wasn’t going to matter.”

A conservative choice. “Of the people I graduated college with, half were going to change the world, some were going to live on a commune, some guys ran away to Canada to avoid the draft, and others were going to a farm in Vermont to grow their own food. Long-term career plans were sort of weird for us—we were all counterculture. Graduate school at the time was a very conservative thing to be doing amongst my friends. When I finished my PhD, I just thought about the next step but didn’t have long-term career goals.” Marder spent a year as a postdoc in David Barker’s laboratory at the University of Oregon and then, in December 1975, with a fellowship from the Helen Hay Whitney Foundation, set off for Paris to work in JacSue Kehoe’s laboratory. “She had written, just head and shoulders, the most beautiful neuropharmacology papers. I had read every paper in the field and these were just so much better than what anyone else was doing.” There, Marder collaborated with Danielle Paupardin-Tritsch, publishing three papers, including a study of the responses of the crab STG to three different neurotransmitters.

More than on/off switches. When she came back to the U.S. in 1978, Marder joined the faculty at Brandeis as an assistant professor in the biology department and has remained there ever since. Along with her first graduate student, Judith Eisen, whom Marder credits with early successes in her lab, Marder provided the initial evidence for the existence of neuromodulators that can prompt longer-term changes influencing how neurons respond to fast-acting neurotransmitters. Eisen initially characterized the pharmacology of the synapses, figuring out which neurons had receptors for which signaling molecules.

Predicting behavior. In 1989, Marder met Larry Abbott, then a theoretical physicist at Brandeis. “It was clear that for a true mechanistic understanding of how the dynamics of the circuit arise from its components, we needed to be able to do modeling,” she recalls. With Abbott, Marder’s lab developed the “dynamic clamp,” a method that uses a computer to introduce a conductance—the ease with which an electric current can pass through a circuit—into neurons to model their behavior. They also worked out thenegative-feedback system within neurons that allows for changes in parameters while maintaining their normal function. “We were building models and they were fragile. We would change a parameter, and the model would crash. I kept saying that the cells don’t crash all the time, so how do cells balance their number of ion channels? What we came up with is a very simple way of thinking about this.”

The bigger picture. “The big themes that have come from the STG model, and partly from our lab, are that neuronal circuits are multiply modulated, that modulators reconfigure circuits, and that there have to be pretty simple global regulatory mechanisms that help neurons maintain stable electrical activity despite the fact that their ion channels—the proteins in the cell membranes that carry out electrical signaling—are being constantly replaced.”

Heterogeneity. For the last 10 years, Marder’s lab has been working to understand the extent of variability within individual nervous systems that still allows the systems to remain stable. “For quite a long time, people thought that all nervous systems had to be very tightly tuned,” she says. “And what we are seeing is that, actually, they can be quite variable and change over time and still work well enough because there is a lot of degeneracy in the way circuits are constructed.”

Marder’s Mind

The new normal. Before 1968, many life-science PhD programs had informal quotas, restricting the number of women accepted, says Marder. But that year, the draft law changed, and graduate school enrollment was no longer a valid way to defer the draft. Life-science graduate programs suddenly switched to gender parity. Marder’s class at UCSD was the first to have a large proportion of women—13 women out of a class of 30; in prior years there were only two or three women per class of 30. “There was a lot of hubbub about it. ‘What are we going to do with all these women? Civilization as we know it will end!’ was how the professors talked about it. But by May that year they had completely forgotten about it,” Marder laughs.

The big picture. “I have always been good at figuring out how to use the same nervous system to ask a bunch of different questions. And I’ve always been pretty good at finding the general principles amongst the idiosyncrasies of a neuronal system. I think that is key if you work on model organisms.”

The benefits of being stuck. “Everything important we did in the lab arose because something wasn’t working as expected. In Judith’s original work, we finally realized after getting different answers from every experiment that there were nine different possible outcomes connecting only three neurons, all consistent with our recordings. So we had to design a completely different experiment. The activity-dependent regulation work arose only because of my total frustration with having to tune conductance-based models. So every time we were really stuck—not trivially, but stuck on a deep intellectual level—that has driven us to rethink, go sideways, or turn the problem inside out and come up with something new.”

A worrisome trend. “I am very concerned with science right now because I think the push to have high-impact papers is having a deleterious effect on the way people design experiments, and it definitely has a deleterious effect on the way researchers write their papers. The most common critique you see from reviewers is that authors are overselling their work, and it’s because the researchers believe high-impact papers are necessary for their careers. So the science we are doing is being warped.”

A scientist in the rough. Marder recently wrote a commentary against the push to decrease the number of students accepted into science PhD programs.“We’re very bad at spotting who is going to be a good scientist and who isn’t [based on graduate-school applications],” she says. “While students are in college, they are fundamentally consumers of knowledge, and becoming a scientist means you have to learn to be a creator of new knowledge. But the tools that allow you to be a great consumer of knowledge don’t set you up for the kind of frustrations and sidewise thinking and problems that you have to confront to be a knowledge creator. You can take the best undergrad students, and they may not be the best graduate students. And the ones who complete the best PhDs may not be the ones that stay in the field. I think it comes down to drive, persistence, willingness to confront failure, creativity, passion—all of these other attributes that we can’t measure easily on an application. So to take just the top undergraduates, you’re going to be missing many of the best ones. The real problem here is that the US government is not funding enough science.”

Potential to fly. “It’s crazy right now because biology in general, and neuroscience specifically, is at this extraordinary moment in time when there is so much really wonderful science that can now be done that was inconceivable even 10 years ago. We are at this incredibly exciting moment for discovery and at the same time the field is being destroyed by extraordinary anxiety about [funding] resources. If we didn’t have this extent of resource anxiety we could be flying, but the funding situation is crippling, and we are crippling ourselves with this incredible collective anxiety.”

Greatest Hits

Discovered that acetylcholine acts as a neurotransmitter in the crustacean stomatogastric ganglion (STG), where it functions as both an excitatory and an inhibitory signal
Among the first to describe neuromodulators that acted differently than neurotransmitters, resulting in long-lasting effects on neuronal circuits
Determined that neurons are robust, maintaining their electrical activity patterns despite the turnover of channels and other changes
With colleagues, developed the “dynamic clamp,” a neurophysiological method that can finely manipulate nerve cells and simulate neuronal and muscle systems using computer-adjusted parameters
Showed that there are multiple sets of parameters in neurons and networks that can produce similar output patterns

Eve Marder, Ph.D.
Professor of Biology
Member, US National Academy of Sciences

Modulation of Neural Networks

B.A., Brandeis University
Ph.D., University of California, San Diego

Contact Information

One of the fundamental problems in neuroscience is understanding how circuit function arises from the intrinsic properties of individual neurons and their synaptic connections. Of particular interest to us today is the extent to which similar circuit outputs can be generated by multiple mechanisms, both in different individual animals, or in the same animal over its life-time. As an experimental preparation we exploit the advantages of the central pattern generating circuits in the crustacean stomatogastric nervous system. Central pattern generators are groups of neurons found in vertebrate and invertebrate nervous systems responsible for the generation of specific rhythmic behaviors such as walking, swimming, and breathing. The central pattern generators in the stomatogastric ganglion (STG) of lobsters and crabs are ideal for many analyses because the STG has only about 30 large neurons, the connectivity is established, the neurons are easy to record from, and when the stomatogastric ganglion is removed from the animal, it continues to produce rhythmic motor patterns.

Work in the lab centers on three main questions:

How do neuromodulators and neuromodulatory neurons reconfigure circuits so that the same group of neurons can produce a variety of behaviorally relevant outputs?
How can networks be both stable over the lifetime of the animal despite ongoing turnover of membrane proteins such as channels and receptors? How is network stability maintained over long time periods? To what extent do similar network outputs result from different underlying mechanisms or solutions?.
How variable are the sets of parameters that govern circuit function across animals? How can animals with disparate sets of circuit parameters respond reliably to perturbations such as neuromodulators and temperature?

To address these questions we employ electrophysiological, biophysical, computational, anatomical, biochemical, and molecular techniques.

See references at bottom

Rewarding Companions

Oxytocin and social contact together modulate endocannabinoid activity in the mouse brain, which could help explain the prosocial effects of marijuana use.

By Rina Shaikh-Lesko | October 26, 2015

http://www.the-scientist.com//?articles.view/articleNo/44338/title/Rewarding-Companions/

The mammalian central nervous system is studded with receptors that bind with a small number of endogenous cannabinoids, as well as those found in marijuana. Marijuana is known to promote heightened social connection, but the mechanism for this phenomenon was not well understood. According to a paper published today (October 26) in PNAS, it is social interaction between mice plus oxytocin—the hormone involved in social bonding—that drive cannabinoid activity in the animals’ brains.

“It was a great surprise to see how powerful the effects of social contact are on production of anandamide in select regions of the brain,” said study coauthor Daniele Piomelli, a neuroscientist at the University of California, Irvine.

The brain has two known cannabinoid receptors, types 1 and 2 (CB1 and CB2). Two main endocannabinoids bind with CB1, anandamide and 2-Arachidonoylglycerol (2-AG), as do plant cannabinoids such as tetrahydrocannabinol (THC), the main psychoactive compound in marijuana.

To study the behavioral effects of oxytocin, endocannabinoids, and social interaction, Poimelli and his colleagues isolated mice for 24 hours, then allowed them to either socialize with other mice or remain in isolation for another three hours. The researchers found that the mice that were allowed to socialize had higher levels of anandamide, but not 2-AG, in their nucleus accumbens.

“I was personally very surprised by the fact that it’s such a simple thing—just taking an animal, isolating it for three hours, then putting it back with its buddies, or not—would make such a difference in a single chemical in a specific area of the brain,” Piomelli told The Scientist.

In mice lacking an enzyme that degrades anandamide, fatty acid amid hydrolase (FAAH), the researchers observed more prosocial behavior. When the researchers used a pharmacological compound to block FAAH, they also saw prosocial behavior, even among the mice that were isolated. Piomelli and his colleagues also found that stimulating oxytocin receptors promoted the action of anandamide, while blocking oxytocin receptors seemed to stifle anandamide. “The effects of oxytocin are virtually entirely dependent on the ability of oxytocin to drive the formation of anandamide,” Piomelli said.

The finding may also point to a possible treatment for autism spectrum disorders, which can involve social-interaction difficulties. (The team initially homed in on the interaction of oxytocin and anandamide while studying autism mouse model of autism.)

“The convincing evidence that enhancing the activation of CB1 receptors by endogenously released anandamide, but not by the endocannabinoid, [2-AG] . . . could be a new strategy for ameliorating some of the unwanted symptoms of disorders like autism,” Roger Pertwee, a neuropharmacologist at the University of Aberdeen, U.K., who was not involved with the study, wrote in an email. “The important next step will be to test the clinical importance of these findings in studies performed with human autistic subjects—to test whether at least some of their autistic symptoms can be ameliorated by a FAAH inhibitor.”

Piomelli said he would like to further explore why anandamide and 2-AG appear to act so differently in relation to oxytocin. The latter endocannabinoid is much more abundant than anandamide. “Anandamide is like the little brother and 2AG is like the big brother,” Piomelli said. He and his colleagues are looking into whether 2-AG plays a role in other forms of reward-oriented behavior.

Wei et al., “Endocannabinoid signaling mediates oxytocin-driven social reward,” PNAS, http://dx.doi.org:/10.1073/pnas.1509795112,2015

Jacob Hooker: Weaver of Brain Science

Director of Radiochemistry, Athinoula A. Martinos Center for Biomedical Imaging; Associate Professor, Harvard Medical School. Age: 35

http://www.the-scientist.com//?articles.view/articleNo/44064/title/Jacob-Hooker–Weaver-of-Brain-Science/

As a kid growing up outside Asheville, North Carolina, Jacob Hooker spent a lot of time tinkering underneath cars with his mechanic father. But it was a guest speaker in his high school chemistry class who provided the spark that propelled him into research. Kent Hester, director of student and career services at North Carolina State University’s College of Textiles, told students about opportunities for studying textile chemistry. Hooker applied to NC State and won a $5,000-per-year North Carolina Textile Foundation merit scholarship, graduating in 2002. “That was one of the largest college-based scholarships on NC State’s campus at the time,” Hester recalls.

Hooker published four papers while at NC State, but when the time came to apply for grad school, he says he was “geographically driven” to branch out from his home state and explore the western half of the country. On a visit to the University of California, Berkeley, he met Matt Francis, a young biochemist who had just set up his lab the year before.

“From the very second I met him, I could just tell he was a very special individual,” says Francis, who develops techniques for the chemical modification of proteins. “He’s got a certain intensity and scientific sophistication to him that supersedes any kind of training he had.” While in Francis’s lab, Hooker published a technique for chemically modifying the interior surface of a viral capsid that would pave the way for viral drug-delivery methods.¹

Near the end of his PhD research, Hooker learned about radioactive labeling and radioisotopes from Jim O’Neil at Lawrence Berkeley National Lab. Then, at a nuclear medicine meeting in the winter of 2006, he attended a presentation given by Brookhaven National Lab organic chemist Joanna Fowler about using specially synthesized radioactive molecules and positron emission tomography (PET) technology to study the neurobiology of addiction. “It was incredibly exciting that a chemist could label a small molecule and observe something fundamental about the brain using that molecule they made,” Hooker says.

He immediately wrote to Fowler, asking if she had an open postdoc position in her lab. “She said she didn’t, but she’d be willing to help me look for money to make one,” Hooker says. He submitted an NIH training proposal as he was finishing his PhD dissertation and got the grant. Hooker moved to Fowler’s lab in 2007. “Getting him was like winning the lottery,” says Fowler, now a professor emeritus at Brookhaven.

Fowler introduced Hooker to PET, which is used in concert with radionucleotide tracers to image molecules in the active brain. Hooker and colleagues built new radiotracers that target novel molecules to gain insights into their in vivo function. He also helped develop a new method for quickly and efficiently labeling the carbamate functional groups with carbon-11 (¹¹C), one of the most important isotopes for PET research.²

Hooker started at the Athinoula A. Martinos Center for Biomedical Imaging at Massachusetts General Hospital in 2009, overseeing the construction of the center’s new MRI/PET facility while setting up his own lab. Earlier this year, Hooker and Mass General neuroscientist Marco Loggia devised a PET imaging strategy to record the activation of glial cells in the brains of chronic-pain patients.³ With other colleagues Hooker developed a novel histone deacetylase–binding radiotracer, [¹¹C]Martinostat, which could open an unprecedented window into DNA expression in the brain.⁴

“He’s the best I’ve ever seen,” Fowler says. “He’s going to ask questions we haven’t thought of before.”

References

J.M. Hooker et al., “Interior surface modification of bacteriophage MS2,” J Am Chem Soc, 126:3718-19, 2004. (Cited 212 times)
J.M. Hooker et al., “One-Pot, direct incorporation of [¹¹C]-CO2 into carbamates,” Angew Chem Int Ed, 48:3482-85, 2009. (Cited 68 times)
M.L. Loggia et al., “Evidence for brain glial activation in chronic pain patients,” Brain, 138:604-15, 2015. (Cited 8 times)
C. Wey et al., “In vivo imaging of histone deacetylases (HDACs) in the central nervous system and major peripheral organs,” J Med Chem, 57:7999-8009, 2014. (Cited 6 times)

Marder Recent Publications:

Swensen, A.M., Golowasch, J., Christie, A.E., Coleman, M.J., Nusbaum, M.P., and Marder, E. (2000) GABA and GABA responses in the stomatogastric ganglion of the crab, Cancer borealis. J. Exp. Biol.,203: 2075-2092. [abstract]

Richards, K.S. and Marder, E. (2000) The actions of crustacean cardioactive peptide on adult and developing stomatogastric ganglion motor patterns. J. Neurobiol. 44: 31-44. [abstract]

Swensen, A.M. and Marder, E. (2000) Multiple peptides converge to activate the same voltage-dependent current in a central pattern generating circuit. J. Neurosci. 20: 6752-6759. [abstract]

Marder, E. (2000) Colored Chalk. Current Biol., 10: R613. [pdf file]

Marder, E. (2000) Models identify hidden assumptions. Nature Neurosci. 3: 1198.

Marder, E. (2000) Motor pattern generation. Curr. Opin. Neurobiol. 10: 691-698. [abstract]

Nusbaum, M.P., Blitz, D.M., Swensen, A.M., Wood, D., and Marder, E. (2001) The roles of co-transmission in neural network modulation. TINS 24: 146-154. [abstract]

Marder, E. (2001) Moving rhythms. Nature, 410: 755.

Soto-Treviño, C., Thoroughman, K.A., Marder, E., and Abbott, L.F. (2001) Activity-dependent modification of inhibitory synapses in models of rhythmic neural networks. Nature Neuroscience, 4: 297-303. [abstract]

Swensen, A.M. and Marder, E. (2001) Modulators with convergent cellular actions elicit distinct circuit outputs. J. Neuroscience, 21: 4050-4058. [abstract]

Goldman, M.S., Golowasch, J., Marder, E., and Abbott, L.F. (2001) Global structure, robustness, and modulation of neuronal models. J. Neuroscience, 21: 5229-5238. [abstract]

Marder, E. and Bucher, D. (2001) Central pattern generators and the control of rhythmic movements. Current Biol., 11: R986-R996. [abstract]

Li, L., Pulver, S.R., Kelley, W.P., Thirumalai, V., Sweedler, J.V., and Marder, E. (2002) Orcokinin peptides in developing and adult crustacean stomatogastric nervous systems and pericardial organs. J. Comp. Neurol., 444:227-244.

Golowasch, J., Goldman, M., Abbott, L.F, and Marder, E. (2002) Failure of averaging in the construction of conductance-based neuron models. J. Neurophysiol., 87: 11291131. [abstract]

Thirumalai, V. and Marder, E. (2002) Colocalized neuropeptides activate a central pattern generator by acting on different circuit targets. J. Neuroscience, 22: 18741882. [abstract]

Pulver, S.R. and Marder, E. (2002) Neuromodulatory complement of the pericardial organ in the embryonic lobster, Homarus americanus. J. Comp. Neurol., 451: 79-90.

Marder, E. (2002) Non-mammalian models for studying neural development and function. Nature, 417: 318-321. [abstract]

Marder, E. and Thirumalai, V. (2002) Cellular, synaptic, and network effects of neuromodulation. Neural Networks, 15: 479-493. [abstract]

Marder, E. and Prinz, A.A. (2002) Modeling stability in neuron and network function: the role of activity in homeostasis. BioEssays, 24:1145-1154. [abstract]

Marder, E. and Prinz, A.A. (2003) Current Compensation in neuronal homeostasis. Neuron, 37: 2-4. [abstract]

Abbott, L.F., Thoroughman, K., Prinz, A., Thirumalai, V. and Marder, E. (2003) Activity-dependent modification of intrinsic and synaptic conductances in neurons and rhythmic networks. In Van Ooyen, A., ed. Modeling Neural Development (MIT Press, Cambridge MA).

Richards, K.S., Simon, D.J., Pulver, S.R., Beltz, B.S. and Marder, E. (2003) Serotonin in the developing stomatogastric system of the lobster, Homarus americanus. J. Neurobiol. 54: 380-392. [abstract]

Prinz, A.A., Thirumalai, V. and Marder, E. (2003) The functional consequences of changes in the strength and duration of synaptic inputs to oscillatory neurons. J. Neurosci. 23: 943-954. [abstract]

Pulver, S.R., Thirumalai, V., Richards, K.S. and Marder, E. (2003) Dopamine and histamine in the developing stomatogastric system of the lobster, Homarus americanus. J. Comp. Neurol 462: 400-414. [abstract]

Bucher, D., Thirumalai, V. and Marder, E. (2003) Axonal dopamine receptors activate peripheral spike initiation in a stomatogastric motor neuron. J. Neurosci., 23: 6866-6875. [abstract]

Luther, JA, Robie AA, Yarotsky J, Reina C, Marder E, and Golowasch J. (2003) Episodic Bouts of Activity Accompany Recovery of Rhythmic Output By a Neuromodulator- and Activity-Deprived Adult Neural Network. J. Neurophysiol. 90:2720-2730. [abstract] [download article]

Li, L., Kelley, W.P., Billimoria, C.P., Christie, A.E., Pulver, S.R., Sweedler, J.V., and Marder, E. (2003) Mass spectrometric investigation of the neuropeptide complement and release in the pericardial organs of the crab, Cancer borealis. J. Neurochem, 87:642-656. [abstract]

Birmingham, J.T., Billimoria, C.P., DeKlotz, T.R., Stewart, R.A. and Marder, E. (2003) Differential and history dependent modulation of a stretch receptor in the stomatogastric system of the crab, Cancer borealis. J. Neurophysiol, 90: 3608 – 3616. [abstract]

Prinz, A.A., Billimoria, C.P, and Marder, E. (2003) An alternative to hand-tuning conductance-based models: construction and analysis of data bases of model neurons. J. Neurophysiol., 90:3998-4015. [abstract] [Download Article]

Christie, A.E., Stein, W., Quinlan, J.E., Beenhakker, M., Marder, E., and Nusbaum, M.P. (2003) Actions of a histaminergic/peptidergic projection neuron on rhythmic motor patterns in the stomatogastric nervous system of the crab, Cancer borealis. J. Comp. Neurol., 469:153 – 169 [abstract]

Mahadevan A, Lappe J, Rhyne RT, Cruz-Bermudez ND, Marder E, Goy MF. (2004) Nitric oxide inhibits the rate and strength of cardiac contractions in the lobster Homarus americanus by acting on the cardiac ganglion. J Neurosci. 24:2813-24. [abstract]

Prinz AA, Abbott LF, Marder E. (2004) The dynamic clamp comes of age. Trends Neurosci. 27:218-24.

Goaillard JM, Schulz DJ, Kilman VL, Marder E. (2004) Octopamine modulates the axons of modulatory projection neurons. J Neurosci. 24(32):7063-73. [abstract]

Pulver SR, Bucher D, Simon DJ, Marder E. (2004) Constant amplitude of postsynaptic responses for single presynaptic action potentials but not bursting input during growth of an identified neuromuscular junction in the lobster, Homarus americanus. J Neurobiol. 2005 Jan;62(1):47-61. [abstract]

Destexhe A, Marder E.(2004) Plasticity in single neuron and circuit computations. Nature. 431:789-95. [abstract]

Prinz AA, Bucher D, Marder E. (2004) Similar network activity from disparate circuit parameters. Nat Neurosci. 7:1345-52. [abstract]

Bucher D, Prinz AA, Marder E. (2005) Animal-to-animal variability in motor pattern production in adults and during growth. J Neurosci. 25:1611-9. [abstract]

Marder E, Rehm KJ.(2005) Development of central pattern generating circuits. Curr Opin Neurobiol. 15:86-93. [abstract]

Soto-Trevino C, Rabbah P, Marder E, Nadim F. (2005) A computational model of electrically coupled, intrinsically distinct pacemaker neurons. J Neurophysiol. 94:590-604. [abstract]

Marder E, Bucher D, Schulz DJ, Taylor AL. (2005) Invertebrate central pattern generation moves along, Curr Biol. 15:R685-99. [abstract]

Billimoria CP, Li L, Marder E. (2005) Profiling of neuropeptides released at the stomatogastric ganglion of the crab, Cancer borealiswith mass spectrometry. J Neurochem. 95:191-9. [abstract]

Thirumalai V, Prinz AA, Johnson CD, Marder E. (2006) Red pigment concentrating hormone strongly enhances the strength of the feedback to the pyloric rhythm oscillator but has little effect on pyloric rhythm period. J Neurophysiol. 95:1762-70. [abstract]

Schulz DJ, Goaillard JM, Marder E. (2006) Variable channel expression in identified single and electrically coupled neurons in different animals, Nat Neurosci. 9:356-62. [abstract]

Cruz-Bermudez ND, Fu Q, Kutz-Naber KK, Christie AE, Li L, Marder E. (2006) Mass spectrometric characterization and physiological actions of GAHKNYLRFamide, a novel FMRFamide-like peptide from crabs of the genus Cancer. J Neurochem. 2006 May;97:784-99. [abstract]

Bucher D, Taylor AL, Marder E. (2006) Central pattern generating neurons simultaneously express fast and slow rhythmic activities in the stomatogastric ganglion. J Neurophysiol. 2006 Jun;95:3617-32. [abstract]

Taylor AL, Hickey TJ, Prinz AA, Marder E. (2006) Structure and visualization of high-dimensional conductance spaces. J Neurophysiol. 2006 Aug;96:891-905. [abstract]

Goaillard JM, Marder E. (2006) Dynamic clamp analyses of cardiac, endocrine, and neural function. Physiology (Bethesda). 2006 Jun; 21:197-207. Review. [abstract]

Billimoria CP, DiCaprio RA, Birmingham JT, Abbott LF, Marder E. (2006) Neuromodulation of spike-timing precision in sensory neurons. J Neurosci. 2006 May 31; 26:5910-9. [abstract]

Marder E, Goaillard JM. (2006) Variability, compensation and homeostasis in neuron and network function. Nat Rev Neurosci. 2006 Jul; 7:563-74. Review. [abstract]

Marder E, Bucher D. (2007) Understanding circuit dynamics using the stomatogastric nervous system of lobsters and crabs. Annu Rev Physiol. 2007; 69:291-316. [abstract]

Bucher D, Johnson CD, Marder E. (2007) Neuronal morphology and neuropil structure in the stomatogastric ganglion of the lobster, Homarus americanus. J Comp Neurol. 2007 Mar 10; 501:185-205. [abstract]

Fu Q, Tang LS, Marder E, Li L. (2007) Mass spectrometric characterization and physiological actions of VPNDWAHFRGSWamide, a novel B type allatostatin in the crab, Cancer borealis. J Neurochem. 2007 May;101:1099-107. [abstract]

Schulz DJ, Goaillard JM, Marder EE. (2007) Quantitative expression profiling of identified neurons reveals cell-specific constraints on highly variable levels of gene expression. Proc Natl Acad Sci U S A. 2007 Aug 7;104:13187-91. [abstract]

Cruz-Bermúdez ND, Marder E. (2007) Multiple modulators act on the cardiac ganglion of the crab, Cancer borealis. J Exp Biol. 2007Aug;210(Pt 16):2873-84. [abstract]

Marder E, Tobin AE, Grashow R. (2007) How tightly tuned are network parameters? Insight from computational and experimental studies in small rhythmic motor networks. Prog Brain Res. 2007;165:193-200. [abstract]

Cape SS, Rehm KJ, Ma M, Marder E, Li L. (2008) Mass spectral comparison of the neuropeptide complement of the stomatogastric ganglion and brain in the adult and embryonic lobster, Homarus americanus. J Neurochem. 2008 May;105:690-702. [abstract]

Rehm KJ, Taylor AL, Pulver SR, Marder E. (2008) Spectral analyses reveal the presence of adult-like activity in the embryonic stomatogastric motor patterns of the lobster, Homarus americanus. J Neurophysiol. 2008 Jun;99:3104-22. [abstract]

Marder E. (2008) The roads not taken.Curr Biol. 2008 Sep 9;18:R725-R726.

Rehm KJ, Deeg KE, Marder E. (2008) Developmental regulation of neuromodulator function in the stomatogastric ganglion of the lobster, Homarus americanus. J Neurosci. 2008 Sep 24;28:9828-39. [abstract] [free PMC article]

Marder E. (2009) Electrical synapses: rectification demystified. Curr Biol. 2009 Jan 13;19:R34-5. Review.

Taylor AL, Goaillard JM, Marder E. (2009) How multiple conductances determine electrophysiological properties in a multicompartment model. J Neurosci. 2009 Apr 29;29:5573-86. [abstract] [free PMC article]

Ma M, Szabo TM, Jia C, Marder E, Li L. (2009) Mass spectrometric characterization and physiological actions of novel crustacean C-type allatostatins.Peptides. 2009 Sep;30:1660-8. [abstract]

Grashow R, Brookings T, Marder E. (2009) Reliable neuromodulation from circuits with variable underlying structure. Proc Natl Acad Sci U S A. 2009 Jul 14;106:11742-6. [abstract] [free PMC article]

Tobin AE, Cruz-Bermúdez ND, Marder E, Schulz DJ. (2009) Correlations in ion channel mRNA in rhythmically active neurons.PLoS One. 2009 Aug 25;4:e6742. [abstract] [free PMC article]

Goaillard JM, Taylor AL, Schulz DJ, Marder E. (2009) Functional consequences of animal-to-animal variation in circuit parameters.Nat Neurosci. 2009 Nov;12:1424-30. [abstract] [free PMC article]

Marder E, Tang LS. Coordinating different homeostatic processes. Neuron. 2010 Apr 29;66(2):161-3. [abstract]

Goaillard JM, Taylor AL, Pulver SR, Marder E. Slow and persistent postinhibitory rebound acts as an intrinsic short-term memory mechanism. J Neurosci. 2010 Mar 31;30(13):4687-92. [abstract]

Marder E. Why so long? Curr Biol. 2010 May 25;20(10):R426.

Grashow R, Brookings T, Marder E. Compensation for variable intrinsic neuronal excitability by circuit-synaptic interactions. J Neurosci. 2010 Jul 7;30(27):9145-56. [abstract]

Tang LS, Goeritz ML, Caplan JS, Taylor AL, Fisek M, Marder E. Precise temperature compensation of phase in a rhythmic motor pattern.PLoS Biol. 2010 Aug 31;8(8). pii: e1000469. [abstract]

Marder E, Kettenmann H, Grillner S. Impacting our young. Proc Natl Acad Sci U S A. 2010 Dec 14; 107(50):21233. Epub 2010 Nov 22.

Marder E, Taylor AL. Multiple models to capture the variability in biological neurons and networks. Nat Neurosci. 2011 Feb;14(2):133-8. [abstract]

Marder E. Variability, compensation, and modulation in neurons and circuits. Proc Natl Acad Sci U S A. 2011 Sep 13;108 Suppl 3:15542-8. [abstract]

Kispersky T, Gutierrez GJ, Marder E. Functional connectivity in a rhythmic inhibitory circuit using Granger causality. (2011) Neural Syst Circuits 1:9. [full paper]

Szabo TM, Chen R, Goeritz ML, Maloney RT, Tang LS, Li L, Marder E. Distribution and physiological effects of B-type allatostatins (myoinhibitory peptides, MIPs) in the stomatogastric nervous system of the crab Cancer borealis. (2011) J Comp Neurol. 2011 Sep 1;519(13):2658-76. [abstract]

Brookings T, Grashow R, Marder E. Statistics of neuronal identification with open- and closed-loop measures of intrinsic excitability. (2012) Front Neural Circuits. 6:19. [abstract]

Callaway, E.M. and Marder, E. (2012) Common Features of Diverse Circuits. Current Opinion in Neurobiology 22: 565-567.

Marder, E. (2012) Neuromodulation of neuronal circuits: back to the future. Neuron, 76: 1-11. PMC3482119 [abstract]

Tang, L.S., Taylor, A.L., Rinberg, A., and Marder, E. (2012) Robustness of a rhythmic circuit to short and long-term temperature changes. J. Neurosci., 32: 10075-10085. NIHMS395465. [abstract]

Kispersky, T., Caplan, J., and Marder, E. (2012) Increase in sodium conductance decreases firing rate and gain in model neurons. J. Neurosci., 32: 10995– 10995. PMC3427781 [abstract]

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microglia and brain maintenance

Posted in Curation, Disease Biology, Metabolism, Metabolomics, Neurodegenerative Diseases, Neurological Diseases, Neuroscience, Nutrition Disorders, Protein-energy malnutrition, Proteins, Proteomics, Signaling & Cell Circuits, Stress Disorders, Translational Science, Unfolded Protein Response (UPR), tagged Autophagy, cell death pathway, signaling pathway, Unfolded Protein Response (UPR) on October 27, 2015| Leave a Comment »

microglia and brain maintenance

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Mapping mosaicism: Tracing subtle mutations in our brains

Posted on January 14, 2015 by Nancy Fliesler

Posted in All Posts, Information technology

More On: brain development, DNA sequencing, genetics, mosaicism, neuroscience, somatic mutations

DNA sequences were once thought to be the same in every cell, but the story is now known to be more complicated than that. The brain is a case in point: Mutations can arise at different times in brain development and affect only a percentage of neurons, forming a mosaic pattern.

Now, thanks to new technology described last week in Neuron, these subtle “somatic” brain mutations can be mapped spatially across the brain and even have their ancestry traced.

Like my family, who lived in Eastern Europe, migrated to lower Manhattan and branched off to Boston, California and elsewhere, brain mutations can be followed from the original mutant cells as they divide and migrate to their various brain destinations, carrying their altered DNA with them.

“Some mutations may occur on one side of the brain and not the other,” says Christopher Walsh, MD, PhD, chief of Genetics and Genomics at Boston Children’s Hospital and co-senior author on the paper. “Some may be ‘clumped,’ affecting just one gyrus [fold] of the brain, disrupting just a little part of the cortex at a time.”

This tracking capability represents a significant advance for genetics research. And for neuroscientists, it provides a new way to study both the normal brain and brain disorders like epilepsy, autism and intellectual disability.

Walsh and colleagues studied normal brain tissue from a teenage boy who had passed away from other causes. Sampling in more than 30 brain locations, they used deep, highly sensitive, whole-genome sequencing of one neuron at a time—unlike usual methods, which sequence thousands or millions of cells mixed together and simply read out an average.

http://vectorblog.org/wp-content/uploads/2015/01/Walsh-figure3B-v2-1024×735.jpg

The blue and green boxes indicate different degrees of mosaicism (based on proportion of cells affected) in the left half of this teen’s normal brain. The blue shaded area indicates that retrotransposon mutation #1 (blue boxes) is limited to a focal area in the middle frontal gyrus. The empty boxes indicate areas where mutation #1 was not detected. (Courtesy Gilad Evrony, PhD, Boston Children’s Hospital)

Next, using technology developed by Alice (Eunjung) Lee in the lab of Peter Park, PhD, at Harvard Medical School’s Center for Biomedical Informatics, they zeroed in on inserted bits of DNA caused by retrotransposons, one type of mutation that can arise as the brain develops. These essentially served as markers that allowed cell lineages to be traced.

http://vectorblog.org/wp-content/uploads/2015/01/Walsh-figure5-1024×509.jpg

This model illustrates the origins of two somatic retrotransposon mutations during prenatal development and their subsequent dissemination in the brain. Insertion #2 (in green) occurred soon after conception; #1 (in blue) happened sometime later during brain development. The ‘pie slices’ show a closeup of the layers of the cerebral cortex. Later in development, additional somatic mutations occurred inside insertions #1 and #2, creating new, smaller sublineages of cells. (Courtesy Gilad Evrony, PhD)

A parallel study from Walsh’s lab in 2014 used single-neuron sequencing to find copy number variants— a different type of mutation affecting the number of copies of chromosomes or chromosome fragments. It, too, found the mutations to be present in normal brains as well as neurologically diseased brains.

Walsh and others speculate that some somatic brain mutations might play a role in autism, epilepsy, schizophrenia and other unsolved neuropsychiatric diseases whose causes are mostly still a mystery.

“It is possible that a whole new class of brain disorders may exist that has not been previously recognized,” says Evrony. “In such disorders, a somatic mutation may subtly affect only one small part of the brain involved in a specific ability, for example language, while sparing the rest of the brain.”

Tracking subtle brain mutations, systematicallyTool can trace and spatially map “mosaic” mutations in the brain

http://www.prnewswire.com/news-releases/tracking-subtle-brain-mutations-systematically-300017369.html

BOSTON, Jan. 7, 2015 /PRNewswire-USNewswire/ — DNA sequences were once thought to be identical from cell to cell, but it’s increasingly understood that mutations can arise during brain development that affect only certain groups of brain cells. A technique developed at Boston Children’s Hospital allows these subtle mutation patterns to be traced and mapped spatially for the first time. This capability is a significant advance for genetics research and provides a new way to study both the normal brain and brain disorders such as epilepsy and autism.

Described in the January 7th issue of Neuron, the technique uses “deep,” highly sensitive whole-genome sequencing of single neurons and a new technology that identifies inserted bits of DNA caused by retrotransposons, one of several kinds of so-called somatic mutations that can arise as the brain develops.

The technique picks up somatic mutations that affect just a fraction of the brain’s cells, in a “mosaic” pattern. It also allows “lineage tracing,” showing when during brain development the mutations arise and how they spread through brain tissue as the mutated cells grow, replicate and migrate, carrying the mutation with them.

“There is a lot of genetic diversity from one neuron to the other, and this work gets at how somatic mutations are distributed in the brain,” says Christopher Walsh, MD, PhD, chief of Genetics and Genomics at Boston Children’s and co-senior author on the paper. “Some mutations may occur on one side of the brain and not the other. Some may be ‘clumped,’ affecting just one gyrus [fold] of the brain, disrupting just a little part of the cortex at a time.”

The study examined brain tissue from a deceased 17-year-old who had been neurologically normal, sampling in more than 30 brain locations. It builds on work published by the Walsh lab in 2012, which developed methods to sequence the genomes of single neurons, and represents the first time single neurons have been sequenced in their entirety. The single-cell technique is better at detecting subtle mosaicism than usual DNA sequencing methods, which sequence many thousands or millions of cells mixed together and read out an average for the sample.

Somatic brain mutations, affecting just pockets of cells, can be harmful, and have been suggested as a possible cause of neurodevelopmental disorders such as autism, epilepsy or intellectual disability (see this review article for further background). But they also can be completely benign or have just a subtle effect.

“Our findings are intriguing because they suggest that every normal brain may in fact be a mosaic patchwork of focal somatic mutations, though in normal individuals most are likely silent or harmless,” says Gilad Evrony, PhD, in the Walsh Lab, co-first author on the Neuron paper. “These same technologies can now be used to study the brains of people who died from unexplained neuropsychiatric diseases to determine whether somatic mutations may be the cause.”

Finally, says Evrony, the findings provide a proof-of-principle for a systematic way of studying how brain cells disperse and migrate during development, “something that has not been possible to do before in humans,” he says.

Co-first author Alice Eunjung Lee, PhD, from the lab of Peter Park, PhD, at the Center for Biomedical Informatics at Harvard Medical School, developed the study’s retrotransposon analysis tool, which detects somatic retrotransposon mutations in single-cell sequencing data.

Mirroring these findings, study published by Walsh’s lab in 2014 used single-neuron sequencing to detect copy number variants—another type of mutation affecting the number of copies of chromosomes or chromosome fragments. The study found that these mutations can occur in both normal and neurologically diseased brains.

Evrony and Lee are first authors on the Neuron paper; Walsh and Park are senior authors. The research was supported by the National Institutes of Health (MSTP grant T32GM007753), the National Institute of Neurological Disorders and Stroke (R01 NS079277 and R01 NS032457), the Louis Lange III Scholarship in Translational Research, the Eleanor and Miles Shore Fellowship, the Research Connection and the Manton Center for Orphan Disease Research at Boston Children’s Hospital, the Paul G. Allen Family Foundation and the Howard Hughes Medical Institute.

SOURCE Boston Children’s Hospital

Beth Stevens: A transformative thinker in neuroscience

Posted on September 29, 2015 by Nancy Fliesler

Posted in All Posts, Drug discovery, Profiles

More On: Alzheimer’s disease, autism, FM Kirby Neurobiology Center, glial cells, neuroscience, synapse development

http://vector.childrenshospital.org/2015/09/beth-stevens-a-transformative-thinker-in-neuroscience/

https://youtu.be/6DOYTpXkLOY

When 2015 MacArthur “genius” grant winner Beth Stevens, PhD, began studying the role of glia in the brain in the 1990s, these cells—“glue” from the Greek—weren’t given much thought. Traditionally, glia were thought to merely protect and support neurons, the brain’s real players.

But Stevens, from the Department of Neurology and the F.M. Kirby Neurobiology Center at Boston Children’s Hospital, has made the case that glia are key actors in the brain, not just caretakers. Her work—at the interface between the nervous and immune systems—is helping transform how neurologic disorders like autism, amyotrophic lateral sclerosis (ALS), Alzheimer’s disease and schizophrenia are viewed.

Soon after college graduation in 1993, without prior experience in neuroscience, she helped discoveran interplay between neurons and glial cells known as Schwann cells that controlled production of the nerve insulation known as myelin It was one of the early pieces of evidence that glia and neurons talk to each other.

In 2007, while still a postdoctoral fellow, Stevens showed how star-shaped glial cells called astrocytes influence the development of synapses, or brain connections. Studying neurons, her lab showed that a gene called C1q was markedly more active when astrocytes were present. C1q is an immune gene, one nobody had expected to see in a normal brain. In the context of disease, it initiates the complement cascade, an immunologic pathway for tagging unwanted cells and debris for clearance by other immune cells.

But in healthy developing brains, Stevens showed, C1q was concentrated at developing synapses, or brain connections, apparently marking certain synapses for pruning.

Then in 2012, the Stevens lab showed that microglia—another type of glia usually thought of as immune cells themselves—actively sculpt the brain’s wiring. They literally trim away unwanted, inappropriate synapses by eating them—in the same way they’d engulf and destroy invading bacteria.

http://19g6dy4by8jx1b5cx74fh0f2.wpengine.netdna-cdn.com/wp-content/uploads/2012/06/Microglial-cell.jpg

That paper was cited by the journal Neuron as the year’s most influential paper.

The same year, she received a Presidential Early Career Award for Scientists and Engineers, honoring her innovative research and scientific leadership.

Stevens’s current investigations are looking at synapse loss—a hallmark of neurodegenerative conditions such as Alzheimer’s—and trying to understand why it occurs. Her lab’s recent work suggests that normal pruning mechanisms that are active during early brain development get re-activated later in life. Intervening with this activation could lead to a new treatment approach, she believes.

Stevens isn’t the only brain researcher at Boston Children’s to become a MacArthur fellow. Neurosurgeon Benjamin Warf, MD, received the honor in 2012.

For more:

Vector Q&A with Beth Stevens
Previous coverage of her Stevens’s work on Vector
Profiles of Stevens on the MacArthur Foundation website and Spectrum News

Immune cells “sculpt” brain circuits — by eating excess connections

Posted on June 5, 2012 by Nancy Fliesler

Posted in All Posts, Drug discovery, Pediatrics

More On: ALS, Alzheimer’s disease, autism, brain development, epilepsy, glaucoma, Huntington’s disease, Lou Gehrig’s disease,Parkinson’s disease, synapse development

https://youtu.be/wb8UAyf8Nhw

The above movie shows an immune cell caught in the act of tending the brain—it’s just eaten away unnecessary connections, or synapses, between neurons.

That’s not something these cells, known as microglia, were previously thought to do. As immune cells, it was thought that their job was to rid the body of unwanted pathogens and debris, by engulfing and digesting them.

The involvement of microglia in the brain’s development has started to be recognized only recently. The latest research finds that microglia tune into the brain’s cues, akin to the way they survey their environment for invading microbes, and get rid of excess synapses the same way they’d dispatch these invaders—by eating them.

It’s a whole other way of understanding how the healthy brain develops—at the hands of cells that were once thought to be merely nerve “glue” (the literal meaning of “glia” from the Greek), playing a protective role to neurons, say investigators Beth Stevens, PhD, and Dori Schafer, PhD, of the F.M. Kirby Neurobiology Center at Boston Children’s Hospital.

“In the field of neuroscience, glia have often been ignored,” says Stevens. “But glia aren’t the nerve glue, they’re actively communicating with neurons. People have gotten a new respect for glia and are hungry to know more about them.”

Such knowledge could eventually shed light on brain disorders ranging from autism to Alzheimer’s.

The “eat me” sign

We’re all born with more brain connections than we need. As we begin to encounter our world, they’re trimmed back to fine-tune our circuitry. It’s a bit of an oversimplification, but Stevens and Schafer demonstrated last week in the journal Neuron that when two neurons start talking to each other less – because their connection is no longer important to our lives– the microglia notice that and prune the synapse away.

To study microglia’s pruning activity, Stevens and Schafer used a time-honored model: the visual system. When you cover one eye soon after birth, you force the brain to rewire: Brain connections with the covered eye weaken and those synapses eventually get eliminated.

Using this model, Stevens and Schafer showed that microglia take their cues from a set of signals also used by the immune system, known as the complement cascade. Specifically, microglia carry receptors that recognize the complement protein C3—the same protein found on synapses that are destined for elimination.

“We think that weaker synapses are being tagged with C3, and that microglia are eliminating them just as macrophages would eliminate bacteria,” says Schafer. “C3 is like an ‘eat me’ signal.”

As a postdoctoral fellow in 2007, Stevens showed that neurons are loaded with complement proteins soon after birth, just when pruning is at its peak. In the new study, she and Schafer deliberately disrupted complement signaling in mice—stripping the microglia of C3 receptors, or blocking those receptors with a drug. When they did so, pruning of irrelevant synapses didn’t occur.

Stevens thinks their findings might have relevance for brain disorders. Developmental brain disorders such as autism, epilepsy or schizophrenia are increasingly seen as disorders of synapse development, and some data suggest that microglia and/or the complement cascade are involved.

At the other end of the spectrum, scientists have noted that microglia—normally in a resting state in adults—are activated in neurodegenerative diseases like glaucoma, Alzheimer’s disease, Lou Gehrig’s disease, Huntington’s disease and Parkinson’s disease. Subtle changes have been found in synapses that might cause them to be targeted for elimination.

So could targeting microglia or the complement cascade prevent synapse loss or alter pruning in these diseases? “All this is still very speculative,” Stevens cautions. “We first need to understand normal brain development.”

Beth Stevens

Neuroscientist

Assistant Professor of Neurology, F. M. Kirby Neurobiology Center, Boston Children’s Hospital

Department of Neurology, Harvard Medical School

Boston, Massachusetts

Age: 45

Published September 28, 2015

https://www.macfound.org/fellows/946/#sthash.GpHuiEC6.dpuf

Beth Stevens is a neuroscientist whose research on microglial cells is prompting a significant shift in thinking about neuron communication in the healthy brain and the origins of adult neurological diseases. Until recently, it was believed that the primary function of microglia was immunological; they protected the brain by reducing inflammation and removing foreign bodies.

Stevens identified an additional, yet critical, role: the microglia are responsible for the “pruning” or removal of synaptic cells during brain development. Synapses form the connections, or means of communication, between nerve cells, and these pathways are the basis for all functions or jobs the brain performs. Using a novel model system that allows direct visualization of synapse pruning at various stages of brain development, Stevens demonstrated that the microglia’s pruning depends on the level of activity of neural pathways. She identified immune proteins called complement that “tag” (or bind) excess synapses with an “eat me” signal in the healthy developing brain. Through a process of phagocytosis, the microglia engulf or “eat” the synapses identified for elimination. This pruning optimizes the brain’s synaptic arrangements, ensuring that it has the most efficient “wiring.”

Stevens’s discoveries indicate that our adult neural circuitry is determined not only by the nerve cells but also by the brain’s immune cells. Her work suggests that adult diseases caused by deficient neural architecture (such as autism and schizophrenia) or states of neurodegeneration (such as Alzheimer’s or Huntington’s disease) may be the result of impaired microglial function and abnormal activation of this pruning pathway. Stevens is redefining our understanding of how the wiring in the brain occurs and changes in early life and shedding new light on how the nervous and immune systems interact in the brain, both in health and disease.

Beth Stevens received B.S. (1993) from Northeastern University and a Ph.D. (2003) from the University of Maryland. She was a postdoctoral fellow (2005–2008) at Stanford University and is currently an assistant professor in the Department of Neurology at Harvard Medical School and the F. M. Kirby Neurobiology Center at Boston Children’s Hospital. She is also an Institute Member of the Broad Institute of MIT and Harvard. Her scientific papers have appeared in such journals as Neuron, Science, Proceedings of the National Academy of Sciences, and Nature Neuroscience, among others.

– See more at: https://www.macfound.org/fellows/946/#sthash.GpHuiEC6.dpuf

Portraits of scientists who are making a mark on autism research.

http://spectrumnews.org/news/profiles/beth-stevens-casting-immune-cells-as-brain-sculptors/

Beth Stevens: Casting immune cells as brain sculptors

BY NICHOLETTE ZELIADT / 24 SEPTEMBER 2015

Shortly after Beth Stevens launched her lab at Boston Children’s Hospital in 2008, she invited students from the Newton Montessori School, in a nearby suburb, to come for a visit. The children peered at mouse and rat brains bobbing in fluid-filled jars. They also learned how to position delicate slices of brain tissue on glass slides and inspect them with a microscope.

This visit sparked a running relationship with the school, with a steady stream of students visiting the growing lab each year. Soon it became too complicated to bring so many children to the lab, so Stevens decided to take her neuroscience lessons on the road, visiting a number of local elementary schools each year. Last year, she dropped in on the classrooms of her 5- and 8-year-old daughters, Zoe and Riley.

“The kids got really excited,” Stevens says. “It’s become such a thing that the principal wants me to come back for the whole school.”

Stevens’ enthusiasm for science has left a lasting impression on researchers, too. Her pioneering work points to a surprise role in brain development formicroglia, a type of cell once considered to simply be the brain’s immune defense system, cleaning up cellular debris, damaged tissue and pathogens. But thanks to Stevens, researchers now appreciate that these non-neuronal cells also play a critical role in shaping brain circuits.

In a 2012 discovery that created a buzz among autism researchers, Stevens and her colleagues discovered that microglia prune neuronal connections, calledsynapses, in the developing mouse brain. The trimming of synapses is thought to go awry in autism. And indeed, emerging work from Stevens’ lab hints at a role for microglia in the disorder.

Stevens has already earned praise and several prizes for her work. In 2012, shereceived the Presidential Early Career Award for Scientists and Engineers, the most prestigious award that the U.S. government bestows on young scientists. And in October, she’ll deliver one of four presidential lectures at the world’s largest gathering of neuroscientists — the annual meeting of the Society for Neuroscience — an honor she shares with three neuroscience heavyweights, including two Nobel laureates.

“The field is probably expecting a lot from Beth,” says Jonathan Kipnis, professor of neuroscience at the University of Virginia. Stevens has put microglia at the forefront, Kipnis says. “What used to be a stepchild of neuroscience research is now getting a lot of attention, and I think in part it’s due to her research.”

Curious mind:

Stevens was born in 1970 in Brockton, Massachusetts, where her mother taught elementary school and her father was the school’s principal. As a child, she was deeply inquisitive, eager to understand how things work. She enjoyed collecting bugs and worms, and would analyze these precious specimens in makeshift labs in her backyard.

But a career in science wasn’t on her radar until high school, when she took a biology class with an inspiring teacher named Anthony Cabral. “He totally made me realize that this could be a career, that I could be a scientist,” Stevens says. “It was that one class that changed it, and I’m like, ‘Okay, I’m going to do this.’”

In 1988, she began studying biology at Northeastern University in Boston, which offered an unusual opportunity. It had a unique cooperative education program that allowed Stevens to spend several semesters working full time in medical labs after finishing her coursework.

After that experience, Stevens knew she wanted to find a job in a research lab. After graduating in 1993, she joined her then-boyfriend Rob Graham, now her husband, in Washington, D.C., where he had landed a job in the U.S. Senate. Stevens headed to the National Institutes of Health (NIH) in Rockville, Maryland, to apply for a job as a research assistant.

At around the same time, neuroscientist R. Douglas Fields was launching his lab at the NIH. He studied how neural impulses influence glia — a class of non-neuronal cells that includes microglia — and shape the structure of the developing brain. Fields readily hired Stevens despite her lack of expertise in neuroscience. “I was impressed with her work ethic, energy and drive,” he says.

Stimulating research:

In Fields’ lab, Stevens used a multi-compartment cell culture system to investigate whether stimulating neurons influences the activity of Schwann cells, glial cells that produce a fatty substance called myelin, which insulates nerves1. She discovered that patterns of neural impulses similar to those that occur during early development influence the maturation of Schwann cells and the production of myelin.

The findings added to mounting evidence that glia and neurons communicate with each other, a newly emerging concept at the time.

“What I loved about the glia research was that there were so few neuroscientists studying it; it was such a mysterious part of neuroscience,” Stevens says. “Those years in Doug’s lab were really exciting because it was a new field.”

Stevens spent five years in Fields’ lab. “She was doing extraordinary work,” Fields says. “She had the potential and the interest to do neuroscience research, and I recommended that she should consider going to graduate school.”

But Stevens didn’t want to give up her position in the lab, and at that time, the NIH did not allow its researchers to have graduate students. So she and Fields convinced the University of Maryland, College Park, just 10 miles away, to allow her to take graduate courses in neuroscience while completing the necessary research for her Ph.D. in Fields’ lab.

In 2000, less than two years after starting graduate school, Stevens published a paper in Science showing that nerves in the peripheral nervous system (located outside the brain and spinal cord) use chemical signals to communicate with Schwann cells2. Two years later, she reported in Neuron that a similar form of communication occurs in the brain, between neurons and oligodendrocytes, the myelin-producing cells in the brain3.

As she was closing in on her Ph.D., Stevens sought career advice from Story Landis, then-director of the National Institute of Neurological Disorders and Stroke. Landis turned Stevens on to the possibility of starting her own lab one day. “I convinced her that she really had the abilities and energy and intelligence to run an independent research program,” Landis says.

In 2004, Stevens sought a postdoctoral fellowship with neurobiologist Ben Barres at Stanford University. “She was already seen as a leading researcher in the glial field,” recalls Barres, who promptly hired her. “She had done all sorts of beautiful work on glia.”

In Barres’ lab, Stevens continued to explore the dialogue between neurons and glia, turning her attention to star-shaped glia called astrocytes. Barres and his team had discovered that astrocytes help neurons form synapses4. To get a better handle on this process, Stevens examined how astrocytes influence gene expression in neurons in the developing mouse brain.

To her surprise, she found that astrocytes trigger neurons to produce a ‘complement’ protein that is best known for its role in the immune system. There, the protein serves as an ‘eat me’ signal, flagging pathogens and debris for removal. She found that neurons deposit this tag around immature synapses, but not mature ones, in mouse brain tissue, and mice that lack this protein have too many immature synapses. The findings suggested that astrocytes might help eliminate synapses by triggering the complement cascade5.

http://spectrumnews.org/wp-content/uploads/2015/09/20150929ProfileBethStevensChild350.jpg

Young recruit: Beth Stevens’ daughter Riley inspects brain tissue during a visit to her mother’s lab. | Courtesy of Beth Stevens

But it was still unclear exactly how the tagged synapses are cleared. The prime suspects were microglia, the only cells in the brain known to have the receptor for the ‘eat me’ signal.

Stevens set out to test this hypothesis in her own lab: After four years as a postdoc, she had decided to branch out on her own. In 2008, neuroscientist Michael Greenberg — chair of the neurobiology department at Harvard — recruited her to the Harvard-affiliated Boston Children’s Hospital. Even when her lab was in its infancy, she had little trouble convincing new staff to join her.

“A lot of people might be a little hesitant to join a new lab,” says Dorothy Schafer, a former postdoctoral fellow in Stevens’ lab who is now assistant professor of neurobiology at the University of Massachusetts-Worcester. “But I was so excited by the research, and she was so energetic and extremely positive, and just seemed like a very nice person.”

One decision Stevens made early on was to continue to studying microglia in mice rather than experiment with new model systems. “You’ll never see her working on songbirds, because she has this aversion to birds,” Schafer says. “I think they think her curly blond hair is a nest or something, and she’s had really bad experiences with many types of birds dive-bombing her head.”

Just four years into her foray as an independent researcher, Stevens found the proof she had been looking for. In 2012, her team published evidence that microglia eat synapses, especially those that are weak and unused6.

The findings pinned down a new role for microglia in wiring the brain. They also helped to explain how the brain, which starts out with a surplus of neurons, trims some of the excess away. Neuron named the paper its most influential publication of 2012.

Stevens continues to study the function of microglia in the healthy brain, most recently uncovering preliminary evidence that a certain protein serves as a ‘don’t eat me’ tag that protects synapses from being engulfed by microglia. She is also exploring the role of microglia in disorders such as autism.

Several studies suggest that microglia are more active and more numerous in the brains of people with autism than in controls. Stevens and her team are looking at whether the activity of microglia is altered during brain development in mouse models of autism.

Immunodulatory Thalidomides in ~ conjugants unleash proteasome degradation on ~ oncoproteins with distinct mechanisms- BRD4,MYC & PIM1 & little collteral damage to 7429 other proteins!

Imagine being able to specifically target a cancer protein for immediate destruction, slipping Robert Louis Stevenson’s notorious black spot into a crevice in the secondary structure and spelling imminent death. Well, this is what Winter et al. (2015) describe in a recent drug discovery report for Science.1 Using phthalimide conjugation, the researchers not only specifically marked BRD4, a transcriptional coactivator important in MYC oncogene upregulation, for proteasomal degradation, but also achieved reduced tumor burdens in vivo.

The research team combined two drugs, thalidomide and JQ1, exploiting the properties of each to create a bifunctional compound, dBET1, that drives the proteasomal degradation of BRD4. JQ1, which in itself is anti-oncogenic, selectively binds BET bromodomains on the transcription factor, thus competitively inhibiting BRD4 activity on chromatin. Thalidomide, a phthalimide-based drug with immunomodulatory properties, binds cereblon (CRBN) in the cullin-RING ubiquitin ligase (CRL) complex, which is important in proteasomal protein degradation.

After confirming that the new phthalimide conjugate, dBET1, retained affinity for BRD4 and that this binding was specific, the team used a human acute myelocytic leukemia (AML) cell line, MV4;11, to show that treatment with the conjugate over 18 hours reduced BRD4 abundance. The researchers also found this with dBET1 treatment of other human cancer cell lines (SUM159, MOLM13). Following this, Winter et al. investigated the mechanisms by which dBET1 inhibits BRD4. By focusing primarily on proteasome function, the researchers determined that the reduction in BRD4 abundance in MV4;11 cells is proteasomal and dependent on CRBN binding activity.

Having established targeted proteasomal degradation using the dBET1 conjugate, Winter et al. then investigated the proteomic consequences of treatment in MV4;11 cells. Scientists at the Thermo Fisher Scientific Center for Multiplexed Proteomics (Harvard Medical School) used quantitative proteomics analysis with an isobaric tagging approach to compare the immediate effects of dBET1 treatment following two hours of incubation with the responses to JQ1 and vehicle control. Spectral data analysis identified 7,429 proteins with few differences in response to either treatment. JQ1 treatment reduced levels of MYC and oncoprotein PIM1 similarly to the response following dBET1 incubation. However, treatment with the latter also reduced BRD2, BRD3 and BRD4 abundance, findings that the research team confirmed with specific immunoblotting. Measuring expression of mRNA showed that both treatments reduced levels of MYC and PIM1 abundance. However, Winter et al. found no difference in BRD3 and BRD4, suggesting that dBET1 reduces the protein levels by post-transcriptional regulation.

Investigating the antiproliferative potential of the phthalimide conjugate, dBET1, Winter and coauthors examined apoptotic response in both MV4;11 and DHL4 lymphoma cells, and in primary human AML blast cultures. Compared to JQ1 treatment, dBET1 stimulated a profound and prolonged apoptotic response in both cell lines, suggesting that targeted degradation could be a more effective treatment than target inhibition.

shapes of proteins as they shift from one stable shape to a different, folded one Protein-structural-changes

Orchestrating the unfolded protein response in health and disease

Randal J. Kaufman Department of Biological Chemistry,
Howard Hughes Medical Institute, University of Michigan Medical Center, Ann Arbor, Michigan, USA J. Clin. Invest. 110:1389–1398 (2002). http://dx.doi.org:/10.1172/JCI200216886

The endoplasmic reticulum (ER), the entrance site for proteins destined to reside in the secretory pathway or the extracellular environment, is also the site of biosynthesis for steroids and for cholesterol and many lipids. Given the considerable number of resident structural proteins and biosynthetic enzymes and the high expression of many secreted proteins, the total concentration of proteins in the this organelle can reach 100 mg/ml. The ER relies on an efficient system of protein chaperones that prevent the accumulation of unfolded or aggregated proteins and correct misfolded proteins that are caught in low-energy kinetic traps (see Horwich, this Perspective series, ref. 1).

These chaperone-mediated processes expend metabolic energy to ensure high-fidelity protein folding in the lumen of the ER. For example, the most abundant ER chaperone, BiP/GRP78, uses the energy from ATP hydrolysis to promote folding and prevent aggregation of proteins within the ER. In addition, the oxidizing environment of the ER creates a constant demand for cellular protein disulfide isomerases to catalyze and monitor disulfide bond formation in a regulated and ordered manner. Operating in parallel with chaperone dependent protein folding are several “quality control” mechanisms, which ensure that, of all proteins translocated into the ER lumen, only those that are properly folded transit to the Golgi compartment. Proteins that are misfolded in the ER are retained until they reach their native conformation or are retrotranslocated back into the cytosol for degradation by the 26S proteasome. The ER has evolved highly specific signaling pathways to ensure that its protein-folding capacity is not overwhelmed. These pathways, collectively termed the unfolded protein response (UPR), are required if the cell is to survive the ER stress (see Ron, this Perspective series, ref. 2) that can result from perturbation in calcium homeostasis or redox status, elevated secretory protein synthesis, expression of misfolded proteins, sugar/glucose deprivation, or altered glycosylation. Upon accumulation of unfolded proteins in the ER lumen, the UPR is activated, reducing the amount of new protein translocated into the ER lumen, increasing retrotranslocation and degradation of ER-localized proteins, and bolstering the protein-folding capacity of the ER. The UPR is orchestrated by the coordinate transcriptional activation of multiple genes, a general decrease in translation initiation, and a concomitant shift in the mRNAs that are translated.

The recent discovery of the mechanisms of ER stress signaling, coupled with the ability to genetically engineer model organisms, has led to major new insights into the diverse cellular and physiological processes that are regulated by the UPR. Here, I summarize current discoveries that have offered insights into the complex regulation of the UPR and its relevance to human physiology and disease.

Glucose and protein folding Early studies demonstrated that both viral transformation and glucose depletion induce transcription of a set of related genes that were termed glucose-regulated proteins (GRPs) (3). Since viral transformation increases both the cellular metabolic rate and ATP utilization, it became evident that, in both cases, this signal emanates from the ER as a consequence of energy deprivation. Because proteins have different ATP requirements for protein folding prior to export, it has been proposed that the threshold for UPR activation might differ among various cell types, depending on their energy stores and the amount and nature of the secretory proteins they produce (4). Glucose not only provides the metabolic energy needed by cells but also participates directly in glycoprotein folding as a component of oligosaccharide structures.

The recognition and modification of oligosaccharide structures in the lumen of the ER is intimately coupled to polypeptide folding (5). As the growing nascent chain is translocated into the lumen of the ER, a 14-oligosaccharide core (GlcNAc2Man9Glc3) is added to consensus asparagine residues. Immediately after the addition of this core, the three terminal glucose residues are cleaved by the sequential action of glucosidases I and II to yield a GlcNAc2Man9 structure. If the polypeptide is not folded properly, a UDP-glucose:glycoprotein glucosyltransferase (UGGT) recognizes the unfolded nature of the glycoprotein and reglucosylates the core structure to re-establish the glucose-α(1, 3)–mannose glycosidic linkage. Monoglucosylated oligosaccharides containing this bond bind to the ER-resident protein chaperones calnexin and calreticulin.

This quality control process ensures that unfolded glycoproteins do not exit the ER. Treatment of cells with castanospermine, a transition-state analogue inhibitor of glucosidases I and II, inhibits this monoglucosylation cycle, prevents interaction of unfolded glycoproteins with calnexin and calreticulin, and activates the UPR. Genetic alterations that reduce the nucleotide sugar precursor pool or glycosyltransferase reactions likewise activate the UPR (6). Therefore, the recognition of altered carbohydrate structures is in some manner linked to UPR activation.

The UPR in yeast and higher eukaryotes On a cellular level, the accumulation of unfolded proteins in the ER lumen induces the transcription of a large set of genes whose products increase the ER’s volume or its capacity for protein folding or promote the degradation of misfolded proteins through the process of ER-associated protein degradation (ERAD) (7). For example, transcription of the ER protein chaperone BiP is a classical marker for UPR activation in yeast and mammalian cells (8). BiP binds hydrophobic exposed patches on the surfaces of unfolded proteins and interactive sites on unassembled protein subunits, and it releases its polypeptide substrates upon ATP binding.

In parallel, as Ron (this Perspective series, ref. 2) details in his accompanying article, translation is attenuated to decrease the protein-folding load. The complex network of physiological responses to ER stress is regulated by only a few ER transmembrane proteins: IRE1, PERK, and ATF6 (9). IRE1, PERK, and ATF6 are proximal sensors that regulate the production and/or quality of basic leucine zipper–containing (bZIP-containing) transcription factors that may form homo- and heterodimers. Combinatorial interactions of these factors generate diversity in responses for different subsets of UPRresponsive genes. In multicellular organisms, if these adaptive responses are not sufficient to relieve ER stress, the cell dies through apoptosis or necrosis.

IRE1-dependent splicing The UPR-signaling pathway was first described less than ten years ago in the budding yeast Saccharomyces cerevisiae. Elegant studies identified IRE1 as the sensor of unfolded proteins in the ER lumen. IRE1 is a type 1 transmembrane Ser/Thr protein kinase that also has a site-specific endoribonuclease (RNase) activity. The presence of unfolded proteins in the ER lumen promotes dimerization and trans-autophosphorylation, rendering IRE1 active as an RNase, and allowing it to cleave a 252-base intron from the mRNA encoding the transcription factor HAC1 (10). The 5′ and 3′ ends of HAC1 mRNA are spliced together by tRNA ligase in a process that is independent of the spliceosome and the usual intranuclear machinery for mRNA splicing. Splicing of HAC1 mRNA increases its translational efficiency and alters sequence of the encoded HAC1 protein, yielding a potent transcriptional activator (11) that can bind and activate the UPR elements (UPREs) upstream of many UPR-inducible genes. In S. cerevisiae, the UPR activates transcription of approximately 381 genes (7).

All eukaryotic cells appear to have maintained the essential and unique properties of the UPR present in S. cerevisiae, but higher eukaryotes possess additional sensors that generate diverse, coordinately regulated responses that promote stress adaptation or cell death. The mammalian genome contains two homologues of yeast IRE1 — IRE1α and IRE1β. Whereas IRE1α is expressed in most cells and tissues, with high-level expression in the pancreas and placenta (12), IRE1β expression is prominent only in intestinal epithelial cells (13). Both IRE1 molecules respond to the accumulation of unfolded proteins in the ER, which activate their kinase and, thereby, their RNase activities. The cleavage specificities of IRE1α and IRE1β are similar, if not identical, suggesting that they do not recognize different sets of substrates but rather generate temporally specific and tissue-specific expression (14, 15).

Searching for transcription factors that mediate the UPR, Yoshida et al. defined a mammalian ER stress response element [ERSEI; CCAAT(N9)CCACG] that is necessary and sufficient for UPR gene activation. Using a yeast one-hybrid screen, these authors isolated XBP1, a bZIP transcription factor X-box DNA binding protein (16). Subsequently, several groups demonstrated that XBP1 mRNA is a substrate for mammalian IRE1, much as the HAC1 mRNA in S. cerevisiae is processed by the yeast IRE1; this pathway is also conserved in Caenorhabditis elegans (17–20). On activation of the UPR, XBP1 mRNA is cleaved by IRE1 to remove a 26-nucleotide intron and generate a translational frameshift. As expected given the precedent of HAC1 regulation in yeast, the resulting processed mRNA encodes a protein with a novel carboxy-terminus that acts as a potent transcriptional activator.

Overexpression of either IRE1α or IRE1β is sufficient to activate transcription from a BiP promoter reporter construct (15). Analysis of a minimal UPRE motif (TGACGTGC/A) (21) uncovered a transcriptional defect in IRE1α-null mouse embryo fibroblasts that could be complemented by expression of spliced XBP1 mRNA (20), and Yoshida et al. (unpublished data) recently identified a UPR-inducible gene that uniquely requires IRE1α-mediated splicing of XBP1 mRNA. However, neither IRE1α nor IRE1β is necessary for transcriptional activation of the BiP gene, as judged by the phenotype of IRE1α/β–deleted murine cells (20, 22, 23). These results indicate that a subset of UPR targets require IRE1 but that at least one IRE1-independent pathway exists for UPR-mediated transcriptional induction. Deletion of IRE1α causes embryonic lethality at embryonic day 10.5 (E10.5) (20, 22, 23). Therefore, although IRE1α is not required for the UPR, it is clearly required for mammalian embryogenesis. XBP1 deletion also causes embryonic lethality, but the mutant embryos can survive up to day E14.5, consistent with the notion that XBP1 acts downstream of IRE1α. XBP1 deletion causes cardiomyopathy and liver hypoplasia (24, 25). In contrast, IRE1β-null mice develop normally but exhibit increased susceptibility to experimentally induced colitis, a phenotype that is consistent with the specific expression of this kinase in the intestinal epithelium (26).

Activation of ATF6 and PERK by ER stress The activating transcription factor ATF6 (16) has been identified as another regulatory protein that, like XBP1, can bind ERSEI elements in the promoters of UPRresponsive genes. There are two forms of ATF6, both synthesized as ER transmembrane proteins. ATF6α (90 kDa) and ATF6β (110 kDa, also known as CREB-RP) both require the presence of the transcription factor CBF (also called NF-Y) to bind ERSEI (27–30).

On activation of the UPR, both forms of ATF6 are processed to generate 50- to 60-kDa cytosolic, bZIP containing transcription factors that migrate to the nucleus (27). Processing of ATF6 by site-1 protease (S1P) and site-2 protease (S2P) occurs within the transmembrane segment and at an adjacent site exposed to the ER lumen. S1P and S2P are the processing enzymes that cleave the ER-associated transmembrane sterolresponse element–binding protein (SREBP) upon cholesterol deprivation (31). The cytosolic fragment of cleaved SREBP migrates to the nucleus to activate transcription of genes required for sterol biosynthesis. Interestingly, although the mechanism regulating ATF6 processing is similar to that regulating SREBP processing (32), the UPR only elicits ATF6 processing, whereas sterol deprivation alone induces SREBP processing. The SREBP cleavage–activating protein (SCAP) confers specificity for SREBP transport to the Golgi compartment, and consequently cleavage in response to sterol deprivation (33). It is unknown whether another cleavage-activating protein, analogous to SCAP but active only following induction of the UPR, promotes the specific cleavage and activation of ATF6 by S1P and S2P.

Transcription of UPR-responsive genes is induced when the cleaved form of ATF6 activates the XBP1 promoter. Therefore, signaling through ATF6 and IRE1 merges to induce XBP1 transcription and mRNA splicing, respectively (Figure 1, a and b). ATF6 increases XBP1 transcription to produce more substrate for IRE1- mediated splicing that generates more active XBP1, providing a positive feedback for UPR activation. However, cells that lack either IRE1α or ATF6 cleavage can induce XBP1 mRNA (20). These two pathways may thus provide parallel signaling pathways for XBP1 transcriptional induction. Alternatively, another pathway — possibly mediated by the ER-localized protein kinase PERK (see Ron, this Perspective series, ref. 2) — may also contribute to induction of XBP1 mRNA. The binding specificities of XBP1 and ATF6 are similar, although ATF6 binding requires CBF binding to an adjacent site, whereas XBP1 binds independently (17, 20, 21, 34). These binding specificities provide another avenue for complementary interaction between the IRE1-XBP1 and ATF6 pathways at the level of transcriptional activation. In addition, these transcription factors might regulate transcription from a second ERSE (ERSEII), which also contains a CCACG motif (35).

In parallel with the activation of ATF6 processing and the consequent changes in gene transcription, the accumulation of unfolded proteins in the ER also alters cellular patterns of translation. The protein kinase PERK has been implicated in this aspect of the ER stress response (see Ron, this Perspective series, ref. 2). Activated PERK phosphorylates the α subunit of eukaryotic translation initiation factor 2 (eIF2α) and attenuates general protein synthesis. Inactivation of the PERK-eIF2α phosphorylation pathway decreases cells’ ability to survive ER stress (36, 37). The PERK pathway promotes cell survival not only by limiting the protein-folding load on the ER, but also by inducing transcription of UPR- activated genes, one-third of which require phosphorylation of eIF2α for their induction (36). Preferential translation of the transcription factor ATF4 allows for continued activation of these genes under conditions of stress, when general protein synthesis is inhibited (36, 37).

A coordinated mechanism for activation One puzzling question about the UPR is how three independent sensors are activated by a common stimulus, the accumulation of unfolded proteins in the ER lumen. BiP, which negatively regulates the UPR, interacts with all three sensors, IRE1, PERK, and ATF6, under nonstressed conditions and may indeed be the master regulator of UPR activation.

Upon accumulation of unfolded proteins in the ER, BiP is released from IRE1, PERK, and ATF6. It is believed that the unfolded proteins bind BiP and sequester it from interacting with IRE1, PERK, and ATF6 to elicit their activation. In this manner, BiP senses both the level of unfolded proteins and the energy (ATP) level in the cell in regulating the UPR. Following release from BiP, IRE1 and PERK are each free to undergo spontaneous homodimerization mediated by their lumenal domains and to become phosphorylated by their endogenous kinase activities (38, 39). BiP interaction with ATF6 prevents trafficking of ATF6 to the Golgi compartment. For this reason, BiP release permits ATF6 transport to the Golgi compartment, where it gains access to S1P and S2P proteases (32). The regulation of signaling through the free level of BiP is an attractive hypothesis providing a direct mechanism by which all three ER stress sensors could be activated by the same stimulus. In addition, the increase in BiP during the UPR would provide a negative feedback to turn off UPR signaling. However, in certain cells, different stress conditions can selectively activate only one or two of the ER stress sensors. For example, in pancreatic β cells, glucose limitation appears to activate PERK prior to activation of IRE1 (D. Scheuner and R.J. Kaufman, unpublished results). It will be important to elucidate how general BiP repression permits the selective activation of individual components of the UPR that mediate various downstream effects.

Signaling the UPR in eukaryotes

Figure 1 Signaling the UPR in eukaryotes.

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Three proximal sensors, IRE1, PERK, and ATF6, coordinately regulate the UPR through their various signaling pathways. Whereas IRE1 and PERK are dispensable for many aspects of the response, ATF6 cleavage is required for UPR transcriptional induction and appears to be the most significant of these effectors in mammalian cells. BiP negatively regulates these pathways. BiP interacts with ATF6 to prevent its transport to the Golgi compartment (a). BiP binds to the lumenal domains of IRE1 (b) and PERK (c) to prevent their dimerization. As unfolded proteins accumulate, they bind BiP and reduce the amount of BiP available to bind and inhibit activation of IRE1, PERK, and ATF6. (a) BiP release from ATF6 permits transport to the Golgi compartment. In the Golgi, ATF6 is cleaved by S1P and S2P proteases to yield a cytosolic fragment that migrates to the nucleus to activate transcription of responsive genes, including XBP1. (b) BiP release from IRE1 permits dimerization to activate its kinase and RNase activities to initiate XBP1 mRNA splicing. XBP1 splicing removes a 26-base intron, creating a translational frameshift to yield a more potent transcriptional activator. (c) BiP release permits PERK dimerization and activation to phosphorylate Ser51 on eIF2α to reduce the frequency of AUG initiation codon recognition. As eIF2α phosphorylation reduces the functional level of eIF2, the general rate of translation initiation is reduced. However, selective mRNAs, such as ATF4 mRNA, are preferentially translated under these conditions, possibly by the presence of open reading frames within the 5′ untranslated region of the mRNA. Upon recovery from the UPR, GADD34 targets PP1 to dephosphorylate eIF2α and increase protein translation.

The UPR as a mediator of programmed cell death In contrast to UPR-signaling adaptation in response to ER stress, prolonged UPR activation leads to apoptotic cell death (Figure 2). The roles of several death-promoting signaling pathways have been shown by analysis of specific gene-deleted cells. Activated IRE1 recruits c-Jun-N-terminal inhibitory kinase (JIK) and the cytosolic adaptor TRAF2 to the ER membrane (22, 40). TRAF2 activates the apoptosis-signaling kinase 1 (ASK1), a mitogen-activated protein kinase kinase kinase (MAPKKK) (41). Activated ASK1 leads to activation of the JNK protein kinase and mitochondriadependent caspase activation (40–42).

ER insults lead to caspase activation by mitochondria/APAF-1–dependent and –independent pathways. ER stress promotes cytochrome c release from mitochondria, possibly by c-ABL kinase (43) or calcium (44). However, APAF1–/– cells are susceptible to ER stress–induced apoptosis, indicating that the mitochondrial pathway is not essential (45). Caspase-12 is an ER-associated proximal effector in the caspase activation cascade, and cells lacking this enzyme are partially resistant to inducers of ER stress (46). ER stress induces TRAF2 release from procaspase 12, allowing it to bind activated IRE1. As shown in Figure 2, release of TRAF2 permits clustering of procaspase-12 at the ER membrane, leading to its activation (40). Caspase-12 can activate caspase-9, which in turn activates caspase- 3 (47). Procaspase-12 can also be activated by m-calpain in response to calcium release from the ER, although the physiological significance of this pathway is not known (48). In addition, upon ER stress, procaspase-7 is activated and recruited to the ER membrane (49). These findings support the notion that ER stress leads to several redundant pathways for caspase activation.

A second death-signaling pathway activated by ER stress is mediated by transcriptional activation of genes encoding proapoptotic functions. Activation of UPR sensor IRE1, PERK, or ATF6 leads to transcriptional activation of CHOP/GADD153, a bZIP transcription factor that potentiates apoptosis (see Ron, this Perspective series, ref. 2).

The UPR in health and disease Primary amino acid sequence contains all the information for a protein to attain its final folded conformation. However, many folding intermediates exist along the folding pathway (see Horwich, this Perspective series, ref. 1), and some of these intermediates can become irreversibly trapped in low-energy states and activate the UPR. Clearance of such misfolded species requires a functional ER-associated degradation (ERAD) pathway, which is regulated by the UPR. Proteasomal degradation of ER-associated misfolded proteins is required to protect from UPR activation. Proteasomal inhibition is sufficient to activate the UPR, and, in turn, genes encoding several components of ERAD are transcriptionally induced by the UPR (7). Therefore, it is to be expected that UPR activation and impaired ERAD function might contribute to a variety of diseases and that polymorphisms affecting the UPR and ERAD responses could modify disease progression. The following examples provide the best available evidence linking the UPR pathway to the natural history of human diseases and animal models of these diseases.

The UPR and ERAD in genetic disease Many recessive inherited genetic diseases are due to loss of-function mutations that disturb productive folding and that produce proteins that are either not secreted or not functional. In other cases, protein-folding mutations can interfere with cellular processes, resulting in a gain of function and a dominant pattern of inheritance. In several instances, UPR activation by the accumulation of unfolded proteins in the ER is known to contribute to disease progression. The distinction between these two classes of genetic disease is important, because gain-of-function protein-misfolding mutations will be less amenable to treatment by gene therapy to deliver a wild-type copy of the mutant gene.

One well-characterized protein-folding defect results from a mutation that leads to type 1 diabetes. The Akita mouse has a gain-of-function Cys96Tyr mutation in the proinsulin 2 (Ins2) gene; this mutation disrupts proinsulin folding. The mutant protein is retained in the ER of the pancreatic β cell and activates the UPR. Crucially, the progressive development of diabetes in this model is not solely due to the lack of insulin but is rather a consequence of the misfolded protein accumulation, UPR activation, and β cell death. When bred into a Chop–/–background, the Akita mutation causes a lesser degree of β cell death and delayed onset of diabetes (50), indicating that the loss of at least one downstream signaling component of the UPR can ameliorate pathogenesis in this setting.

Signaling UPR-mediated cell death

Figure 2 Signaling UPR-mediated cell death.

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The activation of procaspase-12 is likely the major pathway that induces apoptosis in response to ER stress. Upon activation of the UPR, c-Jun-N-terminal inhibitory kinase (JIK) release from procaspase-12 permits clustering and activation of procaspase-12. Caspase-12 activates procaspase-9 to activate procaspase-3, the executioner of cell death. In addition, activated IRE1 binds JIK and recruits TRAF2, which signals through apoptosis-signaling kinase 1 (ASK1) and JNK to promote mitochondria-dependent apoptosis. In addition, in vitro studies suggest that localized calcium release from the ER activates m-calpain to cleave and activate procaspase-12. Upon UPR activation, procaspase-7 is activated and recruited to the ER membrane. Finally, IRE1, PERK, and ATF6 induce transcription of several genes encoding apoptotic functions, including CHOP/GADD153. CSP, caspase; pCSP, procaspase.

Deficiency in α1-proteinase inhibitor (α1-PI, also known as α1-antitrypsin) results in emphysema and destructive lung disease in one out of 1,800 births. However, a subgroup of affected individuals develop chronic liver disease and hepatocellular carcinoma as a consequence of a secretion defect in the misfolded protein at the site of synthesis, the hepatocyte. This is the most common genetic cause of liver disease in children. The Z allele of the α1 gene PI (Glu342Lys mutation) produces a protein that polymerizes and is retained in the ER for degradation by the proteasome (see Lomas and Mahadeva, this Perspective series, ref. 51; and Perlmutter, this series, ref. 52). While α1-PI Z neither binds BiP nor activates the UPR, analysis of fibroblasts obtained from these patients demonstrates that individuals susceptible to liver disease have inherited a second trait that slows degradation of the misfolded protein in the ER (53), consistent with the idea that polymorphisms that reduce ERAD function can exacerbate pathogenesis of certain diseases.

There are numerous additional genetic misfolding diseases that are also likely influenced by UPR signaling. Because BiP release from IRE1, PERK, or ATF6 can activate the UPR, the expression of any wild-type or mutant protein that binds BiP can have a similar effect. In contrast, misfolded proteins that do not bind BiP are unlikely to activate the UPR. For example, cystic fibrosis is due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Approximately 70% of patients with this disease carry a common mutation, deletion of Phe508, that results in a molecule that is retained in the ER and eventually degraded by the proteosome (see Gelman and Kopito, this Perspective series, ref. 54). Although expression of ∆508 CFTR does not activate the UPR in cultured cells, the protein does interact with calnexin, as well as HSP70, and requires ERAD function for cell survival.

Osteogenesis imperfecta (OI) results from misfolding mutations in procollagen that produce molecules that bind BiP and activate the UPR (55). Interestingly, Wolcott-Rallison syndrome is due to inactivating mutations in the PERK gene. Affected individuals, as well as mice with deletions in Perk, display osteoporosis and deficient mineralization throughout the skeletal system (56, 57), the same defects that are observed in OI. Procollagen type I accumulates to high levels and mature collagen is not detected in bone and osteoblasts from PERKnull mice. Osteoblasts from PERK-null humans and mice display fragmented and distended ER that is filled with electron-dense material (56, 57). These observations suggest that procollagen type 1 uniquely requires PERK function to maintain its transport out of the ER, processing, and secretion In this case, PERK may be required to limit procollagen synthesis so that it does not saturate the ER protein-folding capacity.

The UPR and ERAD in conformational diseases Diseases caused by expansion of polyglutamine repeats and neurodegenerative diseases, such as Alzheimer disease and Parkinson disease, represent a large class of conformational diseases associated with accumulation of abnormal protein aggregates in and around affected neurons. Recent evidence indicates that the pathogenesis of these diseases is due to a defect in proteasomal function that results in UPR activation, leading to cell death. The protein aggregates in these diseases are localized to the nucleus or the cytoplasm and would not be predicted to disturb ER function directly. Nevertheless, they have been found in some cases to activate the UPR and to promote cell death. Analysis of the polyglutamine repeat associated with the spinocerebrocellular atrophy protein (SCA3) in Machado-Joseph disease suggests that cytoplasmic accumulation of the SCA3 aggregate can inhibit proteasome function, thereby interfering with ERAD to induce the UPR and elicit caspase-12 activation (41, 58). These findings support the idea that the UPR can signal the accumulation of unfolded proteins in the cytosol via proteasomal inhibition and disruption of ERAD function.

Parkinson disease is the most common movement disorder, affecting about 1% of individuals 65 years of age or older. Autosomal recessive juvenile parkinsonism (AR-JP) results from defects in the Parkin gene (59), which encodes a ubiquitin protein ligase (E3) that functions with ubiquitin-conjugating enzyme UbcH7 or UbcH8 to tag proteins for degradation. Overexpression of Parkin suppresses cell death associated with ER stress (60). Inherited Parkinson disease is associated with the accumulation in the ER of dopaminergic neurons of PAEL-R, a putative transmembrane receptor protein that is detected in an insoluble form in the brains of AR-JP patients (61). The accumulation of PAEL-R results from defective Parkin that does not maintain the proteasome-degrading activity necessary to maintain ER function (62). Other, still-unidentified substrates of the Parkin E3 ligase may also be relevant to the pathogenesis of AR-JP.

The UPR in diabetes The metabolism of glucose is tightly controlled at the levels of synthesis and utilization through hormonal regulation. The most dramatic phenotype in Wolcott-Rallison syndrome is pancreatic β cell death with infancy onset diabetes (56). A similar defect is observed in PERK-null mice; this defect also correlated with increased apoptosis of β cells (57, 63). In addition, mice with a homozygous Ser51Ala mutation at the PERK phosphorylation site in eIF2α display an even greater β cell loss that appears in utero (36). Therefore, translational control through PERK-mediated phosphorylation of eIF2α is required to maintain β cell survival (see Ron, this Perspective series, ref. 2). The more severe β cell loss in mice harboring the Ser51Ala eIF2α mutation suggests that additional eIF2α kinases partially complement the requirement for PERK in β cell function (36)

Glucose not only promotes the secretion of insulin but also stimulates insulin transcription and translation (64–66). Our group has proposed that glucose stimulated proinsulin mRNA translation is regulated by PERK-mediated phosphorylation of eIF2α in response to UPR activation 36). As blood glucose declines, energy may become limiting for protein folding in the ER and therefore activate the UPR to promote PERK-mediated phosphorylation of eIF2α. Conversely, a rise in blood glucose would turn off the UPR so that translation would accelerate, allowing entry of new preproinsulin into the ER. In this manner, PERK mediated phosphorylation of eIF2α provides a brake on protein synthesis, including proinsulin translation. Continual elevation of blood glucose may also prolong elevated proinsulin translation, eventually activating the UPR as the secretion capacity of the ER is overwhelmed. Therefore, a delicate balance between glucose levels and eIF2α phosphorylation needs to be maintained: Disturbances in either direction may lead to excessive UPR activation, with eventual β cell death.

The insulin resistance and hyperglycemia associated with type 2 diabetes is accommodated by an increase in proinsulin translation. Under these conditions the UPR is activated to compensate for the increased protein-folding requirement in the ER. Prolonged activation of the UPR could contribute to the β cell death associated with insulin resistance. Thus, the signaling mechanisms that β cells use for sensing glucose levels, triggering insulin secretion, and rapidly controlling insulin biosynthesis may have coevolved with ER signaling pathways to support these specialized functions. Pancreatic β cells are exquisitely sensitive to physiological fluctuations in blood glucose, because, in contrast to other cell types, they lack hexokinase, an enzyme with a low affinity but a high capacity for binding glucose. Therefore, in β cells, the production of glucose 6-phosphate and the production of ATP through glycolysis are controlled by glucokinase (67), and the ratio of ATP to ADP correlates directly with the blood glucose level. Periodic decreases in blood glucose level (as occurs between meals) would decrease the ATP/ADP ratio and compromise protein folding in the ER so that the UPR may be frequently activated in these cells. Hence, when glucose levels vary within the normal physiological range, the ER compartment of the β cell may be exposed to greater energy fluctuations than is the ER of other cell types, making the β cell uniquely dependent on the UPR for survival during intermittent decreases in blood glucose levels, as happens between meals. Additionally, the high-level expression of PERK and IRE1α in the pancreas may predispose these kinases to dimerization and activation in response to intermittent stress.

The UPR in organelle expansion The UPR is required for ER expansion that occurs upon differentiation of highly specialized secretory cells, but ER membrane expansion can also proceed independently of UPR activation. Overexpression of membrane proteins, such as HMG CoA reductase or the peroxisomal protein Pex15, promotes the expansion of smooth membranes without UPR activation (68, 69), as does overexpression of the p180 ribosome acceptor in the rough ER membrane (70). Conversely, protein overexpression, even under circumstances in which secretory capacity is unchanged (as occurs following the induction of high levels of cytochrome p450), can activate the UPR to induce ER chaperone levels to match the expanded membrane area (71, 72).

During the terminal differentiation of certain secretory cells, such as those in the pancreas or liver, membrane expansion is accompanied by a dramatic increase in protein secretion. Likewise, upon B cell maturation into high-level antibody-secreting plasma cells, the ER compartment expands approximately fivefold to accommodate the large increase in Ig synthesis. The requirement for the UPR in this latter process has been demonstrated in XBP1–/– cells. Since deletion of XBP1 produces an embryonic-lethal phenotype at day E14.5, the role of XBP1 in B and T cell development had to be studied in immunoincompetent RAG1–/– mice reconstituted with XBP1–/– embryonic stem cells (73). Work in these chimeric mice demonstrated that XBP1 is required for high-level Ig production. Interestingly, the induction of Ig heavy-chain and light-chain gene rearrangement and the assembly and transport of Igµ to the surface of the B cells occurred normally. However, plasma cells were not detected, suggesting a role for XBP1 in plasma cell differentiation or survival.

These findings support the hypothesis that induction of Ig synthesis activates the UPR to induce ER expansion to accommodate the high-level antibody expression. Alternatively, activation of the UPR may be part of the differentiation program that occurs prior to induction of high-level antibody synthesis. Plasma cell differentiation is stimulated in vivo by treatment with LPS or by ligation of CD40 receptors, treatments that activate the innate immune response and have been shown to induce XBP1 mRNA splicing (19). Thus, the UPR may contribute to a programmed response to signals that increase a cell’s protein-secretory demand.

The UPR in hyperhomocysteinemia. The association between high levels of serum homocysteine and the development of ischemic heart disease and stroke is supported by substantial epidemiological data. Unfortunately, it is not known whether homocysteine is the underlying cause of atherosclerosis and thrombosis. Severe hyperhomocysteinemia is caused by mutation in the cystathionine β-synthase (CBS) gene, whose product is a vitamin B6–dependent enzyme required for the conversion of homocysteine to cysteine. Elevated homocysteine is also associated with vitamin B deficiency. In cultured vascular endothelial cells, homocysteine induces protein misfolding in the ER by interfering with disulfide bond formation, and it activates the UPR to induce expression of several ER stress response proteins, such as BiP, GRP94, CHOP, and HERP (74–76). Homocysteine also activates apoptosis in a manner that requires an intact IRE1-signaling pathway (76).

These findings suggest that homocysteine acts intracellularly to disrupt ER homoeostasis. Indeed, recent studies confirm that induction of hyperhomocysteinemia elicits UPR activation in the livers of normal or Cbs+/– mice (77). In addition, hyperhomocysteinemia activates SREBP cleavage, leading to intracellular accumulation of cholesterol (77). Increased cholesterol biosynthesis may explain the hepatic steatosis and possibly the atherosclerotic lesions associated with hyperhomocysteinemia. Finally, hyperhomocysteinemia accelerates atherosclerosis in ApoE–/– mice (78, 79), although the molecular mechanisms remain to be elucidated.

Hyperhomocysteinemia is also associated with increased amyloid production and increased amyloid-mediated neuronal death in animal models of Alzheimer disease (80). These observations suggest that the UPR may link the disease etiologies of hyperhomocysteinemia and Alzheimer disease. HERP, a homocysteine-induced ER stress–responsive gene, appears to be involved in amyloid β-protein (Aβ) accumulation, including the formation of senile plaques and vascular Aβ deposits (81), and that it interacts with both presenilin-1 (PS1) and presenilin-2 (PS2), thus regulating presenilin-mediated Aβ generation. Immunohistochemical analysis of brains from patients with Alzheimer disease reveals intense HERP staining in activated microglia in senile plaques.

The UPR in cancer Hypoxia is a common feature of solid tumors that display increased malignancy, resistance to therapy, and poor prognosis. Hypoxia in the tumor results from increased demand due to dysregulated cell growth and from vascular abnormalities associated with cancerous tissue. The importance of hypoxia has been seen in the clinic, since it predicts for poor outcome of treatments, independent of treatment modality. Hypoxia activates the UPR, whose downstream signaling events can undermine the efficacy of treatment. Tumor cells need to adapt to the increasingly hypoxic environment that surrounds them as they grow, and the induction of the UPR is key to this response. Induction of the ER stress response genes, for example BiP and GRP94, in cancerous tissue correlates with malignancy, consistent with their antiapoptotic function (82). In addition, the UPR confers resistance to topoisomerase inhibitors, such as etoposide, and some UPR-induced genes directly mediate drug resistance via the multi-drug-resistance gene MDR. Therefore, approaches to prevent UPR activation in cancerous cells may significantly improve treatment outcome.

The proteasome inhibitor PS-341 is now in earlyphase clinical evaluation for the treatment of multiple myeloma, a clonal B cell tumor of differentiated plasma cells (83). The mechanism of PS-341 function is thought to be inhibition of IκB degradation, which prevents activation of the antiapoptotic transcription factor NF-κB. However, proteasomal inhibition would also prevent ERAD. As high-level heavy- or light-chain Ig production is likely associated with a certain degree of protein misfolding, it is possible that inhibition of ERAD function may be selectively toxic to B cell myelomas through activation of the UPR and apoptosis.

The UPR and viral pathogenesis The two major mediators of the IFN-induced arm of the innate immune response are evolutionarily related to IRE1 and PERK. The kinase/endoribonuclease domain of IRE1 is homologous to RNaseL, and the protein kinase domain of PERK is related to the double-stranded RNA–activated (dsRNA-activated) eIF2α protein kinase PKR. RNase L and PKR mediate the IFN induced antiviral response of the host, which is required to limit viral protein synthesis and pathogenesis. As part of the innate immune response to viral infection, RNase L and PKR are activated by dsRNAs produced as intermediates in viral replication. In contrast to activation by dsRNA, IRE1 and PERK are activated by ER stress, which can be induced by high-level viral glycoprotein expression. All enveloped viruses produce excess glycoproteins that could elicit PERK and IRE1 activation to meet the need for increased folding and secretory capacity. More studies will be required to elucidate the role of the UPR in various viral diseases.

Hepatitis C virus (HCV) is a positive-stranded RNA virus encoding a single polyprotein. Polyprotein cleavage generates at least ten polypeptides, including two glycoproteins, E1 and E2. A large amount of E1 forms disulfide–cross-linked aggregates with E2 in the ER (84). Since the accumulation of misfolded α1-PI elicits UPR activation, with subsequent hepatocyte death and hepatocellular carcinoma, it is possible that the aggregated E1/E2 complexes in the HCV-infected hepatocyte also contribute to hepatitis and hepatocellular carcinoma. Future studies should identify whether these glycoprotein aggregates activate the UPR to mediate the hepatocyte cell death and transformation associated with the pathogenesis of HCV infection.

The UPR in tissue ischemia Finally, neuronal death due to reperfusion after ischemic injury is associated with activation of the UPR (85, 86). Immediately after reperfusion, protein synthesis is inhibited, due at least in part to phosphorylation of eIF2α; this inhibition may represent a protective mechanism to prevent further neuron damage. Recent studies support the idea that eIF2α phosphorylation in response to reperfusion injury is mediated by PERK and hence that it depends on the UPR (87). If so, UPR activation prior to ischemic injury might protect the brain and other tissues from cell death during periods of reperfusion.

Summary A variety of approaches have been employed to identify the UPR signaling components, their function, and their physiological role. Yeast genetics allowed the definition of the basic ER stress–signaling pathway. The identification of homologous and parallel signaling pathways in higher eukaryotes has produced a mechanistic framework the cell uses to sense and compensate for ER over-load and stress. The high-level tissue-specific expression patterns of several ER stress–signaling molecules indicated the pancreas and intestine as organs that require UPR for physiological function. Analysis of UPR-induced gene expression established that protein degradation is required to reduce the stress of unfolded protein accumulation in the ER. Major advances in identifying UPR function and rele vance to disease were derived from mutation of UPR signaling components in model organisms and the identification of mutations in humans.

Despite tremendous progress, our knowledge of the UPR pathway remains incomplete. Further studies promise to expand our understanding of how ER stress impacts the other cellular signaling pathways. It will be very exciting and informative to understand how the UPR varies when critical components are genetically manipulated by deletion or other types of mutations. In addition, although the accumulation of unfolded protein in the ER is now known to contribute to pathogenesis in a variety of diseases, there are still few therapeutic approaches that target these events. With a greater understanding of protein-folding processes, pharmacological intervention with chemical chaperones to promote proper folding becomes feasible, as observed with sodium phenylbutyrate for ∆508 CFTR (see Gelman and Kopito, this Perspective series, ref. 53). Future intervention should consider activation of different subpathways of the UPR or overexpression of appropriate protein chaperones, as in the case of overexpression of the J domain of cytosolic HSP70, which suppresses polyglutamine toxicity in flies (88). Treatments that activate the ERAD response may also ameliorate pathogenesis in a number of the conformational diseases.

Over the past ten years, tremendous progress has been made in understanding the mechanisms and physiological significance of the UPR. The processes of protein folding and secretion, transcriptional and translational activation, and protein degradation are intimately interconnected to maintain homeostasis in the ER. A variety of environmental insults, genetic disease, and underlying genetic modifiers of UPR function contribute to the pathogenesis of different disease states. As we gain a greater understanding of the mechanisms that control UPR activation, it should be possible to discover methods to activate or inhibit the UPR as desired for therapeutic benefit.

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Citation: Cell Death and Disease (2014) 5, e1578; doi:10.1038/cddis.2014.539
Published online 18 December 2014

An activated unfolded protein response promotes retinal degeneration and triggers an inflammatory response in the mouse retina
http://www.nature.com/cddis/journal/v5/n12/full/cddis2014539a.html

T Rana¹, V M Shinde¹, C R Starr¹, A A Kruglov¹, E R Boitet¹, P Kotla¹, S Zolotukhin², A K Gross¹ and M S Gorbatyuk¹

¹Department of Vision Sciences, University of Alabama at Birmingham, AL, USA
²Department of Pediatrics, University of Florida, FL, USA

Correspondence: M Gorbatyuk, Department of Vision Sciences, University of Alabama at Birmingham, 1670 University Boulevard, Birmingham, 35233 AL, USA. Tel: +1 205 934 6762; Fax: +1 205 934 3425; E-mail:mgortk@uab.edu

Received 20 July 2014; Revised 23 October 2014; Accepted 27 November 2014

Edited by P Ekert

Recent studies on the endoplasmic reticulum stress have shown that the unfolded protein response (UPR) is involved in the pathogenesis of inherited retinal degeneration caused by mutant rhodopsin. However, the main question of whether UPR activation actually triggers retinal degeneration remains to be addressed. Thus, in this study, we created a mouse model for retinal degeneration caused by a persistently activated UPR to assess the physiological and morphological parameters associated with this disease state and to highlight a potential mechanism by which the UPR can promote retinal degeneration. We performed an intraocular injection in C57BL6 mice with a known unfolded protein response (UPR) inducer, tunicamycin (Tn) and examined animals by electroretinography (ERG), spectral domain optical coherence tomography (SD-OCT) and histological analyses. We detected a significant loss of photoreceptor function (over 60%) and retinal structure (35%) 30 days post treatment. Analysis of retinal protein extracts demonstrated a significant upregulation of inflammatory markers including interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1) and IBA1. Similarly, we detected a strong inflammatory response in mice expressing either Ter349Glu or T17M rhodopsin (RHO). These mutant rhodopsin species induce severe retinal degeneration and T17M rhodopsin elicits UPR activation when expressed in mice. RNA and protein analysis revealed a significant upregulation of pro- and anti-inflammatory markers such as IL-1β, IL-6, p65 nuclear factor kappa B (NF-kB) and MCP-1, as well as activation of F4/80 and IBA1 microglial markers in both the retinas expressing mutant rhodopsins. We then assessed if the Tn-induced inflammatory marker IL-1β was capable of inducing retinal degeneration by injecting C57BL6 mice with a recombinant IL-1β. We observed ~19%reduction in ERG a-wave amplitudes and a 29% loss of photoreceptor cells compared with control retinas, suggesting a potential link between pro-inflammatory cytokines and retinal pathophysiological effects. Our work demonstrates that in the context of an established animal model for ocular disease, the persistent activation of the UPR could be responsible for promoting retinal degeneration via the UPR-induced pro-inflammatory cytokine IL-1β.

Abbreviations:

ERG, electroretinography; SD-OCT, spectral domain optical coherence tomography; UPR, unfolded protein response; IL-1β, Interleukin-1β; TNF-α, tumor necrosis factor-α; MCP-1, monocyte chemoattractant protein-1; NF-kB, ; nuclear factor kappa B, ; ER, endoplasmic reticulum; ADRP, autosomal dominant retinitis pigmentosa; RHO, rhodopsin; ERAI, ER stress activated indicator; Tn, tunicamycin; ONL, outer nuclear layer; H&E, hematoxylin and eosin; ONH, optic nerve head

ER stress and neuroinflammation: connecting the unfolded protein response to JAK/STAT signaling (P5196)

Gordon Meares,¹ and Etty Benveniste¹

¹Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL

J Immunol May 2013 190 (Meeting Abstract Supplement) 198.5

http://www.jimmunol.org/cgi/content/meeting_abstract/190/1_MeetingAbstracts/198.5

Neuroinflammation and endoplasmic reticulum (ER) stress are associated with many neurological diseases. ER stress is brought on by misfolded proteins. In turn, cells respond with activation of the unfolded protein response (UPR). The UPR is a highly conserved pathway that transmits both adaptive and apoptotic signals to restore homeostasis or eliminate the irreparably damaged cell. Recent evidence indicates that ER stress and inflammation are linked. In this study, we have examined the interaction between ER stress and JAK/STAT-dependent inflammation in astrocytes. The JAK/STAT pathway mediates the biological actions of many cytokines and growth factors. We have found that ER stress leads to the activation of STAT3 in a JAK1-dependent fashion. ER stress-induced activation of the JAK1/STAT3 axis leads to expression of IL-6 and several chemokines. The activation of STAT3 signaling is dependent on the protein kinase PERK, a central component of the UPR. Knockdown of PERK abrogates ER stress-induced activation of STAT3 and overexpression of PERK is sufficient to activate STAT3. Additionally, ER stressed astrocytes, via paracrine signaling, can stimulate activation of microglia leading to production of oncostatin M (OSM). OSM can then synergize with ER stress in astrocytes to drive inflammation. Together, this work describes a new PERK-JAK1-STAT3 signaling pathway that may elicit a feed-forward inflammatory loop involving astrocytes and microglia to drive neuroinflammation.

Neural Plasticity
Volume 2014 (2014), Article ID 610343, 15 pages
http://dx.doi.org/10.1155/2014/610343

Review Article

Surveillance, Phagocytosis, and Inflammation: How Never-Resting Microglia Influence Adult Hippocampal Neurogenesis

Amanda Sierra,^1,2,3 Sol Beccari,^2,3 Irune Diaz-Aparicio,^2,3 Juan M. Encinas,^1,2,3 Samuel Comeau,^4,5 and Marie-Ève Tremblay^4,5

¹Ikerbasque Foundation, 48011 Bilbao, Spain
²Achucarro Basque Center for Neuroscience, Bizkaia Science and Technology Park, 48170 Zamudio, Spain
³Department of Neurosciences, University of the Basque Country, 48940 Leioa, Spain
⁴Centre de Recherche du CHU de Québec, Axe Neurosciences, Canada G1P 4C7
⁵Département de Médecine Moléculaire, Université Laval, Canada G1V 4G2

Received 10 December 2013; Accepted 11 February 2014; Published 19 March 2014

Academic Editor: Carlos Fitzsimons

http://www.hindawi.com/journals/np/2014/610343/

Microglia cells are the major orchestrator of the brain inflammatory response. As such, they are traditionally studied in various contexts of trauma, injury, and disease, where they are well-known for regulating a wide range of physiological processes by their release of proinflammatory cytokines, reactive oxygen species, and trophic factors, among other crucial mediators. In the last few years, however, this classical view of microglia was challenged by a series of discoveries showing their active and positive contribution to normal brain functions. In light of these discoveries, surveillant microglia are now emerging as an important effector of cellular plasticity in the healthy brain, alongside astrocytes and other types of inflammatory cells. Here, we will review the roles of microglia in adult hippocampal neurogenesis and their regulation by inflammation during chronic stress, aging, and neurodegenerative diseases, with a particular emphasis on their underlying molecular mechanisms and their functional consequences for learning and memory.

Microglia: The Resident Immune Cells of the Brain

Microglia were first described in 1919 by the Spanish neuroanatomist Pío del Río Hortega, a disciple of the renowned Santiago Ramón y Cajal, almost half a century later than neurons and astrocytes and just before oligodendrocytes [1]. This delayed appearance into the neuroscience arena is still apparent today, as microglia remain one of the least understood cell types of the brain. Traditionally, microglia were simply considered as “brain macrophages” controlling the inflammatory response during acute insults and neurodegenerative conditions, and only recently was their unique origin revealed. Indeed, microglia were shown to derive from primitive myeloid progenitors of the yolk sac that invade the central nervous system (CNS) during early embryonic development (reviewed in [2]). In contrast, circulating monocytes and lymphocytes, as well as most tissue macrophages, derive from hematopoietic stem cells located initially in the foetal liver and later in the bone marrow [3]. In the adult brain, the microglial population is maintained exclusively by self-renewal during normal physiological conditions [2]. As a consequence, microglia are the only immune cells which permanently reside in the CNS parenchyma, alongside neural tube-derived neurons, astrocytes, and oligodendrocytes.

These past few years, unprecedented insights were also provided into their extreme dynamism and functional behaviour, in health as much as in disease. Indeed, microglia were revealed to be exceptional sensors of their environment, responding on a time scale of minutes to even subtle variations of their milieu, by undergoing concerted changes in morphology and gene expression [4, 5]. During pathological insults, “activated” microglia were particularly shown to thicken and retract their processes, extend filopodia, proliferate and migrate, release factors and compounds influencing neuronal survival (such as proinflammatory cytokines, trophic factors, reactive oxygen species (ROS), etc.), and phagocytose pathogens, degenerating cells and debris, thus providing better understanding of their roles in orchestrating the inflammatory response [6]. These abilities as immune cells are also recruited during normal physiological conditions, where “surveillant” microglia further participate in the remodeling of neuronal circuits by their phagocytic elimination of synapses and their regulation of glutamatergic receptors maturation and synaptic transmission, among other previously unexpected roles [7–9], in addition to their crucial involvement in the phagocytic elimination of newborn cells in the context of adult neurogenesis [10].

Our review will discuss the emerging roles of microglia in adult hippocampal neurogenesis and their regulation by inflammation during chronic stress, aging, and neurodegenerative diseases, with a particular emphasis on their underlying molecular mechanisms and their functional consequences for learning and memory (Figure 1).

http://www.hindawi.com/journals/np/2014/floats/610343/thumbnails/610343.fig.001_th.jpg

Figure 1: The effects of surveillant and inflammatory microglia on the adult hippocampal neurogenic cascade. During physiological conditions, surveillant microglia effectively phagocytose the excess of apoptotic newborn cells and may release antineurogenic factors such as TGF. This anti-inflammatory state is maintained by neuronal (tethered or released) fractalkine. Enriched environment drives microglia towards a phenotype supportive of neurogenesis, via the production of IGF-1. In contrast, inflammatory challenge triggered by LPS, irradiation, aging, or AD induces the production of proinflammatory cytokines such as IL-1, TNF, and IL-6 by microglia as well as resident astrocytes and infiltrating monocytes, neutrophils, and lymphocytes. These cytokines have profound detrimental effects on adult neurogenesis by reducing the proliferation, survival, integration, and differentiation of the newborn neurons and decreasing their recall during learning and memory paradigms.

A Brief Overview of Adult Hippocampal Neurogenesis

Adult hippocampal neurogenesis is continuously maintained by the proliferation of neural stem cells located in the subgranular zone (SGZ) [11–13]. These neuroprogenitors have been named “radial glia-like cells” (rNSCs), or type 1 cells, since they morphologically and functionally resemble the embryonic radial glia. They have also been defined as “quiescent neuroprogenitors” because only a small percentage of the population is actively dividing during normal physiological conditions. The lineage of these cells is frequently traced by using analogs of the nucleotide thymidine, such as bromodeoxyuridine (BrdU) which gets incorporated into the DNA of dividing cells during the S phase and can be detected by immunofluorescence. Alternatively, their lineage can be traced by labeling with fluorescent reporters which are delivered to dividing cells by retroviral vectors or expressed by specific cell type promoters via inducible transgenic mice (for a review of the methods commonly used to study adult neurogenesis, see [14]). The daughter cells of rNSCs, also called type 2 cells or amplifying neuroprogenitors (ANPs), rapidly expand their pool by proliferating before becoming postmitotic neuroblasts. Within a month, these neuroblasts differentiate and integrate as mature neurons into the hippocampal circuitry [15]. They however display unique electrophysiological characteristics during several months, being more excitable than mature neurons [16], and constitute a special cell population that is particularly inclined to undergo synaptic remodeling and activity-dependent plasticity [17].

These unique properties of the newborn neurons and the neurogenic cascade in general suggested that adult hippocampal neurogenesis could play an important role in hippocampal-dependent functions that require extensive neuroplasticity such as learning and memory. Indeed, activity-dependent plasticity and learning are long known for modulating adult neurogenesis in a complex, yet specific manner, with adult hippocampal neurogenesis being influenced by learning tasks which depend on the hippocampus [44, 45]. For instance, hippocampal-dependent learning paradigms were found to regulate the survival of newborn neurons, in a positive manner that depends on the timing between their birth and the phases of learning [46, 47]. Young (1.5–2 months old) newborn neurons were also shown to be preferentially activated during memory recall in a water maze task, compared to mature neurons, as determined by colabeling of BrdU with immediate early genes such as c-Fos and Arc, in which expression correlates with neuronal firing [48]. Nonetheless, it has only been in the last few years that loss-of-function and gain-of-function approaches with inducible transgenic mice were able to confirm that adult hippocampal neurogenesis is necessary for synaptic transmission and plasticity, including the induction of long-term potentiation (LTP) and long-term depression [49], as well as trace learning in conditioned protocols [50], memory retention in spatial learning tasks [51, 52], and encoding of overlapping input patterns, that is, pattern separation [53].

Adult hippocampal neurogenesis and its functional implications for learning and memory are however influenced negatively by a variety of conditions that are commonly associated with microglial activation and inflammation in the brain, such as chronic stress, aging, and neurodegenerative diseases, as we will review herein. Indeed, inflammation caused by irradiation produces a sustained inhibition of neurogenesis, notably by decreasing the proliferation and neuronal differentiation of the progenitors, and therefore, exposure to therapeutic doses of cranial irradiation has been widely used for modulating neurogenesis experimentally before the development of more specific approaches [54].

Regulation of Adult Hippocampal Neurogenesis by Inflammation

Inflammation is a natural bodily response to damage or infection that is generally mediated by proinflammatory cytokines such as interleukin 1 beta (IL-1), interleukin 6 (IL-6), and tumour necrosis factor alpha (TNF), in addition to lipidic mediators such as prostaglandins and leukotrienes. Oftentimes, it is associated with an increased production of ROS, as well as nitric oxide (NO). Together, these proinflammatory mediators lead to an increase in local blood flow, adhesion, and extravasation of circulating monocytes, neutrophils, and lymphocytes [55]. In the brain, microglia are the main orchestrator of the neuroinflammatory response, but other resident cell types, including astrocytes, endothelial cells, mast cells, perivascular and meningeal macrophages, and even neurons, can produce proinflammatory mediators, though perhaps not to the same extent as microglia [56]. In addition, peripheral immune cells invading the CNS during inflammation can further produce proinflammatory mediators, but the respective contribution of microglia versus other cell types in the inflammatory response of the brain is poorly understood.

The harmful effects of inflammation are also widely determined by the actual levels of proinflammatory mediators released, rather than the occurrence or absence of an inflammatory response in itself. For instance, TNF regulates synaptic plasticity by potentiating the cell surface expression of AMPA glutamatergic receptors, thus resulting in a homeostatic scaling following prolonged blockage of neuronal activity during visual system development [57]. However, TNF also produces differential effects at higher concentrations,ranging from an inhibition of long-term potentiation to an enhancement of glutamate-mediated excitotoxicityin vitro [58]. Inflammation induced by chronic ventricular infusion of bacterial lipopolysaccharides (LPS; a main component of the outer membrane of Gram-negative bacteria), that is, the most widely used method for inducing an inflammatory challenge, also increases ex vivo the hippocampal levels of TNF and IL-1, thereby impairing novel place recognition, spatial learning, and memory formation, but all these cognitive deficits can be restored by pharmacological treatment with a TNF protein synthesis inhibitor, a novel analog of thalidomide, 3,6′-dithiothalidomide [59].

The impact of inflammation on adult hippocampal neurogenesis was originally discovered by Olle Lindvall and Theo Palmer’s groups in 2003, showing that systemic or intrahippocampal administration of LPS reduces the formation of newborn neurons in the adult hippocampus, an effect that is prevented by indomethacin, a nonsteroidal anti-inflammatory drug (NSAID) which inhibits the synthesis of proinflammatory prostaglandins [60, 61]. Similarly, inflammation can determine the increase in neurogenesis that is driven by seizures, a context in which neurogenesis can be prevented by LPS and increased by the anti-inflammatory antibiotic minocycline [60]. In these studies, hippocampal proliferation remained unaffected by LPS or minocycline and thus it is likely that inflammation targeted the survival of newborn cells [60, 61], as LPS is known to increase SGZ apoptosis [62]. Inflammation also has further downstream effects on the neurogenic cascade. For instance, LPS increases the number of thin dendritic spines and the expression of the excitatory synapses marker “postsynaptic density protein of 95kDa” (PSD95) in newborn neurons. LPS in addition increases the expression of GABA_A receptors at early stages of synapse formation, leading to suggesting a possible imbalance of excitatory and inhibitory neurotransmission in these young neurons [63]. Finally, LPS also prevents the integration of newborn neurons into behaviourally relevant networks, including most notably their activation during spatial exploration, as determined by the percentage of BrdU cells colabeled with the immediate early gene Arc [64].

Importantly, none of these manipulations is specific to microglia and may directly or indirectly affect other brain cells involved in the inflammatory response of the brain. For instance, both LPS and minocycline affect astrocytic function in vitro and in vivo [65–69]. Furthermore, LPS is known to drive infiltration of monocytes and neutrophils into the brain parenchyma [70]. Monocytes and neutrophils produce major proinflammatory mediators and could therefore act on the neurogenic cascade as well. The implication of microglia in LPS-induced decrease in neurogenesis is nonetheless supported in vivo by the negative correlation between the number of newborn neurons (BrdU+, NeuN+ cells) and the number of “activated” microglia (i.e., expressing ED1) [60]. ED1, also called CD68 or macrosialin, is a lysosomal protein which is overexpressed during inflammatory challenge. While the location of ED1 previously suggested its involvement in phagocytosis, its loss of function did not result in phagocytosis deficits and thus, its function still remains unknown (reviewed in [10]). The number of ED1-positive microglia also negatively correlates with neurogenesis during inflammation provoked by cranial irradiation [61]. While correlation does not involve causation, nor can pinpoint to the underlying mechanism, these experiments were the first to reveal a potential role for “activated” microglia in the regulation of adult hippocampal neurogenesis. More direct evidence of microglial mediation in LPS deleterious effects was obtained from in vitro experiments, as it was shown that conditioned media from LPS-challenged microglia contained IL-6, which in turn caused apoptosis of neuroblasts [61]. Nonetheless, astrocytes can also release IL-6 when stimulated with TNF or IL-1 [71] and chronic astrocytic release of IL-6 in transgenic mice reduced proliferation, survival, and differentiation of newborn cells, thus resulting in a net decrease in neurogenesis [72]. In summary, while the detrimental impact of inflammation on neurogenesis is well established, more work is needed to define the specific roles played by the various inflammatory cells populating the brain.

Inflammation Associated with Chronic Stress

Across health and disease, the most prevalent condition that is associated with neuroinflammation is “chronic stress,” which commonly refers to the repeated or sustained inability to cope with stressful environmental, social, and psychological constraints. Chronic stress is characterized by an imbalanced secretion of glucocorticoids by the hypothalamic-pituitary-adrenal (HPA) axis (most notably cortisol in humans and corticosterone in rodents), which leads to an altered brain remodeling, massive loss of synapses, and compromised cognitive function [73]. In particular, an impairment of spatial learning, working memory, novelty seeking, and decision making has been associated with chronic stress [74]. Glucocorticoids are well known for their anti-inflammatory properties, as they interfere with NF-B-mediated cytokine transcription, ultimately delaying wound healing [75]. They are also potent anti-inflammatory mediators in vivo [76] and in purified microglia cultures [77]. Recently, repeated administration of high doses of glucocorticoids by intraperitoneal injection, to mimic their release by chronic stress, was also shown to induce a loss of dendritic spines in the motor cortex, while impairing learning of a motor task. A transcription-dependent pathway acting downstream of the glucocorticoid receptor GR was proposed [78, 79] but the particular cell types involved were not identified.

Microglia are considered to be a direct target of the glucocorticoids, as they were shown to express GR during normal physiological conditions in vivo [77]. In fact, transgenic mice lacking GR in microglia and macrophages show an increased production of proinflammatory mediators (including TNF and IL-1) and greater neuronal damage in response to an intraparenchymal injection of LPS, compared to wild-type mice [80]. In contrast, glucocorticoids are considered to be proinflammatory in the chronically stressed brain [81], where among other changes they can promote inflammation, oxidative stress, neurodegeneration, and microglial activation [82]. For example, repeated restraint stress induces microglial proliferation and morphological changes, including a hyperramification of their processes in the adult hippocampus following restraint stress [83], but a nearly complete loss of processes in the context of social defeat [84]. Prenatal restraint stress also causes an increase in the basal levels of TNF and IL-1, while increasing the proportion of microglia showing a reactive morphology in the adult hippocampus [85]. Similarly, social defeat leads to an enhanced response to the inflammatory challenge induced by intraperitoneal injection of LPS, including an increased production of TNF and IL-1, and expression of inducible NO synthase (iNOS) by microglia, accompanied by an increased infiltration of circulating monocytes [84, 86]. Therefore, microglia are a strong candidate for mediating some of the effects of stress on adult neurogenesis, as will be discussed below, in synergy with other types of inflammatory cells.

Chronic stress is well known for its negative effects on hippocampal neurogenesis (reviewed in [87, 88]), although not all stress paradigms are equally effective [89]. Several stress paradigms can decrease neuroprogenitors proliferation in the tree shrew [90] and in mice [91, 92], although this effect seems to be compensated by an increased survival of newborn neurons [92] and whether stress results in a net increase or decrease in neurogenesis remains controversial (reviewed in [87, 88]). The effects of stress on adult neurogenesis seem to be mediated at least partially by glucocorticoids, because mice lacking a single copy of the GR gene show behavioural symptoms of depression including learned helplessness, neuroendocrine alterations of the HPA axis, and impaired neurogenesis [93]. In parallel, chronic stress is associated with an increased inflammatory response, which may inhibit neurogenesis as well. For instance, serum levels of IL-1and IL-6 are significantly increased in depressed patients [94]. In mice, restraint stress leads to a widespread activation of NF-B in the hippocampus, including at the level of neuroprogenitors [95] and increased protein levels of IL-1 [96]. In addition to the direct role of glucocorticoids, IL-1 also seems to mediate some of the effects of mild chronic stress, because in vivo manipulations that block IL-1 (either pharmacologically or in null transgenic mice) prevent the anhedonic stress response and the antineurogenic effect of stress [91, 96]. Moreover, the corticoids and IL-1 pathways may regulate each other in a bidirectional manner because the administration of a GR antagonist can blunt the LPS-induced production of hippocampal IL-1 in stressed mice [97], whereas mice knockout for the IL-1 receptor (IL-1R1) fail to display the characteristic elevation of corticosterone induced by mild chronic stress [96]. Another stress-related cytokine, IL-6, induces depressive phenotypes and prevents the antidepressant actions of fluoxetine when administered to mice in vivo [98]. So far the effects of stress on neurogenesis via corticosteroids and inflammation have been assumed to be cell autonomous, as neuroprogenitors express both GR [99] and IL-1R1 [95]. The potential participation of microglia is yet to be determined, but there are some reports of a direct effect of stress on microglial activation. For instance, microglia acutely isolated from mice subjected to acute stress (by inescapable tail shock) showed a primed response to LPS challenge by producing higher levels of IL-1 mRNA ex vivo [100], and the specific loss of expression of GR in microglia leads to a blunted inflammatory response in vitro and to a decreased neuronal damage in vivo in response to LPS [80]. In stress paradigms, these enhanced responses of microglia to inflammatory challenges are similar to their age-related “priming” which has been associated with and is possibly due to an increased basal production of proinflammatory mediators. However, whether microglia express increased levels of IL-1 and other proinflammatory cytokines in response to stressful events is presently unclear [101]. It is thus possible that some of the antineurogenic effects of stress are exerted by means of microglial-dependent inflammation, but this hypothesis remains to be experimentally tested.

Inflammation Associated with Aging and Neurodegenerative Diseases

Inflammation is also commonly associated with normal aging and neurodegenerative diseases and, therefore, could represent a putative underlying mechanism that explains their decrease in hippocampal neurogenesis. Nonetheless, inflammation is also associated with neurological diseases, such as epilepsy or stroke, where neurogenesis is thought to be increased, although the data from rodents and humans is somewhat conflictive [102]. Neurogenesis is well known to decline throughout adulthood and normal aging in rodents and humans [103, 104], but the decay is more pronounced and occurs later in life in mice than in humans [105]. The aging-associated decrease in neurogenesis has been shown to occur mainly as a consequence of exhaustion of the rNSC population which, after being recruited and activated, undergo three rounds of mitosis in average and then terminally differentiate into astrocytes [12, 106]. In addition, a reduced mitotic capacity of the neuroprogenitors could further contribute to decreasing neurogenesis [106], and moreover, an age-related increase in the levels of proinflammatory cytokines could also hinder neurogenesis in the aging brain. Serum levels of IL-1, IL-6, and TNF are elevated in elderly patients [107, 108]. Aged microglia express higher levels of these proinflammatory cytokines and show a greater response to LPS inflammatory challenge, that is, a “primed” response, than their younger counterparts [109]. The origin of this low-grade age-related inflammation (“inflamm-aging” [110]) remains unknown and may be related to both aging and damage to the surrounding neurons, as well as aging of the immune system per se.

At the cellular level, stress to the endoplasmic reticulum (ER) caused by various perturbations, such as nutrient depletion, disturbances in calcium or redox status, or increased levels of misfolded proteins, can induce a cell-autonomous inflammatory response to neurons. Stress to the ER, a multifunctional organelle which is involved in protein folding, lipid biosynthesis, and calcium storage triggers a homeostatic response mechanism named the unfolding protein response (UPR), aiming to clear the unfolded proteins in order to restore normal ER homeostasis [111]. However, if the ER stress cannot be resolved, the UPR also initiates inflammatory and apoptotic pathways via activation of the transcription factor NF-B which controls the expression of most proinflammatory cytokines [112]. In the brain, ER stress is often initiated by the formation of abnormal protein aggregates in several neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), Huntington’s disease (HD), and prion-related disorders [113]. This neurodegeneration-associated ER stress is assumed to occur mostly in neurons, but there are some examples of microglial protein misfolding as well. For instance, both microglia and neurons overexpress CHOP (C/EBP homologous protein), a transcription factor which is activated during ER stress in human patients and mouse models of ALS [114]. Inflammation has been speculated to be a main negative contributor to the pathology of ALS [115], but a direct microglial involvement in mediating the inflammatory response to abnormal protein aggregation in ALS and other neurodegenerative conditions remains to be tested. Finally, ER stress has been linked to a variety of inflammatory conditions [116, 117], including chronic stress, diet-induced obesity, and drug abuse, as well as atherosclerosis and arthritis [118–120]. During normal aging, a progressive decline in expression and activity of key ER molecular chaperones and folding enzymes could also compromise the adaptive response of the UPR, thereby contributing to the age-associated decline in cellular functions [118]. Therefore, aging is strongly associated with a chronic ER stress which leads to increased activation of NF-B [112]; however, the contribution of the different brain cell types to “inflamm-aging” is still poorly understood. The detrimental effects on neurogenesis of increased proinflammatory cytokines in the aging brain are not necessarily related to microglia, but also to stressed neurons. Furthermore, ER stress may also cause a cell-autonomous response in neural stem cells [121], although its impact on neurogenesis remains to be experimentally determined.

In addition, aging is accompanied by an increased level of mitochondrial oxidative stress, which in turn activates the “Inflammasome” [122], a group of multimeric proteins comprising the interleukin 1 converting enzyme (ICE, caspase 1) which serves to release the active form of the cytokine [123]. IL-1 may act directly on rNSCs (visualised by labeling with the Sox2 marker), as they express IL-1R1 in the adult hippocampus [91]. Treatment with IL-1 decreases hippocampal proliferation in young mice [91] and pharmacological inhibition of ICE partially restores the number of newborn neurons in aged mice without significantly affecting their differentiation rate [124]. Transgenic IL-1 overexpression results in chronic inflammation and depletion of doublecortin-labeled neuroblasts, thus mimicking the aging-associated depletion of neurogenesis [125]. The actual mechanism of action of IL-1 on neurogenesis in aged mice, including decreased proliferation of rNSCs/ANPs and survival of newborn neurons, remains undetermined. Microglia are a main source of IL-1in the aging brain, but the hypothesis that microglia-derived IL-1 is responsible for depleting neurogenesis in the aging brain remains to be directly tested.

The regulation of neurogenesis by IL-1 in the aging brain has been further linked to the activity of another cytokine, the chemokine fractalkine, or CX3CL1. Fractalkine has soluble and membrane-tethered forms and is exclusively expressed by neurons, while the fractalkine receptor (CX3CR1) is expressed in the brain by microglia alone [126]. This module forms a unique neuron-microglia signalling unit that controls the extent of microglial inflammation in several neurodegenerative conditions including PD, ALS [127], or AD [128]. In fact, CX3CR1 blocking antibodies increase the production of hippocampal IL-1 when administered to young adult rats [129]. Importantly, chronic treatment with fractalkine increases hippocampal proliferation and the number of neuroblasts in aged (22 months old) but not young (3 months old) or middle-aged rats (12 months old), whereas an antagonists of CX3CR1 has the opposite effects in young, but not in middle-aged nor old rats [129]. Since fractalkine expression is decreased during aging [129], a reduced neuron-microglia signalling might be releasing the brake on microglial contribution to inflammatory responses, although increased levels of fractalkine were instead reported in aged rat hippocampus by other studies [68]. Additional insights into the role of fractalkine signalling come from knock-in mice in which the endogenous CX3CR1 locus is replaced by the fluorescent reporter GFP [126]. The initial studies suggested that (i.e., ) mice have no significant differences in brain development and functions [130], but more systematic investigations recently revealed a long list of hippocampal-dependent changes in young (3 months old) and mice compared to wild-type mice. These changes notably included decreased neuroprogenitors proliferation and neuroblasts number, impaired LTP, performance in contextual fear conditioning and water maze spatial learning and memory, and, importantly, increased IL-1 protein levels [131]. The signalling pathway of fractalkine-IL-1 is functionally relevant, because IL-1R1 antagonists rescued LTP and cognitive function in mice [131]. In sum, even though neuronal fractalkine seems to be sufficient for restraining the inflammatory activity of microglia in young rats, its downregulation during aging could activate the microglial inflammatory response and thereby subsequently reduce the proliferation of remaining neuroprogenitors.

In AD, inflammatory cytokines such as IL-1 are overexpressed in the microglia associated with the amyloid beta (A) plaques of postmortem samples [132] and in transgenic mice modeling the disease [133]. The loss of synapses (from hippocampus to frontal cortex) is one of the main pathological substrates in this disease, but adult neurogenesis is also severely reduced in most mouse models of AD, possibly due to a decreased proliferation of neuroprogenitors and a decreased survival of newborn cells, even though the putative changes in the neurogenic cascade in postmortem samples remain controversial (reviewed in [102]). This lack of agreement is possibly explained by the fact that the vast majority of AD cases have a late onset over 65 years of age, when little neurogenesis remains. In contrast, in most transgenic AD mouse models, the Aaccumulation, cognitive deficits, and changes in neurogenesis are already detectable in young animals (2-3 months old). The study of AD is further hindered by the difficulty in comparing the time course and pathology across different mouse models. For instance, early treatment with minocycline can improve cognition and reduce A burden in mice expressing the human amyloid precursor protein (APP) [134]. In contrast, in mice expressing APP and a mutated form of presenilin 1 (PS1), which is part of the secretase pathway that cleaves A, inflammation is reduced without any detectable changes in A plaques deposition [135]. Concomitantly with a decrease in tissue inflammatory cytokines and number of microglial cells, minocycline restores neurogenesis and hippocampus-dependent memory deficits in these APP/PS1 mice [135], indirectly suggesting that cognitive decay in AD may be at least in part related to a detrimental effect of inflammation on hippocampal neurogenesis. Direct evidence that neurogenesis is associated with the cognitive performance in AD is still lacking. Further research is also necessary to determine the neurogenic targets of AD-related inflammation. One central open question for future therapies aiming at increasing neurogenesis and cognition in AD is whether neuroprogenitors are spared or whether their age-induced loss becomes accelerated. Rather than increasing the proliferation and neurogenic output of the few rNSCs remaining in an old AD brain, it may be more relevant to develop strategies that prevent the age-related loss of neuroprogenitors in presymptomatic patients.

In summary, inflammation associated with a wide variety of experimental models of disease produces strong detrimental effects on hippocampal neurogenesis. These effects on human neurogenesis are however not so well described and, in vitro, IL-1 increases the proliferation of hippocampal embryonic neuroprogenitors but decreases their differentiation into neurons [136]. Novel methods to assess hippocampal neurogenesis in the living human brain, from metabolomics of neuroprogenitors to hippocampal blood brain volume (reviewed in [102]), will help to determine the contribution of inflammation to adult neurogenesis in the healthy and diseased human brain during aging.

Normal Physiological Conditions

In the healthy mature brain, microglia are an essential component of the neurogenic SGZ niche, where they physically intermingle with neuroprogenitors, neuroblasts, and newborn neurons [62]. Here, surveillant microglia effectively and rapidly phagocytose the excess of newborn cells undergoing apoptosis [62]. Importantly, microglial phagocytosis in the adult SGZ is not disturbed by inflammation associated with aging or by LPS challenge, as the phagocytic index (i.e., the proportion of apoptotic cells completely engulfed by microglia) is maintained over 90% in these conditions [62]. Nonetheless, the consequences of microglial phagocytosis on adult hippocampal neurogenesis remain elusive. Treatment of mice with annexin V, which binds to the phosphatidylserine (PS) receptor and prevents the recognition of PS on the surface of apoptotic cells, presumably blocking phagocytosis, increases the number of apoptotic cells in the SGZ [40]. Concomitantly, annexin V reduces neurogenesis by decreasing the survival of neuroblasts without affecting neuroprogenitors proliferation [40]. Similar results were obtained in transgenic mice knock-out for ELMO1, a cytoplasm protein which promotes the internalization of apoptotic cells, although the effects on neurogenesis were ascribed to a decreased phagocytic activity of neuroblasts [40]. The actual phagocytic target of the neuroblasts remains undetermined, but the newborn apoptotic cells in the adult SGZ are exclusively phagocytosed by microglia, at least in physiological conditions [62]. Nevertheless, none of the above manipulations has specifically tested the role of microglial phagocytosis in hippocampal-dependent learning and memory and thus, the functional impact of microglial phagocytosis in adult neurogenic niches during normal physiological conditions remains to be elucidated.

Microglial phagocytosis of apoptotic cells is actively anti-inflammatory, at least in vitro, and thus it has been hypothesized that anti-inflammatory cytokines produced by phagocytic microglia may further regulate neurogenesis [10]. For instance, transforming growth factor beta (TGF), which is produced by phagocytic microglia in vitro [137], inhibits the proliferation of SGZ neuroprogenitors [138]. Microglia are further able to produce proneurogenic factors in vitro [139]. When primed with cytokines associated with T helper cells such as interleukin 4 (IL-4) or low doses of interferon gamma (IFN), cultured microglia support neurogenesis and oligodendrogenesis through decreased production of TNF and increased production of insulin-like growth factor 1 (IGF-1) [139], an inducer of neuroprogenitor proliferation [26]. A list of potential factors produced by microglia and known to act on neuroprogenitor proliferation can be found in Table 1. In addition, recent observations suggest that neuroprogenitor cells may not only regulate their own environment, but also influence microglial functions. For instance, vascular endothelial growth factor (VEGF) produced by cultured neuroprecursor cells directly affects microglial proliferation, migration, and phagocytosis [20]. More potential factors produced by neuroprogenitors shown to be influencing microglial activity and function can be found in Table 2. However, it has to be taken into account that most of these observations were obtained in culture and that further research is needed in order to elucidate whether those factors are also secreted and have the same regulatory responses in vivo.

Table 1: Summary of factors secreted by microglia and the potential effect they have on neuroprogenitors in vitro.


Microglia secreted factors	Reference	Modulation of neural progenitor cells	Reference

BDNF	[18]	Differentiation	[19]
EGF	[20]	Survival, expansion, proliferation, differentiation	[21]
FGF	[22]	Survival and expansion	[23]
GDNF	[24]	Survival, migration, and differentiation	[25]
IGF-1	[21]	Proliferation	[26]
IL-1	[27]	Reduction in migration	[27]
IL-6	[28]	Inhibition of neurogenesis	[29]
IL-7	[20]	Differentiation	[30]
IL-11	[20]	Differentiation	[30]
NT-4	[24]	Differentiation	[31]
PDGF	[32]	Expansion and differentiation	[33]
TGF	[34]	Inhibition of proliferation	[19]

Table 1: Summary of factors secreted by microglia and the potential effect they have on neuroprogenitors in vitro.

http://www.hindawi.com/journals/np/2014/610343/tab1/

Table 2: Summary of factors secreted by neuroprogenitors and the potential effect they have on microglia in vitro.


NPC secreted factors	Reference	Modulation of microglia	Reference

BDNF	[18]	Proliferation and induction of phagocytic activity	[35]
Haptoglobin	[24]	Neuroprotection	[36]
IL-1	[37]	Intracellular Ca⁺² elevation and proliferation	[22]
IL-6	[37]	Increase in proliferation	[38]
M-CSF	[20]	Mitogen	[39]
NGF	[40]	Decrease in LPS-induced NO	[41]
TGF	[37]	Inhibition of TNF secretion	[42]
TNF	[37]	Upregulation of IL-10 secretion	[43]
VEGF	[20]	Induction of chemotaxis and proliferation	[20]

http://www.hindawi.com/journals/np/2014/floats/610343/thumbnails/610343.tab2_th.jpg

Table 2: Summary of factors secreted by neuroprogenitors and the potential effect they have on microglia in vitro.

In addition, microglial capacity to remodel and eliminate synaptic structures during normal physiological conditions has suggested that microglia could also control the synaptic integration of the newborn neurons generated during adult hippocampal neurogenesis [140]. Three main mechanisms were proposed: (1) the phagocytic elimination of nonapoptotic axon terminals and dendritic spines, (2) the proteolytic remodeling of the perisynaptic environment, and (3) the concomitant structural remodeling of dendritic spines [7, 140]. Indeed, microglial contacts with synaptic elements are frequently observed in the cortex during normal physiological conditions, sometimes accompanied by their engulfment and phagocytic elimination [141–143], as in the developing retinogeniculate system [144]. Microglial cells are distinctively surrounded by pockets of extracellular space, contrarily to all the other cellular elements [142], suggesting that microglia could remodel the volume and geometry of the extracellular space, and thus the concentration of various ions, neurotransmitters, and signalling molecules in the synaptic environment. Whether microglia create the pockets of extracellular space themselves or not remains unknown, but these pockets could result from microglial release of extracellular proteases such as metalloproteinases and cathepsins [145], which are well known for influencing the formation, structural remodeling, and elimination of dendritic spines in situ and also experience-dependent plasticity in vivo [7, 146]. More recently, microglial phagocytosis of synaptic components was also observed in the developing hippocampus, in the unique time window of synaptogenesis, a process which is notably regulated by fractalkine-CX3CR1 signalling [147]. Therefore, the attractive hypothesis that microglial sculpts the circuitry of newborn cells in the adult hippocampus deserves further attention.

Lastly, microglia were also involved in increasing adult hippocampal neurogenesis in the enriched environment (EE) experimental paradigm. EE is a paradigm mimicking some features of the normal living circumstances of wild animals, as it gives them access to social interactions, toys, running wheels, and edible treats. EE has long been known to enhance neurogenesis by acting on newborn cells survival, resulting ultimately in an enlargement of the dentate gyrus [148]. Functionally, these changes are accompanied by enhanced spatial learning and memory formation with the water maze paradigm [149]. Similar increases in neurogenesis are obtained by subjecting mice to voluntary running paradigms, although in this case the effect is mediated by increased neuroprogenitor proliferation [150]. During inflammatory conditions, EE is antiapoptotic and neuroprotective [151] and it limits the hippocampal response to LPS challenge by decreasing the expression of several cytokines and chemokines, including IL1- and TNF [152]. In fact, EE is believed to counteract the inflammatory environment and rescue the decreased number of neuroblasts in mice compared to wild-type mice [153]. The effects of EE are independent of the IL-1 signalling pathway, as it increases neurogenesis in mice that are null for IL-1R1 [154]. EE also induces microglial proliferation and expression of the proneurogenic IGF-1 [155], but the full phenotype of microglia in EE compared to standard housing and its impact on the neurogenic cascade remains to be determined.

The mechanisms behind the anti-inflammatory actions of EE are unknown, but they were suggested to involve microglial interactions with T lymphocytes through an increased expression of the major histocompatibility complex of class II (MHC-II) during EE [155]. MHC-II is responsible for presenting the phagocytosed and degraded antigens to the antibodies expressed on the surface of a subtype of T lymphocytes (T helper or CD4+ cells), thus initiating their activation and production of antigen-specific antibodies. Severe combined immunodeficient (SCID) mice lacking either T and B lymphocytes or nude mice lacking only T cells have impaired proliferation and neurogenesis in normal and EE housing compared to wild-type mice [155], as well as impaired performance in the water maze [156]. Similarly, antibody-based depletion of T helper lymphocytes impairs basal and exercise-induced proliferation and neurogenesis [157]. Furthermore, a genetic study in heterogeneous stock mice, which descend from eight inbred progenitor strains, has found a significant positive correlation between genetic loci associated to hippocampal proliferation and to the proportion of CD4+ cells among blood CD3+ lymphocytes [158]. Additional experiments are needed to fully determine the possible interactions between microglia and T cells in neurogenesis, because, at least in normal physiological conditions, (1) T cell surveillance of the brain parenchyma is minimal, (2) microglia are poor antigen presenting cells, and (3) antigen presentation by means of MHC-II family of molecules is thought to occur outside the brain, that is, in the meninges and choroid plexus [159]. In fact, during voluntary exercise, there are no significant changes in T cell surveillance of the hippocampus, nor a direct interaction between T cells and microglia, nor any changes in the gene expression profile of microglia, including that of IGF-1, IL-1, and TNF [160]. The number of microglia is also inversely correlated with the number of hippocampal proliferating cells, rNSCs, and neuroblasts in aged (8 months) mice subjected to voluntary running, as well asin vitro cocultures of microglia and neuroprogenitors, which has been interpreted as resulting from an overall inhibitory effect of microglia on adult neurogenesis [161]. Even though EE is clearly a more complex environmental factor than voluntary running, further research is necessary to disregard nonspecific or indirect effects of genetic or antibody-based T cells depletion on microglia and other brain cell populations, including rNSCs. For instance, adoptive transfer of T helper cells treated with glatiramer acetate, a synthetic analog of myelin basic protein (MBP) approved for the treatment of multiple sclerosis, produces a bystander effect on resident astrocytes and microglia by increasing their expression of anti-inflammatory cytokines such as TGF[162]. Alternatively, it has been suggested that T cells may mediate an indirect effect on adult hippocampal neurogenesis by increasing the production of brain-derived neurotrophic factor (BDNF) [157], which is involved in the proneurogenic actions of EE [163]. Whether BDNF can counteract the detrimental effects of T cell depletion on neurogenesis remains unknown. Overall, the roles of microglia in EE and running-induced neurogenesis are unclear and have to be addressed with more precise experimental designs. In summary, surveillant microglia are part of the physical niche surrounding the neural stem cells and newborn neurons of the mature hippocampus, where they continuously phagocytose the excess of newborn cells. Microglia were also linked to the proneurogenic and anti-inflammatory effects of voluntary running and EE, but direct evidence is missing. The overall contribution of microglia to neurogenesis and learning and memory in normal physiological conditions remains largely unexplored at this early stage in the field.

Conclusion

In light of these observations, microglia are now emerging as important effector cells during normal brain development and functions, including adult hippocampal neurogenesis. Microglia can exert a positive or negative influence on the proliferation, survival, or differentiation of newborn cells, depending on the inflammatory context. For instance, microglia can compromise the neurogenic cascade during chronic stress, aging, and neurodegenerative diseases, by their release of proinflammatory cytokines such as IL-1, IL-6, and TNF. A reduced fractalkine signalling between neurons and microglia could also be involved during normal aging. However, microglia are not necessarily the only cell type implicated because astrocytes, endothelial cells, mast cells, perivascular and meningeal macrophages, and to a lesser extent neurons and invading peripheral immune cells could further contribute by releasing proinflammatory mediators.

Additionally, microglia were shown to phagocytose the excess of newborn neurons undergoing apoptosis in the hippocampal neurogenic niche during normal physiological conditions, while a similar role in the synaptic integration of newborn cells was also proposed in light of their capacity to phagocytose synaptic elements. Lastly, microglial interactions with T cells, leading to the release of anti-inflammatory cytokines, neurotrophic factors, and other proneurogenic mediators (notably during EE and voluntary running), could counteract the detrimental effects of inflammation on adult hippocampal neurogenesis and their functional implications for learning and memory.

However, further research is necessary to assess the relative contribution of microglia versus other types of resident and infiltrating inflammatory cells and to determine the nature of the effector cytokines and other inflammatory mediators involved, as well as their cellular and molecular targets in the neurogenic cascade. Such research will undoubtedly help to develop novel strategies aiming at protecting the neurogenic potential and ultimately its essential contribution to learning and memory.

Abbreviations

AD:	Alzheimer’s disease
ANPs:	Amplifying neuroprogenitors
APP:	Amyloid precursor protein
A:	Amyloid beta
BDNF:	Brain-derived neurotrophic factor
BrdU:	5-Bromo-2′-Deoxyuridine
CX3CL1:	Fractalkine
CX3CR1:	Fractalkine receptor
EAE:	Experimental acute encephalomyelitis
EE:	Enriched environment
EGF:	Epidermal growth factor
FGFb:	Basic fibroblast growth factor
GDNF:	Glial cell line-derived neurotrophic factor
GFAP:	Glial fibrillary acidic protein
GR:	Glucocorticoid receptor
HPA:	Hypothalamic-pituitary-adrenal axis
ICE:	Interleukin 1 converting enzyme
IL-1:	Interleukin 1 beta
IL-1R1:	Interleukin 1 beta receptor
IL-4:	Interleukin 4
IL-6:	Interleukin 6
IL-7:	Interleukin 7
IL-11:	Interleukin 11
IFN:	Interferon gamma
IGF-1:	Insulin-like growth factor 1
iNOS:	Inducible nitric oxide synthase
LPS:	Bacterial lipopolysaccharides
LTP:	Long term potentiation
M-CSF:	Macrophage colony-stimulating factor
MBP:	Myelin basic protein
MHC-II:	Major histocompatibility complex class II
MOG:	Myelin oligodendrocyte glycoprotein
NF-B:	Nuclear factor kappa-light-chain-enhancer of activated B cells
NGF:	Nerve growth factor
NO:	Nitric oxide
NSAID:	Nonsteroidal anti-inflammatory drug
NT-4:	Neurotrophin-4
PDGF:	Platelet-derived growth factor
PS:	Phosphatidylserine
PS1:	Presenilin 1
ROS:	Radical oxygen species
SCID:	Severe combined immunodeficiency
SGZ:	Subgranular zone
TGF:	Transforming growth factor beta
TNF:	Tumor necrosis factor alpha
VEGF:	Vascular endothelial growth factor.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Acknowledgments

This work was supported by grants from the Spanish Ministry of Economy and Competitiveness to Amanda Sierra (BFU2012-32089) and Juan M. Encinas (SAF2012-40085), from Basque Government (Saiotek S-PC 12UN014) and Ikerbasque start-up funds to Juan M. Encinas and Amanda Sierra, and from The Banting Research Foundation, the Scottish Rite Charitable Foundation of Canada, and start-up funds from Université Laval and Centre de recherche du CHU de Québec to Marie-Ève Tremblay.

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……………….

Review

Nature Reviews Molecular Cell Biology 8, 519-529 (July 2007) | doi:10.1038/nrm2199

Signal integration in the endoplasmic reticulum unfolded protein response

David Ron & Peter Walter

http://www.nature.com/nrm/journal/v8/n7/full/nrm2199.html

The endoplasmic reticulum (ER) responds to the accumulation of unfolded proteins in its lumen (ER stress) by activating intracellular signal transduction pathways — cumulatively called the unfolded protein response (UPR). Together, at least three mechanistically distinct arms of the UPR regulate the expression of numerous genes that function within the secretory pathway but also affect broad aspects of cell fate and the metabolism of proteins, amino acids and lipids. The arms of the UPR are integrated to provide a response that remodels the secretory apparatus and aligns cellular physiology to the demands imposed by ER stress.

Figure 1: The unfolded protein response (UPR) signalling pathways.

FromThe impact of the endoplasmic reticulum protein-folding environment on cancer development

Nature Reviews Cancer 14, 581–597 (2014) http://dx.doi.org:/10.1038/nrc3800

(UPR) signalling pathways

http://www.nature.com/nrc/journal/v14/n9/images/nrc3800-f1.jpg

Upon endoplasmic reticulum (ER) stress, unfolded and misfolded proteins bind and sequester immunoglobulin heavy-chain binding protein (BIP), thereby activating the UPR. The UPR comprises three parallel signalling branches: PRKR-like ER kinase (PERK)–eukaryotic translation initiation factor 2α (eIF2α), inositol-requiring protein 1α (IRE1α)–X-box binding protein 1 (XBP1) and activating transcription factor 6α (ATF6α). The outcome of UPR activation increases protein folding, transport and ER-associated protein degradation (ERAD), while attenuating protein synthesis. If protein misfolding is not resolved, cells enter apoptosis. CHOP, C/EBP homologous protein; GADD34, growth arrest and DNA damage-inducible protein 34; JNK, JUN N-terminal kinase; P, phosphorylation; RIDD, regulated IRE1-dependent decay; ROS, reactive oxygen species; XBP1s, transcriptionally active XBP1; XBP1u, unspliced XBP1.

Figure 3: The unfolded protein response (UPR) and inflammation.

(UPR) and inflammation

http://www.nature.com/nrc/journal/v14/n9/images/nrc3800-f3.jpg

The three UPR pathways augment the production of reactive oxygen species (ROS) and activate nuclear factor-κB (NF-κB) and activator protein 1 (AP1) pathways, thereby leading to inflammation. NF-κB, which is a master transcriptional regulator of pro-inflammatory pathways, can be activated through binding to the inositol-requiring protein 1α (IRE1α)–TNF receptor-associated factor 2 (TRAF2) complex in response to endoplasmic reticulum (ER) stress, leading to recruitment of the IκB kinase (IKK), IκB phosphorylation (P) and degradation, and nuclear translocation of NF-κB¹⁹⁶. Moreover, the IRE1α–TRAF2 complex can recruit apoptosis signal-regulating kinase 1 (ASK1) and activate JUN N-terminal kinase (JNK), increasing the expression of pro-inflammatory genes through enhanced AP1 activity¹⁹⁷. The PRKR-like ER kinase (PERK)–eukaryotic translation initiation factor 2α (eIF2α) and activating transcription factor 6α (ATF6α) branches of the UPR activate NF-κB through different mechanisms. Engaging PERK–eIF2α signalling halts overall protein synthesis and increases the ratio of NF-κB to IκB, owing to the short half-life of IκB, thereby freeing NF-κB for nuclear translocation¹⁹⁸^,¹⁹⁹. ATF6α activation following exposure to the bacterial subtilase cytotoxin that cleaves immunoglobulin heavy-chain binding protein (BIP) leads to AKT phosphorylation and consequent NF-κB activation¹⁰⁹^,²⁰⁰.

Figure 4

The cancer-supporting role of the unfolded protein response (UPR).

http://www.nature.com/nrc/journal/v14/n9/images/nrc3800-f4.jpg

cancer-supporting role of the unfolded protein response

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Nutrition and Aging

Posted in Biochemical pathways, Cognition, Curation, Metabolism, Metabolomics, Neurodegenerative Diseases, Neurological Diseases, Neuroscience, Nutrition, Nutrition and Phytochemistry, Population Health Management, tagged Aging, Alzheimers Disease, neurodegenerative diseases, PEM, Protein-energy malnutrition, sulfur amino acids on October 25, 2015| Leave a Comment »

Nutrition and Aging

Curator: Larry H Bernstein, MD, FCAP

UPDATED 10/26/2015

Hospital Malnutrition: Prevalence, Identification and Impact on Patients and the Healthcare System

Lisa A. Barker,^1,* Belinda S. Gout,¹ and Timothy C. Crowe²

Author information ► Article notes ► Copyright and License information ►

Int J Environ Res Public Health. 2011 Feb; 8(2): 514–527.

Published online 2011 Feb 16. doi: 10.3390/ijerph8020514

This article has been cited by other articles in PMC.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3084475/

Malnutrition is a debilitating and highly prevalent condition in the acute hospital setting, with Australian and international studies reporting rates of approximately 40%. Malnutrition is associated with many adverse outcomes including depression of the immune system, impaired wound healing, muscle wasting, longer lengths of hospital stay, higher treatment costs and increased mortality. Referral rates for dietetic assessment and treatment of malnourished patients have proven to be suboptimal, thereby increasing the likelihood of developing such aforementioned complications. Nutrition risk screening using a validated tool is a simple technique to rapidly identify patients at risk of malnutrition, and provides a basis for prompt dietetic referrals. In Australia, nutrition screening upon hospital admission is not mandatory, which is of concern knowing that malnutrition remains under-reported and often poorly documented. Unidentified malnutrition not only heightens the risk of adverse complications for patients, but can potentially result in foregone reimbursements to the hospital through casemix-based funding schemes. It is strongly recommended that mandatory nutrition screening be widely adopted in line with published best-practice guidelines to effectively target and reduce the incidence of hospital malnutrition.

Keywords: diagnosis-related groups, economics, hospital, malnutrition, nutrition assessment, screening

What Is Malnutrition?

Malnutrition is a broad term that can be used to describe any imbalance in nutrition; from over-nutrition often seen in the developed world, to under-nutrition seen in many developing countries, but also in hospitals and residential care facilities in developed nations. Malnutrition can develop as a consequence of deficiency in dietary intake, increased requirements associated with a disease state, from complications of an underlying illness such as poor absorption and excessive nutrient losses, or from a combination of these aforementioned factors [1,2]. Malnutrition is associated with negative outcomes for patients, including higher infection and complication rates [3–6], increased muscle loss [6–8], impaired wound healing [4,9], longer length of hospital stay [10–12] and increased morbidity and mortality [13–17].

Recently, the definition of malnutrition has been clarified by the European Society of Parenteral and Enteral Nutrition (ESPEN) to highlight the differences between cachexia, sarcopenia (loss of muscle mass and function) and malnutrition [18]. Cachexia can be defined as a “multifactorial syndrome characterized by severe body weight, fat and muscle loss and increased protein catabolism due to underlying disease(s)” [18]. Therefore, malnutrition seen in hospitalised patients is often a combination of cachexia (disease-related) and malnutrition (inadequate consumption of nutrients) as opposed to malnutrition alone. Within the context of this review, the definition of malnutrition adopted refers to the complex interplay between underlying disease, disease-related metabolic alterations and the reduced availability of nutrients (because of reduced intake, impaired absorption and/or increased losses or a combination of these) which is a combination of cachexia and malnutrition [18].

In 1859, Florence Nightingale wrote about hospitalised soldiers during the Crimea war, starving amongst plenty of food [19]. Over 100 years later, beginning from the 1970s, numerous authors have reported malnutrition rates in hospital patients to be approximately 35%, with 30 to 55% of patients entering acute hospitals being at risk of malnutrition [20–24]. Studies have also reported on factors which contribute to malnutrition (see Table 1), consequences of malnutrition and the benefit nutrition support can offer malnourished patients [15,25–27].

Protein-Energy Malnutrition in Elderly Medical Patients

Constans MD^†,*, Y. Bacq MD^‡, J.-F. Bréchot MD^§, J.-L. Guilmot MD^‡, P. Choutet MD^‡and F. Lamisse MD^‡

Journal of the American Geriatrics Society Mar 1992; Volume 40, Issue 3: 263–268

online: 27 APR 2015 http://dx.doi.org:/10.1111/j.1532-5415.1992.tb02080.x

Consecutive sample of 324 hospitalized patients ≥70 years (86.4% of eligible patients). Norms of measurements were obtained from a referred sample of healthy control subjects (26 males and 36 females).

Main Outcome Measures Mid-arm circumference, triceps skinfold thickness, serum albumin, prealbumin, and retinol-binding protein levels were measured in patients at admission and on the 15th day.

Results (1) Prevalence of PEM was 30% in male and 41% in female patients. (2) Both mid-arm circumference and serum albumin level decreased over the first 15 days of hospital stay (53 patients, paired t test, P < 0.05). Triceps skinfold thickness did not change. (3) A step-wise discriminant-function analysis determined the utility of the parameters at admission as predictors of in-hospital mortality before the 15th day. Mid-arm circumference, triceps skinfold thickness, albumin, and prealbumin levels, as well as age, are predictors of in-hospital mortality, with 73% sensitivity, 69% specificity, and 70% of correctly classified patients of both sexes.

Conclusions Parameters used are predictors for short-term in-hospital mortality of elderly patients hospitalized in an acute medical unit. The lean body mass is preferentially mobilized for energy during hospitalization.

Downsizing of Lean Body Mass is a Key Determinant of Alzheimer’s Disease
Yves Ingenbleek,∗ and Larry H. Bernstein

Laboratory of Nutrition, Faculty of Pharmacy, University Louis Pasteur, Strasbourg, France; Laboratory of Clinical Pathology, New York Methodist Hospital, Weill-Cornell University, New York, NY, US
Journal of Alzheimer’s Disease 44 (2015) 745–754 http://dx.doi.org:/10.3233/JAD-141950

Lean body mass (LBM) encompasses all metabolically active organs distributed into visceral and structural tissue compartments and collecting the bulk of N and K stores of the human body. Transthyretin (TTR) is a plasma protein mainly secreted by the liver within a trimolecular TTR-RBP-retinol complex revealing from birth to old age strikingly similar evolutionary patterns with LBM in health and disease. TTR is also synthesized by the choroid plexus along distinct regulatory pathways. Chronic dietary methionine (Met) deprivation or cytokine-induced inﬂammatory disorders generates LBM downsizing following differentiated physiopathological processes. Met-restricted regimens downregulate the transsulfuration cascade causing upstream elevation of homocysteine (Hcy) safeguarding Met homeostasis and downstream drop of hydrogen sulﬁde (H2S) impairing anti-oxidative capacities. Elderly persons constitute a vulnerable population group exposed to increasing Hcy burden and declining H2S protection, notably in plant-eating communities or in the course of inﬂammatory illnesses. Appropriate correction of defective protein status and eradication of inﬂammatory processes may restore an appropriate LBM size allowing the hepatic production of the retinol circulating complex to resume, in contrast with the refractory choroidal TTR secretory process. As a result of improved health status, augmented concentrations of plasma-derived TTR and retinol may reach the cerebrospinal ﬂuid and dismantle senile amyloid plaques, contributing to the prevention or the delay of the onset of neurodegenerative events in elderly subjects at risk of Alzheimer’s disease.

Plasma transthyretin (TTR) was initially proposed as an index of protein-depleted states following ﬁeld surveys undertaken in Senegal (West Africa) on children suffering from varying stages of malnutrition ranging from cachectic marasmus to edematous kwashiorkor [1]. The serum analyte is now widely measured in developing areas for the nutritional follow-up of underprivileged populations [2, 3] and in developed countries to screen hospitalized patients who require dietary management [4, 5]. Several neurological investigations have recently reported the innovative observation that the same TTR biomarker impacts on the outcome of Alzheimer’s disease (AD) [6,7], raising the basic premise that alterations of protein status might be implicated in neurodegenerative disorders. Preliminary studies have indeed suggested that the reliability of the TTR indicator is based on its accurately identifying loss of lean body mass (LBM) [8] effecting metabolically active tissues in health and disease. The below review describes the unrecognized correlations linking LBM entity to TTR ﬂuctuations and the mechanisms whereby LBM downsizing, as determined by declining TTR plasma concentrations, generates signiﬁcant public health consequences in neurodeteriorating morbidities, taking AD as exemplary.

Plasma transthyretin as biomarker of lean body mass and catabolic states

Yves Ingenbleek, ¹ MD PhD & Larry H. Bernstein, ² MD

3Laboratory of Nutrition, Faculty of Pharmacy, University Louis Pasteur, Strasbourg, France; and 4Laboratory of Clinical Pathology, New York Methodist Hospital, Weill-Cornell University, New York, NY

Adv Nutr 2015; 6:1–9.

Plasma transthyretin (TTR) is a plasma protein secreted by the liver which circulates bound to retinol-binding protein (RBP4) and its retinol ligand. TTR is the sole plasma protein revealing from birth to old age evolutionary patterns closely superimposable to those of lean body mass (LBM) and working as its best surrogate analyte. Any alteration in energy- to-protein balance impairs the accretion of LBM reserves and causes early depression of TTR production. In acute inflammatory states, cytokines induce urinary leakage of nitrogenous catabolites, deplete LBM stores and cause abrupt drop of TTR-RBP4 values. As a result, thyroxine and retinol ligands are released in free form, creating a second frontline strengthening that primarily initiated by cytokines. Malnutrition and inflammation thus keep 10. in check TTR and RBP4 secretion using distinct and unrelated physiological pathways but operate in concert to downregulate LBM stores. The TTR biomarker integrates these opposite mechanisms at any time, constituting an ideally suited tool to grade residual LBM resources still available for metabolic responses, hence predicting outcome of the most interwoven disease conditions.

Cognitive Impairments in Elderly Diabetic Patients: Understanding the Risks for Better Management

Medscape Medical News from the

European Association for the Study of Diabetes (EASD) 2015 Annual Meeting

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Medscape Diabetes & Endocrinology

COMMENTARY

Lyse Bordier, MD

http://www.medscape.com/viewarticle/852112

Editor’s Note: The following is an edited, translated transcript of a presentation by Professor Lyse Bordier, a diabetologist at Military Hospital Bégin, Saint-Mandé, France, summarizing her lecture at the European Association for the Study of Diabetes (EASD) 2015 AnnualMeeting in Stockholm, Sweden.

Hello. I am Professor Lyse Bordier. I work at the Bégin Military Hospital, in Saint-Mandé, France, and I had the pleasure of participating in a symposium organized by the EASD 2015 conference in Stockholm on elderly patients, specifically on cognitive impairments.

A Public Health Problem

Dementia and cognitive impairments are a major problem; Alzheimer disease accounts for 70% of all cases of dementia. The other main causes are vascular dementias and mixed dementias. They are a real public health problem; it is estimated that, in the United States, 5.2 million people have this condition, and worldwide, every 7 seconds, a new case of dementia is diagnosed.^[1,2] In France, for example, it was estimated in 2010 that 750,000-850,000 people had dementia and that this figure will increase by a factor of 2.4 by the year 2050.

Diabetes is an important contributor to the development of cognitive impairments, all the way up to dementia. In Europe, it is estimated that nearly 25% of people over age 85 years have dementia. Its prevalence and incidence are higher in women than in men.^[2] We know that the complications of diabetes have changed over the years and that acute metabolic complications are, in the end, much less important. With the improvement in life expectancy in our diabetic patients, who are now better treated thanks to better therapeutic management, new complications have arisen, such as renal failure, heart failure, and, of course, geriatric complications, which are, in large part, cognitive disorders.^[3]

Prevalence Underestimated by Physicians

These cognitive impairments are common and largely underestimated. This was clearly shown in the GERODIAB study,^[4] which included a cohort of 987 patients over the age of 70 years. At inclusion, the physicians reported that 11% of their patients had cognitive impairments and that 3% had dementia. In actual fact, 25% of the patients had impaired cognitive functions, with a Mini-Mental State Examination (MMSE) score under 25. The prevalence is therefore significantly underestimated by physicians.

Cognitive impairments are more prevalent and more severe in diabetics than in nondiabetics. It is estimated that the risk for cognitive impairments and that for dementia are 20% to 70% and 60% higher, respectively, in the presence of diabetes.^[5] Furthermore, the risk for Alzheimer dementia is considerable, it being 40% higher in diabetics. As expected (given the combination of the other cardiovascular risk factors), the increase in the risk is even greater for vascular dementia, with an odds ratio of 2.38.^[6]

Mechanisms

What are the mechanisms in the development of cognitive impairments and dementia? There are many mechanisms, and they are often poorly understood. Hyperglycemia plays a very important role as a direct result of oxidative stress, of advanced glycation end-products, but also as a result of micro- and macroangiopathy, hypertension, and dyslipidemia.^[7,8] Other major factors, such as hypoglycemia,^[9-12]play an extremely important role in the development of cognitive impairments. As well, a great deal of literature has been published lately on the role of inflammation^[13] and genetic factors. Another widely known aspect is insulin resistance, which increases the risk for dementia at a fairly early stage by 40%^[14,15]; this already during the metabolic syndrome, even before the onset of type 2 diabetes.

http://img.medscape.com/article/852/112/852112-Figure1.jpg

Figure. Multiple and poorly understood mechanisms of cognitive impairments and dementia. HTA = arterial hypertension. Adapted from Buysschaert M, et al.^[16]

What Are the Consequences of Cognitive Impairments?

Cognitive impairments lead to a number of complications, including a reduction in life expectancy. In the GERODIAB cohort, we found, after 2 years of follow-up, that the mortality rate was twice as high in the patients with an MMSE score <24 compared with those with an MMSE score >24. In this study, the patients with a lower MMSE score had less well-controlled diabetes, were usually treated with insulin, and had heart failure and cerebrovascular complications more often. Very surprisingly, hypoglycemia was not more prevalent in these patients, perhaps because, being less independent, they were better managed by care teams.^[17]

Cognitive impairments lead to geriatric complications, such as malnutrition, falls, and a loss of autonomy. They also promote social and family isolation and iatrogenic accidents, as well as depression, which can both mask cognitive impairments and exacerbate an underlying dementia. Another important aspect is that cognitive impairments increase the risk for hypoglycemia. This has been shown very clearly in all of the studies. There is, in fact, a bidirectional link between dementia and hypoglycemia: Hypoglycemia doubles the risk for dementia, and dementia triples the risk for hypoglycemia.^[18]

Screening and Management

What do we do when a patient presents with cognitive impairments? First, they should be identified so that they can be managed. We need to be vigilant for certain little signs: changes in the patient’s behavior (eg, a patient who forgets his appointments, whose personal hygiene has declined, who is less diligent in keeping his blood glucose diary, and, lastly, who has an unexplained diabetic imbalance). We should also know how to use simple tests, such as the MMSE, which provides an overall assessment of space-time orientation, cognitive functions, language functions, and calculation, and how to assess the patient’s autonomy and loss of autonomy.^[19] Next, we should, as per the recommendations of the American Diabetes Association^[20] and the EASD, individualize the glycemic goals, taking into account, in the most fragile, elderly patients, cognitive status, the level of autonomy, depression, nutritional status—in particular, sarcopenia, which can coexist with obesity, and the risk for hypoglycemia.^[21]

We should therefore avoid overtreating the most fragile patients (those at greatest risk for hypoglycemia), but neither should we undertreat patients who have a long life expectancy and who could develop micro- and macroangiopathic complications.

One last aspect, which is very important, is the family. Help needs to be provided to prevent the patient’s loss of autonomy.^[21] Lastly, I think that cognitive decline should be added to the already long list of degenerative complications of diabetes.

Transthyretin Blocks Retinol Uptake and Cell Signaling by the Holo-Retinol-Binding Protein Receptor STRA6

Daniel C. Berry^a,^b, Colleen M. Croniger^b, Norbert B. Ghyselinck^c and Noa Noy^a,^b

+Author Affiliations

^aDepartments of Pharmacology
^bNutrition, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA
^cInstitut de Génétique et de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique (UMR7104), Institut National de la Santé et de la Recherche Médicale U964, Université de Strasbourg, Illkirch, France

Vitamin A is secreted from cellular stores and circulates in blood bound to retinol-binding protein (RBP). In turn, holo-RBP associates in plasma with transthyretin (TTR) to form a ternary RBP-retinol-TTR complex. It is believed that binding to TTR prevents the loss of RBP by filtration in the kidney. At target cells, holo-RBP is recognized by STRA6, a plasma membrane protein that serves a dual role: it mediates uptake of retinol from extracellular RBP into cells, and it functions as a cytokine receptor that, upon binding holo-RBP, triggers a JAK/STAT signaling cascade. We previously showed that STRA6-mediated signaling underlies the ability of RBP to induce insulin resistance. However, the role that TTR, the binding partner of holo-RBP in blood, plays in STRA6-mediated activities remained unknown. Here we show that TTR blocks the ability of holo-RBP to associate with STRA6 and thereby effectively suppresses both STRA6-mediated retinol uptake and STRA6-initiated cell signaling. Consequently, TTR protects mice from RBP-induced insulin resistance, reflected by reduced phosphorylation of insulin receptor and glucose tolerance tests. The data indicate that STRA6 functions only under circumstances where the plasma RBP level exceeds that of TTR and demonstrate that, in addition to preventing the loss of RBP, TTR plays a central role in regulating holo-RBP/STRA6 signaling.

INTRODUCTION

Vitamin A (retinol [ROH]) plays critical roles both in the embryo and in the adult, where it regulates multiple cellular processes and is essential for embryonic development, reproduction, immune function, and vision (29, 32, 33). The vitamin exerts many of its biological activities by giving rise to active metabolites: the visual chromophore 11-cis-retinaldehyde and retinoic acid (RA), which regulates gene transcription by activating specific nuclear receptors (11, 27). ROH is stored in various tissues, including white adipose tissue (WAT), lung, and retinal pigment epithelium in the eye, but its main storage site is the liver. ROH is secreted from storage into the circulation bound to retinol-binding protein (RBP), a 21-kDa polypeptide that contains one binding site for ROH. In most mammals, ROH-bound RBP (holo-RBP) does not circulate alone but is associated with another protein called transthyretin (TTR), a 56-kDa homotetramer that, in addition to associating with RBP, functions as a carrier for thyroid hormones (23, 24). ROH thus reaches target tissues bound in a holo-RBP-TTR complex that, under normal circumstances, displays a 1:1 molar stoichiometry. It is believed that binding of RBP to TTR serves to prevent the loss of the smaller protein from blood by filtration in the glomeruli. The concentration of the holo-RBP-TTR complex in plasma is kept constant at 1 to 2 μM except in extreme cases of vitamin A deficiency or in disease states. Notably, RBP levels are markedly elevated in blood of obese mice and humans, and it was reported that, under these circumstances, the protein induces insulin resistance (35).

Association with the TTR-RBP complex allows the poorly soluble ROH to circulate in blood, but the vitamin dissociates from RBP prior to entering cells. It was proposed that, due to its hydrophobic nature, ROH can readily move from extracellular RBP into cells by diffusion across the plasma membranes at fluxes that are dictated by its extracellular-to-intracellular concentration gradient (10,14, 20, 21). However, it has also been suggested that uptake of ROH from circulating holo-RBP is mediated by a cell surface receptor (13, 28). Indeed, a plasma membrane protein termed STRA6 (stimulated by retinoic acid 6) was found to bind holo-RBP and transport ROH into cells (15). Our recent studies revealed that, in addition to its function as an ROH transporter, STRA6 is a cytokine receptor. We thus found that binding of holo-RBP triggers phosphorylation of a tyrosine residue in the cytosolic domain of STRA6, resulting in recruitment and activation of the Janus kinase JAK2 and, in a cell-dependent manner, the transcription factors STAT3 or STAT5. Holo-RBP thus activates STRA6-mediated signaling that culminates in upregulation of STAT target genes (2, 4). As STAT target genes in white adipose tissue and muscle include Suppressor of cytokine signaling 3 (Socs3), a potent inhibitor of insulin signaling (8), these findings suggested a rationale for understanding how elevated serum levels of RBP in obese animals induce insulin resistance (35). Additional studies showed that activation of STRA6 is triggered not simply by binding of holo-RBP but by a STRA6-mediated translocation of ROH from extracellular holo-RBP to an intracellular acceptor, the retinol-binding protein CRBP-I. Importantly, this movement was found to be critically linked to the intracellular metabolism of ROH (5). The data further established that ROH uptake and signaling by STRA6 are interdependent, i.e., that activation of a JAK2/STAT cascade by the receptor requires ROH uptake and, conversely, that phosphorylation of STRA6 is essential for enabling ROH transport to proceed (5).

While these recent studies provided surprising new insights into the involvement of STRA6 in vitamin A biology, the role that TTR, the binding partner of holo-RBP in blood, may play in STRA6-mediated functions remained unknown. Here, we show that TTR blocks the ability of holo-RBP to associate with STRA6 and thereby effectively suppresses both STRA6-mediated ROH uptake and STRA6-initiated cell signaling. We show further that, consequently, TTR protects mice from RBP-induced insulin resistance. The data indicate that, in addition to preventing the loss of RBP by filtration in the kidney, TTR plays a central role in regulating holo-RBP/STRA6 signaling.

….

TTR inhibits STRA6-mediated uptake of ROH from holo-RBP. In most mammals, holo-RBP circulates in blood in complex with transthyretin (TTR). To begin to examine the effect of TTR on STRA6 function, hepatocarcinoma HepG2 cells, which endogenously express STRA6, were used to compare the cellular uptake of ROH from holo-RBP and from TTR-bound holo-RBP. Recombinant RBP and TTR were expressed in E. coli and purified (see Materials and Methods). HepG2 cells were treated with RBP complexed with [³H]retinol at a 1 μM concentration, similar to the serum RBP level, or with 1 μM [³H]retinol-labeled RBP complexed with TTR at a 1:1 molar stoichiometry, similar to that found in blood (24). Media were removed, cells washed, and organic compounds extracted from the cells into ethanol, and the amount of [³H]retinol taken up within the incubation period was measured by scintillation counting. The rates of uptake of retinol under the assay conditions were constant during the initial 5 min (Fig. 1a), and subsequent experiments were carried out with a single 3-min time point, well within the initial linear rate. The rate of ROH uptake from the holo-RBP-TTR complex was lower than that of the uptake from holo-RBP alone (Fig. 1a). Moreover, increasing the TTR/RBP ratio by increasing the concentration of TTR inhibited ROH uptake in a dose-dependent manner (Fig. 1b). The dose response of the initial rate of ROH transport from holo-RBP showed a two-phase behavior comprised of an initial saturable component, likely attributable to STRA6-mediated uptake, followed by a nonsaturable phase, reflecting passive diffusion of ROH across the plasma membranes (Fig. 1c). In contrast, uptake of ROH from the holo-RBP-TTR complex displayed a single, nonsaturable phase (Fig. 1c). These observations suggest that TTR does not impede the ability of ROH to enter cells by passive diffusion but effectively blocks ROH transport mediated by STRA6. In agreement with this notion, increasing the expression level of STRA6 in HepG2 cells (Fig. 1d) facilitated ROH uptake from holo-RBP in a dose-responsive manner but had no effect on transport of ROH from TTR-bound holo-RBP (Fig. 1e). Also in agreement, decreasing the expression of STRA6 in HepG2 cells (Fig. 1f) or in NIH 3T3-L1 adipocytes (Fig. 1g) reduced the rate of ROH uptake from holo-RBP but did not affect uptake from TTR-bound holo-RBP. The observation that, in both cell lines, rates of uptake from the holo-RBP-TTR complex were similar to those observed in the absence of STRA6 supports the conclusion that TTR specifically inhibits STRA6-mediated transport.

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FIG 1

TTR inhibits STRA6-mediated uptake of ROH from holo-RBP. (a) Uptake of [³H]ROH by HepG2 cells treated with RBP-[³H]ROH or RBP-[³H]ROH-TTR (1 μM) for denoted times. (b) Uptake of [³H]ROH by HepG2 cells treated with the denoted concentrations of RBP-[³H]ROH or RBP-[³H]ROH-TTR for 3 min. (c) Uptake of [³H]ROH by HepG2 cells following a 3-min incubation with 1 μM RBP-[³H]ROH in the presence of denoted concentrations of TTR. (d) Levels of STRA6 mRNA in HepG2 cells transfected with various amounts of STRA6 cDNA. (e) Effect of increasing the expression level of STRA6 in HepG2 cells on uptake of [³H]ROH from RBP-[³H]ROH or RBP-[³H]ROH-TTR (1 μM, 3 min). (f) Top, expression level of STRA6 in HepG2 cells transfected with an empty vector (e.v.) or vector harboring STRA6shRNA. Bottom, effect of decreasing the expression level of STRA6 in HepG2 cells on uptake of [³H]ROH from RBP-[³H]ROH or from RBP-[³H]ROH-TTR (1 μM, 3 min). (g) Top, expression level of STRA6 in NIH 3T3-L1 cells transfected with an empty vector (e.v.) or a vector harboring STRA6shRNA. Bottom, effect of decreasing the expression level of STRA6 in NIH 3T3-L1 adipocytes on uptake of [³H]ROH from RBP-[³H]ROH or from RBP-[³H]ROH-TTR (1 μM, 3 min). (h) Twelve-week-old WT and STRA6-null male mice were injected intraperitoneally with RBP-[³H]ROH (100 μl, 0.1 mCi, 1 μM). Two hours later, tissues were isolated, weighed, and homogenized, and [³H]ROH was quantified. Data are means ± standard errors of the means; *, P < 0.01 for RBP-ROH-treated versus RBP-ROH-TTR-treated groups. All P values were calculated using a two-tailed Student t test.

The effect of TTR on ROH uptake from holo-RBP was then examined in vivo using our newly generated STRA6-null mice (26). Twelve-week-old wild-type (WT) and STRA6-null male mice were injected intraperitoneally with [³H]ROH-labeled holo-RBP or with holo-RBP complexed with TTR, and ROH uptake into tissues was assessed 2 h later. Uptake of ROH into the STRA6-expressing tissues WAT, skeletal muscle, and the eye was modestly but significantly lower in STRA6-null than in WT mice (Fig. 1h), reflecting that the contribution of STRA6 to overall vitamin A uptake by tissues in vivo is small. ROH uptake from TTR-bound holo-RBP was all but identical to that observed in STRA6^−/− animals (Fig. 1h). Neither ablation of STRA6 nor the presence of TTR affected ROH uptake by the liver, an organ that does not express STRA6 (Fig. 1h). Hence, TTR specifically inhibits STRA6-mediated uptake of ROH in vivo.

TTR inhibits the association of holo-RBP with STRA6.STRA6 may bind the ternary RBP-ROH-TTR complex or, alternatively, it may recognize only free holo-RBP. To dissect these possibilities, we considered that, unlike in most mammals, holo-RBP in zebrafish (Danio rerio) does not associate with TTR. Thus, presumably, zebrafish STRA6 does not contain a TTR-binding region, and while ROH uptake by the mammalian STRA6 may involve recognition of TTR, ROH uptake by zebrafish STRA6 (dSTRA6) will not. In these experiments, NIH 3T3 fibroblasts, which do not endogenously express STRA6, were used. We previously showed that ROH metabolism is essential both for STRA6-mediated ROH transport and for holo-RBP-induced STRA6 signaling (5). Hence, to enable STRA6 action in these cells, an NIH 3T3 line in which ROH metabolism is enhanced by stably overexpressing lecithin:ROH-acyltransferase (LRAT), which catalyzes ROH esterification, was generated. Ectopic overexpression of either hSTRA6 or dSTRA6 in LRAT-expressing NIH 3T3 fibroblasts enhanced ROH uptake from holo-RBP to a similar extent, and introduction of TTR similarly decreased the rate of uptake (Fig. 2a andb). The similarity of the response of dSTRA6, which is unlikely to contain a TTR-binding capability, to that of hSTRA6 suggests that STRA6 in both species recognizes only free and not TTR-bound holo-RBP.

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FIG 2

STRA6 does not bind the holo-RBP-TTR complex. (a) NIH 3T3 cells stably overexpressing LRAT were transfected with an empty vector (e.v.) or with expression vectors encoding human (hSTRA6) or zebrafish (dSTRA6) STRA6, resulting in similar levels of mRNAs. (b) Uptake of [³H]ROH from RBP-[³H]ROH or RBP-[³H]ROH-TTR (1 μM, 3 min) by cells expressing hSTRA6 or dSTRA6. (c) RBP-ROH (R-R) or RBP-ROH-TTR (R-R + TTR) (1 μM) was incubated with the chemical cross-linker bis(sulfosuccinimidyl) suberate (0.5 mM) for 14 h. Proteins were resolved by SDS-PAGE and visualized by Coomassie blue staining. (d) Cross-linked complexes and additional cross-linker (0.5 mM) were added to HepG2 cells transfected with an e.v. or with a vector encoding histidine-tagged STRA6. Following a 15-min incubation, his-STRA6 was immunoprecipitated using antibodies against the tag, and precipitated RBP and STRA6 were visualized by immunoblotting. (e) Fluorescence titrations of RBP and its F96A/L97A mutant (1 μM) with ROH. Progress of titrations was monitored by following the increase in ROH fluorescence upon binding to the protein (λ_ex = 330 nm; λ_em = 460 nm). (f) Fluorescence anisotropy titrations of holo-RBP and holo-RBP-F96A/L97A (3 μM) with TTR. Progress of titrations was monitored by measuring the fluorescence anisotropy of bound ROH (λ_ex = 330 nm; λ_em = 460 nm). (g) Uptake of [³H]ROH from holo-RBP-F96A/L97A (1 μM, 3 min) in the presence or absence of TTR. Data are means ± standard errors of the means (SEM). *, P < 0.01 versus cells transfected with an empty vector; **, P = 0.01 versus cells transfected with an empty vector and treated with RBP-ROH. All Pvalues were calculated using a two-tailed Student t test.

The question of whether STRA6 binds free or TTR-bound holo-RBP was then directly addressed. Recombinant holo-RBP was incubated alone or in the presence of TTR with the chemical cross-linker bis(sulfosuccinimidyl) suberate (0.5 mM, 14 h), resulting in efficient cross-linking of the holo-RBP-TTR complex (Fig. 2c). The mixtures and additional cross-linker were added to NIH 3T3 cells ectopically overexpressing histidine-tagged STRA6. STRA6 was immunoprecipitated, and precipitated proteins were resolved by SDS-PAGE and immunoblotted for RBP-containing complexes (Fig. 2d). Cross-linking of cells with holo-RBP resulted in the appearance of a band with a molecular mass of ∼100 kDa, corresponding to that of an RBP-bound STRA6. No such band was observed in cells cross-linked with the RBP-ROH-TTR complex, and no bands that might correspond to a STRA6-RBP-TTR (∼150 kDa) appeared. The data thus indicate that STRA6 associates only with free holo-RBP and that the presence of TTR prevents the association.

To further examine whether TTR inhibits STRA6-mediated ROH uptake by preventing holo-RBP from binding to the receptor, an RBP mutant defective in its ability to bind TTR was generated. The reported three-dimensional crystal structure of the holo-RBP-TTR complex suggests that the interactions between the two proteins are mediated by several residues, including Phe96 and Leu97 (18). An RBP mutant in which these residues were replaced with alanines (RBP-F96A/L97A) was thus generated. The mutations did not alter the affinity of RBP for retinol (Fig. 2e), indicating that the overall fold of the mutant is intact. As expected, the F96A/L97A mutations disrupted the association of RBP with TTR (Fig. 2f). Measurements of ROH uptake showed that, in contrast with its inhibitory activity on ROH uptake from WT-RBP, TTR had no effect on ROH uptake from RBP-F96A/L97A (Fig. 2g). These observations further establish that TTR inhibits STRA6-mediated ROH uptake by sequestering holo-RBP and not by direct association with the receptor.

TTR inhibits holo-RBP-induced STRA6 signaling.The effect of TTR on RBP-induced STRA6 signaling was then examined using NIH 3T3-L1 adipocytes. We previously showed that in these cells, activation of STRA6 by holo-RBP triggers a JAK2/STAT5 cascade to induce the STAT target genes SOCS3 and PPARγ and inhibit insulin responses (2). Preadipocytes NIH 3T3-L1 cells were grown 2 days past confluence and induced to differentiate using a standard hormone mix (10 μg/ml insulin, 0.5 mM 3-isobutyl-1-methylxanthine [IBMX], 0.25 mM dexamethasone). Three days later, media were replaced and cells grown for 4 days. Differentiation was verified by monitoring lipid accumulation and by examining the expression of the adipocyte marker FABP4 (3). As expected, treatment of differentiated adipocytes with holo-RBP (R-R) increased the phosphorylation levels of JAK2 and STAT5 (Fig. 3a). In contrast, the holo-RBP-TTR complex did not alter the phosphorylation status of these proteins (Fig. 3a). Accordingly, TTR-bound holo-RBP failed to induce the expression of SOCS3 and PPARγ (Fig. 3b). To examine the effect of TTR on the ability of holo-RBP to suppress insulin responses, cells were pretreated with holo-RBP or holo-RBP-TTR for 8 h and treated with insulin for 15 min, and the levels of phosphorylation of the insulin receptor (IR) and its downstream effector AKT were monitored. The data show that inhibition of insulin-induced phosphorylation of IR and AKT by holo-RBP was blunted in the presence of TTR (Fig. 3c). TTR also inhibited the ability of holo-RBP, but not of holo-RBP-F96A/L97A, defective in TTR binding, to trigger STAT5 phosphorylation (Fig. 3d) or to induce the expression of SOCS3 in NIH 3T3-L1 adipocytes (Fig. 3e) or in HepG2 cells (Fig. 3f).

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FIG 3

TTR blocks activation of STRA6 signaling by holo-RBP. (a) NIH 3T3-L1 adipocytes were treated with 1 μM RBP-ROH (R-R), TTR, or RBP-ROH-TTR (R-R-TTR) for 15 min. Cells were lysed, and phosphorylated JAK2 (pJAK2) and STAT5 (pSTAT5) were visualized by immunoblotting. (b) NIH 3T3-L1 adipocytes cells were treated with 1 μM RBP-ROH, TTR, or RBP-ROH-TTR for 4 h, and levels of SOCS3 and PPARγ mRNA were assessed by Q-PCR. Data are means ± SEM. *, P < 0.001 versus nontreated cells. (c) NIH 3T3-L1 adipocytes were pretreated with 1 μM RBP-ROH, TTR, or RBP-ROH-TTR for 8 h and then treated with insulin (25 nM, 15 min.). Phosphorylated IR (pIR) and AKT (pAKT) were visualized by immunoblotting. Bottom, quantitation of band intensities. Means of two independent experiments. (d) NIH 3T3-L1 adipocytes were treated with RBP-ROH or RBP-F96A/L97A-ROH (RBP_96/97-R) in the presence or absence of TTR (1 μM each, 15 min). Lysates were immunoblotted for pSTAT5. (e) NIH 3T3-L1 adipocytes were treated with RBP-ROH or RBP-F96A/L97A-ROH in the presence or absence of TTR (1 μM each, 4 h). Levels of SOCS3 mRNA were assessed by Q-PCR. Data are means ± SEM. *, P < 0.001 versus nontreated cells; **, P < 0.001 versus R-R-TTR-treated cells. (f) HepG2 cells were treated with RBP-ROH in the presence or absence of TTR (1 μM each, 4 h). Levels of SOCS3 mRNA were assessed by Q-PCR. Data are means ± SEM. *, P < 0.001 versus nontreated cells. (g) The phosphotyrosine motifs in mouse, human, and zebrafish STRA6 (mSTRA6, hSTRA6, and dSTRA6). (h) NIH 3T3 fibroblasts stably expressing LRAT were transfected with zebrafish and human STRA6 and treated with 1 μM RBP-ROH or RBP-ROH-TTR for 15 min, and lysates were immunoblotted for pSTAT3. (i) NIH 3T3 fibroblasts stably overexpressing LRAT were transfected with dSTRA6 or hSTRA6 and treated with 1 μM RBP-ROH or RBP-ROH-TTR for 4 h. Levels of SOCS3 mRNA were assessed by Q-PCR. Data are means ± SEM. *, P < 0.001 versus nontreated cells. All P values were calculated using a two-tailed student t test.

The effect of TTR on signaling by the zebrafish STRA6 was then examined. Notably, the phosphotyrosine in the cytosolic domain of STRA6, the STAT recruitment site of the receptor, is present in the dSTRA6, suggesting evolutionary conservation of STRA6 signaling (Fig. 3g). In these experiments, NIH 3T3 fibroblasts that ectopically overexpress LRAT were transfected with expression vectors for either hSTRA6 or dSTRA6. Treatment of cells expressing either hSTRA6 or dSTRA6 with holo-RBP induced phosphorylation of STAT3, the preferred STRA6-activated STAT in these cells (Fig. 3h), and upregulation of SOCS3 (Fig. 3i). TTR suppressed the ability of holo-RBP to induce STAT3 phosphorylation and to upregulate SOCS3 expression in cells expressing either hSTRA6 or dSTRA6 (Fig. 3hand i).

TTR inhibits the ability of holo-RBP to suppress insulin responses in vivo.The effect of TTR on the ability of holo-RBP to promote insulin resistance in vivo was then investigated. Eight-week-old mice were injected with recombinant holo-RBP, TTR, or holo-RBP-TTR. Mice were injected three times at 2-h intervals and sacrificed an hour after the last injection. The treatments resulted in respective elevation of serum levels of RBP, TTR, or both (Fig. 4a and b). As expected, treatment of mice with holo-RBP reduced the phosphorylation levels of the insulin receptor and AKT and induced the expression of SOCS3 and PPARγ in WAT (Fig. 4cand f) and skeletal muscle (Fig. 4d and g) but not in liver (Fig. 4e and h). In contrast, treatment with RBP-ROH-TTR did not affect the phosphorylation of IR and AKT or the expression levels of the STAT target genes (Fig. 4c to h).

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FIG 4

TTR suppresses activation of STRA6 by holo-RBP in vivo. Mice were injected three times with 0.1 μmol RBP-ROH or 0.1 μmol RBP-ROH complexed with TTR and sacrificed 1 h after the last injection. (a, b) Immunoblots of RBP (a) and TTR (b) in serum following the respective injections. Blots from 2 mice of each group are shown. (c to e) Immunoblots of phosphorylated insulin receptor (pIR), AKT (pAKT), and STAT5 (pSTAT5) in WAT (c), skeletal muscle (d), and liver (e) of mice treated as denoted. Total IR served as a loading control. (f to h) Levels of mRNA of SOCS3 and PPARγ in WAT (f), skeletal muscle (g), and liver (h) of treated mice. Data are means ± SEM. *, P < 0.001 for buffer-treated versus RBP-ROH-treated mice.

The observations that only free and not TTR-bound holo-RBP activates STRA6 suggest that the serum RBP/TTR ratio is crucial for regulating STRA6 signaling. In agreement with the report that expression of RBP in adipose tissue increases in obese rodents and humans, resulting in elevation of serum RBP levels (35), feeding mice a high-fat, high-sucrose (HFHS) diet for 10 weeks resulted in upregulation of the expression of RBP in WAT but not in liver (Fig. 5a). In contrast, TTR expression in these organs was not affected by the diet (Fig. 5b). Accordingly, the serum level of RBP was markedly elevated, while the serum level of TTR remained unchanged in obese mice (Fig. 5c). Hence, the RBP/TTR ratio is significantly higher in blood of obese than of lean mice.

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FIG 5

TTR is protective against holo-RBP-induced insulin resistance. (a and b) Levels of mRNA of RBP (a) and TTR (b) in WAT and liver of lean mice and of mice fed an HFHS diet for 10 weeks (obese). (c) Immunoblots of RBP and TTR in serum of mice fed an HFHS diet for 0, 3, 6, and 10 weeks. (d to j) Mice were implanted with an Alzet pump that contained buffer, 0.1 μM holo-RBP, or 0.1 μM holo-RBP complexed with TTR. Implants were replaced once a week for 3 weeks. (d) Immunoblots of RBP (top) and TTR (bottom) in serum following 3 weeks of denoted treatments. (e, f) Immunoblots of pIR and pSTAT5 in WAT (e) and skeletal muscle (f) of mice treated as denoted. (g to i) Levels of SOCS3 mRNA in WAT (g), skeletal muscle (h), and liver (i) of mice treated as denoted. (j) Glucose tolerance tests carried out following 3 weeks of denoted treatments. Data are means ± SEM. *, P < 0.001 for lean versus obese mice; **, P < 0.001 for buffer-treated versus RBP-ROH-treated mice. All P values were calculated using a two-tailed Student ttest.

To directly determine if TTR prevents holo-RBP-induced insulin resistance, mice were treated with holo-RBP or holo-RBP-TTR for 3 weeks prior to the glucose tolerance tests (GTT). Mice were treated by implanting Alzet osmotic pumps containing the appropriate proteins (1 μM), thereby delivering constant amounts of proteins over the 3-week period. Similar to what was seen in the short-term treatments (Fig. 4), 3-week treatment of mice with holo-RBP induced phosphorylation of STAT5, reduced the activation level of IR, and upregulated SOCS3 and PPARγ in WAT (Fig. 5e and g) and muscle (Fig. 5f and h) but not in the liver (Fig. 5i). In contrast, treatment with TTR-bound holo-RBP had no effect on the phosphorylation of STAT5 or IR and did not alter the expression levels of the STAT target genes (Fig. 5e to i). Accordingly, while holo-RBP treatment resulted in a sluggish response in GTT, reflecting the development of insulin resistance, treatment with the holo-RBP-TTR complex did not alter the insulin responses of the mice (Fig. 5j). Hence, association with TTR suppresses the ability of holo-RBP to interfere with insulin signaling.

DISCUSSION

Upon binding of extracellular holo-RBP, STRA6 transports ROH into cells, and it activates a signaling cascade culminating in induction of STAT target genes (4, 5). The observations described here reveal that the binding partner of RBP in blood, TTR, effectively blocks association of holo-RBP with STRA6. Consequently, STRA6 mediates cellular ROH uptake only from free and not from TTR-bound holo-RBP. The data further show that, even in the presence of free holo-RBP, STRA6-mediated ROH uptake by tissues comprises only a small fraction of total uptake by target tissues in vivo (Fig. 1h). The observations thus support the previously proposed model whereby supply of ROH from circulating holo-RBP or holo-RBP-TTR to cells occurs primarily by diffusion through the plasma membranes (10, 14,20, 21). Taken together with the observations that ROH transport by STRA6 is critical for enabling activation of STRA6 signaling (5), the data indicate that, with the exception of the eye (26), the main role of ROH transport by STRA6 is not to provide the vitamin to cells but to couple sensing of circulating free holo-ROH levels to cell signaling. It is worth noting that even in the eye, morphological changes and reduction in visual function in Stra6-null mice are mild, indicating that STRA6 is not the only pathway by which ROH enters the retinal pigment epithelium (26).

The data reveal that, in addition to its function in preventing filtration of the 21-kDa RBP in the kidney, TTR plays an important role in protecting cells from holo-RBP-induced signaling mediated by STRA6. The observations that STRA6 “senses” only free and not TTR-bound RBP establish that the receptor functions only under circumstances in which the serum RBP level exceeds that of TTR. Such circumstances are encountered, for example, in obese animals in which the serum level of RBP is elevated while the TTR level is not (Fig. 5c). The circumstances under which the plasma RBP concentration exceeds that of TTR in healthy lean animals remain to be clarified. In this regard, it is interesting that it has long been known that insulin responsiveness varies in a circadian fashion (17, 31). The molecular basis for these diurnal variations is incompletely understood, but the data presented here raise the intriguing possibility that they may arise from diurnal variations in the plasma RBP/TTR ratio.

The RBP/TTR ratio in blood may be altered by changes in the expression level of RBP, or TTR, or both. TTR is expressed in the central nervous system and in the liver, with the latter serving as the main source for the protein in serum (9). Expression of hepatic TTR is downregulated, and consequently, the serum TTR level dramatically decreases during the acute-phase response (APR), a process characterized by rapid reprogramming of gene expression and metabolism in response to inflammatory cytokine signaling (1, 22). The low serum level of TTR associated with APR may release holo-RBP, thereby activating STRA6. Hence, STRA6 signaling may play a role in APR. It has also been reported that hepatic TTR expression is regulated by sex hormones (12) and is directly controlled by hepatocyte nuclear factor 4α (HNF-4α) (30). The expression of RBP in brown adipose tissue and liver was reported to be regulated by cyclic AMP-mediated pathways and by the nuclear receptors PPARα and PPARγ (6, 25). Whether, by controlling TTR or RBP expression, these factors regulate the RBP-TTR ratio in blood and thus STRA6 signaling remains to be clarified.

Notably, as free holo-RBP is rapidly excreted by glomerular filtration, its lifetime in serum is short. Holo-RBP thus functions like a classical cytokine: its availability to its membrane receptor is tightly regulated, and its signaling activities are constrained by a short half-life in the circulation. These characteristics of the signaling activities of holo-RBP strikingly differ from the characteristics of its role as a shuttling protein that mobilizes ROH from liver stores. Unlike in the former capacity, where holo-RBP functions on its own, delivery of ROH to target tissues is mediated by the holo-RBP-TTR complex. The plasma level of this complex is under tight homeostatic control, and it provides ROH to target cells to support tissue requirement for vitamin A without the need for a specialized receptor.

Biochim Biophys Acta. 1996 May 2; 1294(1):48-54.

Retinoid binding to retinol-binding protein and the interference with the interaction with transthyretin.

Malpeli G¹, Folli C, Berni R.

The retinol carrier retinol-binding protein (RBP) forms a complex with the thyroid hormone binding protein transthyretin in the plasma of a number of vertebrate species. The interactions of retinoid-RBP complexes, as well as of unliganded RBP, with transthyretin have been investigated by means of fluorescence anisotropy studies. The presence of two independent and equivalent RBP binding sites per transthyretin molecule has been established for proteins purified from species distant in evolution. Although the natural ligand retinol participates in the interaction between retinol-RBP and transthyretin, its binding to RBP is not a prerequisite for protein-protein interaction. The dissociation constants of human transthyretin binding liganded and unliganded forms of human RBP were determined to be: all-trans retinol-RBP, Kd approximately 0.2 microM; apoRBP, Kd approximately 1.2 microM; all-trans retinoic acid-RBP, Kd approximately 0.8 microM; all-trans retinyl methyl ether-RBP, Kd approximately 6 microM. The complex of RBP with the synthetic retinoid fenretinide, which bears the bulky hydroxyphenyl end group, exhibits negligible affinity for transthyretin. The replacement of RBP-bound retinol with synthetic retinoids affects RBP-transthyretin recognition to an extent that appears to be well correlated with the nature and steric hindrance of the groups substituting the retinol hydroxyl group, consistent with their location at the interface between the contact areas of RBP and transthyretin.

Methods Mol Biol. 2010; 652:189-207. doi: 10.1007/978-1-60327-325-1_11.

The interaction between retinol-binding protein and transthyretin analyzed by fluorescence anisotropy.

Folli C¹, Favilla R, Berni R.

The retinol carrier retinol-binding protein (RBP) forms in blood a complex with the thyroid hormone carrier transthyretin (TTR). The interactions of retinoid-RBP complexes, as well as of unliganded RBP, with TTR can be investigated by means of fluorescence anisotropy. RBP represents the prototypic lipocalin, in the internal cavity of which the retinol molecule is accommodated. Due to the tight binding of retinol within a substantially apolar binding site, an intense fluorescence emission characterizes the RBP-bound vitamin. The addition of TTR to the retinol-RBP complex (holoRBP) causes a marked increase in the fluorescence anisotropy of the RBP-bound retinol within the system, due to the formation of the holoRBP-TTR complex, which allows the interaction between the two proteins to be monitored. The fluorescence anisotropy technique is also suitable to study the interaction of TTR with apoRBP and RBP in complex with non-fluorescent retinoids. In the latter cases, the fluorescence signal is provided by a fluorescent probe covalently linked to TTR rather than by RBP-bound retinol. We report here on the preparation of recombinant human RBP and TTR, the covalent labeling of TTR with the fluorescent dansyl probe, and fluorescence anisotropy titrations for RBP and TTR.

Vitam Horm. 2004; 69:271-95.

Plasma retinol-binding protein: structure and interactions with retinol, retinoids, and transthyretin.

Zanotti G¹, Berni R.

Retinol-binding protein (RBP) is the retinol-specific transport protein present in plasma. The available crystal structures of different forms of RBP have provided details of the interactions of this binding protein with retinol, retinoids, and transthyretin (TTR, one of the plasma carriers of thyroid hormones). The core of RBP is a beta-barrel, the cavity of which accommodates retinol, establishing with its buried portions apolar contacts. Instead, the retinol hydroxyl is near the protein surface, in the region of the entrance loops surrounding the opening of the binding cavity, and participates in polar interactions. The stability of the retinol-RBP complex appears to be further enhanced when holo-RBP is bound to TTR. Accordingly, the region of the entrance loops represents the contact area of RBP interacting with the TTR counterpart, such that the hydroxyl of the RBP-bound vitamin becomes fully buried in the holo-RBP-TTR complex. Limited protein conformational changes affecting the entrance loops, which lead to a decrease or loss of the binding affinity of RBP for TTR, have been demonstrated for apo-RBP and RBP in complex with retinoids modified in the area of the retinol hydroxyl. A relatively small number of amino acid residues of RBP, essentially confined to the region of the entrance loops, and of TTR appear to play a critical role in the formation of the RBP-TTR complex, as established by crystallographic studies, mutational analysis, and amino acid sequence analysis of phylogenetically distant RBPs and TTRs. Overall, the available evidence indicates the existence of a high degree of complementarity between RBP and TTR, the contact areas of which are highly sensitive to conformational changes and amino acid replacements.

Biochim Biophys Acta. 2004 Dec 1; 1703(1):1-9.

Interactions amongst plasma retinol-binding protein, transthyretin and their ligands: implications in vitamin A homeostasis and transthyretin amyloidosis.

Raghu P¹, Sivakumar B.

Retinol transport complex consisting of retinol-binding protein (RBP) and transthyretin (TTR) is involved in the transport of retinol (vitamin A) and thyroxine (T(4)) in the human plasma. RBP is a 21-kDa single polypeptide chain protein, synthesized in the liver, which binds and transports retinol to the target organs. The circulating RBP binds to another protein called TTR, a 55-kDa homotetrameric T(4) transport protein. Such protein-protein complex formation is thought to prevent glomerular filtration of low molecular mass RBP. Misfolding and aggregation of TTR is implicated in amyloid disorders such as familial amyloid polyneuropathy (FAP) and senile systemic amyloidosis (SSA). Recent observations suggest that both RBP and T(4), the physiological ligands of TTR, prevent its misfolding and amyloid fibril formation, suggesting yet another structure-function relationship to this protein-protein complex. TTR2, a poorly characterized protein, was also found bound to RBP in human and pig plasma but its significance remains to be understood. Furthermore, knockout models of both RBP and TTR unequivocally demonstrated the importance of this protein-protein complex in retinoid transport. Thus, interactions amongst multiple components of retinol transport play critical roles in vitamin A homeostasis and TTR amyloidosis

Retinol Binding Protein and Its Interaction with Transthyretin

Marcia E Newcomer^* and David E. Ong

…………..———————–

Protein Sci. 2001 Nov; 10(11): 2301–2316.

doi: 10.1110/ps.22901

PMCID: PMC2374051

Role of conserved residues in structure and stability: Tryptophans of human serum retinol-binding protein, a model for the lipocalin superfamily

Lesley H. Greene,^1,3 Evangelia D. Chrysina,² Laurence I. Irons,² Anastassios C. Papageorgiou,^2,4 K. Ravi Acharya,²and Keith Brew^1,

The flexible loop and, in particular, Trp 67 is known to be involved in molecular packing interactions at the interface of the human RBP-TTR (transthyretin) complex (Monaco et al. 1995; Naylor and Newcomer 1999). In the two structures reported for this complex, Trp67 and Trp91 have critical roles in heterodimer stabilization, but a more detailed examination of these particular residues is not possible because of the low resolution (3.1 Å). Thus, when the rRBP67L/91H structure is compared with the structure of RBP in the complex, only gross structural differences can be ideied. The most profound differences between the recombinant apo-RBP and RBP-TTR complex are those around residue 62 and at the C terminus (root mean square, 1.05 Å; Fig. 2 ▶); both regions are implicated in interactions with TTR in the complex. In the rRBP structure, Trp67 is disordered, as are the rest of the residues that form the flexible loop, whereas in the rRBP67L/91H structure, the effect of the sequence substitution at position 67 was not investigated in detail because of the poor electron density in this region.

Trp24 is a component of the first (A) of the strands that form the β-barrel, whereas another highly conserved residue, Arg139, is located at the end of the final H strand. The interactions between these two residues contribute significantly to the formation of the barrel cylinder and closing of its base (Tables 1, 22 ).).

Retinol-binding studies with selected tryptophan mutants indicated that the mutations did not eliminate the ability of the protein to bind all-trans retinol (data not shown). After folding and purification, the mutants were isolated in yields of ∼20 mg/L, except for those with substitutions for Trp24 or Arg139, in which the yields were 4- to 20-fold lower.

The folding behavior of RBP has a specific biological interest because only the holo form of the protein is secreted after biosynthesis in mammalian and other cells; RBP molecules that do not acquire a retinol ligand within the cell are retained in the endoplasmic reticulum (Melhus et al. 1992) and appear not to be fully folded (Kaji and Lodish 1993). Previously determined structures for human and bovine apo-RBP show close similarity to the holo-protein. These apo-proteins were prepared from natural holo-protein after extraction with ethyl ether to remove the bound ligand (Zanotti et al. 1993a). Here we find that recombinant human apo-RBP produced by in vitro folding of material extracted from inclusion bodies has a structure and spectroscopic properties that are closely similar to those of apo and holo forms of natural human holo-RBP (Cowan et al. 1990), holo and apo bovine RBP (Zanotti et al. 1993a,c), and RBP in complexes with different retinoids (Zanotti et al. 1993b). Because our preparations of apo-rRBP have never bound a retinol ligand, we can conclude that although retinol may enhance folding yields, the ligand is not necessary for an irreversible maturation step in folding. Thus, the degradation of apo-RBP in vivo must be linked to some specific structural feature or property of the apo- versus holo-protein.

The results described here are necessary for the design and interpretation of unfolding and folding kinetics of rRBP because they establish conditions in which the folded and unfolded conformers are most populated. They also allow comparisons of the effects of mutations on the stability of the transition states for folding and unfolding with those of the native and unfolded states. The spectroscopic properties of the mutants indicate signals that provide information about the structure formation in different parts of the RBP molecule during folding processes. A major focus of our work is to determine if there is a relationship between folding and sequence conservation in functionally divergent paralogous proteins. This is a concept that is recently receiving attention from experimentalists (Martinez and Serrano 1999; Hamill et al. 2000; Nishimura et al. 2000; Plaxco et al. 2000). rRBP, as an experimental model for the large lipocalin superfamily, represents an ideal system to further our understanding between evolution and folding. Toward this end, kinetic and additional X-ray crystallographic studies are in progress with recombinant RBP and mutants.

Protein Synthesis at the Blood-Brain Barrier THE MAJOR PROTEIN SECRETED BY AMPHIBIAN CHOROID PLEXUS IS A LIPOCALIN*

Marc G. Achen, Paul J. Harms, Tim Thomas, Samantha J. Richardson, Richard E. H. Wettenhall, and Gerhard SchreiberS

From the Russell Grimwade School of Biochemistry, University of Melbourne, Parkuille, Victoria 3052, Australia

THE JOURNAL OF BIOLOGICAL CHEMISTRY Nov 15, 1992; 267(32): 23170-23174

Epithelial cells, located at the barriers between extracellular compartments, synthesize and secrete proteins required in these compartments. Examples of such cells are the hepato- cytes providing plasma proteins (for review see Ref. l), the Sertoli cells in the testes synthesizing and secreting cerulo- plasmin (2) and transferrin (3), and the choroid plexus pro- ducing proteins, many of which have transport functions, for the extracellular environment in the brain (Ref. 4, for review see Ref. 1). The epithelial cells of the mammalian (1, 5, 6) and avian (7, 8) choroid plexus, forming the blood-cerebro- spinal fluid barrier, are highly specialized in the synthesis of one particular protein, namely transthyretin. This transthy- retin is secreted exclusively toward the brain (9) and has been proposed to mediate the transport of thyroxine from the bloodstream to the brain (9, 10). Expression of the transthy- retin gene is initiated in the choroid plexus Anlage very early in life (11, 12). Analysis of the proteins synthesized by in vitro incubated choroid plexus from various species showed abundant trans- thyretin synthesis and secretion by choroid plexus from mam- mals, birds, and reptiles (6). The choroid plexus from an amphibian, the cane toad, also synthesized and secreted one predominant polypeptide product (6). However, the size of this product was larger than that of the transthyretin subunit (6). In the following, we describe isolation, properties, cloning, structural analysis of the protein and cDNA, and tissue specificity of expression for the polypeptide most abundantly synthesized and secreted by amphibian choroid plexus. The obtained data also allow a more precise determination of the stage at which the high transthyretin gene expression first occurred in the evolution of the vertebrate brain.

Among the proteins secreted by choroid plexus of vertebrates, one protein is much more abundant than all others. In mammals, birds, and reptiles this protein is transthyretin, a tetramer of identical 15-kDa sub- units. In this study choroid plexus from frogs, tadpoles, and toads incubated in vitro were found to synthesize and secrete one predominant protein. However, this consisted of one single 20-kDa polypeptide chain. It was expressed throughout amphibian metamorphosis. Part of its amino acid sequence was determined and used for construction of oligonucleotides for polymer- ase chain reaction. The amplified DNA was used to screen a toad choroid plexus cDNA library. Full-length cDNA clones were isolated and sequenced. The derived amino acid sequence for the encoded protein was 183 amino acids long, including a 20-amino acid preseg- ment. The calculated molecular weight of the mature protein was 18,500. Sequence comparison with other proteins showed that the protein belonged to the lipo- calin superfamily. Its expression was highest in cho- roid plexus, much lower in other brain areas, and absent from liver. Since no transthyretin was detected in proteins secreted from amphibian choroid plexus, abundant synthesis and secretion of transthyretin in choroid plexus must have evolved only after the stage of the amphibians.

Comparison of the Structure of the Major Protein Synthesized and Secreted by Cane Toad Choroid Plexus with Those of Other Proteins-A protein database search showed that the major amphibian choroid plexus secreted protein belongs to the superfamily of the lipocalins. Lipocalins are small proteins (160-190 residues), most of which are secreted. They possess a common three-dimensional structure (calyx) with a hydro- phobic pocket (25, 26). The binding of small hydrophobic molecules is also a common feature of lipocalins (27). There was variability in the extent of the similarity in amino acid sequence of the cane toad 20-kDa protein with other lipocalins. The most similar was glutathione-independ ent brain prostaglandin D synthetase from humans (28) and rats (29), with this protein from both species exhibiting 41% amino acid identity and 84% similarity for conservative amino acid substitutions to the cane toad 20-kDa protein
FIG. 4. Tissue specificity of the expression of the gene for the major cane toad choroid plexus protein. Northern analysis of mRNAs of different organs. Total cellular RNA from the cane toad, 20 pg from liver, 5 pg from brain without choroid plexus, and 0.1 pg from choroid plexus, was subjected to Northern analysis as describedu nder “Experimental Procedures” using the cDNA de- scribed in Fig. 3 as probe. Autoradiographic exposure was for 48 h at -70 “C. The positions of 28 and 18 S ribosomalR NAb ands are indicated on the right.

However, in contradistinction to the 20-kDa protein described here and to most other lipocalins which are secreted, prostaglandin D synthetase has only been localized intracel- lularly (30). The amino acid sequence identities with some other lipocalins were: rat a2,,-globulin, 32% (31); y component of human complement C8,28% (32); human a,,-globulin, 27% (33); chicken CH21 protein, 25% (34); mouse major urinary protein, 25% (35); and a protein from frog olfactory neuro- epithelium, 25% (36). The sequences for these other lipocalins are not shown in a figure. No other group or single protein, including transthyretins, showed any significant sequence similarity.

Functional and Phylogenetic Implications-The brain possesses its own extracellular environment of specific composition. The blood-brain barrier and the blood-cerebrospinal fluid barrier separate the extracellular spaces of the brain from those in the rest of the body. The cerebrospinal fluid, filling the ventricles and paracerebral spaces in the brain and communicating by bulk-exchange with the fluid in the inter- stitial space of the brain (37), is produced by the choroid plexus, the site of the blood-cerebrospinal fluid barrier. The choroid plexus has been reported to synthesize a number of transport proteins, such as transthyretin (38, 40, 41), trans- ferrin (4, 39, 42), ceruloplasmin (2), and retinol-binding pro- tein (43). Of the proteins synthesized and secreted by the choroid plexus in mammals, birds, and reptiles, transthyretin is by far the most abundant. It is secreted exclusively toward the brain (9) and has been proposed to mediate the transfer of thyroxine to the brain (9, 10).

The data presented in this paper demonstrate that, also in amphibians, the choroid plexus is highly specialized for synthesis and secretion of a specific protein. However, this protein is not transthyretin but is a member of the lipocalin superfamily. Such a lipocalin could possibly have at ransport function across the blood-brain barrier. The absence of transthyretin secretion by the choroid plexus in amphibians, as shown in this paper, suggests that the high expression and secretion of transthyretin, as seen in mammals, birds, and reptiles must have evolved only after the stage of the amphibians. The function in the amphibian brain of the 20-kDa lipocalin abundantly synthesized and secreted by choroid plexus is yet to be elucidated.

Transthyretin and Lean Body Mass in Stable and Stressed State

http://pharmaceuticalintelligence.com/2013/12/01/transthyretin-and-lean-body-mass-in-stable-and-stressed-state/

A Second Look at the Transthyretin Nutrition Inflammatory Conundrum

http://pharmaceuticalintelligence.com/2012/12/03/a-second-look-at-the-transthyretin-nutrition-inflammatory-conundrum/

TTR – amyloidosis

J Neurol Neurosurg Psychiatry 2015;86:159-167 http://dx.doi.org:/10.1136/jnnp-2014-308107

CNS involvement in V30M transthyretin amyloidosis: clinical, neuropathological and biochemical findings

Luís F Maia1,2,3, Rui Magalhães4, Joel Freitas2, Ricardo Taipa5, Manuel Melo Pires5, Hugo Osório6, Daniel Dias7, Helena Pessegueiro8, Manuel Correia2, Teresa Coelho1,9

Correspondence to – Dr Luís F Maia, Serviço de Neurologia, Hospital de Santo António—CHP, Largo Prof. Abel Salazar, Porto 4099-001, Portugal; luis.lf.maia@gmail.com

Online First 4 August 2014

Objectives Since liver transplant (LT) was introduced to treat patients with familial amyloid polyneuropathy carrying the V30M mutation (ATTR-V30M), ocular and cardiac complications have developed. Long-term central nervous system (CNS) involvement was not investigated. Our goals were to: (1) identify and characterise focal neurological episodes (FNEs) due to CNS dysfunction in ATTR-V30M patients; (2) characterise neuropathological features and temporal profile of CNS transthyretin amyloidosis.

Methods We monitored the presence and type of FNEs in 87 consecutive ATTR-V30M and 35 non-ATTR LT patients. FNEs were investigated with CT scan, EEG and extensive neurovascular workup. MRI studies were not performed because all patients had cardiac pacemakers as part of the LT protocol. We characterised transthyretin amyloid deposition in the brains of seven ATTR-V30M patients, dead 3–13 years after polyneuropathy onset.

Results FNEs occurred in 31% (27/87) of ATTR-V30M and in 5.7% (2/35) of the non-ATTR transplanted patients (OR=7.0, 95% CI 1.5 to 33.5). FNEs occurred on average 14.6 years after disease onset (95% CI 13.3 to 16.0) in ATTR-V30M patients, which is beyond the life expectancy of non-transplanted ATTR-V30M patients (10.9, 95% CI 10.5 to 11.3). ATTR-V30M patients with FNEs had longer disease duration (OR=1.24; 95% CI 1.07 to 1.43), renal dysfunction (OR=4.65; 95% CI 1.20 to 18.05) and were men (OR=3.57; 95% CI 1.02 to 12.30). CNS transthyretin amyloidosis was already present 3 years after polyneuropathy onset and progressed from the meninges and its vessels towards meningocortical vessels and the superficial brain parenchyma, as disease duration increased.

Conclusions Our findings indicate that CNS clinical involvement occurs in ATTR-V30M patients regardless of LT. Longer disease duration after LT can provide the necessary time for transthyretin amyloidosis to progress until it becomes clinically relevant. Highly sensitive imaging methods are needed to identify and monitor brain ATTR. Disease modifying therapies should consider brain TTR as a target.

Transthyretin-type cerebral amyloid angiopathy: a serious complication in post-transplant patients with familial amyloid polyneuropathy

Yoshiki Sekijima1,2

1Department of Medicine (Neurology and Rheumatology), Shinshu University School of Medicine, Matsumoto, Japan
2Institute for Biomedical Sciences, Shinshu University, Matsumoto, Japan

J Neurol Neurosurg Psychiatry 2015;86:124 http://dx.doi.org:/10.1136/jnnp-2014-308576

Correspondence to

Dr Yoshiki Sekijima, Department of Medicine (Neurology and Rheumatology), Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Japan; sekijima@shinshu-u.ac.jp

Online First 11 August 2014

Liver transplantation is a well-established treatment for transthyretin (TTR)-type familial amyloid polyneuropathy (TTR-FAP).1 According to data in the Familial Amyloidotic Polyneuropathy World Transplant Registry (http://www.fapwtr.org/ram_fap.htm), more than 2000 liver transplantations have been performed to date in 19 countries. Transplantation replaces the variant TTR gene with the wild-type gene in the liver, the main source of serum circulating TTR. The serum concentration of variant TTR decreases rapidly, reaching almost zero after the operation. The effects of liver transplantation on neuropathy are evident as its progression is …

Linköping Studies in Science and technology Dissertation No. 1179

Molecular Aspects of Transthyretin Amyloid Disease

Karin Sörgjerd

Biochemistry Department of Physics, Chemistry and Biology
Linköping University, SE- 58183 Linköping, Sweden Linköping 2008
ISBN 978-91-7393-906-5 http://liu.diva-portal.org/smash/get/diva2:1717/FULLTEXT01.pdf

This thesis was made to get a deeper understanding of how chaperones interact with unstable, aggregation prone, misfolded proteins involved in human disease. Over the last two decades, there has been much focus on misfolding diseases within the fields of biochemistry and molecular biotechnology research. It has become obvious that proteins that misfold (as a consequence of a mutation or outer factors), are the cause of many diseases. Molecular chaperones are proteins that have been defined as agents that help other proteins to fold correctly and to prevent aggregation. Their role in the misfolding disease process has been the subject for this thesis.

Transthyretin (TTR) is a protein found in human plasma and in cerebrospinal fluid. It works as a transport protein, transporting thyroxin and holo-retinol binding protein. The structure of TTR consists of four identical subunits connected through hydrogen bonds and hydrophobic interactions. Over 100 point mutations in the TTR gene are associated with amyloidosis often involving peripheral neurodegeneration (familial amyloidotic polyneuropathy (FAP)). Amyloidosis represents a group of diseases leading to extra cellular deposition of fibrillar protein known as amyloid. We used human SH-SY5Y neuroblastoma cells as a model for neurodegeneration. Various conformers of TTR were incubated with the cells for different amounts of time. The experiments showed that early prefibrillar oligomers of TTR induced apoptosis when neuroblastoma cells were exposed to these species whereas mature fibrils were not cytotoxic. We also found increased expression of the molecular chaperone BiP in cells challenged with TTR oligomers.

Point mutations destabilize TTR and result in monomers that are unstable and prone to aggregate. TTR D18G is naturally occurring and the most destabilized TTR mutant found to date. It leads to central nervous system (CNS) amyloidosis. The CNS phenotype is rare for TTR amyloid disease. Most proteins associated with amyloid disease are secreted proteins and secreted proteins must pass the quality control check within the endoplasmic reticulum (ER). BiP is a Hsp70 molecular chaperone situated in the ER. BiP is one of the most important components of the quality control system in the cell. We have used TTR D18G as a model for understanding how an extremely aggregation prone protein is handled by BiP. We have shown that BiP can selectively capture TTR D18G during co-expression in both E. coli and during over expression in human 293T cells and collects the mutant in oligomeric states. We have also shown that degradation of TTR D18G in human 293T cells occurs slower in presence of BiP, that BiP is present in amyloid deposition in human brain and mitigates cytotoxicity of TTR D18G oligomers.

Included papers

Paper I: Detection and characterization of aggregates, prefibrillar amyloidogenic oligomers, and protofibrils using fluorescence spectroscopy., Lindgren M, Sörgjerd K, Hammarström P., Biophys J. 2005 Jun;88(6):4200-12.

Paper II: Prefibrillar Amyloid Aggregates and Cold Shocked Tetrameric Wild Type Transthyretin are Cytotoxic. Sörgjerd K, Klingstedt T, Lindgren M, Kågedal K, Hammarström P. In manuscript.

Paper III: Retention of misfolded mutant transthyretin by the chaperone BiP/GRP78 mitigates amyloidogenesis., Sörgjerd K, Ghafouri B, Jonsson BH, Kelly JW, Blond SY, Hammarström P., J Mol Biol. 2006 Feb 17;356(2):469-82.

Paper IV: BiP can function as a molecular shepherd that alleviates oligomer toxicity and amass amyloid. Sörgjerd K.,Wiseman R.L, Kågedal K., Berg I., Klingstedt T., Budka H, Nilsson K.P.R., Ron D., Hammarström P. In manuscript.

Abbreviations
ANS 8-anilino-1-naphthalene sulfonic acid
Bis-ANS 4-4-bis-1-phenylamino-8- naphthalene sulfonate
CNS central nervous system
CtD C-terminal domain
DCVJ 4-(dicyanovinyl)-julolidine
ER endoplasmic reticulum
FAP familial amyloidotic polyneuropathy
LCP luminescent conjugated polymer
MTT 3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
NBC neuroblastoma cells NtD N-terminal domanin
RBP retinol binding protein
SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis
SSA senile systemic amyloidosis
ThT thioflavin T
T4 thyroxin
TEM transmission electron microscopy
TTR transthyretin
TTR D18G transthyretin with amino acid substitution from aspartic acid to glycine at position 18
UPR unfolded protein response

Table of contents

1 Introduction
3 Proteins
5 Protein production- the background story
6 Protein folding
8 Genetic mutations
9 Protein misfolding
10 Transthyretin (TTR)
13 The TTR D18G mutation
18 Molecular chaperones
21 BiP
22 Structure and mechanism of BiP
23 A role for BiP during translocation
25 The role of molecular chaperones in misfolding diseases
25 The ER, cellular stress and cell death
27 The unfolded protein response (UPR)
28 Apoptosis
31 Caspases
31 Methods
33 Cloning, mutagenesis
33 SDS-PAGE and Western blotting
33 Circular Dichroism
33 Fluorescence spectroscopy
34 Chemical cross-linking
35 Transmission Electron Microscopy (TEM) 3
35 Affinity chromatography and immunoprecipitation
36 Analytical ultracentrifugation
36 Results

37 Paper I_
38 Cross-linking to probe formation of aggregates
38 Size and morphology of aggregates and protofibrils
39 Characterization of TTR conformers using molecular probes
40 Different kinetics for different probes

41 Paper II
42 Early oligomeric species of TTR kill human cells
42 Early oligomeric species of TTR induce ER stress

44 Paper III_
45 BiP selectively binds to destabilized variants of TTR
45 Composition of the BiP- TTR D18G complex
46 The BiP- TTR D18G interaction
46 BiP plays a protective role against the toxic effects of TTR D18G

Paper IV
49 BiP interacts with TTR D18G in the mammalian ER
49 The degradation rate of TTR D18G is slowed down in the presence of BiP
49 The BiP- TTR D18G complex was present in a wide distribution of molecular weights
51 BiP can protect cells from TTR D18G cytotoxicity
52 BiP is found in TTR D18G aggregates in patient tissue

53 Conclusions ________________________________________________________55
55 References ________________________________________________________ 57

This thesis summarizes what I have been working on for the past five years and what conclusions I have made from my findings. My main characters are two proteins called BiP; which is a molecular chaperone believed to play a protective role in cells, and transthyretin (TTR); which is associated with human misfolding disease. It has been known for a long time that TTR misfolding disease starts with TTR denaturation and leads to aggregation and fibrillation of TTR, which accumulates in tissues and organs in patients suffering from the disease. Still, there are no cures for most of these kinds of diseases and the pathogenesis and mechanisms are not fully understood.

The aims with my studies have been to elucidate what role BiP plays in TTR misfolding diseases. I have specifically studied a mutant of TTR called TTR D18G, since that mutant is the most destabilized and most unusual form of TTR. I have also aimed to follow the mechanisms for TTR misfolding and to study the consequences in human cells when exposed to misfolded variants of TTR.

I have included four papers in this thesis. In the first, the aggregation process of transthyretin is described, and the different states in the process are characterized. In the second paper, the effect of different conformational states of aggregated TTR variants on human cells has been studied. In the third paper, the interactions of TTR D18G with BiP are characterized and hypothesizes about what role BiP plays in TTR misfolding diseases have been made. In the fourth paper, the BiP TTR D18G interaction is studied from a mammalian point of view and the effects of BiP and TTR D18G on human cells are elucidated.

Proteins are responsible for most of the reactions occurring in the human body such as transport of nutrients and oxygen, defense against microorganisms, control of gene expression, transmission of signals etc. In all organisms and in each cell, they exist and they work. In the human body, over 30 000 different proteins (and around 30 000 protein coding genes) [1] with almost as many different functions are present, however, most of them still have unknown functions. The building blocks for proteins are called amino acids. There are 20 amino acids used for protein synthesis and 12 of them can be produced by human cells whereas eight need to be supplied with the diet. Thus, a versatile diet is important for the body to work properly with all its > 30 000 different proteins to be properly synthesized. An average protein consists of hundreds of amino acids, linked together in different sequences by the peptide bond, and it is the order of the amino acids that dictate the final shape of the protein. The amino acids, the building blocks in a polypeptide, have different properties; they can be polar, non-polar or charged and the hydrophobic ones are usually buried in the interior of the folded protein. The structures of the proteins, i.e., their conformations, differ due to different types of secondary structures, called α-helices or β-sheet and how these structural elements are arranged. It is the conformation that dictates the protein function. Every single amino acid in the folded protein can contribute to and play a role for protein function. A substitution of one amino acid to another might in some cases lead to re-arrangements of the whole protein structure, and thereby induce a new behavior of the protein (often leading to destabilization and degradation). In other cases, an amino acid substitution does not influence the conformation at all. 6

Figure 1) The primary structure of a protein showing amino acids as a string of pearls. The side chains of the amino acids, denoted with R can be polar, non-polar or charged. When the amino acids are connected to form a poly peptide chain, the COO- group of one amino acid reacts with the NH3 + of another, and a peptide bond is formed with release of a water molecule.

Protein molecules can interact with each other, and protein-protein interactions are necessary for many biological functions. Interactions can be prolonged, when a complex is formed, or transient, i.e. when signals are transferred within or between cells. Interactions can also be non-preferred, such as protein aggregation.

Protein production- the background story All proteins begin as linear sequences of amino acids linked together as a string of pearls (Figure 1). The information about the amino acid sequence of the protein, leading to their different conformations is encoded in the deoxyribonucleic acid (DNA), in the specific genes for the proteins of interest, called the genetic code. When synthesis of a polypeptide begins, the DNA information is transferred to a piece of messenger RNA (mRNA). Formation of RNA from DNA is a process called transcription and occurs with help from an enzyme called RNA polymerase. DNA was identified in 1944 and its double helical conformation was revealed in 1953 [2]. Since DNA is situated in the cell nucleus whereas the protein synthesis occurs in the cytoplasm, an intermediate needs to be involved in the transcription. In the fifties, Primary protein structure Amino acid C COONH3 + R H Primary protein structure Amino acid C COONH3 + R H C COONH3 + R H C COONH3 + R H 7 there were discussions about that intermediate and it was proposed that another nucleic acid, single stranded ribonucleic acid (RNA), would be the intermediate responsible for transferring information from the cell nucleus to the cytoplasm. Later, it was formulated that the genetic information in the DNA is transcribed to RNA and then translated from RNA into a protein. The idea was developed over time and in the sixties, it was proposed that a gene is transcribed into a specific RNA species, called mRNA and that a short-lived, non-ribosomal RNA directs the synthesis of proteins. In 1965, Francois Jacob, Jacques Monod and André Lwoff received the Nobel Prize for their research about mRNA. In the seventies, it became known that mRNA could be spliced after transcription, resulting in that the primary transcript can generate different mRNAs and different proteins. In the 1980s, it was found that small RNA molecules could bind to a complementary sequence in mRNA and inhibit translation [3]. mRNA is single stranded and its sequence is called sence because it results in a protein. The complementary sequence is called antisense. When sence mRNA base pair with anti sence mRNA, translation is blocked. The mechanism has not been fully understood until Craig C. Mello coined the term RNA interference in his work from 1997, published in Cell [4]. In 2006, Andrew Z. Fire and Craig C. Mello received the Nobel Prize for uncovering the mechanism of RNA interference. They discovered that genes could be silenced, i.e. gene activity could be turned off, by double-stranded RNA [5].

Protein production is intitiated by transcription from DNA to mRNA. The transcription starts with binding of RNA polymerase to the DNA which, together with different cofactors, unwinds the DNA. The unwinding helps the RNA polymerase to bind the single stranded DNA template. But there is also need for different transcription factors to make the interaction possible. Once RNA polymerase has bound, the elongation starts, which means that an RNA copy of the DNA template is made as RNA polymerase is traversing along the template strand. The copy (mRNA) is transported to the cytoplasm once it is finished. In the cytoplasm, the sequence is translated into amino acids with help from the ribosome. Ribosomes consist of different subunits that surrounds mRNA and use its sequence as a template for amino acid synthesis where the ribosome is constantly fed with amino acids from transport RNA (tRNA) molecules, each specific for one amino acid (Figure 2). When the amino acid sequence is finished, it is released from the ribosome and folds into a three dimensional structure and is transported to its predestined destination.

Figure 2) From DNA to protein. In the nucleus, mRNA is made, which is a copy of the DNA template containing all the genetic information. Protein synthesis is performed in the cytoplasm by the ribosomes.

Translation
Cytoplasm
Mature protein
Folding
Nucleus
DNA
Transcription to mRNA
Mature mRNA
Transport to cytoplasm
Ribosome
Amino acids

Protein folding The normal protein function does not appear until the polypeptide has developed its final three dimensional conformation, i.e. the protein has been folded. Protein folding involves interactions of the amino acids within the polypeptide to form different kinds of secondary structures; α-helices and β-sheet (Figure 3). The secondary structure elements can arrange into a tertiary structure mediated by side chain interactions. The folding process occurs right after the synthesis of the polypeptide and is normally a relatively fast process. It can even occur on a milli or micro second time scale.

American biochemist Christian Anfinsen was the first to show that the order of amino acids in the primary structure is what dictates the final protein conformation [6]. He also found that if a folded protein was denatured, i.e. the non-covalent native Hbonds, the charge-charge interactions and the hydrophobic interactions were broken and the protein became unfolded, it could again find its folded, native conformation, i.e. refold, under permissive conditions. Anfinsen was awarded the Nobel Prize in 1972. However, for many proteins, the process is not as simple as it was initially described by Anfinsen. Some proteins can fold, unfold and refold spontaneously in vitro (usually the smallest ones) in a one-step process [7], some fold through one or many intermediates and most need assistance to adopt their ultimate conformation. The cells are provided with molecules whose major function is to help proteins to fold correctly, these molecules are also proteins, and are called molecular chaperones.

Genetic mutations …

Figure 3. Illustration of secondary and tertiary structures of a protein. a) α-helix b) β-sheet, within a β-hairpin c) tertiary structure of a folded protein consisting of both α-helices and β-sheets and the spacial orientation of the secondary structure elements are dictated by side chain interactions (www.pdb.org).

Protein misfolding When errors occur in the protein folding machinery, it can result in protein misfolding and misfolding disease. Misfolding diseases are often associated with amyloidosis [8]. The reasons for protein misfolding could be mutations in the genes that code for the proteins that are to be misfolded, or outer factors like stress. For some cases, it is unknown why proteins start to misfold. The consequences could be harmful to the surrounding cells and to the organism in general. Protein misfolding diseases strike many people during their lifetime, and it seems like the phenomenon has become more prevalent during the past years. One early disease of this kind was described in 1906, when the German neurologist Alois Alzheimer found a form of amyloidosis that affects the brain. The disease was later named Alzheimer´s disease (AD). Most AD patients get the disease sporadically, i.e., it is usually not inherited. The symptoms for disease include memory disturbance and loss of other intellectual abilities, the symptoms are also called dementia. To date, more than 20 million people are believed to suffer from dementia [9]. In the fifties, the first form of transmissible human amyloid disesase, named kuru, was found among people practicing cannibalism in Papua New Guinea. Shortly afterwards, a disease with similar pathology was discovered in Europe and United States; transmissible spongiform encephalopathy (TSE), among individuals that had been treated with growth hormones extracted from human cadavers. The TSEs include Bovine Spongiform Encephalopathy or Mad Cow Disease (BSE), Creutzfeldt Jakob Disease (vCJD), Gerstmann-Straussler-Scheinker (GSS) disease and fatal familial insomnia (FFI). Certain misfolded proteins, called prions, are implicated in these diseases. The word “prion” stands for “proteinaceous infectious particle”, referring to its pathogenic variants. Stanley B. Pruisiner was the first to identify the molecular mechanisms of prions [10-13] and he was awarded the Nobel Prize in Medicine in 1997 for discovering a new infectious agent- a protein.

All misfolding diseases, whether they are sporadic, inherited or transmissible, are associated with deposition of proteins in different organs, depending on the disease (Table 1). The proteins involved are normally soluble but have become insoluble and aggregated and have developed fibril-like structures. Remarkably, the final fibrils are strikingly similar regardless of the precursor protein, consisting of cross-β-sheet structure with twisted morphology. The mechanisms for developing fibrils are also similar; starting with a conformational change in the protein which becomes a building block for aggregates or clusters that develop long fibril like structures over time and accumulate in tissues or organs in the body with consequences like impaired organ function and cell death [14].

Alzheimer´s, Parkinson´s and Creutzfeldt Jakob disease are examples of notorious misfolding diseases, but there are also less known diseases like Familiar amyloidotic polyneuropathy (FAP) with related pathology (Table 1). The precursor protein for FAP is transthyretin (TTR).

Table 1. A selection of diseases coupled to protein misfolding and amyloidosis and their precursor proteins.

Clinical syndrome Precursor protein

Alzheimer´s disease Aβ-protein
Primary Systemic Amyloidosis   Ig Light Chain
Secondary Systemic Amyloidosis Serum Amyloid A
Senile Systemic Amyloidosis (SSA) Transthyretin
Familial Amyloid Polyneuropathy (FAP) Transthyretin
Finnish Hereditary Systemic Amyloidosis Gelsolin
Type II Diabetes   Islet Amyloid Peptide
Non-Neuropathic Systemic Amyloidosis Lysozyme
Cerebral Amyloid Angiopathy Cystatin C
Atrial Amyloidosis Atrial Natriuretic Factor
Familial Amyloidosis Type III   Apolipoprotein A-1
Hereditary Renal Amyloidosis Fibrinogen

Transthyretin (TTR) Transthyretin (TTR) was discovered in 1942, it became known as prealbumin and was by then detected in the cerebrospinal fluid. In the fifties, prealbumin was identified as a thyroxine (T4) binding protein by Sidney Harold Ingbar, which was published in 1958 in Endocrinology [15]. Kanai et al [16] characterized prealbumin as a retinol binding protein and published their finding in a paper in Journal of Molecular Biology, and prealbumin became Retinol Binding Prealbumin (RBPA). The structure of RBPA was described by Blake and colleagues in 1978 [17] and in 1981, the name transthyretin was accepted [18].

TTR has a molecular weight of 55 kDa and its structure (studied by X-ray crystallography) is a homotetramer with four identical, monomeric subunits, composed of 127 amino acids [19]. Each subunit has a molecular weight of 14 kDa and contains eight β-strands denoted A-H and a helix between strands E and F. The β- strands in each monomer form a β-barrel consisting of two antiparallel four stranded β-sheets containing the DAGH and CBEF strands. Association of two monomers to a dimer forms a β-sandwich stabilized by hydrogen bonds between the H-H (Figure 4) and F-F strands. Association of another dimeric β-sandwich produces the tetrameric conformation. The dimers are connected through hydrophobic interactions between the A-B loop of one monomer and the H-strand of the opposite dimer.

Figure 4. The dimeric form of TTR. The dimer is held together and stabilized by hydrogen bonds between strands H and H and F and F (not shown) in the TTR structure (www.pdb.org, pdb code 1DVQ).

Tetrameric TTR contains two identical T4- binding sites (Figure 5) located in a channel at the center of the molecule [20]. If one of the binding sites is occupied with T4, it becomes harder for the second T4-molecule to bind because of an allosteric effect (negative cooperativity) that takes place in the molecule upon binding of T4. T4 is a thyroid hormone and it plays a role in the metabolism, but is also important for neuronal development [21]. Free T4 is metabolically active. Plasma TTR functions as a transporter of T4 in the blood and transports 15-20 % of serum-T4 and around 80% of CNS-T4 [22]. Other T4 binding proteins and transporters include albumin and thyroxine-binding globulin. TTR is also involved in the transportation of retinol (vitamin A) in complex with retinol binding protein (RBP). The TTR–RBP–retinol complex is formed in the endoplasmic reticulum (ER) of hepatocytes. In TTR, there are four binding sites for RBP, however, only two molecules can bind at the same time because of steric hindrance. In the plasma, most of the TTR does not bind RBP [23, 24]. The source for plasma TTR is the liver. In human plasma, TTR is present at a concentration of 0.25 g/l [23]. The major sites for TTR production are in the liver, the choroid plexus of the brain and the retinal pigment epithelium in the eye.

There are over 100 known mutations in the TTR gene that are associated with amyloid deposition, with varying phenotype depending on the mutation [25] (some are shown in Figure 8). The most common neurodegenerative disease associated with TTR mutations is familial amyloidotic polyneuropathy (FAP) [23, 26-28], which first was described in 1952 by Corino Andrade [29] in Portugal. For more than 20 years, Andrade and colleagues had observed 74 cases from different families suffering from a progressive and mortal, by then unknown disease. ….

Figure 5. Tetrameric TTR (from rat). The binding sites for T4 are pointed out with the arrows (www.pdb.org, pdb code 1IE4).

Although TTR amyloid deposit disease is associated with TTR variants, senile systemic amyloidosis (SSA) is a disease associated with TTR wild type and affects up to 25% of people over the age of 80 and is characterized by amyloid deposits in the heart [36, 37]. The primary structure of TTR is therefore not the only explanation for development of TTR amyloidosis [38]. SSA is usually benign and without symptoms, and mostly men are severely affected [39]. Analysis of the amyloid fibril deposits in SSA patients have revealed that the amyloids contain fragments of TTR and those fragments dominate over full length TTR [40, 41]. The fragmentation has occurred at certain positions (predominantly at positions 46, 49 and 52) in the molecule which makes it tempting to believe that the cleavage of TTR is the cause of disease since the cleavage might expose sequences that are prone to aggregate. However, the cleavage mechanism is not fully understood.

Formation of TTR amyloids starts with dissociation of the tetramer into monomers, that in turn partly unfold and develop aggregates and amyloid fibrils over time [42- 44] (Figure 6). While the structure and properties of amyloid fibrils have been in the focus for diagnosing and understanding the pathogenesis for amyloid disease, there is now increasing evidence that the intermediate states in the amyloid formation pathway, are the most toxic species [45-48].

Another role for TTR, which recently has been published, is the ability to bind the Aβ-protein, which plays the major role in the pathology of Alzheimer´s disease. TTR can act in a chaperone like manner and thereby prevent formation of Aβ amyloid aggregates and thereby possibly halt progression of Alzheimer disease [49].

Figure 6. The amyloid formation process of TTR. The native tetrameric structure of TTR is destabilized and form a rearranged structure that dissociates into monomers. The monomeric species are unstable and aggregation prone and can mature into long, inert fibril structures or unfold. Unfolded TTR can also rearrange to a molten globule like structure (A-state) that has very similar properties as the monomeric amyloidogenic intermediates.

The TTR D18G mutation TTR D18G is a naturally occurring mutation in the TTR gene. The mutation was originally discovered in a Hungarian family, where four definite and three probable affected members were identified. It leads to amyloidosis in the central nervous system (CNS) with disease onset at an average age of 44. The affected family members had extensive amyloid deposition in the leptomeningeal vessels and in the subarachnoid membrane. ….

TTR D18G was identified as the most destabilized TTR mutant found to date. Recently, it has been demonstrated that a combination of thermodynamic and kinetic stability of TTR mutants is strongly correlated to disease progression. …
Analysis of serum and cerebrospinal fluid (CSF) of a heterozygote D18G patient revealed that only TTR wt could be detected [53], which is an indication of degradation or accumulation of D18G within the cell or rapid degradation post secretion. This could explain why patients do not develop disease until 44 years of age.

Figure 7. Position of D18 in the TTR monomer and residues believed to influence the tetramer stability. A) Structure of a TTR monomer. Residues within a radius of B) 5Å C) 7Å

TTR D18G is monomeric (Figure 7) and unable to form tetramers under physiological conditions. The mutant is aggregation prone and aggregates 1000-fold faster that TTR wt under physiological conditions [53]. The location of the D18G mutation is at the end of the A-strand of TTR. The neighboring residues (Figure 7) are known to stabilize the tetrameric structure. For example, the A25T mutation results in destabilization of the TTR tetramer and cause CNS amyloidosis [54], the V20I mutation leads to destabilization of the TTR tetramer and cause cardiac amyloidosis [55], the F87M/L110M mutations engineer the TTR molecule to be monomeric [56] and the L111M mutation leads to cardiac amyloidosis [57].

The region with the D18 mutation obviously has high impact on TTR tetramerization and stability. TTR D18G cannot efficiently form hybrid tetramers with TTR wt. T4 binding was found to facilitate tetramerization of D18G in the choroid plexus, where concentrations of T4 is high, but not in the CSF where the concentrations are lower. That could explain the TTR D18G prevalence to accumulate in the CNS. Expression of TTR D18G in E.coli leads to the formation of inclusion bodies [53].

Figure 8. Primary sequence of TTR with naturally found mutations marked below the wild type sequence (Hou et al 2007).

Molecular chaperones The term “molecular chaperone” was coined by Ron Laskey in 1978. Laskey observed that a nuclear protein, called nucleoplasmin, could solve a misassembly problem during the assembly of histone proteins, termed nucleosomes, in amphibian eggs. Nucleosomes bind DNA by electrostatic interactions. If the interactions are broken, e.g. by changes in the physiological conditions, the nucleoplasmin is not able to rebind the DNA, even if the physiological conditions are readapted, which leads to aggregation of the protein. This can be prevented by the presence of nucleoplasmin, is able to bind the nucleosomes and protect them [58].

The molecular chaperones are today known as folding helpers and it is believed that they are essential for cell survival and for life processes in general. They are present in the mitochondria, the Golgi, the ER and in the cytoplasm of all cells. The chaperones can correct mistakes in the folding machinery, unfold and send misfolded species to be degraded, or hold on to proteins that cannot be folded in a productive way, thereby preventing escapes of misfolded proteins that could cause damage. The chaperones direct their substrates into productive folding, transport or degradation pathways, but they do not become parts of the final structures of the proteins they interact with [59]. The majority of newly synthesized proteins need assistance to adopt their final conformation. Molecular chaperones stabilize non-native proteins, unfold incorrectly folded proteins and send abnormal proteins for degradation. They do not interact with native proteins, only the unfolded or partially unfolded ones. They are interacting with the proteins for a finite time and thereafter release their substrates, often mediated through ATP hydrolysis. Some chaperones interact with a wide variety of polypeptide chains whereas others are very restrictive and only bind to specific targets.

The heat shock response was first discovered in 1962 in Drosophila flies and the heat shock proteins (HSP) were identified as a set of proteins whose expression was induced when the cells were exposed to elevated temperature [60]. Shortly after they had been discovered, it became evident that their synthesis was not only due to elevated temperatures in cells but also to other forms of outer stresses, like radiation (UV or gamma-irradiation), oxidative stress, exposure to heavy metals, amino acid analogues etc. Protein misfolding and aggregation can lead to acute or chronic stress and activation of inappropriate signaling pathways. HSPs have strong cytoprotective effects [61] and are thought to restore the cellular homeostasis when it is disturbed.

Mammalian HSPs are classified according to their molecular weights (in kilodaltons) and are divided into two main groups, the high molecular weight HSPs and the small molecular weight HSPs. The first group includes three major families: Hsp60, Hsp70 and Hsp90. The first group (the heavy HSPs) consists of ATP dependent chaperones whereas the second group (the light HSPs) consists of ATP independent chaperones.

The Hsp70 family is the most studied HSP family, containing proteins from 66 to 78 kDa. Some of the Hsp70 proteins are localized in the cytosol (Hsp70 and Hsp72), one is found in the mitochondrion (mtHsp70) and one in the ER (BiP).

BiP BiP was first defined as glucose regulated protein with a molecular weight of 78 kDa (GRP78) or immunoglobulin heavy chain binding protein. Its function as a molecular chaperone was established by Munro et al [62] in 1986, who demonstrated that BiP is an ATP dependent member of the Hsp70 family, located in the ER lumen. BiP interacts with newly synthesized proteins and chaperones them during transport through the cell and is believed to be one of the most important components in facilitating folding in the ER. BiP has a molecular mass of 74 kDa and its 3D structure is not known but it has been defined by X-ray crystallography for DnaK (an E.coli Hsp70 and BiP homologue).

Structure and mechanism of BiP All the Hsp70 family members have the same structural organization with a 44 kDa N-terminal ATPase domain (NtD), a 18 kDa C-terminal substate binding domain (CtD) (Figure 9) and a third domain, belonging to the C-terminal domain whose function is unknown. The NtD and the CtD communicate allosterically with each other. If the NtD is occupied by an ATP molecule, the affinity for the substrate in the CtD is low but if the NtD is occupied by an ADP molecule, the affinity for substrate is high. If the CtD binding site is occupied by a substrate, the rate of ATP hydrolysis in the NtD increases [63, 64]. Thus, an unfolded polypeptide captured by BiP, can undergo cycles of binding and release, cycles that will proceed until BiP binding motifs no longer are present in the released and folded polypeptide. BiP recognizes a wide variety of nascent polypeptides with no obvious sequence similarity. However, experiments that have been done in order to find sequences that BiP preferentially binds to, have shown that the binding motifs consist of a high proportion of hydrophobic residues, normally located in the interior of a folded protein. It has also been shown that those motifs preferably consist of seven amino acids [63].

Figure 9. The NtD and the CtD of the bacterial BiP homologue DnaK. The NtD binds ATP/ADP (ADP is marked with black, left figure) whereas the CtD is substrate binding (substrate (NRLLLTG) is marked with black, right figure). The domains communicate allosterically with each other. When ATP is bound to the NtD, the CtD releases its substrate (www.pdb.org, pdb code 1S3X for left figure and 1Q5L for right figure).

…..

BiP can self associate into different oligomeric species and it is the CtD that is responsible for oligomerization. The more oligomeric BiP is, the less active is it [65]. BiP can also be post-translationally modified by phosphorylation and by ADP ribosylation. These modifications are believed to play a role in the synthesis and the polypeptide binding of BiP. Accumulation of unfolded proteins in the ER leads to an decreased amount of modified BiP whereas unmodified, monomeric BiP increases [64].

Many chaperones need co-chaperones to be effective. Hsp70 chaperones often need Jdomain containing Hsp40 proteins. The function for the Hsp40 proteins is to stimulate the ATPase activity which is crucial for Hsp70 chaperone activity. BiP can interact with different J-domain proteins [72] which are necessary for the chaperone function. ….

A role for BiP during translocation ….

The role of molecular chaperones in misfolding diseases A current opinion is that the chaperones play important roles in the protein misfolding diseases since they are parts of the control system in the cell [76, 77]. All proteins associated with the classical amyloid diseases are secreted proteins and will therefore pass the quality control checks within the ER, where they interact with a number of proteins facilitating protein folding. In some cases, misfolded proteins are accumulated in the ER [5]. This accumulation causes “ER-stress”, a condition that normal cells respond to by increasing the transcription of genes encoding ERchaperones, such as BiP, to facilitate protein folding or by suppressing the mRNA translation to synthesize proteins. These mechanisms are called “the unfolded protein response” (UPR). Once proteins are aggregated into extracellular amyloid deposits they are quite resistant to degradation.

The ER, cellular stress and cell death The ER is a membrane bound cellular organelle, consisting of tubules, vesicles and cisternae. The environment is oxidizing, which facilitates formation of disulphide bonds in maturing proteins and thereby stabilizing their structures. ER is involved in protein translation, folding, post translational modifications and quality control of proteins that are to be secreted from the cell. The majority of secreted or plasma membrane proteins enter the ER and fold within it. The vesicles of the ER are responsible for transport of proteins to be used in the cell membrane or to be secreted from the cell. Molecular chaperones and folding enzymes assist nascent proteins to fold inside the ER and correctly folded proteins are transported to the Golgi apparatus. Proteins that are not able to fold or that are misfolded, are accumulated in the ER since they cannot be exported. There are different mechanisms responding to accumulation of unfolded or misfolded proteins inside the ER. One of the mechanisms is termed ER-associated degradation (ERAD), which recognizes the misfolded proteins and retrotranslocates them to the cytoplasm and send them for degradation by the ubiquitin-proteasome degradation machinery [78]. Another mechanism that responds to accumulation of unfolded proteins in the ER is the unfolded protein response (UPR). Accumulation of unfolded proteins in the ER may also lead to cell death (apoptosis), if the condition is prolonged and cannot be solved (Figure 10). ER chaperones and ER components play a crucial role for recognition of unfolded proteins and are continuously expressed in the ER. [79].

Figure 10. The ER functions. Proteins entering the ER are facing different destinies. The correctly folded proteins are sent for export, whereas proteins that are not able to fold are sent for degradation. Accumulation of incorrectly folded proteins leads to ER stress, which in turn can result in apoptosis if the condition is prolonged. Most ER processes involve several chaperone systems as indicated in the figure.

The unfolded protein response (UPR) ER has a certain loading capacity, which varies between different cell types and during a cell’s life. When unfolded proteins are accumulated in the ER, the cell becomes stressed and the folding machinery gets perturbed. Unfolded proteins have hydrophobic residues exposed, which normally are buried in the interior of the folded protein. These hydrophobic parts tend to form (protein) aggregates that are toxic to cells. ER stress can also occur as a result of starvation, virus infections or heat, and other outer factors that influence cells negatively, and the condition is either transient or permanent. The cells respond to the stress by activating a pathway of signals leading to transcription of more chaperones, e.g BiP. Simultaneously, the translation of new proteins and the loading of proteins into ER are reduced, and further accumulation of unfolded proteins is decreased. …

ER stress leads mainly to three sets of responses: first, the amount of unfolded proteins that enters the ER is reduced (lowered protein synthesis and translocation into the ER); second, the ER folding capacity is increased (transcriptional activation of UPR target genes) and third, if the homeostasis has not been re-established, cell death (the cells commit suicide (apoptosis) to protect the organism). ER stress leads to activation of different signaling pathways, mediated by trans-membrane proteins, so called stress transducers, which sense the ER overload and transmit a signal to the cytosol where the transcription and translation of proteins take place. Three pathways have been identified (Figure 11), mediated by inositol-requiring protein-1 (IRE1), activating transcription factor-6 (ATF6) or protein kinase RNA (PKR)-like ER kinase (PERK) [82]. ….

Figure 11. The unfolded protein response with a central role for BiP.

Apoptosis Sometimes, cells have to die. They can do it in different ways and for different reasons. One reason for cell death is tissue damage, which results in a process called necrosis. During necrosis, damaged cells swell and burst and release their contents to the surrounding area, which in turn can damage the neighbouring cells and give rise to an inflammation. ….

Caspases Caspases is a family of calcium dependent cysteine proteases and they are able to cleave their substrates after aspartate residues. Robert Horwitz and colleagues identified a gene (in C.elegans) called Ced-3, which coded for a protein with similar properties to the, by then, only known caspase (caspase 1) and what they found was required for cell death [88]. After that discovery, other caspases in different organisms were soon identified and their roles were surveyed [89]. Caspases contain three domains; an N-terminal domain (which vary in size between different caspases), a large domain containing the active site and a small C-terminal domain. ….

Figure 12. The inactive procaspase and the active caspase.

Methods …

Results The findings will be presented in four papers, which are summarized below. The first paper is a study about TTR and its misfolding and fibrillation pathway. The oligomeric intermediates in the process were studied and characterized. In paper II, the different states in the TTR oligomerization pathway were captured, and added to neuroblastoma cells to elucidate which species were toxic to cells when applied from the outside. We could see that early oligomers were toxic to neuroblastoma cells. This resulted in apoptosis and release of BiP into the cytoplasm. In the third paper, the misfolding of TTR is studied from a disease point of view and the role of BiP in misfolding diseases is discussed. The most unstable TTR variant found to date, TTR D18G, was used as a model for the study and we found that BiP strongly interacted with this mutant, which was not the case for TTR wt or other mutants. Paper IV is a study of the role of BiP for TTR D18G misfolding within a eukaryotic cell. Most of the work done previously was in vitro studies and measurements were performed on purified proteins, made in E.coli cells. However, the fourth study was done in an in vivo context, to get a cell biologigal aspect of the work. Human kidney cells were used to express proteins and the interactions inside the cells were studied. We could see that the in vivo results in human cells correlated well to what we had seen earlier from E.coli expressed complexes.

Paper I The purpose with the study in the first paper was to characterize and understand the aggregation process of TTR. We used different techniques to detect structural changes in the aggregation process. An in vitro protocol for creating TTR oligomers was used. Oligomers were studied by using fluorescence spectroscopy, circular dichroism, chemical cross-linking and transmission electron microscopy. ….

Figure 16. Different probes were used to follow the TTR misfolding reaction. Aliquots of the aggregation reaction of TTR were withdrawn and assayed at 2μM probe + 2 μM TTR. Symbols: ANS (circles), Bis-ANS (inverted triangles), DCVJ (triangles) and ThT (squares). The fluorescence intensity of the different probes in the presence of the unfolded monomer state and the burst amplitude from the fit is indicated with horizontal lines labeled with the letter U and “burst” in colors corresponding to the probe.

Paper II In paper I, we characterized different states in the TTR aggregation pathway. In paper II, we harvested the different states in the aggregation pathway (during fibrillation) and challenged with neuroblastoma cells with these species. We exposed the cells to both early, oligomeric TTR species, long and mature fibrils of TTR and native TTR wt. We also wanted to investigate if BiP was upregulated in cells as a marker for UPR activation when exposed to TTR oligomers and used immunostaining for BiP to detect BiP localization in cells.

Early oligomeric species of TTR kill human cells Phase contrast images of human SH-SY5Y neuroblastoma cells exposed to early oligomers or mature fibrils of TTR in 48 hours, revealed that cells that had been exposed to oligomers were dying, whereas cells exposed to mature fibrils were still alive and healthy (Figure 17). …

Figure 17. Phase contrast pictures of cell viability after exposure to TTR. Cells exposed to early TTR oligomers, which had aggregated for 5 min, demonstrated apoptotic morphology, such as decreased cell size and more sparse population compared to non exposed cells (C) or cells exposed to native TTR wt, 22˚C (wt). Cells exposed to TTR that had aggregated for longer than 1 h were more dense. Cells exposed to TTR that had aggregated for more than one day (24 h and 1 week (1 w)) showed similar morphology as control cells (C).

Figure 18. BiP is upregulated in cells stressed with TTR oligomers and cold, native TTR wt. A) Confocal microscopy images of cells immunostained for BiP (green). Top micrograph: cells exposed to vehicle (C). Middle micrograph: cells exposed to early TTR oligomers (5 min). Bottom micrograph: cells exposed to cold native TTR wt (wt 4 °C). B) Western blot analysis of BiP expression in cells following exposure to early oligomers of TTR or cold native TTR wt (4 °C). GAPDH was used as a protein loading control to quantify the level of BiP expression.

Paper III To understand how the chaperone BiP could interact with an unstable, aggregation prone, protein mutant like TTRD18G, plasmids containing genes for His6-BiP or FT2- TTRD18G were introduced into E.coli cells grown in LB media. The proteins were thereafter expressed with IPTG, the cells were harvested and the protein containing lysate was purified on a Ni-NTA-affinity chromatography column for capturing His6- BiP. As controls, BiP was also expressed with FT2-TTRA25T, FT2-TTRL55P or FT2- TTRwt and were treated in a similar way. The protein containing cell lysate was separated on the column and fractions containing column flow through, wash buffer, and protein eluate were analyzed by SDS-PAGE.

BiP selectively binds to destabilized variants of TTR….

Figure 19. Comparison of different TTR mutants in their ability to bind to BiP. TTR wt was compared with the mutants TTR D18G, TTR A25T and TTR L55P. FT= Flow Through, w1= wash 1, w2= wash 2, el= eluate, MW= Molecular Weight marker. ….

Figure 20. The binding site for BiP in the TTR molecule. The F-strand in the TTR molecule was found to be its binding site for BiP. ….

BiP plays a protective role against the toxic effects of TTR D18G ….

Figure 21. BiP protects from TTR D18G cytotoxicity by keeping it in a soluble form. A) Micrographs of TTR D18G complex before (upper left) and after (upper right) addition of ATP. ATP releases substrates from BiP, and release of TTR D18G results in aggregation. B) ThT fluoresecence of BiP/TTR D18G (filled bars) and BiP (open bars) before and after addition of ATP or addition of the competitive peptide 88-103 TTR. Without incubation (w/o inc), without ATP, 37˚C incubation for 18 h (37˚C no add), with ATP, 37˚C, 18h (37˚C ATP), with 88-103 TTR peptide, 37˚C, 18h (37˚C 88-103).

Paper IV In this paper, the idea was to obtain a cell biological aspect of BiP/TTR D18G binding and to investigate if the E.coli derived complexes could be confirmed in a human cellular system. We wanted to study complex formation between BiP and TTR D18G in vivo and used human 293T kidney cells to overexpress the proteins. We also wanted to investigate if BiP would influence the degradation rate of TTR D18G. We could clearly see that the complex was formed in vivo in human cells, which confirmed our previous results. It is also known from before that BiP has the ability to oligomerize. We found that BiP did not prevent aggregation of TTR D18G, but rather oligomerized with it, both in soluble and insoluble aggregates. Surprisingly, aggregates seemed to accumulate inside the ER. We also found that the degradation of TTR D18G was altered in presence of a high amount of BiP.

BiP interacts with TTR D18G in the mammalian ER ….

The degradation rate of TTR D18G is slowed down in the presence of BiP ….

Figure 22. Selective binding of mutant TTR. Cells expressing Flag-BiP and/or TTRwt or TTR D18G were lysed with triton. The triton supernatants (containing the soluble protein fractions) and the triton pellets (containing the insoluble protein fractions) were collected. Immunoprecipitation with anti-TTR antibody shows that Flag-BiP is pulled down with TTR D18G and to a small extent with TTR wt. The amount of soluble TTR D18G seemed to increase when BiP was overexpressed.

Figure 23. The TTR D18G degradation rate is slowed down in presence of BiP. Pulse chase analysis results showed that the degradation of TTR D18G alone (circles) occurred faster than when BiP was overexpressed in the cells (squares).

The BiP- TTR D18G complex was present in a wide distribution of molecular weights ….

Figure 24. Distribution of oligomers in cells expressing TTR D18G alone or TTR D18G and BiP.

BiP can protect cells from TTR D18G cytotoxicity ….

Figure 25. BiP can rescue cells from dying in apoptosis. Phase contrast pictures of human NBC exposed to cold TTR wt (wt), TTR D18G (D18G) or TTR D18G in complex with BiP (D18G + BiP) showed that cells treated with TTR D18G were dead after 48 hours (decreased cell size, modified morphology and sparse population) whereas TTR D18G in complex with BiP were more viable (more dense and normal morphology). ….

BiP is found in TTR D18G aggregates in patient tissue ….

Figure 26. BiP co-localizes with TTR containing amyloid in the brain. Amyloid staining (LCP), TTR, and BiP are all co-localized, however there are patters where the individual signals dominates, indicating that the amyloid composition is layered within the deposits (indicated with arrows, right panel) ….

Conclusions From the studies in this thesis, it could be concluded that:

Fluorescence spectroscopy used intelligently (various molecular probes and time resolves techniques) is a very powerful tool to assay formation of amyloid and prefibrillar oligomers.

Formation of TTR oligomers during acidic conditions from the A-state occurs very fast (within minutes) after the process has been initiated. The formation of amyloid like fibrils occurs via oligomeric intermediates.

Oligomeric, intermediate species in the TTR aggregation pathway are toxic to neuroblastoma cells and cause apoptosis. Mature fibrils are less toxic. Cold stored native, tetrameric TTR is also cytotoxic suggesting an additional pathway for a labile tetramer or monomer to be toxic.

The ER chaperone BiP selectively captures the pathogenic, misfolding prone mutant of TTR; TTR D18G.

The binding site for BiP in TTR is the part in TTR that is involved in formation of the tetrameric structure and is possibly an elongation site in fibrils, comprising residues 88-103. Hence, BiP maintains TTR D18G in a soluble oligomeric form which should be a protection mechanism against oligomer toxicity.

BiP co-aggregates with TTR D18G. The larger the TTR containing aggregates are, the fewer BiP are in the complex. We ascribe this collection role for BiP as a molecular shepherd.

The degradation process of TTR D18G is slowed down in the presence of BiP.

BiP can escape the ER in complexes with TTR D18G and accumulate as extracellular amyloid in human brain.

New Studies Bring Scientists Closer to Combating Dangerous Unstable Proteins

Madeline McCurry-Schmidt

http://www.scripps.edu/newsandviews/e_20141027/wiseman.html

Scientists at The Scripps Research Institute (TSRI) have discovered a way to decrease deadly protein deposits in the heart, kidney and other organs associated with a group of human diseases called the systemic amyloid diseases.

“If we can develop a strategy to reduce the load that’s coming from these proteins, then we can open up treatment options that could be broadly applied to treat multiple systemic amyloid diseases,” said Luke Wiseman, assistant professor at TSRI and a senior author of the new research.

In related studies published recently in the journals Proceedings of the National Academy of Sciences (PNAS) and Chemistry & Biology, Wiseman and his colleagues described a process that can catch unstable proteins before they are released from the cell and form deposits. The process involves a “transcription factor” (which controls genetic expression) called ATF6 that may provide a drug target for future therapies.

Systemic amyloid diseases are caused by the buildup of unstable protein in extracellular environments such as the blood. The accumulation of these proteins damages organs such as the heart, kidney and gut, leading to organ malfunction and, eventually, death. Currently, treatment options for these diseases are limited.

“There has been a lot of work on ATF6, but people haven’t yet asked the functional question—can ATF6 be therapeutically accessed?” said Wiseman.

The Root of the Disease

In the recent PNAS study, the Wiseman lab, in collaboration with Jeffery Kelly’s lab at TSRI, focused on a systemic amyloid disease called light chain amyloidosis, where the unstable proteins are called light chain immunoglobulins.

Current treatments for light chain amyloidosis involve chemotherapy to kill the dysfunctional cells that secrete the disease-associated proteins, but about 30 percent of patients have significant buildup of light chain in the heart, making them too weak for this treatment. The researchers sought to develop a strategy to reduce the buildup of these proteins and increase treatment options for these patients.

Wiseman, Kelly and their teams went to the source of the unstable proteins: a part of the cell called the endoplasmic reticulum (ER). In the ER, proteins, such as immunoglobulin light chains, fold into structures that are then secreted into the blood where they perform important functions in the body. In light chain amyloidosis, mutations in immunoglobulin light chains make the proteins unstable, allowing them to unfold in the blood and form toxic clusters (aggregates) that damage the heart.

Using human cells, the researchers used a library of compounds that target specific biologic pathways to identify mechanisms that would reduce the secretion of unstable light chains from the ER. This approach identified an ER mechanism called the Unfolded Protein Response, or UPR, as a pathway whose activation preferentially reduces secretion of disease-associated light chains.

The UPR regulates ER function through the increased expression of proteins, such as “chaperones,” that directly influence the folding and secretion of destabilized proteins. Although sustained activation of the UPR is toxic, the team wondered if specific aspects of this pathway could be targeted to help cells “catch” these unstable light chains before they are secreted to the blood, where they can cause damage.

Using a chemical biologic approach, the researchers showed that activation of the UPR-associated protein ATF6 increases expression of many ER proteins involved in regulating protein folding and trafficking and reduces secretion of disease-associated light chains without causing toxic consequences. Furthermore, they showed that activating ATF6 decreases the extracellular aggregation of light chains by about 75 percent, suggesting the potential to reduce disease.

“This is an approach to treat light chain amyloidosis that ‘cuts it off at the source’ by not allowing the disease-associated immunoglobulin light chain to get out of cells to aggregate,” said TSRI Research Associate Christina Cooley, co-first author of the new study with Lisa M. Ryno, now an assistant professor at Oberlin College.

One Strategy, Multiple Diseases

In a second study, published October 23 online ahead of print by the journalChemistry and Biology, Wiseman and his team asked if ATF6 activation could be similarly used to reduce secretion and aggregation of transthyretin—a protein that aggregates in association with other systemic amyloid diseases referred to as the transthyretin amyloidoses.

Using a similar approach, the Wiseman lab showed that ATF6 activation reduced the secretion and extracellular aggregation of disease-associated transthyretin variants. Interestingly, the team reported that ATF6 activation increases the ability of the cell to “read” the stability of proteins.

Detecting small variations in stability is crucial because some misfolded proteins can evade the cell’s protective responses. These proteins are only slightly misfolded, and they can slip past the degradation proteins and form dangerous aggregates in the blood.

Wiseman hopes researchers will be able to design therapeutics to take advantage of the body’s natural ability to use AFT6 to decrease secretion and aggregation of multiple amyloid disease-associated proteins.

“It’s very exciting to see if we can treat multiple diseases with one drug, which would really offset the cost of developing a specific drug for each amyloid disease,” said TSRI graduate student John Chen, co-first author of the Chemistry and Biology study with Research Associate Joseph C. Genereux.

Wiseman said the next step in this area of research, which also is being conducted in collaboration with the Kelly lab, is to identify drug candidates that can activate ATF6.

In addition to Cooley, Ryno, Kelly and Wiseman, other contributors to theProceedings of National Academy of Sciences paper, “Unfolded protein response activation reduces secretion and extracellular aggregation of amyloidogenic immunoglobulin light chain,” are Lars Plate, Gareth J. Morgan and John D. Hulleman, all of TSRI. Support for this study came from Arlene and Arnold Goldstein, the Ellison Medical Foundation, the Skaggs Institute for Chemical Biology at TSRI, the Lita Annenberg Hazen Foundation, TSRI and the National Institutes of Health (AG046495, DK075295, NS079882, F32 AG042259). For more information on this study. http://www.pnas.org/content/111/36/13046.full

In addition to Chen, Genereux and Wiseman, other contributors to theChemistry and Biology paper, “ATF6 Activation Reduces the Secretion and Extracellular Aggregation of Destabilized Variants of an Amyloidogenic Protein,” are Song Qu, John D. Hulleman and Matthew D. Shoulders, all of TSRI. Support for this study came from Arlene and Arnold Goldstein, the Ellison Medical Foundation, TSRI, the American Cancer Society and the National Institutes of Health (AG036634, NS079882, DK075295, DK102635, AG046495, HL099245). For more information on this study, http://www.cell.com/chemistry-biology/abstract/S1074-5521(14)00327-5

An insight to the conserved water mediated dynamics of catalytic His88 and its recognition to thyroxin and RBP binding residues in human transthyretin

http://dx.doi.org:/10.1080/07391102.2014.984632

Avik Banerjeea & Bishnu P. Mukhopadhyaya**

online: 08 Dec 2014

Human transthyretin (hTTR) is a multifunctional protein involved in several amyloidogenic diseases. Besides transportation of thyroxin and vitamin-A, its role towards the catalysis of apolipoprotein-A1 and Aβ-peptide are also drawing interest. The role of water molecules in the catalytic mechanism is still unknown. Extensive analyses of 14 high-resolution X-ray structures of human transthyretin and MD simulation studies have revealed the presence of eight conserved hydrophilic centres near its catalytic zone which may be indispensable for the function, dynamics and stability of the protein. Three water molecules (W1, W2 and W3) form a cluster and play an important role in the recognition of the catalytic and RBP-binding residues. They also induce the reorganisation of the His88 for coupling with other catalytic residues (His90, Glu92). Another water molecule (W5) participate in inter-monomer recognition between the catalytic and thyroxin binding sites. The rest four water molecules (W6, W*, W# and W†) form a distorted tetrahedral cluster and impart stability to the catalytic core of hTTR. The conserved water mediated recognition dynamics of the different functional sites may provide some rational clues towards the understanding of the activity and mechanism of hTTR.

Amyloid Formation by Human Carboxypeptidase D Transthyretin-like Domain under Physiological Conditions*

Javier Garcia-Pardo‡,§1, Ricardo Graña-Montes‡,§2, Marc Fernandez-Mendez‡,§, Angels Ruyra‡3, Nerea Roher‡,¶4, Francesc X. Aviles‡,§, Julia Lorenzo‡,§5 and Salvador Ventura‡,§6

↵6 Recipient of an ICREA academia award. To whom correspondence may be addressed. Tel.: 34-935868956; Fax: 34-935811264; E-mail: salvador.ventura@uab.es.

Capsule

Background: Proteins can form amyloid aggregates from initially folded states.

Results: The transthyretin-like domain of human carboxypeptidase D forms amyloid aggregates without extensive unfolding.

Conclusion: The monomeric transthyretin fold has an inherent propensity to aggregate due to the presence of preformed amyloidogenic structural elements.

Significance: Generic aggregation from initially folded states would have a huge impact on cell proteostasis.

Protein aggregation is linked to a growing list of diseases, but it is also an intrinsic property of polypeptides, because the formation of functional globular proteins comes at the expense of an inherent aggregation propensity. Certain proteins can access aggregation-prone states from native-like conformations without the need to cross the energy barrier for unfolding. This is the case of transthyretin (TTR), a homotetrameric protein whose dissociation into its monomers initiates the aggregation cascade. Domains with structural homology to TTR exist in a number of proteins, including the M14B subfamily carboxypeptidases. We show here that the monomeric transthyretin-like domain of human carboxypeptidase D aggregates under close to physiological conditions into amyloid structures, with the population of folded but aggregation-prone states being controlled by the conformational stability of the domain. We thus confirm that the TTR fold keeps a generic residual aggregation propensity upon folding, resulting from the presence of preformed amyloidogenic β-strands in the native state. These structural elements should serve for functional/structural purposes, because they have not been purged out by evolution, but at the same time they put proteins like carboxypeptidase D at risk of aggregation in biological environments and thus can potentially lead to deposition diseases.

The importance of a gatekeeper residue on the aggregation of transthyretin implications for transthyretin-related amyloidoses (Article)

Journal of Biological Chemistry 10 October 2014; 289(41): 28324-28337

Sant’Anna, R.a, Braga, C.a, Varejão, N.a, Pimenta, K.M.a, Granã-Montes, R.d, Alves, A.a, Cortines, J.c, Cordeiro, Y.b, Ventura, S.d , Foguel, D.a

a Instituto de Bioquímica Médica Leopoldo de Meis, Programa de Biologia Estrutural, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

b Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

c Instituto de Microbiologia Professor Paulo de Goés, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

d Institut de Biotecnologia I Biomedicina, Universitat Autònoma de Barcelona, Bellaterra, Spain

Protein aggregation into β-sheet-enriched amyloid fibrils is associated with an increasing number of human disorders. The adoption of such amyloid conformations seems to constitute a generic property of polypeptide chains. Therefore, during evolution, proteins have adopted negative design strategies to diminish their intrinsic propensity to aggregate, including enrichment of gatekeeper charged residues at the flanks of hydrophobic aggregation-prone segments. Wild type transthyretin (TTR) is responsible for senile systemic amyloidosis, and more than 100 mutations in the TTR gene are involved in familial amyloid polyneuropathy. The TTR 26-57 segment bears many of these aggressive amyloidogenic mutations as well as the binding site for heparin. We demonstrate here that Lys-35 acts as a gatekeeper residue in TTR, strongly decreasing its amyloidogenic potential. This protective effect is sequence-specific because Lys-48 does not affect TTR aggregation. Lys-35 is part of the TTR basic heparin-binding motif. This glycosaminoglycan blocks the protective effect of Lys-35, probably by neutralization of its side chain positive charge. A K35L mutation emulates this effect and results in the rapid self-assembly of the TTR 26-57 region into amyloid fibrils. This mutation does not affect the tetrameric protein stability, but it strongly increases its aggregation propensity. Overall, we illustrate how TTR is yet another amyloidogenic protein exploiting negative design to prevent its massive aggregation, and we show how blockage of conserved protective features by endogenous factors or mutations might result in increased disease susceptibility.

Unfolded protein response-induced ERdj3 secretion links ER stress to extracellular proteostasis

Joseph C Genereux^1,2,‡, Song Qu^1,2,3,‡, Minghai Zhou⁴, Lisa M Ryno^1,2, Shiyu Wang⁵, Matthew D Shoulders^2,†, Randal J Kaufman⁵, Corinne I Lasmézas⁴, Jeffery W Kelly^1,2,6 and R Luke Wiseman^1,3,*

EMBO J, 2 Jan 2015; 34(1): 4–19. published online: 31 OCT 2014 http://dx.doi.org:/10.15252/embj.201488896

The Unfolded Protein Response (UPR) indirectly regulates extracellular proteostasis through transcriptional remodeling of endoplasmic reticulum (ER) proteostasis pathways. This remodeling attenuates secretion of misfolded, aggregation-prone proteins during ER stress. Through these activities, the UPR has a critical role in preventing the extracellular protein aggregation associated with numerous human diseases. Here, we demonstrate that UPR activation also directly influences extracellular proteostasis through the upregulation and secretion of the ER HSP40 ERdj3/DNAJB11. Secreted ERdj3 binds misfolded proteins in the extracellular space, substoichiometrically inhibits protein aggregation, and attenuates proteotoxicity of disease-associated toxic prion protein. Moreover, ERdj3 can co-secrete with destabilized, aggregation-prone proteins in a stable complex under conditions where ER chaperoning capacity is overwhelmed, preemptively providing extracellular chaperoning of proteotoxic misfolded proteins that evade ER quality control. This regulated co-secretion of ERdj3 with misfolded clients directly links ER and extracellular proteostasis during conditions of ER stress. ERdj3 is, to our knowledge, the first metazoan chaperone whose secretion into the extracellular space is regulated by the UPR, revealing a new mechanism by which UPR activation regulates extracellular proteostasis.

Synopsis

The unfolded protein response (UPR) triggers secretion of ER chaperone ERdj3 to prevent formation of toxic aSggregates, providing the first mechanistic link between intracellular stress signaling and extracellular proteostasis.

ERdj3 is an ER-stress-induced secreted chaperone
Secretion of ERdj3 is induced by the ATF6 arm of the UPR
Secretion of ERdj3 enhances extracellular proteostasis capacity
ERdj3 can co-secrete as a stable complex with misfolding-prone protein clients
ERdj3 serves as a link between intracellular and extracellular proteostasis capacity during ER stress.

Thumbnail image of graphical abstract

http://onlinelibrary.wiley.com/store/10.15252/embj.201488896/asset/image

Imbalances in extracellular protein homeostasis (or proteostasis) and consequent protein aggregation are inextricably linked to degenerative phenotypes in over 30 human protein misfolding diseases, including Alzheimer’s disease, Creutzfeldt–Jakob disease and the transthyretin (TTR) amyloidoses (Kelly, 1996; Rochet & Lansbury, 2000; Stefani & Dobson, 2003; Buxbaum, 2004; Haass & Selkoe, 2007). Compelling genetic and pharmacologic evidence supports a causal relationship between protein aggregation (including amyloidogenesis) and the degeneration of post-mitotic tissue in these disorders (Tanzi & Bertram, 2005; Aguzzi et al, 2007; Gotz et al, 2011; Coelho et al,2012). Primary determinants of extracellular proteostasis capacity include the spectrum and concentration of secreted proteostasis factors (e.g., chaperones) (Wyatt et al, 2013) and the efficiency of protein folding and quality control in the endoplasmic reticulum (ER) (Wisemanet al, 2007).

Extracellular proteostasis capacity is regulated by secreted proteins that prevent the formation of protein aggregates associated with disease. The best characterized secreted chaperones such as clusterin directly bind misfolded proteins in the extracellular environment and prevent their aggregation through an ATP-independent “holdase” mechanism (Wyatt et al, 2012). Deletion of clusterin predisposes mice to aging-dependent progressive glomerulopathy (Rosenberg et al, 2002) and increases Aβ(1–42) aggregation and deposition in mouse models of Alzheimer’s disease (DeMattos et al, 2004). Furthermore, genome-wide association studies implicate clusterin in the development of Alzheimer’s disease (Harold et al, 2009; Lambert et al, 2009; Wijsman et al, 2011). Small populations of some ER chaperones, such as the HSP70 BiP and the lectin calreticulin, can be trafficked to the plasma membrane, particularly under stress or during apoptosis (Martins et al, 2010; Zhang et al, 2010). Protein disulfide isomerases (PDIs) can also be secreted to promote extracellular disulfide exchange (Jordan & Gibbins, 2006; Hahm et al, 2013). A role for these chaperones in extracellular proteostasis maintenance has not been demonstrated to date, rather their surface expression has been implicated in immunological signaling (Peters & Raghavan, 2011; Lee, 2014).

Extracellular proteostasis is also impacted by proteostasis in the ER, which is responsible for the folding and trafficking of the 1/3 of the human proteome that is targeted to the cellular secretory pathway (Fewell et al, 2001; Braakman & Bulleid, 2011). In the ER, nascent polypeptides interact with components of ER protein folding pathways to facilitate their folding into native three-dimensional conformations (Buck et al, 2007). Folded proteins are then packaged into vesicles at the ER membrane and trafficked to downstream compartments of the secretory pathway or the extracellular space. Proteins unable to attain native three-dimensional conformations in the ER are instead targeted to ER degradation pathways such as ER-associated degradation (ERAD) (Benyair et al, 2011). The partitioning of polypeptides between ER protein folding/trafficking and degradation pathways, also referred to as ER quality control, prevents the secretion of misfolded, aggregation-prone proteins (Powers et al, 2009; Araki & Nagata, 2011).

Despite the typical efficiency of ER quality control, exposure to genetic, environmental or aging-related stresses leads to increased protein misfolding within the ER lumen and imbalances in ER proteostasis. Such stresses can increase secretion of misfolding-prone proteins into the extracellular space, directly challenging extracellular proteostasis capacity and facilitating concentration-dependent protein aggregation into proteotoxic oligomeric conformations. As such, ER stress is pathologically associated with numerous extracellular protein aggregation diseases including the systemic amyloidoses and Alzheimer’s disease (Teixeira et al, 2006; Kim et al, 2008).

To restore ER proteostasis following stress, cells activate the Unfolded Protein Response (UPR). The UPR consists of three integrated stress-responsive signaling pathways activated downstream of the ER stress-sensing proteins IRE1, ATF6, and PERK (Schroder & Kaufman, 2005; Walter & Ron, 2011). These stress sensors are activated by the accumulation of misfolded proteins within the ER lumen (a consequence of ER stress) (Bertolotti et al, 2000; Okamura et al, 2000; Shen et al, 2002). The activation of UPR signaling pathways results in the attenuation of new protein synthesis (Harding et al, 1999) and transcriptional remodeling of ER protein folding, trafficking, and degradation pathways (Lee et al, 2003; Yamamoto et al, 2007; Shoulders et al, 2013), thus enhancing ER proteostasis capacity and quality control. Through these mechanisms, UPR activation reduces accumulation of misfolded proteins in the ER and attenuates the aberrant secretion of aggregation-prone proteins into the extracellular space (Adachi et al, 2008; Shoulders et al, 2013).

In contrast, the functional impact of UPR signaling on extracellular proteostasis capacity remains poorly defined. Not only are the established secreted chaperones not transcriptional targets of the UPR, but clusterin secretion is attenuated during conditions of ER stress, indicating that clusterin secretion is not a protective mechanism to regulate extracellular proteostasis in response to pathologic ER insults (Nizard et al, 2007). Similarly, ER stress reduces secretion of ER proteostasis factors such as protein disulfide isomerase (PDI), suggesting reduced extracellular regulation of disulfide integrity during ER stress (Terada et al, 1995). Thus, we sought to study the functional role for UPR signaling in adapting extracellular proteostasis capacity during conditions of ER stress.

Here, we report that UPR activation regulates extracellular proteostasis directly through the secretion of the ER-targeted HSP40 co-chaperone ERdj3. While ERdj3 is established to function as an HSP40 co-chaperone for BiP within the ER HSP70 chaperoning pathway (Shen & Hendershot, 2005), we show that ERdj3 is also a UPR-induced secreted chaperone, whose extracellular levels increase both in response to ER stress and stress-independent activation of the UPR-associated transcription factor ATF6. Secreted ERdj3 binds to misfolded proteins in the extracellular space, prevents the aggregation of amyloidogenic Aβ_1–40 at substoichiometric concentrations, and ameliorates the toxic effects of misfolded prion protein on neuronal cells. Furthermore, we demonstrate that ERdj3 can co-secrete with destabilized, misfolding-prone clients under conditions where the ER HSP70 chaperoning pathway is overwhelmed, preemptively chaperoning these misfolding-prone secreted proteins that evade ER quality control in the extracellular environment. Thus, the capacity for ERdj3 to function in both ER and extracellular proteostasis provides an unanticipated direct link between these two environments that is regulated by the UPR during conditions of ER stress.

ER stress increases ERdj3 secretion

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We confirmed the ER stress-dependent increase in ERdj3 mRNA in HEK293T cells treated with the small molecule SERCA inhibitor thapsigargin (Tg)—a potent inducer of ER stress (Fig 1B). Upon Tg-induced ER stress, ERdj3 protein levels increased primarily in conditioned media and not intracellularly for HEK293T-Rex (Fig 1C and D). RNAi depletion of ERdj3 reduced intracellular ERdj3 levels > 90% and completely ablated extracellular ERdj3 upon Tg treatment. Tg treatment also selectively increased extracellular, as opposed to intracellular, ERdj3 from Huh7 cells (Supplementary Fig S1D). In stark contrast to ERdj3, BiP and HYOU1, two abundantly expressed, UPR-induced, ER chaperones, were not detected in the conditioned media (Fig 1C), reflecting the presence of ER retention motifs on these proteins. These results are consistent with previous results showing that negligible BiP is secreted to the extracellular space (Munro & Pelham, 1987; Yamamoto et al, 2003; Kern et al, 2009). Rather, BiP that evades the KDEL receptor is typically still retained at the cellular membrane (Wang et al, 2009; Zhang et al, 2010), as is common for other canonical ER-localized chaperones, particularly under apoptotic conditions (Jordan & Gibbins, 2006; Martins et al, 2010; Lee, 2014). HYOU1, the ER resident Hsp110 that both serves as a nucleotide exchange factor for BiP and displays its own chaperone function (Andreasson et al, 2010; Behnke & Hendershot, 2014), has not been implicated in either secretion or presentation at the cellular membrane. Alternatively, intracellular levels of BiP and HYOU1 were significantly increased upon Tg treatment. These data indicate that increased extracellular ERdj3 levels result from constitutive secretion and not from leakage of ER proteins into the extracellular space, as has been proposed for other ER chaperones (Booth & Koch, 1989).

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We next evaluated whether ER stress increases ERdj3 serum levels in mice. In the absence of stress, ERdj3 has a serum concentration of 23 ± 5 nM (Supplementary Fig S1E). Mice subjected to an 18 h fast followed by refeeding on a high-fat diet, which rapidly induces hepatic ER stress (Oyadomari et al, 2008), show a significant twofold increase in serum ERdj3 levels (Fig 1H). ERdj3 serum levels also significantly increased following 7 days on a high-fructose diet (Supplementary Fig S1F and G), which also induces ER stress in hepatic cells (Wanget al, 2012), as indicated by increased BiP, Grp94, and phosphorylated eIF2α in hepatic lysates (Supplementary Fig S1H). These results demonstrate that increased ERdj3 serum levels in mice correlate with hepatic ER stress, strongly indicating that hepatic ER stress increases ERdj3 secretion in vivo.

Stress-independent activation of the UPR-associated transcription factor ATF6 increases ERdj3 secretion

….

Figure 2. ERdj3 is efficiently secreted from cells following stress-independent activation of the UPR-associated transcription factor ATF6

We further characterized the ATF6-dependent increase in ERdj3 secretion using a [³⁵S] metabolic pulse-chase approach. ATF6 preactivation increased the extracellular concentration of newly synthesized ERdj3 fourfold relative to vehicle-treated cells, with nearly 40% of [³⁵S] labeled ERdj3 being secreted following a 4 h incubation in non-radioactive chase media (Fig 2B–D). Despite an increase in ERdj3 synthesis (Supplementary Fig S2B), XBP1s preactivation reduced the fraction of newly synthesized ERdj3 in media, relative to the vehicle treatment (Fig 2D). This decrease in ERdj3 secretion is attributed to an increase in ERdj3 degradation, with ~40% of newly synthesized ERdj3 being degraded after 4 h in cells following XBP1s preactivation (Fig 2E). ATF6 and XBP1s co-activation demonstrated a similar increase in ERdj3 secretion to that observed with ATF6 preactivation alone, demonstrating that ATF6 activation prevents the increased ERdj3 degradation observed upon XBP1s activation (Fig 2B–E). Collectively, these results show that ERdj3 secretion is increased by stress-independent preactivation of the UPR-associated transcription factor ATF6, directly implicating protective UPR signaling in the regulation of the extracellular ERdj3 concentration.

Secreted ERdj3 increases extracellular proteostasis capacity

To evaluate whether ERdj3 secretion is critical for the maintenance of intracellular ER proteostasis, we overexpressed either ERdj3^WT or the non-secreted ERdj3^KDEL and then measured expression of UPR target genes and cellular viability, both in the absence and the presence of Tg-induced ER stress (Supplementary Fig S3A and B). Despite the known role of ERdj3 in folding and degradation of specific clients (Shen & Hendershot, 2005; Hoshino et al, 2007; Jin et al, 2008; Buck et al, 2010; Tan et al, 2014), ERdj3 retention does not significantly impair global ER proteostasis maintenance in the absence or the presence of ER stress.

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Figure 3. Secreted ERdj3 inhibits extracellular protein aggregation

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Figure 4. Secreted ERdj3 attenuates vacuole formation induced by toxic prion protein in mammalian cells

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ERdj3 is co-secreted in complex with secreted destabilized mutant proteins

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http://onlinelibrary.wiley.com/store/10.15252/embj.201488896/asset/image

Figure 5. ERdj3 is co-secreted with destabilized proteins through the secretory pathway

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ERdj3–client co-secretion is regulated by ER proteostasis capacity

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Figure 6. ERdj3–client protein complexes are co-secreted when BiP activity is limiting

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Discussion

Protein synthesis is necessarily an intracellular process. Hence, extracellular metazoan environments such as the blood rely on cellular protein secretion to define extracellular protein concentrations and regulate the integrity of the secreted proteome. The presence of misfolding-prone proteins in the ER directly threatens this environment, as imbalances in ER proteostasis can impair the capacity for ER quality control pathways to prevent secretion of misfolded, aggregation-prone client proteins that in turn challenge the integrity of the extracellular proteome (Hetz & Mollereau, 2014). The UPR indirectly regulates extracellular proteostasis by attenuating the secretion of misfolded protein conformations that can both accumulate during and induce ER stress. Here, we demonstrate a direct role for the UPR in regulating extracellular proteostasis through the increased transcription and secretion of ERdj3. The capacity for UPR activation to influence extracellular proteostasis through ERdj3 secretion links protein misfolding in the secretory pathway to extracellular proteostasis, revealing a new mechanism by which cells coordinate intra- and extracellular environments in response to pathologic insults that induce ER stress (Fig 7).

http://onlinelibrary.wiley.com/store/10.15252/embj.201488896/asset/image

Figure 7. ERdj3 secretion links ER and extracellular proteostasis environments during conditions of ER stress

In response to ER stress, newly synthesized ERdj3 binds misfolded ER client proteins and delivers them to BiP for chaperoning in the Hsp70 cycle. When free BiP becomes limiting, or if repeated BiP cycling cannot productively deplete the levels of the misfolded client, the stable ERdj3–client complex is co-secreted to the extracellular environment, preemptively binding the misfolded protein and preventing the aggregation of the misfolded client protein in the extracellular space. Furthermore, stress-induced ERdj3 can be secreted on its own into the extracellular space where it can bind to misfolded, aggregation-prone client proteins and attenuate pathologic protein aggregation in the extracellular environment.

In its role as a traditional HSP40 co-chaperone to BiP, ERdj3 can influence the trafficking of ER client proteins and is exploited for pathogenic toxin internalization (Yu & Haslam, 2005; Massey et al, 2011) and viral infection (Wen & Damania, 2010; Goodwin et al, 2011) (Fig 7). However, we find that at least half of newly synthesized ERdj3 is secreted. Secreted ERdj3 has the capacity to bind misfolded proteins and prevent their extracellular aggregation and proteotoxicity, suggesting that UPR-dependent increases in ERdj3 secretion offer a new potential mechanism to protect the local extracellular environment from toxic protein conformations that can be secreted during ER stress (Fig 7). Interestingly, ERdj3 also can also influence extracellular proteostasis through its co-secretion with destabilized, misfolding-prone client proteins. This capacity of ERdj3 to be co-secreted with misfolding-prone proteins avoids the problem of diffusion-limited encounter in the extracellular space.

ERdj3–client co-secretion serves as a natural bridge between the dual roles of ERdj3 as an ER HSP40 and in regulating extracellular proteostasis (Fig 7). In the ER, ERdj3 delivers misfolded clients (e.g., ^FTTTR^A25T) into the BiP cycle for chaperoning. If inadequate free BiP is available, ERdj3 remains bound to its client protein throughout the secretory pathway and the client-ERdj3 complex can be secreted into the extracellular space, where lack of BiP prevents ERdj3 release from the substrate. When the ER must deal with high, persistent levels of misfolding-prone proteins, the UPR is activated, increasing the levels of ERdj3, BiP and other chaperones to offer misfolding-prone proteins more ER chaperoning capacity. Simultaneously, increased intracellular ERdj3 is available to bind and co-secrete with misfolding-prone protein clients that evade ER quality control and traverse the secretory pathway, providing preemptive chaperone capacity to the extracellular space. Importantly, clusterin co-secretion, unlike ERdj3 co-secretion, assists destabilized clients in evading ER quality control, providing a potential reason for the reduced secretion of clusterin during conditions of ER stress. Thus, ERdj3 offers a unique link between ER and extracellular proteostasis, employing a mechanism that cannot be achieved with other known secreted chaperones.

The capacity of UPR-dependent activation of ATF6 to influence extracellular proteostasis through ERdj3 secretion indicates a potential role for this pathway in extracellular protein aggregation pathologies. UPR signaling is activated in many extracellular protein aggregation diseases, including Alzheimer’s disease and the systemic amyloidoses (Teixeira et al, 2006; Saxena et al, 2009; Hoozemans et al, 2012; Hetz & Mollereau, 2014). Our results showing that secreted ERdj3 can increase the extracellular chaperoning and prevent extracellular aggregation and/or proteotoxicity of disease-associated proteins such as TPrP, TTR^A25T, and Aβ suggest that ATF6-dependent regulation of ERdj3 secretion is a potential mechanism to protect the extracellular space from proteotoxicity. Consistent with this prediction, presenilin mutations causatively associated with Alzheimer’s disease significantly impair ATF6 activation during stress (Katayama et al, 1999, 2001). Thus, a decreased capacity to regulate extracellular proteostasis through ATF6 activation and consequent ERdj3 secretion could be a contributing factor in the disease pathology of patients harboring these mutations.

We have established that UPR activation directly and adaptively regulates the composition of the extracellular proteostasis network through ERdj3 secretion. In particular, UPR-induced secretion of ERdj3 offers a mechanism for organisms to adapt to the presence of destabilized proteins in the secretory pathway and increase extracellular chaperoning capacity through two mechanisms: (1) the secretion of free ERdj3 available to bind misfolding-prone proteins in the extracellular environment and (2) the co-secretion of ERdj3–client complexes, which preemptively protects the extracellular environment from proteotoxic protein conformations. This potentially provides an endogenous mechanism to prevent ER stress-induced increases in extracellular protein aggregation and proteotoxicity that can lead to degenerative phenotypes. Our identification of ERdj3 as a UPR-induced secreted chaperone demonstrates that targeting adaptive stress responses, particularly the ATF6 arm of the UPR, can enhance the maintenance of extracellular proteostasis, offering a promising approach to better understand and intervene in diseases characterized by extracellular protein aggregation.

References

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Late Onset of Alzheimer’s Disease and One-carbon Metabolism

Reporter and Curator: Dr. Sudipta Saha, Ph.D.

http://pharmaceuticalintelligence.com/2013/05/06/alzheimers-disease-and-one-carbon-metabolism/

The two main molecular signs of AD are:

Extracellular deposits of Amyloid-beta (Aβ) peptides (amyloidogenic pathway) and
Intracellular deposits of phosphorylated protein TAU (fibrillogenic pathway)

For many years, both these two pathways (amyloidogenic and fibrillogenic) contended the role of “responsible” for AD onset in the researchers’ debates, even originating respectively the two groups of “BAptists” and “TAUists” scientists. In the recent years, however, these absolutist hypotheses were confuted by the emerging data evidencing that late onset AD (LOAD) has the characteristics to be considered a multifactorial disease and by scientific reports demonstrating possible interconnection between (but not limited to) the two above-mentioned “pathogenic” pathways.

For example, it was demonstrated that

GSK-3β (glycogen synthase kinase 3-beta), a phosphorylase involved in tau phosphorylation, is also responsible for APP (Amyloid Precursor Protein) phosphorylation and that
Aβ peptides are able to induce GSK-3β.

Among the several possible cocauses and interconnected pathways involved in LOAD onset and progression, a very rapidly emerging topic is related to the role of epigenetics. Moreover, it was hypothesized that methylation impairment could be a common promoter and/or a connection between amyloid and tau pathogenic pathways involving not only DNA methylation but also protein methylation mechanisms. This observation rises from studies on PP2A (protein phosphatase 2A) protein methylation showing that downregulation of neuronal PP2A methylation occurs in affected brain regions from AD patients, causing the accumulation of both phosphorylated tau and APP isoforms and increased secretion of Aβ peptides.

Altered methylation metabolism could represent the connection between B vitamins and LOAD. B vitamins are essential cofactors of homocysteine (HCY) metabolism, also called 1-carbon metabolism. One-carbon metabolism is a complex biochemical pathway regulated by the presence of folate, vitamin B12 and B6 (among other metabolites), and leading to the production of methyl donor molecule S-adenosylmethionine (SAM). High HCY and low B vitamin levels are associated to LOAD, even if a cause-effect relationship is still far to be ascertained; moreover, a clear correlation between HCY and Aβ levels has been found.

In addition, SAM, the principal metabolite in the HCY cycle and the main methyl donor in eukaryotes, appears to be altered in some neurological disorders, including AD. HCY, a thiol containing amino acid produced during the methionine metabolism via the adenosylated compound SAM, once formed is either converted to cysteine by transsulfuration or remethylated to form methionine. In the remethylation pathway HCY is remethylated by the vitamin B12-dependent enzyme methionine synthase (MS) using 5-methyltetrahydrofolate as cosubstrate. Alternatively, mainly in liver, betaine can donate a methyl group in a vitamin B12-independent reaction, catalyzed by betaine-homocysteine methyltransferase (BHMT). In the transsulfuration pathway, HCY can condense with serine to form cystathionine in a reaction catalyzed by the cystathionine beta synthase (CBS), a vitamin B6-dependent enzyme, and the cystathionine is hydrolyzed to cysteine (Cys). Cysteine is used for protein synthesis, metabolized to sulfate, or used for glutathione (GSH) synthesis. The tripeptide GSH is the most abundant intracellular nonprotein thiol, and it is a versatile reductant, serving multiple biological functions, acting, among others, as a quencher of free radicals and a cosubstrate in the enzymatic reduction of peroxides. HCY accumulation causes the accumulation of S-adenosylhomocysteine (SAH) because of the reversibility of the reaction converting SAH to HCY and adenosine (Ado); the equilibrium dynamic favors SAH synthesis. The reaction proceeds in the hydrolytic direction only if HCY and adenosine are efficiently removed. SAH is a strong DNA methyltransferases inhibitor, which reinforces DNA hypomethylation (Chiang et al., 1996). Thus, an alteration of the metabolism through either remethylation or transsulfuration pathways can lead to hyperhomocysteinemia, decrease of SAM/SAH ratio (methylation potential; MP), and alteration of GSH levels, suggesting that hypomethylation is a mechanism through which HCY is involved in vascular disease and AD, together with the oxidative damage. To add insult to injury, oxidative stress also promotes the formation of oxidized derivatives of HCY, like homocysteic acid and homocysteine sulfinic acid. These compounds, through the interaction with glutamate receptors, generate intracellular free radicals.

The first observations about B vitamins or HCY deficiency in neurological disorders were hypothesized in the 80 seconds. Despite this recent acknowledgement, alterations of HCY levels and related compounds were only recently widely recognized as risk factors for LOAD and other forms of dementia. Few mechanisms are suggested as possible protagonists in the toxic pathway of HCY in LOAD onset:

oxidative stress and neurotoxicity,

These results were obtained by using both transgenic and dietary models of hyperhomocysteinemia or altered 1-carbon metabolism. On the one hand, this variety of experimental models allowed to investigate multiple aspects of the biochemical alterations and their consequences; on the other, the lacking of common methods or goals generated a large body of literature in part overlapping for some aspects but fragmentary or incomplete for others. This aspect represents, together with the scarce interplay between clinical/epidemiological and biomolecular research, one of the reasons for the poor relevance given by the scientific community to the role of 1-carbon metabolism in certain diseases like dementia.

A causal connection between 1-carbon alterations:

hyperhomocysteinemia,
low B vitamins,
low SAM, or
high SAH

and biological alterations responsible for LOAD onset and progression is still missing. So, it was previously demonstrated that 1-carbon metabolism was related to AD-like hallmarks (increased Aβ production) via PSEN1 (presenilin 1) and BACE (beta-site APP cleaving enzyme 1) upregulation in cellular and animal models. More recently, it was added to the rising literature body dealing with 1-carbon metabolism and GSK-3β and PP2A modulation; it was also demonstrated that PSEN1 promoter is regulated by site-specific DNA methylation in cell cultures and mice and that this modulation of methylation is dependent on the regulation of the DNA methylation machinery. Although all the proposed pathways of HCY toxicity are possibly involved and nonmutually exclusive, as suggested by the multifactorial origin of LOAD, the recent advances in the connection between epigenetics and LOAD (as discussed above) stress a primary role for methylation dishomeostasis dependent on 1-carbon metabolism alterations.

Protein misfolding and prions

Larry H/ Bernstein, MD, FCAP, Curator

Leaders in Pharmaceutical Intelligence

Series E. 2; 4.9

Revised 9/30/2015

Susan L. Lindquist, Stanley B. Prusiner

http://pharmaceuticalintelligence.com/2015/09/11/protein-misfolding-and-prions-3/

Whitehead Member Susan Lindquist is a pioneer in the study of protein folding. She has shown that changes in protein folding can have profound and unexpected influences in fields as wide-ranging as human disease, evolution and nanotechnology.

Protein misfolding has been implicated as a major mechanism in many severe neurological disorders including Parkinson’s and Huntington’s diseases. Lindquist and colleagues have developed yeast strains that serve as living test tubes in which to study these disorders, unraveling how protein folding contributes to them.

Prions are proteins that can change into a self-perpetuating form. They have only been discovered recently, but one of them is already well known as the cause of mad cow disease. The Lindquist lab investigates both how prions form and the diseases they cause. In addition, Lindquist is convinced that other prion proteins play many important and positive roles in biological processes. The first evidence for this was shown in her work with Nobel Laureate Eric Kandel, which demonstrated that prions may be integral to memory storage in the brain.

Heat shock proteins are a group of molecular chaperone proteins that, as their name might suggest, guide other proteins to fold and mature correctly. Lindquist has established that heat shock protein 90 (Hsp90) can reveal hidden genetic variation in fruit flies and in cress plants (Arabidopsis) under certain environmental conditions.

Lindquist is a Member and former Director (2001-2004) of Whitehead Institute, a Professor of Biology at MIT, and a Howard Hughes Medical Institute investigator. Previously she was the Albert D. Lasker Professor of Medical Sciences from 1999-2001, and a Professor in the Department of Molecular Biology, University of Chicago, since 1978. She received a PhD in Biology from Harvard University in 1976, and was elected to the American Academy of Arts and Sciences in 1997, the National Academy of Sciences in 1997 and the Institute of Medicine in 2006.

Dr. Susan Lindquist – “Alzheimer’s Disease: An Entirely New Point of …

http://www.youtube.com/watch%3Fv%3DZ3tK50LQH_c Nov 15, 2011 …
Whitehead Institute Member Susan Lindquist’s keynote from the 2011 Whitehead Colloquium, November 5, 2011.

Susan Lindquist Lab uploaded a video 1 year ago

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Sue Lindquist Plenary Lecture at AAAS Annual Meeting 2014

by Susan Lindquist Lab

From Yeast Cells to Patient Neurons: A Powerful Discovery Platform for Parkinson’s and Alzheimer’s Disease

Stanley B. Prusiner, MD

Director, Institute for Neurodegenerative Diseases
Professor, Department of Neurology

Prusiner discovered an unprecedented class of pathogens that he named prions. Prions are proteins that acquire an alternative shape that becomes self-propagating. As prions accumulate, they cause neurodegenerative diseases in animals and humans. Prusiner’s discovery lead him to develop a novel disease paradigm: prions cause disorders such as Creutzfeldt-Jakob disease (CJD) in humans that manifest as (1) sporadic, (2) inherited and (3) infectious illnesses. Based on his seminal discovery that prions can assemble into amyloid fibrils, Prusiner proposed that the more common neurodegenerative diseases including Alzheimer’s and Parkinson’s diseases may be caused by prions.

Prusiner’s contributions to scientific research have been internationally recognized: He is a member of the National Academy of Sciences, the Institute of Medicine, the American Academy of Arts and Sciences and the American Philosophical Society, and a foreign member of the Royal Society, London. He is the recipient of numerous prizes, including the Potamkin Prize for Alzheimer’s Disease Research from the American Academy of Neurology (1991); the Richard Lounsbery Award for Extraordinary Scientific Research in Biology and Medicine from the National Academy of Sciences (1993); the Gairdner Foundation International Award (1993); the Albert Lasker Award for Basic Medical Research (1994); the Wolf Prize in Medicine from the State of Israel (1996); the Nobel Prize in Physiology or Medicine (1997); and the United States Presidential National Medal of Science (2009).

Stanley Prusiner – National Medal of Science – YouTube

http://www.youtube.com/watch%3Fv%3DkghMfXrtvAY
Nov 29, 2010 … 2009 Medal of Science Laureate for his discovery of prions — a new class of infectious agents comprised only of proteins. Produced by Evolving ..

2011 Bay Area Council Outlook Conference – Dr. Stanley Prusiner …

http://www.youtube.com/watch%3Fv%3DcSZA8VUXxZ8
Apr 27, 2011 … 2011 Bay Area Council Outlook Conference – Dr. Stanley Prusiner …. President Obama Awards National Medal of Scienceand Medal of …

By Jennifer O’Brien on October 15, 2010

UCSF Nobel laureate Stanley B. Prusiner, MD, UCSF professor of neurology and director of the Institute for Neurodegenerative Diseases, today (Oct. 15, 2010) was named to receive the National Medal of Science, the nation’s highest honor for science and technology.

Prusiner received the medal for his discovery of and ongoing research on a novel infectious agent, which he named the prion (PREE-on). The prion, composed solely of protein, causes bovine spongiform encephalopathy, or “mad cow” disease, and other related fatal neurodegenerative diseases in animals and humans.

Prions

Stanley B. Prusiner †

PNAS Nov 10, 1998; 95(23):13363–13383, http://dx.doi.org:/10.1073/pnas.95.23.13363

Prions are unprecedented infectious pathogens that cause a group of invariably fatal neurodegenerative diseases by an entirely novel mechanism. Prions are transmissible particles that are devoid of nucleic acid and seem to be composed exclusively of a modified protein (PrP^Sc). The normal, cellular PrP (PrP^C) is converted into PrP^Sc through a posttranslational process during which it acquires a high β-sheet content. The species of a particular prion is encoded by the sequence of the chromosomal PrP gene of the mammals in which it last replicated. In contrast to pathogens carrying a nucleic acid genome, prions appear to encipher strain-specific properties in the tertiary structure of PrP^Sc.

The torturous path of the scientific investigation that led to an understanding of familial Creutzfeldt–Jakob disease (CJD) chronicles a remarkable scientific odyssey. By 1930, the high incidence of familial (f) CJD in some families was known (1, 2). Almost 60 years were to pass before the significance of this finding could be appreciated (3–5). CJD remained a curious, rare neurodegenerative disease of unknown etiology throughout this period of three score years (6)(7).

Once CJD was shown to be an infectious disease, relatively little attention was paid to the familial form of the disease since most cases were not found in families(8–12). Libyan Jews living in Israel developed CJD about 30 times more frequently than other Israelis (13). This finding prompted some investigators to propose that the Libyan Jews had contracted CJD by eating lightly cooked brain from scrapie-infected sheep when they lived in Tripoli prior to emigration. Subsequently, the Libyan Jewish patients were all found to carry a mutation at codon 200 in their prion protein (PrP) gene (14–16).

Slow Viruses.

The term “slow virus” had been coined by Bjorn Sigurdsson in 1954 while he was working in Iceland on scrapie and visna of sheep (17). Five years later, William Hadlow had suggested that kuru, a disease of New Guinea highlanders, was similar to scrapie and thus, it, too, was caused by a slow virus (18). Seven more years were to pass before the transmissibility of kuru was established by passaging the disease to chimpanzees inoculated intracerebrally (19). Just as Hadlow had made the intellectual leap between scrapie and kuru, Igor Klatzo made a similar connection between kuru and CJD (20). Neuropathologists were struck by the similarities in light microscopic pathology of the central nervous system (CNS) that kuru exhibited with scrapie or CJD. In 1968, the transmission of CJD to chimpanzees after intracerebral inoculation was reported (7).

In scrapie, kuru, CJD, and all of the other disorders now referred to as prion diseases (Table 1), spongiform degeneration and astrocytic gliosis is found upon microscopic examination of the CNS (Fig. 1) (21–22).

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Table 1

The prion disease

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Figure 1

http://www.pnas.org/content/95/23/13363/F1.medium.gif

Neuropathologic changes in Swiss mice after inoculation with RML scrapie prions. (a) Hematoxylin and eosin stain of a serial section of the hippocampus shows spongiform degeneration of the neuropil, with vacuoles 10–30 μm in diameter.

Prions: A Brief Overview.

Prions are unprecedented infectious pathogens that cause a group of invariably fatal neurodegenerative diseases mediated by an entirely novel mechanism. Prion diseases may present as genetic, infectious, or sporadic disorders, all of which involve modification of the prion protein (PrP), a constituent of normal mammalian cells (23). CJD generally presents as progressive dementia, whereas scrapie of sheep and bovine spongiform encephalopathy (BSE) are generally manifest as ataxic illnesses (Table 1) (24).

Prions are devoid of nucleic acid and seem to be composed exclusively of a modified isoform of PrP designated PrP^Sc.‡ The normal, cellular PrP, denoted PrP^C, is converted into PrP^Sc through a process whereby a portion of its α-helical and coil structure is refolded into β-sheet (25). This structural transition is accompanied by profound changes in the physicochemical properties of the PrP. The amino acid sequence of PrP^Sc corresponds to that encoded by the PrP gene of the mammalian host in which it last replicated. In contrast to pathogens with a nucleic acid genome that encode strain-specific properties in genes, prions encipher these properties in the tertiary structure of PrP^Sc (26–28). Transgenetic studies argue that PrP^Scacts as a template upon which PrP^C is refolded into a nascent PrP^Sc molecule through a process facilitated by another protein.

More than 20 mutations of the PrP gene are now known to cause the inherited human prion diseases, and significant genetic linkage has been established for five of these mutations (4, 16, 29–31). The prion concept readily explains how a disease can be manifest as a heritable as well as an infectious illness.

Families of hypotheses.

Once the requirement for a protein was established, it was possible to revisit the long list of hypothetical structures that had been proposed for the scrapie agent and to eliminate carbohydrates, lipids, and nucleic acids as the infective elements within a scrapie agent devoid of protein (58) (58, 68).

The family of hypotheses that remained after identifying a protein component was still large and required a continued consideration of all possibilities in which a protein was a critical element (49). The prion concept evolved from a family of hypotheses in which an infectious protein was only one of several possibilities. With the accumulation of experimental data on the molecular properties of the prion, it became possible to discard an increasing number of hypothetical structures. (69).

……..

With the discovery of PrP 27–30 and the production of antiserum (87), brains from humans and animals with putative prion diseases were examined for the presence of this protein. In each case, PrP 27–30 was found, and it was absent in other neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis (88–91). The extreme specificity of PrP^Sc for prion disease is an important feature of the protein and is consistent with the postulated role of PrP^Sc in both the transmission and pathogenesis of these illnesses (Table 2) (92).

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Table 2

Arguments for prions being composed largely, if not entirely, of PrP^Sc molecules and devoid of nucleic acid

The accumulation of PrP^Sc contrasts markedly with that of glial fibrillary acidic protein (GFAP) in prion disease. In scrapie, GFAP mRNA and protein levels rise as the disease progresses (93), but the accumulation of GFAP is neither specific nor necessary for either the transmission or the pathogenesis of disease. Mice deficient for GFAP show no alteration in their incubation times (94, 95).

Except for PrP^Sc, no macromolecule has been found in tissues of patients dying of the prion diseases that is specific for these encephalopathies. In searches for a scrapie-specific nucleic acid, cDNAs have been identified that are complementary to mRNAs encoding other proteins with increased expression in prion disease (96–98). Yet none of the proteins has been found to be specific for prion disease.

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Alzheimer’s Outlook 2014

http://pharmaceuticalintelligence.com/2014/08/05/six-johns-hopkins-alzheimers-experts-discuss-the-latest-discoveries/

Six leading experts provide the latest thinking on new and emerging approaches to the prevention, diagnosis and treatment of Alzheimer’s disease and other dementias

* * * * * * * * * * * * * * *

If you or a loved one has been diagnosed with Alzheimer’s disease or another memory disorder…

Or if you are caring for someone with Alzheimer’s and are wondering if there’s a new drug or therapy in the pipeline that might help…

Then it’s vitally important to stay on top of developments in the field — so you can ask your doctor the key questions — and discuss the critical issues that affect the management of the disease.

To help you, we have just published Alzheimer’s Outlook 2014 — a valuable new resource that allows you to sit down with a group of preeminent physicians and listen in as they share their insights and ideas about the future course of Alzheimer’s disease — and provide a clear sense of what caregivers and patients can hope for.

Alzheimer’s Outlook 2014 is part of a series of annual research reports written for concerned lay readers. It gives you special access to information you won’t find anywhere else on the future of Alzheimer’s research.

What’s in the Alzheimer’s pipeline?

In the past few years, researchers have made meaningful strides in the understanding of dementia prevention, diagnosis and treatment. Many important breakthroughs have come from the talented physicians and scientists working here at Johns Hopkins Medicine.

In the pages of Alzheimer’s Outlook 2014 you’ll gain unprecedented access to the insights of Hopkins experts, as well as from colleagues at other renowned research centers.

And there’s so much exciting information to report!

Although we don’t yet have a drug to stop the disease progression, new techniques in molecular biology and genetics are providing remarkable insights into how and why Alzheimer’s begins, how it progresses and how it produces symptoms.

Great progress has also been made in brain imaging and other biomarkers that might allow us to diagnose Alzheimer’s when no or minimal symptoms are present. Thanks to two new radiologic compounds researchers can now see the abnormal proteins in the brain and track the disease from one part of the brain to the next.

Here’s a sample of other key highlights in Alzheimer’s Outlook 2014:

Investigating Causes and Risks by Peter V. Rabins, M.D., M.P.H., Professor of Psychiatry at Johns Hopkins and Medical Editor of the Johns Hopkins Memory Disorders Bulletin. Dr. Rabins takes a close look at the amyloid cascade hypothesis, which predominates Alzheimer’s research and drug development. He also discusses promising new brain tracers, apolipoprotein E as a risk factor for late-onset Alzheimer’s and progress on understanding the genetics of Alzheimer’s.
New Research Efforts to Prevent or Slow Dementia by Peter V. Rabins, M.D., M.P.H. Do vitamin E supplements have any effect on cognitive impairment or Alzheimer’s disease? In his second chapter, Dr. Rabins reviews recent research on this question. He also reports on the role of statins in Alzheimer’s, research on cocoa and enhanced brain health and the status of funding for dementia research.
The Ongoing Search for Drugs That Will Affect Alzheimer’s Disease by Paul Rosenberg, M.D., Associate Director of the Memory and Alzheimer’s Treatment Center at the Johns Hopkins Bayview Medical Center. Researchers now believe that to have any significant benefit, a treatment has to stop the disease long before symptoms of Alzheimer’s appear and before damage to the brain becomes widespread. Dr. Rosenberg describes efforts at early diagnosis, including the A4 trial, the DIAN study, the API study and the SNIFF study.
Noninvasive Brain Stimulation for Aphasia by Argyle Hillis, M.D., Professor of Neurology at the Johns Hopkins University School of Medicine. Primary progressive aphasia causes degeneration of nerve cells in the brain’s left hemisphere, which controls speech and language. It can also be an early symptom of Alzheimer’s. Dr. Hillis describes her work with transcranial direct current stimulation to help aphasia patients recover.
Reducing Risks for Alzheimer’s by Marilyn Albert, Ph.D., Director of the Johns Hopkins Alzheimer’s Disease Research Center. Aerobic exercise promotes better mental functioning by improving cerebral blood flow. But can exercise improve the outlook for Alzheimer’s patients? Dr. Alpert looks at research on exercise and dementia and also reports good news on increased federal funding for dementia research.
Assessing Cognitive Impairment Online by Jason Brandt, Ph.D., Professor of Neurology at the Johns Hopkins University School of Medicine. Low-tech cognitive screening tests offer a quick, inexpensive assessment of a person’s cognitive health. Dr. Brandt has been working on an online assessment tool called the “Dementia Risk Assessment,” which will help patients decide if they should pursue in-person evaluation from a doctor.
Brain Training: The ACTIVE Study by George W. Rebok, Ph.D., Professor of Mental Health at the Johns Hopkins Bloomberg School of Public Health. Dr. Rebok is a principal investigator in the ACTIVE (Advanced Cognitive Training for Independent and Vital Elderly) trial which looks at ways to improve cognitive performance in older adults. In this section, Dr. Rebok explains what he discovered.

Scientists Discover New Disease Caused By Prion Protein

AUGUST 31, 2015 Rob Stein

http://www.npr.org/2015/08/31/436377464/scientists-discover-new-disease-caused-by-prion-protein

Scientists have discovered the first new human disease caused by a “prion” in more than 50 years. Prions are strange, deformed proteins that can act like viruses and bacteria.

Giles and his colleagues at the University of California, San Francisco took a look at a disease called multiple-system atrophy or MSA.

GILES: Initially, it looks like Parkinson’s disease. People have the tremors and the loads of problems of Parkinson’s disease.

STEIN: But MSA destroys the brain even faster than Parkinson’s. Giles and his colleagues suspected MSA might be caused by a misshapen version of a protein called alpha-synuclein. So they created mice that have the human form of that protein in their brains and injected the mice with alpha-synuclein from the brains of 14 people who had died from MSA.

GILES: And in every case, the mice died about four months later of the disease.

STEIN: Under a microscope, their brains looked exactly like the brains of people who died from MSA. And tissue taken from the brains of the dead mice could do the same thing to the brains of other mice.

GILES: So that really shows that this is a transmissible disease and that the protein involved in it is acting as a prion.

STEIN: That has all kinds of implications. For starters, Giles says it raises the disturbing possibility that MSA could spread from one person to another by surgical instruments that have been used on the brains of MSA patients.

GILES: Protein sticks very tightly to the stainless steel. And when you clean it afterwards, you could potentially still have misfolded clumps of protein on the surgical instruments that, if you then do surgery on a second person, could potentially induce that in the other person.

STEIN: And the new research, which is being published in the proceedings of the National Academy of Sciences, could be really important for other reasons.

CORINNE LASMEZAS: The fact that these proteins behave like prions – it has tremendous implications.

STEIN: Corinne Lasmezas is a neuroscientist at the Scripps Research Institute in Florida. If a prion can cause MSA, Lasmezas says that’s a big boost for the idea that prions could cause other much more common diseases like Alzheimer’s, Parkinson’s and Lou Gehrig’s disease.

Another Fatal Brain Disease May Come from the Spread of ‘Prion’ Proteins

by Rachael Rettner,

http://www.livescience.com/52040-prions-multiple-system-atrophy.html

Brain tissue stained to show the features of vCJD.

This photo shows the brain tissue of someone with variant Creutzfeldt-Jakob disease (vCJD), which is caused by abnormal proteins called prions. The disease is believed to have a long incubation period

, of many years, but is ultimately fatal.

A rare and fatal brain disorder called multiple system atrophy (MSA) may be caused by a newly discovered prion, a protein similar to the ones that cause mad cow and Creutzfeldt-Jakob disease (CJD), according to a new study.

The findings may set the stage for new treatments for MSA, a progressive disorder that causes symptoms similar to Parkinson’s disease and has no cure.

What’s more, the researchers say that the prion they believe causes MSA, called alpha-synuclein, is the first new prion to be discovered in half a century. Prions are infectious proteins that fold abnormally and trigger the misfolding of other, similar proteins. Eventually, the buildup of misfolded proteins can cause lesions to form in the brain, leading to disease.

Genomic Promise for Neurodegenerative Diseases, Dementias, Autism Spectrum, Schizophrenia, and Serious Depression

Reporter and writer: Larry H Bernstein, MD, FCAP

There has been an considerable success in the current state of expanding our knowledge in genomics and therapeutic targets in cancer (although clinical remission targets and relapse are a concern), cardiovascular disease, and infectious disease. Our knowledge of prenatal and perinatal events is still at an early stage. The neurology front is by no means unattended. Here there are two prominent drivers of progress –

genomic control of cellular apoptosis by ubiquitin pathways, and
epigenetic investigations,

among a complex sea of sequence-changes. I indicate some of the current status in this. However, as much as we have know, there is an incredible barrier to formulate working models because:

ligand binding between DNA short-sequences is not predictable over time
binding between proteins and DNA is still largely unknown
specific regulatory roles between nucleotide-sequences and histone proeins are still unclear
the relationship between intracellular as well as extracellular cations and the equilibria between cations and anions in intertitial fluid that bathes the cell and between organelles is virgin territory

Consequently, it is quite an accomplishment to have come as far as we have come, and yet, even with the huge compuational power at our disposal, there is insuficient data to unravel the complexity. This may be especially true in the pathway to understanding of neurological and behavioral disorders.

Broad Map of Brain

John Markoff reports in the Feb 18 front-page of New York Times (Project would construct a broad map of the brain) that the Obama administration envisions a decade-long effort to examine the workings of the human brain and construct a map, comparable to what the Human Genome Project did for genetics. It will be a collaboration between universities, the federal government, private foundations, and teams of scientists (neuro-, nano- and whoever else). The goal is to break through the barrier to understanding the brain’s billions of neurons and gain greater insight into

perception
actions
and consciousness.

Essentially, it holds great promise for understanding

Alzheimer’s disease and Parkinson’s, as well as finding therapies for a variety of mental illnesses. An open-ended question is whether it will also advance artificial intelligence research. It is termed the Brain Activity Map project.
http://NYTimes/broad-map-of-brain/

Alzheimer’s Genomic Diagnosis and Treatment

Larry H Bernstein, MD, FCAP

http://pharmaceuticalintelligence.com/2013/03/09/alzheimers-disease-conundrum-are-we-near-the-end-of-the-puzzle/

Gene Mutation Protects Against Alzheimer’s

by Greg Miller on 11 July 2012
Brain preserver. A newly discovered gene mutation appears to protect against Alzheimer’s disease. Credit: Alzheimer’s Disease Education and Referral Center/NIA/NIH
http://news.sciencemag.org/sciencenow/2012/07/gene-mutation-protects-against-a.html

A rare mutation that alters a single letter of the genetic code protects people from the

memory-robbing dementia of Alzheimer’s disease.

The DNA change may inhibit the buildup of β amyloid, the

protein fragment that forms the hallmark plaques in the brains of Alzheimer’s patients.
The mutation affects a gene called APP,
which encodes a protein that gets broken down into pieces,
including β amyloid.

Researchers previously identified more than 30 mutations to APP, none of them good. Several of these changes increase β amyloid formation and cause

• a devastating inherited form of Alzheimer’s that afflicts people in their 30s and 40s—

• much earlier than the far more common “late-onset” form of Alzheimer’s

that typically strikes people their 70s and 80s.

The new mutation, discovered from whole-genome data from 1795 Icelanders for variations in APP that protect against Alzheimer’s, appears to do the opposite. The mutation interferes with one of the enzymes that breaks down the APP protein and causes a 40% reduction in β amyloid formation

New pharmacological strategies for treatment of Alzheimer’s disease: focus on disease modifying drugs.
Salomone S, Caraci F, Leggio GM, Fedotova J, Drago F.
University of Catania, Viale Andrea Doria 6, Catania, Italy.
Br J Clin Pharmacol. 2012 Apr;73(4):504-17. doi: 10.1111/j.1365-2125.2011.04134.x.

Current approved drug treatments for Alzheimer disease (AD) include

cholinesterase inhibitors (donepezil, rivastigmine, galantamine) and the
NMDA receptor antagonist memantine.

These drugs provide symptomatic relief but poorly affect the progression of the disease. Drug discovery has been directed, in the last 10 years, to develop ‘disease modifying drugs’ hopefully able to counteract the progression of AD. Because in a chronic, slow progressing pathological process, such as AD, an early start of treatment enhances the chance of success,

it is crucial to have biomarkers for early detection of AD-related brain dysfunction,
- usable before clinical onset.

Reliable early biomarkers need therefore to be prospectively tested for predictive accuracy,

with specific cut off values validated in clinical practice.

Disease modifying drugs developed so far include drugs to

reduce β amyloid (Aβ) production,
drugs to prevent Aβ aggregation,
drugs to promote Aβ clearance,
drugs targeting tau phosphorylation and assembly

None of these drugs has demonstrated efficacy in phase 3 studies. The failure of clinical trials with disease modifying drugs raises a number of questions, spanning from

methodological flaws to
fundamental understanding of AD pathophysiology and biology.

Diagnostic criteria applicable to presymptomatic stages of AD have now been published.

These new criteria may impact on drug development, such that future trials on disease modifying drugs will include populations susceptible to AD, before clinical onset. http://www.ncbi.nlm.nih.gov/pubmed/22035455

Gene mutation defends against Alzheimer’s disease
Rare genetic variant suggests a cause and treatment for cognitive decline.
Ewen Callaway 11 July 2012
http://www.nature.com/news/gene-mutation-defends-against-alzheimer-s-disease-1.10984

J. NIETH/CORBIS
Almost 30 million people live with Alzheimer’s disease worldwide, a staggering health-care burden that is expected to quadruple by 2050. Yet doctors can offer no effective treatment, and scientists have been unable to pin down the underlying mechanism of the disease.
Research published this week offers some hope on both counts – few people carry a genetic mutation that naturally prevents them from developing the condition – 0.5% of Icelanders have a protective gene, as are 0.2–0.5% of Finns, Swedes and Norwegians. Icelanders who carry it have a 50% better chance of reaching age 85, are more than five times more likely to reach it 85 without Alzheimer’s. The mutation seems to put a brake on the milder mental deterioration that most elderly people experience. Carriers are about 7.5 times more likely than non-carriers to reach the age of 85 without major cognitive decline, and perform better on the cognitive tests that are administered thrice yearly to Icelanders who live in nursing homes.
The discovery not only confirms the principal suspect that is responsible for Alzheimer’s, it also suggests that the disease could be

an extreme form of the cognitive decline seen in many older people.

The mutation — the first ever found to protect against the disease — lies in a gene that produces

amyloid-β precursor protein (APP),
which has an unknown role in the brain

APP was discovered 25 years ago in patients with rare,

inherited forms of Alzheimer’s that strike in middle age.
In the brain, APP is broken down into a smaller molecule called amyloid-β.

Visible clumps, or plaques, of amyloid-β found in the autopsied brains of patients are a hallmark of Alzheimer’s.
Scientists have long debated whether the plaques are a cause of the neurodegenerative condition

or a consequence of other biochemical changes associated with the disease.

The latest finding supports other genetics studies blaming amyloid-β, according to Rudolph Tanzi, a neurologist at the Massachusetts General Hospital in Boston and a member of one of the four teams that discovered APP’s role in the 1980s.
If amyloid-β plaques were confirmed as the cause of Alzheimer’s, it would bolster efforts to develop drugs that block their formation, says Kári Stefánsson, chief executive of deCODE Genetics in Reykjavik, Iceland, who led the latest research. He and his team first discovered the mutation by comparing the complete genome sequences of 1,795 Icelanders with their medical histories. The researchers then studied the variant in nearly 400,000 more Scandinavians.
This suggests that Alzheimer’s disease and cognitive decline are two sides of the same coin, with a common cause — the build-up of amyloid-β plaques in the brain, something seen to a lesser degree in elderly people who do not develop full-blown Alzheimer’s. A drug that mimics the effects of the mutation, might slow cognitive decline as well as prevent Alzheimer’s.
Stefánsson and his team discovered that the mutation introduces a single amino-acid alteration to APP. This amino acid is close to the site where an enzyme called

β-secretase 1 (BACE1) ordinarily snips APP into smaller amyloid-β chunks —
and the alteration is enough to reduce the enzyme’s efficiency.

Stefánsson’s study suggests that blocking β-secretase from cleaving APP has the potential to prevent Alzheimer’s, but Philippe Amouyel, an epidemiologist at the Pasteur Institute in Lille, France, says “it is very difficult to identify the

precise time when this amyloid toxic effect could still be modified”.

“If this effect needs to be blocked as early as possible in life to protect against Alzheimer’s disease, we will need to propose a new design for clinical trials” to identify an effective treatment.

The results demonstrate that whole-genome sequencing can uncover very rare mutations that might offer insight into common diseases.

disease risk, may be determined by genetic variants that slightly tilt the odds of developing disease
In this case a rare mutant may provide very key mechanistic insights into Alzheimer’s

Jonsson, T. et al. Nature http://dx.doi.org/10.1038/nature11283 (2012).
Kang, J. et al. Nature 325, 733–736 (1987).
Goldgaber, D., Lerman, M. I., McBride, O. W., Saffiotti, U. & Gajdusek, D. C. Science 235, 877–880 (1987).

BHCE genetic data combined with brain imaging using agent florbetapir connects the BHCE gene to AD plaque buildup. BHCE is an enzyme that breaks down acetylcholine in the brain, which is depleted early in the disease and results in memory loss. http://www.genengnews.com/

New Alzheimer’s Genes Found
Gigantic Scientific Effort Discovers Clues to Treatment, Diagnosis of Alzheimer’s Disease
By Daniel J. DeNoon
WebMD Health News Reviewed by Laura J. Martin, MD
http://www.webmd.com/alzheimers/news/20110403/new-alzheimers-genes-found

A massive scientific effort has found five new gene variants linked to Alzheimer’s disease. The undertaking involved analyzing the genomes of nearly 40,000 people with and without Alzheimer’s. This study was undertaken by two separate research consortiums in the U.S. and in Europe, which collaborated to confirm each other’s results.
Four genes had previously been linked to Alzheimer’s. Three of them affect only the risk of relatively rare forms of Alzheimer’s. The fourth is APOE, until now the only gene known to affect risk of the common, late-onset form of Alzheimer’s. Roughly 27% of Alzheimer’s disease can be attributed to the five new gene variants. Even though Alzheimer’s is a very complex disease, the new findings represent a large chunk of Alzheimer’s risk, according to Margaret A. Pericak-Vance, PhD, of the U.S. consortium –

20% of the causal risk of Alzheimer’s disease and
32% of the genetic risk.

Alzheimer’s Tied to Mutation Harming Immune Response
By GINA KOLATA Published: November 14, 2012 in NY Times
http://www.nytimes.com/2012/11/15/health/gene-mutation-that-hobbles-immune-response-is-linked-to-alzheimers.html?_r=0
Alzheimer’s researchers and drug companies have for years concentrated on one hallmark of Alzheimer’s disease: the production of toxic shards of a protein that accumulate in plaques on the brain.
Two groups of researchers working from entirely different starting points have converged on a mutated gene involved in another aspect of Alzheimer’s disease:

the immune system’s role in protecting against the disease.

The mutation is suspected of interfering with

the brain’s ability to prevent the buildup of plaque.

When the gene is not mutated, white blood cells in the brain spring into action,

gobbling up and eliminating the plaque-forming toxic protein, beta amyloid.

As a result, Alzheimer’s can be staved off or averted. People with the mutated gene have a threefold to fivefold increase in the likelihood of developing Alzheimer’s disease in old age.

Comparing Differences

Dr. Julie Williams’s, Cardiff, Wales (European team leader) report identified CLU and Picalm. A second study published in Nature Genetics, by Philippe Amouyel from Institut Pasteur de Lille in France, pinpointed CLU and CR1. The greatest inherited risk comes from the APOE gene, discovered in 1993 by a team led by Allen Roses, now director of the Deane Drug Discovery Institute at Duke UMC, in Durham, North Carolina.
The findings “are beginning to give us insight into the biology, but I don’t think you can expect treatments overnight,” Dr. Michael Owen (Cardiff, Wales) said. Instead, the genes will show a mosaic of risk, and “the key issue is what hand of cards you’re dealt,” he said.

Promise for Early Diagnosis
BHCE genetic data combined with brain imaging using agent florbetapir connects the BHCE gene to AD plaque buildup. BHCE is an enzyme that breaks down acetylcholine in the brain, which is depleted early in the disease and results in memory loss.

Dr. Bernstein’s comments:

There has been a long history of failure of drugs to slow down the progression of Alzheimer’s. Regression of the plaques has not corresponded with retention of cognitive ability, which has been behind the arguments over beta amyloid or tau.
We now have two particularly interesting mutations –
1. ApoE gene mutation that increases risk
2. APP mutation that quite dramatically affects retention of cognition

The Alzheimer Scene around the Web

Larry H Bernstein, MD, FCAP, Curator

http://pharmaceuticalintelligence.com/2012/11/02/the-alzheimer-scene-around-the-web/

Neurodegerative Disease
Tumeric-Derived Compound Curcumin May Treat Alzheimer’s
Curry chemical shows promise for treating the memory-robbing disease
By Lauren K. Wolf
Department: Science & Technology
News Channels: Biological SCENE
Keywords: alternative medicine, dietary supplements, curcumin, tumeric, Alzheimer’s disease

CURRY WONDER
Curcumin, derived from the rootstalk of the turmeric plant, not only gives Indian dishes their color but might treat Alzheimer’s.
Credit: Shutterstock
More than 5 million people in the U.S. currently live with Alzheimer’s disease. And according to the Alzheimer’s Association, the situation is only going to get worse.
By 2050, the nonprofit estimates, up to 16 million Americans will have the memory-robbing disease. It will cost the U.S. $1.1 trillion annually to care for them unless a successful therapy is found.
Pharmaceutical companies have invested heavily in developing Alzheimer’s drugs, many of which target amyloid-β, a peptide that misfolds and clumps in the brains of patients. But so far, no amyloid-β-targeted medications have been successful. Expectation for the most advanced drugs—bapineuzumab from Pfizer and Johnson & Johnson and solanezumab from Eli Lilly & Co.—are low on the basis of lackluster data from midstage clinical trials. That sentiment was reinforced last week when bapineuzumab was reported to have failed the first of four Phase III studies.
Even if these late-stage hopefuls do somehow work, they won’t come cheap, says Gregory M. Cole, a neuroscientist at the University of California, Los Angeles. These drugs “would cost patients tens of thousands of dollars per year,” he estimates. That hefty price tag stems from bapineuzumab and solanezumab being costly-to-manufacture monoclonal antibodies against amyloid-β.
“There’s a great need for inexpensive Alzheimer’s treatments,” as well as a backup plan if pharma fails, says Larry W. Baum, a professor in the School of Pharmacy at the Chinese University of Hong Kong. As a result, he says, a great many researchers have turned their attention to less pricy alternatives, such as compounds from plants and other natural sources.
Curcumin, a spice compound derived from the rootstalk of the turmeric plant (Curcuma longa), has stood out among some of the more promising naturally derived candidates.

When administered to mice that develop Alzheimer’s symptoms, curcumin decreases inflammation and reactive oxygen species in the rodents’ brains, researchers have found. The compound also inhibits the aggregation of troublesome amyloid-β strands among the animals’ nerve cells. But the development of curcumin as an Alzheimer’s drug has been stymied, scientists say, both by its low uptake in the body and a lack of funds for effective clinical trials—obstacles researchers are now trying to overcome.
In addition to contributing to curry dishes’ yellow color and pungent flavor, curcumin has been a medicine in India for thousands of years. Doctors practicing traditional Hindu medicine admire turmeric’s active ingredient for its anti-inflammatory properties and have used it to treat patients for ailments including digestive disorders and joint pain.
Only in the 1970s did Western researchers catch up with Eastern practices and confirm curcumin’s anti-inflammatory properties in the laboratory. Scientists also eventually determined that the polyphenolic compound is an antioxidant and has chemotherapeutic activity.

Bharat B. Aggarwal, a professor at the University of Texas M. D. Anderson Cancer Center, says curcumin is an example of a pleiotropic agent: It has a number of different effects and interacts with many targets and biochemical pathways in the body. He and his group have discovered that one important molecule targeted and subsequently suppressed by curcumin is NF-κB, a transcription factor that switches on the body’s inflammatory response when activated (J. Biol. Chem., DOI: 10.1074/jbc.270.42.24995).
Aside from NF-κB, curcumin seems to interact with several other molecules in the inflammatory pathway, a biological activity that Aggarwal thinks is advantageous. “All chronic diseases are caused by dysregulation of multiple targets,” he says. “Chemists don’t yet know how to design a drug that hits multiple targets.” With curcumin, “Mother Nature has already provided a compound that does so.”
Curcumin’s pleiotropy also brought it to the attention of UCLA’s Cole during the early 1990s while he was searching for possible Alzheimer’s therapeutics. “That was before we knew about amyloid-β” and its full role in Alzheimer’s, he says. “We were working on the disease from an oxidative damage and inflammation point of view—two processes implicated in aging.”
When Cole and his wife, Sally A. Frautschy, also at UCLA, searched the literature for compounds that could tackle both of these age-related processes, curcumin jumped out at them. It also didn’t hurt that the incidence of Alzheimer’s in India, where large amounts of curcumin are consumed regularly, is lower than in other parts of the developing world (Lancet Neurol., DOI:10.1016/s1474-4422(08)70169-8).

In 2001, Cole, Frautschy, and colleagues published the first papers that demonstrated curcumin’s potential to treat neurodegenerative disease (Neurobiol. Aging, DOI: 10.1016/s0197-4580(01)00300-1; J. Neurosci.2001, 8370). The researchers studied the effects of curcumin on rats that had amyloid-β injected into their brains, as well as mice engineered to develop amyloid brain plaques. In both cases, curcumin suppressed oxidative tissue damage and reduced amyloid-β deposits.
Those results, Cole says, “turned us into curcuminologists.”
Although the UCLA team observed that curcumin decreased amyloid plaques in animal models, at the time, the researchers weren’t sure of the molecular mechanism involved.
Soon after the team’s first results were published, Cole recalls, a colleague brought to his attention the structural similarity between curcumin and the dyes used to stain amyloid plaques in diseased brain tissue. When Cole and Frautschy tested the spice compound, they saw that it, too, could stick to aggregated amyloid-β. “We thought, ‘Wow, not only is curcumin an antioxidant and an anti-inflammatory, but it also might be an anti-amyloid drug,’ ” he says.
In 2004, a group in Japan demonstrated that submicromolar concentrations of curcumin in solution could inhibit aggregation of amyloid-β and break up preformed fibrils of the stuff (J. Neurosci. Res., DOI: 10.1002/jnr.20025). Shortly after that, the UCLA team demonstrated the same (J. Biol. Chem., DOI: 10.1074/jbc.m404751200).
As an Alzheimer’s drug, however, it’s unclear how important it is that the spice compound inhibits amyloid-β aggregation, Cole says. “When you have something that’s so pleiotropic,” he adds, “it’s hard to know” which of its modes of action is most effective.
Having multiple targets may be what helps curcumin have such beneficial, neuroprotective effects, says David R. Schubert, a neurobiologist at the Salk Institute for Biological Studies, in La Jolla, Calif. But its pleiotropy can also be a detriment, he contends.
The pharmaceutical world, Schubert says, focuses on designing drugs aimed at hitting single-target molecules with high affinity. “But we don’t really know what ‘the’ target for curcumin is,” he says, “and we get knocked for it on grant requests.”
Another problem with curcumin is poor bioavailability. When ingested, UCLA’s Cole says, the compound gets converted into other molecular forms, such as curcumin glucuronide or curcumin sulfate. It also gets hydrolyzed at the alkaline and neutral pHs present in many areas of the body. Not much of the curcumin gets into the bloodstream, let alone past the blood-brain barrier, in its pure, active form, he adds.

Unfortunately, neither Cole nor Baum at the Chinese University of Hong Kong realized the poor bioavailability until they had each launched a clinical trial of curcumin. So the studies showed no significant difference between Alzheimer’s patients taking the spice compound and those taking a placebo (J. Clin. Psychopharmacol., DOI: 10.1097/jcp.0b013e318160862c).
“But we did show curcumin was safe for patients,” Baum says, finding a silver lining to the blunder. “We didn’t see any adverse effects even at high doses.”

Some researchers, such as Salk’s Schubert, are tackling curcumin’s low bioavailability by modifying the compound to improve its properties. Schubert and his group have come up with a molecule, called J147, that’s a hybrid of curcumin and cyclohexyl-bisphenol A. Like Cole and coworkers, they also came upon the compound not by initially screening for the ability to interact with amyloid-β, but by screening for the ability to alleviate age-related symptoms.

The researchers hit upon J147 by exposing cultured Alzheimer’s nerve cells to a library of compounds and then measuring changes to levels of biomarkers for oxidative stress, inflammation, and nerve growth. J147 performed well in all categories. And when given to mice engineered to accumulate amyloid-β clumps in their brains, the hybrid molecule prevented memory loss and reduced formation of amyloid plaques over time (PLoS One, DOI: 10.1371/journal.pone.0027865).

Other researchers have tackled curcumin’s poor bioavailability by reformulating it. Both Baum and Cole have encapsulated curcumin in nanospheres coated with either polymers or lipids to protect the compound from modification after ingestion. Cole tells C&EN that by packaging the curcumin in this way, he and his group have gotten micromolar quantities of it into the bloodstream of humans. The researchers are now preparing for a small clinical trial to test the formulation on patients with mild cognitive impairment, who are at an increased risk of developing Alzheimer’s.

An early-intervention human study such as this one comes with its own set of challenges, Cole says. People with mild cognitive impairment “have good days and bad days,” he says. A large trial over a long period would be the best way to get any meaningful data, he adds. Such a trial can cost up to $100 million, a budget big pharma might be able to scrape together but that is far out of reach for academics funded by grants, Cole says. “If you’re down at the level of what an individual investigator can do, you’re running a small trial,” he says, “and even if the result is positive, it might be inconclusive” because of its small size or short duration. That’s one of the reasons the curcumin work is slow-going, Cole contends.
NIH-Funded Research Provides New Clues on How ApoE4 Affects Alzheimer’s Risk
Published: Tuesday, October 30, 2012
Last Updated: Tuesday, October 30, 2012

Researchers found that ApoE4 triggers an inflammatory reaction that weakens the blood-brain barrier.
Common variants of the ApoE gene are strongly associated with the risk of developing late-onset Alzheimer’s disease, but the gene’s role in the disease has been unclear.

Now, researchers funded by the National Institutes of Health have found that in mice, having the most risky variant of ApoE damages the blood vessels that feed the brain.

The researchers found that the high-risk variant, ApoE4, triggers an inflammatory reaction that weakens the blood-brain barrier, a network of cells and other components that lines brain’s brain vessels.

Normally, this barrier allows nutrients into the brain and keeps harmful substances out.

The study appears in Nature, and was led by Berislav Zlokovic, M.D., Ph.D., director of the Center for Neurodegeneration and Regeneration at the Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles.

“Understanding the role of ApoE4 in Alzheimer’s disease may be one of the most important avenues to a new therapy,” Dr. Zlokovic said. “Our study shows that ApoE4 triggers a cascade of events that damages the brain’s vascular system,” he said, referring to the system of blood vessels that supply the brain.

The ApoE gene encodes a protein that helps regulate the levels and distribution of cholesterol and other lipids in the body. The gene exists in three varieties.

ApoE2 is thought to play a protective role against both Alzheimer’s and heart disease, ApoE3 is believed to be neutral, and ApoE4 confers a higher risk for both conditions.

Outside the brain, the ApoE4 protein appears to be less effective than other versions at clearing away cholesterol; however, inside the brain, exactly how ApoE4 contributes to Alzheimer’s disease has been a mystery.

Dr. Zlokovic and his team studied several lines of genetically engineered mice, including one that lacks the ApoE gene and three other lines that produce only human ApoE2, ApoE3 or ApoE4. Mice normally have only a single version of ApoE.

The researchers found that mice whose bodies made only ApoE4, or made no ApoE at all, had a leaky blood-brain barrier. With the barrier compromised, harmful proteins in the blood made their way into the mice’s brains, and after several weeks, the researchers were able to detect loss of small blood vessels, changes in brain function, and a loss of connections between brain cells.

“The study demonstrates that damage to the brain’s vascular system may play a key role in Alzheimer’s disease, and highlights growing recognition of potential links between stroke and Alzheimer’s-type dementia,” said Roderick Corriveau, Ph.D., a program director at NIH’s National Institute of Neurological Disorders and Stroke (NINDS), which helped fund the research. “It also suggests that we might be able to decrease the risk of Alzheimer’s disease among ApoE4 carriers by improving their vascular health.”

The researchers also found that ApoE2 and ApoE3 help control the levels of an inflammatory molecule called cyclophilin A (CypA), but ApoE4 does not. Levels of CypA were raised about five-fold in blood vessels of mice that produce only ApoE4.

The excess CypA then activated an enzyme, called MMP-9, which destroys protein components of the blood-brain barrier. Treatment with the immunosuppressant drug cyclosporine A, which inhibits CypA, preserved the integrity of the blood-brain barrier and lessened damage to the brain.

An inhibitor of the MMP-9 enzyme had similar beneficial effects. In prior studies, inhibitors of this enzyme have been shown to reduce brain damage after stroke in animal models.

“These findings point to cyclophilin A as a potential new drug target for Alzheimer’s disease,” said Suzana Petanceska, Ph.D., a program director at NIH’s National Institute on Aging (NIA), which also funded Dr. Zlokovic’s study.

“Many population studies have shown an association between vascular risk factors in mid-life, such as high blood pressure and diabetes, and the risk for Alzheimer’s in late-life. We need more research aimed at deepening our understanding of the mechanisms involved and to test whether treatments that reduce vascular risk factors may be helpful against Alzheimer’s.”

Alzheimer’s disease is the most common cause of dementia in older adults, and affects more than 5 million Americans. A hallmark of the disease is a toxic protein fragment called beta-amyloid that accumulates in clumps, or plaques, within the brain.

Gene variations that cause higher levels of beta-amyloid are associated with a rare type of Alzheimer’s that appears early in life, between age 30 and 60.

However, it is the ApoE4 gene variant that is most strongly tied to the more common, late-onset type of Alzheimer’s disease. Inheriting a single copy of ApoE4 from a parent increases the risk of Alzheimer’s disease by about three-fold. Inheriting two copies, one from each parent, increases the risk by about 12-fold.

Dr. Zlokovic’s study and others point to a complex interplay between beta-amyloid and ApoE4. On the one hand, beta-amyloid is known to build up in and damage blood vessels and cause bleeding into the brain.

On the other hand, Dr. Zlokovic’s data suggest that ApoE4 can damage the vascular system independently of beta-amyloid. He theorizes that this damage makes it harder to clear beta-amyloid from the brain.

Some therapies under investigation for Alzheimer’s focus on destroying amyloid plaques, but therapies designed to compensate for ApoE4 might help prevent the plaques from forming, he said.

Compound Could Become Alzheimer’s Treatment
Thu, 10/11/2012 – 1:29pm
A new molecule designed to treat Alzheimer’s disease has significant promise and is potentially the safest to date, according to researchers.

Purdue University professor Arun Ghosh designed the molecule, which is a highly potent beta-secretase inhibitor with unique features that ensure it goes only to its target and does not affect healthy physiological processes, he said.

“This molecule maintains the disease-fighting properties of earlier beta-secretase inhibitors, but is much less likely to cause harmful side effects,” said Ghosh, the Ian P. Rothwell Distinguished Professor of Chemistry and Medicinal Chemistry and Molecular Pharmacology. “The selectivity we achieved is unprecedented, which gives it great promise for the long-term medication required to treat Alzheimer’s. Each time a treatment misses its disease target and instead interacts with a healthy cell or molecule, damage is done that we call toxicity. Even low levels of this toxicity could build up over years and years of treatment, and an Alzheimer’s patient would need to be treated for the rest of his or her life.”

The new molecule shows a 7,000-fold selectivity for its target enzyme, which far surpasses the benchmark of a 1,000-fold selectivity for a viable treatment molecule, and dwarfs the selectivity values in the hundreds for past beta-secretase inhibitors, he said. A paper detailing the work will be published in an upcoming Alzheimer’s research issue of the Journal of Medicinal Chemistry and is currently available online. The National Institutes of Health funded the research.

Beta-secretase inhibitors, which could allow for intervention in the early stages of Alzheimer’s disease, have promise as a potential treatment. Several drugs based on this molecular target have made it to clinical trials, including one based on a molecule Ghosh designed previously. These molecules prevent the first step in a chain of events that leads to the formation of amyloid plaque in the brain, fibrous clumps of toxic proteins that are believed to cause the disease’s devastating symptoms.

The National Institute on Aging estimates that 5.1 million Americans suffer from Alzheimer’s disease, which leads to dementia by affecting parts of the brain that control thought, memory and language.

“Alzheimer’s is a progressive disease that destroys the brain and also destroys the quality of life for those who suffer from it,” Ghosh said. “It eventually robs people of their ability to recognize their own spouse or child and to complete basic tasks necessary for independence, like getting dressed. It is a truly devastating disease for those who suffer from it and for their friends and loved ones.”

Earlier versions of the beta-secretase inhibitor were able to stop and even reverse the progression of amyloid plaques in tests on mice, but potency and selectivity are only two of the three pillars of a viable Alzheimer’s treatment, Ghosh said. It has yet to be shown whether this molecule possesses the third pillar, the ability to be turned into an easily administered drug that passes through the blood-brain barrier.

Ghosh collaborates with Jordan Tang, the J.G. Puterbaugh Chair in Medical Research at the Oklahoma Medical Research Foundation, who in 2000 identified beta-secretase and its role in the progression of Alzheimer’s. Later that year Ghosh designed his first molecule that bound to and inhibited the activity of the enzyme. He has strived to create the needed improvements ever since.

Ghosh bypasses the usual lengthy process of trial and error in finding useful inhibitor molecules by using a structure-based design strategy. He uses the structures of the inhibitor bound to the enzyme as a guide to what molecular features are important for desirable and undesirable characteristics. Then he removes, replaces and adds molecular groups to amplify the desirable and eliminate the undesirable.

“I believe structure-based design is vital to the development of new and improved medicine,” said Ghosh, who also is a member of the Purdue University Center for Cancer Research. “These strategies have the potential to eliminate enormous costs and time needed in traditional random screening protocols for drug development. Structure-based strategies allow us to design molecules that do precisely what we need them to do with fewer undesirable side effects.”

Tang performed the X-ray crystallography and captured the crystal structures to reveal important insights and serve as a guide for Ghosh’s designs.

“Developing inhibitors into clinically useful drugs is an evolutionary process,” Tang said. “We learn what works and what doesn’t along the way, and the knowledge permits us to do better in the next step. The miracles of modern medicine are built on top of excellent scientific findings. We try to do good science and know that the consequence will be a better chance for conquering diseases and improving lives.”

Beta-secretase belongs to a class of enzymes called aspartyl proteases. Research into beta-secretase inhibitors faced setbacks when other aspartyl proteases similar in structure, called memapsin 1 and cathepsin D, were discovered and found to be involved in many important physiological processes. Earlier designed beta-secretase inhibitors were found also to work against the biologically necessary enzymes.

Ghosh’s team focused on developing ways to make the inhibitor more selective so that it would avoid these other, physiologically important enzymes. They compared the structures of beta-secretase and memapsin 1 as they interacted with the inhibitor to find an active area unique only to beta-secretase. Then they added a functional molecular feature that targets and interacts with the unique area, making the inhibitor more attractive to beta-secretase and less attractive to the other enzymes.

“The added feature serves as a bait on the inhibitor molecule that entices beta-secretase and also grabs onto it tightly, greatly enhancing its selectivity,” he said. “This is a fundamental insight into the origins of selectivity and ways to increase it.”
Ghosh said this work highlights an important purpose of academic research.

“Academic research lays out and shares the fundamentals to advance drug discovery,” he said. “Advances in treatment are built upon the basic research happening at universities.”

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Protein Energy Malnutrition and Early Child Development

Posted in Amino acids, Anemia, Biochemical pathways, Developmental biology, Diagnostics and Lab Tests, Disease Biology, Fat soluble vitamins, Folate and B12, History and physical exam, Iron deficiency, Lipid metabolism, Metabolism, Micronutrients, Neurological Diseases, Neuroscience, Nutrition, Nutrition and Phytochemistry, Nutrition Disorders, Protein-energy malnutrition, Proteins, Proteomics, Translational Science, tagged childhood malnutrition and stunting, PEM on October 25, 2015| Leave a Comment »

Protein Energy Malnutrition and Early Child Development

Curator: Larry H. Bernstein, MD, FCAP

In the preceding articles we have seen that poverty and low social class combined with cultural strictures or dependence on a sulfur-poor diet results in childhood stunting and impaired brain development. This is a global health issue.

Protein-Energy Malnutrition

Author: Noah S Scheinfeld, JD, MD, FAAD; Chief Editor: Romesh Khardori, MD, PhD, FACP

http://emedicine.medscape.com/article/1104623-overview

The World Health Organization (WHO)^[1]defines malnutrition as “the cellular imbalance between the supply of nutrients and energy and the body’s demand for them to ensure growth, maintenance, and specific functions.” The term protein-energy malnutrition (PEM) applies to a group of related disorders that includemarasmus, kwashiorkor (see the images below), and intermediate states of marasmus-kwashiorkor. The term marasmus is derived from the Greek wordmarasmos, which means withering or wasting. Marasmus involves inadequate intake of protein and calories and is characterized by emaciation. The term kwashiorkor is taken from the Ga language of Ghana and means “the sickness of the weaning.” Williams first used the term in 1933, and it refers to an inadequate protein intake with reasonable caloric (energy) intake. Edema is characteristic of kwashiorkor but is absent in marasmus.

Studies suggest that marasmus represents an adaptive response to starvation, whereas kwashiorkor represents a maladaptive response to starvation. Children may present with a mixed picture of marasmus and kwashiorkor, and children may present with milder forms of malnutrition. For this reason, Jelliffe suggested the term protein-calorie (energy) malnutrition to include both entities.
Although protein-energy malnutrition affects virtually every organ system, this article primarily focuses on its cutaneous manifestations. Patients with protein-energy malnutrition may also have deficiencies of vitamins, essential fatty acids, and trace elements, all of which may contribute to their dermatosis.

In general, marasmus is an insufficient energy intake to match the body’s requirements. As a result, the body draws on its own stores, resulting in emaciation. In kwashiorkor, adequate carbohydrate consumption and decreased protein intake lead to decreased synthesis of visceral proteins. The resulting hypoalbuminemia contributes to extravascular fluid accumulation. Impaired synthesis of B-lipoprotein produces a fatty liver.

Protein-energy malnutrition also involves an inadequate intake of many essential nutrients. Low serum levels of zinc have been implicated as the cause of skin ulceration in many patients. In a 1979 study of 42 children with marasmus, investigators found that only those children with low serum levels of zinc developed skin ulceration. Serum levels of zinc correlated closely with the presence of edema, stunting of growth, and severe wasting. The classic “mosaic skin” and “flaky paint” dermatosis of kwashiorkor bears considerable resemblance to the skin changes of acrodermatitis enteropathica, the dermatosis of zinc deficiency.

In 2007, Lin et al^[2]stated that “a prospective assessment of food and nutrient intake in a population of Malawian children at risk for kwashiorkor” found “no association between the development of kwashiorkor and the consumption of any food or nutrient.”

Marasmus and kwashiorkor can both be associated with impaired glucose clearance that relates to dysfunction of pancreatic beta-cells.^[3]In utero, plastic mechanisms appear to operate, adjusting metabolic physiology and adapting postnatal undernutrition and malnutrition to define whether marasmus and kwashiorkor will develop.^[4]

In 2012, a report from Texas noted an 18-month-old infant with type 1 glutaric acidemia who had extensive desquamative plaques, generalized nonpitting edema, and red-tinged sparse hair, with low levels of zinc, alkaline phosphatase, albumin, and iron. This patient has a variation on kwashiorkor, and the authors suggest that it be termed acrodermatitis dysmetabolica.^[5]On the same note, a boy aged 18 months with type 1 glutaric acidemia suffered from zinc deficiency and acquired protein energy malnutrition.^[6]

For complex reasons, sickle cell anemia can predispose suffers to protein malnutrition.^[7]

Protein energy malnutrition ramps up arginase activity in macrophages and monocytes.^[8]

Protein energy malnutrition (PEM), brain and various facets of child development.

Udani PM¹.

Indian J Pediatr. 1992 Mar-Apr;59(2):165-86 http://www.ncbi.nlm.nih.gov/pubmed/1383143

Protein energy malnutrition (PEM) is a global problem. Nearly 150 million children under 5 years in the world and 70-80 million in India suffer from PEM, nearly 20 million in the world and 4 million in India suffer from severe forms of PEM, viz., marasmus, kwashiorkor and marasmic kwashiorkor. The studies in experimental animals in the west and children in developing countries have revealed the adverse effects of PEM on the biochemistry of developing brain which leads to tissue damage and tissue contents, growth arrest, developmental differentiation, myelination, reduction of synapses, synaptic transmitters and overall development of dendritic activity. Many of these adverse effects have been described in children in clinical data, biochemical studies, reduction in brain size, histology of the spinal cord, quantitative studies and electron microscopy of sural nerve, neuro -CT scan, magnetic resonance imaging (MRI) and morphological changes in the cerebellar cells. Longer the PEM, younger the child, poorer the maternal health and literacy, more adverse are the effects of PEM on the nervous system. Just like the importance of nutrients on the developing brain, so are the adverse effects on the child development of lack of environmental stimulation, emotional support and love and affection to the child. When both the adverse factors are combined, the impact is severe. Hence prevention of PEM in pregnant and lactating mothers, breast feeding, adequate home based supplements, family support and love will improve the physical growth, mental development, social competence and academic performance of the child. Hence nutritional rehabilitation, psychosocial and psychomotor development of the child should begin in infancy and continue throughout. It should be at all levels, most important being in family, school, community and various intervention programmes, local, regional and national. Moreover medical students, health personnel, all medical disciplines concerned with total health care and school teachers should learn and concentrate on the developmental stimulation and enrichment of the child.

Cognitive development in children with chronic protein energy malnutrition

Bhoomika R Kar,¹ Shobini L Rao,² and B A Chandramouli³

Author information ► Article notes ► Copyright and License information ►

Behav Brain Funct. 2008; 4: 31. http://dx.doi.org:/10.1186/1744-9081-4-31

Background: Malnutrition is associated with both structural and functional pathology of the brain. A wide range of cognitive deficits has been reported in malnourished children. Effect of chronic protein energy malnutrition (PEM) causing stunting and wasting in children could also affect the ongoing development of higher cognitive processes during childhood (>5 years of age). The present study examined the effect of stunted growth on the rate of development of cognitive processes using neuropsychological measures.

Methods: Twenty children identified as malnourished and twenty as adequately nourished in the age groups of 5–7 years and 8–10 years were examined. NIMHANS neuropsychological battery for children sensitive to the effects of brain dysfunction and age related improvement was employed. The battery consisted of tests of motor speed, attention, visuospatial ability, executive functions, comprehension and learning and memory

Results: Development of cognitive processes appeared to be governed by both age and nutritional status. Malnourished children performed poor on tests of attention, working memory, learning and memory and visuospatial ability except on the test of motor speed and coordination. Age related improvement was not observed on tests of design fluency, working memory, visual construction, learning and memory in malnourished children. However, age related improvement was observed on tests of attention, visual perception, and verbal comprehension in malnourished children even though the performance was deficient as compared to the performance level of adequately nourished children.

Conclusion: Chronic protein energy malnutrition (stunting) affects the ongoing development of higher cognitive processes during childhood years rather than merely showing a generalized cognitive impairment. Stunting could result in slowing in the age related improvement in certain and not all higher order cognitive processes and may also result in long lasting cognitive impairments.

Malnutrition is the consequence of a combination of inadequate intake of protein, carbohydrates, micronutrients and frequent infections [1]. In India malnutrition is rampant. WHO report states that for the years 1990–1997 52% of Indian children less than 5 years of age suffer from severe to moderate under nutrition [2]. About 35% of preschool children in sub-Saharan Africa are reported to be stunted [3]. Malnutrition is associated with both structural and functional pathology of the brain. Structurally malnutrition results in tissue damage, growth retardation, disorderly differentiation, reduction in synapses and synaptic neurotransmitters, delayed myelination and reduced overall development of dendritic arborization of the developing brain. There are deviations in the temporal sequences of brain maturation, which in turn disturb the formation of neuronal circuits [1]. Long term alterations in brain function have been reported which could be related to long lasting cognitive impairments associated with malnutrition [4]. A wide range of cognitive deficits has been observed in malnourished children in India. In a study, malnourished children were assessed on the Gessell’s developmental schedule from 4 to 52 weeks of age. Children with grades II and III malnutrition had poor development in all areas of behaviour i.e., motor, adaptive, language and personal social [5]. Rural children studying in primary school between the ages of 6–8 years were assessed on measures of social maturity (Vineland social maturity scale), visuomotor co-ordination (Bender gestalt test), and memory (free recall of words, pictures and objects). Malnutrition was associated with deficits of social competence, visuomotor coordination and memory. Malnutrition had a greater effect on the immediate memory of boys as compared with those of girls. Malnourished boys had greater impairment of immediate memory for words, pictures and objects, while malnourished girls had greater impairment of immediate memory for only pictures. Delayed recall of words and pictures of malnourished boys was impaired. Malnourished girls had an impairment of delayed recall of only words. The same authors measured the intelligence of malnourished children using Malin’s Indian adaptation of the Wechsler’s intelligence scale for children. IQ scores decreased with the severity of malnutrition. Significant decreases were observed in performance IQ, as well as on the subtests of information and digit span among the verbal subtests [6]. The above study has shown that though there is decrease in full scale IQ, yet performance on all the subtests was not affected. This suggests that malnutrition may affect different neuropsychological functions to different degrees. Studies done in Africa and South America have focused on the effect of stunted growth on cognitive abilities using verbal intelligence tests based on assessment of reasoning [7]. Such an assessment does not provide a comprehensive and specific assessment of cognitive processes like attention, memory, executive functions, visuo-spatial functions, comprehension as conducted in the present study. Information about the functional status of specific cognitive processes has implications for developing a cognitive rehabilitation program for malnourished children. A neuropsychological assessment would throw light on functional status of brain behaviour relationships affected by malnutrition. Deficits of cognitive, emotional and behavioural functioning are linked to structural abnormalities of different regions of the brain. Brain structures and brain circuits compute different components of cognitive processes [8]. Malnutrition has long lasting effects in the realm of cognition and behaviour, although the cognitive processes like executive functions have not been fully assessed [9]. The differential nature of cognitive deficits associated with malnutrition suggests that different areas of the brain are compromised to different degrees. A neuropsychological assessment would be able to delineate the pattern of brain dysfunction. Malnutrition is a grave problem in our country as 52% of our children are malnourished. Effects of protein-calorie malnutrition are inextricably blended with the effects of social cultural disadvantage; even within the disadvantaged class, literacy environment at home and parental expectation regarding children’s education are powerful variables. Perhaps membership in a higher caste confers some advantage in regard to home literacy, and parental expectation. Short and tall children do differ in some cognitive tests, but not in all as demonstrated in a study done in Orissa, India [10]. But whether or not stunted growth alone is the causative variable for cognitive weakness is not determined as yet. Moreover, the functional integrity of specific cognitive processes is less clear. Chronic PEM resulting in stunting and wasting could result in delay in the development of cognitive processes or in permanent cognitive impairments. Neuropsychological measures can demonstrate delay in normally developing cognitive processes as well as permanent cognitive deficits.

Children in the age range of 5–10 years attending a corporation school in the city of Bangalore participated in the study. Corporation schools in India are government schools with minimal fee attended by children from lowmiddle class. There were 20 children in adequately nourished group and 20 in the malnourished group. The gender distribution was equal. Children in both the groups were from the same ethnic/language background. They were natives of Karnataka living in Bangalore.

After identifying the malnourished and adequately nourished children the coloured progressive matrices test [12] was administered to rule out mental retardation. Children falling at or below the fifth percentile were excluded from the sample, as the 5th percentile is suggestive of intellectually defective range. The percentile points were calculated from the raw scores using Indian norms [13]. Mental retardation was ruled out as otherwise scores on neuropsychological tests would be uniformly depressed and a differentiation of deficits might not occur. Intelligence was not treated as a covariate in the study. The groups did not differ significantly in their scores on CPM (a screening instrument to rule out intellectual impairment in both the groups).

Table 1: Demographic details of the participants

Adequately nourished N = 20 Malnourished N = 20

Mean age 5–7 years 8–10 years 5–7 years 8–10 years

5.8 years 8.8 years 6.3 years 9.3 years

Gender Girls:10 Boys: 10 Girls:10 Boys: 10

Stunted %

(height for age -2 SD from the median) —- 70%

Stunted and wasted %

(height for age and

weight for height: -2 SD from the median) —- 30%

Exclusion of behaviour problems and history of neurological disorders The children’s behaviour questionnaire form B [14] was administered to the class teachers of the identified children. Children who scored above the cut off score of 9 were not included in the sample. The personal data sheet was filled in consultation with the parents and teachers to rule out any history of any neurological/psychiatric disorders including head injury and epilepsy and one child with epilepsy was excluded. This was one of the exclusion criteria.

Exclusion of behaviour problems and history of neurological disorders The children’s behaviour questionnaire form B [14] was administered to the class teachers of the identified children. Children who scored above the cut off score of 9 were not included in the sample. The personal data sheet was filled in consultation with the parents and teachers to rule out any history of any neurological/psychiatric disorders including head injury and epilepsy and one child with epilepsy was excluded. This was one of the exclusion criteria.

The tests have been grouped under specific cognitive domains on the basis of theoretical rationale and factor analysis. Factor analysis has been done for the battery and the grouping of tests under cognitive functions like executive functions, visuospatial functions, comprehension and learning and memory was done on the basis of the clustering observed in factor analysis as well as on theoretical grounds

The neuropsychological battery consisted of the following tests:

1. Motor speed Finger tapping test [15]

2. Expressive speech Expressive speech test was administered to rule out speech related deficits

3. Attention Color trails test [18] is a measure of focused attention and conceptual tracking.

4. Color cancellation test [21] is a measure of visual scanning/selective attention

5. Executive functions FAS phonemic fluency test is a measure of verbal fluency.

6. Design fluency test [24] is a measure of design fluency, cognitive flexibility and imaginative capacity.

7. Visuo-spatial working memory span task [23]: This test is a measure of visuo-spatial working memory (VSWM) span.

8. Visuospatial functions Motor-free visual perception test [29] is a measure of visuoperceptual ability, having 36 items for visual discrimination, visual closure, figure-ground, perceptual matching and visual memory. Since this test has been originally developed for children between 5–8 years of age, it was modified and items in increasing difficulty level were added by the authors to make it applicable for the children above 8 years. Number of correct responses comprises the score.

9. Picture completion test [30] is a measure of visuoconceptual ability, visual organization and visuo-conceptual reasoning.

10. Block design test [30] is a measure of visuoconstructive ability.

11. Comprehension, learning and memory Token test [31] is a measure of verbal comprehension of commands of increasing complexity.

12. Rey’s auditory verbal learning test (RAVLT) [32] is a measure of verbal learning and memory.

13. Memory for designs test [34] is a measure of visual learning and memory.

Comparison between the performance of adequately nourished children and malnourished children Table 2.0 shows that malnourished group differed significantly from the adequately nourished group on tests of phonemic fluency, design fluency, selective attention, visuospatial working memory, visuospatial functions, verbal comprehension and verbal learning and memory showing poor performance. The two groups did not differ on the test of finger tapping. Since expressive speech was a question answer type assessment looking at repetitive speech, nominative speech and narrative speech, which is like an initial screening for aphasia, like symptoms. Since it did not give a quantitative score, hence was not taken for analysis. As a descriptive account of expressive speech it was observed that malnourished children did not have any difficulty with respect to expressive speech.

Comparison of age related differences in cognitive functions between adequately nourished and malnourished children Data was further subjected to post hoc analysis to compare the two groups across the two age groups to study the rate of improvement with age (Table 2). In both the age groups of 5–7 years and 8–10 years the adequately nourished children performed better than the malnourished children. Figures 1, 2, 3, 4, 5, 6 indicate age related improvement in performance across different cognitive functions in adequately nourished children as compared to malnourished children. Motor speed and coordination was not significantly affected in malnourished children as compared to the adequately nourished children (figure 1). The rate of age related improvement across the two age groups was found rapid on certain functions like selective attention (figure 2) and verbal fluency (figure 3) in malnourished children. However, working memory, design fluency, visuospatial functions, comprehension, learning, and memory showed slowing in terms of age related improvement in malnourished children. Most of the cognitive functions like design fluency (figure 3), working memory (figure 3), Visual perception (figure 4), visuoconceptual reasoning (figure 4), visual construction (figure 4), verbal comprehension (figure 5), verbal and visual memory (figures 6) have shown a very slow rate of improvement with respect to the difference in performance between the two age groups of 5–7 and 8–10 years. On the contrary functions like verbal fluency (figure 3), motor speed (figures 1), and selective attention (figure 2) showed similar rates of improvement in adequately nourished children and malnourished children while comparing the two age groups.

Table 2: Mean comparisons for the cognitive functions across the two age groups of adequately nourished and malnourished children (not shown)

Table 3: Post-hoc comparisons between adequately nourished and malnourished groups across the two age groups (not shown)

Figure 1 Age related comparisons between adequately nourished and malnourished children on motor speed (right and left hand) Age related comparisons between adequately nourished and malnourished children on motor speed (right and left hand). (not shown)

Figure 2 Age related comparisons between adequately nourished and malnourished children on selective attention (color cancellation test). (not shown)

Post-hoc comparisons were computed with Tukey’s posthoc tests to compare the means across age groups between malnourished and adequately nourished children for those test scores that showed significant effects. Hence, post hoc tests were not computed for the finger tapping test scores assessing motor speed. Table 3 presents the post-hoc results with the significance (probability level) levels of the differences across age groups and between adequately nourished and malnourished children. Post hoc results have been done to support our theoretical claims about the lack of age related improvement in certain cognitive functions on one hand and the nature of cognitive impairments on the other in malnourished children. Four comparisons were interpreted i.e., comparing performance between the two age groups of adequately nourished and malnourished children separately. The other comparison was between the adequately nourished and malnourished children for the age group of 5–7 years and similarly for the age group of 8–10 years. Results indicate age related differences within each group as well as between the two groups. Age related differences were found significant for some of the test scores between 5–7 and 8–10 year old children in the adequately nourished group but not for most of the test scores for malnourished group indicative of a delay in development of certain cognitive functions. Differences were found significant between the adequately nourished and malnourished children for the same age group for most of the test scores indicative of a deficit in a particular cognitive function. In few of the tests, performance was not found to be significantly different between the two age groups for both adequately nourished and malnourished children.

Discussion The findings of the present study could be discussed in terms of the effect of chronic malnutrition on neuropsychological performance and with respect to the rate of development of cognitive processes.

Effect of malnutrition on neuropsychological performance Our study indicates that malnourished children perform poor on most of the neuropsychological tests except that of motor speed as compared to adequately nourished children. Malnourished children showed poor performance on tests of higher cognitive functions like cognitive flexibility, attention, working memory, visual perception, verbal comprehension, and memory. These findings are supported by another study on Indian malnourished children, which reported memory impairments in undernourished children and spared fine motor coordination [36]. Malnourished children showed poor performance on novel tasks like tests of executive functions i.e., working memory spatial locations. Poor performance on the tests of fluency and working memory also coincides with very slow rate of improvement between the age groups of 5–7 years and 8–10 years. Poor performance on most of the neuropsychological tests indicated a diffuse impairment including attention, executive functions, visuospatial functions, comprehension and memory.

Effect of malnutrition on cognitive development Both the groups were tested on a neuropsychological battery, which has been found to be sensitive to age related differences in cognitive functions in children (5–15 years). The age trends reported in the present study are based on the assessment that employed the NIMHANS neuropsychological battery for children [13]. The test battery has been standardized based on the growth curve modeling approach for empirical validation of age-related differences in performance on neuropsychological tests. The tests in the battery were found sensitive to show age related differences.

Malnourished children showed poor performance with respect to age as compared to adequately nourished children. The performance of malnourished children in the 5–7 years age group was poor and much lower than the adequately nourished children and did not seem to show much improvement in the 8–10 years age group. The rate of cognitive development was found to be different for different cognitive functions. The rate of development was affected for some of the cognitive functions showing minimal age related improvement across the age range of 5–7 years and 8–10 years such as design fluency, working memory, visual construction, verbal comprehension, learning and memory for verbal and visual material. On the contrary, age related improvement was observed on certain other cognitive functions in malnourished children, where the level of performance was low for both the age groups but the rate of improvement between the two age groups was similar to adequately nourished children.

Not shown

Figure 3 Age related comparisons between adequately nourished and malnourished children on executive functions.

Note: VF: verbal fluency; DF: design fluency; WM: working memory; AN: adequately nourished; MN: malnourished.

MN 5–7 vs 8–10 p > .05 5–7 years AN vs MN p > .05 8–10 years AN vs MN p < .05 Visual memory (memory for designs test) AN 5–7 vs 8–10 p > .05 MN 5–7 vs 8–10 p > .05 5–7 years AN vs MN p < .05 8–10 years AN vs MN p < .05

Figure 4 Age related comparisons between adequately nourished and malnourished children on visuospatial functions.

Figure 5 Age related comparisons between adequately nourished and malnourished children on verbal comprehension and verbal learning.

Motor speed (right and left hand) was not found impaired in malnourished children and the rate of development was also found similar to adequately nourished children.

Executive functions such as design fluency, selective attention and working memory were found deficient in malnourished children also showing poor rate of improvement between the two age groups. All the three tests of executive functions like fluency, selective attention and working memory for spatial locations involved novel stimuli and performance required cognitive flexibility as well as faster information processing which was affected in malnourished children. Results also indicate that malnourished children showed a very slow rate of improvement on these functions.

Visuo-spatial functions like visual perception, visual construction and visuo-conceptual reasoning showed significantly poor performance when compared to the adequately nourished children but showed a steep age related improvement in performance. Performance on functions like visual perception (visual discrimination, perceptual matching, visual closure and visuospatial relationships) and visual construction was severely affected in malnourished children and also showed poor rate of improvement with age.

Verbal comprehension, learning and memory for verbal and visual material was found poor as compared to adequately nourished children but the rate of improvement between 5–7 years age group and 8–10 years age group was similar to that of adequately nourished children. These results suggest that development of comprehension with age might not be affected in malnourished children. However, other than the poor performance on the AVLT test of verbal learning, malnourished children also showed minimal improvement between the two age groups as compared to the greater magnitude of difference between the two age groups in adequately nourished children. Visual memory was most severely affected in malnourished children in terms of the poor performance on delayed recall on design learning test as well as in terms of the difference between the two age groups.

Malnutrition affects brain growth and development and hence future behavioral outcomes [37]. School-age children who suffered from early childhood malnutrition have generally been found to have poorer IQ levels, cognitive function, school achievement and greater behavioral problems than matched controls and, to a lesser extent, siblings. The disadvantages last at least until adolescence. There is no consistent evidence of a specific cognitive deficit [38]. The functional integrity of specific cognitive processes is less clear. Stunting in early childhood is common in developing countries and is associated with poorer cognition and school achievement in later childhood [39]. Deficits in children’s scores have been reported to be smaller at age 11 years than at age 8 years in a longitudinal study on malnourished children stunted children suggesting that adverse effects may decline over time [7]. In our study also all the children in malnourished group were stunted and the cross sectional assessment of age related improvement has shown similar rate of improvement across 5–7 years to 8–10 years age groups as observed in adequately nourished children though the baseline performance was low in malnourished children. These results indicate that the adverse effects of malnutrition (stunting in particular) may decline with age only for certain cognitive functions but the rate of cognitive development for most of the cognitive processes particularly higher cognitive processes including executive processes and visuospatial perception could be severely affected during the childhood years. Decline in the effects of malnutrition overtime has been reported to be independent of differences in educational, socioeconomic and psychosocial resources [7]. Hence, malnutrition (particularly stunting) may result in delayed development of cognitive processes during childhood years rather than a permanent generalized cognitive impairment.

The neuropsychological interpretation of the cognitive processes more severely affected in malnourished children suggests a diffuse cortical involvement. This is with reference to deficits pertaining to functions mediated by dorsolateral prefrontal cortex (poor performance on tests of attention, fluency and working memory), right parietal (poor performance on tests of visuospatial functions) and bilateral temporal cortex (poor performance on tests of comprehension, verbal learning, and memory for verbal and visual material). The prefrontal cortex may be particularly vulnerable to malnutrition [4]. The adverse effects of malnutrition (PEM-stunting) on cognitive development could be related to the delay in certain processes of structural and functional maturation like delayed myelination and reduced overall development of dendritic arborization of the developing brain [1].

The present study highlights two ways in which malnutrition particularly stunting could affect cognitive functions. On one hand age related improvement in cognitive performance is compromised and on the other hand there could be long lasting cognitive impairments as well. However, the effect is nor specific to a particular cognitive domain and is rather more diffuse. Results of the study also indicate that: certain cognitive functions could be vulnerable to the effect of malnutrition in terms of showing impairment but the rate of development of these functions may not be affected. On the other hand, rate of development of certain cognitive functions may be affected and may also show impairment when compared with adequately nourished children.

Conclusion Chronic protein energy malnutrition (stunting) results in cognitive impairments as well as slowing in the rate of the development of cognitive processes. Rate of development of cognitive functions may follow different patterns in children with malnutrition. Chronic protein energy malnutrition affects the development of cognitive processes differently during childhood years rather than merely showing an overall cognitive dysfunction as compared to adequately nourished children. Stunting could result in delay in the development of cognitive functions as well as in permanent cognitive impairments which show minimal improvement with increase in age. Rate of development of attention, executive functions like cognitive flexibility, working memory, visuospatial functions like visual construction is more severely affected by protein energy malnutrition in childhood years, a period that is marked by rapid ongoing development of cognitive functions.

http://www.behavioralandbrainfunctions.com/content/4/1/31

The effects of protein energy malnutrition in early childhood on intellectual and motor abilities in later childhood and adolescence.

Hoorweg J, Stanfield JP.

Dev Med Child Neurol. 1976 Jun;18(3):330-50.

http://www.ncbi.nlm.nih.gov/pubmed/820586

http://dx.doi.org:/10.1111/j.1469-8749.1976.tb03656.x

Three groups of Ugandan children (20 in each group) and one comparison group of 20 children were examined between 11 and 17 years of age. The first three groups had been admitted to hospital for treatment of protein energy malnutrition between the ages of eight to 15, 16 to 21 and 22 to 27 months, respectively. The comparison group had not been clinically malnourished throughout the whole period up to 27 months of age. All the children came from one tribe and were individually matched for sex, age, education and home environment. It was found that the three malnourished groups fell significantly below the comparison group in anthropometric measurements and in tests of intellectual and motor abilities. No evidence was found for a relationship between the deficit and age at admission. Further analysis among the 60 malnourished children revealed that anthropometry and intellectual and motor abilities are the more affected the greater the degree of ‘chronic undernutrition’ at admission, but no correlation was found with the severity of the ‘acute malnutrition’. The results show a general impairment of intellectual abilities, with reasoning and spatial abilities most affected, memory and rote learning intermediately and language ability least, if at all, affected. These findings are discussed in the context of a comprehensive and critical appraisal of the existing literature.

Quake-Hit Nepal Gears up to Tackle Stunting in Children

By Gopal Sharma July 08, 2015 http://www.medscape.com/viewarticle/847572

HECHO, Nepal (Thomson Reuters Foundation) – Shanti Maharjan, who gave birth to a baby girl 10 days ago, has spent the last two months living under corrugated iron sheets with her husband and five others after two major earthquakes reduced her mud-and-brick home to rubble.

Adequate food, drinking water and aid such as tents and blankets have been hard to come by, she says, though scores of aid agencies rushed to the Himalayan nation to help survivors.
What worries the 26-year-old mother most is her inability to produce breastmilk for her new-born daughter, who she fears is at serious risk of malnutrition in the aftermath of the 7.8 and 7.3 magnitude quakes in April and May.

“The earthquake destroyed everything, including our food reserves,” said Maharjan, sitting under the iron sheeting on farmland on the outskirts of the capital, Kathmandu.

“There is not enough food. Getting meat, oil and fruits to eat is difficult in this situation. I am worried about my daughter’s nourishment,” she said as the baby, wrapped in a green cloth, lay sleeping on a wooden bed.

The government, aware that disruption caused by the quakes could worsen the country’s already high rate of child malnutrition is sending out teams of community nurses to give advice and food supplements to women and children in the affected areas.

A 2011 government study showed that more than 40% of Napel’s under-five-year-olds were stunted, showing that the country’s child malnutrition rate was one of the world’s highest.
Experts say the two quakes, which killed 8,895 people and destroyed half a million houses, could make things worse as survivors have inadequate food, water, shelter, healthcare and sanitation.

United Nations officials warn that the rate of stunting among children in the South Asian nation could return to the 2001 level of 57%, if authorities and aid agencies do not respond effectively.

“The risk of malnutrition is high and requires the nutrition and other sectors like agriculture, health, water, sanitation, education and social protection to respond adequately,” said Stanley Chitekwe, UNICEF’s nutrition chief in Nepal.

DRIVE TO NOURISH

Child malnutrition is an underlying cause of death for 3 million children annually around the world – nearly half of all child deaths – most of whom die from preventable illnesses such as diarrhoea due to weak immune systems.

Those lucky enough to survive grow up without enough energy, protein, vitamins and minerals, causing their brains and bodies to be stunted, and they are often unable to fulfill their potential.

Government officials admit the challenges, citing data showing that almost 70% of Nepali children under the age of two suffer from anaemia caused by iron deficiency.

“This shows that (poor) nutrition is a very big problem. The earthquake will further worsen the situation because people simply don’t have enough to eat, let alone have a nutritious diet,” said Health Ministry official Krishna Prasad Paudel.

Supported by UNICEF, authorities have now launched a drive to reach out to more than 500,000 women and children who need supplementary food and medicines.

More than 10,000 female community volunteers will be fanning out across 14 districts affected by the earthquakes, visiting devastated towns and villages and speaking to new and expectant mothers about breast-feeding their infants.

The volunteers will also advise families on eating locally available nutritious foods such as green vegetables and meat and will distribute vitamin A, iron and folic acid, and other micronutrient supplements to pregnant and breastfeeding women.

In Imadole, a prosperous district on the outskirts of the ancient town of Patan, health volunteer Urmila Sharma Dahal found an extremely thin two-year-old boy weighing 7.5 kg (16.5 pounds) last week, suffering from severe acute malnutrition.

Dahal said she provided his family with sachets of ready-to-use therapeutic food – a paste of peanut, sugar, milk powder, vitamin and oil – and the child gained nearly a kilo (2.2 pounds) in weight in just seven days.

“It does not take much. It can be done with small but right interventions,” said Dahal as she sat next to the child in the family’s brick-and-cement home.

Protein-energy malnutrition occurs due to inadequate intake of food and is a major cause of morbidity and mortality in children in developing countries (Grover and Ee 2009).

http://www.wcs-heal.org/global-challenges/public-health-issues-and-costs/malnutrition/protein-energy-malnutrition

http://www.wcs-heal.org/uploads/images/Chris_Golden-malnourished_children_692x513_scaled_cropp.jpg

Protein energy malnutrition (PEM) has significant negative impacts on children’s growth and development (Grover and Ee 2009). Chronic PEM causes children to have stunted growth (low height for age) and to be underweight (low weight for age); it is estimated that among children under age five, one in every four is stunted and one in every six is underweight. PEM also causes two specific conditions in children: marasmus, which is characterized by an emaciated appearance, and kwashiorkor, in which children develop swollen bellies due to edema (abnormal accumulation of fluid) and discoloration of the hair because of pigment loss among other symptoms (UNWFP 2013b, Ahmed et al. 2012). Countries in sub-Saharan Africa and south Asia have the highest proportions of children suffering from PEM (UNWFP 2013a).

PEM causes direct mortality in children and also increases vulnerability to other serious diseases including diarrhea, pneumonia, and malaria. Children suffering from PEM have compromised immune systems, making them particularly susceptible to infectious diseases. Furthermore, PEM has negative impacts on children’s brain development, resulting in issues with memory and delayed motor function; these children have decreased ability to learn and have lower productivity as adults. PEM also has serious and potentially long-term impacts on other organ systems including the cardiovascular, respiratory, and gastrointestinal systems (Grover and Ee 2009).

Many adults in developing countries also suffer from PEM, with women disproportionately impacted compared with men, particularly in south Asian countries (UNWFP 2013a). Pregnant women who are undernourished can fall even further behind in their nutritional status due to the increased demand for nutrients by the developing fetus. Women who don’t gain sufficient weight during pregnancy are at increased risk for complications including maternal morbidity and mortality, low birth weight, and neonatal mortality. These women can also have difficulty providing sufficient quantities of breast milk, leading to malnutrition among neonates (Ahmed et al. 2012).

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Improving learning and memory

Posted in Neurodegenerative Diseases, Neurological Diseases, Neuroscience, tagged Aging, memory on October 25, 2015| Leave a Comment »

Improving learning and memory

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Improving learning and memory in aged mice with cholesterol-binding membrane protein

October 22, 2015

http://www.kurzweilai.net/improving-learning-and-memory-in-aged-mice-with-cholesterol-binding-membrane-protein?utm_source=KurzweilAI+Weekly+Newsletter_147a5a48c1-9a20162408-282099089

SynCav1 gene delivery enhances granule cell neuron dendritic arborization (neuron branching in adult mice. Scale bar: 20 micrometers. (credit: Chitra D. Mandyam et al./Biological Psychiatry)

http://www.kurzweilai.net/images/neuron-arbolization.jpg

Using gene therapy to increase a crucial cholesterol-binding membrane protein called caveolin-1 (Cav-1) in neurons in the hippocampus* of the brain improved learning and memory in aged mice, according to findings from a new study led by scientists at The Scripps Research Institute (TSRI), the Veterans Affairs San Diego Healthcare System (VA) and University of California (UC) San Diego School of Medicine.

The result for treated mice was improved neuron growth and better retrieval of contextual memories — they froze in place, an indication of fear, when placed in a location where they’d once received small electric shocks.

The researchers believe that this type of gene therapy may be a path toward treating age-related memory loss, including loss resulting from alcohol and drug use. The researchers are now testing this gene therapy in mouse models of Alzheimer’s disease and expanding it to possibly treat injuries such as spinal cord injury and traumatic brain injury. ”

The study, published recently online ahead of print in the journal Biological Psychiatry, expands scientists’ understanding of neuroplasticity, the ability of neural pathways to grow in response to new stimuli.

* The hippocampus is a structure in the brain thought to participate in the formation of contextual memories — for example, if one remembers a past picnic when later visiting a park.

Abstract of Neuron-targeted caveolin-1 improves molecular signaling, plasticity and behavior dependent on the hippocampus in adult and aged mice

Background: Studies in vitro demonstrate that neuronal membrane/lipid rafts (MLRs) establish cell polarity by clustering pro-growth receptors and tethering cytoskeletal machinery necessary for neuronal sprouting. However, the effect of MLR and MLR-associated proteins on neuronal aging is unknown.

Methods: Here we assessed the impact of neuron-targeted overexpression of a MLR scaffold protein, caveolin-1 (via a synapsin promoter; SynCav1), in the hippocampus in vivo in adult (6-months-old) and aged (20-month-old) mice on biochemical, morphologic and behavioral changes.

Results: SynCav1 resulted in increased expression of Cav-1, MLRs, and MLR-localization of Cav-1 and tropomyosin-related kinase B (TrkB) receptor independent of age and time post gene transfer. Cav-1 overexpression in adult mice enhanced dendritic arborization within the apical dendrites of hippocampal CA1 and granule cell neurons, effects that were also observed in aged mice, albeit to a lesser extent, indicating preserved impact of Cav-1 on structural plasticity of hippocampal neurons with age. Cav-1 overexpression enhanced contextual fear memory in adult and aged mice demonstrating improved hippocampal function.

Conclusions: Neuron-targeted overexpression of Cav-1 in the adult and aged hippocampus enhances functional MLRs with corresponding roles in cell signaling and protein trafficking. The resultant structural alterations in hippocampal neurons in vivo are associated with improvements in hippocampal dependent learning and memory. Our findings suggest Cav-1 as a novel therapeutic strategy in disorders involving impaired hippocampal function.

references:

Chitra D. Mandyam, Jan M. Schilling, Weihua Cui et al. Neuron-targeted caveolin-1 improves molecular signaling, plasticity and behavior dependent on the hippocampus in adult and aged mice. Biological Psychiatry, October 07, 2015; DOI: 10.1016/j.biopsych.2015.09.020

Topics: Cognitive Science/Neuroscience

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battling depression

Posted in Neurological Diseases, Neuroscience, tagged depression, microglia on October 23, 2015| Leave a Comment »

battling depression

Larry H Bernstein, MD, FCAP, Curator

LPBI

Israeli scientists tout new method for battling depression

By focusing not on the brain’s neurons but on cells known as microglia, researchers may have mapped a path to innovative treatments

BY TIMES OF ISRAEL STAFF October 22, 2015

http://www.timesofisrael.com/israeli-scientists-tout-new-method-for-battling-depression/

Prof. Raz Yirmiya, who heads Hebrew University’s psychoneuroimmunology laboratory, is the senior author of a new paper, “Depression as a microglial disease,” published in the October issue of Trends in Neurosciences.

The paper urges a new focus in depression research away from neurons, the cells that make up the brain’s thinking faculties and allow it to control the body, and toward brain cells called microglia.

About 10 percent of brain cells are microglia, which serve as a kind of infrastructure for the brain, holding neurons in place, passing oxygen and other nutrients to them and fighting brain infections.

Microglia play a special role in repairing brain damage and trauma to neurons, the researchers note.

“Our views on microglia have dramatically changed over the last decade,” Yirmiya said in a Thursday statement by Hebrew University.

“We now know that these cells play a role in the formation and fine-tuning of the connections between neurons [known as synapses] during brain development, as well as in changes [to] these connections throughout life. These roles are important for normal brain and behavioral functions, including pain, mood and cognitive abilities.”

According to Yirmiya, who wrote the paper together with fellow Hebrew University researchers Neta Rimmerman and Ronen Reshef, “Studies in humans, using post-mortem brain tissues or special imaging techniques, as well as studies in animal models of depression, demonstrated that when the structure and function of microglia change, these cells can no longer regulate normal brain and behavior processes and this can lead to depression.”

Major depression “afflicts one in six people at some point in their life,” the university’s statement on the new research noted, and is “the leading global cause of disability – surpassing cardiovascular and respiratory diseases, cancer and HIV/AIDS combined.”

“Progress in the understanding of the biology of depression has been slow,” the researchers note in the paper. New research into the role of malfunctioning microglia could lead to a new push toward finding different and perhaps more effective treatment.

Changes in microglia are found in patients with conditions that correlate with high instances of depression, including “infection, injury, trauma, aging, autoimmune diseases such as multiple sclerosis and neurodegenerative diseases such as Alzheimer’s disease,” the statement explains. Microglia in these conditions take on an “activated” state, growing larger and round while secreting chemicals that lead to inflammation in the brain.

Microglia also change their shape and functioning when exposed to chronic unpredictable psychological stress, which is one of the leading causes of depression. Such conditions have also been found – in research carried out in Yirmiya’s Hebrew University laboratory – to correlate with microglia dying and remaining cells appearing shrunken and degenerated.

Drugs that inhibit overactive microglia or stimulate suppressed microglia may be the key to dramatic new treatments, they argue.

“Prof. Yirmiya’s new research could have a profound impact on the future development of anti-depressant medications. Present drugs do not always have the desired effect on patients, so there is an urgent need to discover novel biological mechanisms and drug targets for diagnosing the root cause of depression and for treating depressed patients appropriately,” the statement read.

New drugs that restore function to diseased microglia, the researchers suggest, may turn out to be as effective as current fast-acting anti-depressants.

https://tpc.googlesyndication.com/pagead/js/r20151014/r20110914/abg.js/

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Critical Gene in ALS

Posted in Neurodegenerative Diseases, Neurological Diseases, Neuroscience, tagged amyotropic lateral sclerosis (ALS), TDP-43 on October 22, 2015| Leave a Comment »

Critical Gene in ALS

Larry H Bernstein, MD, FCAP, Curator

LPBI

Role Found for Critical Gene in 95% of ALS

Thu, 10/22/2015 – Cynthia Fox, Science Writer

http://www.biosciencetechnology.com/articles/2015/10/role-found-critical-gene-95-als

Like a bad teenager, in 95 percent of all amyotrophic lateral sclerosis (ALS) cases, a protein called TDP-43 leaves its home— the nucleus of motor neuron cells—to congregate, in suspect fashion, in the cytoplasm.In a study published in Science this summer, the Johns Hopkins University (JHU) team of pathologist Phillip Wong, Ph.D., offered new insight into this molecular rebellion. It confirmed a function of normal TDP-43 in the nucleus: orchestrating proper RNA splicing and exon formation. It confirmed what lack of nuclear TDP-43 does: creates improper “cryptic” exons. And it identified proteins that mitigate effects of nuclear TDP-43 loss: potential drug leads.

“The recently published work in Science very clearly demonstrates that in the absence of TDP-43, RNA is misspliced, and in many cases targeted for degradation,” University of Michigan neurologist Sami Barmada, M.D., Ph.D., told Bioscience Technology. Barmada was uninvolved with the research. “A very real consequence of such dysfunctional RNA splicing and degradation is an inability to maintain cell health, ultimately resulting in neuron loss. The authors assembled an intriguing story that tells us quite a bit about how TDP-43 functions, and what happens to those functions in diseases such as ALS, and fronto-temporal dementia (FTD). If the mechanism identified in this manuscript does indeed underlie toxicity due to TDP-43 mislocalization, then it may very well contribute to neuron loss in the vast majority of ALS.”

Medical Research Council (MRC) Laboratory of Molecular Biology neurologist Jernej Ule, Ph.D., toldBioscienceTechnology: “Cryptic exons are interesting. I’m glad that this study will raise the interest in them.” Ule was also uninvolved with the new work.

TDP-43’s central role

For about a decade, it has been known that TDP-43 plays a role in ALS. In 2011 and 2012, a series of papers demonstrated “the profound role of TDP-43 in RNA splicing,” Barmada said. Those papers came out of the labs of University of California San Diego neurologist Don Cleveland, Ph.D.; Ule; University of Toronto neurologist Janice Robertson,Ph.D.; University of Texas Southwestern Medical Center neurologist Gang Yu, Ph.D.; and University of Milan neurologist Antonia Ratti, Ph.D.

Since then, while it has remained unclear “precisely how TDP-43 modulates splicing, we know that it many cases it enhances exon inclusion, while in others it reduces exon inclusion,” Barmada said. In that same 2011 to 2012 time period, “[Washington University neurologist] Youhna Ayala and her colleagues showed that TDP-43 regulates the splicing of its own transcript, targeting it for degradation by RNA decay pathways. So we knew that TDP-43-mediated splicing events can stabilize—or destabilize—RNA.”

The Wong group

For the Science paper, Wong’s group silenced the TDP-43 gene in mouse cells in vitro, then sequenced the RNA. They discovered multitudes of exons were erroneously spliced in: “cryptic exons.” Those cells died.

But when they restored cryptic exon suppression function via other proteins, cells without TDP-43 survived.

The team also silenced TDP-43 in human cells in vitro. Many cryptic exons again appeared in the mRNA.

When the team examined autopsied brain tissue of ALS patients, the same cryptic exons were found in mRNA—but none in controls.

The crew also found evidence of a “positive-feedback loop.” Normal proteins left ungenerated thanks to cryptic exons and TDP-43 depletion included RANBP1. RANBP1 enables the import of TDP-43 into the cell nucleus. The team hypothesized that TDP-43 depletion in the nucleus, leading to less cryptic exon suppression, would result in less functional RANBP1. This may then reduce TDP-43 levels in the nucleus even more.

Suppressing cryptic exons: not TDP-43’s main role

As noted, Barmada believes the work confirms that, without working TDP-43, RNA is misspliced, resulting in neuron loss. The study did not, however, convince him the main function of normal TDP-43 is to suppress cryptic exons. “What this manuscript tells me is that inappropriate RNA splicing leads to destabilization,” he told Bioscience Technology. “TDP-43, by virtue of its ability to bind thousands of RNA transcripts, is in a unique position to modulate the splicing of a significant fraction of the expressed genome. Abnormal localization or levels of TDP-43 can therefore have dramatic downstream consequences for RNA splicing and subsequent RNA degradation.”

Barmada said, furthermore, he suspects the link to ALS is even more complicated .

“Cytoplasmic TDP-43 mislocalization is a pathologic hallmark of over 95 percent of ALS, and over half of FTD,” he said. “Cytoplasmic TDP-43 deposition is truly lethal for neurons. The study by Wong and colleagues suggests that nuclear TDP-43 depletion mimics the effects seen in FTD, but there also may be a direct and toxic role for excess TDP-43 in the cytoplasm. TDP-43’s RNA binding pattern is different in the cytoplasm, and it forms ribonucleoprotein particles (RNPs) that may sequester RNA and prevent its translation. My guess is that it’s a combination of both: absence of TDP-43 from the nucleus—resulting in aberrant splicing and RNA decay—and excess cytoplasmic TDP-43, sequestering RNA in RNPs.”

Ule told Bioscience Technology he believes it is important to look at cryptic exons.“We’ve been studying them now for a few years, starting with a study of hnRNP [heterogeneous RNP] C-repressed exons a couple of years ago..”

Does TDP-43 stand out?

However, Ule said, “in the case of TDP-43, I’m still not sure that its role in regulating cryptic exons stands out compared to other hnRNPs. It seems likely to me that most hnRNPs regulate some cryptic exons, though this remains to be fully examined.”

Moreover, said Ule, who (as noted) generated a seminal early paper finding TDP-43 involved in RNA splicing: “TDP-43 regulates many alternative exons as shown by many papers, including one of ours. This study doesn’t convince me that the role of cryptic exons stands out compared to other exons that TDP-43 regulates.”

Finally, he noted, a “huge number of splicing changes occurs in FTD tissues” as shown in his workand that of others “so again, the fact that a few cryptic exons are also misspliced doesn’t convince me that they have a causative role. A more systematic analysis of other model systems would be required for this.”

Mount Sinai Icahn School of Medicine’s Alan Renton, Ph.D., co-wrote a 2014 NatureNeurosciencereview hailing the “landmark” discovery of links between TDP-43, ALS, and FTD. The new paper, he told BioscienceTechnology, “presents good evidence from mouse and human cell culture models that an important normal role of TDP-43 is to suppress the cryptic exon inclusion. The cell model data shows this ability of TDP-43 affects a subset of genes, for some of these genes cryptic exon inclusion appears to neutralize expression from one or both gene copies, and cryptic exon inclusion can sometimes cause cell death.”

The new paper, he continued, “corroborated some of these findings in post-mortem brain tissue obtained from ALS or FTD patients and normal controls. For two genes they detected RNA transcripts that included the predicted cryptic exon in disease cases but not controls. Given that TDP-43 is mislocalized to the cytoplasm of brain cells in the majority of ALS patients, this paper suggests resultant cryptic exon inclusion could be a pathogenic mechanism contributing to the neuronal cell death that characterizes this disease.”

However, he added, “this tentative conclusion would be significantly bolstered by in vivo evidence generated in an animal model system such as mouse.”

“Plenty” to do

Barmada agrees there is “plenty” more to do.

“The first question is what these changes in splicing mean for the proteins encoded by these genes,” he told Bioscience Technology. “Are there changes in protein abundance that reflect the differences in splicing and stability? Following from this, what effect will such changes have on the purported function of these proteins?”

These are some of the downstream questions that “look at the consequences of TDP-43-mediated missplicing on neurodegeneration,” he said. “There are also several upstream questions, concerning how or when TDP-43 is depleted from the nucleus or accumulates in the cytoplasm. One might argue that these are the more clinically relevant questions, since buried in their answers may be ways to prevent such dysfunction from occurring in the first place.”

But he certainly agrees with the authors’ contention in the manuscript that “numerous genetic mutations associated with familial ALS-FTD—VCP, GRN, OPTN, ATXN2, SQSTM1, UBQLN2, PFN1, TBK1, and especially C9ORF72—result in TDP-43 proteinopathy, suggesting a convergent mechanism of neurodegeneration.”

“Absolutely. This is an essential feature of ALS. Nearly 95 percent of all people with ALS demonstrate TDP-43 pathology, whether the disease arises sporadically or if it is inherited. Just about everyone who acquires ALS without a family history of the disease will have TDP-43 pathology, and the majority of those with inherited mutations—including all those listed— will exhibit TDP-43 pathology.”

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Archive for the ‘Neurological Diseases’ Category

Human Genetics and Childhood Diseases

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Insertions and deletions display length and positional preference

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Mutations associated with human disease

3.4. Survival time analyses to develop dementia, psychosis, dyskinesia, and wearing-off

Sequencing of genes associated with BrS

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Nutrition and Aging

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Role Found for Critical Gene in 95% of ALS

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