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Genomics and epigenetics link to DNA structure

Posted in Behavioral Genetics, Biological Networks, Gene Regulation and Evolution, Cancer Informatics, Cell Biology, Signaling & Cell Circuits, Cerebrovascular and Neurodegenerative Diseases, Chemical Genetics, Clinical & Translational, Clinical Genomics, Cognition, Computational Biology/Systems and Bioinformatics, Curation, Developmental biology, Disease Biology, DNA repair, Embryology, Enzyme Induction, Epigenetics and Cardiovascular Risks, Epigenetics and Environmental Factors, Exosomes, Gene Regulation and Evolution, Genome Biology, Genomic Endocrinology, Genomic Expression, Human aging, Mutagenesis, tagged , , , , , , , , , , on April 11, 2016| Leave a Comment »

Genomics and epigenetics link to DNA structure, Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 1: Next Generation Sequencing (NGS)

Genomics and epigenetics link to DNA structure

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Sequence and Epigenetic Factors Determine Overall DNA Structure

http://www.genengnews.com/gen-news-highlights/sequence-and-epigenetic-factors-determine-overall-dna-structure/81252592/

http://www.genengnews.com/Media/images/GENHighlight/Atomiclevelsimulationsshowingelectrostaticforcesbetweeneachatom1259202113.jpg

Atomic-level simulations show electrostatic forces between each atom. [Alek Aksimentiev, University of Illinois at Urbana-Champaign]

 

The traditionally held hypothesis about the highly ordered organization of DNA describes the interaction of various proteins with DNA sequences to mediate the dynamic structure of the molecule. However, recent evidence has emerged that stretches of homologous DNA sequences can associate preferentially with one another, even in the absence of proteins.

Researchers at the University of Illinois Center for the Physics of Living Cells, Johns Hopkins University, and Ulsan National Institute of Science and Technology (UNIST) in South Korea found that DNA molecules interact directly with one another in ways that are dependent on the sequence of the DNA and epigenetic factors, such as methylation.

The researchers described evidence they found for sequence-dependent attractive interactions between double-stranded DNA molecules that neither involve intermolecular strand exchange nor are mediated by DNA-binding proteins.

“DNA molecules tend to repel each other in water, but in the presence of special types of cations, they can attract each other just like nuclei pulling each other by sharing electrons in between,” explained lead study author Hajin Kim, Ph.D., assistant professor of biophysics at UNIST. “Our study suggests that the attractive force strongly depends on the nucleic acid sequence and also the epigenetic modifications.”

The investigators used atomic-level supercomputer simulations to measure the forces between a pair of double-stranded DNA helices and proposed that the distribution of methyl groups on the DNA was the key to regulating this sequence-dependent attraction. To verify their findings experimentally, the scientists were able to observe a single pair of DNA molecules within nanoscale bubbles.

“Here we combine molecular dynamics simulations with single-molecule fluorescence resonance energy transfer experiments to examine the interactions between duplex DNA in the presence of spermine, a biological polycation,” the authors wrote. “We find that AT-rich DNA duplexes associate more strongly than GC-rich duplexes, regardless of the sequence homology. Methyl groups of thymine act as a steric block, relocating spermine from major grooves to interhelical regions, thereby increasing DNA–DNA attraction.”

The findings from this study were published recently in Nature Communications in an article entitled “Direct Evidence for Sequence-Dependent Attraction Between Double-Stranded DNA Controlled by Methylation.”

After conducting numerous further simulations, the research team concluded that direct DNA–DNA interactions could play a central role in how chromosomes are organized in the cell and which ones are expanded or folded up compactly, ultimately determining functions of different cell types or regulating the cell cycle.

“Biophysics is a fascinating subject that explores the fundamental principles behind a variety of biological processes and life phenomena,” Dr. Kim noted. “Our study requires cross-disciplinary efforts from physicists, biologists, chemists, and engineering scientists and we pursue the diversity of scientific disciplines within the group.”

Dr. Kim concluded by stating that “in our lab, we try to unravel the mysteries within human cells based on the principles of physics and the mechanisms of biology. In the long run, we are seeking for ways to prevent chronic illnesses and diseases associated with aging.”

 

Direct evidence for sequence-dependent attraction between double-stranded DNA controlled by methylation

Jejoong Yoo, Hajin Kim, Aleksei Aksimentiev, and Taekjip Ha
Nature Communications 7 11045 (2016)    DOI:10.1038/ncomms11045BibTex

http://bionano.physics.illinois.edu/sites/default/files/styles/large/public/telepathy_figures_0.png?itok=VUJIHX2_

Although proteins mediate highly ordered DNA organization in vivo, theoretical studies suggest that homologous DNA duplexes can preferentially associate with one another even in the absence of proteins. Here we combine molecular dynamics simulations with single-molecule fluorescence resonance energy transfer experiments to examine the interactions between duplex DNA in the presence of spermine, a biological polycation. We find that AT-rich DNA duplexes associate more strongly than GC-rich duplexes, regardless of the sequence homology. Methyl groups of thymine acts as a steric block, relocating spermine from major grooves to interhelical regions, thereby increasing DNA–DNA attraction. Indeed, methylation of cytosines makes attraction between GC-rich DNA as strong as that between AT-rich DNA. Recent genome-wide chromosome organization studies showed that remote contact frequencies are higher for AT-rich and methylated DNA, suggesting that direct DNA–DNA interactions that we report here may play a role in the chromosome organization and gene regulation.

Formation of a DNA double helix occurs through Watson–Crick pairing mediated by the complementary hydrogen bond patterns of the two DNA strands and base stacking. Interactions between double-stranded (ds)DNA molecules in typical experimental conditions containing mono- and divalent cations are repulsive1, but can turn attractive in the presence of high-valence cations2. Theoretical studies have identified the ion–ion correlation effect as a possible microscopic mechanism of the DNA condensation phenomena3, 4, 5. Theoretical investigations have also suggested that sequence-specific attractive forces might exist between two homologous fragments of dsDNA6, and this ‘homology recognition’ hypothesis was supported by in vitro atomic force microscopy7 and in vivo point mutation assays8. However, the systems used in these measurements were too complex to rule out other possible causes such as Watson–Crick strand exchange between partially melted DNA or protein-mediated association of DNA.

Here we present direct evidence for sequence-dependent attractive interactions between dsDNA molecules that neither involve intermolecular strand exchange nor are mediated by proteins. Further, we find that the sequence-dependent attraction is controlled not by homology—contradictory to the ‘homology recognition’ hypothesis6—but by a methylation pattern. Unlike the previous in vitro study that used monovalent (Na+) or divalent (Mg2+) cations7, we presumed that for the sequence-dependent attractive interactions to operate polyamines would have to be present. Polyamine is a biological polycation present at a millimolar concentration in most eukaryotic cells and essential for cell growth and proliferation9, 10. Polyamines are also known to condense DNA in a concentration-dependent manner2, 11. In this study, we use spermine4+(Sm4+) that contains four positively charged amine groups per molecule.

Sequence dependence of DNA–DNA forces

To characterize the molecular mechanisms of DNA–DNA attraction mediated by polyamines, we performed molecular dynamics (MD) simulations where two effectively infinite parallel dsDNA molecules, 20 base pairs (bp) each in a periodic unit cell, were restrained to maintain a prescribed inter-DNA distance; the DNA molecules were free to rotate about their axes. The two DNA molecules were submerged in 100 mM aqueous solution of NaCl that also contained 20 Sm4+molecules; thus, the total charge of Sm4+, 80 e, was equal in magnitude to the total charge of DNA (2 × 2 × 20 e, two unit charges per base pair; Fig. 1a). Repeating such simulations at various inter-DNA distances and applying weighted histogram analysis12 yielded the change in the interaction free energy (ΔG) as a function of the DNA–DNA distance (Fig. 1b,c). In a broad agreement with previous experimental findings13, ΔG had a minimum, ΔGmin, at the inter-DNA distance of 25−30 Å for all sequences examined, indeed showing that two duplex DNA molecules can attract each other. The free energy of inter-duplex attraction was at least an order of magnitude smaller than the Watson–Crick interaction free energy of the same length DNA duplex. A minimum of ΔG was not observed in the absence of polyamines, for example, when divalent or monovalent ions were used instead14, 15.

Figure 1: Polyamine-mediated DNA sequence recognition observed in MD simulations and smFRET experiments.
Polyamine-mediated DNA sequence recognition observed in MD simulations and smFRET experiments.

(a) Set-up of MD simulations. A pair of parallel 20-bp dsDNA duplexes is surrounded by aqueous solution (semi-transparent surface) containing 20 Sm4+ molecules (which compensates exactly the charge of DNA) and 100 mM NaCl. Under periodic boundary conditions, the DNA molecules are effectively infinite. A harmonic potential (not shown) is applied to maintain the prescribed distance between the dsDNA molecules. (b,c) Interaction free energy of the two DNA helices as a function of the DNA–DNA distance for repeat-sequence DNA fragments (b) and DNA homopolymers (c). (d) Schematic of experimental design. A pair of 120-bp dsDNA labelled with a Cy3/Cy5 FRET pair was encapsulated in a ~200-nm diameter lipid vesicle; the vesicles were immobilized on a quartz slide through biotin–neutravidin binding. Sm4+ molecules added after immobilization penetrated into the porous vesicles. The fluorescence signals were measured using a total internal reflection microscope. (e) Typical fluorescence signals indicative of DNA–DNA binding. Brief jumps in the FRET signal indicate binding events. (f) The fraction of traces exhibiting binding events at different Sm4+ concentrations for AT-rich, GC-rich, AT nonhomologous and CpG-methylated DNA pairs. The sequence of the CpG-methylated DNA specifies the methylation sites (CG sequence, orange), restriction sites (BstUI, triangle) and primer region (underlined). The degree of attractive interaction for the AT nonhomologous and CpG-methylated DNA pairs was similar to that of the AT-rich pair. All measurements were done at [NaCl]=50 mM and T=25 °C. (g) Design of the hybrid DNA constructs: 40-bp AT-rich and 40-bp GC-rich regions were flanked by 20-bp common primers. The two labelling configurations permit distinguishing parallel from anti-parallel orientation of the DNA. (h) The fraction of traces exhibiting binding events as a function of NaCl concentration at fixed concentration of Sm4+ (1 mM). The fraction is significantly higher for parallel orientation of the DNA fragments.

Unexpectedly, we found that DNA sequence has a profound impact on the strength of attractive interaction. The absolute value of ΔG at minimum relative to the value at maximum separation, |ΔGmin|, showed a clearly rank-ordered dependence on the DNA sequence: |ΔGmin| of (A)20>|ΔGmin| of (AT)10>|ΔGmin| of (GC)10>|ΔGmin| of (G)20. Two trends can be noted. First, AT-rich sequences attract each other more strongly than GC-rich sequences16. For example, |ΔGmin| of (AT)10 (1.5 kcal mol−1 per turn) is about twice |ΔGmin| of (GC)10 (0.8 kcal mol−1 per turn) (Fig. 1b). Second, duplexes having identical AT content but different partitioning of the nucleotides between the strands (that is, (A)20 versus (AT)10 or (G)20 versus (GC)10) exhibit statistically significant differences (~0.3 kcal mol−1 per turn) in the value of |ΔGmin|.

To validate the findings of MD simulations, we performed single-molecule fluorescence resonance energy transfer (smFRET)17 experiments of vesicle-encapsulated DNA molecules. Equimolar mixture of donor- and acceptor-labelled 120-bp dsDNA molecules was encapsulated in sub-micron size, porous lipid vesicles18 so that we could observe and quantitate rare binding events between a pair of dsDNA molecules without triggering large-scale DNA condensation2. Our DNA constructs were long enough to ensure dsDNA–dsDNA binding that is stable on the timescale of an smFRET measurement, but shorter than the DNA’s persistence length (~150 bp (ref. 19)) to avoid intramolecular condensation20. The vesicles were immobilized on a polymer-passivated surface, and fluorescence signals from individual vesicles containing one donor and one acceptor were selectively analysed (Fig. 1d). Binding of two dsDNA molecules brings their fluorescent labels in close proximity, increasing the FRET efficiency (Fig. 1e).

FRET signals from individual vesicles were diverse. Sporadic binding events were observed in some vesicles, while others exhibited stable binding; traces indicative of frequent conformational transitions were also observed (Supplementary Fig. 1A). Such diverse behaviours could be expected from non-specific interactions of two large biomolecules having structural degrees of freedom. No binding events were observed in the absence of Sm4+ (Supplementary Fig. 1B) or when no DNA molecules were present. To quantitatively assess the propensity of forming a bound state, we chose to use the fraction of single-molecule traces that showed any binding events within the observation time of 2 min (Methods). This binding fraction for the pair of AT-rich dsDNAs (AT1, 100% AT in the middle 80-bp section of the 120-bp construct) reached a maximum at ~2 mM Sm4+(Fig. 1f), which is consistent with the results of previous experimental studies2, 3. In accordance with the prediction of our MD simulations, GC-rich dsDNAs (GC1, 75% GC in the middle 80 bp) showed much lower binding fraction at all Sm4+ concentrations (Fig. 1b,c). Regardless of the DNA sequence, the binding fraction reduced back to zero at high Sm4+ concentrations, likely due to the resolubilization of now positively charged DNA–Sm4+ complexes2, 3, 13.

Because the donor and acceptor fluorophores were attached to the same sequence of DNA, it remained possible that the sequence homology between the donor-labelled DNA and the acceptor-labelled DNA was necessary for their interaction6. To test this possibility, we designed another AT-rich DNA construct AT2 by scrambling the central 80-bp section of AT1 to remove the sequence homology (Supplementary Table 1). The fraction of binding traces for this nonhomologous pair of donor-labelled AT1 and acceptor-labelled AT2 was comparable to that for the homologous AT-rich pair (donor-labelled AT1 and acceptor-labelled AT1) at all Sm4+ concentrations tested (Fig. 1f). Furthermore, this data set rules out the possibility that the higher binding fraction observed experimentally for the AT-rich constructs was caused by inter-duplex Watson–Crick base pairing of the partially melted constructs.

Next, we designed a DNA construct named ATGC, containing, in its middle section, a 40-bp AT-rich segment followed by a 40-bp GC-rich segment (Fig. 1g). By attaching the acceptor to the end of either the AT-rich or GC-rich segments, we could compare the likelihood of observing the parallel binding mode that brings the two AT-rich segments together and the anti-parallel binding mode. Measurements at 1 mM Sm4+ and 25 or 50 mM NaCl indicated a preference for the parallel binding mode by ~30% (Fig. 1h). Therefore, AT content can modulate DNA–DNA interactions even in a complex sequence context. Note that increasing the concentration of NaCl while keeping the concentration of Sm4+ constant enhances competition between Na+ and Sm4+ counterions, which reduces the concentration of Sm4+ near DNA and hence the frequency of dsDNA–dsDNA binding events (Supplementary Fig. 2).

Methylation determines the strength of DNA–DNA attraction

Analysis of the MD simulations revealed the molecular mechanism of the polyamine-mediated sequence-dependent attraction (Fig. 2). In the case of the AT-rich fragments, the bulky methyl group of thymine base blocks Sm4+ binding to the N7 nitrogen atom of adenine, which is the cation-binding hotspot21, 22. As a result, Sm4+ is not found in the major grooves of the AT-rich duplexes and resides mostly near the DNA backbone (Fig. 2a,d). Such relocated Sm4+ molecules bridge the two DNA duplexes better, accounting for the stronger attraction16, 23, 24, 25. In contrast, significant amount of Sm4+ is adsorbed to the major groove of the GC-rich helices that lacks cation-blocking methyl group (Fig. 2b,e).

Figure 2: Molecular mechanism of polyamine-mediated DNA sequence recognition.
Molecular mechanism of polyamine-mediated DNA sequence recognition.

(a–c) Representative configurations of Sm4+ molecules at the DNA–DNA distance of 28 Å for the (AT)10–(AT)10 (a), (GC)10–(GC)10 (b) and (GmC)10–(GmC)10 (c) DNA pairs. The backbone and bases of DNA are shown as ribbon and molecular bond, respectively; Sm4+ molecules are shown as molecular bonds. Spheres indicate the location of the N7 atoms and the methyl groups. (d–f) The average distributions of cations for the three sequence pairs featured in a–c. Top: density of Sm4+ nitrogen atoms (d=28 Å) averaged over the corresponding MD trajectory and the z axis. White circles (20 Å in diameter) indicate the location of the DNA helices. Bottom: the average density of Sm4+ nitrogen (blue), DNA phosphate (black) and sodium (red) atoms projected onto the DNA–DNA distance axis (x axis). The plot was obtained by averaging the corresponding heat map data over y=[−10, 10] Å. See Supplementary Figs 4 and 5 for the cation distributions at d=30, 32, 34 and 36 Å.

If indeed the extra methyl group in thymine, which is not found in cytosine, is responsible for stronger DNA–DNA interactions, we can predict that cytosine methylation, which occurs naturally in many eukaryotic organisms and is an essential epigenetic regulation mechanism26, would also increase the strength of DNA–DNA attraction. MD simulations showed that the GC-rich helices containing methylated cytosines (mC) lose the adsorbed Sm4+ (Fig. 2c,f) and that |ΔGmin| of (GC)10 increases on methylation of cytosines to become similar to |ΔGmin| of (AT)10 (Fig. 1b).

To experimentally assess the effect of cytosine methylation, we designed another GC-rich construct GC2 that had the same GC content as GC1 but a higher density of CpG sites (Supplementary Table 1). The CpG sites were then fully methylated using M. SssI methyltransferase (Supplementary Fig. 3; Methods). As predicted from the MD simulations, methylation of the GC-rich constructs increased the binding fraction to the level of the AT-rich constructs (Fig. 1f).

The sequence dependence of |ΔGmin| and its relation to the Sm4+ adsorption patterns can be rationalized by examining the number of Sm4+ molecules shared by the dsDNA molecules (Fig. 3a). An Sm4+ cation adsorbed to the major groove of one dsDNA is separated from the other dsDNA by at least 10 Å, contributing much less to the effective DNA–DNA attractive force than a cation positioned between the helices, that is, the ‘bridging’ Sm4+ (ref. 23). An adsorbed Sm4+ also repels other Sm4+ molecules due to like-charge repulsion, lowering the concentration of bridging Sm4+. To demonstrate that the concentration of bridging Sm4+ controls the strength of DNA–DNA attraction, we computed the number of bridging Sm4+ molecules, Nspm (Fig. 3b). Indeed, the number of bridging Sm4+ molecules ranks in the same order as |ΔGmin|: Nspm of (A)20>Nspm of (AT)10≈Nspm of (GmC)10>Nspm of (GC)10>Nspm of (G)20. Thus, the number density of nucleotides carrying a methyl group (T and mC) is the primary determinant of the strength of attractive interaction between two dsDNA molecules. At the same time, the spatial arrangement of the methyl group carrying nucleotides can affect the interaction strength as well (Fig. 3c). The number of methyl groups and their distribution in the (AT)10 and (GmC)10 duplex DNA are identical, and so are their interaction free energies, |ΔGmin| of (AT)10≈|ΔGmin| of (GmC)10. For AT-rich DNA sequences, clustering of the methyl groups repels Sm4+ from the major groove more efficiently than when the same number of methyl groups is distributed along the DNA (Fig. 3b). Hence, |ΔGmin| of (A)20>|ΔGmin| of (AT)10. For GC-rich DNA sequences, clustering of the cation-binding sites (N7 nitrogen) attracts more Sm4+ than when such sites are distributed along the DNA (Fig. 3b), hence |ΔGmin| is larger for (GC)10 than for (G)20.

Figure 3: Methylation modulates the interaction free energy of two dsDNA molecules by altering the number of bridging Sm4+.
Methylation modulates the interaction free energy of two dsDNA molecules by altering the number of bridging Sm4+.

(a) Typical spatial arrangement of Sm4+ molecules around a pair of DNA helices. The phosphates groups of DNA and the amine groups of Sm4+ are shown as red and blue spheres, respectively. ‘Bridging’ Sm4+molecules reside between the DNA helices. Orange rectangles illustrate the volume used for counting the number of bridging Sm4+ molecules. (b) The number of bridging amine groups as a function of the inter-DNA distance. The total number of Sm4+ nitrogen atoms was computed by averaging over the corresponding MD trajectory and the 10 Å (x axis) by 20 Å (y axis) rectangle prism volume (a) centred between the DNA molecules. (c) Schematic representation of the dependence of the interaction free energy of two DNA molecules on their nucleotide sequence. The number and spatial arrangement of nucleotides carrying a methyl group (T or mC) determine the interaction free energy of two dsDNA molecules.

Genome-wide investigations of chromosome conformations using the Hi–C technique revealed that AT-rich loci form tight clusters in human nucleus27, 28. Gene or chromosome inactivation is often accompanied by increased methylation of DNA29 and compaction of facultative heterochromatin regions30. The consistency between those phenomena and our findings suggest the possibility that the polyamine-mediated sequence-dependent DNA–DNA interaction might play a role in chromosome folding and epigenetic regulation of gene expression.

  1. Rau, D. C., Lee, B. & Parsegian, V. A. Measurement of the repulsive force between polyelectrolyte molecules in ionic solution: hydration forces between parallel DNA double helices. Proc. Natl Acad. Sci. USA 81, 2621–2625 (1984).
  2. Raspaud, E., Olvera de la Cruz, M., Sikorav, J. L. & Livolant, F. Precipitation of DNA by polyamines: a polyelectrolyte behavior. Biophys. J. 74, 381–393 (1998).
  3. Besteman, K., Van Eijk, K. & Lemay, S. G. Charge inversion accompanies DNA condensation by multivalent ions. Nat. Phys. 3, 641–644 (2007).
  4. Lipfert, J., Doniach, S., Das, R. & Herschlag, D. Understanding nucleic acid-ion interactions.Annu. Rev. Biochem. 83, 813–841 (2014).
  5. Grosberg, A. Y., Nguyen, T. T. & Shklovskii, B. I. The physics of charge inversion in chemical and biological systems. Rev. Mod. Phys. 74, 329–345 (2002).
  6. Kornyshev, A. A. & Leikin, S. Sequence recognition in the pairing of DNA duplexes. Phys. Rev. Lett. 86, 3666–3669 (2001).
  7. Danilowicz, C. et al. Single molecule detection of direct, homologous, DNA/DNA pairing.Proc. Natl Acad. Sci. USA 106, 19824–19829 (2009).
  8. Gladyshev, E. & Kleckner, N. Direct recognition of homology between double helices of DNA in Neurospora crassa. Nat. Commun. 5, 3509 (2014).
  9. Tabor, C. W. & Tabor, H. Polyamines. Annu. Rev. Biochem. 53, 749–790 (1984).
  10. Thomas, T. & Thomas, T. J. Polyamines in cell growth and cell death: molecular mechanisms and therapeutic applications. Cell. Mol. Life Sci. 58, 244–258 (2001).

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Schizophrenia, broken-links

Posted in Autism Spectrum Disorders, Cell Biology, Signaling & Cell Circuits, Cerebrovascular and Neurodegenerative Diseases, Chemical Biology and its relations to Metabolic Disease, Chemical Genetics, Child and Adolescent Psychiatry, Clinical & Translational, Cognition, Genetics & Innovations in Treatment, Genome Biology, MRI, Neurodegenerative Diseases, Neurohumoral Transmission, Neurological Diseases, Neuroscience, tagged , , , , , , , on April 5, 2016| Leave a Comment »

Schizophrenia, broken-links

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Runs in the Family

 New findings about schizophrenia rekindle old questions about genes and identity.
BY Annals of Science MARCH 28, 2016 ISSUE      http://www.newyorker.com/magazine/2016/03/28/the-genetics-of-schizophrenia

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The author and his father have seen several relatives succumb to mental illness.CREDIT PHOTOGRAPH BY DAYANITA SINGH FOR THE NEW YORKER

In the winter of 2012, I travelled from New Delhi, where I grew up, to Calcutta to visit my cousin Moni. My father accompanied me as a guide and companion, but he was a sullen and brooding presence, lost in a private anguish. He is the youngest of five brothers, and Moni is his firstborn nephew—the eldest brother’s son. Since 2004, Moni, now fifty-two, has been confined to an institution for the mentally ill (a “lunatic home,” as my father calls it), with a diagnosis of schizophrenia. He is kept awash in antipsychotics and sedatives, and an attendant watches, bathes, and feeds him through the day.

My father has never accepted Moni’s diagnosis. Over the years, he has waged a lonely campaign against the psychiatrists charged with his nephew’s care, hoping to convince them that their diagnosis was a colossal error, or that Moni’s broken psyche would somehow mend itself. He has visited the institution in Calcutta twice—once without warning, hoping to see a transformed Moni, living a secretly normal life behind the barred gates. But there was more than just avuncular love at stake for him in these visits. Moni is not the only member of the family with mental illness. Two of my father’s four brothers suffered from various unravellings of the mind. Madness has been among the Mukherjees for generations, and at least part of my father’s reluctance to accept Moni’s diagnosis lies in a grim suspicion that something of the illness may be buried, like toxic waste, in himself.

Rajesh, my father’s third-born brother, had once been the most promising of the Mukherjee boys—the nimblest, the most charismatic, the most admired. But in the summer of 1946, at the age of twenty-two, he began to behave oddly, as if a wire had been tripped in his brain. The most obvious change in his personality was a volatility: good news triggered uncontained outbursts of joy; bad news plunged him into inconsolable desolation. By that winter, the sine curve of Rajesh’s psyche had tightened in its frequency and gained in its amplitude. My father recalls an altered brother: fearful at times, reckless at others, descending and ascending steep slopes of mood, irritable one morning and overjoyed the next. When Rajesh received news of a successful performance on his college exams, he vanished, elated, on a two-night excursion, supposedly “exercising” at a wrestling camp. He was feverish and hallucinating when he returned, and died of pneumonia soon afterward. Only years later, in medical school, did I realize that Rajesh was likely in the throes of an acute manic phase. His mental breakdown was the result of a near-textbook case of bipolar disorder.

Jagu, the fourth-born of my father’s siblings, came to live with us in Delhi in 1975, when I was five years old and he was forty-five. His mind, too, was failing. Tall and rail thin, with a slightly feral look in his eyes and a shock of matted, overgrown hair, he resembled a Bengali Jim Morrison. Unlike Rajesh, whose illness had surfaced in his twenties, Jagu had been troubled from his adolescence. Socially awkward, withdrawn from everyone except my grandmother, he was unable to hold a job or live by himself. By 1975, he had visions, phantasms, and voices in his head that told him what to do. He was still capable of extraordinary bursts of tenderness—when I accidentally smashed a beloved Venetian vase at home, he hid me in his bedclothes and informed my mother that he had “mounds of cash” stashed away, enough to buy “a thousand” replacement vases. But this episode was symptomatic: even his love for me extended the fabric of his psychosis and confabulation.

Unlike Rajesh, Jagu was formally diagnosed. In the late nineteen-seventies, a physician in Delhi examined him and determined that he had schizophrenia. But no medicines were prescribed. Instead, Jagu continued to live at home, half hidden away in my grandmother’s room. (As in many families in India, my grandmother lived with us.) For nearly a decade, she and my father maintained a fragile truce, with Jagu living under her care, eating meals in her room and wearing clothes that she stitched for him. At night, when Jagu was consumed by his fears and fantasies, she put him to bed like a child, with her hand on his forehead. She was his nurse, his housekeeper, his only friend, and, more important, his public defender. When my grandmother died, in 1985, Jagu joined a religious sect in Delhi and disappeared, until his death, a dozen years later.

……

at schizophrenia runs in families was evident even to the person who first defined the illness. In 1911, Eugen Bleuler, a Swiss-German psychiatrist, published a book describing a series of cases of men and women, typically in their teens and early twenties, whose thoughts had begun to tangle and degenerate. “In this malady, the associations lose their continuity,” Bleuler wrote. “The threads between thoughts are torn.” Psychotic visions and paranoid thoughts flashed out of nowhere. Some patients “feel themselves weak, their spirit escapes, they will never survive the day. There is a growth in their heads. Their bones have turned liquid; their hearts have turned into stone. . . . The patient’s wife must not use eggs in cooking, otherwise he will grow feathers.” His patients were often trapped between flickering emotional states, unable to choose between two radically opposed visions, Bleuler noted. “You devil, you angel, you devil, you angel,” one woman said to her lover.

Bleuler tried to find an explanation for the mysterious symptoms, but there was only one seemingly common element: schizophrenic patients tended to have first-degree relatives who were also schizophrenic. He had no tools to understand the mechanism behind the heredity. The word “gene” had been coined just two years before Bleuler published his book. The notion that a mental illness could be carried across generations by unitary, indivisible factors—corpuscles of information threading through families—would have struck most of Bleuler’s contemporaries as mad in its own right. Still, Bleuler was astonishingly prescient about the complex nature of inheritance. “If one is looking for ‘theheredity,’ one can nearly always find it,” he wrote. “We will not be able to do anything about it even later on, unless the single factor of heredity can be broken down into many hereditary factors along specific lines.”

In the nineteen-sixties, Bleuler’s hunch was confirmed by twin studies. Psychiatrists determined that if an identical twin was schizophrenic the other twin had a forty-to-fifty-per-cent chance of developing the disease—fiftyfold higher than the risk in the general population. By the early two-thousands, large population studies had revealed a strong genetic link between schizophrenia and bipolar disorder. Some of the families described in these studies had a crisscrossing history that was achingly similar to my own: one sibling affected with schizophrenia, another with bipolar disorder, and a nephew or niece also schizophrenic.

“The twin studies clarified two important features of schizophrenia and bipolar disorder,” Jeffrey Lieberman, a Columbia University psychiatrist who has studied schizophrenia for thirty years, told me. “First, it was clear that there wasn’t a single gene, but dozens of genes involved in causing schizophrenia—each perhaps exerting a small effect. And, second, even if you inherited the entire set of risk genes, as identical twins do, you still might not develop the disease. Obviously, there were other triggers or instigators involved in releasing the illness.” But while these studies established that schizophrenia had a genetic basis, they revealed nothing about the nature of the genes involved. “For doctors, patients, and families in the schizophrenia community, genetics became the ultimate mystery,” Lieberman said. “If we knew the identity of the genes, we would find the causes, and if we found the causes we could find medicines.”

In 2006, an international consortium of psychiatric geneticists launched a genomic survey of schizophrenia, hoping to advance the search for the implicated genes. With 3,322 patients and 3,587 controls, this was one of the largest and most rigorous such studies in the history of the disease. Researchers scanned through the nearly seven thousand genomes to find variations in gene segments that were correlated with schizophrenia. This strategy, termed an “association study,” does not pinpoint a gene, but it provides a general location where a disease-linked gene may be found, like a treasure map with a large “X” scratched in a corner of the genome.

The results, reported in 2009 (and updated in 2014) in the journal Nature, were a dispiriting validation of Bleuler’s hunch about multiple hereditary factors: more than a hundred independent segments of the genome were associated with schizophrenia. “There are lots of small, common genetic effects, scattered across the genome,” one researcher said. “There are many different biological processes involved.” Some of the putative culprits made biological sense—if dimly. There were genes linked to transmitters that relay messages between neurons, and genes for molecular channels that move electrical signals up and down nerve cells. But by far the most surprising association involved a gene segment on chromosome 6. This region of the genome—termed the MHC region—carries hundreds of genes typically associated with the immune system.

“The MHC-segment finding was so strange and striking that you had to sit up and take notice,” Lieberman told me. “Here was the most definitive evidence that something in the immune system might have something to do with schizophrenia. There had been hints about an immunological association before, but this was impossible to argue with. It raised an endlessly fascinating question: what was the link between immune-response genes and schizophrenia?”

 …..  for more see http://www.newyorker.com/magazine/2016/03/28/the-genetics-of-schizophrenia
The Rogue Immune Cells That Wreck the Brain

Beth Stevens thinks she has solved a mystery behind brain disorders such as Alzheimer’s and schizophrenia.

by Adam Piore   April 4, 2016            https://www.technologyreview.com/s/601137/the-rogue-immune-cells-that-wreck-the-brain/

In the first years of her career in brain research, Beth Stevens thought of microglia with annoyance if she thought of them at all. When she gazed into a microscope and saw these ubiquitous cells with their spidery tentacles, she did what most neuroscientists had been doing for generations: she looked right past them and focused on the rest of the brain tissue, just as you might look through specks of dirt on a windshield.

“What are they doing there?” she thought. “They’re in the way.’”

Stevens never would have guessed that just a few years later, she would be running a laboratory at Harvard and Boston’s Children’s Hospital devoted to the study of these obscure little clumps. Or that she would be arguing in the world’s top scientific journals that microglia might hold the key to understanding not just normal brain development but also what causes Alzheimer’s, Huntington’s, autism, schizophrenia, and other intractable brain disorders.

Microglia are part of a larger class of cells—known collectively as glia—that carry out an array of functions in the brain, guiding its development and serving as its immune system by gobbling up diseased or damaged cells and carting away debris. Along with her frequent collaborator and mentor, Stanford biologist Ben Barres, and a growing cadre of other scientists, Stevens, 45, is showing that these long-overlooked cells are more than mere support workers for the neurons they surround. Her work has raised a provocative suggestion: that brain disorders could somehow be triggered by our own bodily defenses gone bad.

A type of glial cell known as an oligodendrocyte

In one groundbreaking paper, in January, Stevens and researchers at the Broad Institute of MIT and Harvard showed that aberrant microglia might play a role in schizophrenia—causing or at least contributing to the massive cell loss that can leave people with devastating cognitive defects. Crucially, the researchers pointed to a chemical pathway that might be targeted to slow or stop the disease. Last week, Stevens and other researchers published a similar finding for Alzheimer’s.

This might be just the beginning. Stevens is also exploring the connection between these tiny structures and other neurological diseases—work that earned her a $625,000 MacArthur Foundation “genius” grant last September.

All of this raises intriguing questions. Is it possible that many common brain disorders, despite their wide-ranging symptoms, are caused or at least worsened by the same culprit, a component of the immune system? If so, could many of these disorders be treated in a similar way—by stopping these rogue cells?

Nature. 2016 Feb 11;530(7589):177-83. http://dx.doi.org:/10.1038/nature16549. Epub 2016 Jan 27.   Schizophrenia risk from complex variation of complement component 4.
Sekar A, Bialas AR, de Rivera H, Davis A, Hammond TR, Kamitaki N, Tooley K, Presumey J, Baum M, Van Doren V, Genovese G,Rose SA, Handsaker RE; Schizophrenia Working Group of the Psychiatric Genomics Consortium, Daly MJ, Carroll MC, Stevens B,McCarroll SA.   Collaborators (307)

Schizophrenia is a heritable brain illness with unknown pathogenic mechanisms. Schizophrenia’s strongest genetic association at a population level involves variation in the major histocompatibility complex (MHC) locus, but the genes and molecular mechanisms accounting for this have been challenging to identify. Here we show that this association arises in part from many structurally diverse alleles of the complement component 4 (C4) genes. We found that these alleles generated widely varying levels of C4A and C4B expression in the brain, with each common C4 allele associating with schizophrenia in proportion to its tendency to generate greater expression of C4A. Human C4 protein localized to neuronal synapses, dendrites, axons, and cell bodies. In mice, C4 mediated synapse elimination during postnatal development. These results implicate excessive complement activity in the development of schizophrenia and may help explain the reduced numbers of synapses in the brains of individuals with schizophrenia.

 

Science  31 Mar 2016;        http://dx.doi.org:/10.1126/science.aad8373      Complement and microglia mediate early synapse loss in Alzheimer mouse models.
Soyon Hong1, Victoria F. Beja-Glasser1,*, Bianca M. Nfonoyim1,*,…., Ben A. Barres6, Cynthia A. Lemere,2, Dennis J. Selkoe2,7, Beth Stevens1,8,†

 Synapse loss in Alzheimer’s disease (AD) correlates with cognitive decline. Involvement of microglia and complement in AD has been attributed to neuroinflammation, prominent late in disease. Here we show in mouse models that complement and microglia mediate synaptic loss early in AD. C1q, the initiating protein of the classical complement cascade, is increased and associated with synapses before overt plaque deposition. Inhibition of C1q, C3 or the microglial complement receptor CR3, reduces the number of phagocytic microglia as well as the extent of early synapse loss. C1q is necessary for the toxic effects of soluble β-amyloid (Aβ) oligomers on synapses and hippocampal long-term potentiation (LTP). Finally, microglia in adult brains engulf synaptic material in a CR3-dependent process when exposed to soluble Aβ oligomers. Together, these findings suggest that the complement-dependent pathway and microglia that prune excess synapses in development are inappropriately activated and mediate synapse loss in AD.
Genome-wide association studies (GWAS) implicate microglia and complement-related pathways in AD (1). Previous research has demonstrated both beneficial and detrimental roles of complement and microglia in plaque-related neuropathology (2, 3); however, their roles in synapse loss, a major pathological correlate of cognitive decline in AD (4), remain to be identified. Emerging research implicates microglia and immune-related mechanisms in brain wiring in the healthy brain (1). During development, C1q and C3 localize to synapses and mediate synapse elimination by phagocytic microglia (5–7). We hypothesized that this normal developmental synaptic pruning pathway is activated early in the AD brain and mediates synapse loss.

 

Complex machinery

It’s not surprising that scientists for years have ignored microglia and other glial cells in favor of neurons. Neurons that fire together allow us to think, breathe, and move. We see, hear, and feel using neurons, and we form memories and associations when the connections between different neurons strengthen at the junctions between them, known as synapses. Many neuroscientists argue that neurons create our very consciousness.

Glia, on the other hand, have always been considered less important and interesting. They have pedestrian duties such as supplying nutrients and oxygen to neurons, as well as mopping up stray chemicals and carting away the garbage.

Scientists have known about glia for some time. In the 1800s, the pathologist Rudolf Virchow noted the presence of small round cells packing the spaces between neurons and named them “nervenkitt” or “neuroglia,” which can be translated as nerve putty or glue. One variety of these cells, known as astrocytes, was defined in 1893. And then in the 1920s, the Spanish scientist Pio del Río Hortega developed novel ways of staining cells taken from the brain. This led him to identify and name two more types of glial cells, including microglia, which are far smaller than the others and are characterized by their spidery shape and multiple branches. It is only when the brain is damaged in adulthood, he suggested, that microglia spring to life—rushing to the injury, where it was thought they helped clean up the area by eating damaged and dead cells. Astrocytes often appeared on the scene as well; it was thought that they created scar tissue.

This emergency convergence of microglia and astrocytes was dubbed “gliosis,” and by the time Ben Barres entered medical school in the late 1970s, it was well established as a hallmark of neurodegenerative diseases, infection, and a wide array of other medical conditions. But no one seemed to understand why it occurred. That intrigued Barres, then a neurologist in training, who saw it every time he looked under a microscope at neural tissue in distress. “It was just really fascinating,” he says. “The great mystery was: what is the point of this gliosis? Is it good? Is it bad? Is it driving the disease process, or is it trying to repair the injured brain?”

 https://youtu.be/6DOYTpXkLOY

Barres began looking for the answer. He learned how to grow glial cells in a dish and apply a new recording technique to them. He could measure their electrical qualities, which determine the biochemical signaling that all brain cells use to communicate and coördinate activity.

“From the second I started recording the glial cells, I thought ‘Oh, my God!’” Barres recalls. The electrical activity was more dynamic and complex than anyone had thought. These strange electrical properties could be explained only if the glial cells were attuned to the conditions around them, and to the signals released from nearby neurons. Barres’s glial cells, in other words, had all the machinery necessary to engage in a complex dialogue with neurons, and presumably to respond to different kinds of conditions in the brain.

Why would they need this machinery, though, if they were simply involved in cleaning up dead cells? What could they possibly be doing? It turns out that in the absence of chemicals released by glia, the neurons committed the biochemical version of suicide. Barres also showed that the astrocytes appeared to play a crucial role in forming synapses, the microscopic connections between neurons that encode memory. In isolation, neurons were capable of forming the spiny appendages necessary to reach the synapses. But without astrocytes, they were incapable of connecting to one another.

Hardly anyone believed him. When he was a young faculty member at Stanford in the 1990s, one of his grant applications to the National Institutes of Health was rejected seven times. “Reviewers kept saying, ‘Nah, there’s no way glia could be doing this,’” Barres recalls. “And even after we published two papers in Science showing that [astrocytes] had profound, almost all-or-nothing effects in controlling synapses’ formation or synapse activity, I still couldn’t get funded! I think it’s still hard to get people to think about glia as doing anything active in the nervous system.”

Marked for elimination

Beth Stevens came to study glia by accident. After graduating from Northeastern University in 1993, she followed her future husband to Washington, D.C., where he had gotten work in the U.S. Senate. Stevens had been pre-med in college and hoped to work in a lab at the National Institutes of Health. But with no previous research experience, she was soundly rebuffed. So she took a job waiting tables at a Chili’s restaurant in nearby Rockville, Maryland, and showed up at NIH with her résumé every week.

After a few months, Stevens received a call from a researcher named Doug Fields, who needed help in his lab. Fields was studying the intricacies of the process in which neurons become insulated in a coating called myelin. That insulation is essential for the transmission of electrical impulses.

As Stevens spent the following years pursuing a PhD at the University of Maryland, she was intrigued by the role that glial cells played in insulating neurons. Along the way, she became familiar with other insights into glial cells that were beginning to emerge, especially from the lab of Ben Barres. Which is why, soon after completing her PhD in 2003, Stevens found herself a postdoc in Barres’s lab at Stanford, about to make a crucial discovery.

Barres’s group had begun to identify the specific compounds astrocytes secreted that seemed to cause neurons to grow synapses. And eventually, they noticed that these compounds also stimulated production of a protein called C1q.

Conventional wisdom held that C1q was activated only in sick cells—the protein marked them to be eaten up by immune cells—and only outside the brain. But Barres had found it in the brain. And it was in healthy neurons that were arguably at their most robust stage: in early development. What was the C1q protein doing there?

https://d267cvn3rvuq91.cloudfront.net/i/images/glia33.jpg?sw=590&cx=0&cy=0&cw=2106&ch=2106

A stained astrocyte.

The answer lies in the fact that marking cells for elimination is not something that happens only in diseased brains; it is also essential for development. As brains develop, their neurons form far more synaptic connections than they will eventually need. Only the ones that are used are allowed to remain. This pruning allows for the most efficient flow of neural transmissions in the brain, removing noise that might muddy the signal.

But it was unknown how exactly the process worked. Was it possible that C1q helped signal the brain to prune unused synapses? Stevens focused her postdoctoral research on finding out. “We could have been completely wrong,” she recalls. “But we went for it.”

It paid off. In a 2007 paper, Barres and Stevens showed that C1q indeed plays a role in eliminating unneeded neurons in the developing brain. And they found that the protein is virtually absent in healthy adult neurons.

Now the scientists faced a new puzzle. Does C1q show up in brain diseases because the same mechanism involved in pruning a developing brain later goes awry? Indeed, evidence was already growing that one of the earliest events in neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and Huntington’s was significant loss of synapses.

When Stevens and Barres examined mice bred to develop glaucoma, a neurodegenerative disease that kills neurons in the optic system, they found that C1q appeared long before any other detectable sign that the disease was taking hold. It cropped up even before the cells started dying.

This suggested the immune cells might in fact cause the disease, or at the very least accelerate it. And that offered an intriguing possibility: that something could be made to halt the process. Barres founded a company, Annexon Biosciences, to develop drugs that could block C1q. Last week’s paper published by Barres, Stevens, and other researchers shows that a compound being tested by Annexon appears to be able to prevent the onset of Alzheimer’s in mice bred to develop the disease. Now the company hopes to test it in humans in the next two years.

Paths to treatments

To better understand the process that C1q helps trigger, Stevens and Barres wanted to figure out what actually plays the role of Pac-Man, eating up the synapses marked for death. It was well known that white blood cells known as macrophages gobbled up diseased cells and foreign invaders in the rest of the body. But macrophages are not usually present in the brain. For their theory to work, there had to be some other mechanism. And further research has shown that the cells doing the eating even in healthy brains are those mysterious clusters of material that Beth Stevens, for years, had been gazing right past in the microscope—the microglia that Río Hortega identified almost 100 years ago.

Now Stevens’s lab at Harvard, which she opened in 2008, devotes half its efforts to figuring out what microglia are doing and what causes them to do it. These cells, it turns out, appear in the mouse embryo at day eight, before any other brain cell, which suggests they might help guide the rest of brain development—and could contribute to any number of neurodevelopmental diseases when they go wrong.

Meanwhile, she is also expanding her study of the way different substances determine what happens in the brain. C1q is actually just the first in a series of proteins that accumulate on synapses marked for elimination. Stevens has begun to uncover evidence that there is a wide array of protective “don’t eat me” molecules too. It’s the balance between all these cues that regulates whether microglia are summoned to destroy synapses. Problems in any one could, conceivably, mess up the system.

Evidence is now growing that microglia are involved in several neurodevelopmental and psychiatric problems. The potential link to schizophrenia that was revealed in January emerged after researchers at the Broad Institute, led by Steven McCarroll and a graduate student named Aswin Sekar, followed a trail of genetic clues that led them directly to Stevens’s work. In 2009, three consortia from around the globe had published papers comparing DNA in people with and without schizophrenia. It was Sekar who identified a possible pattern: the more a specific type of protein was present in synapses, the higher the risk of developing the disease. The protein, C4, was closely related to C1q, the one first identified in the brain by Stevens and Barres.

McCarroll knew that schizophrenia strikes in late adolescence and early adulthood, a time when brain circuits in the prefrontal cortex undergo extensive pruning. Others had found that areas of the prefrontal cortex are among those most ravaged by the disease, which leads to massive synapse loss. Could it be that over-pruning by rogue microglia is part of what causes schizophrenia?

To find out, Sekar and McCarroll got in touch with Stevens, and the two labs began to hold joint weekly meetings. They soon demonstrated that C4 also had a role in pruning synapses in the brains of young mice, suggesting that excessive levels of the protein could indeed lead to over-pruning—and to the thinning out of brain tissue that appears to occur as symptoms such as psychotic episodes grow worse.

If the brain damage seen in Parkinson’s and Alzheimer’s stems from over-pruning that might begin early in life, why don’t symptoms of those diseases show up until later? Barres thinks he knows. He notes that the brain can normally compensate for injury by rewiring itself and generating new synapses. It also contains a lot of redundancy. That would explain why patients with Parkinson’s disease don’t show discernible symptoms until they have lost 90 percent of the neurons that produce dopamine.

It also might mean that subtle symptoms could in fact be detected much earlier. Barres points to a study of nuns published in 2000. When researchers analyzed essays the nuns had written upon entering their convents decades before, they found that women who went on to develop Alzheimer’s had shown less “idea density” even in their 20s. “I think the implication of that is they could be lifelong diseases,” Barres says. “The disease process could be going on for decades and the brain is just compensating, rewiring, making new synapses.” At some point, the microglia are triggered to remove too many cells, Barres argues, and the symptoms of the disease begin to manifest fully.

Turning this insight into a treatment is far from straightforward, because much remains unclear. Perhaps an overly aggressive response from microglia is determined by some combination of genetic variants not shared by everyone. Stevens also notes that diseases like schizophrenia are not caused by one mutation; rather, a wide array of mutations with small effects cause problems when they act in concert. The genes that control the production of C4 and other immune-system proteins may be only part of the story. That may explain why not everyone who has a C4 mutation will go on to develop schizophrenia.

Nonetheless, if Barres and Stevens are right that the immune system is a common mechanism behind devastating brain disorders, that in itself is a fundamental breakthrough. Because we have not known the mechanisms that trigger such diseases, medical researchers have been able only to alleviate the symptoms rather than attack the causes. There are no drugs available to halt or even slow neurodegeneration in diseases like Alzheimer’s. Some drugs elevate neurotransmitters in ways that briefly make it easier for individuals with dementia to form new synaptic connections, but they don’t reduce the rate at which existing synapses are destroyed. Similarly, there are no treatments that tackle the causes of autism or schizophrenia. Even slowing the progress of these disorders would be a major advance. We might finally go after diseases that have run unchecked for generations.

“We’re a ways away from a cure,” Stevens says. “But we definitely have a path forward.”

Adam Piore is a freelance writer who wrote “A Shocking Way to Fix the Brain”  in November/December 2015.

 

Int Immunopharmacol. 2001 Mar;1(3):365-92.

Genetic, structural and functional diversities of human complement components C4A and C4B and their mouse homologues, Slp and C4.

Blanchong CA1, Chung EK, Rupert KL, Yang Y, Yang Z, Zhou B, Moulds JM, Yu CY.

Author information

Abstract

The complement protein C4 is a non-enzymatic component of the C3 and C5 convertases and thus essential for the propagation of the classical complement pathway. The covalent binding of C4 to immunoglobulins and immune complexes (IC) also enhances the solubilization of immune aggregates, and the clearance of IC through complement receptor one (CR1) on erythrocytes. Human C4 is the most polymorphic protein of the complement system. In this review, we summarize the current concepts on the 1-2-3 loci model of C4A and C4B genes in the population, factors affecting the expression levels of C4 transcripts and proteins, and the structural, functional and serological diversities of the C4A and C4B proteins. The diversities and polymorphisms of the mouse homologues Slp and C4 proteins are described and contrasted with their human homologues. The human C4 genes are located in the MHC class III region on chromosome 6. Each human C4 gene consists of 41 exons coding for a 5.4-kb transcript. The long gene is 20.6 kb and the short gene is 14.2 kb. In the Caucasian population 55% of the MHC haplotypes have the 2-locus, C4A-C4B configurations and 45% have an unequal number of C4A and C4B genes. Moreover, three-quarters of C4 genes harbor the 6.4 kb endogenous retrovirus HERV-K(C4) in the intron 9 of the long genes. Duplication of a C4 gene always concurs with its adjacent genes RP, CYP21 and TNX, which together form a genetic unit termed an RCCX module. Monomodular, bimodular and trimodular RCCX structures with 1, 2 and 3 complement C4 genes have frequencies of 17%, 69% and 14%, respectively. Partial deficiencies of C4A and C4B, primarily due to the presence of monomodular haplotypes and homo-expression of C4A proteins from bimodular structures, have a combined frequency of 31.6%. Multiple structural isoforms of each C4A and C4B allotype exist in the circulation because of the imperfect and incomplete proteolytic processing of the precursor protein to form the beta-alpha-gamma structures. Immunofixation experiments of C4A and C4B demonstrate > 41 allotypes in the two classes of proteins. A compilation of polymorphic sites from limited C4 sequences revealed the presence of 24 polymophic residues, mostly clustered C-terminal to the thioester bond within the C4d region of the alpha-chain. The covalent binding affinities of the thioester carbonyl group of C4A and C4B appear to be modulated by four isotypic residues at positions 1101, 1102, 1105 and 1106. Site directed mutagenesis experiments revealed that D1106 is responsible for the effective binding of C4A to form amide bonds with immune aggregates or protein antigens, and H1106 of C4B catalyzes the transacylation of the thioester carbonyl group to form ester bonds with carbohydrate antigens. The expression of C4 is inducible or enhanced by gamma-interferon. The liver is the main organ that synthesizes and secretes C4A and C4B to the circulation but there are many extra-hepatic sites producing moderate quantities of C4 for local defense. The plasma protein levels of C4A and C4B are mainly determined by the corresponding gene dosage. However, C4B proteins encoded by monomodular short genes may have relatively higher concentrations than those from long C4A genes. The 5′ regulatory sequence of a C4 gene contains a Spl site, three E-boxes but no TATA box. The sequences beyond–1524 nt may be completely different as the C4 genes at RCCX module I have RPI-specific sequences, while those at Modules II, III and IV have TNXA-specific sequences. The remarkable genetic diversity of human C4A and C4B probably promotes the exchange of genetic information to create and maintain the quantitative and qualitative variations of C4A and C4B proteins in the population, as driven by the selection pressure against a great variety of microbes. An undesirable accompanying byproduct of this phenomenon is the inherent deleterious recombinations among the RCCX constituents leading to autoimmune and genetic disorders.

 

C4A isotype is responsible for effective binding to form amide bonds with immune aggregates or protein antigens, while C4B isotype catalyzes the transacylation of the thioester carbonyl group to form ester bonds with carbohydrate antigens.

Derived from proteolytic degradation of complement C4, C4a anaphylatoxin is a mediator of local inflammatory process.

 

Schizophrenia and the Synapse

Genetic evidence suggests that overactive synaptic pruning drives development of schizophrenia.

By Ruth Williams | January 27, 2016

http://www.the-scientist.com/?articles.view/articleNo/45189/title/Schizophrenia-and-the-Synapse/

Compared to the brains of healthy individuals, those of people with schizophrenia have higher expression of a gene called C4, according to a paper published inNature today (January 27). The gene encodes an immune protein that moonlights in the brain as an eradicator of unwanted neural connections (synapses). The findings, which suggest increased synaptic pruning is a feature of the disease, are a direct extension of genome-wide association studies (GWASs) that pointed to the major histocompatibility (MHC) locus as a key region associated with schizophrenia risk.

“The MHC [locus] is the first and the strongest genetic association for schizophrenia, but many people have said this finding is not useful,” said psychiatric geneticist Patrick Sullivan of the University of North Carolina School of Medicine who was not involved in the study. “The value of [the present study is] to show that not only is it useful, but it opens up new and extremely interesting ideas about the biology and therapeutics of schizophrenia.”

Schizophrenia has a strong genetic component—it runs in families—yet, because of the complex nature of the condition, no specific genes or mutations have been identified. The pathological processes driving the disease remain a mystery.

Researchers have turned to GWASs in the hope of finding specific genetic variations associated with schizophrenia, but even these have not provided clear candidates.

“There are some instances where genome-wide association will literally hit one base [in the DNA],” explained Sullivan. While a 2014 schizophrenia GWAS highlighted the MHC locus on chromosome 6 as a strong risk area, the association spanned hundreds of possible genes and did not reveal specific nucleotide changes. In short, any hope of pinpointing the MHC association was going to be “really challenging,” said geneticist Steve McCarroll of Harvard who led the new study.

Nevertheless, McCarroll and colleagues zeroed in on the particular region of the MHC with the highest GWAS score—the C4 gene—and set about examining how the area’s structural architecture varied in patients and healthy people.

The C4 gene can exist in multiple copies (from one to four) on each copy of chromosome 6, and has four different forms: C4A-short, C4B-short, C4A-long, and C4B-long. The researchers first examined the “structural alleles” of the C4 locus—that is, the combinations and copy numbers of the different C4 forms—in healthy individuals. They then examined how these structural alleles related to expression of both C4Aand C4B messenger RNAs (mRNAs) in postmortem brain tissues.

…………..

Schizophrenia risk from complex variation of complement component 4

Aswin Sekar, Allison R. Bialas, Heather de Rivera, …, Schizophrenia Working Group of the Psychiatric Genomics Consortium, Mark J. Daly, Michael C. Carroll, Beth Stevens & Steven A. McCarroll

Nature (11 Feb 2016); 530: 177–183 http://dx.doi.org:/10.1038/nature16549

Schizophrenia is a heritable brain illness with unknown pathogenic mechanisms. Schizophrenia’s strongest genetic association at a population level involves variation in the major histocompatibility complex (MHC) locus, but the genes and molecular mechanisms accounting for this have been challenging to identify. Here we show that this association arises in part from many structurally diverse alleles of the complement component 4 (C4) genes. We found that these alleles generated widely varying levels of C4A and C4B expression in the brain, with each common C4 allele associating with schizophrenia in proportion to its tendency to generate greater expression of C4A. Human C4 protein localized to neuronal synapses, dendrites, axons, and cell bodies. In mice, C4 mediated synapse elimination during postnatal development. These results implicate excessive complement activity in the development of schizophrenia and may help explain the reduced numbers of synapses in the brains of individuals with schizophrenia.

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The strongest genetic association found in schizophrenia is its association to genetic markers across the major histocompatibility complex (MHC) locus, first described in three Nature papers in 2009. …

 

Schizophrenia: From genetics to physiology at last

Ryan S. Dhindsa & David B. Goldstein
Nature  (11 Feb 2016); 530:162–163   http://dx.doi.org:/10.1038/nature16874

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 Jianxin Shi1, et al.   Common variants on chromosome 6p22.1 are associated with schizophrenia.  Nature 460, 753-757 (6 August 2009) | doi:10.1038/nature08192; Received 29 May 2009; Accepted 10 June 2009; Published online 1 July 2009; Corrected 6 August 2009

Schizophrenia, a devastating psychiatric disorder, has a prevalence of 0.5–1%, with high heritability (80–85%) and complex transmission1. Recent studies implicate rare, large, high-penetrance copy number variants in some cases2, but the genes or biological mechanisms that underlie susceptibility are not known. Here we show that schizophrenia is significantly associated with single nucleotide polymorphisms (SNPs) in the extended major histocompatibility complex region on chromosome 6. We carried out a genome-wide association study of common SNPs in the Molecular Genetics of Schizophrenia (MGS) case-control sample, and then a meta-analysis of data from the MGS, International Schizophrenia Consortium and SGENE data sets. No MGS finding achieved genome-wide statistical significance. In the meta-analysis of European-ancestry subjects (8,008 cases, 19,077 controls), significant association with schizophrenia was observed in a region of linkage disequilibrium on chromosome 6p22.1 (P = 9.54 × 10-9). This region includes a histone gene cluster and several immunity-related genes—possibly implicating aetiological mechanisms involving chromatin modification, transcriptional regulation, autoimmunity and/or infection. These results demonstrate that common schizophrenia susceptibility alleles can be detected. The characterization of these signals will suggest important directions for research on susceptibility mechanisms.

Editor’s Summary   6 August 2009
Schizophrenia risk: link to chromosome 6p22.1

A genome-wide association study using the Molecular Genetics of Schizophrenia case-control data set, followed by a meta-analysis that included over 8,000 cases and 19,000 controls, revealed that while common genetic variation that underlies risk to schizophrenia can be identified, there probably are few or no single common loci with large effects. The common variants identified here lie on chromosome 6p22.1 in a region that includes a histone gene cluster and several genes implicated in immunity.

Letter

Hreinn Stefansson1,48, et al. Common variants conferring risk of schizophrenia.
Nature 460, 744-747 (6 August 2009) | doi:10.1038/nature08186; Received 16 March 2009; Accepted 5 June 2009; Published online 1 July 2009

Schizophrenia is a complex disorder, caused by both genetic and environmental factors and their interactions. Research on pathogenesis has traditionally focused on neurotransmitter systems in the brain, particularly those involving dopamine. Schizophrenia has been considered a separate disease for over a century, but in the absence of clear biological markers, diagnosis has historically been based on signs and symptoms. A fundamental message emerging from genome-wide association studies of copy number variations (CNVs) associated with the disease is that its genetic basis does not necessarily conform to classical nosological disease boundaries. Certain CNVs confer not only high relative risk of schizophrenia but also of other psychiatric disorders1, 2, 3. The structural variations associated with schizophrenia can involve several genes and the phenotypic syndromes, or the ‘genomic disorders’, have not yet been characterized4. Single nucleotide polymorphism (SNP)-based genome-wide association studies with the potential to implicate individual genes in complex diseases may reveal underlying biological pathways. Here we combined SNP data from several large genome-wide scans and followed up the most significant association signals. We found significant association with several markers spanning the major histocompatibility complex (MHC) region on chromosome 6p21.3-22.1, a marker located upstream of the neurogranin gene (NRGN) on 11q24.2 and a marker in intron four of transcription factor 4 (TCF4) on 18q21.2. Our findings implicating the MHC region are consistent with an immune component to schizophrenia risk, whereas the association with NRGN and TCF4 points to perturbation of pathways involved in brain development, memory and cognition.

 

Letter

The International Schizophrenia Consortium. Common polygenic variation contributes to risk of schizophrenia and bipolar disorder.  Nature 460, 748-752 (6 August 2009) | doi:10.1038/nature08185; Received 11 February 2009; Accepted 8 June 2009; Published online 1 July 2009; Corrected 6 August 2009

Schizophrenia is a severe mental disorder with a lifetime risk of about 1%, characterized by hallucinations, delusions and cognitive deficits, with heritability estimated at up to 80%1, 2. We performed a genome-wide association study of 3,322 European individuals with schizophrenia and 3,587 controls. Here we show, using two analytic approaches, the extent to which common genetic variation underlies the risk of schizophrenia. First, we implicate the major histocompatibility complex. Second, we provide molecular genetic evidence for a substantial polygenic component to the risk of schizophrenia involving thousands of common alleles of very small effect. We show that this component also contributes to the risk of bipolar disorder, but not to several non-psychiatric diseases.

 

The Psychiatric GWAS Consortium Steering Committee. A framework for interpreting genome-wide association studies of psychiatric disorders.  Molecular Psychiatry (2009) 14, 10–17; doi:10.1038/mp.2008.126; published online 11 November 2008

Genome-wide association studies (GWAS) have yielded a plethora of new findings in the past 3 years. By early 2009, GWAS on 47 samples of subjects with attention-deficit hyperactivity disorder, autism, bipolar disorder, major depressive disorder and schizophrenia will be completed. Taken together, these GWAS constitute the largest biological experiment ever conducted in psychiatry (59 000 independent cases and controls, 7700 family trios and >40 billion genotypes). We know that GWAS can work, and the question now is whether it will work for psychiatric disorders. In this review, we describe these studies, the Psychiatric GWAS Consortium for meta-analyses of these data, and provide a logical framework for interpretation of some of the conceivable outcomes.

Keywords: genome-wide association, attention-deficit hyperactivity disorder, autism, bipolar disorder, major depressive disorder, schizophrenia

The purpose of this article is to consider the ‘big picture’ and to provide a logical framework for the possible outcomes of these studies. This is not a review of GWAS per se as many excellent reviews of this technically and statistically intricate methodological approach are available.7, 8, 9, 10, 11, 12 This is also not a review of the advantages and disadvantages of different study designs and sampling strategies for the dissection of complex psychiatric traits. We would like to consider how the dozens of GWAS papers that will soon be in the literature can be synthesized: what can integrated mega-analyses (meta-analysis is based on summary data (for example, odds ratios) from all available studies whereas ‘mega-analysis’ uses individual-level genotype and phenotype data) of all available GWAS data tell us about the etiology of these psychiatric disorders? This is an exceptional opportunity as positive or negative results will enable us to learn hard facts about these critically important psychiatric disorders. We suggest that it is not a matter of ‘success versus failure’ or ‘optimism versus pessimism’ but rather an opportunity for systematic and logical approaches to empirical data whereby both positive and appropriately qualified negative findings are informative.

The studies that comprise the Psychiatric GWAS Consortium (PGC; http://pgc.unc.edu) are shown in Table 1. GWAS data for ADHD, autism, bipolar disorder, major depressive disorder and schizophrenia from 42 samples of European subjects should be available for mega-analyses by early 2009 (>59 000 independent cases and controls and >7700 family trios). To our knowledge, the PGC will have access to the largest set of GWAS data available.

A major change in human genetics in the past 5 years has been in the growth of controlled-access data repositories, and individual phenotype and genotype data are now available for many of the studies in Table 1. When the PGC mega-analyses are completed, most data will be available to researchers via the NIMH Human Genetics Initiative (http://nimhgenetics.org). Although the ready availability of GWAS data is a benefit to the field by allowing rapid application of a wide range of analytic strategies to GWAS data, there are potential disadvantages. GWAS mega-analysis is complex and requires considerable care and expertise to be done validly. For psychiatric phenotypes, there is the additional challenge of working with disease entities based largely on clinical description, with unknown biological validity and having both substantial clinical variation within diagnostic categories as well as overlaps across categories.13 Given the urgent need to know if there are replicable genotype–phenotype associations, a new type of collaboration was required.

The purpose of the PGC is to conduct rigorous and comprehensive within- and cross-disorder GWAS mega-analyses. The PGC began in early 2007 with the principal investigators of the four GAIN GWAS,14 and within six months had grown to 110 participating scientists from 54 institutions in 11 countries. The PGC has a coordinating committee, five disease-working groups, a cross-disorder group, a statistical analysis and computational group, and a cluster computer for statistical analysis. It is remarkable that almost all investigators approached agreed to participate and that no one has left the PGC. Most effort is donated but we have obtained funding from the NIMH, the Netherlands Scientific Organization, Hersenstichting Nederland and NARSAD.

The PGC has two major specific aims. (1) Within-disorder mega-analyses: conduct separate mega-analyses of all available GWAS data for ADHD, autism, bipolar disorder, major depressive disorder, and schizophrenia to attempt to identify genetic variation convincingly associated with any one of these five disorders. (2) Cross-disorder mega-analyses: the clinically-derived DSM-IV and ICD-10 definitions may not directly reflect the fundamental genetic architecture.15 There are two subaims. (2a) Conduct mega-analysis to identify genetic variation convincingly associated with conventional definitions of two or more disorders. This nosological aim could assist in delineating the boundaries of this set of disorders. (2b) An expert working group will convert epidemiological and genetic epidemiological evidence into explicit hypotheses about overlap among these disorders, and then conduct mega-analyses based on these definitions (for example, to examine the lifetime presence of idiopathic psychotic features without regard to diagnostic context).

The goal of the PGC is to identify convincing genetic variation-disease associations. A convincing association would be extremely unlikely to result from chance, show consistent effect sizes across all or almost all samples and be impervious to vigorous attempts to disprove the finding (for example, by investigating sources of bias, confirmatory genotyping, and so on). Careful attention will be paid to the impact of potential sources of heterogeneity17 with the goal of assessing its impact without minimizing its presence.

Biological plausibility is not an initial requirement for a convincing statistical association, as there are many examples in human genetics of previously unsuspected candidate genes nonetheless showing highly compelling associations. For example, multiple SNPs in intron 1 of the FTO gene were associated with body mass index in 13 cohorts with 38 759 participants18 and yet ‘FTO’ does not appear in an exhaustive 116 page compilation of genetic studies of obesity.19 Some strong associations are in gene deserts: multiple studies have found convincing association between prostate cancer and a region on 8q24 that is ~250 kb from the nearest annotated gene.20 Both of these examples are being intensively investigated and we suspect that a compelling mechanistic ‘story’ will emerge in the near future. The presence of a compelling association without an obvious biological mechanism establishes a priority research area for molecular biology and neuroscience of a psychiatric disorder.

The PGC will use mega-analysis as the main analytic tool as individual-level data will be available from almost all samples. To wield this tool appropriately, a number of preconditions must be met. First, genotype data from different GWAS platforms must be made comparable as the direct overlap between platforms is often modest. This requires meticulous quality control for the inclusion of both SNPs and subjects and attention to the factors that can cause bias (for example, population stratification, cryptic relatedness or genotyping batch effects). Genotype harmonization can be accomplished using imputation (21, 22, for example) so that the same set of ~2 million23, 24 directly or imputed SNP genotypes are available for all subjects. Second, phenotypes need to be harmonized across studies. This is one of the most crucial components of the PGC and we are fortunate to have world experts directing the work. Third, the mega-analyses will assess potential heterogeneity of associations across samples.

A decision-tree schematic of the potential outcomes of the PGC mega-analyses is shown in Figure 1. Note that many of the possibilities in Figure 1 are not mutually exclusive and different disorders may take different paths through this framework. It is possible that there eventually will be dozens or hundreds of sequence variants strictly associated with these disorders with frequencies ranging from very rare to common.

………

 

GWAS has the potential to yield considerable insights but it is no panacea and may well perform differently for psychiatric disorders. Even if these psychiatric GWAS efforts are successful, the outcomes will be complex. GWAS may help us learn that clinical syndromes are actually many different things—for example, proportions of individuals with schizophrenia might evidence associations with rare CNVs of major effect,5, 6 with more common genetic variation in dozens (perhaps hundreds) of genomic regions, between genetic variation strongly modified by environmental risk factors, and some proportion may be genetically indistinguishable from the general population. Moreover, as fuel to long-standing ‘lumper versus splitter’ debates in psychiatric nosology, empirical data might show that some clinical disorders or identifiable subsets of subjects might overlap considerably.

The critical advantage of GWAS is the search of a ‘closed’ hypothesis space. If the large amount of GWAS data being generated are analyzed within a strict and coherent framework, it should be possible to establish hard facts about the fundamental genetic architecture of a set of important psychiatric disorders—which might include positive evidence of what these disorders are or exclusionary evidence of what they are not. Whatever the results, these historically large efforts should yield hard facts about ADHD, autism, bipolar disorder, major depressive disorder and schizophrenia that may help guide the next era of psychiatric research.

  1. Pe’er I, Yelensky R, Altshuler D, Daly MJ. Estimation of the multiple testing burden for genomewide association studies of nearly all common variants. Genet Epidemiol 2008; 32: 381–385. | Article | PubMed |
  2. Weiss LA, Shen Y, Korn JM, Arking DE, Miller DT, Fossdal R et al. Association between microdeletion and microduplication at 16p11.2 and autism. N Engl J Med 2008; 358: 667–675. | Article | PubMed | ChemPort |

 

Letter

Hreinn Stefansson1,36, et al. Large recurrent microdeletions associated with schizophrenia. Nature 455, 232-236 (11 September 2008) | doi:10.1038/nature07229; Received 17 April 2008; Accepted 8 July 2008; Corrected 11 September 2008

Reduced fecundity, associated with severe mental disorders1, places negative selection pressure on risk alleles and may explain, in part, why common variants have not been found that confer risk of disorders such as autism2, schizophrenia3 and mental retardation4. Thus, rare variants may account for a larger fraction of the overall genetic risk than previously assumed. In contrast to rare single nucleotide mutations, rare copy number variations (CNVs) can be detected using genome-wide single nucleotide polymorphism arrays. This has led to the identification of CNVs associated with mental retardation4, 5 and autism2. In a genome-wide search for CNVs associating with schizophrenia, we used a population-based sample to identify de novoCNVs by analysing 9,878 transmissions from parents to offspring. The 66 de novo CNVs identified were tested for association in a sample of 1,433 schizophrenia cases and 33,250 controls. Three deletions at 1q21.1, 15q11.2 and 15q13.3 showing nominal association with schizophrenia in the first sample (phase I) were followed up in a second sample of 3,285 cases and 7,951 controls (phase II). All three deletions significantly associate with schizophrenia and related psychoses in the combined sample. The identification of these rare, recurrent risk variants, having occurred independently in multiple founders and being subject to negative selection, is important in itself. CNV analysis may also point the way to the identification of additional and more prevalent risk variants in genes and pathways involved in schizophrenia.

 

The C4 gene can exist in multiple copies (from one to four) on each copy of chromosome 6, and has four different forms: C4A-short, C4B-short, C4A-long, and C4B-long. The researchers first examined the “structural alleles” of the C4 locus—that is, the combinations and copy numbers of the different C4 forms—in healthy individuals. They then examined how these structural alleles related to expression of both C4Aand C4B messenger RNAs (mRNAs) in postmortem brain tissues.

From this the researchers had a clear picture of how the architecture of the C4 locus affected expression ofC4A and C4B. Next, they compared DNA from roughly 30,000 schizophrenia patients with that from 35,000 healthy controls, and a correlation emerged: the alleles most strongly associated with schizophrenia were also those that were associated with the highest C4A expression. Measuring C4A mRNA levels in the brains of 35 schizophrenia patients and 70 controls then revealed that, on average, C4A levels in the patients’ brains were 1.4-fold higher.

C4 is an immune system “complement” factor—a small secreted protein that assists immune cells in the targeting and removal of pathogens. The discovery of C4’s association to schizophrenia, said McCarroll, “would have seemed random and puzzling if it wasn’t for work . . . showing that other complement components regulate brain wiring.” Indeed, complement protein C3 locates at synapses that are going to be eliminated in the brain, explained McCarroll, “and C4 was known to interact with C3 . . . so we thought well, actually, this might make sense.”

McCarroll’s team went on to perform studies in mice that revealed C4 is necessary for C3 to be deposited at synapses. They also showed that the more copies of the C4 gene present in a mouse, the more the animal’s neurons were pruned.

Synaptic pruning is a normal part of development and is thought to reflect the process of learning, where the brain strengthens some connections and eradicates others. Interestingly, the brains of deceased schizophrenia patients exhibit reduced neuron density. The new results, therefore, “make a lot of sense,” said Cardiff University’s Andrew Pocklington who did not participate in the work. They also make sense “in terms of the time period when synaptic pruning is occurring, which sort of overlaps with the period of onset for schizophrenia: around adolescence and early adulthood,” he added.

“[C4] has not been on anybody’s radar for having anything to do with schizophrenia, and now it is and there’s a whole bunch of really neat stuff that could happen,” said Sullivan. For one, he suggested, “this molecule could be something that is amenable to therapeutics.”

 

 

UniProtKB

Derived from proteolytic degradation of complement C4, C4a anaphylatoxin is a mediator of local inflammatory process. It induces the contraction of smooth muscle, increases vascular permeability and causes histamine release from mast cells and basophilic leukocytes.

Non-enzymatic component of C3 and C5 convertases and thus essential for the propagation of the classical complement pathway. Covalently binds to immunoglobulins and immune complexes and enhances the solubilization of immune aggregates and the clearance of IC through CR1 on erythrocytes. C4A isotype is responsible for effective binding to form amide bonds with immune aggregates or protein antigens, while C4B isotype catalyzes the transacylation of the thioester carbonyl group to form ester bonds with carbohydrate antigens.

 

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Making Sense of Hypertensive Retinopathy

Posted in Cerebrovascular and Neurodegenerative Diseases, Clinical Diagnostics, Curation, Dialectic, Discovery process, Disease Biology, Experimental validation, Explanatory, Historical relevance, Neurological Diseases, Neuroscience, Pharmaceutical Drug Discovery, Pharmacodynamics and Pharmacokinetics, Pharmacotherapy of Cardiovascular Disease, Systemic Inflammatory Response Related Disorders, Translational Science, Vision on February 21, 2016| Leave a Comment »

High blood pressure can damage the retina’s blood vessels and limit the retina’s function. It can also put pressure on the optic nerve.

Sourced through Scoop.it from: www.healthline.com

See on Scoop.it – Cardiovascular Disease: PHARMACO-THERAPY

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Cerebral Perfusion Pressure (CPP) | Regulation | Calculation

Posted in Cerebrovascular and Neurodegenerative Diseases, Chemical Biology and its relations to Metabolic Disease, Medical Devices R&D Investment, Medical Imaging Technology, Image Processing/Computing, MRI, CT, Nuclear Medicine, Ultra Sound, Stroke, Technology Transfer: Biotech and Pharmaceutical, Transarterial Chemoembolization, Translational Science on February 21, 2016| Leave a Comment »

Cerebral Perfusion Pressure (CPP) | Regulation | Calculation

Reporter: Aviva Lev-Ari, PhD, RN

 

Watch Video

https://www.youtube.com/v/fyYT8wozccw?fs=1&hl=fr_FR

Nursing School Doesn’t Have to be so DAMN Hard! CPP=MAP-ICP Normal range should be greater than 70 mmHg How to calculate, regulate, and manage CPP or cerebra…

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Prevention of Transient Ischemic Attack, and What are The Risk Factors ?

Posted in Cerebrovascular and Neurodegenerative Diseases, Stroke on February 21, 2016| Leave a Comment »

Prevention of Transient Ischemic Attack, and What are The Risk Factors ?

Reporter: Aviva Lev-Ari, PhD, RN

 

Watch Video

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Transient ischemic attack (tia) prevention mayo clinic . , . . . . Knowing your risk factors and living healthfully are the best things you can do to prevent…

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Graphene Interaction with Neurons

Posted in Biomedical Measurement Science, Ca2+ triggered activation, Calcium Signaling, Cell Biology, Signaling & Cell Circuits, Cerebrovascular and Neurodegenerative Diseases, Cognition, Curation, Innovations in Neurophysiology & Neuropsychology, Materials Science & Engineering, Neurodegenerative Diseases, Neurohumoral Transmission, Neurological Diseases, Neuroscience, tagged , , , , , on January 29, 2016| Leave a Comment »

Graphene Interaction with Neurons

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Graphene Shown to Safely Interact with Neurons in the Brain

University of Cambridge

(Source: University of Cambridge)

http://www.biosciencetechnology.com/sites/biosciencetechnology.com/files/bt1601_cambridge_graphene.png

 

Researchers have successfully demonstrated how it is possible to interface graphene – a two-dimensional form of carbon – with neurons, or nerve cells, while maintaining the integrity of these vital cells. The work may be used to build graphene-based electrodes that can safely be implanted in the brain, offering promise for the restoration of sensory functions for amputee or paralyzed patients, or for individuals with motor disorders such as epilepsy or Parkinson’s disease.

The research, published in the journal ACS Nano, was an interdisciplinary collaboration coordinated by the University of Trieste in Italy and the Cambridge Graphene Centre.

Previously, other groups had shown that it is possible to use treated graphene to interact with neurons. However the signal to noise ratio from this interface was very low. By developing methods of working with untreated graphene, the researchers retained the material’s electrical conductivity, making it a significantly better electrode.

“For the first time we interfaced graphene to neurons directly,” said Professor Laura Ballerini of the University of Trieste in Italy. “We then tested the ability of neurons to generate electrical signals known to represent brain activities, and found that the neurons retained their neuronal signaling properties unaltered. This is the first functional study of neuronal synaptic activity using uncoated graphene based materials.”

Our understanding of the brain has increased to such a degree that by interfacing directly between the brain and the outside world we can now harness and control some of its functions. For instance, by measuring the brain’s electrical impulses, sensory functions can be recovered. This can be used to control robotic arms for amputee patients or any number of basic processes for paralyzed patients – from speech to movement of objects in the world around them. Alternatively, by interfering with these electrical impulses, motor disorders (such as epilepsy or Parkinson’s) can start to be controlled.

Scientists have made this possible by developing electrodes that can be placed deep within the brain. These electrodes connect directly to neurons and transmit their electrical signals away from the body, allowing their meaning to be decoded.

However, the interface between neurons and electrodes has often been problematic: not only do the electrodes need to be highly sensitive to electrical impulses, but they need to be stable in the body without altering the tissue they measure.

Too often the modern electrodes used for this interface (based on tungsten or silicon) suffer from partial or complete loss of signal over time. This is often caused by the formation of scar tissue from the electrode insertion, which prevents the electrode from moving with the natural movements of the brain due to its rigid nature.

Graphene has been shown to be a promising material to solve these problems, because of its excellent conductivity, flexibility, biocompatibility and stability within the body.

Based on experiments conducted in rat brain cell cultures, the researchers found that untreated graphene electrodes interfaced well with neurons. By studying the neurons with electron microscopy and immunofluorescence the researchers found that they remained healthy, transmitting normal electric impulses and, importantly, none of the adverse reactions which lead to the damaging scar tissue were seen.

According to the researchers, this is the first step towards using pristine graphene-based materials as an electrode for a neuro-interface. In future, the researchers will investigate how different forms of graphene, from multiple layers to monolayers, are able to affect neurons, and whether tuning the material properties of graphene might alter the synapses and neuronal excitability in new and unique ways. “Hopefully this will pave the way for better deep brain implants to both harness and control the brain, with higher sensitivity and fewer unwanted side effects,” said Ballerini.

“We are currently involved in frontline research in graphene technology towards biomedical applications,” said Professor Maurizio Prato from the University of Trieste. “In this scenario, the development and translation in neurology of graphene-based high-performance biodevices requires the exploration of the interactions between graphene nano- and micro-sheets with the sophisticated signalling machinery of nerve cells. Our work is only a first step in that direction.”

“These initial results show how we are just at the tip of the iceberg when it comes to the potential of graphene and related materials in bio-applications and medicine,” said Professor Andrea Ferrari, Director of the Cambridge Graphene Centre. “The expertise developed at the Cambridge Graphene Centre allows us to produce large quantities of pristine material in solution, and this study proves the compatibility of our process with neuro-interfaces.”

The research was funded by the Graphene Flagship, a European initiative which promotes a collaborative approach to research with an aim of helping to translate graphene out of the academic laboratory, through local industry and into society.

Source: University of Cambridge

 

Remembering to Remember Supported by Two Distinct Brain Processes

http://www.biosciencetechnology.com/news/2013/08/remembering-remember-supported-two-distinct-brain-processes

To investigate how prospective memory is processed in the brain, psychological scientist Mark McDaniel of Washington University in St. Louis and colleagues had participants lie in an fMRI scanner and asked them to press one of two buttons to indicate whether a word that popped up on a screen was a member of a designated category.  In addition to this ongoing activity, participants were asked to try to remember to press a third button whenever a special target popped up. The task was designed to tap into participants’ prospective memory, or their ability to remember to take certain actions in response to specific future events.

When McDaniel and colleagues analyzed the fMRI data, they observed that two distinct brain activation patterns emerged when participants made the correct button press for a special target.

When the special target was not relevant to the ongoing activity—such as a syllable like “tor”—participants seemed to rely on top-down brain processes supported by the prefrontal cortex. In order to answer correctly when the special syllable flashed up on the screen, the participants had to sustain their attention and monitor for the special syllable throughout the entire task. In the grocery bag scenario, this would be like remembering to bring the grocery bags by constantly reminding yourself that you can’t forget them.

When the special target was integral to the ongoing activity—such as a whole word, like “table”—participants recruited a different set of brain regions, and they didn’t show sustained activation in these regions. The findings suggest that remembering what to do when the special target was a whole word didn’t require the same type of top-down monitoring. Instead, the target word seemed to act as an environmental cue that prompted participants to make the appropriate response—like reminding yourself to bring the grocery bags by leaving them near the front door.

“These findings suggest that people could make use of several different strategies to accomplish prospective memory tasks,” says McDaniel.

McDaniel and colleagues are continuing their research on prospective memory, examining how this phenomenon might change with age.

Co-authors on this research include Pamela LaMontagne, Michael Scullin, Todd Braver of Washington University in St. Louis; and Stefanie Beck of Technische Universität Dresden.

This research was funded by the National Institute on Aging, the Washington University Institute of Clinical and Translation Sciences, the National Center for Advancing Translational Sciences, and the German Science Foundation.

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Mindful Discoveries

Posted in Academic Publishing, Alzheimer's Disease, Artificial Intelligence - Breakthroughs in Theories and Technologies, Behavioral Genetics, Big Data, Cancer - General, Cell Biology, Cerebrovascular and Neurodegenerative Diseases, Child and Adolescent Psychiatry, Clinical & Translational, Cognition, Curation, Disease Biology, Explanatory, Gene Regulation, Genetics & Innovations in Treatment, Genetics & Pharmaceutical, Genome Biology, Human aging, Immunology, Innovations in Neurophysiology & Neuropsychology, Intelligent Information Systems, Neurodegenerative Diseases, Neurological Diseases, Neuroscience, Regenerative Biology and Medicine, RNA Biology, Cancer and Therapeutics, Schizophrenia, Stem Cells for Regenerative Medicine, tagged , , , , on January 28, 2016| Leave a Comment »

Mindful Discoveries

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Schizophrenia and the Synapse

Genetic evidence suggests that overactive synaptic pruning drives development of schizophrenia.

By Ruth Williams | January 27, 2016 … more follows)

http://www.the-scientist.com/?articles.view/articleNo/45189/title/Schizophrenia-and-the-Synapse/

3.2.4

3.2.4   Mindful Discoveries, Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 2: CRISPR for Gene Editing and DNA Repair

http://www.the-scientist.com/images/News/January2016/Schizophrenia.jpg

C4 (green) at synapses of human neurons

Compared to the brains of healthy individuals, those of people with schizophrenia have higher expression of a gene called C4, according to a paper published inNature today (January 27). The gene encodes an immune protein that moonlights in the brain as an eradicator of unwanted neural connections (synapses). The findings, which suggest increased synaptic pruning is a feature of the disease, are a direct extension of genome-wide association studies (GWASs) that pointed to the major histocompatibility (MHC) locus as a key region associated with schizophrenia risk.

“The MHC [locus] is the first and the strongest genetic association for schizophrenia, but many people have said this finding is not useful,” said psychiatric geneticist Patrick Sullivan of the University of North Carolina School of Medicine who was not involved in the study. “The value of [the present study is] to show that not only is it useful, but it opens up new and extremely interesting ideas about the biology and therapeutics of schizophrenia.”

Schizophrenia has a strong genetic component—it runs in families—yet, because of the complex nature of the condition, no specific genes or mutations have been identified. The pathological processes driving the disease remain a mystery.

Researchers have turned to GWASs in the hope of finding specific genetic variations associated with schizophrenia, but even these have not provided clear candidates.

“There are some instances where genome-wide association will literally hit one base [in the DNA],” explained Sullivan. While a 2014 schizophrenia GWAS highlighted the MHC locus on chromosome 6 as a strong risk area, the association spanned hundreds of possible genes and did not reveal specific nucleotide changes. In short, any hope of pinpointing the MHC association was going to be “really challenging,” said geneticist Steve McCarroll of Harvard who led the new study.

Nevertheless, McCarroll and colleagues zeroed in on the particular region of the MHC with the highest GWAS score—the C4 gene—and set about examining how the area’s structural architecture varied in patients and healthy people.

The C4gene can exist in multiple copies (from one to four) on each copy of chromosome 6, and has four different forms: C4A-short, C4B-short, C4A-long, and C4B-long. The researchers first examined the “structural alleles” of the C4 locus—that is, the combinations and copy numbers of the different C4 forms—in healthy individuals. They then examined how these structural alleles related to expression of both C4Aand C4B messenger RNAs (mRNAs) in postmortem brain tissues.From this the researchers had a clear picture of how the architecture of the C4 locus affected expression ofC4A and C4B. Next, they compared DNA from roughly 30,000 schizophrenia patients with that from 35,000 healthy controls, and a correlation emerged: the alleles most strongly associated with schizophrenia were also those that were associated with the highest C4A expression. Measuring C4A mRNA levels in the brains of 35 schizophrenia patients and 70 controls then revealed that, on average, C4A levels in the patients’ brains were 1.4-fold higher.C4 is an immune system “complement” factor—a small secreted protein that assists immune cells in the targeting and removal of pathogens. The discovery of C4’s association to schizophrenia, said McCarroll, “would have seemed random and puzzling if it wasn’t for work . . . showing that other complement components regulate brain wiring.” Indeed, complement protein C3 locates at synapses that are going to be eliminated in the brain, explained McCarroll, “and C4 was known to interact with C3 . . . so we thought well, actually, this might make sense.”McCarroll’s team went on to perform studies in mice that revealed C4 is necessary for C3 to be deposited at synapses. They also showed that the more copies of the C4 gene present in a mouse, the more the animal’s neurons were pruned.Synaptic pruning is a normal part of development and is thought to reflect the process of learning, where the brain strengthens some connections and eradicates others. Interestingly, the brains of deceased schizophrenia patients exhibit reduced neuron density. The new results, therefore, “make a lot of sense,” said Cardiff University’s Andrew Pocklington who did not participate in the work. They also make sense “in terms of the time period when synaptic pruning is occurring, which sort of overlaps with the period of onset for schizophrenia: around adolescence and early adulthood,” he added.

“[C4] has not been on anybody’s radar for having anything to do with schizophrenia, and now it is and there’s a whole bunch of really neat stuff that could happen,” said Sullivan. For one, he suggested, “this molecule could be something that is amenable to therapeutics.”

A. Sekar et al., “Schizophrenia risk from complexvariation of complement component 4,”Nature,   http://dx.doi.com:/10.1038/nature16549, 2016.     

Tags schizophrenia, neuroscience, gwas, genetics & genomics, disease/medicine and cell & molecular biology

Schizophrenia: From genetics to physiology at last

Ryan S. Dhindsa, & David B. Goldstein

Nature (2016)  http://dx.doi.org://10.1038/nature16874

The identification of a set of genetic variations that are strongly associated with the risk of developing schizophrenia provides insights into the neurobiology of this destructive disease.

http://www.nytimes.com/2016/01/28/health/schizophrenia-cause-synaptic-pruning-brain-psychiatry.html

Genetic study provides first-ever insight into biological origin of schizophrenia

Suspect gene may trigger runaway synaptic pruning during adolescence — NIH-funded study

NIH/NATIONAL INSTITUTE OF MENTAL HEALTH

IMAGE

http://media.eurekalert.org/multimedia_prod/pub/web/107629_web.jpg

The site in Chromosome 6 harboring the gene C4 towers far above other risk-associated areas on schizophrenia’s genomic “skyline,” marking its strongest known genetic influence. The new study is the first to explain how specific gene versions work biologically to confer schizophrenia risk.  CREDIT  Psychiatric Genomics Consortium

Versions of a gene linked to schizophrenia may trigger runaway pruning of the teenage brain’s still-maturing communications infrastructure, NIH-funded researchers have discovered. People with the illness show fewer such connections between neurons, or synapses. The gene switched on more in people with the suspect versions, who faced a higher risk of developing the disorder, characterized by hallucinations, delusions and impaired thinking and emotions.

“Normally, pruning gets rid of excess connections we no longer need, streamlining our brain for optimal performance, but too much pruning can impair mental function,” explained Thomas Lehner, Ph.D., director of the Office of Genomics Research Coordination of the NIH’s National Institute of Mental Health (NIMH), which co-funded the study along with the Stanley Center for Psychiatric Research at the Broad Institute and other NIH components. “It could help explain schizophrenia’s delayed age-of-onset of symptoms in late adolescence/early adulthood and shrinkage of the brain’s working tissue. Interventions that put the brakes on this pruning process-gone-awry could prove transformative.”

The gene, called C4 (complement component 4), sits in by far the tallest tower on schizophrenia’s genomic “skyline” (see graph below) of more than 100 chromosomal sites harboring known genetic risk for the disorder. Affecting about 1 percent of the population, schizophrenia is known to be as much as 90 percent heritable, yet discovering how specific genes work to confer risk has proven elusive, until now.

A team of scientists led by Steve McCarroll, Ph.D., of the Broad Institute and Harvard Medical School, Boston, leveraged the statistical power conferred by analyzing the genomes of 65,000 people, 700 postmortem brains, and the precision of mouse genetic engineering to discover the secrets of schizophrenia’s strongest known genetic risk. C4’s role represents the most compelling evidence, to date, linking specific gene versions to a biological process that could cause at least some cases of the illness.

“Since schizophrenia was first described over a century ago, its underlying biology has been a black box, in part because it has been virtually impossible to model the disorder in cells or animals,” said McCarroll. “The human genome is providing a powerful new way in to this disease. Understanding these genetic effects on risk is a way of prying open that block box, peering inside and starting to see actual biological mechanisms.”

McCarroll’s team, including Harvard colleagues Beth Stevens, Ph.D., Michael Carroll, Ph.D., and Aswin Sekar, report on their findings online Jan. 27, 2016 in the journal Nature.

A swath of chromosome 6 encompassing several genes known to be involved in immune function emerged as the strongest signal associated with schizophrenia risk in genome-wide analyses by the NIMH-funded Psychiatric Genomics Consortium over the past several years. Yet conventional genetics failed to turn up any specific gene versions there linked to schizophrenia.

To discover how the immune-related site confers risk for the mental disorder, McCarroll’s team mounted a search for “cryptic genetic influences” that might generate “unconventional signals.” C4, a gene with known roles in immunity, emerged as a prime suspect because it is unusually variable across individuals. It is not unusual for people to have different numbers of copies of the gene and distinct DNA sequences that result in the gene working differently.

The researchers dug deeply into the complexities of how such structural variation relates to the gene’s level of expression and how that, in turn, might relate to schizophrenia. They discovered structurally distinct versions that affect expression of two main forms of the gene in the brain. The more a version resulted in expression of one of the forms, called C4A, the more it was associated with schizophrenia. The more a person had the suspect versions, the more C4 switched on and the higher their risk of developing schizophrenia. Moreover, in the human brain, the C4 protein turned out to be most prevalent in the cellular machinery that supports connections between neurons.

Adapting mouse molecular genetics techniques for studying synaptic pruning and C4’s role in immune function, the researchers also discovered a previously unknown role for C4 in brain development. During critical periods of postnatal brain maturation, C4 tags a synapse for pruning by depositing a sister protein in it called C3. Again, the more C4 got switched on, the more synapses got eliminated.

In humans, such streamlining/pruning occurs as the brain develops to full maturity in the late teens/early adulthood – conspicuously corresponding to the age-of-onset of schizophrenia symptoms.

Future treatments designed to suppress excessive levels of pruning by counteracting runaway C4 in at risk individuals might nip in the bud a process that could otherwise develop into psychotic illness, suggest the researchers. And thanks to the head start gained in understanding the role of such complement proteins in immune function, such agents are already in development, they note.

“This study marks a crucial turning point in the fight against mental illness. It changes the game,” added acting NIMH director Bruce Cuthbert, Ph.D. “Thanks to this genetic breakthrough, we can finally see the potential for clinical tests, early detection, new treatments and even prevention.”

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VIDEO: Opening Schizophrenia’s Black Box https://youtu.be/s0y4equOTLg

Reference: Sekar A, Biala AR, de Rivera H, Davis A, Hammond TR, Kamitaki N, Tooley K Presumey J Baum M, Van Doren V, Genovese G, Rose SA, Handsaker RE, Schizophrenia Working Group of the Psychiatric Genomics Consortium, Daly MJ, Carroll MC, Stevens B, McCarroll SA. Schizophrenia risk from complex variation of complement component 4.Nature. Jan 27, 2016. DOI: 10.1038/nature16549.

Schizophrenia risk from complex variation of complement component 4

Aswin Sekar, Allison R. Bialas, Heather de Rivera, Avery Davis, Timothy R. Hammond, …., Michael C. Carroll, Beth Stevens & Steven A. McCarroll

Nature(2016)   http://dx.doi.org:/10.1038/nature16549

Schizophrenia is a heritable brain illness with unknown pathogenic mechanisms. Schizophrenia’s strongest genetic association at a population level involves variation in the major histocompatibility complex (MHC) locus, but the genes and molecular mechanisms accounting for this have been challenging to identify. Here we show that this association arises in part from many structurally diverse alleles of the complement component 4 (C4) genes. We found that these alleles generated widely varying levels of C4A and C4B expression in the brain, with each common C4 allele associating with schizophrenia in proportion to its tendency to generate greater expression of C4A. Human C4 protein localized to neuronal synapses, dendrites, axons, and cell bodies. In mice, C4 mediated synapse elimination during postnatal development. These results implicate excessive complement activity in the development of schizophrenia and may help explain the reduced numbers of synapses in the brains of individuals with schizophrenia.

Figure 1: Structural variation of the complement component 4 (C4) gene.

http://www.nature.com/nature/journal/vaop/ncurrent/carousel/nature16549-f1.jpg

a, Location of the C4 genes within the major histocompatibility complex (MHC) locus on human chromosome 6. b, Human C4 exists as two paralogous genes (isotypes), C4A and C4B; the encoded proteins are distinguished at a key site

http://www.nature.com/nature/journal/vaop/ncurrent/carousel/nature16549-f3.jpg

http://www.nature.com/nature/journal/vaop/ncurrent/carousel/nature16549-sf8.jpg

Gene Study Points Toward Therapies for Common Brain Disorders

University of Edinburgh    http://www.dddmag.com/news/2016/01/gene-study-points-toward-therapies-common-brain-disorders

Scientists have pinpointed the cells that are likely to trigger common brain disorders, including Alzheimer’s disease, Multiple Sclerosis and intellectual disabilities.

It is the first time researchers have been able to identify the particular cell types that malfunction in a wide range of brain diseases.

Scientists say the findings offer a roadmap for the development of new therapies to target the conditions.

The researchers from the University of Edinburgh’s Centre for Clinical Brain Sciences used advanced gene analysis techniques to investigate which genes were switched on in specific types of brain cells.

They then compared this information with genes that are known to be linked to each of the most common brain conditions — Alzheimer’s disease, anxiety disorders, autism, intellectual disability, multiple sclerosis, schizophrenia and epilepsy.

Their findings reveal that for some conditions, the support cells rather than the neurons that transmit messages in the brain are most likely to be the first affected.

Alzheimer’s disease, for example, is characterised by damage to the neurons. Previous efforts to treat the condition have focused on trying to repair this damage.

The study found that a different cell type — called microglial cells — are responsible for triggering Alzheimer’s and that damage to the neurons is a secondary symptom of disease progression.

Researchers say that developing medicines that target microglial cells could offer hope for treating the illness.

The approach could also be used to find new treatment targets for other diseases that have a genetic basis, the researchers say.

Dr Nathan Skene, who carried out the study with Professor Seth Grant, said: “The brain is the most complex organ made up from a tangle of many cell types and sorting out which of these cells go wrong in disease is of critical importance to developing new medicines.”

Professor Seth Grant said: “We are in the midst of scientific revolution where advanced molecular methods are disentangling the Gordian Knot of the brain and completely unexpected new pathways to solving diseases are emerging. There is a pressing need to exploit the remarkable insights from the study.”

Quantitative multimodal multiparametric imaging in Alzheimer’s disease

Qian Zhao, Xueqi Chen, Yun Zhou      Brain Informatics  http://link.springer.com/article/10.1007/s40708-015-0028-9

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder, causing changes in memory, thinking, and other dysfunction of brain functions. More and more people are suffering from the disease. Early neuroimaging techniques of AD are needed to develop. This review provides a preliminary summary of the various neuroimaging techniques that have been explored for in vivo imaging of AD. Recent advances in magnetic resonance (MR) techniques, such as functional MR imaging (fMRI) and diffusion MRI, give opportunities to display not only anatomy and atrophy of the medial temporal lobe, but also at microstructural alterations or perfusion disturbance within the AD lesions. Positron emission tomography (PET) imaging has become the subject of intense research for the diagnosis and facilitation of drug development of AD in both animal models and human trials due to its non-invasive and translational characteristic. Fluorodeoxyglucose (FDG) PET and amyloid PET are applied in clinics and research departments. Amyloid beta (Aβ) imaging using PET has been recognized as one of the most important methods for the early diagnosis of AD, and numerous candidate compounds have been tested for Aβ imaging. Besides in vivo imaging method, a lot of ex vivo modalities are being used in the AD researches. Multiphoton laser scanning microscopy, neuroimaging of metals, and several metal bioimaging methods are also mentioned here. More and more multimodality and multiparametric neuroimaging techniques should improve our understanding of brain function and open new insights into the pathophysiology of AD. We expect exciting results will emerge from new neuroimaging applications that will provide scientific and medical benefits.

Keywords –   Alzheimer’s disease Neuroimaging PET MRI Amyloid beta Multimodal

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that gradually destroys brain cells, causing changes in memory, thinking, and other dysfunction of brain functions [1]. AD is considered to a prolonged preclinical stage where neuropathological changes precede the clinical symptoms [2]. An estimation of 35 million people worldwide is living with this disease. If effective treatments are not discovered in a timely fashion, the number of AD cases is anticipated to rise to 113 million by 2050 [3].

Amyloid beta (Aβ) and tau are two of the major biomarkers of AD, and have important and different roles in association with the progression of AD pathophysiology. Jack et al. established hypothetical models of the major biomarkers of AD. By renewing and modifying the models, they found that the two major proteinopathies underlying AD biomarker changes, Aβ and tau, may be initiated independently in late onset AD where they hypothesize that an incident Aβ pathophysiology can accelerate an antecedent limbic and brainstem tauopathy [4]. MRI technique was used in the article, which revealed that the level of Aβ load was associated with a shorter time-to-progression of AD [5]. This warrants an urgent need to develop early neuroimaging techniques of AD neuropathology that can detect and predict the disease before the onset of dementia, monitor therapeutic efficacy in halting and slowing down progression in the earlier stage of the disease.

There have been various reports on the imaging assessments of AD. Some measurements reflect the pathology of AD directly, including positron emission tomography (PET) amyloid imaging and cerebrospinal fluid (CSF) beta-amyloid 42 (Aβ42), while others reflect neuronal injury associated with AD indirectly, including CSF tau (total and phosphorylated tau), fluorodeoxy-d-glucose (FDG)-PET, and MRI. AD Neuroimaging Initiative (ADNI) has been to establish the optimal panel of clinical assessments, MRI and PET imaging measures, as well as other biomarkers from blood and CSF, to inform clinical trial design for AD therapeutic development. At the same time, it has been highly productive in generating a wealth of data for elucidating disease mechanisms occurring during early stages of preclinical and prodromal AD [6].

Single neuroimaging often reflects limit information of AD. As a result, multimodal neuroimaging is widely used in neuroscience researches, as it overcomes the limitations of individual modalities. Multimodal multiparametric imaging mean the combination of different imaging techniques, such as PET, MRI, simultaneously or separately. The multimodal multiparametric imaging enables the visualization and quantitative analysis of the alterations in brain structure and function, such as PET/CT, and PET/MRI. [7]. In this review article, we summarize and discuss the main applications, findings, perspectives as well as advantages and challenges of different neuroimaging in AD, especially MRI and PET imaging.

2 Magnetic resonance imaging

MRI demonstrates specific volume loss or cortical atrophy patterns with disease progression in AD patients [8–10]. There are several MRI techniques and analysis methods used in clinical and scientific research of AD. Recent advances in MR techniques, such as functional MRI (fMRI) and diffusion MRI, depict not only anatomy and atrophy of the medial temporal lobe (MTL), but also microstructural alterations or perfusion disturbance within this region.

2.1 Functional MRI

Because of the cognitive reserve (CR), the relationship between severity of AD patients’ brain damage and corresponding clinical symptoms is not always paralleled [11, 12]. Recently, resting-state fMRI (RS-fMRI) is popular for its ability to map brain functional connectivity non-invasively [13]. By using RS-fMRI, Bozzali et al. reported that the CR played a role in modulating the effect of AD pathology on default mode network functional connectivity, which account for the variable clinical symptoms of AD [14]. Moreover, AD patients with higher educated experience were able to recruit compensatory neural mechanisms, which can be measured using RS-fMRI. Arterial spin-labeled (ASL) MRI is another functional brain imaging modality, which measures cerebral blood flow (CBF) by magnetically labeled arterial blood water following through the carotid and vertebral arteries as an endogenous contrast medium. Several studies have concluded the characteristics of CBF changes in AD patients using ASL-MRI [15–17].

At some point in time, sufficient brain damage accumulates to result in cognitive symptoms and impairment. Mild cognitive impairment (MCI) is a condition in which subjects are usually only mildly impaired in memory with relative preservation of other cognitive domains and functional activities and do not meet the criteria for dementia [18], or as the prodromal state AD [19]. MCI patients are at a higher risk of developing AD and up to 15 % convert to AD per year [18]. Binnewijzend et al. have reported the pseudocontinuous ASL could distinguish both MCI and AD from healthy controls, and be used in the early diagnosis of AD [20]. In their continuous study, they used quantitative whole brain pseudocontinuous ASL to compare regional CBF (rCBF) distribution patterns in different types of dementia, and concluded that ASL-MRI could be a non-invasive and easily accessible alternative to FDG-PET imaging in the assessment of CBF of AD patients [21].

2.2 Structure MRI

Structural MRI (sMRI) has already been a reliable imaging method in the clinical diagnosis of AD, characterized as gray matter reduction and ventricular enlargement in standard T1-weighted sequences [9]. Locus coeruleus (LC) and substantia nigra (SN) degeneration was seen in AD. By using new quantitative calculating method, Chen et al. presented a new quantitative neuromelanin MRI approach for simultaneous measurement of locus LC and SN of brainstem in living human subjects [22]. The approach they used demonstrated advantages in image acquisition, pre-processing, and quantitative analysis. Numerous transgenic animal models of amyloidosis are available, which can manipulate a lot of neuropathological features of AD progression from the deposition of β-amyloid [23]. Braakman et al. demonstrated the dynamics of amyloid plaque formation and development in a serial MRI study in a transgenic mouse model [24]. Increased iron accumulation in gray matter is frequently observed in AD. Because of the paramagnetic nature of iron, MRI shows nice potential in the investigating iron levels in AD [25]. Quantitative MRI was shown high sensitivity and specificity in mapping cerebral iron deposition, and helped in the research on AD diagnosis [26].

The imaging patterns are always associated with the pathologic changes, such as specific protein markers. Spencer et al. manifested the relationship between quantitative T1 and T2 relaxation time changes and three immunohistochemical markers: β-amyloid, neuron-specific nuclear protein (a marker of neuronal cell load), and myelin basic protein (a marker of myelin load) in AD transgenic mice [27].

High-field MRI has been successfully applied to imaging plaques in transgenic mice for over a decade without contrast agents [24, 28–30]. Sillerud et al. devised a method using blood–brain barrier penetrating, amyloid-targeted, superparamagnetic iron oxide nanoparticles (SPIONs) for better imaging of amyloid plaque [31]. Then, they successfully used this SPION-MRI to assess the drug efficacy on the 3D distribution of Aβ plaques in transgenic AD mouse [32].

2.3 Diffusion MRI

Diffusion-weighted imaging (DWI) is a sensitive tool that allows quantifying of physiologic alterations in water diffusion, which result from microscopic structural changes.

Diffusion tensor imaging (DTI) is a well-established and commonly employed diffusion MRI technique in clinical and research on neuroimaging studies, which is based on a Gaussian model of diffusion processes [33]. In general, AD is associated with widespread reduced fractional anisotropy (FA) and increased mean diffusivity (MD) in several regions, most prominently in the frontal and temporal lobes, and along the cingulum, corpus callosum, uncinate fasciculus, superior longitudinal fasciculus, and MTL-associated tracts than healthy controls [34–37]. Acosta-Cabronero et al. reported increased axial diffusivity and MD in the splenium, which were the earliest abnormalities in AD [38]. FA and radial diffusivity (DR) differences in the corpus callosum, cingulum, and fornix were found to separate individuals with MCI who converted to AD from non-converters [39]. DTI was also found to be a better predictor of AD-specific MTL atrophy when compared to CSF biomarkers [40]. These findings suggested the potential clinical utility of DTI as early biomarkers of AD and its progression. However, an increase in MD and DR and a decrease in FA with advancing age in selective brain regions have been previously reported [41, 42]. Diffusion MRI can be also used in the classifying of various stages of AD. Multimodal classification method, which combined fMRI and DTI, separated more MCI from healthy controls than single approaches [43].

In recent years, tau has emerged as a potential target for therapeutic intervention. Tau plays a critical role in the neurodegenerative process forming neurofibrillary tangles, which is a major hallmark of AD and correlates with clinical disease progression. Wells et al. applied multiparametric MRI, containing high-resolution structure MRI (sMRI), a novel chemical exchange saturation transfer (CEST) MRI, DTI, and ASL, and glucose CEST to measure changes of tau pathology in AD transgenic mouse [44].

Besides DWI MRI, perfusion-weighted imaging (PWI) is another advanced MR technique, which could measure the cerebral hemodynamics at the capillary level. Zimny et al. evaluated the correlation of MTL with both DWI and PWI in AD and MCI patients [45].

3 Positron emission tomography

PET is a specific imaging technique applying in researches of brain function and neurochemistry of small animals, medium-sized animals, and human subjects [46–48]. As a particular brain imaging technique, PET imaging has become the subject of intense research for the diagnosis and facilitation of drug development of AD in both animal models and human trials due to its non-invasive and translational characteristic. PET with various radiotracers is considered as a standard non-invasive quantitative imaging technique to measure CBF, glucose metabolism, and β-amyloid and tau deposition.

3.1 FDG-PET

To date, 18F-FDG is one of the best and widely used neuroimaging tracers of PET, which employed for research and clinical assessment of AD [49]. Typical lower FDG metabolism was shown in the precuneus, posterior cingulate, and temporal and parietal cortex with progression to whole brain reductions with increasing disease progress in AD brains [50, 51]. FDG-PET imaging reflects the cerebral glucose metabolism, neuronal injury, which provides indirect evidence on cognitive function and progression that cannot be provided by amyloid PET imaging.

Schraml et al. [52] identified a significant association between hypometabolic convergence index and phenotypes using ADNI data. Some researchers also used 18F-FDG-PET to analyze genetic information with multiple biomarkers to classify AD status, predicting cognitive decline or MCI to AD conversion [53–55]. Trzepacz et al. [56] reported multimodal AD neuroimaging study, using MRI, 11C-PiB PET, and 18F-FDG-PET imaging to predict MCI conversion to AD along with APOE genotype. Zhang et al. [57] compared the genetic modality single-nucleotide polymorphism (SNP) with sMRI, 18F-FDG-PET, and CSF biomarkers, which were used to differentiate healthy control, MCI, and AD. They found FDG-PET is the best modality in terms of accuracy.

3.2 Amyloid beta PET

Aβ, the primary constituent of senile plaques, and tau tangles are hypothesized to play a primary role in the pathogenesis of AD, but it is still hard to identify the fundamental mechanisms [58–60]. Aβ plaque in brain is one of the pathological hallmarks of AD [61,62]. Accumulation of Aβ peptide in the cerebral cortex is considered one cause of dementia in AD [63]. Numerous studies have involved in vivo PET imaging assessing cortical β-amyloid burden [64–66].

Aβ imaging using PET has been recognized as one of the most important methods for the early diagnosis of AD [67]. Numerous candidate compounds have been tested for Aβ imaging, such as 11C-PiB [68], 18F-FDDNP [69], 11C-SB-13 [70], 18F-BAY94-9172 [71], 18F-AV-45 [72], 18F-flutemetamol [73, 74], 11C-AZD2184 [75], and 18F-ADZ4694 [76], 11C-BF227 and 18F-FACT [77].

Several amyloid PET studies examined genotypes, phenotypes, or gene–gene interactions. Ramanan et al. [78] reported the GWAS results with 18F-AV-45 reflecting the cerebral amyloid metabolism in AD for the first time. Swaminathan et al. [79] revealed the association between plasma Aβ from peripheral blood and cortical amyloid deposition on 11C-PiB. Hohman et al. [80] reported the relationship between SNPs involved in amyloid and tau pathophysiology with 18F-AV-45 PET.

Among the PET tracers, 11C-PiB, which has a high affinity for fibrillar Aβ, is a reliable biomarker of underlying AD pathology [68, 81]. It shows cortical uptake well paralleled with AD pathology [82, 83], has recently been approved for use by the Food and Drug Administration (FDA, April 2012) and the European Medicines Agency (January 2013). 18F-GE-067 (flutemetamol) and 18F-BAY94-9172 (florbetaben) have also been approved by the US FDA in the last 2 years [84, 85].

18F-Florbetapir (also known as 18F-AV-45) exhibits high affinity specific binding to amyloid plaques. 18F-AV-45 labels Aβ plaques in sections from patients with pathologically confirmed AD [72].

It was reported in several research groups that 18F-AV-45 PET imaging showed a reliability of both qualitative and quantitative assessments in AD patients, and Aβ+ increased with diagnostic category (healthy control < MCI < AD) [82, 86, 87]. Johnson et al. used 18F-AV-45 PET imaging to evaluate the amyloid deposition in both MCI and AD patients qualitatively and quantitatively, and found that amyloid burden increased with diagnostic category (MCI < AD), age, and APOEε4 carrier status [88]. Payoux et al. reported the equivocal amyloid PET scans using 18F-AV-45 associated with a specific pattern of clinical signs in a large population of non-demented older adults more than 70 years old [89].

More and more researchers consider combination and comparison of multiple PET tracers targeting amyloid plaque imaging together. Bruck et al. compared the prognostic ability of 11C-PiB PET, 18F-FDG-PET, and quantitative hippocampal volumes measured with MR imaging in predicting MCI to AD conversion. They found that the FDG-PET and 11C-PiB PET imaging are better in predicting MCI to AD conversion [90]. Hatashita et al. used 11C-PiB and FDG-PET imaging to identify MCI due to AD, 11C-PiB showed a higher sensitivity of 96.6 %, and FDG-PET added diagnostic value in predicting AD over a short period [91].

Besides, new Aβ imaging agents were radiosynthesized. Yousefi et al. radiosynthesized a new Aβ imaging agent 18F-FIBT, and compared the three different Aβ-targeted radiopharmaceuticals for PET imaging, including 18F-FIBT, 18F-florbetaben, and 11C-PiB [92]. 11C-AZD2184 is another new PET tracer developed for amyloid senile plaque imaging, and the kinetic behavior of 11C-AZD2184 is suitable for quantitative analysis and can be used in clinical examination without input function [75,93, 94].

4 Multimodality imaging: PET/MRI

Several diagnostic techniques, including MRI and PET, are employed for the diagnosis and monitoring of AD [95]. Multimodal imaging could provide more information in the formation and key molecular event of AD than single method. It drives the progression of neuroimaging research due to the recognition of the clinical benefits of multimodal data [96], and the better access to hybrid devices, such as PET/MRI [97].

Maier et al. evaluated the dynamics of 11C-PiB PET, 15O-H2O-PET, and ASL-MRI in transgenic AD mice and concluded that the AD-related decline of rCBF was caused by the cerebral Aβ angiopathy [98]. Edison et al. systematically compared 11C-PiB PET and MRI in AD, MCI patients, and controls. They thought that 11C-PiB PET was adequate for clinical diagnostic purpose, while MRI remained more appropriate for clinical research [99]. Zhou et al. investigated the interactions between multimodal PET/MRI in elder patients with MCI, AD, and healthy controls, and confirmed the invaluable application of amyloid PET and MRI in early diagnosis of AD [100]. Kim et al. reported that Aβ-weighted cortical thickness, which incorporates data from both MRI and amyloid PET imaging, is a consistent and objective imaging biomarker in AD [101].

5 Other imaging modalities

Multiphoton non-linear optical microscope imaging systems using ultrafast lasers have powerful advantages such as label-free detection, deep penetration of thick samples, high sensitivity, subcellular spatial resolution, 3D optical sectioning, chemical specificity, and minimum sample destruction [102, 103]. Coherent anti-Stokes–Raman scattering (CARS), two-photon excited fluorescence (TPEF), and second-harmonic generation (SHG) microscopy are the most widely used biomedical imaging techniques [104–106].

Quantitative electroencephalographic and neuropsychological investigation of an alternative measure of frontal lobe executive functions: the Figure Trail Making Test

 Paul S. Foster, Valeria Drago, Brad J. Ferguson, Patti Kelly Harrison,David W. Harrison 

Brain Informatis    http://dx.doi.org:/10.1007/s40708-015-0025-z    http://link.springer.com/article/10.1007/s40708-015-0025-z/fulltext.html

The most frequently used measures of executive functioning are either sensitive to left frontal lobe functioning or bilateral frontal functioning. Relatively little is known about right frontal lobe contributions to executive functioning given the paucity of measures sensitive to right frontal functioning. The present investigation reports the development and initial validation of a new measure designed to be sensitive to right frontal lobe functioning, the Figure Trail Making Test (FTMT). The FTMT, the classic Trial Making Test, and the Ruff Figural Fluency Test (RFFT) were administered to 42 right-handed men. The results indicated a significant relationship between the FTMT and both the TMT and the RFFT. Performance on the FTMT was also related to high beta EEG over the right frontal lobe. Thus, the FTMT appears to be an equivalent measure of executive functioning that may be sensitive to right frontal lobe functioning. Applications for use in frontotemporal dementia, Alzheimer’s disease, and other patient populations are discussed.

Keywords – Frontal lobes, Executive functioning, Trail making test, Sequencing, Behavioral speed, Designs, Nonverbal, Neuropsychological assessment, Regulatory control, Effortful control

A recent survey indicated that the vast majority of neuropsychologists frequently assess executive functioning as part of their neuropsychological evaluations [1]. Surveys of neuropsychologists have indicated that the Trail Making Test (TMT), Controlled Oral Word Association Test (COWAT), Wisconsin Card Sorting Test (WCST), and the Stroop Color-Word Test (SCWT) are among the most commonly used instruments [1,2]. Further, the Rabin et al. [1] survey indicated that these same tests are among the most frequently used by neuropsychologists when specifically assessing executive or frontal lobe functioning. The frequent use of the TMT, WCST, and the SCWT, as well as the assumption that they are measures of executive functioning, led Demakis (2003–2004) to conduct a series of meta-analyses to determine the sensitivity of these test to detect frontal lobe dysfunction, particularly lateralized frontal lobe dysfunction. The findings indicated that the SCWT and Part A of the TMT [3], as well as the WCST [4], were all sensitive to frontal lobe dysfunction. However, only the SCWT differentiated between left and right frontal lobe dysfunction, with the worst performance among those with left frontal lobe dysfunction [3].

The finding of the Demakis [4] meta-analysis, that the WCST was not sensitive to lateralized frontal lobe dysfunction, is not surprising given the equivocal findings that have been reported. Whereas performance on the WCST is sensitive to frontal lobe dysfunction [5, 6], demonstration of lateralized frontal dysfunction has been quite problematic. Unilateral left or right dorsolateral frontal dysfunction has been associated with impaired performance on the WCST [6]. Fallgatter and Strik [7] found bilateral frontal lobe activation during performance of the WCST. However, other imaging studies have found right lateralized frontal lobe activation [8] and left lateralized frontal activation [9] in response to performance on the WCST. Further, left frontal lobe alpha power is negatively correlated with performance on the WCST [10]. Finally, patients with left frontal lobe tumors exhibit more impaired performance on the WCST than those with right frontal tumors [11].

Unlike the data for the WCST, more consistent findings have been reported regarding lateralized frontal lobe functioning for the other commonly used measures of executive functioning. For instance, as with the Demakis [3] study, many investigations have found the SCWT to be sensitive to left frontal lobe functioning, although the precise localization within the left frontal lobe has varied. Impaired performance on the SCWT results from left frontal lesions [12] and specifically from lesions localized to the left dorsolateral frontal lobe [13, 14], though bilateral frontal lesions have also yielded impaired performance [13, 14]. Further, studies using neuroimaging to investigate the neural basis of performance on the SCWT have indicated involvement of the left anterior cingulated cortex [15], left lateral prefrontal cortex [16], left inferior precentral sulcus [17], and the left dorsolateral frontal lobe [18].

Wide agreement exists among investigations of the frontal lateralization of verbal or lexical fluency to confrontation. Specifically, patients with left frontal lobe lesions are known to exhibit impaired performance on lexical fluency to confrontation tasks, relative to either patients with right frontal lesions [12, 19, 20] or controls [21]. A recent meta-analysis also indicated that the largest deficits in performance on measures of lexical fluency are associated with left frontal lobe lesions [22]. Troster et al. [23] found that, relative to patients with right pallidotomy, patients with left pallidotomy exhibited more impaired lexical fluency. Several neuroimaging investigations have further supported the role of the left frontal lobe in lexical fluency tasks [15, 24–27]. Performance on lexical fluency tasks also varies as a function of lateral frontal lobe asymmetry, as assessed by electroencephalography [28].

The Trail Making Test is certainly among the most widely used tests [1] and perhaps the most widely researched. Various norms exist for the TMT (see [29]), with Tombaugh [30] providing the most recent comprehensive set of normative data. Different methods of analyzing and interpreting the data have also been proposed and used, including error analysis [13, 14, 31–33], subtraction scores [13, 14, 34], and ratio scores [13, 14, 35].

Several different language versions of the test have been developed and reported, including Arabic [36], Chinese [37, 38], Greek [39], and Hebrew [40]. Numerous alternative versions of the TMT have been developed to address perceived shortcomings of the original TMT. For instance, the Symbol Trail Making Test [41] was developed to reduce the cultural confounds associated with the use of the Arabic numeral system and English alphabet in the original TMT. The Color Trails Test (CTT; [42]) was also developed to control for cultural confounds, although mixed results have been reported regarding whether the CTT is indeed analogous to the TMT [43–45]. A version of the TMT for preschool children, the TRAILS-P, has also been reported [46].

Additionally, the Comprehensive Trail Making Test [47] was developed to control for perceived psychometric shortcomings of the original TMT (for a review see [48] and the Oral Trail Making Test (OTMT; [49]) was developed to reduce confounds associated with motor speed and visual search abilities, with research supporting the OTMT as an equivalent measure [50, 51]. Alternate forms of the TMT have also been developed to permit successive administrations [32, 52] and to assess the relative contributions of the requisite cognitive skills [53].

Delis et al. [54] stated that the continued development of new instrumentation for improving diagnosis and treatment is a critical undertaking in all health-related fields. Further, in their view, the field of neuropsychology has recognized the importance of continually striving to develop new clinical measures. Delis and colleagues developed the extensive Delis-Kaplan Executive Functioning System (D-KEFS; [55]) in the spirit of advancing the instrumentation of neuropsychology. The D-KEFS includes a Trail Making Test consisting of five separate conditions. The Number-Letter Switching condition involves a sequencing procedure similar to that of the classic TMT. The other four conditions are designed to assess the component processes involved in completing the Number-Letter Switching condition so that a precise analysis of the nature of any underlying dysfunction may be accomplished. Specifically, these additional components include Visual Scanning, Number Sequencing, Letter Sequencing, and Motor Speed.

Given that the TMT comprises numbers and letters and is a measure of executive functioning, it may preferentially involve the left frontal lobe. Although the literature is somewhat controversial, neuropsychological and neuroimaging studies seem to provide support for the sensitivity of the TMT to detect left frontal dysfunction [56]. Recent clinically oriented studies investigating frontal lobe involvement of the TMT using transcranial magnetic stimulation (TMS) and near-infrared spectroscopy (NIRS) also support this localization [57]. Performance on Part B of the TMT improved following repetitive TMS applied to the left dorsolateral frontal lobe [57].

With 9–13-year-old boys performing TMT Part B, Weber et al. [58] found a left lateralized increase in the prefrontal cortex in deoxygenated hemoglobin, an indicator of increased oxygen consumption. Moll et al. [59] demonstrated increased activation specific to the prefrontal cortex, especially the left prefrontal region, in healthy controls performing Part B of the TMT. Foster et al. [60] found a significant positive correlation between performance on Part A of the TMT and low beta (13–21 Hz) magnitude (μV) at the left lateral frontal lobe, but not at the right lateral frontal lobe. Finally, Stuss et al. [13, 14] found that patients with left dorsolateral frontal dysfunction evidenced more errors than patients with lesions in other areas of the frontal lobes and those patients with left frontal lesions were the slowest to complete the test.

Taken together, the possibility exists that the aforementioned tests are largely associated with left frontal lobe activity and the TMT, in particular, provides information concerning mental processing speed as well as cognitive flexibility and set-shifting. While some studies have found that deficits in visuomotor set-shifting are specific to the frontal lobe damage [61], others investigators have reported such impairment in patients with posterior brain lesions and widespread cerebral dysfunctions, including cerebellar damage [62] and Alzheimer disease [63]. Thus, it remains unclear whether impairments in visuomotor set-shifting are specific to frontal lobe dysfunction or whether they are non-specific and can result from more posterior or widespread brain dysfunction.

Compared to the collective knowledge we have regarding the cognitive roles of the left frontal lobe, relatively little is known about right frontal lobe contributions to executive functioning. This is likely a result of the dearth of tests that are associated with right frontal activity. The Ruff Figural Fluency Test (RFFT; [64]) is among the few standardized tests of right frontal lobe functioning and was listed as the 14th most commonly used instrument to assess executive functioning in the Rabin et al. [1] survey. The RFFT is known to be sensitive to right frontal lobe functioning [65, 66]; see also [67] pp. 297–298), as is a measure based on the RFFT [19].

The present investigation, with the same intent and spirit as that reported by Delis et al. [54], sought to develop and initially validate a measure of right frontal lobe functioning in an effort to attain a greater understanding of right frontal contributions to executive functioning and to advance the instrumentation of neuropsychology. To meet this objective, a version of the Trail Making Test comprising figures, as opposed to numbers and letters, was developed. The TMT was used as a model for the new test, referred to as the Figure Trail Making Test (FTMT), due to the high frequency of use, the volume of research conducted, and the ease of administration of the TMT. Given that the TMT and the FTMT are both measuring executive functioning, we felt that a moderate correlation would exist between these two measures. Specifically, we hypothesized that performance on the FTMT would be positively correlated with performance on the TMT, in terms of the total time required to complete each part of the tests, an additive and subtractive score, and a ratio score. The total time required to complete each part of the FTMT was also hypothesized to be negatively correlated with the total number of unique designs produced on the RFFT and positively correlated with the number of perseverative errors committed on the RFFT and the perseverative error ratio. We also sought to determine whether the TMT and the FTMT were measuring different constructs by conducting a factor analysis, anticipating that the two tests would load on separate factors.

Additionally, we sought to obtain neurophysiological evidence that the FTMT is sensitive to right frontal lobe functioning. Specifically, we used quantitative electroencephalography (QEEG) to measure electrical activity over the left and right frontal lobes. A previous investigation we conducted found that performance on Part A of the TMT was related to left frontal lobe (F7) low beta magnitude [60]. For the present investigation, we predicted that significant negative correlations would exist between performance on Parts A and B of the TMT and both low and high beta magnitude at the F7 electrode site. We further predicted that significant negative correlations would exist between performance on Parts C and D of the FTMT and both low and high beta magnitude at the F8 electrode site.

3 Discussion

The need for additional measures of executive functions and especially instruments which may provide implications relevant to cerebral laterality is clear. There remains especially a void for neuropsychological instruments using a TMT format, which may provide information pertaining to the functional integrity of the right frontal region. Consistent with the hypotheses forwarded, significant correlations were found between performance on the TMT and the FTMT, in terms of the raw time required to complete each respective part of the tests as well as the additive and subtraction scores. The fact that the ratio scores were not significantly correlated is not surprising given that research has generally indicated a lack of clinical utility for this score [13, 14, 35]. Given the present findings, the TMT and the FTMT appear to be equivalent measures of executive functioning. Further, the present findings not only suggest that the FTMT may be a measure of executive functioning but also extend the realm of executive functioning to the sequencing and set-shifting of nonverbal stimuli.

However, the finding of significant correlations between the TMT and the FTMT represents somewhat of a caveat in that the TMT has been found to be sensitive to left frontal lobe functioning [13, 14, 57, 59]. This would seem to suggest the possibility that the FTMT is also sensitive to left frontal lobe functioning. The possibility that FTMT is related to left frontal lobe functioning is tempered, though, by the fact that the many of the hypothesized correlations between performance on the RFFT and the FTMT were also significant. Performance on the RFFT is related to right frontal lobe functioning [65,66]. Thus, the significant correlations between the RFFT and the FTMT suggest that the FTMT may also be sensitive to right frontal lobe functioning. Additionally, it should also be noted that the TMT was not significantly correlated with performance on the RFFT, with the exception of the significant correlation between performance on the TMT Part A and the total number of unique designs produced on the RFFT. Taken together, the results suggest that the FTMT may be a measure of right frontal executive functioning.

Additional support for the sensitivity of the FTMT to right frontal lobe functioning is provided by the finding of a significant negative correlation between performance on Part D of the FTMT and high beta magnitude. We have previously used QEEG to provide neurophysiological validation of the RFFT [65] and the Rey Auditory Verbal Learning Test [70] and the present findings provide further support for the use of QEEG in validating neuropsychological tests. The lack of significant correlations between the TMT and either low or high beta magnitude may be related to a restricted range of scores on the TMT. As a whole, performance on the FTMT was more variable than performance on the TMT and this relatively restricted range for the TMT may have impacted the obtained correlations. Given the present findings, together with those of the Foster et al. [65, 70] investigations, further support is also provided for the use of EEG in establishing neurophysiological validation for neuropsychological tests.

The results from the factor analysis provide support for the contention that the FMT may be a measure of right frontal lobe activity and also provide initial discriminant validity for the FTMT. Specifically, Parts C and D of the FTMT were found to load on the same factor as the number of designs generated on the RFFT, although the time required to complete Part A of the TMT is also included. Additionally, the number of errors committed on Parts C and D of the FTMT comprises a single factor, separate from either the TMT or the RFFT. Although these results support the FTMT as a measure of nonverbal executive functioning, it would be helpful to conduct an additional factor analysis including additional measures of right frontal functioning, and perhaps other measures of right hemisphere functioning as marker variables.

We sought to develop a measure sensitive to right frontal lobe functioning due to the paucity of such tests and the potentially important uses that right frontal lobe tests may have clinically. Tests of right frontal lobe functioning may, for instance, be useful in identifying and distinguishing left versus right frontotemporal dementia (FTD). Research has indicated that FTD is associated with cerebral atrophy at the right dorsolateral frontal and left premotor cortices [71]. Fukui and Kertesz [72] found right frontal lobe volume reduction in FTD relative to Alzheimer’s disease and progressive nonfluent aphasia. Some have suggested that FTD should not be considered as a unitary disorder and that neuropsychological testing may aid in differentially diagnosing left versus right FTD [73].

Whereas right FTD has been associated with more errors and perseverative responses on the Wisconsin Card Sorting Test (WCST), left FTD has been associated with significantly worse performance on the Boston Naming Test (BNT) and the Stroop Color-Word test [73]. Razani et al. [74] also distinguished between left and right FTD in finding that left FTD performed worse on the BNT and the right FTD patients performed worse on the WCST. However, as noted earlier, the WCST has been associated with left frontal activity [9], right frontal activation [8], and bilateral frontal activation [7]. Further, patients with left frontal tumors perform worse than those with right frontal tumors [11].

Patients with FTD that predominantly involves the right frontotemporal region have behavioral and emotional abnormalities and those with predominantly left frontotemporal region damage have a loss of lexical semantic knowledge. Patients, in whom neural degeneration begins on the left side, often present to the clinicians at an early stage of the disease due to the presence of language abnormalities, but maintain their emotion processing abilities, being preserved the right anterior temporal lobe. However, as this disease advances, the disease may progress to the right frontotemporal regions. Tests sensitive to right frontal lobe functioning may be useful tools to identify in advance the course of the disease, providing immediate and specific treatments and informing the caregivers on the possible prospective frame of the disease.

A potentially more important use of tests sensitive to right frontal lobe functioning, though, may be in predicting dementia patients that will develop significant and disruptive behavioral deficits. Research has found that approximately 92 % of right-sided FTD patients exhibit socially undesirable behaviors as their initial symptom, as compared to only 11 % of left-sided FTD patients [75]. Behavioral deficits in FTD are associated with gray matter loss at the dorsomedial frontal region, particularly on the right [76].

Alzheimer’s disease (AD) is also often associated with significant behavioral disturbances. Even AD patients with mild dementia are noted to exhibit behavioral deficits such as delusions, hallucinations, agitation, dysphoria, anxiety, apathy, and irritability [77]. Indeed, Shimabukuro et al. [77] found that regardless of dementia severity, over half of all AD patients exhibited apathy, delusions, irritability, dysphoria, and anxiety. Delusions in AD patients are associated with relative right frontal hypoperfusion as indicated by SPECT imaging [78, 79]. Further, positron emission tomography (PET) has indicated that AD patients exhibiting delusions exhibit hypometabolism at the right superior dorsolateral frontal and right inferior frontal pole [80].

Although research clearly implicates right frontal lobe dysfunction in the expression of behavioral deficits, data from neuropsychological testing are not as clear. Negative symptoms in patients with AD and FTD have been related to measures of nonverbal and verbal executive functioning as well as verbal memory [81]. Positive symptoms, in contrast, were related to constructional skills and attention. However, Staff et al. [78] failed to dissociate patients with delusions from those without delusions based on neuropsychological test performance, despite significant differences existing in right frontal and limbic functioning as revealed by functional imaging. The inclusion of other measures of right frontal lobe functioning may result in improved neuropsychological differentiation of dementia patients with and without significant behavioral disturbances. Further, it may be possible to predict early in the disease process those patients that will ultimately develop behavioral disturbances with improved measures of right frontal functioning. Predicting those that may develop behavioral problems will permit earlier treatment and will provide the family with more time to prepare for the potential emergence of such difficulties. Certainly, future research needs to be conducted that incorporates measures of right and left frontal lobe functioning in regression analyses to determine the plausibility of such prediction.

Tests sensitive to right frontal lobe functioning may also be useful in identifying more subtle right frontal lobe dysfunction and the cognitive and behavioral changes that follow. The right frontal lobe mediates language melody or prosody and forms a cohesive discourse, interprets abstract communication in spoken and written languages, and interprets the inferred relationships involved in communications. Subtle difficulties in interpreting abstract meaning in communication, comprehending metaphors, and even understanding jokes that are often seen in right frontal lobe stroke patients may not be detected by the family and may also be under diagnosed by clinicians [82]. Further, patients with right frontal lobe lesions are generally more euphoric and unconcerned, often minimizing their symptoms [82] or denying the illness, which may delay referral to a clinician and diagnosis.

Attention deficit hyperactivity disorder (ADHD) is a neurological disease characterized by motor inhibition deficit, problems with cognitive flexibility, social disruption, and emotional disinhibition [83, 84]. Functional MRI studies reveal reduced right prefrontal activation during “frontal tasks,” such as go/no go [85], Stroop [86], and attention task performance [87]. The right frontal lobe deficit hypothesis is further supported by structural studies [88, 89]. Tests of right frontal lobe functioning may be useful in further characterizing the nature of this deficit and in specifying the likely hemispheric locus of dysfunction.

To summarize, we feel that right frontal lobe functioning has been relatively neglected in neuropsychological assessment and that many uses for such tests exist. Our intent was to develop a test purportedly sensitive to right frontal functioning that would be easy and quick to administer in a clinical setting. However, we are certainly not meaning to assert that our FTMT would be applicable in all the aforementioned conditions. Additional research should be conducted to determine the precise clinical utility of the FTMT.

Further validation of the FTMT should also be undertaken. Establishing convergent validation may involve correlating tests measuring the same domain, such as executive functioning. This was initially accomplished in the present investigation through the significant correlations between the TMT and the FTMT. Additionally, convergent validation may also involve correlating tests that purportedly measure the same region of the brain. This was also initially accomplished in the present investigation through the significant correlations between the FTMT and the RFFT. However, additional convergent validation certainly needs to be obtained, as well as validation using patient populations and neurophysiological validation.

We are currently collecting data that hopefully will provide neurophysiological validation of the FTMT. Certainly, though, it is hoped that the present investigation will not only stimulate further research seeking to validate the FTMT and provide more comprehensive normative data, but also stimulate research investigating whether the FTMT or other measures of right frontal lobe functioning may be used to predict patients that will develop behavioral disturbances.

World’s Greatest Literature Reveals Multifractals, Cascades of Consciousness

http://www.scientificcomputing.com/news/2016/01/worlds-greatest-literature-reveals-multifractals-cascades-consciousness

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Multifractal analysis of Finnegan’s Wake by James Joyce. The ideal shape of the graph is virtually indistinguishable from the results for purely mathematical multifractals. The horizontal axis represents the degree of singularity, and the vertical axis shows the spectrum of singularity. Courtesy of IFJ PAN

Arthur Conan Doyle, Charles Dickens, James Joyce, William Shakespeare and JRR Tolkien. Regardless of the language they were working in, some of the world’s greatest writers appear to be, in some respects, constructing fractals. Statistical analysis, however, revealed something even more intriguing. The composition of works from within a particular genre was characterized by the exceptional dynamics of a cascading (avalanche) narrative structure. This type of narrative turns out to be multifractal. That is, fractals of fractals are created.

As far as many bookworms are concerned, advanced equations and graphs are the last things which would hold their interest, but there’s no escape from the math. Physicists from the Institute of Nuclear Physics of the Polish Academy of Sciences (IFJ) in Cracow, Poland, performed a detailed statistical analysis of more than one hundred famous works of world literature, written in several languages and representing various literary genres. The books, tested for revealing correlations in variations of sentence length, proved to be governed by the dynamics of a cascade. This means that the construction of these books is, in fact, a fractal. In the case of several works, their mathematical complexity proved to be exceptional, comparable to the structure of complex mathematical objects considered to be multifractal. Interestingly, in the analyzed pool of all the works, one genre turned out to be exceptionally multifractal in nature.

Fractals are self-similar mathematical objects: when we begin to expand one fragment or another, what eventually emerges is a structure that resembles the original object. Typical fractals, especially those widely known as the Sierpinski triangle and the Mandelbrot set, are monofractals, meaning that the pace of enlargement in any place of a fractal is the same, linear: if they at some point were rescaled x number of times to reveal a structure similar to the original, the same increase in another place would also reveal a similar structure.

Multifractals are more highly advanced mathematical structures: fractals of fractals. They arise from fractals ‘interwoven’ with each other in an appropriate manner and in appropriate proportions. Multifractals are not simply the sum of fractals and cannot be divided to return back to their original components, because the way they weave is fractal in nature. The result is that, in order to see a structure similar to the original, different portions of a multifractal need to expand at different rates. A multifractal is, therefore, non-linear in nature.

“Analyses on multiple scales, carried out using fractals, allow us to neatly grasp information on correlations among data at various levels of complexity of tested systems. As a result, they point to the hierarchical organization of phenomena and structures found in nature. So, we can expect natural language, which represents a major evolutionary leap of the natural world, to show such correlations as well. Their existence in literary works, however, had not yet been convincingly documented. Meanwhile, it turned out that, when you look at these works from the proper perspective, these correlations appear to be not only common, but in some works they take on a particularly sophisticated mathematical complexity,” says Professor Stanislaw Drozdz, IFJ PAN, Cracow University of Technology.

The study involved 113 literary works written in English, French, German, Italian, Polish, Russian and Spanish by such famous figures as Honore de Balzac, Arthur Conan Doyle, Julio Cortazar, Charles Dickens, Fyodor Dostoevsky, Alexandre Dumas, Umberto Eco, George Elliot, Victor Hugo, James Joyce, Thomas Mann, Marcel Proust, Wladyslaw Reymont, William Shakespeare, Henryk Sienkiewicz, JRR Tolkien, Leo Tolstoy and Virginia Woolf, among others. The selected works were no less than 5,000 sentences long, in order to ensure statistical reliability.

To convert the texts to numerical sequences, sentence length was measured by the number of words (an alternative method of counting characters in the sentence turned out to have no major impact on the conclusions). The dependences were then searched for in the data — beginning with the simplest, i.e. linear. This is the posited question: if a sentence of a given length is x times longer than the sentences of different lengths, is the same aspect ratio preserved when looking at sentences respectively longer or shorter?

“All of the examined works showed self-similarity in terms of organization of the lengths of sentences. Some were more expressive — here The Ambassadors by Henry James stood out — while others to far less of an extreme, as in the case of the French seventeenth-century romance Artamene ou le Grand Cyrus. However, correlations were evident and, therefore, these texts were the construction of a fractal,” comments Dr. Pawel Oswiecimka (IFJ PAN), who also noted that fractality of a literary text will, in practice, never be as perfect as in the world of mathematics. It is possible to magnify mathematical fractals up to infinity, while the number of sentences in each book is finite and, at a certain stage of scaling, there will always be a cut-off in the form of the end of the dataset.

Things took a particularly interesting turn when physicists from IFJ PAN began tracking non-linear dependence, which in most of the studied works was present to a slight or moderate degree. However, more than a dozen works revealed a very clear multifractal structure, and almost all of these proved to be representative of one genre, that of stream of consciousness. The only exception was the Bible, specifically the Old Testament, which has, so far, never been associated with this literary genre.

“The absolute record in terms of multifractality turned out to be Finnegan’s Wakeby James Joyce. The results of our analysis of this text are virtually indistinguishable from ideal, purely mathematical multifractals,” says Drozdz.

The most multifractal works also included A Heartbreaking Work of Staggering Genius by Dave Eggers, Rayuela by Julio Cortazar, The US Trilogy by John Dos Passos, The Waves by Virginia Woolf, 2666 by Roberto Bolano, and Joyce’sUlysses. At the same time, a lot of works usually regarded as stream of consciousness turned out to show little correlation to multifractality, as it was hardly noticeable in books such as Atlas Shrugged by Ayn Rand and A la recherche du temps perdu by Marcel Proust.

“It is not entirely clear whether stream of consciousness writing actually reveals the deeper qualities of our consciousness, or rather the imagination of the writers. It is hardly surprising that ascribing a work to a particular genre is, for whatever reason, sometimes subjective. We see, moreover, the possibility of an interesting application of our methodology: it may someday help in a more objective assignment of books to one genre or another,” notes Drozdz.

Multifractal analyses of literary texts carried out by the IFJ PAN have been published in Information Sciences, the journal of computer science. The publication has undergone rigorous verification: given the interdisciplinary nature of the subject, editors immediately appointed up to six reviewers.

Citation: “Quantifying origin and character of long-range correlations in narrative texts” S. Drożdż, P. Oświęcimka, A. Kulig, J. Kwapień, K. Bazarnik, I. Grabska-Gradzińska, J. Rybicki, M. Stanuszek; Information Sciences, vol. 331, 32–44, 20 February 2016; DOI: 10.1016/j.ins.2015.10.023

New Quantum Approach to Big Data could make Impossibly Complex Problems Solvable

David L. Chandler, MIT

http://www.scientificcomputing.com/news/2016/01/new-quantum-approach-big-data-could-make-impossibly-complex-problems-solvable

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This diagram demonstrates the simplified results that can be obtained by using quantum analysis on enormous, complex sets of data. Shown here are the connections between different regions of the brain in a control subject (left) and a subject under the influence of the psychedelic compound psilocybin (right). This demonstrates a dramatic increase in connectivity, which explains some of the drug’s effects (such as “hearing” colors or “seeing” smells). Such an analysis, involving billions of brain cells, would be too complex for conventional techniques, but could be handled easily by the new quantum approach, the researchers say. Courtesy of the researchers

From gene mapping to space exploration, humanity continues to generate ever-larger sets of data — far more information than people can actually process, manage or understand.

Machine learning systems can help researchers deal with this ever-growing flood of information. Some of the most powerful of these analytical tools are based on a strange branch of geometry called topology, which deals with properties that stay the same even when something is bent and stretched every which way.

Such topological systems are especially useful for analyzing the connections in complex networks, such as the internal wiring of the brain, the U.S. power grid, or the global interconnections of the Internet. But even with the most powerful modern supercomputers, such problems remain daunting and impractical to solve. Now, a new approach that would use quantum computers to streamline these problems has been developed by researchers at MIT, the University of Waterloo, and the University of Southern California.

The team describes their theoretical proposal this week in the journal Nature Communications. Seth Lloyd, the paper’s lead author and the Nam P. Suh Professor of Mechanical Engineering, explains that algebraic topology is key to the new method. This approach, he says, helps to reduce the impact of the inevitable distortions that arise every time someone collects data about the real world.

In a topological description, basic features of the data (How many holes does it have? How are the different parts connected?) are considered the same no matter how much they are stretched, compressed, or distorted. Lloyd explains that it is often these fundamental topological attributes “that are important in trying to reconstruct the underlying patterns in the real world that the data are supposed to represent.”

It doesn’t matter what kind of dataset is being analyzed, he says. The topological approach to looking for connections and holes “works whether it’s an actual physical hole, or the data represents a logical argument and there’s a hole in the argument. This will find both kinds of holes.”

Using conventional computers, that approach is too demanding for all but the simplest situations. Topological analysis “represents a crucial way of getting at the significant features of the data, but it’s computationally very expensive,” Lloyd says. “This is where quantum mechanics kicks in.” The new quantum-based approach, he says, could exponentially speed up such calculations.

Lloyd offers an example to illustrate that potential speedup: If you have a dataset with 300 points, a conventional approach to analyzing all the topological features in that system would require “a computer the size of the universe,” he says. That is, it would take 2300 (two to the 300th power) processing units — approximately the number of all the particles in the universe. In other words, the problem is simply not solvable in that way.

“That’s where our algorithm kicks in,” he says. Solving the same problem with the new system, using a quantum computer, would require just 300 quantum bits — and a device this size may be achieved in the next few years, according to Lloyd.

“Our algorithm shows that you don’t need a big quantum computer to kick some serious topological butt,” he says.

There are many important kinds of huge datasets where the quantum-topological approach could be useful, Lloyd says, for example understanding interconnections in the brain. “By applying topological analysis to datasets gleaned by electroencephalography or functional MRI, you can reveal the complex connectivity and topology of the sequences of firing neurons that underlie our thought processes,” he says.

The same approach could be used for analyzing many other kinds of information. “You could apply it to the world’s economy, or to social networks, or almost any system that involves long-range transport of goods or information,” Lloyd says. But the limits of classical computation have prevented such approaches from being applied before.

While this work is theoretical, “experimentalists have already contacted us about trying prototypes,” he says. “You could find the topology of simple structures on a very simple quantum computer. People are trying proof-of-concept experiments.”

Ignacio Cirac, a professor at the Max Planck Institute of Quantum Optics in Munich, Germany, who was not involved in this research, calls it “a very original idea, and I think that it has a great potential.” He adds “I guess that it has to be further developed and adapted to particular problems. In any case, I think that this is top-quality research.”

The team also included Silvano Garnerone of the University of Waterloo in Ontario, Canada, and Paolo Zanardi of the Center for Quantum Information Science and Technology at the University of Southern California. The work was supported by the Army Research Office, Air Force Office of Scientific Research, Defense Advanced Research Projects Agency, Multidisciplinary University Research Initiative of the Office of Naval Research, and the National Science Foundation.

Beyond Chess: Computer Beats Human in Ancient Chinese Game

http://www.rdmag.com/news/2016/01/beyond-chess-computer-beats-human-ancient-chinese-game

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A player places a black stone while his opponent waits to place a white one as they play Go, a game of strategy, in the Seattle Go Center, Tuesday, April 30, 2002. The game, which originated in China more than 2,500 years ago, involves two players who take turns putting markers on a grid. The object is to surround more area on the board with the markers than one’s opponent, as well as capturing the opponent’s pieces by surrounding them. A paper released Wednesday, Jan. 27, 2016 describes how a computer program has beaten a human master at the complex board game, marking significant advance for development of artificial intelligence. (AP Photo/Cheryl Hatch)

A computer program has beaten a human champion at the ancient Chinese board game Go, marking a significant advance for development of artificial intelligence.

The program had taught itself how to win, and its developers say its learning strategy may someday let computers help solve real-world problems like making medical diagnoses and pursuing scientific research.

The program and its victory are described in a paper released Wednesday by the journal Nature.

Computers previously have surpassed humans for other games, including chess, checkers and backgammon. But among classic games, Go has long been viewed as the most challenging for artificial intelligence to master.

Go, which originated in China more than 2,500 years ago, involves two players who take turns putting markers on a checkerboard-like grid. The object is to surround more area on the board with the markers than one’s opponent, as well as capturing the opponent’s pieces by surrounding them.

While the rules are simple, playing it well is not. It’s “probably the most complex game ever devised by humans,” Dennis Hassabis of Google DeepMind in London, one of the study authors, told reporters Tuesday.

The new program, AlphaGo, defeated the European champion in all five games of a match in October, the Nature paper reports.

In March, AlphaGo will face legendary player Lee Sedol in Seoul, South Korea, for a $1 million prize, Hassabis said.

Martin Mueller, a computing science professor at the University of Alberta in Canada who has worked on Go programs for 30 years but didn’t participate in AlphaGo, said the new program “is really a big step up from everything else we’ve seen…. It’s a very, very impressive piece of work.”

Biological Origin of Schizophrenia

Excessive ‘pruning’ of connections between neurons in brain predisposes to disease

http://hms.harvard.edu/sites/default/files/uploads/news/McCarroll_C4_600x400.jpg

Imaging studies showed C4 (in green) located at the synapses of primary human neurons. Image: Heather de Rivera, McCarroll lab

 PAUL GOLDSMITH    http://hms.harvard.edu/news/biological-origin-schizophrenia

The risk of schizophrenia increases if a person inherits specific variants in a gene related to “synaptic pruning”—the elimination of connections between neurons—according to a study from Harvard Medical School, the Broad Institute and Boston Children’s Hospital. The findings were based on genetic analysis of nearly 65,000 people.

The study represents the first time that the origin of this psychiatric disease has been causally linked to specific gene variants and a biological process.

Get more HMS news here

It also helps explain two decades-old observations: synaptic pruning is particularly active during adolescence, which is the typical period of onset for symptoms of schizophrenia, and the brains of schizophrenic patients tend to show fewer connections between neurons.

The gene, complement component 4 (C4), plays a well-known role in the immune system. It has now been shown to also play a key role in brain development and schizophrenia risk. The insight may allow future therapeutic strategies to be directed at the disorder’s roots, rather than just its symptoms.

The study, which appears online Jan. 27 in Nature, was led by HMS researchers at the Broad Institute’s Stanley Center for Psychiatric Research and Boston Children’s. They include senior author Steven McCarroll, HMS associate professor of genetics and director of genetics for the Stanley Center; Beth Stevens, HMS assistant professor of neurology at Boston Children’s and institute member at the Broad; Michael Carroll, HMS professor of pediatrics at Boston Children’s; and first author Aswin Sekar, an MD-PhD student at HMS.

The study has the potential to reinvigorate translational research on a debilitating disease. Schizophrenia afflicts approximately 1 percent people worldwide and is characterized by hallucinations, emotional withdrawal and a decline in cognitive function. These symptoms most frequently begin in patients when they are teenagers or young adults.

“These results show that it is possible to go from genetic data to a new way of thinking about how a disease develops—something that has been greatly needed.”

First described more than 130 years ago, schizophrenia lacks highly effective treatments and has seen few biological or medical breakthroughs over the past half-century.

In the summer of 2014, an international consortium led by researchers at the Stanley Center identified more than 100 regions in the human genome that carry risk factors for schizophrenia.

The newly published study now reports the discovery of the specific gene underlying the strongest of these risk factors and links it to a specific biological process in the brain.

“Since schizophrenia was first described over a century ago, its underlying biology has been a black box, in part because it has been virtually impossible to model the disorder in cells or animals,” said McCarroll. “The human genome is providing a powerful new way in to this disease. Understanding these genetic effects on risk is a way of prying open that black box, peering inside and starting to see actual biological mechanisms.”

“This study marks a crucial turning point in the fight against mental illness,” said Bruce Cuthbert, acting director of the National Institute of Mental Health. “Because the molecular origins of psychiatric diseases are little-understood, efforts by pharmaceutical companies to pursue new therapeutics are few and far between. This study changes the game. Thanks to this genetic breakthrough we can finally see the potential for clinical tests, early detection, new treatments and even prevention.”

The path to discovery

The discovery involved the collection of DNA from more than 100,000 people, detailed analysis of complex genetic variation in more than 65,000 human genomes, development of an innovative analytical strategy, examination of postmortem brain samples from hundreds of people and the use of animal models to show that a protein from the immune system also plays a previously unsuspected role in the brain.

Over the past five years, Stanley Center geneticists and collaborators around the world collected more than 100,000 human DNA samples from 30 different countries to locate regions of the human genome harboring genetic variants that increase the risk of schizophrenia. The strongest signal by far was on chromosome 6, in a region of DNA long associated with infectious disease. This caused some observers to suggest that schizophrenia might be triggered by an infectious agent. But researchers had no idea which of the hundreds of genes in the region was actually responsible or how it acted.

Based on analyses of the genetic data, McCarroll and Sekar focused on a region containing the C4 gene. Unlike most genes, C4 has a high degree of structural variability. Different people have different numbers of copies and different types of the gene.

McCarroll and Sekar developed a new molecular technique to characterize the C4 gene structure in human DNA samples. They also measured C4 gene activity in nearly 700 post-mortem brain samples.

They found that the C4 gene structure (DNA) could predict the C4 gene activity (RNA) in each person’s brain. They then used this information to infer C4 gene activity from genome data from 65,000 people with and without schizophrenia.

These data revealed a striking correlation. People who had particular structural forms of the C4 gene showed higher expression of that gene and, in turn, had a higher risk of developing schizophrenia.

Connecting cause and effect through neuroscience

But how exactly does C4—a protein known to mark infectious microbes for destruction by immune cells—affect the risk of schizophrenia?

Answering this question required synthesizing genetics and neurobiology.

Stevens, a recent recipient of a MacArthur Foundation “genius grant,” had found that other complement proteins in the immune system also played a role in brain development. These results came from studying an experimental model of synaptic pruning in the mouse visual system.

“This discovery enriches our understanding of the complement system in brain development and in disease, and we could not have made that leap without the genetics.”

Carroll had long studied C4 for its role in immune disease, and developed mice with different numbers of copies of C4.

The three labs set out to study the role of C4 in the brain.

They found that C4 played a key role in pruning synapses during maturation of the brain. In particular, they found that C4 was necessary for another protein—a complement component called C3—to be deposited onto synapses as a signal that the synapses should be pruned. The data also suggested that the more C4 activity an animal had, the more synapses were eliminated in its brain at a key time in development.

The findings may help explain the longstanding mystery of why the brains of people with schizophrenia tend to have a thinner cerebral cortex (the brain’s outer layer, responsible for many aspects of cognition) with fewer synapses than do brains of unaffected individuals. The work may also help explain why the onset of schizophrenia symptoms tends to occur in late adolescence.

The human brain normally undergoes widespread synapse pruning during adolescence, especially in the cerebral cortex. Excessive synaptic pruning during adolescence and early adulthood, due to increased complement (C4) activity, could lead to the cognitive symptoms seen in schizophrenia.

“Once we had the genetic findings in front of us we started thinking about the possibility that complement molecules are excessively tagging synapses in the developing brain,” Stevens said.

“This discovery enriches our understanding of the complement system in brain development and in disease, and we could not have made that leap without the genetics,” she said. “We’re far from having a treatment based on this, but it’s exciting to think that one day we might be able to turn down the pruning process in some individuals and decrease their risk.”

Opening a path toward early detection and potential therapies

Beyond providing the first insights into the biological origins of schizophrenia, the work raises the possibility that therapies might someday be developed that could turn down the level of synaptic pruning in people who show early symptoms of schizophrenia.

This would be a dramatically different approach from current medical therapies, which address only a specific symptom of schizophrenia—psychosis—rather than the disorder’s root causes, and which do not stop cognitive decline or other symptoms of the illness.

The researchers emphasize that therapies based on these findings are still years down the road. Still, the fact that much is already known about the role of complement proteins in the immune system means that researchers can tap into a wealth of existing knowledge to identify possible therapeutic approaches. For example, anticomplement drugs are already under development for treating other diseases.

“In this area of science, our dream has been to find disease mechanisms that lead to new kinds of treatments,” said McCarroll. “These results show that it is possible to go from genetic data to a new way of thinking about how a disease develops—something that has been greatly needed.”

This work was supported by the Broad Institute’s Stanley Center for Psychiatric Research and by the National Institutes of Health (grants U01MH105641, R01MH077139 and T32GM007753).

Adapted from a Broad Institute news release.

 

Scientists open the ‘black box’ of schizophrenia with dramatic genetic discovery

Amy Ellis Nutt    https://www.washingtonpost.com/news/speaking-of-science/wp/2016/01/27/scientists-open-the-black-box-of-schizophrenia-with-dramatic-genetic-finding/

Scientists Prune Away Schizophrenia’s Hidden Genetic Mechanisms

http://www.genengnews.com/gen-news-highlights/scientists-prune-away-schizophrenia-s-hidden-genetic-mechanisms/81252297/

https://youtu.be/s0y4equOTLg

A landmark study has revealed that a person’s risk of schizophrenia is increased if they inherit specific variants in a gene related to “synaptic pruning”—the elimination of connections between neurons. The findings represent the first time that the origin of this devastating psychiatric disease has been causally linked to specific gene variants and a biological process.

http://www.genengnews.com/Media/images/GENHighlight/thumb_107629_web2209513618.jpg

The site in Chromosome 6 harboring the gene C4 towers far above other risk-associated areas on schizophrenia’s genomic “skyline,” marking its strongest known genetic influence. The new study is the first to explain how specific gene versions work biologically to confer schizophrenia risk. [Psychiatric Genomics Consortium]

  • A new study by researchers at the Broad Institute’s Stanley Center for Psychiatric Research, Harvard Medical School, and Boston Children’s Hospital genetically analyzed nearly 65,000 people and revealed that an individual’s risk of schizophrenia is increased if they inherited distinct variants in a gene related to “synaptic pruning”—the elimination of connections between neurons. This new data represents the first time that the origin of this psychiatric disease has been causally linked to particular gene variants and a biological process.

The investigators discovered that versions of a gene commonly thought to be involved in immune function might trigger a runaway pruning of an adolescent brain’s still-maturing communications infrastructure. The researchers described a scenario where patients with schizophrenia show fewer such connections between neurons or synapses.

“Normally, pruning gets rid of excess connections we no longer need, streamlining our brain for optimal performance, but too much pruning can impair mental function,” explained Thomas Lehner, Ph.D., director of the Office of Genomics Research Coordination at the NIH’s National Institute of Mental Health (NIMH), which co-funded the study along with the Stanley Center for Psychiatric Research at the Broad Institute and other NIH components. “It could help explain schizophrenia’s delayed age-of-onset of symptoms in late adolescence and early adulthood and shrinkage of the brain’s working tissue. Interventions that put the brakes on this pruning process-gone-awry could prove transformative.”

The gene the research team called into question, dubbed C4 (complement component 4), was associated with the largest risk for the disorder. C4’s role represents some of the most compelling evidence, to date, linking specific gene versions to a biological process that could cause at least some cases of the illness.

The findings from this study were published recently in Nature through an article entitled “Schizophrenia risk from complex variation of complement component 4.”

“Since schizophrenia was first described over a century ago, its underlying biology has been a black box, in part because it has been virtually impossible to model the disorder in cells or animals,” noted senior study author Steven McCarroll, Ph.D., director of genetics for the Stanley Center and an associate professor of genetics at Harvard Medical School. “The human genome is providing a powerful new way into this disease. Understanding these genetic effects on risk is a way of prying open that block box, peering inside and starting to see actual biological mechanisms.”

Dr. McCarroll and his colleagues found that a stretch of chromosome 6 encompassing several genes known to be involved in immune function emerged as the strongest signal associated with schizophrenia risk in genome-wide analyses. Yet conventional genetics failed to turn up any specific gene versions there that were linked to schizophrenia.

In order to uncover how the immune-related site confers risk for the mental disorder, the scientists mounted a search for cryptic genetic influences that might generate unconventional signals. C4, a gene with known roles in immunity, emerged as a prime suspect because it is unusually variable across individuals.

Upon further investigation into the complexities of how such structural variation relates to the gene’s level of expression and how that, in turn, might link to schizophrenia, the team discovered structurally distinct versions that affect expression of two main forms of the gene within the brain. The more a version resulted in expression of one of the forms, called C4A, the more it was associated with schizophrenia. The greater number of copies an individual had of the suspect versions, the more C4 switched on and the higher their risk of developing schizophrenia. Furthermore, the C4 protein turned out to be most prevalent within the cellular machinery that supports connections between neurons.

“Once we had the genetic findings in front of us we started thinking about the possibility that complement molecules are excessively tagging synapses in the developing brain,” remarked co-author Beth Stevens, Ph.D. a neuroscientist and assistant professor of neurology at Boston Children’s Hospital and institute member at the Broad. “This discovery enriches our understanding of the complement system in brain development and disease, and we could not have made that leap without the genetics. We’re far from having a treatment based on this, but it’s exciting to think that one day we might be able to turn down the pruning process in some individuals and decrease their risk.”

“This study marks a crucial turning point in the fight against mental illness. It changes the game,” added acting NIMH director Bruce Cuthbert, Ph.D. “Because the molecular origins of psychiatric diseases are little-understood, efforts by pharmaceutical companies to pursue new therapeutics are few and far between. This study changes the game. Thanks to this genetic breakthrough, we can finally see the potential for clinical tests, early detection, new treatments, and even prevention.”

Connecting cause and effect through neuroscience

But how exactly does C4—a protein known to mark infectious microbes for destruction by immune cells—affect the risk of schizophrenia?

Answering this question required synthesizing genetics and neurobiology.

Stevens, a recent recipient of a MacArthur Foundation “genius grant,” had found that other complement proteins in the immune system also played a role in brain development. These results came from studying an experimental model of synaptic pruning in the mouse visual system.

“This discovery enriches our understanding of the complement system in brain development and in disease, and we could not have made that leap without the genetics.”

Carroll had long studied C4 for its role in immune disease, and developed mice with different numbers of copies of C4.

The three labs set out to study the role of C4 in the brain.

They found that C4 played a key role in pruning synapses during maturation of the brain. In particular, they found that C4 was necessary for another protein—a complement component called C3—to be deposited onto synapses as a signal that the synapses should be pruned. The data also suggested that the more C4 activity an animal had, the more synapses were eliminated in its brain at a key time in development.

The findings may help explain the longstanding mystery of why the brains of people with schizophrenia tend to have a thinner cerebral cortex (the brain’s outer layer, responsible for many aspects of cognition) with fewer synapses than do brains of unaffected individuals. The work may also help explain why the onset of schizophrenia symptoms tends to occur in late adolescence.

The human brain normally undergoes widespread synapse pruning during adolescence, especially in the cerebral cortex. Excessive synaptic pruning during adolescence and early adulthood, due to increased complement (C4) activity, could lead to the cognitive symptoms seen in schizophrenia.

“Once we had the genetic findings in front of us we started thinking about the possibility that complement molecules are excessively tagging synapses in the developing brain,” Stevens said.

“This discovery enriches our understanding of the complement system in brain development and in disease, and we could not have made that leap without the genetics,” she said. “We’re far from having a treatment based on this, but it’s exciting to think that one day we might be able to turn down the pruning process in some individuals and decrease their risk.”

Opening a path toward early detection and potential therapies

Beyond providing the first insights into the biological origins of schizophrenia, the work raises the possibility that therapies might someday be developed that could turn down the level of synaptic pruning in people who show early symptoms of schizophrenia.

This would be a dramatically different approach from current medical therapies, which address only a specific symptom of schizophrenia—psychosis—rather than the disorder’s root causes, and which do not stop cognitive decline or other symptoms of the illness.

The researchers emphasize that therapies based on these findings are still years down the road. Still, the fact that much is already known about the role of complement proteins in the immune system means that researchers can tap into a wealth of existing knowledge to identify possible therapeutic approaches. For example, anticomplement drugs are already under development for treating other diseases.

“In this area of science, our dream has been to find disease mechanisms that lead to new kinds of treatments,” said McCarroll. “These results show that it is possible to go from genetic data to a new way of thinking about how a disease develops—something that has been greatly needed.”

This work was supported by the Broad Institute’s Stanley Center for Psychiatric Research and by the National Institutes of Health (grants U01MH105641, R01MH077139 and T32GM007753).

Adapted from a Broad Institute news release.

 

https://img.washingtonpost.com/wp-apps/imrs.php?src=https://img.washingtonpost.com/rf/image_908w/2010-2019/WashingtonPost/2011/09/27/Production/Sunday/SunBiz/Images/mental2b.jpg&w=1484

This post has been updated.

For the first time, scientists have pinned down a molecular process in the brain that helps to trigger schizophrenia. The researchers involved in the landmark study, which was published Wednesday in the journal Nature, say the discovery of this new genetic pathway probably reveals what goes wrong neurologically in a young person diagnosed with the devastating disorder.

The study marks a watershed moment, with the potential for early detection and new treatments that were unthinkable just a year ago, according to Steven Hyman, director of the Stanley Center for Psychiatric Research at the Broad Institute at MIT. Hyman, a former director of the National Institute of Mental Health, calls it “the most significant mechanistic study about schizophrenia ever.”

“I’m a crusty, old, curmudgeonly skeptic,” he said. “But I’m almost giddy about these findings.”

The researchers, chiefly from the Broad Institute, Harvard Medical School and Boston Children’s Hospital, found that a person’s risk of schizophrenia is dramatically increased if they inherit variants of a gene important to “synaptic pruning” — the healthy reduction during adolescence of brain cell connections that are no longer needed.

[Schizophrenic patients have different oral bacteria than non-mentally ill individuals]

In patients with schizophrenia, a variation in a single position in the DNA sequence marks too many synapses for removal and that pruning goes out of control. The result is an abnormal loss of gray matter.

The genes involved coat the neurons with “eat-me signals,” said study co-author Beth Stevens, a neuroscientist at Children’s Hospital and Broad. “They are tagging too many synapses. And they’re gobbled up.

The Institute’s founding director, Eric Lander, believes the research represents an astonishing breakthrough. “It’s taking what has been a black box…and letting us peek inside for the first time. And that is amazingly consequential,” he said.

The timeline for this discovery has been relatively fast. In July 2014, Broad researchers published the results of the largest genomic study on the disorder and found more than 100 genetic locations linked to schizophrenia. Based on that research, Harvard and Broad geneticist Steven McCarroll analyzed data from about 29,000 schizophrenia cases, 36,000 controls and 700 post mortem brains. The information was drawn from dozens of studies performed in 22 countries, all of which contribute to the worldwide database called the Psychiatric Genomics Consortium.

[Influential government-appointed panel recommends depression screening for everyone]

One area in particular, when graphed, showed the strongest association. It was dubbed the “Manhattan plot” for its resemblance to New York City’s towering buildings. The highest peak was on chromosome 6, where McCarroll’s team discovered the gene variant. C4 was “a dark corner of the human genome,” he said, an area difficult to decipher because of its “astonishing level” of diversity.

C4 and numerous other genes reside in a region of chromosome 6 involved in the immune system, which clears out pathogens and similar cellular debris from the brain. The study’s researchers found that one of C4’s variants, C4A, was most associated with a risk for schizophrenia.

More than 25 million people around the globe are affected by schizophrenia, according to the World Health Organization, including 2 million to 3 million Americans. Highly hereditable, it is one of the most severe mental illnesses, with an annual economic burden in this country of tens of billions of dollars.

“This paper is really exciting,” said Jacqueline Feldman, associate medical director of the National Alliance on Mental Illness. “We as scientists and physicians have to temper our enthusiasm because we’ve gone down this path before. But this is profoundly interesting.”

There have been hundreds of theories about schizophrenia over the years, but one of the enduring mysteries has been how three prominent findings related to each other: the apparent involvement of immune molecules, the disorder’s typical onset in late adolescence and early adulthood, and the thinning of gray matter seen in autopsies of patients.

[A low-tech way to help treat young schizophrenic patients]

“The thing about this result,” said McCarroll, the lead author, ” it makes a lot of other things understandable. To have a result to connect to these observations and to have a molecule and strong level of genetic evidence from tens of thousands of research participants, I think that combination sets [this study] apart.”

The authors stressed that their findings, which combine basic science with large-scale analysis of genetic studies, depended on an unusual level of cooperation among experts in genetics, molecular biology, developmental neurobiology and immunology.

“This could not have been done five years ago,” said Hyman. “This required the ability to reference a very large dataset . …When I was [NIMH] director, people really resisted collaborating. They were still in the Pharaoh era. They wanted to be buried with their data.”

The study offers a new approach to schizophrenia research, which has been largely stagnant for decades.  Most psychiatric drugs seek to interrupt psychotic thinking, but experts agree that psychosis is just a single symptom — and a late-occurring one at that. One of the chief difficulties for psychiatric researchers, setting them apart from most other medical investigators, is that they can’t cut schizophrenia out of the brain and look at it under a microscope. Nor are there any good animal models.

All that now has changed, according to Stevens. “We now have a strong molecular handle, a pathway and a gene, to develop better models,” he said.

Which isn’t to say a cure is right around the corner.

“This is the first exciting  clue, maybe even the most important we’ll ever have, but it will be decades” before a true cure is found,” Hyman said. “Hope is a wonderful thing. False promise is not.”

Insight Pharma Report

Three neurodegenerative disorders that are heavily focused on in this report include: Alzheimer’s Disease/Mild Cognitive Impairment, Parkinson’s Disease, and Amyotrophic Lateral Sclerosis. Part II of the report will include all three of these disorders, highlighting specifics including background, history, and development of the disease. Deeper into the chapters, the report will unfold biomarkers under investigation, genetic targets, and an analysis of multiple studies investigating these elements.

Experts interviewed in these chapters include:

  • Dr. Jens Wendland, Head of Neuroscience Genetics, Precision Medicine, Clinical Research, Pfizer Worldwide R&D
  • Dr. Howard J. Federoff, Executive Vice President for Health Sciences, Georgetown University
  • Dr. Andrew West, Associate Professor of Neurology and Neurobiology and Co-Director, Center for Neurodegeneration and Experimental Therapeutics
  • Dr. Merit Ester Cudkowicz, Chief of Neurology at Massachusetts General Hospital

Part III of the report makes a shift from neurobiomarkers to neurodiagnostics. This section highlights several diagnostics in play and in the making from a number of companies, identifying company strategies, research underway, hypotheses, and institution goals. Elite researchers and companies highlighted in this part include:

  • Dr. Xuemei Huang, Professor and Vice Chair, Department of Neurology; Professor of Neurosurgery, Radiology,  Pharmacology, and Kinesiology Director; Hershey Brain Analysis Research Laboratory for Neurodegenerative Disorders, Penn State University-Milton, S. Hershey Medical Center Department of Neurology
  • Dr. Andreas Jeromin, CSO and President of Atlantic Biomarkers
  • Julien Bradley, Senior Director, Sales & Marketing, Quanterix
  • Dr. Scott Marshall, Head of Bioanalytics, and Dr. Jared Kohler, Head of Biomarker Statistics, BioStat Solutions, Inc.

Further analysis appears in Part IV. This section includes a survey exclusively conducted for this report. With over 30 figures and graphics and an in depth analysis, this part features insight into targets under investigation, challenges, advantages, and desired features of future diagnostic applications. Furthermore, the survey covers more than just the featured neurodegenerative disorders in this report, expanding to Multiple Sclerosis and Huntington’s Disease.

Finally, Insight Pharma Reports concludes this report with clinical trial and pipeline data featuring targets and products from over 300 companies working in Alzheimer’s Disease, Parkinson’s Disease and Amyotrophic Lateral Sclerosis.

Epigenome Tapped to Understand Rise of Subtype of Brain Medulloblastoma

http://www.genengnews.com/gen-news-highlights/epigenome-tapped-to-understand-rise-of-subtype-of-brain-medulloblastoma/81252294/

Scientists have identified the cells that likely give rise to the brain tumor subtype Group 4 medulloblastoma. [V. Yakobchuk/ Fotolia]

http://www.genengnews.com/Media/images/GENHighlight/thumb_Jan_28_2016_Fotolia_6761569_ColorfulBrain_4412824411.jpg

An international team of scientists say they have identified the cells that likely give rise to the brain tumor subtype Group 4 medulloblastoma. The believe their study (“Active medulloblastoma enhancers reveal subgroup-specific cellular origins”), published in Nature, removes a barrier to developing more effective targeted therapies against the brain tumor’s most common subtype.

Medulloblastoma occurs in infants, children, and adults, but it is the most common malignant pediatric brain tumor. The disease includes four biologically and clinically distinct subtypes, of which Group 4 is the most common. In children, about half of medulloblastoma patients are of the Group 4 subtype. Efforts to improve patient outcomes, particularly for those with high-risk Group 4 medulloblastoma, have been hampered by the lack of accurate animal models.

Evidence from this study suggests Group 4 tumors begin in neural stem cells that are born in a region of the developing cerebellum called the upper rhomic lip (uRL), according to the researchers.

“Pinpointing the cell(s) of origin for Group 4 medulloblastoma will help us to better understand normal cerebellar development and dramatically improve our chances of developing genetically faithful preclinical mouse models. These models are desperately needed for learning more about Group 4 medulloblastoma biology and evaluating rational, molecularly targeted therapies to improve patient outcomes,” said Paul Northcott, Ph.D., an assistant member of the St. Jude department of developmental neurobiology. Dr. Northcott, Stefan Pfister, M.D., of the German Cancer Research Center (DKFZ), and James Bradner, M.D., of Dana-Farber Cancer Institute, are the corresponding authors.

The discovery and other findings about the missteps fueling tumor growth came from studying the epigenome. Researchers used the analytic tool ChiP-seq to identify and track medulloblastoma subtype differences based on the activity of epigenetic regulators, which included proteins known as master regulator transcription factors. They bind to DNA enhancers and super-enhancers. The master regulator transcription factors and super-enhancers work together to regulate the expression of critical genes, such as those responsible for cell identity.

Those and other tools helped investigators identify more than 3,000 super-enhancers in 28 medulloblastoma tumors as well as evidence that the activity of super-enhancers varied by subtype. The super-enhancers switched on known cancer genes, including genes like ALK, MYC, SMO, and OTX2 that are associated with medulloblastoma, the researchers reported.

Knowledge of the subtype super-enhancers led to identification of the transcription factors that regulate their activity. Using computational methods, researchers applied that information to reconstruct the transcription factor networks responsible for medulloblastoma subtype diversity and identity, providing previously unknown insights into the regulatory landscape and transcriptional output of the different medulloblastoma subtypes.

The approach helped to discover and nominate Lmx1A as a master regulator transcription factor of Group 4 tumors, which led to the identification of the likely Group 4 tumor cells of origin. Lmx1A was known to play an important role in normal development of cells in the uRL and cerebellum. Additional studies performed in mice with and without Lmx1A in this study supported uRL cells as the likely source of Group 4 tumors.

“By studying the epigenome, we also identified new pathways and molecular dependencies not apparent in previous gene expression and mutational studies,” explained Dr. Northcott. “The findings open new therapeutic avenues, particularly for the Group 3 and 4 subtypes where patient outcomes are inferior for the majority of affected children.”

For example, researchers identified increased enhancer activity targeting the TGFbeta pathway. The finding adds to evidence that the pathway may drive Group 3 medulloblastoma, currently the subtype with the worst prognosis. The pathway regulates cell growth, cell death, and other functions that are often disrupted in cancer, but it’s role in medulloblastoma is poorly understood.

The analysis included samples from 28 medulloblastoma tumors representing the four subtypes. Researchers believe it is the largest epigenetic study yet for any single cancer type and, importantly, the first to use a large cohort of primary patient tumor tissues instead of cell lines grown in the laboratory. Previous studies have suggested that cell lines may be of limited use for studying the tumor epigenome. The three Group 3 medulloblastoma cell lines used in this study reinforced the observation, highlighting significant differences in epigenetic regulators at work in medulloblastoma cell lines versus tumor samples.

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Beyond tau and amyloid

Posted in Alzheimer's Disease, Ca2+ triggered activation, Calcium Signaling, Cell Biology, Signaling & Cell Circuits, Cerebrovascular and Neurodegenerative Diseases, Clinical & Translational, Cognition, Disease Biology, Etiology, Gene Regulation, Innovations in Neurophysiology & Neuropsychology, Neurodegenerative Diseases, Pharmacotherapy and Cell Activity, Signaling & Cell Circuits, Small Molecules in Development of Therapeutic Drugs, tagged , , , , , , on January 26, 2016| Leave a Comment »

Beyond tau and amyloid

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

 

BEYOND AΒ AND TAU: OTHER TOXIC INSULTS AND AD PATHOLOGY

 

Neurovascular pathways to neurodegeneration in Alzheimer’s disease and other disorders.

Berislav V. Zlokovic

Nature Reviews Neuroscience 12, 723-738 (December 2011) |   http:dx.doi.org:/10.1038/nrn3114

The neurovascular unit (NVU) comprises brain endothelial cells, pericytes or vascular smooth muscle cells, glia and neurons. The NVU controls blood–brain barrier (BBB) permeability and cerebral blood flow, and maintains the chemical composition of the neuronal ‘milieu’, which is required for proper functioning of neuronal circuits. Recent evidence indicates that BBB dysfunction is associated with the accumulation of several vasculotoxic and neurotoxic molecules within brain parenchyma, a reduction in cerebral blood flow, and hypoxia. Together, these vascular-derived insults might initiate and/or contribute to neuronal degeneration. This article examines mechanisms of BBB dysfunction in neurodegenerative disorders, notably Alzheimer’s disease, and highlights therapeutic opportunities relating to these neurovascular deficits.

 

Summary

The neurovascular unit comprises vascular cells (endothelial cells, pericytes and vascular smooth muscle cells (VSMCs)), glial cells (astrocytes, microglia and oliogodendroglia) and neurons.
Neurodegenerative disorders such as Alzheimer’s disease and amyotrophic lateral sclerosis (ALS) are associated with microvascular dysfunction and/or degeneration in the brain, neurovascular disintegration, defective blood–brain barrier (BBB) function and/or vascular factors.
The interactions between endothelial cells and pericytes are crucial for the formation and maintenance of the BBB. Indeed, pericyte deficiency leads to BBB breakdown and extravasation of multiple vasculotoxic and neurotoxic circulating macromolecules, which can contribute to neuronal dysfunction, cognitive decline and neurodegenerative changes.
Alterations in cerebrovascular metabolic functions can also lead to the secretion of multiple neurotoxic and inflammatory factors.
BBB dysfunction and/or breakdown and cerebral blood flow (CBF) reductions and/or dysregulation may occur in sporadic Alzheimer’s disease and experimental models of this disease before cognitive decline, amyloid-β deposition and brain atrophy. In patients with ALS and in some experimental models of ALS, CBF dysregulation, blood–spinal cord barrier breakdown and spinal cord hypoperfusion have been reported prior to motor neuron cell death.
Several studies in animal models of Alzheimer’s disease and, more recently, in patients with this disorder have shown diminished amyloid-β clearance from brain tissue. The recognition of amyloid-β clearance pathways opens exciting new therapeutic opportunities for this disease.
‘Multiple-target, multiple-action’ agents will stand a better chance of controlling the complex disease mechanisms that mediate neurodegeneration in disorders such as Alzheimer’s disease than will agents that have only one target. According to the vasculo-neuronal-inflammatory triad model of neurodegenerative disorders, in addition to neurons, brain endothelium, VSMCs, pericytes, astrocytes and activated microglia all represent important therapeutic targets.

 

Neurons depend on blood vessels for their oxygen and nutrient supplies, and for the removal of carbon dioxide and other potentially toxic metabolites from the brain’s interstitial fluid (ISF). The importance of the circulatory system to the human brain is highlighted by the fact that although the brain comprises ~2% of total body mass, it receives up to 20% of cardiac output and is responsible for ~20% and ~25% of the body’s oxygen consumption and glucose consumption, respectively1. To underline this point, when cerebral blood flow (CBF) stops, brain functions end within seconds and damage to neurons occurs within minutes2.

Neurodegenerative disorders such as Alzheimer’s disease and amyotrophic lateral sclerosis (ALS) are associated with microvascular dysfunction and/or degeneration in the brain, neurovascular disintegration, defective blood–brain barrier (BBB) function and/or vascular factors1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12. Microvascular deficits diminish CBF and, consequently, the brain’s supply of oxygen, energy substrates and nutrients. Moreover, such deficits impair the clearance of neurotoxic molecules that accumulate and/or are deposited in the ISF, non-neuronal cells and neurons. Recent evidence suggests that vascular dysfunction leads to neuronal dysfunction and neurodegeneration, and that it might contribute to the development of proteinaceous brain and cerebrovascular ‘storage’ disorders. Such disorders include cerebral β-amyloidosis and cerebral amyloid angiopathy (CAA), which are caused by accumulation of the peptide amyloid-β in the brain and the vessel wall, respectively, and are features of Alzheimer’s disease1.

In this Review, I will discuss neurovascular pathways to neurodegeneration, placing a focus on Alzheimer’s disease because more is known about neurovascular dysfunction in this disease than in other neurodegenerative disorders. The article first examines transport mechanisms for molecules to cross the BBB, before exploring the processes that are involved in BBB breakdown at the molecular and cellular levels, and the consequences of BBB breakdown, hypoperfusion, and hypoxia and endothelial metabolic dysfunction for neuronal function. Next, the article reviews evidence for neurovascular changes during normal ageing and neurovascular BBB dysfunction in various neurodegenerative diseases, including evidence suggesting that vascular defects precede neuronal changes. Finally, the article considers specific mechanisms that are associated with BBB dysfunction in Alzheimer’s disease and ALS, and therapeutic opportunities relating to these neurovascular deficits.

The neurovascular unit

The neurovascular unit (NVU) comprises vascular cells (that is, endothelium, pericytes and vascular smooth muscle cells (VSMCs)), glial cells (that is, astrocytes, microglia and oliogodendroglia) and neurons1,2, 13 (Fig. 1). In the NVU, the endothelial cells together form a highly specialized membrane around blood vessels. This membrane underlies the BBB and limits the entry of plasma components, red blood cells (RBCs) and leukocytes into the brain. The BBB also regulates the delivery into the CNS of circulating energy metabolites and essential nutrients that are required for proper neuronal and synaptic function. Non-neuronal cells and neurons act in concert to control BBB permeability and CBF. Vascular cells and glia are primarily responsible for maintenance of the constant ‘chemical’ composition of the ISF, and the BBB and the blood–spinal cord barrier (BSCB) work together with pericytes to prevent various potentially neurotoxic and vasculotoxic macromolecules in the blood from entering the CNS, and to promote clearance of these substances from the CNS1.

In the brain, pial arteries run through the subarachnoid space (SAS), which contains the cerebrospinal fluid (CSF). These vessels give rise to intracerebral arteries, which penetrate into brain parenchyma. Intracerebral arteries are separated from brain parenchyma by a single, interrupted layer of elongated fibroblast-like cells of the pia and the astrocyte-derived glia limitans membrane that forms the outer wall of the perivascular Virchow–Robin space. These arteries branch into smaller arteries and subsequently arterioles, which lose support from the glia limitans and give rise to pre-capillary arterioles and brain capillaries. In an intracerebral artery, the vascular smooth muscle cell (VSMC) layer occupies most of the vessel wall. At the brain capillary level, vascular endothelial cells and pericytes are attached to the basement membrane. Pericyte processes encase most of the capillary wall, and they communicate with endothelial cells directly through synapse-like contacts containing connexins and N-cadherin. Astrocyte end-foot processes encase the capillary wall, which is composed of endothelium and pericytes. Resting microglia have a ‘ramified’ shape and can sense neuronal injury.

Figure 2 | Blood–brain barrier transport mechanisms.

Small lipophilic drugs, oxygen and carbon dioxide diffuse across the blood–brain barrier (BBB), whereas ions require ATP-dependent transporters such as the (Na++K+)ATPase. Transporters for nutrients include the glucose transporter 1 (GLUT1; also known as solute carrier family 2, facilitated glucose transporter member 1 (SLC2A1)), the lactate transporter monocarboxylate transporter 1 (MCT1) and the L1 and y+ transporters for large neutral and cationic essential amino acids, respectively. These four transporters are expressed at both the luminal and albuminal membranes. Non-essential amino acid transporters (the alanine, serine and cysteine preferring system (ASC), and the alanine preferring system (A)) and excitatory amino acid transporter 1 (EAAT1), EAAT2 and EAAT3 are located at the abluminal side. The ATP-binding cassette (ABC) efflux transporters that are found in the endothelial cells include multidrug resistance protein 1 (ABCB1; also known as ATP-binding cassette subfamily B member 1) and solute carrier organic anion transporter family member 1C1 (OATP1C1). Finally, transporters for peptides or proteins include the endothelial protein C receptor (EPCR) for activated protein C (APC); the insulin receptors (IRs) and the transferrin receptors (TFRs), which are associated with caveolin 1 (CAV1); low-density lipoprotein receptor-related protein 1 (LRP1) for amyloid-β, peptide transport system 1 (PTS1) for encephalins; and the PTS2 and PTS4–vasopressin V1a receptor (V1AR) for arginine vasopressin.

 

Transport across the blood–brain barrier. The endothelial cells that form the BBB are connected by tight and adherens junctions, and it is the tight junctions that confer the low paracellular permeability of the BBB1. Small lipophilic molecules, oxygen and carbon dioxide diffuse freely across the endothelial cells, and hence the BBB, but normal brain endothelium lacks fenestrae and has limited vesicular transport.

The high number of mitochondria in endothelial cells reflects a high energy demand for active ATP-dependent transport, conferred by transporters such as the sodium pump ((Na++K+)ATPase) and the ATP-binding cassette (ABC) efflux transporters. Sodium influx and potassium efflux across the abluminal side of the BBB is controlled by (Na++K+)ATPase (Fig. 2). Changes in sodium and potassium levels in the ISF influence the generation of action potentials in neurons and thus directly affect neuronal and synaptic functions1, 12.

Brain endothelial cells express transporters that facilitate the transport of nutrients down their concentration gradients, as described in detail elsewhere1, 14 (Fig. 2). Glucose transporter 1 (GLUT1; also known as solute carrier family 2, facilitated glucose transporter member 1 (SLC2A1)) — the BBB-specific glucose transporter — is of special importance because glucose is a key energy source for the brain.

Monocarboxylate transporter 1 (MCT1), which transports lactate, and the L1 and y+ amino acid transporters are expressed at the luminal and abluminal membranes12, 14. Sodium-dependent excitatory amino acid transporter 1 (EAAT1), EAAT2 and EAAT3 are expressed at the abluminal side of the BBB15 and enable removal of glutamate, an excitatory neurotransmitter, from the brain (Fig. 2). Glutamate clearance at the BBB is essential for protecting neurons from overstimulation of glutaminergic receptors, which is neurotoxic16.

ABC transporters limit the penetration of many drugs into the brain17. For example, multidrug resistance protein 1 (ABCB1; also known as ATP-binding cassette subfamily B member 1) controls the rapid removal of ingested toxic lipophilic metabolites17 (Fig. 2). Some ABC transporters also mediate the efflux of nutrients from the endothelium into the ISF. For example, solute carrier organic anion transporter family member 1C1 (OATP1C1) transports thyroid hormones into the brain. MCT8 mediates influx of thyroid hormones from blood into the endothelium18 (Fig. 2).

The transport of circulating peptides across the BBB into the brain is restricted or slow compared with the transport of nutrients19. Carrier-mediated transport of neuroactive peptides controls their low levels in the ISF20, 21, 22, 23, 24 (Fig. 2). Some proteins, including transferrin, insulin, insulin-like growth factor 1 (IGF1), leptin25, 26, 27 and activatedprotein C (APC)28, cross the BBB by receptor-mediated transcytosis (Fig. 2).

Circumventricular organs. Several small neuronal structures that surround brain ventricles lack the BBB and sense chemical changes in blood or the cerebrospinal fluid (CSF) directly. These brain areas are known as circumventricular organs (CVOs). CVOs have important roles in multiple endocrine and autonomic functions, including the control of feeding behaviour as well as regulation of water and salt metabolism29. For example, the subfornical organ is one of the CVOs that are capable of sensing extracellular sodium using astrocyte-derived lactate as a signal for local neurons to initiate neural, hormonal and behavioural responses underlying sodium homeostasis30. Excessive sodium accumulation is detrimental, and increases in plasma sodium above a narrow range are incompatible with life, leading to cerebral oedema (swelling), seizures and death29.

Vascular-mediated pathophysiology

The key pathways of vascular dysfunction that are linked to neurodegenerative diseases include BBB breakdown, hypoperfusion–hypoxia and endothelial metabolic dysfunction (Fig. 3). This section examines processes that are involved in BBB breakdown at the molecular and cellular levels, and explores the consequences of all three pathways for neuronal function and viability.

Figure 3 | Vascular-mediated neuronal damage and neurodegeneration.

a | Blood–brain barrier (BBB) breakdown that is caused by pericyte detachment leads to leakage of serum proteins and focal microhaemorrhages, with extravasation of red blood cells (RBCs). RBCs release haemoglobin, which is a source of iron. In turn, this metal catalyses the formation of toxic reactive oxygen species (ROS) that mediate neuronal injury. Albumin promotes the development of vasogenic oedema, contributing to hypoperfusion and hypoxia of the nervous tissue, which aggravates neuronal injury. A defective BBB allows several potentially vasculotoxic and neurotoxic proteins (for example, thrombin, fibrin and plasmin) to enter the brain. b | Progressive reductions in cerebral blood flow (CBF) lead to increasing neuronal dysfunction. Mild hypoperfusion, oligaemia, leads to a decrease in protein synthesis, whereas more-severe reductions in CBF, leading to hypoxia, cause an array of detrimental effects.


Blood–brain barrier breakdown. Disruption to tight and adherens junctions, an increase in bulk-flow fluid transcytosis, and/or enzymatic degradation of the capillary basement membrane cause physical breakdown of the BBB.

The levels of many tight junction proteins, their adaptor molecules and adherens junction proteins decrease in Alzheimer’s disease and other diseases that cause dementia1, 9, ALS31, multiple sclerosis32 and various animal models of neurological disease8, 33. These decreases might be partly explained by the fact that vascular-associated matrix metalloproteinase (MMP) activity rises in many neurodegenerative disorders and after ischaemic CNS injury34, 35; tight junction proteins and basement membrane extracellular matrix proteins are substrates for these enzymes34. Lowered expression of messenger RNAs that encode several key tight junction proteins, however, has also been reported in some neurodegenerative disorders, such as ALS31.

Endothelial cell–pericyte interactions are crucial for the formation36, 37and maintenance of the BBB33, 38. Pericyte deficiency can lead to a reduction in expression of certain tight junction proteins, including occludin, claudin 5 and ZO1 (Ref. 33), and to an increase in bulk-flow transcytosis across the BBB, causing BBB breakdown38. Both processes can lead to extravasation of multiple small and large circulating macromolecules (up to 500 kDa) into the brain parenchyma33, 38. Moreover, in mice, an age-dependent progressive loss of pericytes can lead to BBB disruption and microvasular degeneration and, subsequently, neuronal dysfunction, cognitive decline and neurodegenerative changes33. In their lysosomes, pericytes concentrate and degrade multiple circulating exogenous39 and endogenous proteins, including serum immunoglobulins and fibrin33, which amplify BBB breakdown in cases of pericyte deficiency.

BBB breakdown typically leads to an accumulation of various molecules in the brain. The build up of serum proteins such as immunoglobulins and albumin can cause brain oedema and suppression of capillary blood flow8, 33, whereas high concentrations of thrombin lead to neurotoxicity and memory impairment40, and accelerate vascular damage and BBB disruption41. The accumulation of plasmin (derived from circulating plasminogen) can catalyse the degradation of neuronal laminin and, hence, promote neuronal injury42, and high fibrin levels accelerate neurovascular damage6. Finally, an increase in the number of RBCs causes deposition of haemoglobin-derived neurotoxic products including iron, which generates neurotoxic reactive oxygen species (ROS)8, 43(Fig. 3a). In addition to protein-mediated vasogenic oedema, local tissue ischaemia–hypoxia depletes ATP stores, causing (Na++K+)ATPase pumps and Na+-dependent ion channels to stop working and, consequently, the endothelium and astrocytes to swell (known as cytotoxic oedema)44. Upregulation of aquaporin 4 water channels in response to ischaemia facilitates the development of cytotoxic oedema in astrocytes45.

Hypoperfusion and hypoxia. CBF is regulated by local neuronal activity and metabolism, known as neurovascular coupling46. The pial and intracerebral arteries control the local increase in CBF that occurs during brain activation, which is termed ‘functional hyperaemia’. Neurovascular coupling requires intact pial circulation, and for VSMCs and pericytes to respond normally to vasoactive stimuli33, 46, 47. In addition to VSMC-mediated constriction and vasodilation of cerebral arteries, recent studies have shown that pericytes modulate brain capillary diameter through constriction of the vessel wall47, which obstructs capillary flow during ischaemia48. Astrocytes regulate the contractility of intracerebral arteries49, 50.

Progressive CBF reductions have increasingly serious consequences for neurons (Fig. 3b). Briefly, mild hypoperfusion — termed oligaemia — affects protein synthesis, which is required for the synaptic plasticity mediating learning and memory46. Moderate to severe CBF reductions and hypoxia affect ATP synthesis, diminishing (Na++K+)ATPase activity and the ability of neurons to generate action potentials9. In addition, such reductions can lower or increase pH, and alter electrolyte balances and water gradients, leading to the development of oedema and white matter lesions, and the accumulation of glutamate and proteinaceous toxins (for example, amyloid-β and hyperphopshorylated tau) in the brain. A reduction of greater than 80% in CBF results in neuronal death2.

The effect of CBF reductions has been extensively studied at the molecular and cellular levels in relation to Alzheimer’s disease. Reduced CBF and/or CBF dysregulation occurs in elderly individuals at high risk of Alzheimer’s disease before cognitive decline, brain atrophy and amyloid-β accumulation10, 46, 51, 52, 53, 54. In animal models, hypoperfusion can induce or amplify Alzheimer’s disease-like neuronal dysfunction and/or neuropathological changes. For example, bilateral carotid occlusion in rats causes memory impairment, neuronal dysfunction, synaptic changes and amyloid-β oligomerization55, leading to accumulation of neurotoxic amyloid-β oligomers56. In a mouse model of Alzheimer’s disease, oligaemia increases neuronal amyloid-β levels and neuronal tau phosphophorylation at an epitope that is associated with Alzheimer’s disease-type paired helical filaments57. In rodents, ischaemia leads to the accumulation of hyperphosphorylated tau in neurons and the formation of filaments that resemble those present in human neurodegenerative tauopathies and Alzheimer’s disease58. Mice expressing amyloid-β precursor protein (APP) and transforming growth factor β1 (TGFβ1) develop deficient neurovascular coupling, cholinergic denervation, enhanced cerebral and cerebrovascular amyloid-β deposition, and age-dependent cognitive decline59.

Recent studies have shown that ischaemia–hypoxia influences amyloidogenic APP processing through mechanisms that increase the activity of two key enzymes that are necessary for amyloid-β production; that is, β-secretase and γ-secretase60, 61, 62, 63. Hypoxia-inducible factor 1α (HIF1α) mediates transcriptional increase in β-secretase expression61. Hypoxia also promotes phosphorylation of tau through the mitogen-activated protein kinase (MAPK; also known as extracellular signal-regulated kinase (ERK)) pathway64, downregulates neprilysin — an amyloid-β-degrading enzyme65 — and leads to alterations in the expression of vascular-specific genes, including a reduction in the expression of the homeobox protein MOX2 gene mesenchyme homeobox 2 (MEOX2) in brain endothelial cells5 and an increase in the expression of the myocardin gene (MYOCD) in VSMCs66. In patients with Alzheimer’s disease and in models of this disorder, these changes cause vessel regression, hypoperfusion and amyloid-β accumulation resulting from the loss of the key amyloid-β clearance lipoprotein receptor (see below). In addition, hypoxia facilitates alternative splicing of Eaat2 mRNA in Alzheimer’s disease transgenic mice before amyloid-β deposition67 and suppresses glutamate reuptake by astrocytes independently of amyloid formation68, resulting in glutamate-mediated neuronal injury that is independent of amyloid-β.

In response to hypoxia, mitochondria release ROS that mediate oxidative damage to the vascular endothelium and to the selective population of neurons that has high metabolic activity. Such damage has been suggested to occur before neuronal degeneration and amyloid-β deposition in Alzheimer’s disease69, 70. Although the exact triggers of hypoxia-mediated neurodegeneration and the role of HIF1α in neurodegeneration versus preconditioning-mediated neuroprotection remain topics of debate, mitochondria-generated ROS seem to have a primary role in the regulation of the HIF1α-mediated transcriptional switch that can activate an array of responses, ranging from mechanisms that increase cell survival and adaptation to mechanisms inducing cell cycle arrest and death71. Whether inhibition of hypoxia-mediated pathogenic pathways will delay onset and/or control progression in neurodegenerative conditions such as Alzheimer’s disease remains to be determined.

When comparing the contributions of BBB breakdown and hypoperfusion to neuronal injury, it is interesting to consider Meox2+/− mice. Such animals have normal pericyte coverage and an intact BBB but a substantial perfusion deficit5 that is comparable to that found in pericyte-deficient mice that develop BBB breakdown33 Notably, however, Meox2+/− mice show less pronounced neurodegenerative changes than pericyte-deficient mice, indicating that chronic hypoperfusion–hypoxia alone can cause neuronal injury, but not to the same extent as hypoperfusion–hypoxia combined with BBB breakdown.

Endothelial neurotoxic and inflammatory factors. Alterations in cerebrovascular metabolic functions can lead to the secretion of multiple neurotoxic and inflammatory factors72, 73. For example, brain microvessels that have been isolated from individuals with Alzheimer’s disease (but not from neurologically normal age-matched and young individuals) and brain microvessels that have been treated with inflammatory proteins release neurotoxic factors that kill neurons74, 75. These factors include thrombin, the levels of which increase with the onset of Alzheimer’s disease76. Thrombin can injure neurons directly40and indirectly by activating microglia and astrocytes73. Compared with those from age-matched controls, brain microvessels from individuals with Alzheimer’s disease secrete increased levels of multiple inflammatory mediators, such as nitric oxide, cytokines (for example, tumour necrosis factor (TNF), TGFβ1, interleukin-1β (IL-1β) and IL-6), chemokines (for example, CC-chemokine ligand 2 (CCL2; also known as monocyte chemoattractant protein 1 (MCP1)) and IL-8), prostaglandins, MMPs and leukocyte adhesion molecules73. Endothelium-derived neurotoxic and inflammatory factors together provide a molecular link between vascular metabolic dysfunction, neuronal injury and inflammation in Alzheimer’s disease and, possibly, in other neurodegenerative disorders.

Neurovascular changes

This section examines evidence for neurovascular changes during normal ageing and for neurovascular and/or BBB dysfunction in various neurodegenerative diseases, as well as the possibility that vascular defects can precede neuronal changes.

Age-associated neurovascular changes. Normal ageing diminishes brain circulatory functions, including a detectable decay of CBF in the limbic and association cortices that has been suggested to underlie age-related cognitive changes77. Alterations in the cerebral microvasculature, but not changes in neural activity, have been shown to lead to age-dependent reductions in functional hyperaemia in the visual system in cats78 and in the sensorimotor cortex in pericyte-deficient mice33. Importantly, a recent longitudinal CBF study in neurologically normal individuals revealed that people bearing the apolipoprotein E (APOE) ɛ4allele — the major genetic risk factor for late-onset Alzheimer’s disease79, 80, 81 — showed greater regional CBF decline in brain regions that are particularly vulnerable to pathological changes in Alzheimer’s disease than did people without this allele82.

A meta-analysis of BBB permeability in 1,953 individuals showed that neurologically healthy humans had an age-dependent increase in vascular permeability83. Moreover, patients with vascular or Alzheimer’s disease-type dementia and leucoaraiosis — a small-vessel disease of the cerebral white matter — had an even greater age-dependent increase in vascular permeability83. Interestingly, an increase in BBB permeability in brain areas with normal white matter in patients with leukoaraiosis has been suggested to play a causal part in disease and the development of lacunar strokes84. Age-related changes in the permeability of the blood–CSF barrier and the choroid plexus have been reported in sheep85.

Vascular pathology. Patients with Alzheimer’s disease or other dementia-causing diseases frequently show focal changes in brain microcirculation. These changes include the appearance of string vessels (collapsed and acellular membrane tubes), a reduction in capillary density, a rise in endothelial pinocytosis, a decrease in mitochondrial content, accumulation of collagen and perlecans in the basement membrane, loss of tight junctions and/or adherens junctions3, 4, 5, 6, 9,46, 86, and BBB breakdown with leakage of blood-borne molecules4, 6,7, 9. The time course of these vascular alterations and how they relate to dementia and Alzheimer’s disease pathology remain unclear, as no protocol that allows the development of the diverse brain vascular pathology to be scored, and hence to be tracked with ageing, has so far been developed and widely validated87. Interestingly, a recent study involving 500 individuals who died between the ages of 69 and 103 years showed that small-vessel disease, infarcts and the presence of more than one vascular pathological change were associated with Alzheimer’s disease-type pathological lesions and dementia in people aged 75 years of age87. These associations were, however, less pronounced in individuals aged 95 years of age, mainly because of a marked ageing-related reduction in Alzheimer’s disease neuropathology relative to a moderate but insignificant ageing-related reduction in vascular pathology87.

Accumulation of amyloid-β and amyloid deposition in pial and intracerebral arteries results in CAA, which is present in over 80% of Alzheimer’s disease cases88. In patients who have Alzheimer’s disease with established CAA in small arteries and arterioles, the VSMC layer frequently shows atrophy, which causes a rupture of the vessel wall and intracerebral bleeding in about 30% of these patients89, 90. These intracerebral bleedings contribute to, and aggravate, dementia. Patients with hereditary cerebral β-amyloidosis and CAA of the Dutch, Iowa, Arctic, Flemish, Italian or Piedmont L34V type have accelerated VSMC degeneration resulting in haemorrhagic strokes and dementia91. Duplication of the gene encoding APP causes early-onset Alzheimer’s disease dementia with CAA and intracerebral haemorrhage92.

Early studies of serum immunoglobulin leakage reported that patients with ALS had BSCB breakdown and BBB breakdown in the motor cortex93. Microhaemorrhages and BSCB breakdown have been shown in the spinal cord of transgenic mice expressing mutant variants of human superoxide dismutase 1 (SOD1), which in mice cause an ALS-like disease8, 94, 95. In mice with ALS-like disease and in patients with ALS, BSCB breakdown has been shown to occur before motor neuron degeneration or brain atrophy8, 11, 95.

BBB breakdown in the substantia nigra and the striatum has been detected in murine models of Parkinson’s disease that are induced by administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)96, 97, 98. However, the temporal relationship between BBB breakdown and neurodegeneration in Parkinson’s disease is currently unknown. Notably, the prevalence of CAA and vascular lesions increases in Parkinson’s disease99, 100. Vascular lesions in the striatum and lacunar infarcts can cause vascular parkinsonism syndrome101. A recent study reported BBB breakdown in a rat model of Huntington’s disease that is induced with the toxin 3-nitropropionic acid102.

Several studies have established disruption of BBB with a loss of tight junction proteins during neuroinflammatory conditions such as multiple sclerosis and its murine model, experimental allergic encephalitis. Such disruption facilitates leukocyte infiltration, leading to oliogodendrocyte death, axonal damage, demyelination and lesion development32.

Functional changes in the vasculature. In individuals with Alzheimer’s disease, GLUT1 expression at the BBB decreases103, suggesting a shortage in necessary metabolic substrates. Studies using18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) have identified reductions in glucose uptake in asymptomatic individuals with a high risk of dementia104, 105. Several studies have suggested that reduced glucose uptake across the BBB, as seen by FDG PET, precedes brain atrophy104, 105, 106, 107, 108.

Amyloid-β constricts cerebral arteries109. In a mouse model of Alzheimer’s disease, impairment of endothelium-dependent regulation of neocortical microcirculation110, 111 occurs before amyloid-β accumulation. Recent studies have shown that CD36, a scavenger receptor that binds amyloid-β, is essential for the vascular oxidative stress and diminished functional hyperaemia that occurs in response to amyloid-β exposure112. Neuroimaging studies in patients with Alzheimer’s disease have shown that neurovascular uncoupling occurs before neurodegenerative changes10, 51, 52, 53. Moreover, cognitively normal APOE ɛ4 carriers at risk of Alzheimer’s disease show impaired CBF responses to brain activation in the absence of neurodegenerative changes or amyloid-β accumulation54. Recently, patients with Alzheimer’s disease as well as mouse models of this disease with high cerebrovascular levels of serum response factor (SRF) and MYOCD, the two transcription factors that control VSMC differentiation, have been shown to develop a hypercontractile arterial phenotype resulting in brain hypoperfusion, diminished functional hyperaemia and CAA66, 113. More work is needed to establish the exact role of SRF and MYOCD in the vascular dysfunction that results in the Alzheimer’s disease phenotype and CAA.

PET studies with 11C-verapamil, an ABCB1 substrate, have indicated that the function of ABCB1, which removes multiple drugs and toxins from the brain, decreases with ageing114 and is particularly compromised in the midbrain of patients with Parkinson’s disease, progressive supranuclear palsy or multiple system atrophy115. More work is needed to establish the exact roles of ABC BBB transporters in neurodegeneration and whether their failure precedes the loss of dopaminergic neurons that occurs in Parkinson’s disease.

In mice with ALS-like disease and in patients with ALS, hypoperfusion and/or dysregulated CBF have been shown to occur before motor neuron degeneration or brain atrophy8, 116. Reduced regional CBF in basal ganglia and reduced blood volume have been reported in pre-symptomatic gene-tested individuals at risk for Huntington’s disease117. Patients with Huntington’s disease display a reduction in vasomotor activity in the cerebral anterior artery during motor activation118.

Vascular and neuronal common growth factors. Blood vessels and neurons share common growth factors and molecular pathways that regulate their development and maintenance119, 120. Angioneurins are growth factors that exert both vasculotrophic and neurotrophic activities121. The best studied angioneurin is vascular endothelial growth factor (VEGF). VEGF regulates vessel formation, axonal growth and neuronal survival120. Ephrins, semaphorins, slits and netrins are axon guidance factors that also regulate the development of the vascular system121. During embryonic development of the neural tube, blood vessels and choroid plexus secrete IGF2 into the CSF, which regulates the proliferation of neuronal progenitor cells122. Genetic and pharmacological manipulations of angioneurin activity yielded various vascular and cerebral phenotypes121. Given the dual nature of angioneurin action, these studies have not been able to address whether neuronal dysfunction results from a primary insult to neurons and/or whether it is secondary to vascular dysfunction.

Increased levels of VEGF, a hypoxia-inducible angiogenic factor, were found in the walls of intraparenchymal vessels, perivascular deposits, astrocytes and intrathecal space of patients with Alzheimer’s disease, and were consistent with the chronic cerebral hypoperfusion and hypoxia that were observed in these individuals73. In addition to VEGF, brain microvessels in Alzheimer’s disease release several molecules that can influence angiogenesis, including IL-1β, IL-6, IL-8, TNF, TGFβ, MCP1, thrombin, angiopoietin 2, αVβ3 and αVβ5 integrins, and HIF1α73. However, evidence for increased vascularity in Alzheimer’s disease is lacking. On the contrary, several studies have reported that focal vascular regression and diminished microvascular density occur in Alzheimer’s disease4, 5, 73 and in Alzheimer’s disease transgenic mice123. The reason for this discrepancy is not clear. The anti-angiogenic activity of amyloid-β, which accumulates in the brains of individuals with Alzheimer’s disease and Alzheimer’s disease models, may contribute to hypovascularity123. Conversely, genome-wide transcriptional profiling of brain endothelial cells from patients with Alzheimer’s disease revealed that extremely low expression of vascular-restricted MEOX2 mediates aberrant angiogenic responses to VEGF and hypoxia, leading to capillary death5. This finding raises the interesting question of whether capillary degeneration in Alzheimer’s disease results from unsuccessful vascular repair and/or remodelling. Moreover, mice that lack one Meox2 allele have been shown to develop a primary cerebral endothelial hypoplasia with chronic brain hypoperfusion5, resulting in secondary neurodegenerative changes33.

Does vascular dysfunction cause neuronal dysfunction? In summary, the evidence that is discussed above clearly indicates that vascular dysfunction is tightly linked to neuronal dysfunction. There are many examples to illustrate that primary vascular deficits lead to secondary neurodegeneration, including CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts), an hereditary small-vessel brain disease resulting in multiple small ischaemic lesions, neurodegeneration and dementia124; mutations in SLC2A1 that cause dysfunction of the BBB-specific GLUT1 transporter in humans resulting in seizures; cognitive impairment and microcephaly125; microcephaly and epileptiform discharges in mice with genetic deletion of a single Slc2a1allele126; and neurodegeneration mediated by a single Meox2 homebox gene deletion restricted to the vascular system33. Patients with hereditary cerebral β-amyloidosis and CAA of the Dutch, Iowa, Arctic, Flemish, Italian or Piedmont L34V type provide another example showing that primary vascular dysfunction — which in this case is caused by deposition of vasculotropic amyloid-β mutants in the arterial vessel wall — leads to dementia and intracerebral bleeding. Moreover, as reviewed in the previous sections, recent evidence suggests that BBB dysfunction and/or breakdown, and CBF reductions and/or dysregulation may occur in sporadic Alzheimer’s disease and experimental models of this disease before cognitive decline, amyloid-β deposition and brain atrophy. In patients with ALS and in some experimental models of ALS, CBF dysregulation, BSCB breakdown and spinal cord hypoperfusion have been reported to occur before motor neuron cell death. Whether neurological changes follow or precede vascular dysfunction in Parkinson’s disease, Huntington’s disease and multiple sclerosis remains less clear. However, there is little doubt that vascular injury mediates, amplifies and/or lowers the threshold for neuronal dysfunction and loss in several neurological disorders.

Disease-specific considerations

This section examines how amyloid-β levels are kept low in the brain, a process in which the BBB has a central role, and how faulty BBB-mediated clearance mechanisms go awry in Alzheimer’s disease. On the basis of this evidence and the findings discussed elsewhere in the Review, a new hypothesis for the pathogenesis of Alzheimer’s disease that incorporates the vascular evidence is presented. ALS-specific disease mechanisms relating to the BBB are then examined.

Alzheimer’s disease. Amyloid-β clearance from the brain by the BBB is the best studied example of clearance of a proteinaceous toxin from the CNS. Multiple pathways regulate brain amyloid-β levels, including its production and clearance (Fig. 4). Recent studies127, 128, 129 have confirmed earlier findings in multiple rodent and non-human primate models demonstrating that peripheral amyloid-β is an important precursor of brain amyloid-β130, 131, 132, 133, 134, 135, 136. Moreover, peripheral amyloid-β sequestering agents such as soluble LRP1 (ref.137), anti-amyloid-β antibodies138, 139, 140, gelsolin and the ganglioside GM1 (Ref. 141), or systemic expression of neprilysin142, 143have been shown to reduce the amyloid burden in Alzheimer’s disease mice by eliminating contributions of the peripheral amyloid-β pool to the total brain pool of this peptide.

Figure 4 | The role of blood–brain barrier transport in brain homeostasis of amyloid-β.

Amyloid-β (Aβ) is produced from the amyloid-β precursor protein (APP), both in the brain and in peripheral tissues. Clearance of amyloid-β from the brain normally maintains its low levels in the brain. This peptide is cleared across the blood–brain barrier (BBB) by the low-density lipoprotein receptor-related protein 1 (LRP1). LRP1 mediates rapid efflux of a free, unbound form of amyloid-β and of amyloid-β bound to apolipoprotein E2 (APOE2), APOE3 or α2-macroglobulin (not shown) from the brain’s interstitial fluid into the blood, and APOE4 inhibits such transport. LRP2 eliminates amyloid-β that is bound to clusterin (CLU; also known as apolipoprotein J (APOJ)) by transport across the BBB, and shows a preference for the 42-amino-acid form of this peptide. ATP-binding cassette subfamily A member 1 (ABCA1; also known as cholesterol efflux regulatory protein) mediates amyloid-β efflux from the brain endothelium to blood across the luminal side of the BBB (not shown). Cerebral endothelial cells, pericytes, vascular smooth muscle cells, astrocytes, microglia and neurons express different amyloid-β-degrading enzymes, including neprilysin (NEP), insulin-degrading enzyme (IDE), tissue plasminogen activator (tPA) and matrix metalloproteinases (MMPs), which contribute to amyloid-β clearance. In the circulation, amyloid-β is bound mainly to soluble LRP1 (sLRP1), which normally prevents its entry into the brain. Systemic clearance of amyloid-β is mediated by its removal by the liver and kidneys. The receptor for advanced glycation end products (RAGE) provides the key mechanism for influx of peripheral amyloid-β into the brain across the BBB either as a free, unbound plasma-derived peptide and/or by amyloid-β-laden monocytes. Faulty vascular clearance of amyloid-β from the brain and/or an increased re-entry of peripheral amyloid-β across the blood vessels into the brain can elevate amyloid-β levels in the brain parenchyma and around cerebral blood vessels. At pathophysiological concentrations, amyloid-β forms neurotoxic oligomers and also self-aggregates, which leads to the development of cerebral β-amyloidosis and cerebral amyloid angiopathy.


The receptor for advanced glycation end products (RAGE) mediates amyloid-β transport in brain and the propagation of its toxicity. RAGE expression in brain endothelium provides a mechanism for influx of amyloid-β144, 145 and amyloid-β-laden monocytes146 across the BBB, as shown in Alzheimer’s disease models (Fig. 4). The amyloid-β-rich environment in Alzheimer’s disease and models of this disorder increases RAGE expression at the BBB and in neurons147, 148, amplifying amyloid-β-mediated pathogenic responses. Blockade of amyloid-β–RAGE signalling in Alzheimer’s disease is a promising strategy to control self-propagation of amyloid-β-mediated injury.

Several studies in animal models of Alzheimer’s disease and, more recently, in patients with this disorder149 have shown that diminished amyloid-β clearance occurs in brain tissue in this disease. LRP1 plays an important part in the three-step serial clearance of this peptide from brain and the rest of the body150 (Fig. 4). In step one, LRP1 in brain endothelium binds brain-derived amyloid-β at the abluminal side of the BBB, initiating its clearance to blood, as shown in many animal models151, 152, 153, 154, 155, 156 and BBB models in vitro151, 157,158. The vasculotropic mutants of amyloid-β that have low binding affinity for LRP1 are poorly cleared from the brain or CSF151, 159, 160. APOE4, but not APOE3 or APOE2, blocks LRP1-mediated amyloid-β clearance from the brain and, hence, promotes its retention161, whereas clusterin (also known as apolipoprotein J (APOJ)) mediates amyloid-β clearance across the BBB via LRP2 (Ref. 153). APOE and clusterin influence amyloid-β aggregation162, 163. Reduced LRP1 levels in brain microvessels, perhaps in addition to altered levels of ABCB1, are associated with amyloid-β cerebrovascular and brain accumulation during ageing in rodents, non-human primates, humans, Alzheimer’s disease mice and patients with Alzheimer’s disease66, 151, 152, 164, 165, 166. Moreover, recent work has shown that brain LRP1 is oxidized in Alzheimer’s disease167, and may contribute to amyloid-β retention in brain because the oxidized form cannot bind and/or transport amyloid-β137. LRP1 also mediates the removal of amyloid-β from the choroid plexus168.

In step two, circulating soluble LRP1 binds more than 70% of plasma amyloid-β in neurologically normal humans137. In patients with Alzheimer’s disease or mild cognitive impairment (MCI), and in Alzheimer’s disease mice, amyloid-β binding to soluble LRP1 is compromised due to oxidative changes137, 169, resulting in elevated plasma levels of free amyloid-β isoforms comprising 40 or 42 amino acids (amyloid-β1–40 and amyloid-β1–42). These peptides can then re-enter the brain, as has been shown in a mouse model of Alzheimer’s disease137. Rapid systemic removal of amyloid-β by the liver is also mediated by LRP1 and comprises step three of the clearance process170.

In brain, amyloid-β is enzymatically degraded by neprilysin171, insulin-degrading enzyme172, tissue plasminogen activator173 and MMPs173,174 in various cell types, including endothelial cells, pericytes, astrocytes, neurons and microglia. Cellular clearance of this peptide by astrocytes and VSMCs is mediated by LRP1 and/or another lipoprotein receptor66, 175. Clearance of amyloid-β aggregates by microglia has an important role in amyloid-β-directed immunotherapy176 and reduction of the amyloid load in brain177. Passive ISF–CSF bulk flow and subsequent clearance through the CSF might contribute to 10–15% of total amyloid-β removal152, 153, 178. In the injured human brain, increasing soluble amyloid-β concentrations in the ISF correlated with improvements in neurological status, suggesting that neuronal activity might regulate extracellular amyloid-β levels179.

The role of BBB dysfunction in amyloid-β accumulation, as discussed above, underlies the contribution of vascular dysfunction to Alzheimer’s disease (see Fig. 5 for a model of vascular damage in Alzheimer’s disease). The amyloid hypothesis for the pathogenesis of Alzheimer’s disease maintains that this peptide initiates a cascade of events leading to neuronal injury and loss and, eventually, dementia180, 181. Here, I present an alternative hypothesis — the two-hit vascular hypothesis of Alzheimer’s disease — that incorporates the vascular contribution to this disease, as discussed in this Review (Box 1). This hypothesis states that primary damage to brain microcirculation (hit one) initiates a non-amyloidogenic pathway of vascular-mediated neuronal dysfunction and injury, which is mediated by BBB dysfunction and is associated with leakage and secretion of multiple neurotoxic molecules and/or diminished brain capillary flow that causes multiple focal ischaemic or hypoxic microinjuries. BBB dysfunction also leads to impairment of amyloid-β clearance, and oligaemia leads to increased amyloid-β generation. Both processes contribute to accumulation of amyloid-β species in the brain (hit two), where these peptides exert vasculotoxic and neurotoxic effects. According to the two-hit vascular hypothesis of Alzheimer’s disease, tau pathology develops secondary to vascular and/or amyloid-β injury.

Figure 5 | A model of vascular damage in Alzheimer’s disease.

a | In the early stages of Alzheimer’s disease, small pial and intracerebral arteries develop a hypercontractile phenotype that underlies dysregulated cerebral blood flow (CBF). This phenotype is accompanied by diminished amyloid-β clearance by the vascular smooth muscle cells (VSMCs). In the later phases of Alzheimer’s disease, amyloid deposition in the walls of intracerebral arteries leads to cerebral amyloid angiopathy (CAA), pronounced reductions in CBF, atrophy of the VSMC layer and rupture of the vessels causing microbleeds. b | At the level of capillaries in the early stages of Alzheimer’s disease, blood–brain barrier (BBB) dysfunction leads to a faulty amyloid-β clearance and accumulation of neurotoxic amyloid-β oligomers in the interstitial fluid (ISF), microhaemorrhages and accumulation of toxic blood-derived molecules (that is, thrombin and fibrin), which affect synaptic and neuronal function. Hyperphosphorylated tau (p-tau) accumulates in neurons in response to hypoperfusion and/or rising amyloid-β levels. At this point, microglia begin to sense neuronal injury. In the later stages of the disease in brain capillaries, microvascular degeneration leads to increased deposition of basement membrane proteins and perivascular amyloid. The deposited proteins and amyloid obstruct capillary blood flow, resulting in failure of the efflux pumps, accumulation of metabolic waste products, changes in pH and electrolyte composition and, subsequently, synaptic and neuronal dysfunction. Neurofibrillary tangles (NFTs) accumulate in response to ischaemic injury and rising amyloid-β levels. Activation of microglia and astrocytes is associated with a pronounced inflammatory response. ROS, reactive oxygen species.


Amyotrophic lateral sclerosis. The cause of sporadic ALS, a fatal adult-onset motor neuron neurodegenerative disease, is not known182. In a relatively small number of patients with inherited SOD1 mutations, the disease is caused by toxic properties of mutant SOD1 (Ref. 183). Mutations in the genes encoding ataxin 2 and TAR DNA-binding protein 43 (TDP43) that cause these proteins to aggregate have been associated with ALS182, 184. Some studies have suggested that abnormal SOD1 species accumulate in sporadic ALS185. Interestingly, studies in ALS transgenic mice expressing a mutant version of human SOD1 in neurons, and in non-neuronal cells neighbouring these neurons, have shown that deletion of this gene from neurons does not influence disease progression186, whereas deletion of this gene from microglia186 or astrocytes187 substantially increases an animal’s lifespan. According to an emerging hypothesis of ALS that is based on studies in SOD1 mutant mice, the toxicity that is derived from non-neuronal neighbouring cells, particularly microglia and astrocytes, contributes to disease progression and motor neuron degeneration186, 187, 188, 189, 190, whereas BBB dysfunction might be critical for disease initiation8, 43, 94, 95. More work is needed to determine whether this concept of disease initiation and progression may also apply to cases of sporadic ALS.

Human data support a role for angiogenic factors and vessels in the pathogenesis of ALS. For example, the presence of VEGF variations (which were identified in large meta-analysis studies) has been linked to ALS191. Angiogenin is another pro-angiogenic gene that is implicated in ALS because heterozygous missense mutations in angiogenin cause familial and sporadic ALS192. Moreover, mice with a mutation that eliminates hypoxia-responsive induction of the Vegf gene (Vegfδ/δ mice) develop late-onset motor neuron degeneration193. Spinal cord ischaemia worsens motor neuron degeneration and functional outcome in Vegfδ/δmice, whereas the absence of hypoxic induction of VEGF in mice that develop motor neuron disease from expression of ALS-linked mutant SOD1G93A results in substantially reduced survival191.

Therapeutic opportunities

Many investigators believe that primary neuronal dysfunction resulting from an intrinsic neuronal disorder is the key underlying event in human neurodegenerative diseases. Thus, most therapeutic efforts for neurodegenerative diseases have so far been directed at the development of so-called ‘single-target, single-action’ agents to target neuronal cells directly and reverse neuronal dysfunction and/or protect neurons from injurious insults. However, most preclinical and clinical studies have shown that such drugs are unable to cure or control human neurological disorders2, 181, 183, 194, 195. For example, although pathological overstimulation of glutaminergic NMDA receptors (NMDARs) has been shown to lead to neuronal injury and death in several disorders, including stroke, Alzheimer’s disease, ALS and Huntington’s disease16, NMDAR antagonists have failed to show a therapeutic benefit in the above-mentioned human neurological disorders.

Recently, my colleagues and I coined the term vasculo-neuronal-inflammatory triad195 to indicate that vascular damage, neuronal injury and/or neurodegeneration, and neuroinflammation comprise a common pathological triad that occurs in multiple neurological disorders. In line with this idea, it is conceivable that ‘multiple-target, multiple-action’ agents (that is, drugs that have more than one target and thus have more than one action) will have a better chance of controlling the complex disease mechanisms that mediate neurodegeneration than agents that have only one target (for example, neurons). According to the vasculo-neuronal-inflammatory triad model, in addition to neurons, brain endothelium, VSMCs, pericytes, astrocytes and activated microglia are all important therapeutic targets.

Here, I will briefly discuss a few therapeutic strategies based on the vasculo-neuronal-inflammatory triad model. VEGF and other angioneurins may have multiple targets, and thus multiple actions, in the CNS120. For example, preclinical studies have shown that treatment of SOD1G93A rats with intracerebroventricular VEGF196 or intramuscular administration of a VEGF-expressing lentiviral vector that is transported retrogradely to motor neurons in SOD1G93A mice197 reduced pathology and extended survival, probably by promoting angiogenesis and increasing the blood flow through the spinal cord as well as through direct neuronal protective effects of VEGF on motor neurons. On the basis of these and other studies, a phase I–II clinical trial has been initiated to evaluate the safety of intracerebroventricular infusion of VEGF in patients with ALS198. Treatment with angiogenin also slowed down disease progression in a mouse model of ALS199.

IGF1 delivery has been shown to promote amyloid-β vascular clearance and to improve learning and memory in a mouse model of Alzheimer’s disease200. Local intracerebral implantation of VEGF-secreting cells in a mouse model of Alzheimer’s disease has been shown to enhance vascular repair, reduce amyloid burden and improve learning and memory201. In contrast to VEGF, which can increase BBB permeability, TGFβ, hepatocyte growth factor and fibroblast growth factor 2 promote BBB integrity by upregulating the expression of endothelial junction proteins121 in a similar way to APC43. However, VEGF and most growth factors do not cross the BBB, so the development of delivery strategies such as Trojan horses is required for their systemic use25.

A recent experimental approach with APC provides an example of a neurovascular medicine that has been shown to favourably regulate multiple pathways in non-neuronal cells and neurons, resulting in vasculoprotection, stabilization of the BBB, neuroprotection and anti-inflammation in several acute and chronic models of the CNS disorders195 (Box 2).

The recognition of amyloid-β clearance pathways (Fig. 4), as discussed above, opens exciting new therapeutic opportunities for Alzheimer’s disease. Amyloid-β clearance pathways are promising therapeutic targets for the future development of neurovascular medicines because it has been shown both in animal models of Alzheimer’s disease1 and in patients with sporadic Alzheimer’s disease149 that faulty clearance from brain and across the BBB primarily determines amyloid-β retention in brain, causing the formation of neurotoxic amyloid-β oligomers56 and the promotion of brain and cerebrovascular amyloidosis3. The targeting of clearance mechanisms might also be beneficial in other diseases; for example, the clearance of extracellular mutant SOD1 in familial ALS, the prion protein in prion disorders and α-synuclein in Parkinson’s disease might all prove beneficial. However, the clearance mechanisms for these proteins in these diseases are not yet understood.

Conclusions and perspectives

Currently, no effective disease-modifying drugs are available to treat the major neurodegenerative disorders202, 203, 204. This fact leads to a question: are we close to solving the mystery of neurodegeneration? The probable answer is yes, because the field has recently begun to recognize that, first, damage to neuronal cells is not the sole contributor to disease initiation and progression, and that, second, correcting disease pathways in vascular and glial cells may offer an array of new approaches to control neuronal degeneration that do not involve targeting neurons directly. These realizations constitute an important shift in paradigm that should bring us closer to a cure for neurodegenerative diseases. Here, I raise some issues concerning the existing models of neurodegeneration and the new neurovascular paradigm.

The discovery of genetic abnormalities and associations by linkage analysis or DNA sequencing has broadened our understanding of neurodegeneration204. However, insufficient effort has been made to link genetic findings with disease biology. Another concern for neurodegenerative research is how we should interpret findings from animal models202. Genetically engineered models of human neurodegenerative disorders in Drosophila melanogaster andCaenorhabditis elegans have been useful for dissecting basic disease mechanisms and screening compounds. However, in addition to having much simpler nervous systems, insects and avascular species do not have cerebrovascular and immune systems that are comparable to humans and, therefore, are unlikely to replicate the complex disease pathology that is found in people.

For most neurodegenerative disorders, early steps in the disease processes remain unclear, and biomarkers for these stages have yet to be identified. Thus, it is difficult to predict whether mammalian models expressing human genes and proteins that we know are implicated in the intermediate or later stages of disease pathophysiology, such as amyloid-β or tau in Alzheimer’s disease7, 181, will help us to discover therapies for the early stages of disease and for disease prevention, because the exact role of these pathological accumulations during disease onset remains uncertain. According to the two-hit vascular hypothesis of Alzheimer’s disease, incorporating vascular factors that are associated with Alzheimer’s disease into current models of this disease may more faithfully replicate dementia events in humans. Alternatively, by focusing on the comorbidities and the initial cellular and molecular mechanisms underlying early neurovascular dysfunction that are associated with Alzheimer’s disease, new models of dementia and neurodegeneration may be developed that do not require the genetic manipulation of amyloid-β or tau expression.

The proposed neurovascular triad model of neurodegenerative diseases challenges the traditional neurocentric view of such disorders. At the same time, this model raises a set of new important issues that require further study. For example, the molecular basis of the neurovascular link with neurodegenerative disorders is poorly understood, in terms of the adhesion molecules that keep the physical association of various cell types together, the molecular crosstalk between different cell types (including endothelial cells, pericytes and astrocytes) and how these cellular interactions influence neuronal activity. Addressing these issues promises to create new opportunities not only to better understand the molecular basis of the neurovascular link with neurodegeneration but also to develop novel neurovascular-based medicines.

The construction of a human BBB molecular atlas will be an important step towards understanding the role of the BBB and neurovascular interactions in health and disease. Achievement of this goal will require identifying new BBB transporters by using genomic and proteomic tools, and by cloning some of the transporters that are already known. Better knowledge of transporters at the human BBB will help us to better understand their potential as therapeutic targets for disease.

Development of higher-resolution imaging methods to evaluate BBB integrity, key transporters’ functions and CBF responses in the microregions of interest (for example, in the entorhinal region of the hippocampus) will help us to understand how BBB dysfunction correlates with cognitive outcomes and neurodegenerative processes in MCI, Alzheimer’s disease and related disorders.

The question persists: are we missing important therapeutic targets by studying the nervous system in isolation from the influence of the vascular system? The probable answer is yes. However, the current exciting and novel research that is based on the neurovascular model has already begun to change the way that we think about neurodegeneration, and will continue to provide further insights in the future, leading to the development of new neurovascular therapies.

References

  1. Zlokovic, B. V. The blood–brain barrier in health and chronic neurodegenerative disorders. Neuron 57, 178–201 (2008).

  2. Moskowitz, M. A., Lo, E. H. & Iadecola, C. The science of stroke: mechanisms in search of treatments. Neuron 67, 181–198 (2010).
    A comprehensive review describing mechanisms of ischaemic injury to the neurovascular unit.

  3. Zlokovic, B. V. Neurovascular mechanisms of Alzheimer’s neurodegeneration. Trends Neurosci. 28, 202–208 (2005).

  4. Brown, W. R. & Thore, C. R. Review: cerebral microvascular pathology in ageing and neurodegeneration. Neuropathol. Appl. Neurobiol. 37, 56–74 (2011).

  5. Wu, Z. et al. Role of the MEOX2 homeobox gene in neurovascular dysfunction in Alzheimer disease. Nature Med. 11, 959–965 (2005).
    A study demonstrating that low expression of MEOX2 in brain endothelium leads to aberrant angiogenesis and vascular regression in Alzheimer’s disease.

  6. Paul, J., Strickland, S. & Melchor, J. P. Fibrin deposition accelerates neurovascular damage and neuroinflammation in mouse models of Alzheimer’s disease. J. Exp. Med. 204, 1999–2008 (2007).
    A study showing BBB breakdown in models of Alzheimer’s disease.

  7. Zipser, B. D. et al. Microvascular injury and blood–brain barrier leakage in Alzheimer’s disease. Neurobiol. Aging 28, 977–986 (2007).

  8. Zhong, Z. et al. ALS-causing SOD1 mutants generate vascular changes prior to motor neuron degeneration. Nature Neurosci. 11, 420–422 (2008).
    A study demonstrating that BSCB defects precede motor neuron degeneration in mice that develop an ALS-like disease.

  9. Kalaria, R. N. Vascular basis for brain degeneration: faltering controls and risk factors for dementia. Nutr. Rev. 68, S74–S87 (2010).

  10. Knopman, D. S. & Roberts, R. Vascular risk factors: imaging and neuropathologic correlates. J. Alzheimers Dis. 20, 699–709 (2010).

  11. Miyazaki, K. et al. Disruption of neurovascular unit prior to motor neuron degeneration in amyotrophic lateral sclerosis. J. Neurosci. Res. 89, 718–728 (2011).

  12. Neuwelt, E. A. et al. Engaging neuroscience to advance translational research in brain barrier biology. Nature Rev. Neurosci. 12, 169–182 (2011).

  13. Guo, S. & Lo, E. H. Dysfunctional cell–cell signaling in the neurovascular unit as a paradigm for central nervous system disease.Stroke 40, S4–S7 (2009).

  14. Redzic, Z. Molecular biology of the blood–brain and the blood–cerebrospinal fluid barriers: similarities and differences. Fluids Barriers CNS 8, 3 (2011).

  15. O’Kane, R. L., Martinez-Lopez, I., DeJoseph, M. R., Vina, J. R. & Hawkins, R. A. Na+-dependent glutamate transporters (EAAT1, EAAT2, and EAAT3) of the blood–brain barrier. A mechanism for glutamate removal. J. Biol. Chem. 274, 31891–31895 (1999).

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Author affiliations

  1. Department of Physiology and Biophysics, and Center for Neurodegeneration and Regeneration at the Zilkha Neurogenetic Institute, University of Southern California, Keck School of Medicine, 1501 San Pablo Street, Los Angeles, California 90089, USA.
    Email: bzlokovi@usc.edu

 

Retromer in Alzheimer disease, Parkinson disease and other neurological disorders.

Scott A. Small and Gregory A. Petsko

Nature Reviews Neuroscience  2015; 16:126-132.   http://dx.doi.org:/10.1038/nrn3896

 

Retromer is a protein assembly that has a central role in endosomal trafficking, and retromer dysfunction has been linked to a growing number of neurological disorders. First linked to Alzheimer disease, retromer dysfunction causes a range of pathophysiological consequences that have been shown to contribute to the core pathological features of the disease. Genetic studies have established that retromer dysfunction is also pathogenically linked to Parkinson disease, although the biological mechanisms that mediate this link are only now being elucidated. Most recently, studies have shown that retromer is a tractable target in drug discovery for these and other disorders of the nervous system.

Yeast has proved to be an informative model organism in cell biology and has provided early insight into much of the molecular machinery that mediates the intracellular transport of proteins1,2. Indeed, the term ‘retromer’ was first introduced in a yeast study in 1998 (Ref. 3). In this study, retromer was referred to as a complex of proteins that was dedicated to transporting cargo in a retrograde direction, from the yeast endosome back to the Golgi.

By 2004, a handful of studies had identified the molecular4 and the functional5, 6 homologies of the mammalian retromer, and in 2005 retromer was linked to its first human disorder, Alzheimer disease (AD)7. At the time, the available evidence suggested that the mammalian retromer might match the simplicity of its yeast homologue. Since then, a dramatic and exponential rise in research focusing on retromer has led to more than 300 publications. These studies have revealed the complexity of the mammalian retromer and its functional diversity in endosomal transport, and have implicated retromer in a growing number of neurological disorders.

New evidence indicates that retromer is a ‘master conductor’ of endosomal sorting and trafficking8. Synaptic function heavily depends on endosomal trafficking, as it contributes to the presynaptic release of neurotransmitters and regulates receptor density in the postsynaptic membrane, a process that is crucial for neuronal plasticity9. Therefore, it is not surprising that a growing number of studies are showing that retromer has an important role in synaptic biology10, 11, 12, 13. These observations may account for why the nervous system seems particularly sensitive to genetic and other defects in retromer. In this Progress article, we briefly review the molecular organization and the functional role of retromer, before discussing studies that have linked retromer dysfunction to several neurological diseases — notably, AD and Parkinson disease (PD).

Function and organization

The endosome is considered a hub for intracellular transport. From the endosome, transmembrane proteins can be actively sorted and trafficked to various intracellular sites via distinct transport routes (Fig. 1a). Studies have shown that the mammalian retromer mediates two of the three transport routes out of endosomes. First, retromer is involved in the retrieval of cargos from endosomes and in their delivery, in a retrograde direction, to the trans-Golgi network (TGN)5,6. Retrograde transport has many cellular functions but, as we describe, it is particularly important for the normal delivery of hydrolases and proteases to the endosomal–lysosomal system. The second transport route in which retromer functions is the recycling of cargos from endosomes back to the cell surface14, 15 (Fig. 1a). It is this transport route that is particularly important for neurons, as it mediates the normal delivery of glutamate and other receptors to the plasma membrane during synaptic remodelling and plasticity10, 11, 12, 13.

Figure 1: Retromer’s endosomal transport function and molecular organization.
Retromer's endosomal transport function and molecular organization.

a | Retromer mediates two transport routes out of endosomes via tubules that extend out of endosomal membranes. The first is the retrograde pathway in which cargo is retrieved from the endosome and trafficked to the trans-Golgi network (TGN). The second is the recycling pathway in which cargo is trafficked back from the endosome to the cell surface. The degradation pathway, which is not mediated by retromer, involves the trafficking of cargo from endosomes to lysosomes for degradation. b | The retromer assembly of proteins can be organized into distinct functional modules, all of which work together as part of retromer’s transport role. The ‘cargo-recognition core’ is the central module of the retromer assembly and comprises a trimer of proteins, in which vacuolar protein sorting-associated protein 26 (VPS26) and VPS29 bind VPS35. The ‘tubulation’ module includes protein complexes that bind the cargo-recognition core and aid in the formation and stabilization of tubules that extend out of endosomes, directing the transport of cargos towards their final destinations. The ‘membrane-recruiting’ proteins recruit the cargo-recognition core to the endosomal membrane. The WAS protein family homologue (WASH) complex of proteins also binds the cargo-recognition core and is involved in endosomal ‘actin remodelling’ to form actin patches, which are important for directing cargos towards retromer’s transport pathways. Retromer cargos includes a range of receptors — which bind the cargo-recognition core — and their ligands. PtdIns3P, phosphatidylinositol-3-phosphate.

As well as extending the endosomal transport routes, recent studies have considerably expanded the number of molecular constituents and what is known about the functional organization of the mammalian retromer. Following this expansion in knowledge of the molecular diversity and organizational complexity, retromer might be best described as a multimodular protein assembly. The protein or group of proteins that make up each module can vary, but each module is defined by its distinct function, and the modules work in unison in support of retromer’s transport role.

Two modules are considered central to the retromer assembly. First and foremost is a trimeric complex that functions as a ‘cargo-recognition core’, which selects and binds to the transmembrane proteins that need to be transported and that reside in endosomal membranes5, 6. This trimeric core comprises vacuolar protein sorting-associated protein 26 (VPS26), VPS29 and VPS35; VPS35 functions as the core’s backbone to which the other two proteins bind16. VPS26 is the only member of the core that has been found to have two paralogues, VPS26a and VPS26b17,18, and studies suggest that VPS26b might be differentially expressed in the brain19, 20. Some studies suggest that VPS26a and VPS26b are functionally redundant21, whereas others suggest that they might form distinct cargo-recognition cores20, 22.

The second central module of the retromer assembly is the ‘tubulation’ module, which is made up of proteins that work together in the formation and the stabilization of tubules that extend out of endosomes and that direct the transport of cargo towards its final destination (Fig. 1b). The proteins in this module, which directly binds the cargo-recognition core, are members of the subgroup of the sorting nexin (SNX) family that are characterized by the inclusion of a carboxy-terminal BIN–amphiphysin–RVS (BAR) domain23. These members include SNX1, SNX2, SNX5 and SNX6 (Refs 24,25). As part of the tubulation module, these SNX-BAR proteins exist in different dimeric combinations, but typically SNX1 interacts with SNX5 or SNX6, and SNX2 interacts with SNX5 or SNX6 (Refs 26,27). The EPS15-homology domain 1 (EHD1) protein can be included in this module, as it is involved in stabilizing the tubules formed by the SNX-BAR proteins28.

A third module of the retromer assembly functions to recruit the cargo-recognition core to endosomal membranes and to stabilize the core once it is there (Fig. 1b). Proteins that are part of this ‘membrane-recruiting’ module include SNX3 (Ref. 29), the RAS-related protein RAB7A30, 31,32 and TBC1 domain family member 5 (TBC1D5), which is a member of the TRE2–BUB2–CDC16 (TBC) family of RAB GTPase-activating proteins (GAPs)28. In addition, the lipid phosphatidylinositol-3-phosphate (PtdIns3P), which is found on endosomal membranes, contributes to recruiting most of the retromer-related SNXs through their phox homology domains33. Interestingly, another SNX with a phox homology domain, SNX27, was recently linked to retromer and its function15, 34. SNX27 functions as an adaptor for binding to PDZ ligand-containing cargos that are destined for transport to the cell surface via the recycling pathway. Thus, according to the functional organization of the retromer assembly, SNX27 belongs to the module that engages in cargo recognition and selection.

Recent studies have identified a fourth module of the retromer assembly. The five proteins in this module — WAS protein family homologue 1 (WASH1), FAM21, strumpellin, coiled-coil domain-containing protein 53 (CCDC53) and KIAA1033 (also known as WASH complex subunit 7) — form the WASH complex and function as an ‘actin-remodelling’ module28, 35, 36 (Fig. 1b). Specifically, the WASH complex functions in the rapid polymerization of actin to create patches of actin filaments on endosomal membranes. The complex is recruited to endosomal membranes by binding VPS35 (Ref. 28), and together they divert cargo towards retromer transport pathways and away from the degradation pathway.

The cargos that are transported by retromer include the receptors that directly bind the cargo-recognition core and the ligands of these receptors that are co-transported with the receptors. The receptors that are transported by retromer that have so far been identified to be the most relevant to neurological diseases are the family of VPS10 domain-containing receptors (including sortilin-related receptor 1 (SORL1; also known as SORLA), sortilin, and SORCS1, SORCS2 and SORCS3)7; the cation-independent mannose-6-phosphate receptor (CIM6PR)6, 5; glutamate receptors10; and phagocytic receptors that mediate the clearing function of microglia37. The most disease-relevant ligand to be identified that is trafficked as retromer cargo is the β-amyloid precursor protein (APP)7, 38, 39, 40, 41, which binds SORL1 and perhaps other VPS10 domain-containing receptors42 at the endosomal membrane.

Retromer dysfunction

Guided by retromer’s established function, and on the basis of empirical evidence, there are three well-defined pathophysiological consequences of retromer dysfunction that have proven to be relevant to AD and nervous system disorders. First, retromer dysfunction can cause cargos that typically transit rapidly through the endosome to reside in the endosome for longer than normal durations, such that they can be pathogenically processed into neurotoxic fragments (for example, APP, when stalled in the endosome, is more likely to be processed into amyloid-β, which is implicated in AD43 (Fig. 2a)). Second, by reducing endosomal outflow via impairment of the recycling pathway, retromer dysfunction can lead to a reduction in the number of cell surface receptors that are important for brain health (for example, microglia phagocytic receptors37 (Fig. 2b)).

Figure 2: The pathophysiology of retromer dysfunction.
The pathophysiology of retromer dysfunction.

Retromer dysfunction has three established pathophysiological consequences. In the examples shown, the left graphic represents a cell with normal retromer function and the right graphic represents a cell with a deficit in retromer function. a | Retromer dysfunction causes increased levels of cargo to reside in endosomes. For example, in primary neurons, retromer transports the β-amyloid precursor protein (APP) out of endosomes. Accordingly, retromer dysfunction increases APP levels in endosomes, leading to accelerated APP processing, resulting in an accumulation of neurotoxic fragments of APP (namely, β-carboxy-terminal fragment (βCTF) and amyloid-β) that are pathogenic in Alzheimer disease. b | Retromer dysfunction causes decreased cargo levels at the cell surface. For example, in microglia, retromer mediates the transport of phagocytic receptors to the cell surface and retromer dysfunction results in a decrease in the delivery of these receptors. Studies suggest that this cellular phenotype might have a pathogenic role in Alzheimer disease. c | Retromer dysfunction causes decreased delivery of proteases to the endosome. Retromer is required for the normal retrograde transport of the cation-independent mannose-6-phosphate receptor (CIM6PR) from the endosome back to the trans-Golgi network (TGN). It is in the TGN that this receptor binds cathepsin D and other proteases, and transports them to the endosome, to support the normal function of the endosomal–lysosomal system. By impairing the retrograde transport of the receptor, retromer dysfunction ultimately leads to reduced delivery of cathepsin D to this system. Cathepsin D deficiency has been shown to disrupt the endosomal–lysosomal system and to trigger tau pathology either within endosomes or secondarily in the cytosol.

The third consequence (Fig. 2c) is a result of the established role that retromer has in the retrograde transport of receptors, such as CIM6PR5, 6 or sortilin44, after these receptors transport proteases from the TGN to the endosome. Once at the endosome, the proteases disengage from the receptors, are released into endosomes and migrate to lysosomes. These proteases function in the endosomal–lysosomal system to degrade proteins, protein oligomers and aggregates45. Retromer functions to transfer the ‘naked’ receptor from the endosome back to the TGN via the retrograde pathway5, 6, allowing the receptors to continue in additional rounds of protease delivery. Accordingly, by reducing the normal retrograde transport of these receptors, retromer dysfunction has been shown to reduce the proper delivery of proteases to the endosomal–lysosomal system5,6, which, as discussed below, is a pathophysiological state linked to several brain disorders.

Although requiring further validation, recent studies suggest that retromer dysfunction might be involved in two other mechanisms that have a role in neurological disease. One study suggested that retromer might be involved in trafficking the transmembrane protein autophagy-related protein 9A (ATG9A) to recycling endosomes, from where it can then be trafficked to autophagosome precursors — a trafficking step that is crucial in the formation and the function of autophagosomes46. Autophagy is an important mechanism by which neurons clear neurotoxic aggregates that accumulate in numerous neurodegenerative diseases47. A second study has suggested that retromer dysfunction might enhance the seeding and the cell-to-cell spread of intracellular neurotoxic aggregates48, which have emerged as novel pathophysiological mechanisms that are relevant to AD49, PD50 and other neurodegenerative diseases.

Alzheimer disease

Retromer was first implicated in AD in a molecular profiling study that relied on functional imaging observations in patients and animal models to guide its molecular analysis7. Collectively, neuroimaging studies confirmed that the entorhinal cortex is the region of the hippocampal circuit that is affected first in AD, even in preclinical stages, and suggested that this effect was independent of ageing (as reviewed in Ref. 51). At the same time, neuroimaging studies identified a neighbouring hippocampal region, the dentate gyrus, that is relatively unaffected in AD52. Guided by this information, a study was carried out in which the two regions of the brain were harvested post mortem from patients with AD and from healthy individuals, intentionally covering a broad range of ages. A statistical analysis was applied to the determined molecular profiles of the regions that was designed to address the following question: among the thousands of profiled molecules, which are the ones that are differentially affected in the entorhinal cortex versus the dentate gyrus, in patients versus controls, but that are not affected by age? The final results led to the determination that the brains of patients with AD are deficient in two core retromer proteins — VPS26 and VPS35 (Ref. 7).

Little was known about the receptors of the neuronal retromer, so to understand how retromer deficiency might be mechanistically linked to AD, an analysis was carried out on the molecular data set that looked for transmembrane molecules for which expression levels correlated with VPS35 expression. The top ‘hit’ was the transcript encoding the transmembrane protein SORL1 (Ref. 43). As SORL1 belongs to the family of VPS10-containing receptors and as VPS10 is the main retromer receptor in yeast3, it was postulated that SORL1 and the family of other VPS10-containing proteins (sortillin, SORCS1, SORCS2 and SORCS3) might function as retromer receptors in neurons7. In addition, SORL1 had recently been reported to bind APP53, so if SORL1 was assumed to be a receptor that is trafficked by retromer, then APP might be the cargo that is co-trafficked by retromer. This led to a model in which retromer traffics APP out of endosomes7, which are the organelles in which APP is most likely to be cleaved by βAPP-cleaving enzyme 1 (BACE1; also known as β-secretase 1)43; this is the initial enzymatic step in the pathogenic processing of APP.

Subsequent studies were required to further establish the pathogenic link between retromer and AD, and to test the proposed model. The pathogenic link was further supported by human genetic studies. First, a genetic study investigating the association between AD, the genes encoding the components of the retromer cargo-recognition core and the family of VPS10-containing receptors found that variants of SORL1 increase the risk of developing AD38. This finding was confirmed by numerous studies, including a recent large-scale AD genome-wide association study54. Other genetic studies identified AD-associated variants in genes encoding proteins that are linked to nearly all modules of the retromer assembly55, including genes encoding proteins of the retromer tubulation module (SNX1), genes encoding proteins of the retromer membrane-recruiting module (SNX3 and RAB7A) and genes encoding proteins of the retromer actin-remodelling module (KIAA1033). In addition, nearly all of the genes encoding the family of VPS10-containing retromer receptors have been found to have variants that associate with AD56. Finally, a study found that brain regions that are differentially affected in AD are deficient in PtdIns3P, which is the phospholipid required for recruiting many sorting nexins to endosomal membranes57. Thus, together with the observation that the brains of patients with AD are deficient in VPS26a and VPS35 (Refs 7,37), all modules in the retromer assembly are implicated in AD.

Studies in mice39, 58, 59, flies39 and cells in culture34, 40, 41, 60, 61 have investigated how retromer dysfunction leads to the pathogenic processing of APP. Although rare discrepancies have been observed among these studies62, when viewed in total, the most consistent findings are that retromer dysfunction causes increased pathogenic processing of APP by increasing the time that APP resides in endosomes. Moreover, these studies have confirmed that SORL1 and other VPS10-containing proteins function as APP receptors that mediate APP trafficking out of endosomes.

Retromer has unexpectedly been linked to microglial abnormalities37 — another core feature of AD — which, on the basis of recent genetic findings, seem to have an upstream role in disease pathogenesis54, 63. A recent study found that microglia harvested from the brains of individuals with AD are deficient in VPS35 and provided evidence suggesting that retromer’s recycling pathway regulates the normal delivery of various phagocytic receptors to the cell surface of microglia37, including the phagocytic receptor triggering receptor expressed on myeloid cells 2 (TREM2) (Fig. 2b). Mutations in TREM2 have been linked to AD63, and a recent study indicates that these mutations cause a reduction in its cell surface delivery and accelerate TREM2 degradation, which suggests that the mutations are linked to a recycling defect64. While they are located at the microglial cell surface, these phagocytic receptors function in the clearance of extracellular proteins and other molecules from the extracellular space65. Taken together, these recent studies suggest that defects in the retromer’s recycling pathway can, at least in part, account for the microglial defects observed in the disease.

The microtubule-associated protein tau is the key element of neurofibrillary tangles, which are the other hallmark histological features of AD. Although a firm link between retromer dysfunction and tau toxicity remains to be established, recent insight into tau biology suggests several plausible mechanisms that are worth considering. Tau is a cytosolic protein, but nonetheless, through mechanisms that are still undetermined, it is released into the extracellular space from where it gains access to neuronal endosomes via endocytosis66, 67. In fact, recent studies suggest that the pathogenic processing of tau is triggered after it is endocytosed into neurons and while it resides in endosomes67. Of note, it still remains unknown which specific tau processing step — its phosphorylation, cleavage or aggregation — is an obligate step towards tau-related neurotoxicity. Accordingly, if defects in microglia or in other phagocytic cells reduce their capacity to clear extracellular tau, this would accelerate tau endocytosis in neurons and its pathogenic processing.

A second possibility comes from the established role retromer has in the proper delivery of cathepsin D and other proteases to the endosomal–lysosomal system via CIM6PR or sortilin (Fig. 2c). Studies in sheep, mice and flies68 have shown that cathepsin D deficiency can enhance tau toxicity and that this is mediated by a defective endosomal–lysosomal system68. Whether this mechanism leads to abnormal processing of tau within endosomes or in the cytosol via caspase activation68 remains unclear. As discussed above, retromer dysfunction will lead to a decrease in the normal delivery of cathepsin D to the endosome and will result in endosomal–lysosomal system defects. Retromer dysfunction can therefore be considered as a functional phenocopy of cathepsin D deficiency, which suggests a plausible link between retromer dysfunction and tau toxicity. Nevertheless, although these recent insights establish plausibility and support further investigation into the link between retromer and tau toxicity, whether this link exists and how it may be mediated remain open and outstanding questions.

Parkinson disease

The pathogenic link between retromer and PD is singular and straightforward: exome sequencing has identified autosomal-dominant mutations in VPS35 that cause late-onset PD69, 70, one of a handful of genetic causes of late-onset disease. However, the precise mechanism by which these mutations cause the disease is less clear.

Among a group of recent studies, all46, 48, 71, 72, 73, 74, 75, 76 but one77 strongly suggest that these mutations cause a loss of retromer function. At the molecular level, the mutations do not seem to disrupt mutant VPS35 from interacting normally with VPS26 and VPS29, and from forming the cargo-recognition core. Rather, two studies suggest that the mutations have a restricted effect on the retromer assembly but reduce the ability of VPS35 to associate with the WASH complex46, 75. Studies disagree about the pathophysiological consequences of the mutations. Four studies suggest that the mutations affect the normal retrograde transport of CIM6PR71, 73, 75, 76 from the endosome back to the TGN (Fig. 2c). In this scenario, the normal delivery of cathepsin D to the endosomal–lysosomal system should be reduced and this has been empirically shown73. Cathepsin D has been shown to be the dominant endosomal–lysosomal protease for the normal processing of α-synuclein76, and mutations could therefore lead to abnormal α-synuclein processing and to the formation of α-synuclein aggregates, which are thought to have a key pathogenic role in PD.

A separate study suggested that the mutation might cause a mistrafficking of ATG9, and thereby, as discussed above, reduce the formation and the function of autophagosomes46. Autophagosomes have also been implicated as an intracellular site in which α-synuclein aggregates are cleared. Thus, although future studies are needed to resolve these discrepant findings (which may in fact not be mutually exclusive), these studies are generally in agreement that retromer defects will probably increase the neurotoxic levels of α-synuclein aggregates48.

Several studies in flies71, 74 and in rat neuronal cultures71 provide strong evidence that increasing retromer function by overexpressing VPS35 rescues the neurotoxic effects of the most common PD-causing mutations in leucine-rich repeat kinase 2 (LRRK2). Moreover, a separate study has shown that increasing retromer levels rescues the neurotoxic effect of α-synuclein aggregates in a mouse model48. These findings have immediate therapeutic implications for drugs that increase VPS35 and retromer function, as discussed in the next section, but they also offer mechanistic insight. LRRK2 mutations were found to phenocopy the transport defects caused either by theVPS35 mutations or by knocking down VPS35 (Ref. 71). Together, this and other studies78suggest that LRRK2 might have a role in retromer-dependent transport, but future studies are required to clarify this role.

Other neurological disorders

Besides AD and PD, in which a convergence of findings has established a strong pathogenic link, retromer is being implicated in an increasing number of other neurological disorders. Below, we briefly review three disorders for which the evidence of the involvement of retromer in their pathophysiology is currently the most compelling.

The first of these disorders is Down syndrome (DS), which is caused by an additional copy of chromosome 21. Given the hundreds of genes that are duplicated in DS, it has been difficult to identify which ones drive the intellectual impairments that characterize this condition. A recent elegant study provides strong evidence that a deficiency in the retromer cargo-selection protein SNX27 might be a primary driver for some of these impairments79. This study found that the brains of individuals with DS were deficient in SNX27 and that this deficiency may be caused by an extra copy of a microRNA (miRNA) encoded by human chromosome 21 (the miRNA is produced at elevated levels and thereby decreases SNX27 expression). Consistent with the known role of SNX27 in retromer function, decreased expression of this protein in mice disrupted glutamate receptor recycling in the hippocampus and led to dendritic dysfunction. Importantly, overexpression of SNX27 rescued cognitive and other defects in animal models79, which not only strengthens the causal link between retromer dysfunction and cognitive impairment in DS but also has important therapeutic implications.

Hereditary spastic paraplegia (HSP) is another disorder linked to retromer. HSP is caused by genetic mutations that affect upper motor neurons and is characterized by progressive lower limb spasticity and weakness. Although there are numerous mutations that cause HSP, most are unified by their effects on intracellular transport80. One HSP-associated gene in particular encodes strumpellin81, which is a member of the WASH complex.

The third disorder linked to retromer is neuronal ceroid lipofuscinosis (NCL). NCL is a young-onset neurodegenerative disorder that is part of a larger family of lysosomal storage diseases and is caused by mutations in one of ten identified genes — nine neuronal ceroid lipofuscinosis (CLN) genes and the gene encoding cathepsin D82. Besides cathepsin D, for which the link to retromer has been discussed above, CLN3 seems to function in the normal trafficking of CIM6PR83. However, the most direct link to retromer has been recently described for CLN5, which seems to function, at least in part, as a retromer membrane-recruiting protein84.

Retromer as a therapeutic target

As suggested by the first study implicating retromer in AD7, and in several subsequent studies71,85, increasing the levels of retromer’s cargo-recognition core enhances retromer’s transport function. Motivated by this observation and after a decade-long search86, we identified a novel class of ‘retromer pharmacological chaperones’ that can bind and stabilize retromer’s cargo-recognition core and increase retromer levels in neurons61.

Validating the motivating hypothesis, the chaperones were found to enhance retromer function, as shown by the increased transport of APP out of endosomes and a reduction in the accumulation of APP-derived neurotoxic fragments61. Although there are numerous other pharmacological approaches for enhancing retromer function, this success provides the proof-of-principle that retromer is a tractable therapeutic target.

As retromer functions in all cells, a general concern is whether enhancing its function will have toxic adverse effects. However, studies have found that in stark contrast to even mild retromer deficiencies, increasing retromer levels has no obvious negative consequences in yeast, neuronal cultures, flies or mice40, 48, 61, 71. This might make sense because unlike drugs that, for example, function as inhibitors, simply increasing the normal flow of transport through the endosome might not be cytotoxic.

If retromer drugs are safe and can effectively enhance retromer function in the nervous system — which are still outstanding issues — there are two general indications for considering their clinical application. One rests on the idea that these agents will only be efficacious in patients who have predetermined evidence of retromer dysfunction. The most immediate example is that of individuals with PD that is caused by LRRK2 mutations. As discussed above, several ‘preclinical’ studies in flies and neuronal cultures have already established that increasing retromer levels71, 74can reverse the neurotoxic effects of such mutations and, thus, if this approach is proven to be safe, LRRK2-linked PD might be an appropriate indication for clinical trials.

Alternatively, the pathophysiology of a disease might be such that retromer-enhancing drugs would be efficacious regardless of whether there is documented evidence of retromer dysfunction. AD illustrates this point. As reviewed above, current evidence suggests that retromer-enhancing drugs will, at the very least, decrease pathogenic processing of APP in neurons and enhance microglial function, even if there are no pre-existing defects in retromer.

More generally, histological studies comparing the entorhinal cortex of patients with sporadic AD to age-matched controls have documented that enlarged endosomes are a defining cellular abnormality in AD87, 88. Importantly, enlarged endosomes are uniformly observed in a broad range of patients with sporadic AD, which suggests that enlarged endosomes reflect an intracellular site at which molecular aetiologies converge87. In addition, because they are observed in early stages of the disease in regions of the brain without evidence of amyloid pathology87, enlarged endosomes are thought to be an upstream event. Mechanistically, the most likely cause of enlarged endosomes is either too much cargo flowing into endosomes — as occurs, for example, with apolipoprotein E4 (APOE4), which has been shown to accelerate endocytosis89, 90 — or too little cargo flowing out, as observed in retromer dysfunction40, 61 and related transport defects57. By any mechanism, retromer-enhancing drugs might correct this unifying cellular defect and might be expected to be beneficial regardless of the specific aetiology.

Conclusions

The fact that retromer defects, including those derived from bona fide genetic mutations, seem to differentially target the nervous system suggests that the nervous system is differentially dependent on retromer for its normal function. We think that this reflects the unique cellular properties of neurons and how synaptic biology heavily depends on endosomal transport and trafficking. Although plausible, future studies are required to confirm and to test the details of this hypothesis.

However, currently, it is the clinical rather than the basic neuroscience of retromer that is much better understood, with the established pathophysiological consequences of retromer dysfunction providing a mechanistic link to the disorders in which retromer has been implicated. Nevertheless, many questions remain. The two most interesting questions, which are in fact inversions of each other, relate to regional vulnerability in the nervous system. First, why does retromer dysfunction target specific neuronal populations? Second, how can retromer dysfunction cause diseases that target different regions of the nervous system? Recent evidence hints at answers to both questions, which must somehow be rooted in the functional and molecular diversity of retromer.

The type and the extent of retromer defects linked to different disorders might provide pathophysiological clues as well as reasons for differential vulnerability. As discussed, in AD there seem to be across-the-board defects in retromer, such that each module of the retromer assembly as well as multiple retromer cargos have been pathogenically implicated. By contrast, the profile of retromer defects in PD seems to be more circumscribed, involving selective disruption of the interaction between VPS35 and the WASH complex. These insights might agree with histological87, 88 and large-scale genetic studies54 that suggest that endosomal dysfunction is a unifying focal point in the cellular pathogenesis of AD. In contrast, genetics and other studies91suggest that the cellular pathobiology of PD is more distributed, implicating the endosome but other organelles as well, in particular the mitochondria.

Interestingly, studies suggest that the entorhinal cortex — a region that is differentially vulnerable to AD — has unique dendritic structure and function92, which are highly dependent on endosomal transport. We speculate that it is the unique synaptic biology of the entorhinal cortex that can account for why it might be particularly sensitive to defects in endosomal transport in general and retromer dysfunction in particular, and for why this region is the early site of disease. Future studies are required to investigate this hypothesis, as well as to understand why the substantia nigra or other regions that are differentially vulnerable to PD would be particularly sensitive to the more circumscribed defect in retromer.

Perhaps the most important observation for clinical neuroscience is the now well-established fact that increasing levels of retromer proteins enhances retromer function and has already proved capable of reversing defects associated with AD, PD and DS in either cell culture or in animal models. The relationships between protein levels and function are not always simple, but emerging pharmaceutical technologies that selectively and safely increase protein levels are now a tractable goal in drug discovery93. With the evidence mounting that retromer has a pathogenic role in two of the most common neurodegenerative diseases, we think that targeting retromer to increase its functional activity is an important goal that has strong therapeutic promise.

References

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……. 93

Affiliations   

Taub Institute for Research on Alzheimer’s Disease and the Ageing Brain, Departments of Neurology, Radiology, and Psychiatry, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA.

Scott A. Small

Helen and Robert Appel Alzheimer’s Disease Research Institute, Department of Neurology and Feil Family Brain and Mind Research Institute, Weill Cornell Medical College, New York, New York 10065, USA.

Gregory A. Petsko

 

See also:

Neurobiol Aging. 2011 Nov;32(11):2109.e1-14. doi: 10.1016/j.neurobiolaging.2011.05.025.
Altered intrinsic neuronal excitability and reduced Na+ currents in a mouse model of Alzheimer’s disease.
Brown JT, Chin J, Leiser SC, Pangalos MN, Randall AD.

Trends Neurosci. 2013 Jun;36(6):325-35. doi: 10.1016/j.tins.2013.03.002.
Why size matters – balancing mitochondrial dynamics in Alzheimer’s disease.
DuBoff B, Feany M, Götz J.

Neuron. 2014 Dec 3;84(5):1023-33. doi: 10.1016/j.neuron.2014.10.024.
Dendritic structural degeneration is functionally linked to cellular hyperexcitability in a mouse model of Alzheimer’s disease.
Šišková Z, Justus D, Kaneko H, Friedrichs D, Henneberg N, Beutel T, Pitsch J, Schoch S, Becker A, von der Kammer H, Remy S.

 

 

Video: How can we tease out the role of other toxic insults in AD pathogenesis?

https://neuroalzheimerscommunity.nature.com/videos/3896-other-toxic-insults/download.mp4

 

 

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Reinforced disordered cell expression

Posted in Alzheimer's Disease, Biochemical pathways, Biological Networks, Cell Biology, Signaling & Cell Circuits, Cerebrovascular and Neurodegenerative Diseases, Chemical Biology and its relations to Metabolic Disease, Curation, Developmental biology, Diabetes Mellitus, Disease Biology, Etiology, Gene Regulation, Human aging, Metabolism, Metabolomics, Neurodegenerative Diseases, Neuroscience, Proteomics, S-nitrosylation, Signaling, Signaling & Cell Circuits, tagged , , , on January 9, 2016| Leave a Comment »

Reinforced disordered cell expression

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Diabetes, Alzheimer’s Share Molecular Pathways, Part of Same Vicious Cycle

http://www.genengnews.com/gen-news-highlights/diabetes-alzheimer-s-share-molecular-pathways-part-of-same-vicious-cycle/81252206/

http://www.genengnews.com/Media/images/GENHighlight/thumb_Jan8_2016_Fotolia_30836005_JigsawPuzzleBrainAndHead1904910113.jpg

A molecular-level link has been found that helps explain the poorly understood association between diabetes and Alzheimer’s disease. Both disorders can drive and be driven by the same pathological process, the disruption of a particular kind of post-translational modification called S-nitrosylation. Thus, the disorders can reinforce each other. [© freshidea/Fotolia]

 

Though they appear to be distinct, diabetes and Alzheimer’s disease have much in common at the molecular level. In fact, recent findings indicate that either disease can worsen the other by disrupting the same chemical process—S-nitrosylation, a form of post-translational modification that is necessary for the proper functioning of multiple enzymes.

S-nitrosylation, it turns out, can be disrupted by excess sugar or β-amyloid protein, either of which can wreak havoc by increasing the levels of nitric oxide and other free radical species. Once S-nitrosylation is disturbed and poorly functioning enzymes are produced, the downstream effects include abnormal increases in both insulin and β-amyloid protein.

Thus, diabetes and Alzheimer’s can drive, and be driven by, the same vicious cycle. Furthermore, either can contribute to the other’s progress. These results emerged from a study completed by researchers based at the Sanford Burnham Prebys Medical Discovery Institute and the Scintillon Institute. The research team was led by Stuart A. Lipton, M.D., Ph.D., a physician-scientist affiliated with both institutions.

“This work points to a new common pathway to attack both type 2 diabetes, along with its harbinger, metabolic syndrome, and Alzheimer’s disease,” stated Dr. Lipton.

The researchers published their work January 8 in the journal Nature Communications in an article entitled, “Elevated glucose and oligomeric β-amyloid disrupt synapses via a common pathway of aberrant protein S-nitrosylation.” This article describes how the scientists used a so-called “disease-in-a-dish” model to discover molecular pathways that are in common in both diabetes and Alzheimer’s.

Specifically, the scientists genetically reprogrammed the skin of human patients to make induced pluripotent stem cells, which were then used to derive nerve cells. They also used mouse models of each disease to analyze the combined effects of high blood sugar and β-amyloid protein in living animals.

“[We] report in human and rodent tissues that elevated glucose, as found in [metabolic syndrome and type 2 diabetes] and oligomeric β-amyloid (Aβ) peptide, thought to be a key mediator of [Alzheimer’s disease], coordinately increase neuronal Ca2+ and nitric oxide (NO) in an NMDA receptor-dependent manner,” wrote the authors of the Nature Communications article. “The increase in NO results in S-nitrosylation of insulin-degrading enzyme (IDE) and dynamin-related protein 1 (Drp1), thus inhibiting insulin and Aβ catabolism as well as hyperactivating mitochondrial fission machinery.”

The scientists also found that the changes in enzyme activity led to damage of synapses, the region where nerve cells communicate with one another in the brain. The combination of high sugar and β-amyloid protein caused the greatest loss of synapses. Since loss of synapses correlates with cognitive decline in Alzheimer’s, high sugar and β-amyloid coordinately contribute to memory loss.

“The NMDA receptor antagonist memantine attenuates these effects,” the authors continued. “Our studies show that redox-mediated posttranslational modification of brain proteins link Aβ and hyperglyaemia to cognitive dysfunction in [metabolic syndrome/type 2 diabetes] and [Alzheimer’s disease].”

“[Our work] means that we now know these diseases are related on a molecular basis, and hence, they can be treated with new drugs on a common basis,” stated Dr. Ambasudhan, a senior author of the study and an assistant professor at Scintillon.

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3-D videogames boost memory

Posted in Behavior, Cell Biology, Signaling & Cell Circuits, Cerebrovascular and Neurodegenerative Diseases, Clinical & Translational, Cognition, Curation, Human aging, tagged , , , on December 14, 2015| Leave a Comment »

3-D videogames boost memory

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

 

Playing 3-D video games can boost memory formation

http://www.kurzweilai.net/playing-3-d-video-games-can-boost-memory-formation

Video games used in the experiment : screenshot of 2-D Angry Birds (left) and Super Mario 3D World (right) (credit: Gregory D. Clemenson and Craig E.L. Stark/The Journal of Neuroscience)

http://www.kurzweilai.net/images/2D-vs-3D-video-games.jpg

 

Playing three-dimensional video games can boost the formation of memories, especially for people who lose memory as they age or suffer from dementia, according to University of California, Irvine (UCI) neurobiologists.

Craig Stark and Dane Clemenson of UCI’s Center for the Neurobiology of Learning & Memory recruited non-gamer college students to play either a video game with a passive, two-dimensional environment (“Angry Birds”) or one with an intricate, 3-D setting (“Super Mario 3D World”) for 30 minutes per day over two weeks.

Before and after the two-week period, the students took memory tests that engaged the brain’s hippocampus, the region associated with complex learning and memory. They were given a series of pictures of everyday objects to study. Then they were shown images of the same objects, new ones, and others that differed slightly from the original items and asked to categorize them.

Students playing the 3-D video game improved their scores on the memory test by about 12 percent, the same amount it normally decreases between the ages of 45 and 70, while the 2-D gamers did not improve.

 

https://youtu.be/t1YfgMVhhdA

UC Irvine | 3D Video Games and Memory – UC Irvine

 

Role of the hippocampus

Recognition of the slightly altered images requires the hippocampus, Stark said, and his earlier research had demonstrated that the ability to do this clearly declines with age. This is a large part of why it’s so difficult to learn new names or remember where you put your keys as you get older.

In previous studies on rodents, postdoctoral scholar Clemenson and others showed that exploring the environment resulted in the growth of new neurons that became entrenched in the hippocampus’ memory circuit and increased neuronal signaling networks. Stark noted some commonalities between the 3-D game the humans played and the environment the rodents explored — qualities lacking in the 2-D game. “First, the 3-D games have … a lot more spatial information in there to explore. Second, they’re much more complex, with a lot more information to learn,” Stark noted.

Stark added that it’s unclear whether the overall amount of information and complexity in the 3-D game or the spatial relationships and exploration is stimulating the hippocampus. “This is one question we’re following up on,” he said.

Myths of “brain training”

“Results from this study add to the existing literature that playing video games may provide meaningful stimulation to the brain. However, it is important to be cautious when generalizing these results to other instances. Recently, 70 neuroscientists from universities and institutions around the world published a letter discussing the myths of “brain training” (Max Planck Institute for Human Development/Stanford Center on Longevity, 2014. A consensus on the brain training industry from the scientific community. Stanford, CA: Stanford Center on Longevity).

“In contrast to typical brain training, typical video games are not created with specific cognitive processes in mind but rather designed to captivate and immerse the user into charactersand adventure. Rather than isolate single brain processes, modern video games can naturally draw on or require many cognitive processes, including visual, spatial, emotional, motivational, attentional, critical thinking, problem solving, and working memory. It’s quite possible that by explicitly avoiding a narrow focus on a single … cognitive domain and by more closely paralleling natural experience, immersive video games may be better suited to provide enriching experiences that translate into functional gains.”

— Gregory D. Clemenson and Craig E.L. Stark. Virtual Environmental Enrichment through Video Games Improves Hippocampal-Associated Memory. The Journal of Neuroscience.

 

The next step is to determine if environmental enrichment — either through 3-D video games or real-world exploration experiences — can reverse the hippocampal-dependent cognitive deficits present in older populations.

“Can we use this video game approach to help improve hippocampus functioning?” Stark asked. “It’s often suggested that an active, engaged lifestyle can be a real factor in stemming cognitive aging. While we can’t all travel the world on vacation, we can do many other things to keep us cognitively engaged and active. Video games may be a nice, viable route.”

The research is described in a paper published today (Dec. 9) in The Journal of Neuroscience and is funded by a $300,000 Dana Foundation grant.

references:

 

Virtual Environmental Enrichment through Video Games Improves Hippocampal-Associated Memory

Gregory D. Clemenson and Craig E.L. Stark
The Journal of Neuroscience, 9 Dec 2015, 35(49):16116-16125;      http://dx.doi.org:/10.1523/JNEUROSCI.2580-15.2015

 

The positive effects of environmental enrichment and their neural bases have been studied extensively in the rodent (van Praag et al., 2000). For example, simply modifying an animal’s living environment to promote sensory stimulation can lead to (but is not limited to) enhancements in hippocampal cognition and neuroplasticity and can alleviate hippocampal cognitive deficits associated with neurodegenerative diseases and aging. We are interested in whether these manipulations that successfully enhance cognition (or mitigate cognitive decline) have similar influences on humans. Although there are many “enriching” aspects to daily life, we are constantly adapting to new experiences and situations within our own environment on a daily basis. Here, we hypothesize that the exploration of the vast and visually stimulating virtual environments within video games is a human correlate of environmental enrichment. We show that video gamers who specifically favor complex 3D video games performed better on a demanding recognition memory task that assesses participants’ ability to discriminate highly similar lure items from repeated items. In addition, after 2 weeks of training on the 3D video game Super Mario 3D World, naive video gamers showed improved mnemonic discrimination ability and improvements on a virtual water maze task. Two control conditions (passive and training in a 2D game, Angry Birds), showed no such improvements. Furthermore, individual performance in both hippocampal-associated behaviors correlated with performance in Super Mario but not Angry Birds, suggesting that how individuals explored the virtual environment may influence hippocampal behavior.

SIGNIFICANCE STATEMENT The hippocampus has long been associated with episodic memory and is commonly thought to rely on neuroplasticity to adapt to the ever-changing environment. In animals, it is well understood that exposing animals to a more stimulating environment, known as environmental enrichment, can stimulate neuroplasticity and improve hippocampal function and performance on hippocampally mediated memory tasks. Here, we suggest that the exploration of vast and visually stimulating environments within modern-day video games can act as a human correlate of environmental enrichment. Training naive video gamers in a rich 3D, but not 2D, video game, resulted in a significant improvement in hippocampus-associated cognition using several behavioral measures. Our results suggest that modern day video games may provide meaningful stimulation to the human hippocampus.

 

I enjoyed reading the Stanford “consensus on brain training” letter.
http://longevity3.stanford.edu/blog/2014/10/15/the-consensus-on-the-brain-training-industry-from-the-scientific-community-2/

One quote stood out right away about “brain games”: “… In fact, the notion that performance on a single task cannot stand in for an entire ability is a cornerstone of scientific psychology …”

But playing a challenging 3-D video game or exploring a virtual reality like Second Life is far more “educational” and experiential, involving all sorts of cognitive (and, for that matter, psycho-motor and affective) skill domains. Good for the brain and often good for the spirit too.

 

A Consensus on the Brain Training Industry from the Scientific Community

http://longevity3.stanford.edu/blog/2014/10/15/the-consensus-on-the-brain-training-industry-from-the-scientific-community-2/

As the baby boomers enter their golden years with mounting concerns about the potential loss of cognitive abilities, markets are responding with products promising to allay anxieties about potential decline. Computer-based cognitive-training software –popularly known as brain games– claim a growing share of the marketplace. The promotion of these products reassures and entices a worried public.

Consumers are told that playing brain games will make them smarter, more alert, and able to learn faster and better. In other words, the promise is that if you adhere to a prescribed regimen of cognitive exercise, you will reduce cognitive slowing and forgetfulness, and will fundamentally improve your mind and brain.

brain training

http://longevity3.stanford.edu/wp-content/uploads/2014/10/iStock_000018505398XSmall-347×320-copy-300×276.jpg

 

It is customary for advertising to highlight the benefits and overstate potential advantages of their products. In the brain-game market, advertisements also reassure consumers that claims and promises are based on solid scientific evidence, as the games are “designed by neuroscientists” at top universities and research centers. Some companies present lists of credentialed scientific consultants and keep registries of scientific studies pertinent to cognitive training. Often, however, the cited research is only tangentially related to the scientific claims of the company, and to the games they sell. In addition, even published peer-reviewed studies merit critical evaluation. A prudent approach calls for integrating findings over a body of research rather than relying on single studies that often include only a small number of participants.

The Stanford Center on Longevity and the Berlin Max Planck Institute for Human Development gathered many of the world’s leading cognitive psychologists and neuroscientists –people who have dedicated their careers to studying the aging mind and brain– to share their views about brain games and offer a consensus report to the public. What do expert scientists think about these claims and promises? Do they have specific recommendations for effective ways to boost cognition in healthy, older adults? Are there merits to the claimed benefits of the brain games and if so, do older adults benefit from brain-game learning in the same ways younger people do? How large are the gains associated with computer-based cognitive exercises? Are the gains restricted to specific skills or does general cognitive aptitude improve? How does playing games compare with other proposed means of mitigating age-related declines, such as physical activity and exercise, meditation, or social engagement?

The search for effective means of mitigating or postponing age-related cognitive declines has taught most of us to recognize the enormous complexity of the subject matter. Like many challenging scientific topics, this is a devil of many details. The consensus of the group is that claims promoting brain games are frequently exaggerated and at times misleading. Cognitive training produces statistically significant improvement in practiced skills that sometimes extends to improvement on other cognitive tasks administered in the lab. In some studies, such gains endure, while other reports document dissipation over time. In commercial promotion, these small, narrow, and fleeting advances are often billed as general and lasting improvements of mind and brain. The aggressive advertising entices consumers to spend money on products and to take up new behaviors, such as gaming, based on these exaggerated claims. As frequently happens, initial findings, based on small samples, generate understandable excitement by suggesting that some brain games may enhance specific aspects of behavior and even alter related brain structures and functions. However, as the findings accumulate, compelling evidence of general and enduring positive effects on the way people’s minds and brains age has remained elusive.

Mind_circle copyThese conclusions do not mean that the brain does not remain malleable, even in old age. Any mentally effortful new experience, such as learning a language, acquiring a motor skill, navigating in a new environment, and, yes, playing commercially available computer games, will produce changes in those neural systems that support acquisition of the new skill. For example, there may be an increase in the number of synapses, the number of neurons and supporting cells, or a strengthening of the connections among them. This type of brain plasticity is possible throughout the life span, though younger brains seem to have an advantage over the older ones. It would be appropriate to conclude from such work that the potential to learn new skills remains intact throughout the life span. However at this point it is not appropriate to conclude that training-induced changes go significantly beyond the learned skills, that they affect broad abilities with real-world relevance, or that they generally promote “brain health”.

As we take a closer look at the evidence on brain games, one issue needs to be kept in mind: It is not sufficient to test the hypothesis of training-induced benefits against the assumption that training brings no performance increases at all. Rather, we need to establish that observed benefits are not easily and more parsimoniously explained by factors that are long known to benefit performance, such as the acquisition of new strategies or changes in motivation. It is well established, for example, that improvements on a particular memory task often result from subtle changes instrategy thatreflect improvement in managing the demands of that particular task. Such improvement is rewarding for players (the fun factor) but does not imply a general improvement in memory. In fact, the notion that performance on a single task cannot stand in for an entire ability is a cornerstone of scientific psychology. Claims about brain games often ignore this tenet. In psychology, it is good scientific practice to combine information provided by many tasks to generate an overall index representing a given ability. According to the American Psychological Association, newly developed psychological tests must meet specific psychometric standards, including reliability and validity. The same standards should be extended into the brain game industry, but this is not the state of affairs today.

To date, there is little evidence that playing brain games improves underlying broad cognitive abilities, or that it enables one to better navigate a complex realm of everyday life. Some intriguing isolated reports do inspire additional research, however. For instance, some studies suggest that both non-computerized reasoning and computerized speed-of-processing training are associated with improved driving in older adults and a reduction in the number of accidents. Another study revealed, for a sample of younger adults, that 100 days of practicing 12 different computerized cognitive tasks resulted in small general improvements in the cognitive abilities of reasoning and episodic memory, some of which were maintained over a period of two years. In other studies, older adults have reported that they felt better about everyday functioning after cognitive training, but no objective measures supported that impression. Additional systematic research is needed to replicate, clarify, consolidate, and expand such results. To be fully credible, an empirical test of the usefulness of brain games needs to address the following questions. Does the improvement encompass a broad array of tasks that constitute a particular ability, or does it just reflect the acquisition of specific skills? Do the gains persist for a reasonable amount of time? Are the positive changes noticed in real life indices of cognitive health? What role do motivation and expectations play in bringing about improvements in cognition when they are observed?

iStock_000000218284XSmall-180x180 copyIn a balanced evaluation of brain games, we also need to keep in mind opportunity costs. Time spent playing the games is time not spent reading, socializing, gardening, exercising, or engaging in many other activities that may benefit cognitive and physical health of older adults. Given that the effects of playing the games tend to be task-specific, it may be advisable to train an activity that by itself comes with benefits for everyday life. Another drawback of publicizing computer games as a fix to deteriorating cognitive performance is that it diverts attention and resources from prevention efforts. The promise of a magic bullet detracts from the message that cognitive vigor in old age, to the extent that it can be influenced by the lives we live, reflects the long-term effects of a healthy and active lifestyle.

We also must keep in mind that studies reporting positive effects of brain games on cognition are more likely to be published than studies with null results –the so-called “file drawer effect”– such that even the available evidence is likely to draw an overly positive picture of the true state of affairs. Statistical methods such meta-analysis, which integrates the results of many studies in a given field of inquiry, allow estimation of effect magnitude as well as the likelihood of the file-drawer effect. While some meta-analyses report small positive effects of training on cognition, others note substantial disparities in methodological rigor among the studies that cast doubt on any firm conclusion. Further, the problems that haunt individual studies do not simply disappear when results from such studies are summarized in a meta-analysis. In particular, the practice of assessing specific tests rather than broader assays of ability is just as problematic on the level of meta-analytic integration as it is on the level of individual studies.

In summary, research on aging has shown that the human mind is malleable throughout life span. In developed countries around the world, later-born cohorts live longer and reach old age with higher levels of cognitive functioning than those who were born in earlier times. When researchers follow people across their adult lives, they find that those who live cognitively active, socially connected lives and maintain healthy lifestyles are less likely to suffer debilitating illness and early cognitive decline in their golden years than their sedentary, cognitively and socially disengaged counterparts. The goal of research on the effectiveness of computer-based cognitive exercise is to provide experimental evidence to support or qualify these observations. Some of the initial results are promising and make further research highly desirable. However, at present, these findings do not provide a sound basis for the claims made by commercial companies selling brain games. Many scientists cringe at exuberant advertisements claiming improvements in the speed and efficiency of cognitive processing and dramatic gains in “intelligence”, in particular when these appear in otherwise trusted news sources. In the judgment of the signatories below, exaggerated and misleading claims exploit the anxiety of adults facing old age for commercial purposes. Perhaps the most pernicious claim, devoid of any scientifically credible evidence, is that brain games prevent or reverse Alzheimer’s disease.

In closing, we offer five recommendations. Some of these recommendations reflect experimental findings in human populations, whereas others are based on a synthesis of correlational evidence in humans and mechanistic knowledge about risks and protective factors.

  • Much more research needs to be done before we understand whether and what types of challenges and engagements benefit cognitive functioning in everyday life. In the absence of clear evidence, the recommendation of the group, based largely on correlational findings, is that individuals lead physically active, intellectually challenging, and socially engaged lives, in ways that work for them. Before investing time and money on brain games, consider what economists call opportunity costs: If an hour spent doing solo software drills is an hour not spent hiking, learning Italian, making a new recipe, or playing with your grandchildren, it may not be worth it. But if it replaces time spent in a sedentary state, like watching television, the choice may make more sense for you.
  • Physical exercise is a moderately effective way to improve general health, including brain fitness. Scientists have found that regular aerobic exercise increases blood flow to the brain, and helps to support formation of new neural and vascular connections. Physical exercise has been shown to improve attention, reasoning, and components of memory. All said, one can expect small but noticeable gains in cognitive performance, or attenuation of loss, from taking up aerobic exercise training.
  • A single study, conducted by researchers with financial interests in the product, or one quote from a scientist advocating the product, is not enough to assume that a game has been rigorously examined. Findings need to be replicated at multiple sites, based on studies conducted by independent researchers who are funded by independent sources. Moreover, participants of training programs should show evidence of significant advantage over a comparison group that does not receive the treatment but is otherwise treated exactly the same as the trained group.
  • No studies have demonstrated that playing brain games cures or prevents Alzheimer’s disease or other forms of dementia.
  • Do not expect that cognitively challenging activities will work like one-shot treatments or vaccines; there is little evidence that you can do something once (or even for a concentrated period) and be inoculated against the effects of aging in an enduring way. In all likelihood, gains won’t last long after you stop the challenge.

In summary: We object to the claim that brain games offer consumers a scientifically grounded avenue to reduce or reverse cognitive decline when there is no compelling scientific evidence to date that they do. The promise of a magic bullet detracts from the best evidence to date, which is that cognitive health in old age reflects the long-term effects of healthy, engaged lifestyles. In the judgment of the signatories, exaggerated and misleading claims exploit the anxiety of older adults about impending cognitive decline. We encourage continued careful research and validation in this field.

 

 

 

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