Posts Tagged ‘neurodegeneration’

Brain Science

Larry H Bernstein, MD, FCAP, Curator



A Protein Atlas of the Brain


What looks like an island is actually a schematic representation of a mouse brain. Researchers have now analyzed the mouse brain proteome and summarized the data in an atlas. (Image:  MPI of Biochemistry/ K. Scharma )


What looks like an island is actually a schematic representation of a mouse brain. Researchers have now analyzed the mouse brain proteome and summarized the data in an atlas. (Image: MPI of Biochemistry/ K. Scharma )


Just as in the Middle Ages when there were still many uncharted areas on Earth, researchers today are aware that there is still a great deal to learn about cells in our microcosm. But instead of sextants and compasses, researchers nowadays use modern methods such as mass spectrometry to look into the world of protein molecules. Neuroscientists are focussed particularly on resolving brain complexity with its billions of specialized cells. To understand the brain’s functions, scientists from the Max Planck Institutes of Biochemistry in Martinsried and Experimental Medicine in Göttingen have for the first time quantified the entire set of proteins ‒ the proteome ‒ in the adult mouse brain. The information about which proteins and how many of them are found in the various cell types and regions has been summarized in a protein atlas.

The brain consists of hundreds of billions of interconnected cells which communicate with one another. Different cell types specialize in different functions. Nerve cells transmit and process stimuli from outside; distinct glial cells supply them with nutrients, regulate the flow of blood in the brain, help in isolating nerve fibres and perform tasks in the immune system.

Cells are comprised of proteins which are the functional building blocks. They act as small molecular machines and give the cell its structure. The information for synthesis of protein molecules is encoded in DNA and RNA; biomolecules which have been extensively examined in the brain. “Up to now, however, it was not known which and how many proteins are produced in the different, highly specialized cells or even how the numbers of proteins in the individual regions differ”, explains neuroscientist Mikael Simons. “To examine this, we needed modern measuring and analysis methods in order to be able to record and evaluate these enormous numbers of proteins.” Together with protein research specialists, a team headed by Matthias Mann in Martinsried, the scientists further developed the mass spectrometry technology for in-depth profiling of brain proteins in a rapid, reproducible and a quantitative fashion.

They were able to show that there are around 13,000 different proteins in the adult mouse brain. The quantity of proteins in the different cell types and brain regions, and how they differ from one another can now be found in the recently established protein atlas at http://www.mousebrainproteome.com. The protein data presented there from five different cell types and ten regions in the mouse brain constitute the most comprehensive collection to date.

This deep proteome investigation should serve as a rich resource for analyses of brain development and function. “Surprisingly, only 10 per cent of all proteins are cell type-specific”, explains Kirti Sharma, lead author of the study. “These cell-specific proteins are mostly found on the surface of the cell.” The large majority – 90 per cent of all proteins – are found in all cell types. As in a satellite view of previously uncharted landscapes, the researchers have created a protein atlas based on the most comprehensive data collection that should help in the development of new treatments for alleviating brain diseases.

Source: Max Planck Institute



New Computational Strategy Finds Brain Tumor-shrinking Molecules



These are MRI renderings of mouse brain tumors. Tumors treated with SKOG102 (lower panels) shrank by about half compared to tumors treated with a control (upper panels). (Credit: UC San Diego Health)


These are MRI renderings of mouse brain tumors. Tumors treated with SKOG102 (lower panels) shrank by about half compared to tumors treated with a control (upper panels). (Credit: UC San Diego Health)


Patients with glioblastoma, a type of malignant brain tumor, usually survive fewer than 15 months following diagnosis. Since there are no effective treatments for the deadly disease, University of California, San Diego researchers developed a new computational strategy to search for molecules that could be developed into glioblastoma drugs. In mouse models of human glioblastoma, one molecule they found shrank the average tumor size by half. The study is published October 30 byOncotarget.

The newly discovered molecule works against glioblastoma by wedging itself in the temporary interface between two proteins whose binding is essential for the tumor’s survival and growth. This study is the first to demonstrate successful inhibition of this type of protein, known as a transcription factor.

“Most drugs target stable pockets within proteins, so when we started out, people thought it would be impossible to inhibit the transient interface between two transcription factors,” said first author Igor Tsigelny, Ph.D., research scientist at UC San Diego Moores Cancer Center, as well as the San Diego Supercomputer Center and Department of Neurosciences at UC San Diego. “But we addressed this challenge and created a new strategy for drug design — one that we expect many other researchers will immediately begin implementing in the development of drugs that target similar proteins, for the treatment of a variety of diseases.”

Transcription factors control which genes are turned “on” or “off” at any given time. For most people, transcription factors labor ceaselessly in a highly orchestrated system. In glioblastoma, one misfiring transcription factor called OLIG2 keeps cell growth and survival genes “on” when they shouldn’t be, leading to quick-growing tumors.

In order to work, transcription factors must buddy up, with two binding to each other and to DNA at same time. If any of these associations are disrupted, the transcription factor is inhibited.

In this study, Tsigelny and team aimed to disrupt the OLIG2 buddy system as a potential treatment for glioblastoma. Based on the known structure of related transcription factors, study co-author Valentina Kouznetsova, Ph.D., associate project scientist at UC San Diego, developed a computational strategy to search databases of 3D molecular structures for those small molecules that might engage the hotspot between two OLIG2 transcription factors. The team used the Molecular Operation Environment (MOE) program produced by the Chemical Computing Group in Montreal, Canada and high-performance workstations at the San Diego Supercomputer Center to run the search.

With this approach, the researchers identified a few molecules that would likely fit the OLIG2 interaction. They then tested the molecules for their ability to kill glioblastoma tumors in the Moores Cancer Center lab of the study’s senior author, Santosh Kesari, M.D., Ph.D.. The most effective of these candidate drug molecules, called SKOG102, shrank human glioblastoma tumors grown in mouse models by an average of 50 percent.

“While the initial pre-clinical findings are promising,” Kesari cautioned, “it will be several years before a potential glioblastoma therapy can be tested in humans. SKOG102 must first undergo detailed pharmacodynamic, biophysical and mechanistic studies in order to better understand its efficacy and possible toxicity.”

To this end, SKOG102 has been licensed to Curtana Pharmaceuticals, which is currently developing the inhibitor for clinical applications. Kesari is a co-founder, has an equity interest in and is chair of the scientific advisory board for Curtana Pharmaceuticals. Co-authors Rajesh Mukthavaram, Ph.D., and Wolfgang Wrasidlo, Ph.D., also own stock in Curtana Pharmaceuticals.

his research was funded, in part, by the National Institutes of Health, Voices Against Brain Cancer Foundation, Christopher and Bronwen Gleeson Family Trust and American Brain Tumor Association Drug Discovery Grant.

Source: University of California San Diego

Musical Rhythms in the Brain



Researchers at Max Planck Institute for Empirical Aesthetics in Frankfurt and of New York University have identified how brain rhythms are used to process music, a finding that also contributes to a better understanding of the auditory system. Furthermore, the study suggests that musical training can enhance the functional role of brain rhythms.

The paper, which appears in the journal Proceedings of the National Academy of Sciences, points to a newfound role the brain’s cortical oscillations play in the detection of musical sequences. The term “cortical oscillations” refers to the rhythmic electrical activity generated spontaneously and in response to stimuli by neural tissue in the central nervous system. The importance of brain oscillations in sensory-cognitive processes has become increasingly evident.

“We’ve isolated the rhythms in the brain that match rhythms in music,” explains Keith Doelling, lead author. “Specifically, our findings show that the presence of these rhythms enhances our perception of music and of pitch changes.”

 Headbanging is most common in the rock, punk and heavy metal music genres. Scientist have now identified how rhythms inside the human brain are used to process music. (Image: Wikimedia / Małgorzata Miłaszewska)


Headbanging is most common in the rock, punk and heavy metal music genres. Scientist have now identified how rhythms inside the human brain are used to process music. (Image: Wikimedia / Małgorzata Miłaszewska)


Previous research has shown that brain rhythms very precisely synchronize with speech, enabling us to parse continuous streams of speech — in other words, how we can isolate syllables, words, and phrases from speech, which is not, when we hear it, marked by spaces or punctuation.

However, it has not been clear what role such cortical brain rhythms, or oscillations, play in processing other types of complex sounds, such as music.

To address these questions, the researchers conducted three experiments using magnetoencephalography (MEG), which allows the tiny magnetic fields generated by brain activity to be measured. The study’s subjects were asked to detect short pitch distortions in 13-second clips of classical piano music (by Bach, Beethoven, Brahms) that varied in tempo — from half a note to eight notes per second. The study’s authors divided the subjects into musicians (those with at least six years of musical training and who were currently practicing music) and non-musicians (those with two or fewer years of musical training and who were no longer involved in it).

Not surprisingly, the study found that musicians have more potent oscillatory mechanisms than non-musicians do. “What this shows is we can be trained, in effect, to make more efficient use of our auditory-detection systems,” observes study co-author David Poeppel, director of the Max Planck Institute for Empirical Aesthetics. “Musicians, through their experience, are simply better at this type of processing.”

For music that is faster than one note per second, both musicians and non-musicians showed cortical oscillations that synchronized with the note rate of the clips. The researchers therefore conclude that these oscillations were effectively employed by everyone to process the sounds they heard, although musicians’ brains synchronized more to the musical rhythms. Only musicians, however, showed oscillations that synchronized with unusually slow clips. This difference, the researchers say, may suggest that non-musicians are less able to process the music as a continuous melody rather than as individual notes. Moreover, musicians are able to detect pitch distortions much more accurately — as evidenced by corresponding cortical oscillations.

Thus, brain rhythms appear to play a role in parsing and grouping sound streams into ‘chunks’ that are then analyzed as speech or music, the scientists add.

Source: Max Planck Institute



New Three-Minute Test Detects Lewy Body Disease


Lewy Body disease is the second most common type of progressive dementia, according to the Mayo Clinic, and affects approximately 1.3 million Americans.

The Lewy Body Dementia Association says the disease is widely accepted to be highly underdiagnosed and is the most frequently misdiagnosed form of dementia.

The new test, called the “Lewy Body Composite Risk Score” (LBCRS), developed by James E. Galvin, M.D., M.P.H., professor of clinical biomedical science at FAU, is a simple, one page-survey, that includes yes or no questions for a clinician to complete.  The structured questions look at six non-motor features that are present in patients with LBD, but are much less common in other forms of dementia.  The tool helps clinician assess whether a patient has rest tremor, postural instability, rigidity, or bradykensia, without having to grade each extremity.

Bioscience Bulletin: Potential Alzheimer’s Test, and Stress Linked to Stroke


N.J. Researchers Closing in on Alzheimer’s, Parkinson’s Tests
Researchers from the Rowan University School of Osteopathic Medicine, have developed a test that could detect Alzheimer’s before symptoms start by identifying a series of obscure antibodies. Scientists narrowed down the auto-antibodies he was looking at from a sample of nearly 10,000 to just 10.

Role Found for Critical Gene in 95% of ALS
Cynthia Fox interviewed experts about a recent Science study that offered new insight surrounding a protein called TDP-43 in relation to amyotrophic lateral sclerosis.  The study found that in 95 percent of ALS cases the protein leaves its home – the nucleus of motor neuron cells – resulting in the creation of improper “cryptic” exons.

Like a bad teenager, in 95 percent of all amyotrophic lateral sclerosis (ALS) cases, a protein called TDP-43 leaves its home— the nucleus of motor neuron cells—to congregate, in suspect fashion, in the cytoplasm.

In a study published in Science this summer, the Johns Hopkins University (JHU) team of pathologist Phillip Wong, Ph.D., offered new insight into this molecular rebellion. It confirmed a function of normal TDP-43 in the nucleus: orchestrating proper RNA splicing and exon formation. It confirmed what lack of nuclear TDP-43 does: creates improper “cryptic” exons. And it identified proteins that mitigate effects of nuclear TDP-43 loss: potential drug leads.

“The recently published work in Science very clearly demonstrates that in the absence of TDP-43, RNA is misspliced, and in many cases targeted for degradation,” University of Michigan neurologist Sami Barmada, M.D., Ph.D., told Bioscience Technology. Barmada was uninvolved with the research. “A very real consequence of such dysfunctional RNA splicing and degradation is an inability to maintain cell health, ultimately resulting in neuron loss. The authors assembled an intriguing story that tells us quite a bit about how TDP-43 functions, and what happens to those functions in diseases such as ALS, and fronto-temporal dementia (FTD). If the mechanism identified in this manuscript does indeed underlie toxicity due to TDP-43 mislocalization, then it may very well contribute to neuron loss in the vast majority of ALS.”

High Stress Jobs May be Linked to Increased Stroke Risk
The final story in our round up this week takes a look at a new Neurology study, which found there may be a link between high stress jobs and an increased risk for stroke. After analyzing six studies, comprised of a total of 138,782 participants, researchers concluded that people with high stress jobs (such as waitresses and nursing aides) were 58 percent more likely to have an ischemic stroke than those in low stress jobs (such as natural scientists and architects).

New Scanner to Help Uncover Causes of Dementia


The funding from the Medical Research Council will allow the SIGNA PET/MR scanner, made by GE Healthcare, to be installed in Central Manchester University Hospitals NHS Foundation Trust.  Currently there are only two of these scanners in the UK, but following the Manchester funding and money to other university centers in the UK, this number will increase to seven, from a number of manufacturers.

The new scanner will help scientists and clinicians understand the causes and progression of dementia, and provide ways to test the effects of new treatments.  Molecular changes in the brain are believed to be responsible for dementia and the scanners have the potential to link these with the brain changes that they cause – leading to new understanding and new treatments.

Professor Nigel Hooper is the University’s Director for Dementia Research.  He said: “Dementia is a condition that is poorly understood and difficult to treat effectively.  It’s going to become more of a problem in the coming decades, so our research response needs to pick up as well.

“This scanner and the wider network will give us that ability to understand dementia better and to develop more treatments.”

The scanner is expected to be operational from July 2016 and work is currently underway to refurbish a shelled space adjacent to the Nuclear Medicine Centre to house the scanner suite which will have three treatment rooms, a research office and a radiopharmacy room. CMFT was chosen as the ideal location for the scanner due to its central Manchester location and close proximity to the main University campus, the co-location of the suite adjacent to the clinical PET/CT scanner at CMFT and the opportunity to take research into clinical practice.

Christine Tonge, the Director of Medical Physics at Central Manchester said: “We are excited by this opportunity to contribute to this important area of research. This scanner will put Manchester at the forefront of dementia research and we look forward to collaborating not only with our colleagues from the University, but also  from other hospitals in Greater Manchester and beyond.”

The scanner is being funded as part of the Dementias Platform UK – a multi-million pound public-private partnership, developed by the Medical Research Council, to accelerate progress in dementias research. DPUK’s aims are early detection, improved treatment and, ultimately, prevention of dementias. It is the world’s largest study group for use in dementias research, pulling together two million well-characterized participants from over 30 national population studies.

The Manchester scanner will be supervised by Professor Alan Jackson, who is the director of the University’s Wolfson Molecular Imaging Centre, which hosts two PET scanners and one MR scanner and produces radiotracers for use in PET scanning.  He said: “Manchester now has a range of scanning facilities which mean that clinicians and scientists can produce high quality research across a range of conditions.

“With the growing urgency of developing treatments for dementia, this new equipment is vital in addressing a major growing health concern.

“Most importantly, being linked with the other four universities which are also purchasing PET-MR scanners will mean Manchester has the ability to become involved in multi-centre trials and research grants and contracts – increasing the effectiveness of the UK’s research in this area.”

Source: University of Manchester


Trends Mol Med. 2014 Feb;20(2):66-71. doi: 10.1016/j.molmed.2013.11.003. Epub 2013 Dec 16.
TDP-43-mediated neurodegeneration: towards a loss-of-function hypothesis?
Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are clinically distinct fatal neurodegenerative disorders. Increasing molecular evidence indicates that both disorders are linked in a continuous spectrum (ALS-FTD spectrum). Neuronal cytoplasmic inclusions consisting of the nuclear TAR DNA-binding protein 43 (TDP-43) are found in the large majority of patients in the ALS-FTD spectrum and dominant mutations in the TDP-43 gene cause ALS. A major unresolved question is whether TDP-43-mediated neuronal loss is caused by toxic gain of function of cytoplasmic aggregates, or by a loss of its normal function in the nucleus. Here we argue that based on recent genetic studies in worms, flies, fish, and rodents, loss of function of TDP-43, rather than toxic aggregates, is the key factor in TDP-43-related proteinopathies.
Gains or losses: molecular mechanisms of TDP43-mediated neurodegeneration

Edward B. Lee, Virginia M.-Y. Lee & John Q. Trojanowski

Nature Reviews Neuroscience 13, 38-50 (January 2012) |   http://dx.doi.org:/10.1038/nrn3121

RNA-binding proteins, and in particular TAR DNA-binding protein 43 (TDP43), are central to the pathogenesis of motor neuron diseases and related neurodegenerative disorders. Studies on human tissue have implicated several possible mechanisms of disease and experimental studies are now attempting to determine whether TDP43-mediated neurodegeneration results from a gain or a loss of function of the protein. In addition, the distinct possibility of pleotropic or combined effects — in which gains of toxic properties and losses of normal TDP43 functions act together — needs to be considered.


Transposable Elements in TDP-43-Mediated Neurodegenerative Disorders


Published: September 5, 2012   DOI: http://dx.doi.org:/10.1371/journal.pone.0044099

Elevated expression of specific transposable elements (TEs) has been observed in several neurodegenerative disorders. TEs also can be active during normal neurogenesis. By mining a series of deep sequencing datasets of protein-RNA interactions and of gene expression profiles, we uncovered extensive binding of TE transcripts to TDP-43, an RNA-binding protein central to amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Second, we find that association between TDP-43 and many of its TE targets is reduced in FTLD patients. Third, we discovered that a large fraction of the TEs to which TDP-43 binds become de-repressed in mouse TDP-43 disease models. We propose the hypothesis that TE mis-regulation contributes to TDP-43 related neurodegenerative diseases.

Citation: Li W, Jin Y, Prazak L, Hammell M, Dubnau J (2012) Transposable Elements in TDP-43-Mediated Neurodegenerative Disorders. PLoS ONE 7(9): e44099. doi:10.1371/journal.pone.0044099

Editor: Koichi M. Iijima, Thomas Jefferson University, United States of America


Accumulation of TAR DNA-binding protein 43 (TDP-43) containing cytoplasmic inclusions is a shared pathological hallmark in a broad spectrum of neurodegenerative disorders, including ALS, FTLD and Alzheimer’s disease [1]. Mutations in this multifunctional RNA binding protein are also known to underlie some familial and sporadic cases of ALS [1]. Despite considerable progress, the mechanisms that link TDP-43 to neurodegeneration still are unclear. We conducted a meta-analysis of TDP-43 protein:RNA target binding datasets and of mRNA expression datasets. All previous analyses of such data focused on sequence reads that uniquely map to the reference genome, thereby excluding transcripts derived from transposable elements (TEs). In contrast, we included sequences that map to multiple locations and examined reads that align to TEs. Our analyses lead to the striking hypothesis that TE over-expression may contribute to TDP-43 mediated neurodegeneration.

Transposable elements (TEs) are highly abundant mobile genetic elements that constitute a large fraction of most eukaryotic genomes. Retrotransposons, which copy themselves through an RNA intermediate, represent approximately 40% of the human genome [2], [3]. Although the majority of TE copies are nonfunctional, a subset have retained the ability to mobilize and even the immobile copies can be expressed [4]. Because of their potential to copy themselves and insert into new genomic locations as well as to generate enormous levels of expression, transposable elements present a massive endogenous reservoir of genomic instability and cellular toxicity [3]. The impacts of these parasitic genetic elements normally are stifled by potent cellular mechanisms involving small interfering RNAs that act via the RNA induced silencing complex (RISC) to inhibit transposon expression ([5] for review). Although most investigations have naturally focused on the germline, where new insertions are heritable and thus favored by transposon evolution, somatic tissues also have an active transposon silencing mechanism whose functional significance is less understood. An emerging literature has established that certain TEs are normally active in brain [6], [7], [8], [9] and elevated expression of some LINE, SINE (which are non-LTR retrotransposons) and LTR elements have been correlated with several neurodegenerative disorders [10], [11], [12], [13], [14], [15], [16]. We therefore investigated whether the RNA targets of TDP-43 include transposon-derived transcripts.

Several recent studies used deep sequencing to profile the RNA targets that co-purify with immunoprecipitated mouse, rat or human TDP-43 and also to profile gene expression changes in mouse after knockdown or over-expression of TDP-43 [17], [18], [19], [20]. In each case, however, these studies analyzed annotated protein coding sequences and excluded TE-derived transcripts and other repetitive elements due to the difficulties inherent in working with ambiguously mapped reads from short read technologies [e.g. [21]]. Despite efforts to develop new algorithms for analyzing multiple alignments of short reads [22], these algorithms have not been applied systematically for analyzing TE-derived transcripts in any neurodegenerative disease. Because each of the above mentioned TDP-43 related studies provided public access to their raw data, we were able to use this resource to search for TDP-43 targets and for transcript mis-expression when we included sequence reads that map to multiple genomic locations, the majority of which are TE derived transcripts in these datasets. Our meta-analysis supports three main conclusions. First, TDP-43 broadly targets TE-derived transcripts, including many SINE, LINE and LTR classes as well as some DNA elements. Second, the association between TDP-43 and TE-derived RNA targets is reduced in FTLD patients relative to healthy subjects, consistent with the idea that loss of TE control might be part of the disease pathology. Third, we observe broad over-expression of TE derived transcripts in each of two different mouse models with TDP-43 dysfunction. Finally there is a striking overlap between the TE transcripts identified as targets and those that are over-expressed with TDP-43 misexpression.



We first re-analyzed raw data from the rat TDP-43 RNA immunoprecipitation sequencing (RIP-seq) dataset [17] and the mouse and human TDP-43 in vivo crosslinking-immunoprecipitation sequencing (CLIP-seq) datasets [18], [19]. We tested three different analysis methods to examine effects on TEs (Fig. 1A–C; Methods and Figs. S1 and Tables S1, S2, S3). Because reads could potentially map to many regions, we first used an analysis in which each location was weighted based on the number of alignments (Figs. 1A,B) see methods). This analysis method (MULTI), which included both unique and multi mapped reads, assigns an enrichment level for each element, but does not distinguish contributions of individual instances of each element. Although this method can potentially include effects from TEs that are difficult to map with short read sequence, a disadvantage is that it does not distinguish which instances of a given TE are detected. In addition, because many TE copies are present within introns of genes, the MULTI method does not distinguish whether the TE sequences are co-expressed with genes or expressed from TEs per se. To address these issues, and to test the robustness of our observations, we also tested two additional mapping methods for the rat and human datasets (Figs. 1C and S1E,F; Methods). First, we examined only the subset of reads that map uniquely to the genome (UNIQ). This method does bias the results to the fraction of TEs that have diverged enough to have unique sequences, but provides confidence that signal derives from unique chromosomal locations. As a third mapping strategy (UNIQ+SameEle), we examined the effects of including both uniquely mapped sequences and those that map to multiple locations so long as they map to the same element (weighted for their contribution to each instance as above – see Methods).


Figure 1. TDP-43 binds broadly to transposable element (TE)-derived transcripts.

Magnitude (log2-fold) of enrichments (up) or depletions (down) are shown (A, rat; B, mouse) for significantly bound repeat elements grouped by class. MULTI method (see text) was used for A and B. (C) The majority of rat TE targets identified with MULTI also are identified (Left Panel, Rat) when analysis is restricted to reads that map uniquely (UNIQ) or when both uniquely mapped and multi-mapped reads that map to the same TE were included (UNIQ+SameEle). These conclusions also hold for TE targets whose binding is reduced in FTLD samples from human tissue relative to healthy controls (Left panel, Human). Most rat TE targets and differentially bound human TE targets identified from uniquely mapped reads are intergenic (Right panel). (D) For TDP-43, peaks (UNIQ+SameEle) over TE targets are tall and sharp with mean peak height of 158 counts/peak. In contrast, peak heights are lower for FUS (mean peak height of 17).


With all three mapping strategies we find a dramatic enrichment of sequences that derive from each major class of TE (Figs. 1A–C; S1; Table S3). With the MULTI method, we find 271 significantly enriched or depleted (most were enriched) repeat element sub-families in the rat TDP-43-IP samples versus control (Fig. 1A), of which 245 correspond to TEs. In the mouse dataset (Fig. 1B), MULTI detects significant enrichment of 352 repeat element sub-families of which 334 correspond to TEs (Table S3). These comprise all major classes of TEs, including LINE, SINE, LTR and some DNA elements [3]. For instance, 85 out of the 122 known mouse LINE elements and 6 out of the 7 known rat LINE elements are identified as TDP-43 targets. Similarly 26 out of 41 mouse SINE elements and 36 out of 37 rat SINE elements also were detected as TDP-43 targets. One caveat to the mouse clip-seq analysis was the lack of a control IP to use in estimating background counts for this single dataset, which could potentially lead to a larger false positive rate in the detected peaks (see Methods); however, the similarity in the results obtained for this dataset as compared to the well-controlled studies for rat (Fig. 1A) and human datasets (see below) argues for the inclusion of this dataset despite its caveats.

Overall, we detect the most extensive binding to TEs with the MULTI method, and these findings are not an artifact of the way we assigned weights with the MULTI method because even with the more restricted UNIQ analysis, we identify ∼80% of the rat elements that are differentially enriched when all mappable reads are included (Figs. 1C, S1F). Moreover, among the uniquely mapped subset of TE instances that we identify as TDP-43 targets, greater than 80% map to intergenic regions rather than to elements contained within genes (Fig. 1C). When we include both unique mappers and multi mappers from the same element (UNIQ+SameEle), we detect enrichment for 95% of the TE sub-families that were identified as TDP-43 targets with the MULTI method (Figs. 1C, S1F). The concordant results from these three different mapping strategies provide confidence that identification of TE derived transcripts as TDP-43 targets is a robust effect that is detected with a variety of methods for dealing with multi-copy elements.

As a test of the biological specificity of our finding that TDP-43 selectively binds to TE derived transcripts, we applied the UNIQ mapping method to a CLIP-seq dataset for an unrelated RNA binding protein. For this purpose we chose fused in sarcoma (FUS), which like TDP-43, is an hnRNP RNA binding protein that plays diverse roles in RNA biology, including splicing [23]. FUS is a relevant control for specificity because like TDP-43, it is implicated in neurodegenerative disorders including ALS [24]. The result with FUS is in stark contrast with TDP-43 (Fig. 1D). For TDP-43, peaks (defined within a 500 bp window) that map to TEs are relatively large, with a mean peak height of 158 counts. In contrast, with FUS we only see small peaks over TEs with a height of just a few counts (mean peak height of 17; Fig. 1D for distribution). Peaks that map over RefGene annotations, on the other hand, are similarly distributed for both FUS and TDP-43 (Mean height of 32 and 68 respectively, Fig. S1H). The distributions of mean peak heights (see histogram, Fig. 1D) shows a clear separation between TDP-43 peaks and those obtained with FUS and this separation between peak heights is statistically significant (Wilcoxon rank sum p-value<2.2e−16). Thus our findings show specificity for TDP-43 and are not a byproduct of inherent biases in library construction or analysis.

Because TDP-43 has a known binding motif among its mRNA targets, we used MEME ([25]and see Methods) to identify enriched motifs among both the RefGene and repetitive targets. We identify a UGUGU pentamer motif that is equivalently enriched in uniquely mapped and repetitive targets (Fig. S1C; Methods). This motif is consistent with the binding specificity of TDP-43 that has previously been observed for uniquely mapped sequences [17], [18], [19], [20]. Thus TDP-43 binds TE derived transcripts via a similar sequence motif as identified for RefGene targets.

Because the human dataset [18] includes samples from healthy and FTLD patients (which exhibit TDP-43 positive cytoplasmic inclusions), it also provided an opportunity to identify differences in the TDP-43 targets between FTLD and healthy controls. As in rat and mouse, we observe in human samples a dramatic and significant enrichment in target sequences that derive from many classes of TEs. As with the mouse and rat data, the distribution of peak heights for TE and RefGene targets of TDP-43 are similar (Fig. S1I), indicating that the targeting of TE transcripts is as robust as it is for RefGene targets. More striking, however, is the comparison between healthy subjects and FTLD patients. When we examine the relative enrichment for each repeat element within healthy vs. FTLD samples, we detect a dramatic difference in binding to TE derived RNAs (Fig. 1E–H). Overall, the association between TDP-43 and TE transcripts is significantly reduced in FTLD patients, which leads to a relative enrichment of 38 repeat elements in healthy versus FTLD, 28 of which correspond to transcripts derived from TEs (Fig. 2 and Table S3; See Methods for statistical analyses). We see reduced binding of TDP-43 to transcripts from all major classes of TE including SINE, LINE, LTR and a few DNA elements. Here too, we observe that the majority of the TE targets whose binding to TDP-43 was reduced in FTLD are consistently identified with all three methods (Fig. 1C). Most of the TE targets that show reduced binding to TDP-43 in FTLD samples are intergenic rather than contained within genes (Fig. 1C). Example peaks are shown for one RefGene control (Fig. 1F) as well as two differentially targeted TEs (Figs. 1G,H).


Figure 2. TDP-43 binding to TEs is selectively lost in FTLD patients.

(A) In the human CLIP-seq data from FTLD versus healthy control, 38 repeat elements showed significant (p-value< = 1e-5 and fold changes> = 2) differential binding. Log2 fold binding differences are shown for significantly enriched/depleted elements. (B,C,D) Peaks are shown in genome browser for one RefGene control (B) and two differentially targeted TEs (C,D) in Healthy (top) versus FTLD (bottom). (E) Enrichment for the UGUGU motif relative to its prevalence in the genome is shown across a 51-nt window surrounding binding sites (−25 nt, 25 nt). Healthy samples (Blue) show similar enrichment for the UGUGU pentamer motif among RefGene (solid) and repeat (dashed) sequences (RefGene/repeat motif enrichment ratio ≈1.3). In contrast, motif enrichment in FTLD samples (Red) is significantly reduced among repeat (dashed) annotations relative to RefGene (solid; p-value< = 0.01; RefGene/repeat motif enrichment ratio ≈2.0).


This reduced binding in FTLD patients of TDP-43 to TE-derived transcripts also is apparent when we examine over-all enrichment for the UGUGU pentamer motif (Figs. 2E and S1) relative to the genome. In the rat and mouse samples as well as in the dataset from healthy human brain samples, we observe equivalent enrichment of UGUGU binding motifs among uniquely mapped (RefGene) versus repetitively mapped (repeat) TDP-43 targets (RefGene/repeat enrichment ratio near 1.0; Fig. S1D; see Methods). In the FTLD-TDP-43-CLIP samples, we also see enrichment for the UGUGU motif among RefGene targets that is equivalent to that seen in healthy subjects (Fig. 2E), but the level of enrichment for this UGUGU motif is significantly lower among the sequences that map to repeat elements. In the FTLD samples, the RefGene/repeat enrichment ratio is increased to 2.0 (Fig. 2E; p-value< = 0.01, p-values were assigned with 100 iterations on randomly chosen sets containing 50% of original data; see Methods). In other words, FTLD samples exhibit a selective reduction of binding to TE transcripts and also exhibit reduced UGUGU motif enrichment among the remaining repetitive sequences that still co-purify with TDP-43. This difference in motif enrichment between FTLD and control samples is only manifested among repeat annotations.

The reduced binding of TE transcripts in FTLD patients suggested that TDP-43 pathology might include a loss of TE regulation. We investigated this possibility in two ways. First, we analyzed the repetitive sequence reads from two different mRNA-seq datasets from mouse models of TDP-43 pathology.

The first mRNA-seq study that we analyzed [20] used over-expression of human TDP-43 in transgenic mice. Overexpression of this aggregation prone protein is associated with toxic TDP-43 pathological effects and is thought to act as a dominant-negative, causing reduction in the normal functions of TDP-43. The second mRNA-seq study [19] used antisense oligonucleotide-mediated depletion of TDP-43 in mouse striatum to test the effects of TDP-43 loss of function. Both studies identified transcripts that are differentially expressed or spliced in response to these TDP-43 manipulations. To ask if the above TDP-43 depletion and over-expression/dominant-negative impacted TE derived transcripts, we again analyzed sequence reads including those that map to multiple locations. We found broad elevations of TE derived transcripts in both the over-expression transgenic mouse model and in the striatal depletion of TDP-43 (Figs. 3A,B). TDP-43 over-expression was associated with elevated expression of 86 repetitive elements (Fig. 3A), whereas TDP-43 depletion results in increased expression levels of 223 repetitive element species (Fig. 3B). In both cases, most of these correspond to LINE, SINE and LTR elements. Overall, the affected TE transcripts are expressed at comparable levels to those of the differentially expressed RefGene transcripts (Fig. S1J), suggesting that these are robust effects on transcripts whose expression levels are not at the limit of detection. More importantly, when TDP-43 function is compromised, we observe a striking degree of concordance between the TE transcripts that are elevated and the ones that we identified as RNA targets of TDP-43 in normal tissue (Red in Fig. 3; See Table S3). Indeed the majority of elevated TE transcripts in both mouse mRNA-seq datasets also were detected as TDP-43 targets in the iCLIP-seq binding dataset (Fig. 3; Table S3). This remarkable concordance between the transcripts that are targeted by TDP-43 and those that are elevated in response to TDP-43 misexpression is unique to the repetitive elements in the genome. In contrast, CLIP targets identified from the RefGene fraction of the transcriptome have little overlap with those that show over-expression when TDP-43 function is compromised suggesting that the coding gene expression increases are largely indirect effects [19]. RefGene transcripts whose expression is reduced show good concordance with direct target identification.


Figure 3. Concordance between mis-regulated TE transcripts upon TDP-43 manipulation and TDP-43 bound TE transcripts.

(A,B) Over-expression [20] of TDP-43 in transgenic mice and depletion [19] of TDP-43 in mouse striatum each result in elevated expression of many TE derived transcripts. The majority of over-expressed TEs also were detected (Table S3) as binding targets by CLIP-seq (RED). A few showed elevated expression but were not detected as binding targets (BLUE).



TDP-43 aggregation and neuropathology plays a fundamental role in a broad spectrum of neurodegenerative disorders [1], [26], [27]. This hnRNP-like RNA binding protein already has been implicated in a remarkable number of cellular functions including repression of HIV-1, alternative splicing, regulation of mRNA stability and microRNA biogenesis [26], [27]. Importantly, a large number of cellular targets of TDP-43 have been characterized, leading to the hypothesis that one key role of this multi-functional protein is to regulate alternative splicing of mRNA targets with a preference for those with large UG rich introns [17], [18], [19], [26], [28]. Our findings support the novel hypothesis that TDP-43 also targets the mobile element derived transcriptome. This association is defective in FTLD patients and the TE transcriptome is broadly over-expressed in mouse models of TDP-43 pathology.

A large fraction of the genetic material of multicellular organisms is made up of mobile elements as well as inactivated TEs. A fraction of these TEs retain the capacity to copy themselves and insert at new genomic locations. During the co-evolution of TEs with their host genomes, organisms have evolved elaborate and efficient mechanisms to prevent or at least regulate such transposition events. As a result, even the potentially active TE copies rarely mobilize within the germline and are also largely constrained in somatic tissue. Several recent studies demonstrate, however, that LINE-1 elements are normally active and mobile during neurogenesis in both rodent and human tissue [7], [8], [9]. Somatic mobilization of Alu and SVA elements as well as LINEs also has recently been detected in several different human brain regions [6]. This raises the intriguing hypothesis that active mobilization of some TEs plays a role in normal brain development or physiology. On the other hand, there also is emerging evidence that unregulated activation of TEs is associated with neuropathology. TE activation in brain has been observed in macular degeneration [14], Rett syndrome [11], Prion diseases [13],[29], Fragile-X associated tremor/ataxia syndrome (FXTAS) [15] and ALS [12]. Moreover, for the cases of macular degeneration and FXTAS, there is evidence that activation of SINEs and an LTR-retrotransposon respectively may contribute to the observed pathology [14], [15].

Our findings support three conclusions. First, that TDP-43 broadly targets TE-derived transcripts, including many SINE, LINE and LTR classes as well as some DNA elements. This conclusion is replicated in three independent datasets from rat, mouse and human. Second, the association between TDP-43 and TE-derived RNA targets is reduced in FTLD patients relative to healthy subjects, consistent with the idea that loss of TE control might be part of the disease pathology. Third, we observe broad over-expression of TE derived transcripts in each of two different mouse models with TDP-43 dysfunction. And there is a striking overlap between the TE targets identified in the CLIP study and those that are over-expressed with TDP-43 misexpression. Taken together, our findings raise the hypothesis that TDP-43 normally functions to silence or regulate TE expression. When TDP-43 protein function is compromised, TEs become over-expressed. Unregulated TE expression can have a number of detrimental impacts including genome instability, activation of DNA-damage stress response or toxic effects from accumulation of TE-derived RNAs or proteins. Such toxicity from activation of mobile genetic elements may contribute to TDP-43-mediated neurodegenerative disorders.


Does a loss of TDP-43 function cause neurodegeneration?

Zuo-Shang Xu

Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation St, 817 LRB, Worcester, MA, 01605, USA

Molecular Neurodegeneration 2012, 7:27  doi:10.1186/1750-1326-7-27     http://www.molecularneurodegeneration.com/content/7/1/27

In 2006, TAR-DNA binding protein 43 kDa (TDP-43) was discovered to be in the intracellular aggregates in the degenerating cells in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), two fatal neurodegenerative diseases [1,2]. ALS causes motor neuron degeneration leading to paralysis [3,4]. FTLD causes neuronal degeneration in the frontal and temporal cortices leading to personality changes and a loss of executive function [5]. The discovery triggered a flurry of research activity that led to the discovery of TDP-43 mutations in ALS patients and the widespread presence of TDP-43 aggregates in numerous neurodegenerative diseases. A key question regarding the role of TDP-43 is whether it causes neurotoxicity by a gain of function or a loss of function. The gain-of-function hypothesis has received much attention primarily based on the striking neurodegenerative phenotypes in numerous TDP-43-overexpression models. In this review, I will draw attention to the loss-of-function hypothesis, which postulates that mutant TDP-43 causes neurodegeneration by a loss of function, and in addition, by exerting a dominant-negative effect on the wild-type TDP-43 allele. Furthermore, I will discuss how a loss of function can cause neurodegeneration in patients where TDP-43 is not mutated, review the literature in model systems to discuss how the current data support the loss-of-function mechanism and highlight some key questions for testing this hypothesis in the future.


Amyotrophic lateral sclerosis (ALS) is a disorder where progressive degeneration of large motor neurons in the spinal cord and cerebral cortex leads to paralysis and death [3,4]. Frontotemporal lobar degeneration (FTLD) causes degeneration of neurons in frontal and temporal cortices, leading to deterioration of executive, cognitive and social functions, as well as loss of emotional control[5]. Although clinically distinct, a significant overlap exists between these two diseases in the patient population, resulting in a continuous spectrum ranging from patients with one disease at either end and patients with varying degrees of both diseases in the middle [6,7]. Recent genetic data has reaffirmed the connection between these two diseases. Some genetic mutations cause one disease but rarely the other, e.g. SOD1, FUS and TDP-43 for ALS, and tau, progranulin and CHMP2B for FTLD. Other mutations cause either or both diseases in the same patient or family, e.g. ubiquilin 2 and C9ORF72. In a significant population of patients (~95 % ALS and ~50 % FTLD), TDP-43 positive intracellular inclusions are present in the CNS even though the TDP-43 gene is not mutated [811], raising the question of how wild-type TDP-43 is involved in the pathogenesis of these cases.

TDP-43 is a RNA binding protein containing two RNA-recognition motifs (RRM), a nuclear localization signal (NLS) and a nuclear export signal (NES) [12]. The protein is normally concentrated in the nucleus but also shuttles back and forth between the nucleus and cytoplasm[13]. TDP-43 is a global regulator of gene expression and is involved in regulation of transcription and multiple aspects of RNA processing and functioning, including splicing, stability, transport, translation and microRNA maturation [1417]. TDP-43 interacts with many proteins and RNAs and functions in multi-protein/RNA complexes [1821]. TDP-43 maintains its protein expression at a constant level within a tight range by auto-feedback mechanisms, which involve TDP-43 binding to its own 3’ untranslated region [15,22]. Overexpression of TDP-43 leads to down-regulation of the endogenous TDP-43 [23,24], and blocking expression of one allele leads to a compensatory increase in the expression of the other allele [2527]. The tight regulation of TDP-43 levels is suggestive of its crucial role in the functioning of multi-protein/RNA complexes, where maintaining a certain stoichiometry between TDP-43 and the other components may be critical.

Because mutations in TDP-43 lead to ALS, a causal role of TDP-43 for neurodegeneration is firmly established [12,28,29]. Therefore, understanding how the mutants cause neurodegeneration offers a convenient entry point for exploring how TDP-43 plays this role. The first question is whether a gain, a loss of function or a dominant-negative effect mediates neurotoxicity. A resolution to this question is of critical importance because it sets the direction of further research on the disease mechanism and on the design of therapeutic strategies. To answer this question, model systems of both gain or loss of function must be employed (Table 1). Gain-of-function models are usually achieved by gene overexpression and loss-of-function models by gene knockout or knockdown. Based on the phenotypic readouts, the mechanism whereby the mutants cause neurodegeneration can be deduced (Table 1).

Table 1.Assay for disease mechanism using transgenic animals

A gain-of-function (Table 1, GF column) mechanism includes two scenarios: first, the mutant gene gains a novel toxic activity that is independent of the normal function of the gene, and second, the mutant becomes hyperactive in one of its normal functions leading to toxicity. In the first scenario, overexpression of the mutant gene, but not the wild type, will cause the disease phenotype. In the second scenario, overexpression of either the mutant or wild-type gene will cause the disease phenotype. In both gain-of-function scenarios, knockout or knockdown of the gene is not expected to cause the disease phenotype.

A loss of function (haploinsufficiency; Table 1, LF column) means that the mutant gene has no function or a reduced function but does not interfere with the function of the wild-type allele. In this scenario, neither overexpression of the mutant nor the wild type is expected to cause the disease phenotype. But knockout or knockdown reproduces the loss of function, and therefore, is expected to generate the disease phenotype.

A dominant-negative mechanism (Table 1, DN column) denotes the condition where the mutant allele is dysfunctional and inhibitory to the function of the wild-type allele. In this scenario, overexpression of the mutant gene is expected to cause the disease phenotype because it dominant-negatively inhibits the function of the endogenous wild-type protein. On the other hand, overexpression of the wild type is generally not expected to generate the disease phenotype because the wild-type gene can function normally and does not inhibit the function of the normal endogenous allele. However, there are exceptions under certain circumstances, for example, if the protein functions in a multi-protein complex (see details below). Knockout or knockdown of the gene is expected to reproduce the disease phenotype because this reduces the function of the wild-type gene. Thus, in model systems, the dominant-negative mechanism can display characteristics of both a gain and a loss of function—it is a loss of function in essence, yet its effect can dominate over the endogenous wild-type allele.

In the case of TDP-43, an abundance of gain-of-function models have been generated in various species, including worm, fly, fish and rodents [12]. In all models with rare exceptions, a consistent finding is that overexpression of both mutant and wild type TDP-43 can cause a neurodegenerative phenotype (Table 1, TDP-43 columns), thus supporting a gain-of-function mechanism and a potential overactivation of TDP-43 in the mutants [12]. Loss-of-function models have also been generated in non-mammalian species and all except the worm showed neurological and neurodegenerative phenotypes [3033,35,44]. The difference between worm and the other species may reflect some species difference, since TDP-43 is dispensable for survival in the worm but not so in other species. In general, the degenerative phenotypes in the loss-of-function models appear less overwhelming than the overexpression models and are often difficult to separate from the developmental effects stemming from a lack of TDP-43 function. Importantly, there is a lack of evidence in mammalian models that a loss of TDP-43 function causes neurodegeneration. This is largely due to the failure in generating such a model using a gene knockout approach [2527,36]. As a result, the current literature leans towards a gain-of-function mechanism as far as the role of TDP-43 in neurodegeneration is concerned.

Yet despite the preponderance of evidence for the gain-of-function mechanism, it has not been sufficient to rule out the loss-of-function mechanism, because the gain-of-function mechanism does not explain well a phenomenon that is consistently observed in numerous pathological studies, i.e. the nuclear clearance of TDP-43 that accompanies the presence of TDP-43 intracellular aggregates[1,2,45]. The question whether the depletion of TDP-43 in the nucleus is consequential in the pathogenesis remains unanswered. In addition, although the aggregates in the cytoplasm may generate gain-of-function type of toxicity, it is also conceivable that the aggregation of TDP-43 renders TDP-43 non-functional, and as such, causes TDP-43 dysfunction. In this review, I propose a model that is centered on the loss-of-function mechanism whereby TDP-43 plays its role in neurodegeneration. I will highlight the evidence in the current literature that is consistent with this model and the evidence that is still needed from future experiments to test this model.

A model for the loss of TDP-43 function as a central mechanism of pathogenesis in human disease

The TDP-43 protein is normally expressed through transcription and translation, and once produced, it regulates its own expression by a feedback mechanism, i.e., upregulating its own expression when the protein level is too low and inhibiting its expression when the protein level is too high [15,2227]. By this auto-regulatory mechanism, the intracellular level of TDP-43 is maintained within a narrow range (Figure 1, #1 normal). This tightly maintained TDP-43 level may be important because TDP-43 functions in multiprotein/RNA complexes [1821], where a proper structure and function of the complex requires a certain stoichiometric ratio between TDP-43 and its protein and RNA partners (Figure 1, #1 normal). Such a requirement is not unique to TDP-43 complexes as it has been demonstrated in other protein-RNA or protein complexes. For example, in the primary micro RNA (pri-miRNA) processing Drosha complex, overexpression of one subunit DGCR8 leads to an inhibition in the processing activity [46]. As another example, in the kinesin-2 heterotrimeric complex that drives the antegrade transport of late endosomes and lysosomes, overexpression of one subunit KAP3 inhibited the transport similar to the KAP3 knockdown [47].

Figure 1 .
Mechanisms that can cause TDP-43 dysfunction in ALS, FTLD and other neurodegenerative conditions. AD means Alzheimer’s disease, PD Parkinson’s disease, HD Huntington’s disease, LBD Lewy body dementia, DS Down syndrome, HSD hippocampal sclerosis dementia, FBD familial British dementia, and SCA spinal cerebellar ataxia. See the section subtitled “A model for the loss of TDP-43 function as a central mechanism of pathogenesis in human disease” for a detailed description of this diagram.

Xu Molecular Neurodegeneration 2012 7:27   doi:10.1186/1750-1326-7-27   Download authors’ original image


In the disease situation, conditions in patients’ cells become conducive for TDP-43 aggregation. For example, TDP-43 mutants and its C-terminal fragments associated with ALS and FTLD have enhanced aggregation propensity [4851], and therefore, can drive TDP-43 aggregation. The aggregation can lead to a reduction in the pool of TDP-43 that can be incorporated into the TDP-43 protein/RNA complexes (Figure 1, #2 aggregation), thereby reducing the complex function and leading to neurodegeneration.

In model systems where TDP-43 is overexpressed (Figure 1, #3), the function of TDP-43 can be inhibited because an oversupply of exogenous TDP-43 mismatches with a limited supply of its endogenous interacting protein/RNA partners, resulting in the formation of incomplete and dysfunctional complexes. Below I highlight the evidence in the current literature that is consistent with this model and the future experiments that are need to test this model.

TDP-43 performs functions of vital importance, but the consequence of its dysfunction in neurodegeneration remains unclear

A crucial piece of evidence for a loss-of-function mechanism would be demonstration that a loss of TDP-43 function can cause neurodegeneration. This has not yet been experimentally achieved in a convincing manner, particularly in mammalian species. Knockouts in rodents cause early embryonic lethality [2527,36]. Inducible knockout in adult mice causes a rapid loss of fat tissue and lethality [36]. These results have not been informative as to the consequences of TDP-43 dysfunction in the nervous system. Nevertheless, the severity of the phenotype in the knockout models suggests a critical functional importance of TDP-43 in the health and survival of mammalian cells. Indeed, the conditional knockout of TDP-43 in mouse embryonic stem cells causes cell death [36]. Therefore, it is conceivable that TDP-43 function may also be vital for the survival and function of neurons. Supporting this notion are the experiments where TDP-43 knockdown causes morphological abnormalities and cell death in cultured neurons [50,52,53] and a large change in gene expression in cells of the CNS [15,16].

Experimental data from non-mammalian species have also been consistent with the critical functional importance of TDP-43. In C. Elegans, TDP-43 deletion mutants are viable, but show low fertility, slow growth and locomotor defects [44]. In Drosophila, TDP-43 knockout causes abortive embryonic development and lethality [30,31]. Although some escape the lethality and develop to adults, they display severe locomotor defects, premature death and abnormal neuronal morphology [30,31]. Evidence for progressive axonal degeneration and locomotor defects has also been reported in adult TDP-43 knockdown flies [32]. In zebrafish, TDP-43 knockdown during embryonic development causes selective defects in motor axonal growth and results in motor behavioral abnormalities [35]. These results do not conclusively demonstrate a role of TDP-43 dysfunction in neurodegeneration in ALS and FTLD, but do indicate that TDP-43 is important in the development and functioning of the nervous system, thus leaving open the possibility that TDP-43 dysfunction could play a role in neurodegeneration.

How a loss of TDP-43 function explains the pathogenic mechanism of TDP-43 mutants

Mutations in TDP-43 cause motor neuron degeneration and ALS [28,29]. The overwhelming majority of the mutations are located in the C-terminal glycine-rich domain [12], which is unstructured and responsible for interactions with other proteins [17,21,54]. How mutant TDP-43 causes neurodegeneration is not known. Overexpression models support a gain of function, but the reliance of overexpression to elicit neurodegenerative phenotypes risks over-interpretation. A lack of convincing evidence that TDP-43 levels are elevated in human disease leaves open the question of whether the results from the overexpression models are relevant for the human disease.

While there is room for doubt for the gain-of-function mechanism, evidence for the loss-of-function mechanism is also weak, primarily because few experiments have generated data directly relevant to this question, especially in mammalian systems. Nevertheless, reasonable scenarios for this mechanism can be formulated based on the current, albeit fragmented and incomplete, experimental literature. First, wild-type TDP-43 is an aggregation-prone protein and mutant TDP-43 is even more so [48,51,55]. Therefore, TDP-43 mutants can initiate and drive protein aggregation, leading to TDP-43 depletion from the cell nucleus, as has been observed in patients [1,2,56]. In addition, mutant TDP-43 may have an enhanced susceptibility for polypeptide fragmentation, which generates the patient-specific 25-kDa fragments [29,57]. These fragments have a high propensity for aggregation [50,55,58] and can coaggregate with wild-type TDP-43, thereby sequestering wild-type TDP-43 into the aggregates and depleting TDP-43 from the nucleus [50].

Second, the mutant may be functionally less active or inactive but may still retain its autoregulation capability. As a result, the overall TDP-43 level would be maintained but the function of TDP-43 would be reduced because the protein expressed from the mutant allele is dysfunctional. Some experimental data support this scenario. In mice, overexpression of mutant TDP-43 inhibited the expression of the endogenous TDP-43 to the same extent as wild type overexpression [23,37,38], suggesting that the disease-causing mutants retain their autoregulatory function. In Drosophila, wild-type TDP-43 is capable of promoting growth of dendrites and increasing the size of synaptic terminals at the neuromuscular junction. However, these activities are lost in the ALS-causing mutants [31,34], suggesting that the mutants have lost some of the wild-type functions.

Third, mutant TDP-43 may form defective TDP-43 protein/RNA complexes, thereby poisoning the function of the complex. In this capacity, the mutant TDP-43 can act dominant-negatively to inhibit the function of the wild-type allele. There is evidence that TDP-43 forms a homodimer [59] and that multiple TDP-43 molecules are incorporated into each complex [19]. Therefore, if a mutant TDP-43 molecule were capable of rendering dysfunction to the whole complex that contains both mutant and wild-type TDP-43 molecules, then the function of the wild-type allele would be inhibited.

These scenarios are consistent with a model where TDP-43 mutants cause a loss of TDP-43 function by a dominant negative mechanism. Notably, while the first scenario requires the formation of aggregates for cellular toxicity, the second and third scenarios make such a requirement unnecessary. Indeed, in both cellular and animal models, toxicity induced by mutant TDP-43 does not require its aggregation [33,37,39,60].

How TDP-43 dysfunction could contribute to neurotoxicity from overexpression of either mutant or wild-type TDP-43 in model systems

The prevailing interpretation for the observation that overexpression of mutant TDP-43 causes neurodegeneration is that mutant TDP-43 exert its toxicity by a gain of function. However, these results are also consistent with a dominant-negative mechanism, as discussed above (also see Table 1). The dominant-negative model predicts that overexpression of the mutant in sufficient quantities will inhibit the function of the two endogenous wild-type alleles in the model systems.

A puzzling observation is that overexpression of wild-type TDP-43 causes similar neurotoxic phenotypes in model systems [23,33,35,37,38,4043,60,61]. Because of the autoregulatory mechanism, overexpression of human wild-type TDP-43 leads to a suppression of the endogenous TDP-43 [23,24]. This has led to a proposal that a loss of the endogenous TDP-43 caused neurotoxicity [24]. While this proposal can reasonably explain the toxicity of the mutants on the premise that they are dysfunctional, the toxicity from the wild-type TDP-43 poses a problem because several studies have shown that the human wild-type TDP-43 gene can substitute the function of its homologue in species as distant as Drosophila and C. Elegans[30,44]. A more plausible explanation can be derived from the fact that TDP-43 functions in multiprotein/RNA complexes, whose function may depend on a certain stoichiometric composition of the different protein/RNA components. Overexpression of wild-type TDP-43 provides an amount of TDP-43 in excess of the other components that form the complexes, thereby sequestering those components into incomplete and dysfunctional complexes (Figure 1, #3 overexpression). Therefore, both overexpression of the mutants and the wild-type TDP-43 can cause neurodegeneration by dominant-negatively inhibiting the normal function of TDP-43 complexes so long as it interacts with two or more components in the complexes simultaneously and with near equal binding affinities.

While the above interpretation of the literature remains to be confirmed by further experimentation, some of the predictions from this loss-of-function/dominant-negative hypothesis are supported by observations in the current literature. First, overexpression of mutant should be more potent in causing neurodegeneration than overexpression of the wild type, which has been the case in several overexpression models [35,40,60,61]. Although this finding is not inconsistent with the gain-of-function mechanism, the result can also be explained readily by the dominant-negative mechanism outlined above. Overexpression of mutants can inhibit normal TDP-43 function by three mechanisms: (1) displacing the endogenous TDP-43 through the autoregulation mechanism, (2) inserting itself into the TDP-43 complexes in the place of the wild-type protein, and (3) forming dysfunctional complexes by disruption of the stoichiometry between TDP-43 and other protein/RNA components. In contrast, overexpression of the wild-type TDP-43 can inhibit TDP-43 function only through the third mechanism because unlike the mutant protein, it has full function. Therefore, to inhibit TDP-43 function to the same degree, a higher level of expression will be required for the wild-type TDP-43 than the mutant.

Second, if the dominant-negative hypothesis is correct, overexpression and knockout or knockdown of the gene can cause similar phenotypes. Currently, data from mammalian species is lacking to address this point. However, evidence can be drawn from other species. For example, overexpression of either mutant or the wild-type TDP-43 in Drosophila motor neurons causes progressive locomotor defects and a shortening of lifespan [33]. These phenotypes are similar to those caused by TDP-43 knockdown [33]. As another example, expression of human TDP-43 mutants but not the wild type in zebrafish embryos compromised motor axonal growth and caused locomotor defects. Similarly as in flies, knocking down the endogenous TDP-43 caused the same phenotypes [35]. Importantly, the phenotypes in the knockdown fish are rescued by the expression of human wild-type TDP-43 but not the mutants. These results are consistent with the view that the ALS-relevant TDP-43 mutants are dysfunctional and are capable of inhibiting TDP-43 function in a dominant negative manner.

Third, the loss-of-function/dominant-negative hypothesis predicts that ALS-causing mutants should be loss-of-function alleles. As discussed above, the observations that the mutants lost their ability to stimulate the growth of dendrites and axons in flies [31,34,35] and their inability to rescue phenotypes from TDP-43 knockdown in zebrafish [35] supports the loss-of-function proposition. However, key evidence from mammalian species remains to be produced.

While the case for a loss of function by a dominant-negative mechanism can be argued for, it may be overly simplistic to argue that a gain of function does not contribute to the phenotypes caused by TDP-43 overexpression in the model systems. Some evidence indicate that TDP-43 is capable of causing cellular toxicity by a gain of function under ectopic and overexpressed conditions. For example, TDP-43 causes toxicity in yeast, which does not possess an endogenous TDP-43 homologue [62]. Similarly, TDP-43 is not essential in C. Elegans, yet overexpression of human TDP-43 can still cause toxicity that is not observed in knockouts [44,61,63,64]. Therefore, in model systems where TDP-43 performs vital functions, phenotypes caused by TDP-43 overexpression are likely derived from both an interference of endogenous TDP-43 function and a gain of function. Given the complexity in the protein/RNA interaction networks of TDP-43, perhaps this would not be surprising. Overexpression is likely to generate new aberrant interactions as well as to disrupt the authentic interactions that are vital for the cell. Therefore, disentangling these effects will be complex in the overexpression models.

What is the role of wild type TDP-43 in human neurodegeneration

While the case for a loss of function in the TDP-43 mutants and in the overexpression model systems can be made, can the loss-of-function mechanism play a role in patients where TDP-43 is not mutated and not overexpressed? This is an important question because the vast majority of patients with ALS and FTLD-TDP do not have TDP-43 mutations. The answer to this question is yes because even though the primary trigger of the degenerative process lies not in TDP-43 but elsewhere, the same kind of TDP-43 aggregation and nuclear clearance is observed in the CNS of these patients [1,2,45] (Figure 1). The loss-of-function/dominant-negative model will predict that the nuclear clearance and the cytoplasmic aggregation of TDP-43 are probably a significant contributor to neurodegeneration by causing a loss of TDP-43 function. However, the experimental data for testing this prediction is scarce. In Drosophila and zebrafish, knockout or knockdown of TDP-43 produced similar neurodegenerative phenotypes [33,35]. However, further analysis is needed to differentiate the effects of TDP-43 dysfunction on neurodegeneration from those on neurodevelopment, and the relevance of these observations to human neurodegeneration remains to be established. A mammalian model with TDP-43 dysfunction in the mature CNS is urgently needed to understand the effects from a loss of TDP-43-function.

Based on the loss-of-function/dominant-negative hypothesis outlined above, what triggers TDP-43 aggregation will be one of the most intriguing and important questions in understanding the pathogenic mechanisms in ALS and FTLD. Recent investigations have shown that multiple causes can trigger secondary TDP-43 aggregation and nuclear clearance. These causes can be classified into several categories: (1) Gene mutations that enhance the mutant protein aggregation propensity and cause ALS-FTLD with TDP-43 aggregation. Examples in this category include VCP, optineurin, dynactin, ataxin 2 and ubiquilin 2. All the mutant proteins form aggregates and some form coaggregates with wild-type TDP-43 [9,6569]. The mechanism whereby these mutants cause TDP-43 aggregation is not understood. One possibility is that the aggregation of these proteins weakens the capacity of cellular proteostasis [70], which creates an environment conducive for aggregation-prone proteins such as TDP-43 to aggregate. Some of the proteins such as VCP and ubiquilin may be involved in TDP-43 degradation [71,72]. Therefore, mutations in these proteins may directly alter the TDP-43 economy and cause TDP-43 aggregation. (2) Gene mutations that cause ALS and FTLD with TDP-43 aggregation, but the mutant proteins are not involved in protein aggregation themselves. Examples in this category include progranulin, angiogenin and C9ORF72[1,11,73,74]. At present, it is not known how these mutations lead to TDP-43 aggregation. (3) Traumatic brain injury that lead to ALS-FTLD without gene mutations. Repetitive traumatic brain injury has been shown to be associated with ALS and FTLD with intracellular TDP-43 aggregation[75,76]. (4) Other neurodegenerative diseases that are not ALS-FTLD but trigger secondary TDP-43 aggregation. Examples of this category include some of the most common neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease and numerous others [8,77,78] (Figure1). Aggregation of TDP-43 in these cases may also be attributed to a disruption of proteostasis environment due to the aggregation of other proteins, although direct experimental evidence for this hypothesis is not yet in existence. (5) Unknown causes in sporadic ALS and FTLD cases. Some of the speculated causes include genetic predisposition in combination with environmental stress, e.g. environmental toxins, trauma and high physical activity [7982].

Recent studies have suggested that a redistribution of TDP-43 to the cytoplasm may be a precursor to TDP-43 aggregation. In ALS and FTLD patients, some neurons show an increase in cytoplasmic TDP-43 immunoreactivity with diffused or granular appearance, which may represent an early stage of TDP-43 aggregation [8386]. The cause for the cytoplasmic redistribution is not clear. However, a recent study demonstrate that a single traumatic brain injury can be followed by a persistent increase in the cytoplasmic levels of TDP-43 [87], suggesting that injuries to the CNS can be an initial trigger for increased levels of cytoplasmic TDP-43. In model systems, the redistribution of TDP-43 can be triggered by various stresses, including neuronal injury [8890], overexpression of disease-associated mutant TDP-43 and VCP [9193], oxidative stress [93,94] and proteasome inhibition [53]. The functional consequence of the cytoplasmic localization of TDP-43 will require further characterization. Nevertheless, some studies suggest that the cytoplasm-localized TDP-43 is recruited to stress granules before being transformed into aggregates that can persist independent of stress granules [9395]. Another study demonstrated that a modest knockdown of TDP-43 exacerbated, rather than alleviated, cell death that is induced by proteasome inhibition and associated with TDP-43 cytoplasmic translocation [53], suggesting that any toxicity that might be associated with TDP-43 cytoplasmic translocation is derived from a loss of TDP-43 function. These data are consistent with the hypothesis that an increased cytoplasmic level of TDP-43, which follows the initial cellular stress, can lead to TDP-43 aggregation and nuclear depletion.

Therapeutic implications from the dominant-negative model

Discussion on therapeutic implication based on the loss-of-function hypothesis may be premature since the hypothesis remains to be tested. However, such an exercise may be helpful for illustration of the critical importance for a resolution of this question. In the case of a gain of function, strategies that reduce the function should be effective. This may be achieved by lowering the protein levels through an inhibition of its synthesis or a stimulation of its degradation. If the toxic activity is known, strategies that inhibit the specific toxic activity may also be effective. In the case of a loss of function, on the other hand, strategies that increase the function should be effective. This may be achieved by increasing expression and stability of the protein, or stimulating its activity.

The therapeutic strategy for the dominant negative mechanism differs from both purely gain- or loss-of-function mechanisms and will be most challenging. We cannot simply increase the level of TDP-43 because uncontrolled increase of TDP-43 may inhibit the function of TDP-43 rather than improving it. High levels of TDP-43 could also further accelerate its aggregation and produce aberrant interactions with other proteins and RNA. Moreover, we do not understand why TDP-43 stays in the cytoplasm and becomes depleted from the nucleus in the disease. Therefore, it is not clear whether a simple increase of TDP-43 will replenish its level in the nucleus. In the case of mutant TDP-43, allele-specific inhibition of the mutant TDP-43 may be helpful but may not be sufficient to compensate for the lost function of the mutant allele. If the hypothesis that TDP-43 aggregation drives nuclear depletion of the TDP-43 is correct, preventing or reversing the aggregation may be a rational and safe approach to mitigate the loss of TDP-43 function. To achieve this, we need to understand how TDP-43 aggregation is triggered and propagated. We also need to understand the TDP-43 aggregation process at molecular and structural levels. Alternatively, strategies that enhance the function of TDP-43 without resorting to increase the protein level, or retain TDP-43 in the nucleus may also be effective.


TDP-43 aggregation and nuclear depletion have been observed widely in neurodegenerative diseases. The role of TDP-43 in neurodegeneration remains to be defined. Chief among the questions is whether a gain of function, a loss of function or a dominant-negative mechanism is responsible for neurotoxicity. The answer to this question is of critical importance because it guides the future direction of research and sets the foundation for therapeutic strategies. Current experimental data from model systems has been predominantly invoked to support the gain-of-function mechanism. However, a careful review of the data suggests that a loss of TDP-43 function caused by its mutations, its aggregation and nuclear depletion, and the inhibition of TDP-43 function by a dominant-negative mechanism in the overexpression models, are at least as plausible as the gain-of-function theory, if not more so. Therefore, in our future research, we need to gain a more detailed understanding of the normal function of TDP-43, particularly in the cells of the CNS. We need models of loss of TDP-43 function in the CNS, particularly in mammalian species, to understand the consequence of TDP-43 dysfunction. In such a pursuit, models with a partial loss of TDP-43 function may be especially desirable because in humans, it is unlikely that the TDP-43 function is totally lost. We need evidence from human diseases to determine whether the conditions are more in tune with a gain or a loss of TDP-43 function. Lastly, we need to design strategies to address the difficult problem of how to restore the normal levels of TDP-43 function as a therapy.

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Endocrine Action on Midbrain

Writer and Curator: Larry H. Bernstein, MD, FCAP

  • Brain’s Role in Browning White Fat
  • Insulin and leptin act on specialized neurons in the mouse hypothalamus to promote conversion of white to beige fat.

By Anna Azvolinsky | January 15, 2015


Ever since energy-storing white fat has been shown to convert to metabolically active beige fat, through a process called browning, scientists have been trying to understand how this switch occurs. The immune system has been shown to contribute to activation of brown fat cells. Now, researchers from Monash University in Australia and their colleagues have shown that insulin and leptin—two hormones that regulate glucose metabolism and satiety and hunger cues—activate “satiety” neurons in the mouse hypothalamus to promote the conversion of white fat to beige. The results are published today (January 15) in Cell.

Hypothalamic appetite-suppressing proopiomelanocortin (POMC) neurons are known to relay the satiety signals in the bloodstream to other parts of the brain and other tissues to promote energy balance. “What is new here is that one way that these neurons promote calorie-burning is to stimulate the browning of white fat,” said Xiaoyong Yang, who studies the molecular mechanisms of metabolism at the Yale University School of Medicine, but was not involved in the work. “The study identifies how the brain communicates to fat tissue to promote energy dissipation.”

“The authors show that [insulin and leptin] directly interact in the brain to produce nervous-system signaling both to white and brown adipose tissue,” said Jan Nedergaard, a professor of physiology at Stockholm University who also was not involved in the study. “This is a nice demonstration of how the acute and chronic energy status talks to the thermogenic tissues.”

Although the differences between beige and brown fat are still being defined, the former is currently considered a metabolically active fat—which converts the energy of triglycerides into heat—nestled within white fat tissue. Because of their energy-burning properties, brown and beige fat are considered superior to white fat, so understanding how white fat can be browned is a key research question. Exposure to cold can promote the browning of white fat, but the ability of insulin and leptin to act in synergy to signal to the brain to promote browning was not known before this study, according the author Tony Tiganis, a biochemist at Monash.

White fat cells steadily produce leptin, while insulin is produced by cells of the pancreas in response to a surge of glucose into the blood. Both hormones are known to signal to the brain to regulate satiety and body weight. To explore the connection between this energy expenditure control system and fat tissue, Garron Dodd, a postdoctoral fellow in Tiganis’s laboratory, and his colleagues deleted one or both of two phosphatase enzymes in murine POMC neurons. These phosphatase enzymes were previously known to act in the hypothalamus to regulate both glucose metabolism and body weight, each regulating either leptin or insulin signaling. When both phosphatases were deleted, mice had less white fat tissue and increased insulin and leptin signaling.

“These [phosphatase enzymes] work in POMC neurons by acting as ‘dimmer switches,’ controlling the sensitivity of leptin and insulin receptors to their endogenous ligands,” Dodd told The Scientist in an e-mail. The double knockout mice also had an increase in beige fat and more active heat-generating brown fat. When fed a high-fat diet, unlike either the single knockout or wild-type mice, the double knockout mice did not gain weight, suggesting that leptin and insulin signaling to POMC neurons is important for controlling body weight and fat metabolism.

The researchers also infused leptin and insulin directly into the hypothalami of wild-type mice, which promoted the browning of white fat. But when these hormones were infused but the neuronal connections between the white fat and the brain were physically severed, browning was prevented. Moreover, hormone infusion and cutting the neuronal connection to only a single fat pad resulted in browning only in the fat pad that maintained signaling ties to the brain. “This really told us that direct innervation from the brain is necessary and that these hormones are acting together to regulate energy expenditure,” said Tiganis.

These results are “really exciting as, perhaps, resistance to the actions of leptin and insulin in POMC neurons is a key feature underlying obesity in people,” said Dodd.

Another set of neurons in the hypothalamus, the agouti-related protein expressing (AgRP) or “hunger” neurons, are activated by hunger signals and promote energy storage. Along with Tamas Horvath, Yale’s Yang recently showed that fasting activates AgRP neurons that then suppress the browning of white fat. “These two stories are complimentary, providing a bigger picture: that the hunger and satiety neurons control browning of fat depending on the body’s energy state,” said Yang. Activation of POMC neurons during caloric intake protects against diet-induced obesity while activation of AgRP neurons tells the body to store energy during fasting.

Whether these results hold up in humans has yet to be explored. Expression of the two phosphatases in the hypothalamus is known to be higher in obese people, but it is not clear whether this suppresses the browning of white fat.

“One of the next big questions is whether this increased expression and prevention of insulin plus leptin signaling, and conversion of white to brown fat perturbs energy balance and promotes obesity,” said Tiganis. Another, said Dodd, is whether other parts of the brain are involved in signaling to and from adipose tissue.

  1. Dodd et al., “Leptin and insulin act on POMC neurons to promote the browning of white fat,”

Cell, 2015.    http://dx.doi.org:/10.1016/j.cell.2014.12.022   http://medicine.yale.edu/lab/horvath/index.aspx

Our main interest is the neuroendocrine regulation of homeostasis with particular emphasis on metabolic disorders, such as obesity and diabetes, and the effect of metabolic signals on higher brain functions and neurodegeneration. We have active research programs to pursue the role of synaptic plasticity in the mediation of peripheral hormones’ effects on the central nervous system.

We also study the role of mitochondrial membrane potential in normal and pathological brain functions with particular emphasis on the acute effect of mitochondria in neuronal transmission and neuroprotection. We combine classical neurobiological approaches, including electrophysiology and neuroanatomy, with endocrine and genetic techniques to better understand biological events at the level of the organism.

Leptin and Insulin Act on POMC Neurons to Promote the Browning of White Fat

Garron T. Dodd, Stephanie Decherf, Kim Loh, Stephanie E. Simonds, Florian Wiede, Eglantine Balland, Troy L. Merry, et al.



  • Insulin and leptin act synergistically on POMC neurons to promote WAT browning
  • Increased POMC-mediated WAT browning prevents diet-induced obesity
  • PTP1B and TCPTP attenuate leptin and insulin signaling in POMC neurons
  • Combined PTP1B and TCPTP deficiency in POMC neurons promotes white fat browning

The primary task of white adipose tissue (WAT) is the storage of lipids. However, “beige” adipocytes also exist in WAT. Beige adipocytes burn fat and dissipate the energy as heat, but their abundance is diminished in obesity. Stimulating beige adipocyte development, or WAT browning, increases energy expenditure and holds potential for combating metabolic disease and obesity. Here, we report that insulin and leptin act together on hypothalamic neurons to promote WAT browning and weight loss. Deletion of the phosphatases PTP1B and TCPTP enhanced insulin and leptin signaling in proopiomelanocortin neurons and prevented diet-induced obesity by increasing WAT browning and energy expenditure. The coinfusion of insulin plus leptin into the CNS or the activation of proopiomelanocortin neurons also increased WAT browning and decreased adiposity. Our findings identify a homeostatic mechanism for coordinating the status of energy stores, as relayed by insulin and leptin, with the central control of WAT browning.  http://www.cell.com/cms/attachment/2023992410/2043906325/fx1.jpg

Light on the Brain

Researchers find that photoreceptors expressed in zebrafish hypothalamus contribute to light-dependent behavior.

By Sabrina Richards | September 20, 2012

A 21 day old zebrafish. Their optical clarity and relatively easy maintenance make them a favorite for geneticists and developmental biologists. In this fish, the muscles can be seen as chevron shapes in the tail, the swim bladder as a “bubble” just behind the head, and the food that the fish has been eating as a brown patch just below the swim bladder.

Juvenile zebrafish. Shawn Burgess, NHGRI

Zebrafish larvae without eyes or pineal glands can still respond to light using photopigments located deep within their brains.  Published today (September 20) in Current Biology, the findings are the first to link opsins, photoreceptors in the hypothalamus and other brain areas, to increased swimming in response to darkness, a behavior researchers hypothesize may help the fish move toward better-lit environments.

“[It’s a] strong demonstration that opsin-dependent photoreceptors in deep brain areas affect behaviors,” said Samer Hattar, who studies light reception in mammals at Johns Hopkins University but did not participate in the research.

Photoreceptors in eyes enable vision, and photoreceptors in the pineal gland, a small endocrine gland located in the center of the vertebrate brain, regulate circadian rhythms. But photoreceptors are also found in other brain areas of both invertebrates and vertebrate lineages. The function of these extraocular photoreceptors has been best studied in birds, where they regulate seasonal reproduction, explained Harold Burgess, a behavioral neurogeneticist at the Eunice Kennedy Shriver National Institute for Child Health and Human Development. Many opsins have been reported in the brains of tiny and transparent larval zebrafish, raising the possibility that light could be stimulating the photoreceptors even deep in the brain. To test for behaviors that may be regulated by deep brain photoreceptors, Burgess and his colleagues in Wolfgang Driever’s lab at the University of Freiburg removed the eyes of zebrafish larvae, and compared their behavior to larvae that retained their eyes. Although most light-dependent behavior required eyes, the eyeless larvae did respond when the lights were turned off, increasing their activity for a several minutes, though to a somewhat lesser extent than control larvae. But the fact that they responded at all suggests that non-retinal photoreceptors contributed to the behavior.

To confirm the role of the deep brain photoreceptors, the researchers also tested eyeless larvae that had been genetically modified to block expression of photoreceptors in the pineal gland. This fish still showed this jump in activity for several minutes after entering darkness.

Two different types of opsins—melanopsin and multiple tissue opsin—are expressed in the same type of neuron in zebrafish hypothalamus. Burgess and his colleagues looked at zebrafish missing the transcription factor Orthopedia, which is unique to these neurons, and found that the darkness-induced activity boost is nearly absent in these fish. To further narrow the search for the responsible photoreceptors, the researchers overexpressed melanopsin in hypothalamus neurons that co-express Orthopedia and melanopsin, and found that it increased the sensitivity of eyeless zebrafish to reductions in light. The results point to both melanopsin and Orthopedia as key players in modulating this behavior and pinpoint the location to neurons that coexpress these factors in the zebrafish hypothalamus.

Interestingly, the hypothalamus is one of the oldest parts of the vertebrate brain, said Detlev Arendt, a developmental biologist at the European Molecular Biology Laboratory in Heidelberg. “It’s very possible that this is one of the oldest functions”—one that evolved in “non-visual organisms” that had no eyes but still needed to sense light.

Although not as directed and efficient as eye-dependent behaviors that help fish swim toward light, Burgess speculates that deep brain opsins can still benefit zebrafish larvae. “You could imagine situation where it can’t see light, if a leaf falls on it and it doesn’t know where to swim. I think this behavior puts it in a hyperactive state where it swims wildly for several minutes until it reaches enough light for eyes to take over,” he explained, noting that such behavior is common in invertebrates.

It remains to be seen whether these deep brain opsins regulate other behaviors, perhaps in similar fashion to seasonal hormonal regulation in birds, but Hattar believes it is likely. “It’s beyond reasonable doubt there are many functions for these deep brain photoreceptors.”

Fernandes et al., “Deep brain photoreceptors control light-seeking behavior in zebrafish larvae,” Current Biology, 22:1-6, 2012.

Neuroendocrine basis of sexuality, mood, anxiety, social consciousness

Physiology, signaling, and pharmacology of galanin peptides and receptors: Three decades of emerging diversity

Lang, R., Gundlach, A.L., Holmes, F.E., (…), Hökfelt, T., Kofler, B.
Pharmacological Reviews 2015: 67 (1), pp. 118-175

Galanin was first identified 30 years ago as a “classic neuropeptide,” with actions primarily as a modulator of neurotransmission in the brain and peripheral nervous system. Other structurally-related peptides—galanin-like peptide and alarin—with diverse biologic actions in brain and other tissues have since been identified, although, unlike galanin, their cognate receptors are currently unknown. Over the last two decades, in addition to many neuronal actions, a number of nonneuronal actions of galanin and other galanin family peptides have been described. These include actions associated with neural stem cells, nonneuronal cells in the brain such as glia, endocrine functions, effects on metabolism, energy homeostasis, and paracrine effects in bone. Substantial new data also indicate an emerging role for galanin in innate immunity, inflammation, and cancer. Galanin has been shown to regulate its numerous physiologic and pathophysiological processes through interactions with three G protein–coupled receptors, GAL1, GAL2, and GAL3, and signaling via multiple transduction pathways, including inhibition of cAMP/PKA (GAL1, GAL3) and stimulation of phospholipase C (GAL2). In this review, we emphasize the importance of novel galanin receptor–specific agonists and antagonists. Also, other approaches, including new transgenic mouse lines (such as a recently characterized GAL3 knockout mouse) represent, in combination with viral-based techniques, critical tools required to better evaluate galanin system physiology. These in turn will help identify potential targets of the galanin/galanin-receptor systems in a diverse range of human diseases, including pain, mood disorders, epilepsy, neurodegenerative conditions, diabetes, and cancer.

Estradiol regulates responsiveness of the dorsal premammillary nucleus of the hypothalamus and affects fear- and anxiety-like behaviors in female rats

Litvin, Y., Cataldo, G., Pfaff, D.W., Kow, L.-M.
European Journal of Neuroscience 2014; 40 (2), pp. 2344-2351

Research suggests a causal link between estrogens and mood. Here, we began by examining the effects of estradiol (E2) on rat innate and conditioned defensive behaviors in response to cat odor. Second, we utilized whole-cell patch clamp electrophysiological techniques to assess noradrenergic effects on neurons within the dorsal premammillary nucleus of the hypothalamus (PMd), a nucleus implicated in fear reactivity, and their regulation by E2. Our results show that E2 increased general arousal and modified innate defensive reactivity to cat odor. When ovariectomized females treated with E2 as opposed to oil were exposed to cat odor, they showed elevations in risk assessment and reductions in freezing, indicating a shift from passive to active coping. In addition, animals previously exposed to cat odor showed clear cue + context conditioning 24 h later. However, although E2 persisted in its effects on general arousal in the conditioning task, its effects on fear disappeared. In the patch clamp experiments noradrenergic compounds that typically induce fear clearly excited PMd neurons, producing depolarizations and action potentials. E2 treatment shifted some excitatory effects of noradrenergic agonists to inhibitory, possibly by differentially affecting α- and β-adrenoreceptors. In summary, our results implicate E2 in general arousal and fear reactivity, and suggest these may be governed by changes in noradrenergic responsivity in the PMd. These effects of E2 may have ethological relevance, serving to promote mate seeking even in contexts of ambiguous threat and shed light on the involvement of estrogen in mood and its associated disorders.

Endogenous opiates and behavior: 2013

Richard J. Bodnar
Peptides 62 (2014) 67–136

This paper is the thirty-sixth consecutive installment of the annual review of research concerning the endogenous opioid system. It summarizes papers published during 2013 that studied the behavioral effects of molecular, pharmacological and genetic manipulation of opioid peptides, opioid receptors, opioid agonists and opioid antagonists. The particular topics that continue to be covered include the molecular-biochemical effects and neurochemical localization studies of endogenous opioids and their receptors related to behavior, and the roles of these opioid peptides and receptors in pain and analgesia; stress and social status; tolerance and dependence; learning and memory; eating and drinking; alcohol and drugs of abuse; sexual activity and hormones, pregnancy, development and endocrinology; mental illness and mood; seizures and neurologic disorders; electrical-related activity and neurophysiology; general activity and locomotion; gastrointestinal, renal and hepatic functions; cardiovascular responses; respiration and thermoregulation; and immunological responses.

Brain aromatase (cyp19a1b) and gonadotropin releasing hormone (gnrh2 and gnrh3) expression during reproductive development and sex change in black sea bass (Centropristis striata)

Timothy S Breton, Matthew A DiMaggio, Stacia A Sowe, David L Berlinsky, et al.
Comparative Biochemistry and Physiology, Part A 181 (2015) 45–53

Teleost fish exhibit diverse reproductive strategies, and some species are capable of changing sex. The influence of many endocrine factors, such as gonadal steroids and neuropeptides, has been studied in relation to sex change, but comparatively less research has focused on gene expression changes within the brain in temperate grouper species with non-haremic social structures. The purpose of the present study was to investigate gonadotropin releasing hormone (GnRH) and brain aromatase (cyp19a1b) gene expression patterns during reproductive development and sex change in protogynous (female to male) black sea bass (Centropristis striata). Partial cDNA fragments for cyp19a1b and eef1a (a reference gene) were identified, and included with known gnrh2 and gnrh3 sequences in real time quantitative PCR. Elevated cyp19a1b expression was evident in the olfactory bulbs, telencephalon, optic tectum, and hypothalamus/
midbrain region during vitellogenic growth, which may indicate changes in the brain related to neurogenesis or sexual behavior. In contrast, gnrh2 and gnrh3 expression levels were largely similar among gonadal states, and all three genes exhibited stable expression during sex change. Although sex change in black sea bass is not associated with dramatic changes in GnRH or cyp19a1b gene expression among brain regions, these genes may mediate processes at other levels, such as within individual hypothalamic nuclei, or through changes in neuron size, that warrant further research.

Evaluation for roles of neurosteroids in modulating forebrain mechanisms controlling vasopressin secretion and related phenomena in conscious rats

Ken’ichi Yamaguchi
Neuroscience Research xxx (2015) xxx–xxx

Anteroventral third ventricular region (AV3V) regulates autonomic functions through a GABAergic mechanism that possesses neuroactive steroid (NS)-synthesizing ability. Although NS can exert effects by acting on a certain type of GABAA-receptor (R), it is not clear whether NS may operate to modulateAV3V GABAergic activity for controlling autonomic functions. This study aimed to investigate the issue.AV3V infusion with a GABAA antagonist bicuculline increased plasma vasopressin (AVP), glucose, blood pressure (BP), and heart rate in rats. These events were abolished by preinjecting its agonist muscimol, whereas the infusion with allopregnanolone, a NS capable of potentiating GABAA-R function, affectednone of the variables in the absence or presence of such bicuculline actions. Similarly, AV3V infusion with pregnanolone sulfate, a NS capable of antagonizing GABAA-R, produced no effect on those variables.AV3V infusion with muscimol was effective in inhibiting the responses of plasma AVP or glucose, orBP to an osmotic loading or bleeding. However, AV3V infusion with aminoglutethimide, a NS synthesis inhibitor, did not affect any of the variables in the absence or presence of those stimuli. These results suggest that NS may not cause acute effects on the AV3V GABAergic mechanism involved in regulating AVP release and other autonomic function.

Novel receptor targets for production and action of allopregnanolone in the central nervous system: a focus on pregnane xenobiotic receptor

Cheryl A. Frye, Carolyn J. Koonce, and Alicia A. Walf
Front in Cell Neurosci Apr 2014; 8(106)

Neurosteroids are cholesterol-based hormones that can be produced in the brain, independent of secretion from peripheral endocrine glands, such as the gonads and adrenals. A focus in our laboratory for over 25 years has been how production of the pregnane neurosteroid, allopregnanolone, is regulated and the novel (i.e., non steroid receptor) targets for steroid action for behavior. One endpoint of interest has been lordosis, the mating posture of female rodents. Allopregnanolone is necessary and sufficient for lordosis, and the brain circuitry underlying it, such as actions in the midbrain ventral tegmental area (VTA), has been well-characterized. Published and recent findings supporting a dynamic role of allopregnanolone are included in this review. First, contributions of ovarian and adrenal sources of precursors of allopregnanolone, and the requisite enzymatic actions for de novo production in the central nervous system will be discussed.
Second, how allopregnanolone produced in the brain has actions on behavioral processes that are independent of binding to steroid receptors, but instead involve rapid modulatory actions via neurotransmitter targets (e.g., g-amino butyric acid-GABA, Nmethyl-D-aspartate- NMDA) will be reviewed.
Third, a recent focus on characterizing the role of a promiscuous nuclear receptor, pregnane xenobiotic receptor (PXR), involved in cholesterol metabolism and expressed in the VTA, as a target for allopregnanolone and how this relates to both actions and production of allopregnanolone will be addressed. For example, allopregnanolone can bind PXR and knocking down expression of PXR in the midbrain VTA attenuates actions of allopregnanolone via NMDA and/or GABAA for lordosis. Our understanding of allopregnanolone’s actions in the VTA for lordosis has been extended to reveal the role of allopregnanolone for broader, clinically-relevant questions, such as neurodevelopmental processes, neuropsychiatric disorders, epilepsy, and aging.

Long-term dysregulation of brain corticotrophin and glucocorticoid receptors and stress reactivity by single early-life pain experience in male and female rats

Nicole C. Victoria, Kiyoshi Inoue, Larry J. Young, Anne Z. Murphy
Psychoneuroendocrinology (2013) 38, 3015—3028

Inflammatory pain experienced on the day of birth (postnatal day 0: PD0) significantly dampens behavioral responses to stress- and anxiety-provoking stimuli in adult rats. However, to date, the mechanisms by which early life pain permanently alters adult stress responses remain unknown. The present studies examined the impact of inflammatory pain, experienced on the day of birth, on adult expression of receptors or proteins implicated in the activation and termination of the stress response, including corticotrophin releasing factor receptors (CRFR1 and CRFR2) and glucocorticoid receptor (GR). Using competitive receptor autoradiography, we show that Sprague Dawley male and female rat pups administered 1% carrageenan into the intraplantar surface of the hindpaw on the day of birth have significantly decreased CRFR1 binding in the basolateral amygdala and midbrain periaqueductal gray in adulthood. In contrast, CRFR2 binding, which is associated with stress termination, was significantly increased in the lateral septum and cortical amygdala. GR expression, measured with in situ hybridization and immunohistochemistry, was significantly increased in the paraventricular nucleus of the hypothalamus and significantly decreased in the hippocampus of neonatally injured adults. In parallel, acute stress-induced corticosterone release was significantly attenuated and returned to baseline more rapidly in adults injured on PD0 in comparison to controls. Collectively, these data show that early life pain alters neural circuits that regulate responses to and neuroendocrine recovery from stress, and suggest that pain experienced by infants in the Neonatal Intensive Care Unit may permanently alter future responses to anxiety- and stress provoking stimuli.

Dysruption of Corticotropin Releasing Factor in hypocampal region

Stress and trauma: BDNF control of dendritic-spine formation and regression

M.R. Bennett, J. Lagopoulos
Progress in Neurobiology 112 (2014) 80–99

Chronic restraint stress leads to increases in brain derived neurotrophic factor (BDNF) mRNA and protein in some regions of the brain, e.g. the basal lateral amygdala (BLA) but decreases in other regions such as the CA3 region of the hippocampus and dendritic spine density increases or decreases in line with these changes in BDNF. Given the powerful influence that BDNF has on dendritic spine growth, these observations suggest that the fundamental reason for the direction and extent of changes in dendritic spine density in a particular region of the brain under stress is due to the changes in BDNF there.
The most likely cause of these changes is provided by the stress initiated release of steroids, which readily enter neurons and alter gene expression, for example that of BDNF. Of particular interest is how glucocorticoids and mineralocorticoids tend to have opposite effects on BDNF gene expression offering the possibility that differences in the distribution of their receptors and of their downstream effects might provide a basis for the differential transcription of the BDNF genes. Alternatively, differences in the extent of methylation and acetylation in the epigenetic control of BDNF transcription are possible in different parts of the brain following stress.
Although present evidence points to changes in BDNF transcription being the major causal agent for the changes in spine density in different parts of the brain following stress, steroids have significant effects on downstream pathways from the TrkB receptor once it is acted upon by BDNF, including those that modulate the density of dendritic spines.
Finally, although glucocorticoids play a canonical role in determining BDNF modulation of dendritic spines, recent studies have shown a role for corticotrophin releasing factor (CRF) in this regard. There is considerable improvement in the extent of changes in spine size and density in rodents with forebrain specific knockout of CRF receptor 1 (CRFR1) even when the glucocorticoid pathways are left intact. It seems then that CRF does have a role to play in determining BDNF control of dendritic spines.

Central CRF system perturbation in an Alzheimer’s disease knockin mouse model

Qinxi Guo, Hui Zheng, Nicholas John Justice
Neurobiology of Aging 33 (2012) 2678–2691

Alzheimer’s disease (AD) is often accompanied by changes in mood as well as increases in circulating cortisol levels, suggesting that regulation of the stress responsive hypothalamic-pituitary-adrenal (HPA) axis is disturbed. Here, we show that amyloid precursor protein (APP) is endogenously expressed in important limbic, hypothalamic, and midbrain nuclei that regulate hypothalamic-pituitary-adrenal axis activity. Furthermore, in a knockin mouse model of AD that expresses familial AD (FAD) mutations of both APP with humanized amyloid beta (hA), and presenilin 1 (PS1), in their endogenous patterns (APP/hA/PS1 animals), corticotropin releasing factor (CRF) levels are increased in key stress-related nuclei, resting corticosteroid levels are elevated, and animals display increased anxiety-related behavior. Endocrine and behavioral phenotypes can be normalized by loss of 1 copy of CRF receptor type-1 (Crfr1), consistent with a perturbation of central CRF signaling in APP/hA/PS1 animals. However, reductions in anxiety and corticosteroid levels conferred by heterozygosity of CRF receptor type-1 do not improve a deficit in working memory observed in APP/hA/PS1 mice, suggesting that perturbations of the CRF system are not the primary cause of decreased cognitive performance.

Alzheimer’s disease-like neuropathology of gene-targeted APP-SLxPS1mut mice expressing the amyloid precursor protein at endogenous levels

Christoph Kohler, Ulrich Ebert, Karlheinz Baumann, and Hannsjorg Schroeder
Neurobiology of Disease 20 (2005) 528 – 540

Most transgenic mice used for preclinical evaluation of potential disease-modifying treatments of Alzheimer’s disease develop major histopathological features of this disease by several-fold overexpression of the human amyloid precursor protein. We studied the phenotype of three different strains of gene-targeted mice which express the amyloid precursor protein at endogenous levels. Only further crossing with transgenic mice overexpressing mutant human presenilin1 led to the deposition of extracellular amyloid, accompanied by the deposition of apolipoprotein E, an astrocyte and microglia reaction, and the occurrence of dilated cholinergic terminals in the cortex. Features of neurodegeneration, however, were absent. The pattern of plaque development and deposition in these mice was similar to that of amyloid precursor protein overproducing strains if crossed to presenilin1-transgenics. However, plaque development started much later and developed slowly until the age of 18 months but then increased more rapidly.

Central Cholinergic Functions In Human Amyloid Precursor Protein Knock-In/Presenilin-1 Transgenic Mice

Hartmann, C. Erb, U. Ebert, K. H. Baumann, A. Popp, G. Koenig, J. Klein
Neuroscience 125 (2004) 1009–1017

Alzheimer’s disease is characterized by amyloid peptide formation and deposition, neurofibrillary tangles, central cholinergic dysfunction, and dementia; however, the relationship between these parameters is not well understood. We studied the effect of amyloid peptide formation and deposition on central cholinergic function in knock-in mice carrying the human amyloid precursor protein (APP) gene with the Swedish/London double mutation (APP-SL mice) which were crossbred with transgenic mice overexpressing normal (PS1wt) or mutated (M146L; PS1mut) human presenilin-1. APP-SLxPS1mut mice had increased levels of Aβ peptides at 10 months of age and amyloid plaques at 14 months of age while APP-SLPS1wt mice did not have increased peptide levels and did not develop amyloid plaques. We used microdialysis in 15–27 months old mice to compare hippocampal acetylcholine (ACh) levels in the two mouse lines and found that extracellular ACh levels were slightly but significantly reduced in the APP-SLPS1mut mice (-26%; P=0.044). Exploratory activity in the open field increased hippocampal ACh release by two-fold in both mouse lines; total and relative increases were not significantly different for the two strains under study. Similarly, infusion of scopolamine (1 µM) increased hippocampal ACh release to a similar extent (3–5-fold) in both groups. High-affinity choline uptake, a measure of the ACh turnover rate, was identical in both mouse lines. Neurons expressing choline acetyltransferase were increased in the septum of APP-SLPS1mut mice (26%; P =0.046). We conclude that amyloid peptide production causes a small decrease of extracellular ACh levels. The deposition of amyloid plaques, however, does not impair stimulated ACh release and proceeds without major changes of central cholinergic function.

Glutamate Neurotoxicity

Glutamate Neurotoxicity and Diseases of the Nervous System

Dennis W. Choi
Neuron. Oct, 1988; 1: 623-634

A growing number of studies now suggest that the cellular mechanisms which normally participate in signaling in the central nervous system (CNS) can be transformed by disease into instruments of neuronal cell destruction. Excitatory synaptic transmission in the mammalian CNS is principally mediated by L-glutamate. In fact, glutamate excites virtually all central neurons and is present in nerve terminals at millimolar levels (Curtis and Johnston, 1974). Normally, the extracellular levels of glutamate rise to high levels only in the brief and spatially localized fashion appropriate to synaptic transmission. This is fortunate, because as Lucas and Newhouse first showed in 1957, sustained exposure to glutamate can destroy retinal neurons. In a subsequent set of pioneering experiments, Olney (Olney and Sharpe, 1969; Olney et al., 1971) established that this toxicity, which he later called excitotoxicity, was not unique to glutamate or to retinal neurons, but was a feature common to the actions of all excitatory amino acids on central neurons. He postulated therefore that glutamate, or related compounds, might be the cause of the neuronal cell loss found in certain neurological diseases. In recent years, this hypothesis has gathered considerable support, fueled by new insights into glutamate receptor function and the development of effective glutamate antagonist drugs. The evidence is most convincing in diseases involving an acute insult to the brain, as occurs in a stroke, with abrupt deprivation of blood supply. But neurotoxicity due to excitatory amino acids may also be involved in slowly progressive degenerative diseases such as Huntington’s disease. Although the detailed molecular basis of glutamate neurotoxicity is not known, it appears that Ca2+ influx may play a critical role.
Glutamate interacts with at least three classes of membrane receptors, each commonly referred to by preferred pharmacological agonists: N-methyl-o-aspartate (NMDA), quisqualate, and kainate (Watkins and Olverman, 1987) (Figure I). These three classes are linked to membrane cation channels. A second type of quisqualate receptor has been additionally linked to a second messenger system (see below). It has been suggested that all three classes might actually be substates of a single molecular complex, but binding studies and newer physiological studies favor separate structures.

Quisqualate                         NMDA                       Kainate

Three Classes of Glutamate Receptors

Three Classes of Glutamate Receptors

Three Classes of Glutamate Receptors

One type of quisqualate receptor stimulates the formation of inositol 1,4,5-trisphosphate UPS) and diacylglycerol (DAG) from phosphatidylinositol-4,5-biphosphate (PIP,); the other is linked directly to a Na+ ionophore. Activation of the quisqualate receptor-ionophore complex can be potentiated by Zn2+. The NMDA receptor opens a channel permeable to Ca2+ as well as Na+; this receptor-channel complex has several modulatory sites discussed in the text. The kainate receptor opens an ionophore permeable to Na+.

Best defined is the NMDA receptor. This receptor opens a distinctive membrane channel characterized by high conductance (main state about 50 pS), voltage dependent Mgz+ blockade and permeability to both Ca2+ and Na+. The NMDA receptor can be selectively activated by several endogenous compounds, including L-aspartate, homocysteate, and quinolinate. Activation requires the coavailability of glycine in near micromolar concentrations. The action of glutamate at the NMDA receptor can be selectively antagonized: competitively by 2-amino-5-phosphonovalerate (APV) and 2-amino-5-phosphonoheptanoate (APH), or noncompetitively by drugs that bind to the phencyclidine site within the open channel (such as phencyclidine, MK-801, dextrorphan, or ketamine. The NMDA receptor-activated channel can also be blocked noncompetitively by Znz+, most likely at a site different from that which binds Mg2.
Although glutamate has high affinity for all three classes of postsynaptic receptors, it is not easy to demonstrate its neurotoxicity in vivo. Even when directly injected into brain, bypassing the blood-brain barrier, extremely high doses of glutamate are required to create lesions.  Mangano & Schwartz found that they could infuse 0.5 crl/hr of a 300 mM glutamate solution into the hippocampus of a rat for 2 weeks without producing neuronal injury. This apparent low in vivo neurotoxic potency of glutamate may represent one reason why Olney’s “glutamate hypothesis” of neurological disease did not initially achieve a more widespread following. However, in fact, glutamate is a potent and rapidly acting neurotoxin; its neurotoxicity in vivo is likely masked by the efficiency of normal cellular uptake mechanisms in removing glutamate from the extracellular space. Glutamate neurotoxicity can be most directly studied in cell culture where bath exposure is not limited by cellular uptake.
The toxic changes produced by glutamate or related excitatory amino acids in vivo are of two sorts:

  1. acute swelling of neuronal dendrites and cell bodies and a
  2. more slowly evolving neuronal degeneration (Olney, 1986).

Axons and glia are relatively spared, although high levels of excitatory amino acids can produce some swelling of glia. A hallmark of excitatory amino acid neurotoxicity is its cellular selectivity, with distinctive patterns of neuronal loss produced by different excitatory amino acids and different routes of administration. For example, Nadler and co-workers (1978) found that intraventricular kainate preferentially destroys hippocampal CA3 neurons but spares dentate granule neurons. Different neuronal subpopulations
may differ in their intrinsic vulnerability to damage.

Possible Mechanisms Involved in Glutamate Neurotoxicity

How Ca*+ may mediate glutamate-induced neuronal degeneration. Glutamate acts on NMDA, non-NMDA, and “metabotropic” receptors (the quisqualate receptor linked to a second messenger system) to produce an increase in cytosolic free Ca*+. This cytosolic Ca *+, in concert with diacylglycerol liberated by the quisqualate-triggered second messenger system, activates protein kinase C, which acts via a number of mechanisms (primarily by altering membrane ion channels) to increase neuronal excitability and further increase cytosolic Ca*+. Elevated cytosolic Ca2+ then activates several enzymes capable of either directly or indirectly (through free radical formation) destroying cellular structure. Glutamate released from synaptic terminals or leaking nonspecifically from ruptured neurons contributes to additional injury propagation.

Glutamate Neurotoxicity in Perspective

The hypothesis that excitatory amino acids may specifically mediate pathological neuronal injury gives new form to this age-old enemy and raises the tantalizing possibility that current molecular and cellular insights into excitatory amino acid transmitter systems might be harnessed to develop an efficacious clinical therapy. Some points of attack are already apparent; others will likely be defined as the biology of excitatory amino acids continues to be unraveled. An intriguing area for investigation is the relationship between excitatory amino acid neurotoxicity and normal neuronal processes such as maturation, neurite outgrowth, and synaptic plasticity.

Glutamate Toxicity in a Neuronal Cell line Involves Inhibition of Cystine Transport Leading to Oxidative Stress

Timothy H. Murphy, M Miyamoto, A Sastre, R Schnaar and JT Coyle
Neuron 1989: 2: 1547-88.

Glutamate binds to both excitatory neurotransmitter binding sites and a W-dependent, quisqualate- and cystine-inhibited transport site on brain neurons. The neuroblastoma-primary retina hybrid cells (NWRE-105) are susceptible to glutamate-induced cytotoxicity. The Cl–dependent transport site to which glutamate and quisqualate (but not kainate or NMDA) bind has a higher affinity for cystine than for glutamate. Towering cystine concentrations in the cell culture medium results in cytotoxicity similar to that induced by glutamate addition in its morphology, kinetics, and CaZ+ dependence. Glutamate-induced cytotoxicity is directly proportional to its ability to inhibit cystine uptake. Exposure to glutamate (or lowered cystine) causes a decrease in glutathione levels and an accumulation of intracellular peroxides. Like NW-RE-105 cells, primary rat hippocampal neurons (but not glia) in culture degenerate in medium with lowered cystine concentration. Thus, glutamate-induced cytotoxicity in N18-RE-105 cells is due to inhibition of cystine uptake, resulting in lowered glutathione levels leading to oxidative stress and cell death.

Mechanism of glutamate-induced neurotoxicity in HT22 mouse hippocampal cells

Masayuki Fukui, Ji-Hoon Song, Jinyoung Choi, Hye Joung Choi, Bao Ting Zhu
European Journal of Pharmacology 617 (2009) 1–11

Glutamate is an endogenous excitatory neurotransmitter. At high concentrations, it is neurotoxic and contributes to the development of certain neurodegenerative diseases. There is considerable controversy in the literature with regard to whether glutamate-induced cell death in cultured HT22 cells (an immortalized mouse hippocampal cell line) is apoptosis, necrosis, or a new form of cell death. The present study focused on investigating the mechanism of glutamate-induced cell death. We found that glutamate induced, in a time dependent manner, both necrosis and apoptosis in HT22 cells. At relatively early time points (8–12 h), glutamate induced mostly necrosis, whereas at late time points (16–24 h), it induced mainly apoptosis. Glutamate-induced mitochondrial oxidative stress and dysfunction were crucial early events required for the induction of apoptosis through the release of the mitochondrial apoptosis-inducing factor (AIF), which catalyzed DNA fragmentation (an ATP-independent process). Glutamate-induced cell death proceeded independently of the Bcl-2 family proteins and caspase activation. The lack of caspase activation likely resulted from the lack of intracellular ATP when the mitochondrial functions were rapidly disrupted by the mitochondrial oxidative stress. In addition, it was observed that activation of JNK, p38, and ERK signaling molecules was also involved in the induction of apoptosis by glutamate. In conclusion, glutamate-induced apoptosis is AIF-dependent but caspase-independent, and is accompanied by DNA ladder formation but not chromatin condensation.

Understanding Low Reliability of Memories for Neutral Information Encoded under Stress: Alterations in Memory-Related Activation in the Hippocampus and Midbrain

Shaozheng Qin, EJ Hermans, HJF van Marle, and G Fernandez, et al.
The Journal of Neuroscience, Mar 21, 2012; 32(12): 4032–4041

Exposure to an acute stressor can lead to unreliable remembrance of intrinsically neutral information, as exemplified by low reliability of eyewitness memories, which stands in contrast with enhanced memory for the stressful incident itself. Stress-sensitive neuromodulators (e.g., catecholamines) are believed to cause this low reliability by altering neurocognitive processes underlying memory formation. Using event-related functional magnetic resonance imaging, we investigated neural activity during memory formation in 44 young, healthy human participants while incidentally encoding emotionally neutral, complex scenes embedded in either a stressful or neutral context.
We recorded event-related pupil dilation responses as an indirect index of phasic noradrenergic activity. Autonomic, endocrine, and psychological measures were acquired to validate stress manipulation. Acute stress during encoding led to a more liberal response bias (more hits and false alarms) when testing memory for the scenes 24 h later. The strength of this bias correlated negatively with pupil dilation responses and positively with stress-induced heart rate increases at encoding. Acute stress, moreover, reduced subsequent memory effects (SMEs; items later remembered vs forgotten) in hippocampus and midbrain, and in pupil dilation responses.
The diminished SMEs indicate reduced selectivity and specificity in mnemonic processing during memory formation. This is in line with a model in which stress-induced catecholaminergic hyperactivation alters phasic neuromodulatory signaling in memory-related circuits, resulting in generalized (gist-based) processing at the cost of specificity. Thus, one may speculate that loss of specificity may yield less discrete memory representations at time of encoding, thereby causing a more liberal response bias when probing these memories.

Neuroendocrinology – Signaling, neuron plasticity and memory

Leptin Signaling Modulates the Activity of Urocortin 1 Neurons in the Mouse Nonpreganglionic Edinger-Westphal Nucleus

Lu Xu, Wim J. J. M. Scheenen, Rebecca L. Leshan, Christa M. Patterson, et al.
Endocrinology 152(3): 979–988, 2011

A recent study systematically characterized the distribution of the long form of the leptin receptor (LepRb) in the mouse brain and showed substantial LepRb mRNA expression in the nonpreganglionic Edinger-Westphal nucleus (npEW) in the rostroventral part of the midbrain. This nucleus hosts the majority of urocortin 1 (Ucn1) neurons in the rodent brain, and because Ucn1 is a potent satiety hormone and electrical lesioning of the npEW strongly decreases food intake, we have hypothesized a role of npEW-Ucn1 neurons in leptin-controlled food intake. Here, we show by immunohistochemistry that npEW-Ucn1 neurons in the mouse contain LepRb and respond to leptin administration with induction of the Janus kinase 2-signal transducer and activator of transcription 3 pathway, both in vivo and in vitro. Furthermore, systemic leptin administration increases the Ucn1 content of then pEW significantly, whereas in mice that lack LepRb (db/db mice), then pEW contains considerably reduced amount of Ucn1. Finally, we reveal by patch clamping of midbrain Ucn1 neurons that leptin administration reduces the electrical firing activity of the Ucn1 neurons. In conclusion, we provide ample evidence for leptin actions that go beyond leptin’s well-known targets in the hypothalamus and propose that leptin can directly influence the activity of the midbrain Ucn1 neurons.

Leptin regulation of hippocampal synaptic function in health and disease

Andrew J. Irving and Jenni Harvey
Trans. R. Soc. B 369: 20130155 http://dx.doi.org/10.1098/rstb.2013.0155

The endocrine hormone leptin plays a key role in regulating food intake and body weight via its actions in the hypothalamus. However, leptin receptors are highly expressed in many extra-hypothalamic brain regions and evidence is growing that leptin influences many central processes including cognition. Indeed, recent studies indicate that leptin is a potential cognitive enhancer as it markedly facilitates the cellular events underlying hippocampal-dependent learning and memory, including effects on glutamate receptor trafficking, neuronal morphology and activity-dependent synaptic plasticity. However, the ability of leptin to regulate hippocampal synaptic function markedly declines with age and aberrant leptin function has been linked to neurodegenerative disorders such as Alzheimer’s disease (AD). Here, we review the evidence supporting a cognitive enhancing role for the hormone leptin and discuss the therapeutic potential of using leptin-based agents to treat AD.

The Y2 receptor agonist PYY3–36 increases the behavioral response to novelty and acute dopaminergic drug challenge in mice

Ulrike Stadlbauer, Elisabeth Weber, Wolfgang Langhans and Urs Meyer
International Journal of Neuropsychopharmacology (2014), 17, 407–419

The gastrointestinal hormone PYY3–36 is a preferential Y2 neuropeptide Y (NPY) receptor agonist. Recent evidence indicates that PYY3–36 acts on central dopaminergic pathways, but its influence on dopamine-dependent behaviors remains largely unknown. We therefore explored the effects of peripheral PYY3–36 treatment on the behavioral responses to novelty and to dopamine-activating drugs in mice. In addition, we examined whether PYY3–36 administration may activate distinct dopamine and γ-aminobutyric acid (GABA) cell populations in the mesoaccumbal and nigrostriatal pathways. We found that i.p. PYY3–36 injection led to a dose-dependent increase in novel object exploration. The effective dose of PYY3–36 (1 μg/100 g body weight) also potentiated the locomotor reaction to the indirect dopamine receptor agonist amphetamine and increased stereotyped climbing/leaning responses following administration of the direct dopamine receptor agonist apomorphine. PYY3–36 administration did not affect activity of midbrain dopaminergic cells as evaluated by double immuno-enzyme staining of the neuronal early gene product c-Fos with tyrosine hydroxylase. PYY3–36 did, however, lead to a marked increase in the number of cells co-expressing c-Fos with glutamic acid decarboxylase in the nucleus accumbens and caudate putamen, indicating activation of GABAergic cells in dorsal and ventral striatal areas. Our results support the hypothesis that acute administration of the preferential Y2 receptor agonist PYY3–36 modulates dopamine-dependent behaviours. These effects do not seem to involve direct activation of midbrain dopamine cells but instead are associated with neuronal activation in the major input areas of the mesoaccumbal and nigrostriatal pathways.

Somatostatin and nociceptin inhibit neurons in the central nucleus of amygdala that project to the periaqueductal grey

Billy Chieng, MacDonald J. Christie
Neuropharmacology 59 (2010) 425e430

The central nucleus of amygdala (CeA) plays an important role in modulation of the descending antinociceptive pathways. Using whole-cell patch clamp recordings from brain slices, we found that CeA neurons responded to the endogenous ligands somatostatin (SST) and nociceptin/orphanin FQ (OFQ) via an increased K-conductance. Co-application with selective antagonists suggested that SST and OFQ act on SSTR2 and ORL1 receptors, respectively. Taking account of anatomical localisation of recorded neurons, the present study showed that many responsive neurons were located within the medial subdivision of CeA and all CeA projection neurons to the midbrain periaqueductal grey invariably responded to these peptides. Randomly selected agonist-responsive neurons in CeA predominantly classified physiologically as low-threshold spiking neurons. The similarity of SST, OFQ and, as previously reported, opioid responsiveness in a sub-population of CeA neurons suggests converging roles of these peptides to inhibit the activity of projections from CeA to vlPAG, and potentially similar antinociceptive actions in this pathway.

In vitro identification and electrophysiological characterization of dopamine neurons in the ventral tegmental area

Tao A. Zhang, Andon N. Placzek, John A. Dani
Neuropharmacology 59 (2010) 431e436

Dopamine (DA) neurons in the ventral tegmental area (VTA) have been implicated in brain mechanisms related to motivation, reward, and drug addiction. Successful identification of these neurons in vitro has historically depended upon the expression of a hyperpolarization-activated current (Ih) and immunohistochemical demonstration of the presence of tyrosine hydroxylase (TH), the rate-limiting enzyme for DA synthesis. Recent findings suggest that electrophysiological criteria may be insufficient for distinguishing DA neurons from non-DA neurons in the VTA. In this study, we sought to determine factors that could potentially account for the apparent discrepancies in the literature regarding DA neuron identification in the rodent brain slice preparation. We found that confirmed DA neurons from the lateral VTA generally displayed a larger amplitude Ih relative to DA neurons located in the medial VTA. Measurement of a large amplitude Ih (>100 pA) consistently indicated a dopaminergic phenotype, but non-dopamine neurons also can have Ih current. The data also showed that immunohistochemical TH labeling of DA neurons can render false negative results after relatively long duration (>15 min) wholecell patch clamp recordings. We conclude that whole-cell patch clamp recording in combination with immunohistochemical detection of TH expression can guarantee positive but not negative DA identification in the VTA.

Dopamine Enables In Vivo Synaptic Plasticity Associated with the Addictive Drug Nicotine

Jianrong Tang and John A. Dani
Neuron, Sept 10, 2009; 63, 673–682

Addictive drugs induce a dopamine signal that contributes to the initiation of addiction, and the dopamine signal influences drug-associated memories that perpetuate drug use. The addiction process shares many commonalities with the synaptic plasticity mechanisms normally attributed to learning and memory. Environmental stimuli repeatedly linked to addictive drugs become learned associations, and those stimuli come to elicit memories or sensations that motivate continued drug use. Applying in vivo recording techniques to freely moving mice, we show that physiologically relevant concentrations of the addictive drug nicotine directly cause in vivo hippocampal synaptic potentiation of the kind that underlies learning and memory. The drug-induced long-term synaptic plasticity required a local hippocampal dopamine signal. Disrupting general dopamine signaling prevented the nicotine-induced synaptic plasticity and conditioned place preference. These results suggest that dopaminergic signaling serves as a functional label of salient events by enabling and scaling synaptic plasticity that underlies drug-induced associative memory.

NCS-1 in the Dentate Gyrus Promotes Exploration, Synaptic Plasticity, and Rapid Acquisition of Spatial Memory

Bechara J. Saab, John Georgiou, Arup Nath, Frank J.S. Lee, et al.
Neuron, Sept 10, 2009; 63, 643–656

The molecular underpinnings of exploration and its link to learning and memory remain poorly understood. Here we show that inducible, modest overexpression of neuronal calcium sensor 1 (Ncs1) selectively in the adult murine dentate gyrus (DG) promotes a specific form of exploratory behavior. The mice also display a selective facilitation of longterm potentiation (LTP) in the medial perforant path and a selective enhancement in rapid-acquisition spatial memory, phenotypes that are reversed by direct application of a cell-permeant peptide (DNIP) designed to interfere with NCS-1 binding to the dopamine type-2 receptor (D2R). Moreover, the DNIP and the D2R-selective antagonist L-741,626 attenuated exploratory behavior, DG LTP, and spatial memory in control mice. These data demonstrate a role for NCS-1 and D2R in DG plasticity and provide insight for understanding how the DG contributes to the origin of exploration and spatial memory acquisition.

Neuroligin 2 Drives Postsynaptic Assembly at Perisomatic Inhibitory Synapses through Gephyrin and Collybistin

Alexandros Poulopoulos, Gayane Aramuni, Guido Meyer, Tolga Soykan, et al.
Neuron 63, 628–642, Sept 10, 2009

In the mammalian CNS, each neuron typically receives thousands of synaptic inputs from diverse classes of neurons. Synaptic transmission to the postsynaptic neuron relies on localized and transmitter-specific differentiation of the plasma membrane with postsynaptic receptor, scaffolding, and adhesion proteins accumulating in precise apposition to presynaptic sites of transmitter release. We identified protein interactions of the synaptic adhesion molecule neuroligin 2 that drive postsynaptic differentiation at inhibitory synapses. Neuroligin 2 binds the scaffolding protein gephyrin through a conserved cytoplasmic motif and functions as a specific activator of collybistin, thus guiding membrane tethering of the inhibitory postsynaptic scaffold. Complexes of neuroligin 2, gephyrin and collybistin are sufficient for cell-autonomous clustering of inhibitory neurotransmitter receptors. Deletion of neuroligin 2 in mice perturbs GABAergic and glycinergic synaptic transmission and leads to a loss of postsynaptic specializations specifically at perisomatic inhibitory synapses.

A Subset of Ventral Tegmental Area Neurons is Inhibited by Dopamine, 5-Hydroxytryptamine and Opioids

L. Cameron, M. W. Wessendorf and J. T. Williams
Neuroscience 1997; 77(1), pp. 155–166 PII: S0306-4522(96)00444-7

Neurons originating in the ventral tegmental area are thought to play a key role in the formation of addictive behaviors, particularly in response to drugs such as cocaine and opioids. In this study we identified different populations of ventral tegmental area neurons by the pharmacology of their evoked synaptic potentials and their response to dopamine, 5-hydroxytryptamine and opioids. Intracellular recordings were made from ventral tegmental area neurons in horizontal slices of guinea-pig brain and electrical stimulation was used to evoke synaptic potentials. The majority of cells (61.3%) hyperpolarized in response to dopamine, depolarized to 5-hydroxytryptamine, failed to respond to [Met]5enkephalin and exhibited a slow GABAB-mediated inhibitory postsynaptic potential. A smaller proportion of cells (11.3%) hyperpolarized in response to [Met]5enkephalin, depolarized to 5-hydroxytryptamine, failed to respond to dopamine and did not exhibit a slow inhibitory postsynaptic potential. These two groups of cells corresponded to previously described ‘‘principal’’ and ‘‘secondary’’ cells, respectively. A further group of cells (27.4%) was identified that, like the principal cells, hyperpolarized to dopamine.

However, these ‘‘tertiary cells’’ also hyperpolarized to both 5-hydroxytryptamine and [Met]5enkephalin and exhibited a slow, cocaine-sensitive 5-hydroxytryptamine1A-mediated inhibitory postsynaptic potential. When principal and tertiary cells were investigated immuno-histochemically, 82% of the principal cells were positive for tyrosine hydroxylase compared
with only 29% of the tertiary cells. The 5-hydroxytryptamine innervation of both these cell types was investigated and a similar density of putative contacts was observed near the somata and dendrites in both groups. This latter finding suggests that the existence of a 5-hydroxytryptamine-mediated inhibitory postsynaptic potential in the tertiary cells may be determined by the selective expression of 5-hydroxytryptamine receptors, rather than the distribution or density of the 5-hydroxytryptamine innervation.
We conclude that tertiary cells are a distinct subset of ventral tegmental area neurons where cocaine and μ-opioids both mediate inhibition.

Dopamine reward circuitry: Two projection systems from the ventral midbrain to the nucleus accumbens–olfactory tubercle complex

Satoshi Ikemoto
Brain Research Reviews 56 (2007) 27–78

Anatomical and functional refinements of the meso-limbic dopamine system
of the rat are discussed. Present experiments suggest that dopaminergic neurons localized in the posteromedial ventral tegmental area (VTA) and central linear nucleus raphe selectively project to the ventromedial striatum (medial olfactory tubercle and medial nucleus accumbens shell), whereas
the anteromedial VTA has few if any projections to the ventral striatum,
and the lateral VTA largely projects to the ventrolateral striatum (accumbens
core, lateral shell and lateral tubercle). These findings complement the recent behavioral findings that cocaine and amphetamine are more rewarding when administered into the ventromedial striatum than into the ventrolateral striatum. Drugs such as nicotine and opiates are more rewarding when administered into the posterior VTA or the central linear nucleus than into
the anterior VTA. A review of the literature suggests that
(1) the midbrain has corresponding zones for the accumbens core and medial shell;
(2) the striatal portion of the olfactory tubercle is a ventral extension of the nucleus accumbens shell; and
(3) a model of two dopamine projection systems from the ventral midbrain to the ventral striatum is useful for understanding reward function.
The medial projection system is important in the regulation of arousal characterized by affect and drive and plays a different role in goal directed learning than the lateral projection system, as described in the variation–selection hypothesis of striatal functional organization.

Metabolic hormones, dopamine circuits, and feeding

Nandakumar S. Narayanan, Douglas J. Guarnieri, Ralph J. DiLeone
Frontiers in Neuroendocrinology 31 (2010) 104–112

Recent evidence has emerged demonstrating that metabolic hormones such as ghrelin and leptin can act on ventral tegmental area (VTA) midbrain dopamine neurons to influence feeding. The VTA is the origin of mesolimbic dopamine neurons that project to the nucleus accumbens (NAc) to influence behavior. While blockade of dopamine via systemic antagonists or targeted gene delete can impair food intake, local NAc dopamine manipulations have little effect on food intake. Notably, non-dopaminergic manipulations in the VTA and NAc produce more consistent effects on feeding and food choice. More recent genetic evidence supports a role for the substantia nigra-striatal dopamine pathways in food intake, while the VTA–NAc circuit is more likely involved in higher-order aspects of food acquisition, such as motivation and cue associations. This rich and complex literature should be considered in models of how peripheral hormones influence feeding behavior via action on the midbrain circuits.

Control of brain development and homeostasis by local and systemic insulin signaling

Liu, P. Speder & A. H. Brand
Diabetes, Obesity and Metabolism 16 (Suppl. 1): 16–20, 2014

Insulin and insulin-like growth factors (IGFs) are important regulators of growth and metabolism. In both vertebrates and invertebrates, insulin/IGFs are made available to various organs, including the brain, through two routes: the circulating systemic insulin/IGFs act on distant organs via endocrine signaling, whereas insulin/IGF ligands released by local tissues act in a paracrine or autocrine fashion. Although the mechanisms governing the secretion and action of systemic insulin/IGF have been the focus of extensive investigation, the significance of locally derived insulin/IGF has only more recently come to the fore. Local insulin/IGF signaling is particularly important for the development and homeostasis of the central nervous system, which is insulated from the systemic environment by the blood–brain barrier. Local insulin/IGF signaling from glial cells, the blood–brain barrier and the cerebrospinal fluid has emerged as a potent regulator of neurogenesis. This review will address the main sources of local insulin/IGF and how they affect neurogenesis during development. In addition, we describe how local insulin/IGF signaling couples neural stem cell proliferation with systemic energy state in Drosophila and in mammals.

Pharmacology, Physiology, and Mechanisms of Action of Dipeptidyl Peptidase-4 Inhibitors

Erin E. Mulvihill and Daniel J. Drucker
Endocrine Reviews 35: 992–1019, 2014

Dipeptidyl peptidase-4 (DPP4) is a widely expressed enzyme transducing actions through an anchored transmembrane molecule and a soluble circulating protein. Both membrane-associated and soluble DPP4 exert
catalytic activity, cleaving proteins containing a position 2 alanine or proline. DPP4-mediated enzymatic cleavage alternatively inactivates peptides or generates new bioactive moieties that may exert competing or novel activities. The widespread use of selective DPP4 inhibitors for the treatment of type 2 diabetes has heightened interest in the molecular mechanisms through which DPP4 inhibitors exert their pleiotropic actions. Here we review the biology ofDPP4with a focus on:
1) identification of pharmacological vs physiological DPP4 substrates; and
2) elucidation of mechanisms of actions of DPP4 in studies employing genetic elimination or chemical reduction of DPP4 activity.
We review data identifying the roles of key DPP4 substrates in transducing the glucoregulatory, anti-inflammatory, and cardiometabolic actions of DPP4  inhibitors in both preclinical and clinical studies. Finally, we highlight experimental pitfalls and technical challenges encountered in studies designed to understand the mechanisms of action and downstream targets activated by inhibition of DPP4.
Dipeptidyl peptidase-4 (DPP4) is a multifunctional protein that exerts biological activity through pleiotropic actions including:

  • protease activity (1),
  • association with adenosine deaminase (ADA) (2),
  • interaction with the extracellular matrix (3),
  • cell surface coreceptor activity mediating viral entry (4), and
  • regulation of intracellular signal transduction coupled to control of cell migration and proliferation (5).

The complexity of DPP4 action is amplified by the panoply of bioactive DPP4 substrates, which in turn act as elegant biochemical messengers in multiple tissues, including the immune and neuroendocrine systems.

DPP4 transmits signals across cell membranes and interacts with other membrane proteins (Figure). Remarkably, most of the protein is extracellular, including the C-terminal catalytic domain, a cysteine-rich area, and a large glycosylated region linked by a flexible stalk to the transmembrane segment. Only six N-terminal amino acids are predicted to extend into the cytoplasm. The active site, Ser 630, is flanked by the classic serine peptidase motif Gly-Trp-Ser630-Tyr-Gly-Gly-Tyr-Val.

Membrane-bound DPP4

Membrane-bound DPP4

Membrane-bound DPP4 contains residues 1–766, whereas sDPP4 contains residues 39–766. sDPP4 is lacking the cytoplasmic domain [residues 1–6], transmembrane domain [residues 7–28], and the flexible stalk [residues 29–39]. Both membrane-bound and circulating sDPP4 share many domains including the glycosylated region [residues 101–535, specific residues 85,92, 150], ADA binding domain [340–343], fibronectin binding domain [468–479], cysteine-rich domain [351–506, disulfide bonds are formed from 385–394, 444–472, and 649–762], and the catalytic domain [507–766 including residues composing the catalytic active site 630, 708, and 740].

DPP4 activity is subject to regulation at many levels, including control of gene and protein expression, interaction with binding partners, and modulation of enzyme activity. The DPP4 gene does not contain conventional TATAA or CCAAT promoter sequences but is characterized by a cytosine/guanine-rich promoter region.
DPP4 contains eight to 11 potential N-glycosylation sites, which can contribute to its folding and stability. Although glycosylation may contribute approximately 18–25% of the total molecular weight, mutational analysis has determined that the glycosylation sites are not required for dimerization, catalytic activity, or ADA binding. However, N-terminal sialylation facilitates trafficking of DPP4 to the apical membrane. Interestingly, molecular analysis of DPP4 isoforms isolated from the rat kidney brush border membrane reveals extensive heterogeneity in the oligosaccharides of DPP4.DPP4 was first investigated for its role in hydrolysis of dietary prolyl peptides (58); subsequent studies using DPP4 isolated using immunoaffinity chromatography and ADA binding identified DPP4 as the primary enzyme responsible for the generation of Gly-Prop-nitroanilide substrates in human serum. It is now known that DPP4 can cleave dozens of peptides, including chemokines, neuropeptides, and regulatory peptides, most containing a proline or alanine residue at position 2 of the amino-terminal region. Despite the preference for a position 2 proline, alternate residues (hydroxyproline, dehydroproline > alanine >,  glycine, threonine, valine, or leucine) at the penultimate position are also cleaved by DPP4, suggesting a required stereochemistry. The DPP4 cleavage at postproline peptide bonds inactivates peptides and/or generates new bioactive peptides (see Figure), thereby regulating diverse biological processes.

DPP4 cleavage regulates substrate-receptor interactions

DPP4 cleavage regulates substrate-receptor interactions

DPP4 cleavage regulates substrate/receptor interactions. A, DPP4 cleaves NPY [1–36] and PYY [1–36]. The intact forms of these peptides signal through Y1R-Y5R. After DPP4 cleavage, NPY [3–36] and PYY [3–36] are generated and preferentially signal through the Y2R and Y5R. B, DPP4 cleaves SP [1–11], which signals through the NK1R receptor to generate SP [5–11], which can signal through (NK1R, -2R, -3R).

GHRH and IGF-1

GHRH [1–44] and [1–40] are produced in the arcuate nucleus of the hypothalamus and bind its receptor on the anterior pituitary to stimulate the release of GH, and in turn, GH stimulates hepatic IGF-1 release. GHRH was among the first peptides to be identified as a DPP4 substrate; it is rapidly degraded in rodent and human plasma to GHRH [3–44]/GHRH [3–40], and this cleavage was blocked upon incubation of human plasma with the DPP4 inhibitor, diprotin A (99).GHRH[1–44] or [1–40] exhibits a very short half-life (6 min) andDPP4 cleavage was initially perceived to be a critical regulator of GHRH bioactivity and, in turn, the GH-IGF-1 axis. IGF-1, the downstream effector of GHRH and GH, is a 105-amino acid protein produced mainly by the liver.
IGF-1 was identified as a pharmacological DPP4 substrate by matrix-assisted laser desorption/ionization-time of flight analysis of molecular forms of IGF-1 generated after incubation with DPP4 purified from baculovirus-infected insect cells. However, studies in pigs treated with sitagliptin at doses inhibiting 90% of DPP4 activity failed to demonstrate an increase in active intact IGF-1.
Clinically, treatment of healthy human male subjects with sitagliptin (25–600 mg) for 10 days did not produce increased concentrations of serum IGF-1 or IGF-binding protein 3 as measured by ELISA. Furthermore, Dpp4/ mice or rats do not exhibit increased organ growth or body size. Hence, the available data suggest that although DPP4 cleaves and inactivates both GHRH and IGF-1, enzymatic inactivation by DPP4 is not the major mechanism regulating the bioactivity of the GHRH-IGF-1 axis.

The role of acute cortisol and DHEAS in predicting acute and chronic PTSD symptoms

Joanne Mouthaan, Marit Sijbrandij, Jan S.K. Luitse
Psychoneuroendocrinology (2014) 45, 179—186

Background: Decreased activation of the hypothalamus—pituitary—adrenal (HPA) axis in response to stress is suspected to be a vulnerability factor for posttraumatic stress disorder (PTSD). Previous studies showed inconsistent findings regarding the role of cortisol in predicting PTSD. In addition, no prospective studies have examined the role of dehydroepiandrosterone (DHEA), or its sulfate form DHEAS, and the cortisol-to-DHEA(S) ratio in predicting PTSD. In this study, we tested whether acute plasma cortisol, DHEAS and the cortisol-to-DHEAS ratio predicted PTSD symptoms at 6 weeks and 6 months post-trauma. Methods: Blood samples of 397 adult level-1 trauma center patients, taken at the trauma resuscitation room within hours after the injury, were analyzed for cortisol and DHEAS levels. PTSD symptoms were assessed at 6 weeks and 6 months post-trauma with the Clinician Administered PTSD Scale. Results: Multivariate linear regression analyses showed that lower cortisol predicted PTSD symptoms at both 6 weeks and 6 months, controlling for age, gender, time of blood sampling, injury, trauma history, and admission to intensive care. Higher DHEAS and a smaller cortisol-to-DHEAS ratio predicted PTSD symptoms at 6 weeks, but not after controlling for the same variables, and not at 6 months. Conclusions: Our study provides important new evidence on the crucial role of the HPA-axis in response to trauma by showing that acute cortisol and DHEAS levels predict PTSD symptoms in survivors of recent trauma.
Neurobiology of DHEA and effects on sexuality, mood and cognition

  1. Pluchino, P.Drakopoulos, F.Bianchi-Demicheli, J.M.Wenger
    J Steroid Biochem & Molec Biol 145 (2015) 273–280

Dehydroepiandrosterone (DHEA) and its sulfate ester, DHEAS, are the most abundant steroid hormones in the humans. However, their physiological significance, their mechanisms of action and their possible roles as treatment are not fully clarified. Biological actions of DHEA(S) in the brain involve neuroprotection, neurite growth, neurogenesis and neuronal survival, apoptosis, catecholamine synthesis and secretion, as well as anti-oxidant, anti- inflammatory and antiglucocorticoid effects. In addition, DHEA affects neurosteroidogen is and endorphin synthesis/release. We also demonstrated in a model of ovariectomized rats that DHEA therapy increases proceptive behaviors, already after 1 week of treatment, affecting central function of sexual drive. In women, the analyses of clinical outcomes are far from being conclusive and many issues should still be addressed. Although DHEA preparations have been available in the market since the 1990s, there are very few definitive reports on the biological functions of this steroid. We demonstrate that 1 year DHEA administration at the dose of 10mg provided a significant improvement in comparison with vitamin D in sexual function
and in frequency of sexual intercourse in early postmenopausal women. Among symptomatic women, the spectrum of symptoms responding to DHEA requires further investigation, to define the type of sexual symptoms (e.g. decreased sexual function or hypoactive sexual desire disorder) and the degree of mood/cognitive symptoms that could be responsive to hormonal treatment.
In this regard, our findings are promising, although they need further exploration with a larger and more representative sample size.
Although adrenal cortex is considered to be the primary source of DHEAS in the brain, it was reported that DHEAS did not dis- appear or decrease in the brain 15 days neither after orchiectomy, adrenalectomy, or both, nor after the inhibition of adrenal secretion by dexamethasone. DHEA and DHEAS were among the first neurosteroids identified in rat brains. Cytochrome P450c17 was found in a subset of neurons of embryonic rodent brains. While P450c17 protein was readily detected in the brain, the abundance of P450c17 mRNA transcripts in the embryonic mouse brain or hippocampus of adult male rats was low, and was approximated to be 1/200th of the expression in testis.
DHEAS may be synthesized in the brain from DHEA. Sulfation of DHEA has been observed in the brains of rhesus monkeys in vivo and in human fetal brain slices in vitro. DHEA sulfotransferase (HSTor SULT2A1) is an enzyme that sulfonates DHEA (in addition to pregnenolone).Western blotting and immune-histochemistry showed protein expression of an HST in adult Wistar rat brain. In addition SULT2A1 mRNA expression has been shown in rat brains. DHEAS is predominately transported out of the brain across the blood–brain barrier and DHEAS found in the brain is most likely due to local synthesis . DHEA(S) may mediate some of its actions through conversion into more potent sex steroids and activation of androgen or estrogen receptors in tissue.
According to existing assumption of the biology of depression, DHEA(S) ability to modulate many neurobiological actions could underlie relationships between endogenous and/or exogenously- supplemented DHEA(S) concentrations and depression. There is evidence that DHEAS concentrations are negatively correlated with ratings of depressed mood. However, the remaining literature examining plasma and serum DHEA(S) concentrations in depression is still inconsistent and other plasma indexes were studied in order to more accurately discriminate depressed from nondepressed individuals. Hypothalamic–pituitary–adrenal axis (HPA) hyperactivity has
been demonstrated in chronic diseases affecting nervous system disorders like depression. The end products of HPA axis, glucocorticoids (GCs), regulate many physiological functions and play an important role in affective regulation and dysregulation. Despite DHEAS levels which markedly decrease throughout adulthood, an increase in circulating cortisol with advanced age has been observed in human and nonhuman primates.
The most relevant aspect meriting attention is certainly the controversial finding among the studies that investigate the correlation of the endogenous DHEA sulfate (DHEAS) level, the aging process or organ illness with the results coming from studies focusing on the effects of exogenous DHEAS administration on brain function, sexuality, cardiovascular health and metabolic syndrome. Indeed, the marked age-related decline in serum DHEA and DHEAS has suggested that a deficiency of these steroids may be causally related to the development of a series of diseases that are generally associ- ated with aging. The postulated consequences of low DHEA levels include insulin resistance, obesity, cardiovascular disease, cancer, reduction of the immune defense system as well as psychosocial problems such as depression and a general deterioration in the sensation of well-being and cognitive function, DHEA replacement may seem an attractive treatment opportunity. Nevertheless, the analyses of clinical outcomes are far from being conclusive.

Dehydroepiandrosterone, its metabolites and ion channels

Hill, M. Dusková, L. Stárka
J Steroid Biochem & Molec Biology 145(2015)293–314

This review is focused on the physiological and pathophysiological relevance of steroids influencing the activities of the central and peripheral nervous systems with regard to their concentrations in body fluids and tissues in various stages of human life like the fetal development or pregnancy. The data summarized in this review shows that DHEA and its unconjugated and sulfated metabolites are physiologically and pathophysiologically relevant in modulating numerous ion channels and participate in vital functions of the human organism. DHEA and its unconjugated and sulfated metabolites including 5 _/ _-reduced androstane steroids participate in various physiological and pathophysiological processes like the management of GnRH cyclic release, regulation of glandular and neurotransmitter secretions, maintenance of glucose homeostasis on one hand and insulin insensitivity on the other hand, control of skeletalmuscle and smooth muscle activities including vasoregulation, promotion of tolerance to ischemia and other neuroprotective effects. In respect of prevalence of steroid sulfates over unconjugated steroids in the periphery and the opposite situation in the CNS, the sulfated androgens and androgen metabolites reach relevance in peripheral organs. The unconjugated androgens and estrogens are relevant in periphery and so much the more in the CNS due to higher concentrations of most unconjugated steroids in the CNS tissues than in circulation and peripheral organs.

Neurotrophins are proteins found within a broad range of cell types in the brain and periphery that facilitate neuronal growth, survival, and plasticity. The neurotrophin ‘‘superfamily’’ includes nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), neurotrophin-4/5 (NT4/5), and neurotrophin-6. Target tissues are hypothesized to regulate neuron survival by making neurotrophins available in limited amounts, resulting in selection of neurons with the best connectivity to the target tissue. NGF, in particular, is released by the target tissue and taken up in responsive neurons by receptor-mediated endocytosis. It is then transported retrogradedly into the cell where it exerts trophic effects. Lu et al. proposed a ‘‘Yin and Yang model,’’ whereby neurotrophic action is mediated by two principal classes of transmembrane receptor systems: the tyrosine kinase (Trk) receptors (including TrkA [selective for NGF], TrkB [selective for BDNF and NT4/5], and TrkC [selective for NT3]) and the neurotrophin receptor p75NTR. Each receptor type binds mature neurotrophins and/or neurotrophin precursors (proneurotrophins), creating a complex ‘‘balance’’ that then causes neuronal survival or death.
DHEA has been shown to evoke NGF mRNA expression in target cells. In a study of pregnant women, Schulte-Herbrüggen et al. showed no relationships between serum DHEAS and NGF. In contrast, we showed that DHEAS independently associated with salivary NGF (sNGF) in military men under baseline conditions, while DHEA did not. We now know that both DHEA(S) and NGF respond affirmatively to stressful insult, yet the association between these analytes during stress exposure is not understood. Characterization of this relationship has implications for prevention and treatment of traumatic stress and injury, degenerative disease management, and nerve repair. In this report, we extended our prior study of neuroprotective properties of DHEAS in men under baseline conditions to a prospective paradigm involving intense stress exposure in both men and women. We hypothesized that

(a) robust associations would prevail between total output of DHEAS and sNGF across the stress trajectory and at each time point,
(b) changes in DHEAS would predict corresponding changes in sNGF, and
(c) baseline DHEAS would positively predict total sNGF output across the stress trajectory.
We also explored the roles of testosterone and cortisol. In light of less definitive prior literature, directional hypotheses were not stated regarding these analytes.

In the first regression model, total hormone output (AUCG) of the independent variables (DHEAS, testosterone, and cortisol) combined to explain 63.7% of variance in sNGF output (F = 65.4, p < 0.001). Standardized beta coefficients revealed that testosterone exerted an independent effect (b = 0.80, p < 0.001), while the other predictors were not significant. In light of this unexpected finding, we then used regression-based causal steps modeling to evaluate whether testosterone mediated a hypothesized direct effect of DHEAS on sNGF. Following this approach, DHEAS predicted sNGF in an initial regression model (b = 0.45, p < 0.001). When testosterone was added, the direct effect of DHEAS (path c0) on sNGF was nearly eradicated and no longer significant (b = .04, p = .57), thus suggesting a mediated effect. An alternate statistical test (Sobel Test; 34) evaluating the hypothesized difference between the total effect (path c) and the direct effect (path c0) of DHEAS on sNGF produced a similar result (test statistic = 4.0, p < 0.001). Fig. 1 depicts positive association of DHEAS to sNGF, while Fig. 2 depicts Positive association of testosterone to sNGF.

Positive association of DHEAS total output and sNGF total output

Positive association of DHEAS total output and sNGF total output

Positive association of DHEAS total output and sNGF total output

Positive association of testosterone total output and sNGF total output

Positive association of testosterone total output and sNGF total output

Positive association of testosterone total output and sNGF total output.
The models were then decomposed at each time point. At baseline, the independent variables (DHEAS, testosterone, and cortisol) combined to account for 10.2% of variance in sNGF (F = 5.3, p < 0.01). Standardized beta coefficients showed that DHEAS exerted an independent effect on sNGF (b = 0.39, p < 0.001), while the other predictors were not significant. During stress exposure, the independent variables combined to account for 28.0% of variance in NGF (F = 15.8, p < 0.001). Again, DHEAS exerted an independent effect (b = 0.56, p < 0.001) while the other predictors were not significant. During recovery, the predictor set accounted for 18.0% of variance in sNGF (F = 9.2, p < 0.001), and DHEAS exerted an independent effect (b = 0.47, p < 0.001) while the other predictors did not.
The models were then decomposed relative to each change index. In terms of reactivity, the independent variables (DHEAS, testosterone, and cortisol reactivity) and covariate (sex) combined to account for 20.3% of variance in sNGF reactivity (F = 8.2, p < 0.001). Standardized beta coefficients revealed that DHEAS reactivity exerted an independent effect (b = 0.39, p < 0.001), while the other predictors were not significant. In terms of recovery, the predictors combined to account for 28.2% of variance in sNGF recovery (F = 15.5, p < 0.001); DHEAS recovery exerted an independent effect (b = 0.52, p < 0.001), as did testosterone recovery (b = [1]0.27, p < 0.01). In terms of residual elevation/depression, the independent variables explained 12.4% of variance in sNGF residual elevation (F = 6.2, p < 0.001). DHEAS residual elevation exerted an independent effect (b = 0.35, p < 0.001), while the other predictors did not.

Endocrine-Disrupting Chemicals and Human Growth and Maturation: A Focus on Early Critical Windows of Exposure

Julie Fudvoye, Jean-Pierre Bourguignon, Anne-Simone Parent
Vitamins and Hormones, 2014; 94: Chapt 1. 1-25.

Endocrine-disrupting chemicals (EDCs) are exogenous substances that interfere with hormone synthesis, metabolism, or action. In addition, some of them could cause epigenetic alterations of DNA that can be transmitted to the following generations. Because the developing organism is highly dependent on sex steroids and thyroid hormones for its maturation, the fetus and the child are very sensitive to any alteration of their hormonal environment. An additional concern about that early period of life comes from the shaping of the homeostatic mechanisms that takes place also at that time with involvement of epigenetic mechanisms along with the concept of fetal origin of health and disease. In this chapter, we will review the studies reporting effects of EDCs on human development. Using a translational approach, we will review animal studies that can shed light on some mechanisms of action of EDCs on the developing organism. We will focus on the major hormone-dependent stages of development: fetal growth, sexual differentiation, puberty, brain development, and energy balance. We will also discuss the possible epigenetic effects of EDCs on human development.

Several studies have reported that prenatal or early postnatal exposure to some EDCs is associated with alterations of cognitive or motor functions in children. Knowing the fundamental role played by thyroid hormones and sex steroids in cortex development, one can hypothesize that disruption of those hormones could cause alteration of the development of the cerebral cortex and of its functions later in life. We will review here the human data suggesting a causal effect for endocrine disrupters on impairment of cortical functions and approach some EDC mechanisms of action using animal models.

Thyroid hormones are known to be essential for brain development. They regulate progenitor proliferation and differentiation, neuron migration, and dendrite outgrowth (Parent, Naveau, Gerard, Bourguignon, & Westbrook, 2011). Even mild thyroid hormone insufficiency in humans can produce measurable deficits in cognitive functions (Zoeller & Rovet, 2004). Thyroid hormone action is mediated by two classes of nuclear receptors (Forrest & Vennstro¨m, 2000) that exhibit differential spatial and temporal expressions in the brain, suggesting that thyroid hormones have variable functions during brain development. This differential expression of thyroid hormone receptors explains the critical period of thyroid hormone action on brain development as suggested by models of maternal hypothyroidism or congenital hypothyroidism.

Depending on the timing of onset of hypothyroidism, the offspring will display problems of visual attention, gross or fine motor skills, or language and memory skills. Similarly, one can hypothesize that disruption of thyroid function by EDCs will have different effects based on the timing of exposure. However, few studies focused on that aspect. Polychlorinated biphenyls (PCBs) form a group of widespread environmental contaminants composed of 209 different congeners used in a wide variety of applications. Their production was banned in the 1970s but PCBs are still present in the environment due to their high stability. PCBs were among the first EDCs identified as responsible for alterations of cognitive functions. Indeed, impaired memory and altered learning abilities have been associated with prenatal exposure to EDCs in humans and In animal models, perinatal exposure to PCBs has been consistently associated with a decrease of thyroid hormone concentration in maternal serum as well as pup serum. Some but not all epidemiological studies in human have found an association between PCB body burden and thyroid hormone levels. This disruption of thyroid function could explain some of the effects of PCBs on the developing brain. Indeed, animal models have shown that the ototoxic effects of PCBs could be partially ameliorated by thyroxin replacement and PCBs seem to alter some of the developmental processes in the cortex and the cerebellum that are dependent on thyroid hormones. However, recent publications raise important issues.

As it is the case for other EDCs, some windows of susceptibility have been identified during pre- and postnatal brain development. Recent studies have shown that exposure to PBDEs causes alteration of thyroid hormone levels in pregnant women and infants as it is the case in rodents. Only very few studies, however, have focused on the molecular or cellular effects of perinatal exposure to PBDEs in vivo. Viberg et al. have reported a decrease of cholinergic nicotinic receptors in the hippocampus after exposure to BDE-99 and BDE-153. However, the link between such a decrease and the behavioral effects of PBDEs is still unclear. Other teams have reported that exposure to PBDEs reduced hippocampal long term potentiation and decreased brain-derived neurotrophic factor expression in the brain. While several studies have reported negative effect of PBDEs on brain development and cognitive function in animals, there is relatively little information about adverse health effects of PBDEs in humans. Some very recent studies have identified a correlation between prenatal exposure to PBDEs and alteration of cognitive functions.

Endocrine-Disrupting Chemicals: Elucidating Our Understanding of Their Role in Sex and Gender-Relevant End Points

Cheryl A. Frye
Vitamins and Hormones, 2014; 94: 41-98

Endocrine-disrupting chemicals (EDCs) are diverse and pervasive and may have significant consequence for health, including reproductive development and expression of sex-/gender-sensitive parameters. This review chapter discusses what is known about common EDCs and their effects on reproductively relevant end points. It is proposed that one way that EDCs may exert such effects is by altering steroid levels (androgens or 17-estradiol, E2) and/or intracellular E2 receptors (ERs) in the hypothalamus and/or hippocampus. Basic research findings that demonstrate developmentally sensitive end points to androgens and E2 are provided. Furthermore, an approach is suggested to examine differences in EDCs that diverge in their actions at ERs to elucidate their role in sex-/gender-sensitive parameters.

Reproductive dysfunction among adults and emotional, attentional, and behavioral disorders among children are on the rise. Sperm counts and fertility have declined in the last 50 years . Incidence of attention-deficit hyperactivity disorder (ADHD) and autism has increased in the last 30 years. These increases in reproductive dysfunction and developmental disorders may be due to increased exposure to environmental contaminants, although there is controversy about the relationship between exposure and these effects.
Many contaminants in the environment, including polychlorinated biphenyls (PCBs), dioxins, and metals, accumulate in exposed individuals and may have adverse consequences due to effects as endocrine-disrupting chemicals (EDCs). EDCs may have effects by altering steroid levels (androgens or 17β-estradiol, E2) and/or intracellular E2 receptors (ERs) in the hypothalamus and/or hippocampus.
Steroid hormones, during critical periods of development, organize sexual dimorphisms in brain and behavior and give rise to sex differences in later responses to steroid hormones. EDCs can profoundly disrupt reproductive responses following adult exposure and result in pervasive effects that extend throughout the life of their offspring. Many nonreproductive behaviors, such
as spatial performance, activity, and arousal, are also sexually dimorphic and organized and activated by steroid hormones. Thus, EDCs may affect reproductive and the aforementioned nonreproductive parameters by altering E2 levels and/or ER binding in the hypothalamus and/or hippocampus.
Results from the literature and preliminary data will be presented that demonstrate our use of a whole-animal model to begin to investigate effects of exposure (in adulthood and/or development) to EDCs on steroid levels (androgens and E2), actions at ERs (in hypothalamus and hippocampus), and reproductive-sensitive measures (anogenital distance, accessory structure weight, onset of puberty and sexual maturity, and reproductive behavior) and nonreproductive behaviors (spatial performance, play behavior, and arousal) throughout development.

A common feature of many environmental contaminants is their estrogenic effects. Some contaminants can alter production of E2 and/or androgens or act as agonists or antagonists for intracellular or membrane ERs. Thus, the term “endocrine-disrupting chemicals” (EDCs) in this chapter is used to refer to contaminants with these effects. An important question considered here is the extent to which EDCs’ actions to alter E2 levels and/or ER binding in the hypothalamus or hippocampus mitigates effects on reproductive or nonreproductive processes. There are potential pervasive, negative effects of endocrine disrupters on steroid sensitive tissues, which may confer risk to disease states, such as cancer, heart disease, and neurodegenerative disorders. The following discussion provides evidence that exposure to EDCs during development may result in permanent, lifelong differences in sexual function and reproductive ability, as well as cognitive function and/or emotional reactivity/arousal. Gonad development, sex determination, and reproductive success of offspring are highly dependent on sex hormone systems. The developing organism is exquisitely sensitive to alterations in hormone function. In the early embryonic state, the gonads of human males and females are morphologically identical. Sexual differentiation begins under hormonal influence during the fifth and sixth weeks of fetal development, and thus, alterations in hormones during this highly sensitive period can have profound consequences. Disruption of the sex steroid system during fetal stages of life results in profound adverse developmental reproductive effects, as is well known from the effects of DES. The balance of estrogens and androgens is critical for normal development, growth, and functioning of the reproductive system. Although especially important during development, this balance is important throughout life for the preservation of normal feminine or masculine traits, as well as the expression of some sexually dimorphic behaviors (sex, spatial performance, and arousal).

Proposed negative effects of exposure to endocrine disrupters during development in people and in animals. The focus here is on vulnerability to sexually dimorphic processes that are estrogen-sensitive, such as reproductive, cognitive, and emotional development and associated behavioral processes

The existing data clearly indicate that developmental exposure to EDCs can adversely affect sexual development of people and animals; however, there are different effects depending upon the EDCs and when in development exposure occurs. Therefore, we consider the next effects of EDCs exposure at different point in development and the consequences for reproductive development and behavior, as well as E2 levels and hypothalamic ER binding.
Steroid hormones also play a critical role in neurodevelopment that influences not only reproductive but also nonreproductive behaviors that show sex differences. Specific behavioral differences in nonreproductive behaviors between males and females include differences in spatial learning, play, exploration, activity levels, novelty-seeking behavior, and emotional reactivity. These sex dimorphisms are thought to reflect adaptive differences for behavioral strategies in coping as a result of sexual selection. Moreover, these sexually dimorphic behaviors may be relevant for concerns regarding increased developmental, cognitive, or emotional disabilities over the past 30 years. Also, behaviors are particularly sensitive measures of effects of EDCs.
EDCs can alter cognitive development. Some, but not all, studies have shown a predictive relationship between prenatal PCB exposure and cognitive development in infancy through preschool years. EDCs have direct effects on nervous system function. Long-term potentiation (LTP), a form of synaptic plasticity used as a model system for study of cognitive potential, is altered by PCBs and lead. The protein kinase C (PKC)-signaling pathway is involved in the modulation of learning, memory, and motor behavior and may be a target of E2’s actions. PCBs also alter PKC signaling. Although findings provide evidence that EDCs can alter cognitive performance, these measures of cognition are neither sexually dimorphic nor E2- or ER-dependent.
There are sex-specific effects of perinatal PCB and dioxin exposure on spatial learning. Yu-Cheng boys that were prenatally exposed to high levels of PCBs and PCDFs when their mothers were accidentally exposed to these contaminants in rice oil show more disrupted cognitive development, mainly spatial function, than did exposed girls. In animal studies, spatial learning that favors males is mediated by perinatal exposure to androgens. Gestational and lactational exposure to ortho-substituted PCBs produces spatial deficits at adolescence in male mice or adulthood in male rats. The sparse data suggest that developmental exposure to EDCs disrupts spatial memory. Furthermore, Exposure during adulthood to EDCs can also have activational effects on spatial memory. Females exposed to a phytoestrogen-rich diet exhibit “masculinized” spatial performance in a radial arm maze, while males fed with a phytoestrogen-free diet show “feminized” performance.
An important question is what are the mechanisms by which developmental and/or adult exposure to EDCs alters spatial performance? There is evidence for sex differences in spatial performance and activational effects of E2 in adulthood to alter spatial performance of rats. Systemic or intrahippocampal administration of E2 improves spatial performance of female rats. Further, E2’s actions at intracellular ERs in the hippocampus of adults do not seem to be required to mediate these effects on spatial performance.
EDCs may have effects on E2 metabolism in a number of ways. First, some EDCs can alter serum lipid concentrations. Cholesterol is the precursor for the production of E2 and other steroid hormones (see Fig. 3.3). Second, there is also evidence that some EDCs can alter metabolism enzymes that are necessary for converting cholesterol to steroid hormones. Induction of CYP occurs when EDCs, such as TCDD, bind the aromatic hydrocarbon receptor (AhR). There is a firm link between PCBs, enzyme induction, and AhR. The binding of EDCs with AhR can result in antiestrogenic activity through increased metabolism and depletion of endogenous E2. Elevated levels of CYP enzymes, primarily expressed not only in the liver but also in the brain and other tissues, result in increased E2 metabolism and excretion. Alternatively, compounds that are metabolized by P450s may result in a net estrogenic effect if they inhibit endogenous estrogens from being metabolized.
Steroid hormones are lipid molecules with limited solubility in plasma and are accordingly carried through the plasma compartment to target cells by specific plasma transporter proteins. Each transporter protein has a specific ligand-binding domain for its associated hormone. It is generally accepted that the “free” formof the steroid hormone, and not the conjugate of the hormone with its plasma transport protein, enters target cells and binds with the appropriate receptor. Receptors for the steroid hormones are proteins located primarily in the cell nucleus or partitioned between the cytoplasm and the nucleus. The unoccupied steroid receptors may reside in the cell as heterodimeric complexes with the 90 kDa heat-shock protein, which prevents the receptor from binding with the DNA until the receptor has first bound with its steroid hormone. Once the hormone binds to the receptor, the hormone receptor complexes with the heterodimeric heat-shock protein and undergoes a conformational change and is activated. The activated receptor binds with DNA at a specific site, initiating gene transcription.

Traditional effects of steroid hormones at their cognate steroid receptors

Traditional effects of steroid hormones at their cognate steroid receptors

Traditional effects of steroid hormones at their cognate steroid receptors, which act as transcription factors. In this example, effects of steroid hormones, such as estradiol, to bind estrogen receptor (ER) subtypes, referred to as ERa and ERb, are shown.

Beyond traditional actions solely through intracellular cognate estrogen receptors (ERs; ERa and ERb), steroids, such as estradiol, and estradiol-mimetics (endocrine disrupters) may have novel actions involving membrane bound ERs, other neurotransmitter systems (e.g., NMDA receptor), and signal transduction cascades (e.g., growth factors, MAPK).

To date, there has been little investigation in a whole-animal model of the effects of EDCs on E2 levels and/or activity at intracellular ERs in the brain. Thus, changes in E2 levels and ER activity in the hypothalamus and hippocampus, concomitant with alterations in endocrine parameters and reproductive behavior and nonreproductive behavior, respectively, are
needed to elucidate tissue specificity of EDCs’ functions and mechanisms.

Low-Dose Effects of Hormones and Endocrine Disruptors

Laura N. Vandenberg
Vitamins and Hormones, 2014; 94: 129-165

Endogenous hormones have effects on tissue morphology, cell physiology, and behaviors at low doses. In fact, hormones are known to circulate in the part-per-trillion and part-per-billion concentrations, making them highly effective and potent signaling molecules.

Many endocrine-disrupting chemicals (EDCs) mimic hormones, yet there is strong debate over whether these chemicals can also have effects at low doses. In the 1990s, scientists proposed the “low-dose hypothesis,” which postulated that EDCs affect humans and animals at environmentally relevant doses. This chapter focuses on data that support and refute the low-dose hypothesis. A case study examining the highly controversial example of bisphenol A and its low-dose effects on the prostate is examined through the lens of endocrinology. Finally, the chapter concludes with a discussion of factors that can influence the ability of a study to detect and interpret low-dose effects appropriately.

Since EDCs began to be studied in depth in the 1990s, there has been intense debate over whether the public should be concerned about low level exposures to these chemicals. The low-dose hypothesis, proposed at that time, has steadily accumulated evidence that EDCs have actions at low doses, and these effects are not necessarily predicted from high-dose toxicology testing. In 2002, the NTP expert panel reported evidence for low-dose effects for a small number of EDCs and estradiol. In 2012, an updated approach identified several dozen additional EDCs with evidence for low-dose effects. Further, epidemiology studies continue to find relationships between EDC exposure levels and diseases in the general public, which has raised concerns because the general public is exposed to a large number of environmental chemicals at low doses. For decades, hormones have been known to produce striking changes in tissue morphology, physiology, and behaviors at exceedingly low doses.

A relatively large body of evidence suggests that EDCs, and in particular those environmental chemicals that mimic endogenous hormones, have similar effects at low doses. Although there is still no consensus about the universality of “low-dose effects” in the toxicology community, the Endocrine Society (Diamanti-Kandarakis et al., 2009; Zoeller et al., 2012) believes not only that there is sufficient evidence in support of this phenomenon but also that it is time for public health agencies to make changes to risk assessment paradigms and give greater consideration to studies that specifically identify low-dose effects when considering risks from chemical exposures.

Bisphenol A interferes with synaptic remodeling

Tibor Hajszan, Csaba Leranth
Frontiers in Neuroendocrinology 31 (2010) 519–530

The potential adverse effects of Bisphenol A (BPA), a synthetic xenoestrogen, have long been debated. Although standard toxicology tests have revealed no harmful effects, recent research highlighted what was missed so far: BPA-induced alterations in the nervous system. Since 2004, our laboratory has been investigating one of the central effects of BPA, which is interference with gonadal steroid-induced synaptogenesis and the resulting loss of spine synapses. We have shown in both rats and nonhuman primates that BPA completely negates the ~70–100% increase in the number of hippocampal and prefrontal spine synapses induced by both estrogens and androgens. Synaptic loss of this magnitude may have significant consequences, potentially causing cognitive decline, depression, and schizophrenia, to mention those that our laboratory has shown to be associated with synaptic loss. Finally, we discuss why children may particularly be vulnerable to BPA, which represents future direction of research in our laboratory.

Bisphenol-A rapidly promotes dynamic changes in hippocampal dendritic morphology through estrogen receptor-mediated pathway by concomitant phosphorylation of NMDA receptor subunit NR2B

Xiaohong Xu ⁎, Yinping Ye, Tao Li, Lei Chen, Dong Tian, Qingqing Luo, Mei Lu
Toxicology and Applied Pharmacology 249 (2010) 188–196

Bisphenol-A (BPA) is known to be a potent endocrine disrupter. Evidence is emerging that estrogen exerts a rapid influence on hippocampal synaptic plasticity and the dendritic spine density, which requires activation of NMDA receptors. In the present study, we investigated the effects of BPA (ranging from 1 to 1000 nM), focusing on the rapid dynamic changes in dendritic filopodia and the expressions of estrogen receptor (ER) β and NMDA receptor, as well as the phosphorylation of NMDA receptor subunit NR2B in the cultured hippocampal neurons. A specific ER antagonist ICI 182,780 was used to examine the potential involvement of ERs. The results demonstrated that exposure to BPA (ranging from 10 to 1000 nM) for 30 min rapidly enhanced the motility and the density of dendritic filopodia in the cultured hippocampal neurons, as well as the phosphorylation of NR2B (pNR2B), though the expressions of NMDA receptor subunits NR1, NR2B, and ERβ were not changed. The antagonist of ERs completely inhibited the BPA-induced increases in the filopodial motility and the number of filopodia extending from dendrites. The increased pNR2B induced by BPA (100 nM) was also completely eliminated. Furthermore, BPA attenuated the effects of 17β-estradiol (17β-E2) on the dendritic filopodia outgrowth and the expression of pNR2B when BPA was co-treated with 17β-E2. The present results suggest that BPA, like 17β-E2, rapidly results in the enhanced motility and density of dendritic filopodia in the cultured hippocampal neurons with the concomitant activation of NMDA receptor subunit NR2B via an ER-mediated signaling pathway. Meanwhile, BPA suppressed the enhancement effects of 17β-E2 when it coexists with 17β-E2. These results provided important evidence suggesting the neurotoxicity of the low levels of BPA during the early postnatal development of the brain.

Bisphenol-A rapidly enhanced passive avoidance memory and phosphorylation of NMDA receptor subunits in hippocampus of young rats

Xiaohong Xu⁎, Tao Li, Qingqing Luo, Xing Hong, Lingdan Xie, Dong Tian
Toxicology and Applied Pharmacology 255 (2011) 221–228

Bisphenol-A (BPA), an endocrine disruptor, is found to influence development of brain and behaviors in rodents. The previous study indicated that perinatal exposure to BPA impaired learning-memory and inhibited N-methyl-D-aspartate receptor (NMDAR) subunits expressions in hippocampus during the postnatal development in rats; and in cultured hippocampal neurons, BPA rapidly promotes dynamic changes in dendritic morphology through estrogen receptor-mediated pathway by concomitant phosphorylation of NMDAR subunit NR2B. In the present study, we examined the rapid effect of BPA on passive avoidance memory and NMDAR in the developing hippocampus of Sprague–Dawley rats at the age of postnatal day 18. The results showed that BPA or estradiol benzoate (EB) rapidly extended the latency to step down from the platform 1 h after foot shock and increased the phosphorylation levels of NR1, NR2B, and mitogen-activated extracellular signal-regulated kinase (ERK) in hippocampus within 1 h. While 24 h after BPA or EB treatment, the improved memory and the increased phosphorylation levels of NR1, NR2B, ERK disappeared. Furthermore, pre-treatment with an estrogen receptors (ERs) antagonist, ICI182, 780, or an ERK-activating kinase inhibitor, U0126, significantly attenuated EB- or BPA-induced phosphorylations of NR1, NR2B, and ERK within 1 h. These data suggest that BPA rapidly enhanced short-term passive avoidance memory in the developing rats. A non-genomic effect via ERs may mediate the modulation of the phosphorylation of NMDAR subunits NR1 and NR2B through ERK signaling pathway.

Bisphenol A promotes dendritic morphogenesis of hippocampal neurons through estrogen receptor-mediated ERK1/2 signal pathway

Xiaohong Xu, Yang Lu, Guangxia Zhang, Lei Chen, Dong Tian, et al.
Chemosphere 96 (2014) 129–137

Bisphenol A (BPA), an environmental endocrine disruptor, has attracted increasing attention to its adverse effects on brain developmental process. The previous study indicated that BPA rapidly increased motility and density of dendritic filopodia and enhanced the phosphorylation of N-methyl-D-aspartate (NMDA) receptor subunit NR2B in cultured hippocampal neurons within 30 min. The purpose of the present study was further to investigate the effects of BPA for 24 h on dendritic morphogenesis and the underlying mechanisms. After cultured for 5 d in vitro, the hippocampal neurons from 24 h-old rat were infected by AdV-EGFP to indicate time-lapse imaging of living neurons. The results demonstrated that the exposure of the cultured hippocampal neurons to BPA (10, 100 nM) or 17β-estradiol (17β-E2, 10 nM) for 24 h significantly promoted dendritic development, as evidenced by the increased total length of dendrite and the enhanced motility and density of dendritic filopodia. However, these changes were suppressed by an ERs antagonist, ICI182,780, a non-competitive NMDA receptor antagonist, MK-801, and a mitogen activated ERK1/2-activating kinase (MEK1/2) inhibitor, U0126. Meanwhile, the increased F-actin (filamentous actin) induced by BPA (100 nM) was also completely eliminated by these blockers. Furthermore, the result of western blot analyses showed that, the exposure of the cultures to BPA or 17β-E2 for 24 h promoted the expression of Rac1/Cdc42 but inhibited that of RhoA, suggesting Rac1 (Ras related C3 botulinum toxinsubstrate 1)/Cdc42 (cell divisioncycle 42) and RhoA (Ras homologous A), the Rho family of small GTPases, were involved in BPA- or 17β-E2-induced changes in the dendritic morphogenesis of neurons. These BPA- or 17b-E2-induced effects were completely blocked by ICI182,780, and were partially suppressed by U0126. These results reveal that, similar to 17β-E2, BPA exerts its effects on dendritic morphogenesis by eliciting both nuclear actions and extranuclear-initiated actions that are integrated to influence the development of dendrite in hippocampal neurons.

Tyreoliberin (Trh) – The Regulatory Neuropeptide Of Cns Homeostasis
Danuta Jantas
Advances In Cell Biology 2;(4)/2010 (139–154)

The physiological role of thyreoliberin (TRH) is the preservation of homeostasis within four systems
(i) the hypothalamic-hypophsysiotropic neuroendocrine system,
(ii) the brain stem/midbrain/spinal cord system,
(iii) the limbic/cortical system, and
(iv) the chronobiological system.

Thus TRH, via various cellular mechanisms, regulates a wide range of biological processes (arousal, sleep, learning, locomotive activity, mood) and possesses the potential for unique and widespread applications for treatment of human illnesses. Since the therapeutic potential of TRH is limited by its pharmacological profile (enzymatic instability, short half-life, undesirable effects), several synthetic analogues of TRH were constructed and studied in mono- or adjunct therapy of central nervous system (CNS) disturbances. The present article summarizes the current state of understanding of the physiological role of TRH and describes its putative role in clinical indications in CNS maladies with a focus on the action of TRH analogues.

Breakthrough in neuroendocrinology by discovering novel neuropeptides and neurosteroids: 2. Discovery of neurosteroids and pineal neurosteroids

Kazuyoshi Tsutsui, Shogo Haraguchi
General and Comparative Endocrinology 205 (2014) 11–22

Bargmann–Scharrer’s discovery of ‘‘neurosecretion’’ in the first half of the 20th century has since matured into the scientific discipline of neuroendocrinology. Identification of novel neurohormones, such as neuropeptides and neurosteroids, is essential for the progress of neuroendocrinology. Our studies over the past two decades have significantly broadened the horizons of this field of research by identifying novel neuropeptides and neurosteroids in vertebrates that have opened new lines of scientific investigation in neuroendocrinology. We have established de novo synthesis and functions of neurosteroids in the brain of various vertebrates. Recently, we discovered 7α-hydroxypregnenolone (7α-OH PREG), a novel bioactive neurosteroid that acts as a key regulator for inducing locomotor behavior by means of the dopaminergic system. We further discovered that the pineal gland, an endocrine organ located close to the brain, is an important site of production of neurosteroids de novo from cholesterol (CHOL). The pineal gland secretes 7α-OH PREG and 3α,5α-tetrahydroprogesterone (3α,5α-THP; allopregnanolone) that are involved in locomotor rhythms and neuronal survival, respectively. Subsequently, we have demonstrated their mode of action and functional significance. This review summarizes the discovery of these novel neurosteroids and its contribution to the progress of neuroendocrinology.

Mechanisms of crosstalk between endocrine systems: Regulation of sex steroid hormone synthesis and action by thyroid hormones

Paula Duarte-Guterman, Laia Navarro-Martín, Vance L. Trudeau
General and Comparative Endocrinology 203 (2014) 69–85

Thyroid hormones (THs) are well-known regulators of development and metabolism in vertebrates. There is increasing evidence that THs are also involved in gonadal differentiation and reproductive function. Changes in TH status affect sex ratios in developing fish and frogs and reproduction (e.g., fertility), hormone levels, and gonad morphology in adults of species of different vertebrates. In this review, we have summarized and compared the evidence for cross-talk between the steroid hormone and thyroid axes and present a comparative model. We gave special attention to TH regulation of sex steroid synthesis and action in both the brain and gonad, since these are important for gonad development and brain sexual differentiation and have been studied in many species. We also reviewed research showing that there is a TH system, including receptors and enzymes, in the brains and gonads in developing and adult vertebrates. Our analysis shows that THs influences sex steroid hormone synthesis in vertebrates, ranging from fish to pigs. This concept of crosstalk and conserved hormone interaction has implications for our understanding of the role of THs in reproduction, and how these processes may be dysregulated by environmental endocrine disruptors.

Insights into the structure of class B GPCRs

Kaspar Hollenstein, Chris de Graaf, Andrea Bortolato, Ming-Wei Wang, et al.
Trends in Pharmacological Sciences, Jan 2014; 35(1)

The secretin-like (class B) family of G protein-coupled receptors (GPCRs) are key players in hormonal homeostasis and are interesting drug targets for the treatment of several metabolic disorders (such as type 2 diabetes, osteoporosis, and obesity) and nervous system diseases (such as migraine, anxiety, and depression). The recently solved crystal structures of the transmembrane domains of the human glucagon receptor and human corticotropin-releasing factor receptor 1 have opened up new opportunities to study the structure and function of class B GPCRs. The current review shows how these structures offer more detailed explanations to previous biochemical and pharmacological studies of class B GPCRs, and provides new insights into their interactions with ligands.

Class B G protein-coupled receptors (GPCRs), also referred to as the secretin family of GPCRs, include receptors for 15 peptide hormones, which can be grouped into five subfamilies based on their physiological role (see Table 1 for an overview) [1]. These receptors are important drug targets in many human diseases, including diabetes, osteoporosis, obesity, cancer, neurodegeneration, cardiovascular disease, headache, and psychiatric disorders. However, the identification of small-molecule oral drugs for this family has proved extremely challenging.

(A,B) Crystal structures of the class B G protein-coupled receptors corticotropin-releasing factor receptor 1 (CRF1) [Protein Data Bank (PDB) identifier: 4K5Y] and glucagon receptor (PDB identifier: 4L6R) are shown in blue and orange ribbons, respectively, in two different views from within the membrane. Transmembrane (TM) helices and helix 8 are labelled. The disulfide bond tethering extracellular loop 2 (ECL2) to the tip of TM3 is shown as purple sticks. In CRF1 the small-molecule antagonist CP-376395 is shown in stick representation with carbon, nitrogen, and oxygen atoms colored magenta, blue, and red, respectively, and as skeletal formula in an inset. (C) Superposition of the two structures, with insets highlighting regions of particular interest. To highlight the structural differences in the extracellular halves of CRF1 and glucagon receptor, the distance of approximately 11 A° between the Ca-atoms of residues 7.33b at the N-terminal end of TM7 is indicated with a red arrow. The small molecule binding pocket is shown as a superposition of the two receptors around CP-376395, illustrating the antagonist binding mode and the substantial structural differences observed for TM6 of the two receptors.

  • Overview of NMR and crystal structures of class B G protein-coupled

receptor (GPCR) extracellular domains (ECDs; magenta) and their complexes with peptide ligands (different colors). A complete overview of corresponding Protein Data Bank identifiers is presented in Table 1 (not shown). (B) Structure-based sequence alignment of representative peptide ligands of class B GPCR, adopted from Parthier et al. [6]. The residues of the peptide ligands solved in ECD–ligand complex crystal structures are marked using the same colour as in Figure 2A. The residues that are boxed black are found in an α-helical conformation in the complex. Peptide ligand residues that covalently bind receptors in photo-crosslinking or cysteine-trapping studies are colored and boxed green, whereas peptide ligand residues that have been mutated and studied in combination with receptor mutants are colored and boxed red. Note that the first residue of glucagon-like peptide-1 (GLP-1) is His7. A complete overview of all ECD structures and important peptide ligands for all class B GPCRs is presented in Table 1. Putative helix-capping residues [6] are coloured blue and cysteines involved in a disulfide-bridge (calcitonin) are coloured orange. D-phenylalanine (f), and norleucine (m) residues are indicated in stressin and astressin. The last 41 and 99 residues of parathyroid hormone (PTH) and PTH-related protein.  (PTHrP), respectively, are not displayed. Abbreviations: CGRP, calcitonin gene-related peptide; CLR, calcitonin receptor-like receptor; CRF, corticotropin-releasing factor; CT, calcitonin; Ext-4, exendin-4; GHRHR, growth hormone releasing hormone receptor; GIP, glucose-dependent insulinotropic peptide; PAC, pituitary adenylate cyclase; PACAP, pituitary adenylate cyclase activating polypeptide; RAMP, receptor-activity modifying proteins; SCTR, secretin receptor; Ucn, urocortin; VPAC, vasoactive pituitary adenylate cyclase.

Figure 3. (not shown) (A) The spatial correspondence between residues in transmembrane (TM) helices of class A and class B G protein-coupled receptors (GPCRs) makes it possible to align the most conserved residues in class A (designated X.50, Ballesteros–Weinstein numbering) and class B (designated X.50b, Wootten numbering) for comparisons between GPCR classes (Box 1). (B) Structural alignment of corticotropin-releasing factor receptor 1 (CRF1; blue) and glucagon receptor (GCGR; orange) to two representative class A GPCRs, histamine H1 receptor (H1R; Protein Data Bank identifier: 3RZE) and CXC-chemokine receptor 4 (CXCR4; Protein Data Bank identifier: 3ODU/3OE0) (in grey). Helices are depicted as cylinders, and the ligands glucagon (for GCGR), CP-376395 (for CRF1), doxepin (for H1R), and IT1t and CVX15 (for CXCR4) are shown as sticks. The

location of the Ca-atoms of the most conserved residues of TM1–3 and TM5 among class A and class B GPCRs (Box 1) are indicated by spheres (TM4 is not depicted for clarity).

The GCGR and CRF1 crystal structures show distinct structural features and different binding pockets compared to class A GPCRs, and give new insights into the molecular details of peptide and small-molecule binding to class B GPCRs. The first two crystal structures of the TM domains of class B GPCRs provide a structural framework that will enable the design of biochemical and biophysical experiments detailing the complex structure of this class of receptors, and facilitate the design of stabilized constructs that might lead to the solution of full-length class B GPCR–ligand complexes. The structures furthermore present more reliable structural templates for the design of specific and potent small molecules for the treatment of type 2 diabetes (GCGR) and depression (CRF1) in particular, and open new avenues for structure-based small-molecule drug discovery for class B GPCRs as a whole.

Novel receptor targets for production and action of allopregnanolone in the central nervous system: a focus on pregnane xenobiotic receptor

Cheryl A. Frye, Carolyn J. Koonce and Alicia A. Walf
Front in Cell Neurosci  Apr 2014; 8(106): 1-13.

Neurosteroids are cholesterol-based hormones that can be produced in the brain,

independent of secretion from peripheral endocrine glands, such as the gonads and

adrenals. A focus in our laboratory for over 25 years has been how production of the

pregnane neurosteroid, allopregnanolone, is regulated and the novel (i.e., non steroid

receptor) targets for steroid action for behavior. One endpoint of interest has been lordosis, the mating posture of female rodents. Allopregnanolone is necessary and sufficient for lordosis, and the brain circuitry underlying it, such as actions in the midbrain ventral tegmental area (VTA), has been well-characterized. Published and recent findings supporting a dynamic role of allopregnanolone are included in this review.
First, contributions of ovarian and adrenal sources of precursors of allopregnanolone, and the requisite enzymatic actions for de novo production in the central nervous system will be discussed.
Second, how allopregnanolone produced in the brain has actions on behavioral processes that are independent of binding to steroid receptors, but instead involve rapid modulatory actions via neurotransmitter targets (e.g., g-amino butyric acid-GABA, N methyl-D-aspartate- NMDA) will be reviewed.
Third, a recent focus on characterizing the role of a promiscuous nuclear receptor, pregnane xenobiotic receptor (PXR), involved in cholesterol metabolism and expressed in the VTA, as a target for allopregnanolone and how this relates to both actions and production of allopregnanolone will be addressed. For example, allopregnanolone can bind PXR and knocking down expression of PXR in the midbrain VTA attenuates actions of allopregnanolone via NMDA and/or GABAA for lordosis. Our understanding of allopregnanolone’s actions in the VTA for lordosis has been extended to reveal the role of allopregnanolone for broader, clinically-relevant questions, such as neurodevelopmental processes, neuropsychiatric disorders, epilepsy, and aging.

Genetically Encoded Chemical Probes in Cells Reveal the Binding Path of Urocortin-I to CRF Class B GPCR

Irene Coin, Vsevolod Katritch, Tingting Sun, Zheng Xiang, Fai Yiu Siu
Cell  Dec 2013; 155, 1258–1269

Molecular determinants regulating the activation of class B G-protein-coupled receptors (GPCRs) by native peptide agonists are largely unknown. We have investigated here the interaction between the corticotropin releasing factor receptor type 1 (CRF1R) and its native 40-mer peptide ligand Urocortin- I directly in mammalian cells. By incorporating unnatural amino acid photochemical and new click chemical probes into the intact receptor expressed in the native membrane of live cells, 44 intermolecular spatial constraints have been derived for the ligand-receptor interaction. The data were analyzed in the context of the recently resolved crystal structure of
CRF1R transmembrane domain and existing extracellular domain structures, yielding a complete conformational model for the peptide-receptor complex. Structural features of the receptor-ligand complex yield molecular insights
on the mechanism of receptor activation and the basis for discrimination between agonist and antagonist function.

Investigation of GPCR-Ligand Interactions under Native Conditions Using Genetically Encoded Chemical Probes GPCRs are integral membrane proteins containing multiple domains and various posttranslational modifications. To understand GPCR-ligand interactions by crystallography, receptors have to be extracted from the cell membrane and modified with a series of expedients such as deglycosylation, therm-stabilizing mutations, fusions with soluble proteins, or complexes with stabilizing nanobodies. We present here a method to investigate GPCR-ligand interactions at the intact fully posttranslationally modified receptor bound to its WT ligand on the membrane of the live cell, which mimics the native conditions for GPCR function. We first genetically incorporated into the receptor the photocrosslinking Uaa Azi, which served as
a proximity probe to provide an overall map of the ligand binding sites on the receptor. We then determined the relative position of the ligand in the binding pocket using a residue-specific chemical crosslinking reaction between Ffact genetically incorporated into the receptor and Cys introduced into the ligand. The derived intermolecular spatial constraints served eventually to build a detailed conformational model for the receptor-ligand complex.

Glutamate Neurons within the Midbrain Dopamine Regions

  1. Morales and D. H. Root
    Neuroscience 282 (2014) 60–68

Midbrain dopamine (DA) neurons are hypothesized to play roles in reward-based behavior and addiction, reward prediction and learning by error detection, effort-based decision making, flexible reward-directed behaviors,

incentive salience, stimulus salience (e.g., prediction of rewarding and aversive events), aversion, depression, and fear. The extensive, divergent behavioral roles of midbrain dopamine neurons, predominantly from the ventral tegmental area (VTA), indicate that this system is highly heterogeneous.
This heterogeneity may be reflected in part by the diverse phenotypic characteristics among DAergic neurons and their interactive brain structures.

Midbrain dopamine systems play important roles in Parkinson’s disease, schizophrenia, addiction, and depression. The participation of midbrain dopamine systems in diverse clinical contexts suggests these systems are highly complex. Midbrain dopamine regions contain at least three neuronal phenotypes: dopaminergic, GABAergic, and glutamatergic. Here, we review the locations, subtypes, and functions of glutamatergic neurons within midbrain dopamine regions. Vesicular glutamate transporter 2 (VGluT2) mRNA-expressing neurons are observed within each midbrain dopamine system. Within rat retrorubral field (RRF), large populations of VGluT2 neurons are observed throughout its anteroposterior extent. Within rat substantia nigra pars compacta (SNC), VGluT2 neurons are observed centrally and caudally, and are most dense within the laterodorsal subdivision. RRF and SNC rat VGluT2 neurons lack tyrosine hydroxylase (TH), making them an entirely distinct population of neurons from dopaminergic neurons. The rat ventral tegmental area (VTA) contains the most heterogeneous populations of VGluT2 neurons. VGluT2 neurons are found in each VTA subnucleus but are most dense within the anterior midline subnuclei. Some subpopulations of rat VGluT2 neurons co-express TH or glutamic acid decarboxylase (GAD), but most of the VGluT2 neurons lack TH or GAD. Different subsets of rat VGluT2-TH neurons exist based on the presence or absence of vesicular monoamine transporter 2, dopamine transporter, or D2 dopamine receptor. Thus, the capacity by which VGluT2-TH neurons may release dopamine will differ based on their capacity to accumulate vesicular dopamine, uptake extracellular dopamine, or be autoregulated by dopamine. Rat VTA VGluT2 neurons exhibit intrinsic VTA projections and extrinsic projections to the accumbens and to the prefrontal cortex. Mouse VTA VGluT2 neurons project to accumbens shell, prefrontal cortex, ventral pallidum, amygdala, and lateral habenula. Given their molecular diversity and participation in circuits involved in addiction, we hypothesize that individual VGluT2 subpopulations of neurons play unique roles in addiction and other disorders. This article is part of a Special Issue entitled: Ventral Tegmentum & Dopamine. Published by Elsevier Ltd. On behalf of IBRO.

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Summary and Perspectives: Impairments in Pathological States: Endocrine Disorders, Stress Hypermetabolism and Cancer

Summary and Perspectives: Impairments in Pathological States: Endocrine Disorders, Stress Hypermetabolism and Cancer

Author and Curator: Larry H. Bernstein, MD, FCAP

This summary is the last of a series on the impact of transcriptomics, proteomics, and metabolomics on disease investigation, and the sorting and integration of genomic signatures and metabolic signatures to explain phenotypic relationships in variability and individuality of response to disease expression and how this leads to  pharmaceutical discovery and personalized medicine.  We have unquestionably better tools at our disposal than has ever existed in the history of mankind, and an enormous knowledge-base that has to be accessed.  I shall conclude here these discussions with the powerful contribution to and current knowledge pertaining to biochemistry, metabolism, protein-interactions, signaling, and the application of the -OMICS to diseases and drug discovery at this time.

The Ever-Transcendent Cell

Deriving physiologic first principles By John S. Torday | The Scientist Nov 1, 2014

Both the developmental and phylogenetic histories of an organism describe the evolution of physiology—the complex of metabolic pathways that govern the function of an organism as a whole. The necessity of establishing and maintaining homeostatic mechanisms began at the cellular level, with the very first cells, and homeostasis provides the underlying selection pressure fueling evolution.

While the events leading to the formation of the first functioning cell are debatable, a critical one was certainly the formation of simple lipid-enclosed vesicles, which provided a protected space for the evolution of metabolic pathways. Protocells evolved from a common ancestor that experienced environmental stresses early in the history of cellular development, such as acidic ocean conditions and low atmospheric oxygen levels, which shaped the evolution of metabolism.

The reduction of evolution to cell biology may answer the perennially unresolved question of why organisms return to their unicellular origins during the life cycle.

As primitive protocells evolved to form prokaryotes and, much later, eukaryotes, changes to the cell membrane occurred that were critical to the maintenance of chemiosmosis, the generation of bioenergy through the partitioning of ions. The incorporation of cholesterol into the plasma membrane surrounding primitive eukaryotic cells marked the beginning of their differentiation from prokaryotes. Cholesterol imparted more fluidity to eukaryotic cell membranes, enhancing functionality by increasing motility and endocytosis. Membrane deformability also allowed for increased gas exchange.

Acidification of the oceans by atmospheric carbon dioxide generated high intracellular calcium ion concentrations in primitive aquatic eukaryotes, which had to be lowered to prevent toxic effects, namely the aggregation of nucleotides, proteins, and lipids. The early cells achieved this by the evolution of calcium channels composed of cholesterol embedded within the cell’s plasma membrane, and of internal membranes, such as that of the endoplasmic reticulum, peroxisomes, and other cytoplasmic organelles, which hosted intracellular chemiosmosis and helped regulate calcium.

As eukaryotes thrived, they experienced increasingly competitive pressure for metabolic efficiency. Engulfed bacteria, assimilated as mitochondria, provided more bioenergy. As the evolution of eukaryotic organisms progressed, metabolic cooperation evolved, perhaps to enable competition with biofilm-forming, quorum-sensing prokaryotes. The subsequent appearance of multicellular eukaryotes expressing cellular growth factors and their respective receptors facilitated cell-cell signaling, forming the basis for an explosion of multicellular eukaryote evolution, culminating in the metazoans.

Casting a cellular perspective on evolution highlights the integration of genotype and phenotype. Starting from the protocell membrane, the functional homolog for all complex metazoan organs, it offers a way of experimentally determining the role of genes that fostered evolution based on the ontogeny and phylogeny of cellular processes that can be traced back, in some cases, to our last universal common ancestor.  ….

As eukaryotes thrived, they experienced increasingly competitive pressure for metabolic efficiency. Engulfed bacteria, assimilated as mitochondria, provided more bioenergy. As the evolution of eukaryotic organisms progressed, metabolic cooperation evolved, perhaps to enable competition with biofilm-forming, quorum-sensing prokaryotes. The subsequent appearance of multicellular eukaryotes expressing cellular growth factors and their respective receptors facilitated cell-cell signaling, forming the basis for an explosion of multicellular eukaryote evolution, culminating in the metazoans.

Casting a cellular perspective on evolution highlights the integration of genotype and phenotype. Starting from the protocell membrane, the functional homolog for all complex metazoan organs, it offers a way of experimentally determining the role of genes that fostered evolution based on the ontogeny and phylogeny of cellular processes that can be traced back, in some cases, to our last universal common ancestor.

Given that the unicellular toolkit is complete with all the traits necessary for forming multicellular organisms (Science, 301:361-63, 2003), it is distinctly possible that metazoans are merely permutations of the unicellular body plan. That scenario would clarify a lot of puzzling biology: molecular commonalities between the skin, lung, gut, and brain that affect physiology and pathophysiology exist because the cell membranes of unicellular organisms perform the equivalents of these tissue functions, and the existence of pleiotropy—one gene affecting many phenotypes—may be a consequence of the common unicellular source for all complex biologic traits.  …

The cell-molecular homeostatic model for evolution and stability addresses how the external environment generates homeostasis developmentally at the cellular level. It also determines homeostatic set points in adaptation to the environment through specific effectors, such as growth factors and their receptors, second messengers, inflammatory mediators, crossover mutations, and gene duplications. This is a highly mechanistic, heritable, plastic process that lends itself to understanding evolution at the cellular, tissue, organ, system, and population levels, mediated by physiologically linked mechanisms throughout, without having to invoke random, chance mechanisms to bridge different scales of evolutionary change. In other words, it is an integrated mechanism that can often be traced all the way back to its unicellular origins.

The switch from swim bladder to lung as vertebrates moved from water to land is proof of principle that stress-induced evolution in metazoans can be understood from changes at the cellular level.


A MECHANISTIC BASIS FOR LUNG DEVELOPMENT: Stress from periodic atmospheric hypoxia (1) during vertebrate adaptation to land enhances positive selection of the stretch-regulated parathyroid hormone-related protein (PTHrP) in the pituitary and adrenal glands. In the pituitary (2), PTHrP signaling upregulates the release of adrenocorticotropic hormone (ACTH) (3), which stimulates the release of glucocorticoids (GC) by the adrenal gland (4). In the adrenal gland, PTHrP signaling also stimulates glucocorticoid production of adrenaline (5), which in turn affects the secretion of lung surfactant, the distension of alveoli, and the perfusion of alveolar capillaries (6). PTHrP signaling integrates the inflation and deflation of the alveoli with surfactant production and capillary perfusion.  THE SCIENTIST STAFF

From a cell-cell signaling perspective, two critical duplications in genes coding for cell-surface receptors occurred during this period of water-to-land transition—in the stretch-regulated parathyroid hormone-related protein (PTHrP) receptor gene and the β adrenergic (βA) receptor gene. These gene duplications can be disassembled by following their effects on vertebrate physiology backwards over phylogeny. PTHrP signaling is necessary for traits specifically relevant to land adaptation: calcification of bone, skin barrier formation, and the inflation and distention of lung alveoli. Microvascular shear stress in PTHrP-expressing organs such as bone, skin, kidney, and lung would have favored duplication of the PTHrP receptor, since sheer stress generates radical oxygen species (ROS) known to have this effect and PTHrP is a potent vasodilator, acting as an epistatic balancing selection for this constraint.

Positive selection for PTHrP signaling also evolved in the pituitary and adrenal cortex (see figure on this page), stimulating the secretion of ACTH and corticoids, respectively, in response to the stress of land adaptation. This cascade amplified adrenaline production by the adrenal medulla, since corticoids passing through it enzymatically stimulate adrenaline synthesis. Positive selection for this functional trait may have resulted from hypoxic stress that arose during global episodes of atmospheric hypoxia over geologic time. Since hypoxia is the most potent physiologic stressor, such transient oxygen deficiencies would have been acutely alleviated by increasing adrenaline levels, which would have stimulated alveolar surfactant production, increasing gas exchange by facilitating the distension of the alveoli. Over time, increased alveolar distension would have generated more alveoli by stimulating PTHrP secretion, impelling evolution of the alveolar bed of the lung.

This scenario similarly explains βA receptor gene duplication, since increased density of the βA receptor within the alveolar walls was necessary for relieving another constraint during the evolution of the lung in adaptation to land: the bottleneck created by the existence of a common mechanism for blood pressure control in both the lung alveoli and the systemic blood pressure. The pulmonary vasculature was constrained by its ability to withstand the swings in pressure caused by the systemic perfusion necessary to sustain all the other vital organs. PTHrP is a potent vasodilator, subserving the blood pressure constraint, but eventually the βA receptors evolved to coordinate blood pressure in both the lung and the periphery.

Gut Microbiome Heritability

Analyzing data from a large twin study, researchers have homed in on how host genetics can shape the gut microbiome.
By Tracy Vence | The Scientist Nov 6, 2014

Previous research suggested host genetic variation can influence microbial phenotype, but an analysis of data from a large twin study published in Cell today (November 6) solidifies the connection between human genotype and the composition of the gut microbiome. Studying more than 1,000 fecal samples from 416 monozygotic and dizygotic twin pairs, Cornell University’s Ruth Ley and her colleagues have homed in on one bacterial taxon, the family Christensenellaceae, as the most highly heritable group of microbes in the human gut. The researchers also found that Christensenellaceae—which was first described just two years ago—is central to a network of co-occurring heritable microbes that is associated with lean body mass index (BMI).  …

Of particular interest was the family Christensenellaceae, which was the most heritable taxon among those identified in the team’s analysis of fecal samples obtained from the TwinsUK study population.

While microbiologists had previously detected 16S rRNA sequences belonging to Christensenellaceae in the human microbiome, the family wasn’t named until 2012. “People hadn’t looked into it, partly because it didn’t have a name . . . it sort of flew under the radar,” said Ley.

Ley and her colleagues discovered that Christensenellaceae appears to be the hub in a network of co-occurring heritable taxa, which—among TwinsUK participants—was associated with low BMI. The researchers also found that Christensenellaceae had been found at greater abundance in low-BMI twins in older studies.

To interrogate the effects of Christensenellaceae on host metabolic phenotype, the Ley’s team introduced lean and obese human fecal samples into germ-free mice. They found animals that received lean fecal samples containing more Christensenellaceae showed reduced weight gain compared with their counterparts. And treatment of mice that had obesity-associated microbiomes with one member of the Christensenellaceae family, Christensenella minuta, led to reduced weight gain.   …

Ley and her colleagues are now focusing on the host alleles underlying the heritability of the gut microbiome. “We’re running a genome-wide association analysis to try to find genes—particular variants of genes—that might associate with higher levels of these highly heritable microbiota.  . . . Hopefully that will point us to possible reasons they’re heritable,” she said. “The genes will guide us toward understanding how these relationships are maintained between host genotype and microbiome composition.”

J.K. Goodrich et al., “Human genetics shape the gut microbiome,” Cell,  http://dx.doi.org:/10.1016/j.cell.2014.09.053, 2014.

Light-Operated Drugs

Scientists create a photosensitive pharmaceutical to target a glutamate receptor.
By Ruth Williams | The Scentist Nov 1, 2014

light operated drugs MO1

light operated drugs MO1


The desire for temporal and spatial control of medications to minimize side effects and maximize benefits has inspired the development of light-controllable drugs, or optopharmacology. Early versions of such drugs have manipulated ion channels or protein-protein interactions, “but never, to my knowledge, G protein–coupled receptors [GPCRs], which are one of the most important pharmacological targets,” says Pau Gorostiza of the Institute for Bioengineering of Catalonia, in Barcelona.

Gorostiza has taken the first step toward filling that gap, creating a photosensitive inhibitor of the metabotropic glutamate 5 (mGlu5) receptor—a GPCR expressed in neurons and implicated in a number of neurological and psychiatric disorders. The new mGlu5 inhibitor—called alloswitch-1—is based on a known mGlu receptor inhibitor, but the simple addition of a light-responsive appendage, as had been done for other photosensitive drugs, wasn’t an option. The binding site on mGlu5 is “extremely tight,” explains Gorostiza, and would not accommodate a differently shaped molecule. Instead, alloswitch-1 has an intrinsic light-responsive element.

In a human cell line, the drug was active under dim light conditions, switched off by exposure to violet light, and switched back on by green light. When Gorostiza’s team administered alloswitch-1 to tadpoles, switching between violet and green light made the animals stop and start swimming, respectively.

The fact that alloswitch-1 is constitutively active and switched off by light is not ideal, says Gorostiza. “If you are thinking of therapy, then in principle you would prefer the opposite,” an “on” switch. Indeed, tweaks are required before alloswitch-1 could be a useful drug or research tool, says Stefan Herlitze, who studies ion channels at Ruhr-Universität Bochum in Germany. But, he adds, “as a proof of principle it is great.” (Nat Chem Biol, http://dx.doi.org:/10.1038/nchembio.1612, 2014)

Enhanced Enhancers

The recent discovery of super-enhancers may offer new drug targets for a range of diseases.
By Eric Olson | The Scientist Nov 1, 2014

To understand disease processes, scientists often focus on unraveling how gene expression in disease-associated cells is altered. Increases or decreases in transcription—as dictated by a regulatory stretch of DNA called an enhancer, which serves as a binding site for transcription factors and associated proteins—can produce an aberrant composition of proteins, metabolites, and signaling molecules that drives pathologic states. Identifying the root causes of these changes may lead to new therapeutic approaches for many different diseases.

Although few therapies for human diseases aim to alter gene expression, the outstanding examples—including antiestrogens for hormone-positive breast cancer, antiandrogens for prostate cancer, and PPAR-γ agonists for type 2 diabetes—demonstrate the benefits that can be achieved through targeting gene-control mechanisms.  Now, thanks to recent papers from laboratories at MIT, Harvard, and the National Institutes of Health, researchers have a new, much bigger transcriptional target: large DNA regions known as super-enhancers or stretch-enhancers. Already, work on super-enhancers is providing insights into how gene-expression programs are established and maintained, and how they may go awry in disease.  Such research promises to open new avenues for discovering medicines for diseases where novel approaches are sorely needed.

Super-enhancers cover stretches of DNA that are 10- to 100-fold longer and about 10-fold less abundant in the genome than typical enhancer regions (Cell, 153:307-19, 2013). They also appear to bind a large percentage of the transcriptional machinery compared to typical enhancers, allowing them to better establish and enforce cell-type specific transcriptional programs (Cell, 153:320-34, 2013).

Super-enhancers are closely associated with genes that dictate cell identity, including those for cell-type–specific master regulatory transcription factors. This observation led to the intriguing hypothesis that cells with a pathologic identity, such as cancer cells, have an altered gene expression program driven by the loss, gain, or altered function of super-enhancers.

Sure enough, by mapping the genome-wide location of super-enhancers in several cancer cell lines and from patients’ tumor cells, we and others have demonstrated that genes located near super-enhancers are involved in processes that underlie tumorigenesis, such as cell proliferation, signaling, and apoptosis.

Super-enhancers cover stretches of DNA that are 10- to 100-fold longer and about 10-fold less abundant in the genome than typical enhancer regions.

Genome-wide association studies (GWAS) have found that disease- and trait-associated genetic variants often occur in greater numbers in super-enhancers (compared to typical enhancers) in cell types involved in the disease or trait of interest (Cell, 155:934-47, 2013). For example, an enrichment of fasting glucose–associated single nucleotide polymorphisms (SNPs) was found in the stretch-enhancers of pancreatic islet cells (PNAS, 110:17921-26, 2013). Given that some 90 percent of reported disease-associated SNPs are located in noncoding regions, super-enhancer maps may be extremely valuable in assigning functional significance to GWAS variants and identifying target pathways.

Because only 1 to 2 percent of active genes are physically linked to a super-enhancer, mapping the locations of super-enhancers can be used to pinpoint the small number of genes that may drive the biology of that cell. Differential super-enhancer maps that compare normal cells to diseased cells can be used to unravel the gene-control circuitry and identify new molecular targets, in much the same way that somatic mutations in tumor cells can point to oncogenic drivers in cancer. This approach is especially attractive in diseases for which an incomplete understanding of the pathogenic mechanisms has been a barrier to discovering effective new therapies.

Another therapeutic approach could be to disrupt the formation or function of super-enhancers by interfering with their associated protein components. This strategy could make it possible to downregulate multiple disease-associated genes through a single molecular intervention. A group of Boston-area researchers recently published support for this concept when they described inhibited expression of cancer-specific genes, leading to a decrease in cancer cell growth, by using a small molecule inhibitor to knock down a super-enhancer component called BRD4 (Cancer Cell, 24:777-90, 2013).  More recently, another group showed that expression of the RUNX1 transcription factor, involved in a form of T-cell leukemia, can be diminished by treating cells with an inhibitor of a transcriptional kinase that is present at the RUNX1 super-enhancer (Nature, 511:616-20, 2014).

Fungal effector Ecp6 outcompetes host immune receptor for chitin binding through intrachain LysM dimerization 
Andrea Sánchez-Vallet, et al.   eLife 2013;2:e00790 http://elifesciences.org/content/2/e00790#sthash.LnqVMJ9p.dpuf

LysM effector

LysM effector


While host immune receptors

  • detect pathogen-associated molecular patterns to activate immunity,
  • pathogens attempt to deregulate host immunity through secreted effectors.

Fungi employ LysM effectors to prevent

  • recognition of cell wall-derived chitin by host immune receptors

Structural analysis of the LysM effector Ecp6 of

  • the fungal tomato pathogen Cladosporium fulvum reveals
  • a novel mechanism for chitin binding,
  • mediated by intrachain LysM dimerization,

leading to a chitin-binding groove that is deeply buried in the effector protein.

This composite binding site involves

  • two of the three LysMs of Ecp6 and
  • mediates chitin binding with ultra-high (pM) affinity.

The remaining singular LysM domain of Ecp6 binds chitin with

  • low micromolar affinity but can nevertheless still perturb chitin-triggered immunity.

Conceivably, the perturbation by this LysM domain is not established through chitin sequestration but possibly through interference with the host immune receptor complex.

Mutated Genes in Schizophrenia Map to Brain Networks
From www.nih.gov –  Sep 3, 2013

Previous studies have shown that many people with schizophrenia have de novo, or new, genetic mutations. These misspellings in a gene’s DNA sequence

  • occur spontaneously and so aren’t shared by their close relatives.

Dr. Mary-Claire King of the University of Washington in Seattle and colleagues set out to

  • identify spontaneous genetic mutations in people with schizophrenia and
  • to assess where and when in the brain these misspelled genes are turned on, or expressed.

The study was funded in part by NIH’s National Institute of Mental Health (NIMH). The results were published in the August 1, 2013, issue of Cell.

The researchers sequenced the exomes (protein-coding DNA regions) of 399 people—105 with schizophrenia plus their unaffected parents and siblings. Gene variations
that were found in a person with schizophrenia but not in either parent were considered spontaneous.

The likelihood of having a spontaneous mutation was associated with

  • the age of the father in both affected and unaffected siblings.

Significantly more mutations were found in people

  • whose fathers were 33-45 years at the time of conception compared to 19-28 years.

Among people with schizophrenia, the scientists identified

  • 54 genes with spontaneous mutations
  • predicted to cause damage to the function of the protein they encode.

The researchers used newly available database resources that show

  • where in the brain and when during development genes are expressed.

The genes form an interconnected expression network with many more connections than

  • that of the genes with spontaneous damaging mutations in unaffected siblings.

The spontaneously mutated genes in people with schizophrenia

  • were expressed in the prefrontal cortex, a region in the front of the brain.

The genes are known to be involved in important pathways in brain development. Fifty of these genes were active

  • mainly during the period of fetal development.

“Processes critical for the brain’s development can be revealed by the mutations that disrupt them,” King says. “Mutations can lead to loss of integrity of a whole pathway,
not just of a single gene.”

These findings support the concept that schizophrenia may result, in part, from

  • disruptions in development in the prefrontal cortex during fetal development.

James E. Darnell’s “Reflections”

A brief history of the discovery of RNA and its role in transcription — peppered with career advice
By Joseph P. Tiano

James Darnell begins his Journal of Biological Chemistry “Reflections” article by saying, “graduate students these days

  • have to swim in a sea virtually turgid with the daily avalanche of new information and
  • may be momentarily too overwhelmed to listen to the aging.

I firmly believe how we learned what we know can provide useful guidance for how and what a newcomer will learn.” Considering his remarkable discoveries in

  • RNA processing and eukaryotic transcriptional regulation

spanning 60 years of research, Darnell’s advice should be cherished. In his second year at medical school at Washington University School of Medicine in St. Louis, while
studying streptococcal disease in Robert J. Glaser’s laboratory, Darnell realized he “loved doing the experiments” and had his first “career advancement event.”
He and technician Barbara Pesch discovered that in vivo penicillin treatment killed streptococci only in the exponential growth phase and not in the stationary phase. These
results were published in the Journal of Clinical Investigation and earned Darnell an interview with Harry Eagle at the National Institutes of Health.

Darnell arrived at the NIH in 1956, shortly after Eagle  shifted his research interest to developing his minimal essential cell culture medium, still used. Eagle, then studying cell metabolism, suggested that Darnell take up a side project on poliovirus replication in mammalian cells in collaboration with Robert I. DeMars. DeMars’ Ph.D.
adviser was also James  Watson’s mentor, so Darnell met Watson, who invited him to give a talk at Harvard University, which led to an assistant professor position
at the MIT under Salvador Luria. A take-home message is to embrace side projects, because you never know where they may lead: this project helped to shape
his career.

Darnell arrived in Boston in 1961. Following the discovery of DNA’s structure in 1953, the world of molecular biology was turning to RNA in an effort to understand how
proteins are made. Darnell’s background in virology (it was discovered in 1960 that viruses used RNA to replicate) was ideal for the aim of his first independent lab:
exploring mRNA in animal cells grown in culture. While at MIT, he developed a new technique for purifying RNA along with making other observations

  • suggesting that nonribosomal cytoplasmic RNA may be involved in protein synthesis.

When Darnell moved to Albert Einstein College of Medicine for full professorship in 1964,  it was hypothesized that heterogenous nuclear RNA was a precursor to mRNA.
At Einstein, Darnell discovered RNA processing of pre-tRNAs and demonstrated for the first time

  • that a specific nuclear RNA could represent a possible specific mRNA precursor.

In 1967 Darnell took a position at Columbia University, and it was there that he discovered (simultaneously with two other labs) that

  • mRNA contained a polyadenosine tail.

The three groups all published their results together in the Proceedings of the National Academy of Sciences in 1971. Shortly afterward, Darnell made his final career move
four short miles down the street to Rockefeller University in 1974.

Over the next 35-plus years at Rockefeller, Darnell never strayed from his original research question: How do mammalian cells make and control the making of different
mRNAs? His work was instrumental in the collaborative discovery of

  • splicing in the late 1970s and
  • in identifying and cloning many transcriptional activators.

Perhaps his greatest contribution during this time, with the help of Ernest Knight, was

  • the discovery and cloning of the signal transducers and activators of transcription (STAT) proteins.

And with George Stark, Andy Wilks and John Krowlewski, he described

  • cytokine signaling via the JAK-STAT pathway.

Darnell closes his “Reflections” with perhaps his best advice: Do not get too wrapped up in your own work, because “we are all needed and we are all in this together.”

Darnell Reflections - James_Darnell

Darnell Reflections – James_Darnell


Recent findings on presenilins and signal peptide peptidase

By Dinu-Valantin Bălănescu

γ-secretase and SPP

γ-secretase and SPP

Fig. 1 from the minireview shows a schematic depiction of γ-secretase and SPP


GxGD proteases are a family of intramembranous enzymes capable of hydrolyzing

  • the transmembrane domain of some integral membrane proteins.

The GxGD family is one of the three families of

  • intramembrane-cleaving proteases discovered so far (along with the rhomboid and site-2 protease) and
  • includes the γ-secretase and the signal peptide peptidase.

Although only recently discovered, a number of functions in human pathology and in numerous other biological processes

  • have been attributed to γ-secretase and SPP.

Taisuke Tomita and Takeshi Iwatsubo of the University of Tokyo highlighted the latest findings on the structure and function of γ-secretase and SPP
in a recent minireview in The Journal of Biological Chemistry.

  • γ-secretase is involved in cleaving the amyloid-β precursor protein, thus producing amyloid-β peptide,

the main component of senile plaques in Alzheimer’s disease patients’ brains. The complete structure of mammalian γ-secretase is not yet known; however,
Tomita and Iwatsubo note that biochemical analyses have revealed it to be a multisubunit protein complex.

  • Its catalytic subunit is presenilin, an aspartyl protease.

In vitro and in vivo functional and chemical biology analyses have revealed that

  • presenilin is a modulator and mandatory component of the γ-secretase–mediated cleavage of APP.

Genetic studies have identified three other components required for γ-secretase activity:

  1. nicastrin,
  2. anterior pharynx defective 1 and
  3. presenilin enhancer 2.

By coexpression of presenilin with the other three components, the authors managed to

  • reconstitute γ-secretase activity.

Tomita and Iwatsubo determined using the substituted cysteine accessibility method and by topological analyses, that

  • the catalytic aspartates are located at the center of the nine transmembrane domains of presenilin,
  • by revealing the exact location of the enzyme’s catalytic site.

The minireview also describes in detail the formerly enigmatic mechanism of γ-secretase mediated cleavage.

SPP, an enzyme that cleaves remnant signal peptides in the membrane

  • during the biogenesis of membrane proteins and
  • signal peptides from major histocompatibility complex type I,
  • also is involved in the maturation of proteins of the hepatitis C virus and GB virus B.

Bioinformatics methods have revealed in fruit flies and mammals four SPP-like proteins,

  • two of which are involved in immunological processes.

By using γ-secretase inhibitors and modulators, it has been confirmed

  • that SPP shares a similar GxGD active site and proteolytic activity with γ-secretase.

Upon purification of the human SPP protein with the baculovirus/Sf9 cell system,

  • single-particle analysis revealed further structural and functional details.

HLA targeting efficiency correlates with human T-cell response magnitude and with mortality from influenza A infection

From www.pnas.org –  Sep 3, 2013 4:24 PM

Experimental and computational evidence suggests that

  • HLAs preferentially bind conserved regions of viral proteins, a concept we term “targeting efficiency,” and that
  • this preference may provide improved clearance of infection in several viral systems.

To test this hypothesis, T-cell responses to A/H1N1 (2009) were measured from peripheral blood mononuclear cells obtained from a household cohort study
performed during the 2009–2010 influenza season. We found that HLA targeting efficiency scores significantly correlated with

  • IFN-γ enzyme-linked immunosorbent spot responses (P = 0.042, multiple regression).

A further population-based analysis found that the carriage frequencies of the alleles with the lowest targeting efficiencies, A*24,

  • were associated with pH1N1 mortality (r = 0.37, P = 0.031) and
  • are common in certain indigenous populations in which increased pH1N1 morbidity has been reported.

HLA efficiency scores and HLA use are associated with CD8 T-cell magnitude in humans after influenza infection.
The computational tools used in this study may be useful predictors of potential morbidity and

  • identify immunologic differences of new variant influenza strains
  • more accurately than evolutionary sequence comparisons.

Population-based studies of the relative frequency of these alleles in severe vs. mild influenza cases

  • might advance clinical practices for severe H1N1 infections among genetically susceptible populations.

Metabolomics in drug target discovery

J D Rabinowitz et al.

Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ.
Cold Spring Harbor Symposia on Quantitative Biology 11/2011; 76:235-46.

Most diseases result in metabolic changes. In many cases, these changes play a causative role in disease progression. By identifying pathological metabolic changes,

  • metabolomics can point to potential new sites for therapeutic intervention.

Particularly promising enzymatic targets are those that

  • carry increased flux in the disease state.

Definitive assessment of flux requires the use of isotope tracers. Here we present techniques for

  • finding new drug targets using metabolomics and isotope tracers.

The utility of these methods is exemplified in the study of three different viral pathogens. For influenza A and herpes simplex virus,

  • metabolomic analysis of infected versus mock-infected cells revealed
  • dramatic concentration changes around the current antiviral target enzymes.

Similar analysis of human-cytomegalovirus-infected cells, however, found the greatest changes

  • in a region of metabolism unrelated to the current antiviral target.

Instead, it pointed to the tricarboxylic acid (TCA) cycle and

  • its efflux to feed fatty acid biosynthesis as a potential preferred target.

Isotope tracer studies revealed that cytomegalovirus greatly increases flux through

  • the key fatty acid metabolic enzyme acetyl-coenzyme A carboxylase.
  • Inhibition of this enzyme blocks human cytomegalovirus replication.

Examples where metabolomics has contributed to identification of anticancer drug targets are also discussed. Eventual proof of the value of

  • metabolomics as a drug target discovery strategy will be
  • successful clinical development of therapeutics hitting these new targets.

 Related References

Use of metabolic pathway flux information in targeted cancer drug design. Drug Discovery Today: Therapeutic Strategies 1:435-443, 2004.

Detection of resistance to imatinib by metabolic profiling: clinical and drug development implications. Am J Pharmacogenomics. 2005;5(5):293-302. Review. PMID: 16196499

Medicinal chemistry, metabolic profiling and drug target discovery: a role for metabolic profiling in reverse pharmacology and chemical genetics.
Mini Rev Med Chem.  2005 Jan;5(1):13-20. Review. PMID: 15638788 [PubMed – indexed for MEDLINE] Related citations

Development of Tracer-Based Metabolomics and its Implications for the Pharmaceutical Industry. Int J Pharm Med 2007; 21 (3): 217-224.

Use of metabolic pathway flux information in anticancer drug design. Ernst Schering Found Symp Proc. 2007;(4):189-203. Review. PMID: 18811058

Pharmacological targeting of glucagon and glucagon-like peptide 1 receptors has different effects on energy state and glucose homeostasis in diet-induced obese mice. J Pharmacol Exp Ther. 2011 Jul;338(1):70-81. http://dx.doi.org:/10.1124/jpet.111.179986. PMID: 21471191

Single valproic acid treatment inhibits glycogen and RNA ribose turnover while disrupting glucose-derived cholesterol synthesis in liver as revealed by the
[U-C(6)]-d-glucose tracer in mice. Metabolomics. 2009 Sep;5(3):336-345. PMID: 19718458

Metabolic Pathways as Targets for Drug Screening, Metabolomics, Dr Ute Roessner (Ed.), ISBN: 978-953-51-0046-1, InTech, Available from: http://www.intechopen.com/books/metabolomics/metabolic-pathways-as-targets-for-drug-screening

Iron regulates glucose homeostasis in liver and muscle via AMP-activated protein kinase in mice. FASEB J. 2013 Jul;27(7):2845-54.
http://dx.doi.org:/10.1096/fj.12-216929. PMID: 23515442

Metabolomics and systems pharmacology: why and how to model the human metabolic network for drug discovery

Drug Discov. Today 19 (2014), 171–182     http://dx.doi.org:/10.1016/j.drudis.2013.07.014


  • We now have metabolic network models; the metabolome is represented by their nodes.
  • Metabolite levels are sensitive to changes in enzyme activities.
  • Drugs hitchhike on metabolite transporters to get into and out of cells.
  • The consensus network Recon2 represents the present state of the art, and has predictive power.
  • Constraint-based modelling relates network structure to metabolic fluxes.

Metabolism represents the ‘sharp end’ of systems biology, because changes in metabolite concentrations are

  • necessarily amplified relative to changes in the transcriptome, proteome and enzyme activities, which can be modulated by drugs.

To understand such behaviour, we therefore need (and increasingly have) reliable consensus (community) models of

  • the human metabolic network that include the important transporters.

Small molecule ‘drug’ transporters are in fact metabolite transporters, because

  • drugs bear structural similarities to metabolites known from the network reconstructions and
  • from measurements of the metabolome.

Recon2 represents the present state-of-the-art human metabolic network reconstruction; it can predict inter alia:

(i) the effects of inborn errors of metabolism;

(ii) which metabolites are exometabolites, and

(iii) how metabolism varies between tissues and cellular compartments.

However, even these qualitative network models are not yet complete. As our understanding improves

  • so do we recognise more clearly the need for a systems (poly)pharmacology.

Introduction – a systems biology approach to drug discovery

It is clearly not news that the productivity of the pharmaceutical industry has declined significantly during recent years

  • following an ‘inverse Moore’s Law’, Eroom’s Law, or
  • that many commentators, consider that the main cause of this is
  • because of an excessive focus on individual molecular target discovery rather than a more sensible strategy
  • based on a systems-level approach (Fig. 1).
drug discovery science

drug discovery science

Figure 1.

The change in drug discovery strategy from ‘classical’ function-first approaches (in which the assay of drug function was at the tissue or organism level),
with mechanistic studies potentially coming later, to more-recent target-based approaches where initial assays usually involve assessing the interactions
of drugs with specified (and often cloned, recombinant) proteins in vitro. In the latter cases, effects in vivo are assessed later, with concomitantly high levels of attrition.

Arguably the two chief hallmarks of the systems biology approach are:

(i) that we seek to make mathematical models of our systems iteratively or in parallel with well-designed ‘wet’ experiments, and
(ii) that we do not necessarily start with a hypothesis but measure as many things as possible (the ’omes) and

  • let the data tell us the hypothesis that best fits and describes them.

Although metabolism was once seen as something of a Cinderella subject,

  • there are fundamental reasons to do with the organisation of biochemical networks as
  • to why the metabol(om)ic level – now in fact seen as the ‘apogee’ of the ’omics trilogy –
  •  is indeed likely to be far more discriminating than are
  • changes in the transcriptome or proteome.

The next two subsections deal with these points and Fig. 2 summarises the paper in the form of a Mind Map.

metabolomics and systems pharmacology

metabolomics and systems pharmacology


Metabolic Disease Drug Discovery— “Hitting the Target” Is Easier Said Than Done

David E. Moller, et al.   http://dx.doi.org:/10.1016/j.cmet.2011.10.012

Despite the advent of new drug classes, the global epidemic of cardiometabolic disease has not abated. Continuing

  • unmet medical needs remain a major driver for new research.

Drug discovery approaches in this field have mirrored industry trends, leading to a recent

  • increase in the number of molecules entering development.

However, worrisome trends and newer hurdles are also apparent. The history of two newer drug classes—

  1. glucagon-like peptide-1 receptor agonists and
  2. dipeptidyl peptidase-4 inhibitors—

illustrates both progress and challenges. Future success requires that researchers learn from these experiences and

  • continue to explore and apply new technology platforms and research paradigms.

The global epidemic of obesity and diabetes continues to progress relentlessly. The International Diabetes Federation predicts an even greater diabetes burden (>430 million people afflicted) by 2030, which will disproportionately affect developing nations (International Diabetes Federation, 2011). Yet

  • existing drug classes for diabetes, obesity, and comorbid cardiovascular (CV) conditions have substantial limitations.

Currently available prescription drugs for treatment of hyperglycemia in patients with type 2 diabetes (Table 1) have notable shortcomings. In general,

Therefore, clinicians must often use combination therapy, adding additional agents over time. Ultimately many patients will need to use insulin—a therapeutic class first introduced in 1922. Most existing agents also have

  • issues around safety and tolerability as well as dosing convenience (which can impact patient compliance).

Pharmacometabolomics, also known as pharmacometabonomics, is a field which stems from metabolomics,

  • the quantification and analysis of metabolites produced by the body.

It refers to the direct measurement of metabolites in an individual’s bodily fluids, in order to

  • predict or evaluate the metabolism of pharmaceutical compounds, and
  • to better understand the pharmacokinetic profile of a drug.

Alternatively, pharmacometabolomics can be applied to measure metabolite levels

  • following the administration of a pharmaceutical compound, in order to
  • monitor the effects of the compound on certain metabolic pathways(pharmacodynamics).

This provides detailed mapping of drug effects on metabolism and

  • the pathways that are implicated in mechanism of variation of response to treatment.

In addition, the metabolic profile of an individual at baseline (metabotype) provides information about

  • how individuals respond to treatment and highlights heterogeneity within a disease state.

All three approaches require the quantification of metabolites found

relationship between -OMICS

relationship between -OMICS


Pharmacometabolomics is thought to provide information that

Looking at the characteristics of an individual down through these different levels of detail, there is an

  • increasingly more accurate prediction of a person’s ability to respond to a pharmaceutical compound.
  1. the genome, made up of 25 000 genes, can indicate possible errors in drug metabolism;
  2. the transcriptome, made up of 85,000 transcripts, can provide information about which genes important in metabolism are being actively transcribed;
  3. and the proteome, >10,000,000 members, depicts which proteins are active in the body to carry out these functions.

Pharmacometabolomics complements the omics with

  • direct measurement of the products of all of these reactions, but with perhaps a relatively
  • smaller number of members: that was initially projected to be approximately 2200 metabolites,

but could be a larger number when gut derived metabolites and xenobiotics are added to the list. Overall, the goal of pharmacometabolomics is

  • to more closely predict or assess the response of an individual to a pharmaceutical compound,
  • permitting continued treatment with the right drug or dosage
  • depending on the variations in their metabolism and ability to respond to treatment.

Pharmacometabolomic analyses, through the use of a metabolomics approach,

  • can provide a comprehensive and detailed metabolic profile or “metabolic fingerprint” for an individual patient.

Such metabolic profiles can provide a complete overview of individual metabolite or pathway alterations,

This approach can then be applied to the prediction of response to a pharmaceutical compound

  • by patients with a particular metabolic profile.

Pharmacometabolomic analyses of drug response are

Pharmacogenetics focuses on the identification of genetic variations (e.g. single-nucleotide polymorphisms)

  • within patients that may contribute to altered drug responses and overall outcome of a certain treatment.

The results of pharmacometabolomics analyses can act to “inform” or “direct”

  • pharmacogenetic analyses by correlating aberrant metabolite concentrations or metabolic pathways to potential alterations at the genetic level.

This concept has been established with two seminal publications from studies of antidepressants serotonin reuptake inhibitors

  • where metabolic signatures were able to define a pathway implicated in response to the antidepressant and
  • that lead to identification of genetic variants within a key gene
  • within the highlighted pathway as being implicated in variation in response.

These genetic variants were not identified through genetic analysis alone and hence

  • illustrated how metabolomics can guide and inform genetic data.


Benznidazole Biotransformation and Multiple Targets in Trypanosoma cruzi Revealed by Metabolomics

Andrea Trochine, Darren J. Creek, Paula Faral-Tello, Michael P. Barrett, Carlos Robello
Published: May 22, 2014   http://dx.doi.org:/10.1371/journal.pntd.0002844

The first line treatment for Chagas disease, a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi,

  • involves administration of benznidazole (Bzn).

Bzn is a 2-nitroimidazole pro-drug which requires nitroreduction to become active. We used a

  • non-targeted MS-based metabolomics approach to study the metabolic response of T. cruzi to Bzn.

Parasites treated with Bzn were minimally altered compared to untreated trypanosomes, although the redox active thiols

  1. trypanothione,
  2. homotrypanothione and
  3. cysteine

were significantly diminished in abundance post-treatment. In addition, multiple Bzn-derived metabolites were detected after treatment.

These metabolites included reduction products, fragments and covalent adducts of reduced Bzn

  • linked to each of the major low molecular weight thiols:
  1. trypanothione,
  2. glutathione,
  3. g-glutamylcysteine,
  4. glutathionylspermidine,
  5. cysteine and
  6. ovothiol A.

Bzn products known to be generated in vitro by the unusual trypanosomal nitroreductase, TcNTRI,

  • were found within the parasites,
  • but low molecular weight adducts of glyoxal, a proposed toxic end-product of NTRI Bzn metabolism, were not detected.

Our data is indicative of a major role of the

  • thiol binding capacity of Bzn reduction products
  • in the mechanism of Bzn toxicity against T. cruzi.



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Larry H Bernstein, MD, FCAP, Curator

http://pharmaceuticalintelligence/7/8/2014/Proteins and cellular adaptation to stress

There are two recent articles that are, if not interesting, possibly important in the direction of cellular regulation, adaptation, and decline.  One deals with apoptosis, or cell death, which is synchronized with recovery of membrane and protein breakdown for reuse in synthesis and maintenance.  The other is a new perspective to Alzhemier’s Disease, for which there is no effective pharmacotherapy. In both cases, the stresses of the cell are critical to the responce to the environment.  This is not just about the classical transcriptomics story. This is a perfect followup to the just posted research on the regulatory role of a small RNA that is related to, but distinct from silencing RNA, and also the revelations about lncRNA.

Protein Helps Cells Adapt—or Die

Scientists show how cell stress both prevents and promotes cell suicide in a study that’s equally divisive.

By Ruth Williams | July 3, 2014

A cellular stress pathway called the unfolded-protein-response (UPR) both activates and degrades death receptor 5 protein (DR5), which can promote or prevent cell suicide, according to a paper published in Science today (July 3). The theory is that initial stress blocks cell suicide, or apoptosis, to give the cell a chance to adapt, but that if the stress persists, it eventually triggers apoptosis.

“This work has made the most beautiful simplification of all this big complex mess. Basically, they identified and pinpointed the specific protein involved in the switching decision and explain how the decision is made,” said Alexei Korennykh, a professor of molecular biology at Princeton University, who was not involved in the work.

But Randal Kaufman of the Sanford-Burnham Medical Research Institute in La Jolla, California, was not impressed. He questioned the physiological relevance of the experiments supporting the authors’ main conclusions about this key cellular process.

Protein folding in a cell takes place largely in the endoplasmic reticulum (ER), but if the process goes awry, unfolded proteins accumulate, stressing the ER. This triggers the UPR, which shuts down translation, degrades unfolded proteins, and increases production of protein-folding machinery. If ER stress is not resolved, however, the UPR can also induce apoptosis.

Two main factors control the UPR—IRE1a and PERK. IRE1a promotes cell survival by activating the transcription factor XBP1, which drives expression of cell-survival genes. PERK, on the other hand, activates a transcription factor called CHOP, which in turn drives expression of the proapoptotic factor DR5.

Peter Walter of the University of California, San Francisco, and his colleagues have now confirmed that CHOP activates DR5, showing that it is a cell-autonomous process. But they have also found that IRE1a suppresses DR5, directly degrading its mRNA through a process called regulated IRE1a-dependent degradation (RIDD). Inhibition of IRE1a in a human cancer cell line undergoing ER stress both prevented DR5 mRNA decay and increased apoptosis.

However, in an e-mail to The Scientist, Kaufman expressed concern that “the significance of RIDD has not been demonstrated in a physiologically-relevant context.”

Walter insisted that the evidence for RIDD’s existence is “crystal clear.” His only concession was that “the effects aren’t 100 percent,” he said, because “RIDD degrades mRNA by a few-fold,” making it difficult to measure.

This RIDD debate aside, the researchers have also sparked a rumpus with their finding that IRE1a expression switches off just 24 hours after ER stress initiation, leaving PERK to drive the cell toward apoptosis. “We and others have evidence that suggests another model,” said Scott Oakes, a professor of pathology at the University of California, San Francisco, “which is that both PERK and IRE1a under high stress will send out death signals.”

Whether IRE1a promotes or inhibits apoptosis under extreme stress “is controversial,” said Ira Tabas, a professor at Columbia University in New York City. But it’s essential that scientists figure it out. Cell death from ER stress is a pathological process in many major diseases, Tabas said, and there are IRE1a inhibitors in pharmaceutical development. “It is very important because under high stress you have two different views here,” said Oakes. “One is that you want to keep IRE1a on, the other is that you want to shut it off.”

Because ER stress is central to many diseases, “a lot of people are passionate about it,” said Tabas, explaining the polemic views. “Who’s right? . . . I think it depends on the context in which the experiments are done—one pathway may be important in some settings, and another pathway may be important in different settings,” he suggested. What might help to resolve the issues, he said, will be “in vivo causation studies using actual disease models.”

Researchers will continue to debate. So, said Walter, “we’ll have to see what holds-up five years from now.”

M. Lu et al., “Opposing unfolded-protein-response signals converge on death receptor 5 to control apoptosis,” Science, 345:98-101, 2014.

Tags stress responseprotein foldingdisease/medicinecell & molecular biology and apoptosis


Protein May Hold the Key to Who Gets Alzheimer’s


By PAM BELLUCK     MARCH 19, 2014


It is one of the big scientific mysteries of Alzheimer’s disease: Why do some people whose brains accumulate the plaques and tangles so strongly associated with Alzheimer’s not develop the disease?


Now, a series of studies by Harvard scientists suggests a possible answer, one that could lead to new treatments if confirmed by other research.


The memory and thinking problems of Alzheimer’s disease and other dementias, which affect an estimated seven million Americans, may be related to a failure in the brain’s stress response system, the new research suggests. If this system is working well, it can protect the brain from abnormal Alzheimer’s proteins; if it gets derailed, critical areas of the brain start degenerating.

“This is an extremely important study,” said Li-Huei Tsai, director of the Picower Institute for Learning and Memory at the Massachusetts Institute of Technology, who was not involved in the research but wrote a commentary accompanying the study. “This is the first study that is really starting to provide a plausible pathway to explain why some people are more vulnerable to Alzheimer’s than other people.”

An image of tau tangles in the brain, often a hallmark of Alzheimer’s disease.

An image of tau tangles in the brain, often a hallmark of Alzheimer’s disease.




The research, published on Wednesday in the journal Nature, focuses on a protein previously thought to act mostly in the brains of developing fetuses. The scientists found that the protein also appears to protect neurons in healthy older people from aging-related stresses. But in people with Alzheimer’s and other dementias, the protein is sharply depleted in key brain regions.

Experts said if other scientists could replicate and expand upon the findings, the role of the protein, called REST, could spur development of new drugs for dementia, which has so far been virtually impossible to treat. But they cautioned that much more needed to be determined, including whether the decline of REST was a cause, or an effect, of brain deterioration, and whether it was specific enough to neurological diseases that it could lead to effective therapies.

“You’re going to see a lot of papers now following up on it,” said Dr. Eric M. Reiman, executive director of the Banner Alzheimer’s Institute in Phoenix, who was not involved in the study. “While it’s a preliminary finding, it raises an avenue that hasn’t been considered before. And if this provides a handle on which to understand normal brain aging, that will be great, too.”

REST, a regulator that switches off certain genes, is primarily known to keep fetal neurons in an immature state until they develop to perform brain functions, said Dr. Bruce A. Yankner, a professor of genetics at Harvard Medical School and the lead author of the new study. By the time babies are born, REST becomes inactive, he said, except in some areas outside the brain like the colon, where it seems to suppress cancer.

While investigating how different genes in the brain change as people age, Dr. Yankner’s team was startled to find that REST was the most active gene regulator in older brains. The researchers have found that this protein, normally active in fetuses, may also protect the neurons in older people.  It is not yet possible to measure the levels of this protein that is a gene regulator called REST, in living people.

“Why should a fetal gene be coming on in an aging brain?” he wondered. He hypothesized that it was because in aging, as in birth, brains encounter great stress, threatening neurons that cannot regenerate if harmed.

His team discovered that REST appears to switch off genes that promote cell death, protecting neurons from normal aging processes like energy decrease, inflammation and oxidative stress.

Analyzing brains from brain banks and dementia studies, the researchers found that brains of young adults ages 20 to 35 contained little REST, while healthy adults between the ages of 73 and 106 had plenty. REST levels grew the older people got, so long as they did not develop dementia, suggesting that REST is related to longevity.

But in people with Alzheimer’s, mild cognitive impairment, frontotemporal dementia and Lewy body dementia, the brain areas affected by these diseases contained much less REST than healthy brains.

This was true only in people who actually had memory and thinking problems. People who remained cognitively healthy, but whose brains had the same accumulation of amyloid plaques and tau tangles as people with Alzheimer’s, had three times more REST than those suffering Alzheimer’s symptoms. About a third of people who have such plaques will not develop Alzheimer’s symptoms, studies show.

REST levels dropped as symptoms worsened, so people with mild cognitive impairment had more REST than Alzheimer’s patients. And only key brain regions were affected. In Alzheimer’s, REST steeply declined in the prefrontal cortex and hippocampus, areas critical to learning, memory and planning. Other areas of the brain not involved in Alzheimer’s showed no REST drop-off.

It is not yet possible to analyze REST levels in the brains of living people, and several Alzheimer’s experts said that fact limited what the new research could prove.

John Hardy, an Alzheimer’s researcher at University College London, cautioned in an email that information from post-mortem brains could not prove that a decline in REST caused dementia because death might produce unrelated damage to brain cells.

To investigate further, the team conducted what both Dr. Tsai and Dr. Reiman called a “tour de force” of research, examining REST in mice, roundworms and cells in the lab.

“We wanted to make sure the story was right,” Dr. Yankner said. “It was difficult to believe at first, to be honest with you.”

Especially persuasive was that mice genetically engineered to lack REST lost neurons as they aged in brain areas afflicted in Alzheimer’s.

Dr. Yankner said REST appeared to work by traveling to a neuron’s nucleus when the brain was stressed. In dementia, though, REST somehow gets diverted, traveling with toxic dementia-related proteins to another part of the neuron where it is eventually destroyed.

Experts said the research, while intriguing, left many unanswered questions. Bradley Wise of the National Institute on Aging’s neuroscience division, which helped finance the studies, said REST’s role needed further clarification. “I don’t think you can really say if it’s a cause of Alzheimer’s or a consequence of Alzheimer’s” yet, he said.

Dr. Samuel E. Gandy, an Alzheimer’s researcher at Mount Sinai Medical Center, wondered if REST figured only in neurodegenerative diseases or in other diseases, too, which could make it difficult to use REST to develop specific treatments or diagnostic tests for dementia.

“My ambivalence is, is this really a way that advances our understanding of the disease or does this just tell us this is even more complicated than we thought?” he said.

Dr. Yankner’s team is looking at REST in other neurological diseases, like Parkinson’s. He also has thoughts about a potential treatment, lithium, which he said appears to stimulate REST function, and is considered relatively safe.

But he and other experts said it was too early. “I would hesitate to start rushing into lithium treatment” unless rigorous studies showed that it could forestall dementia, said Dr. John C. Morris, an Alzheimer’s researcher at Washington University in St. Louis.

Still, Dr. Morris said, the REST research the team conducted so far is “very well done, and certainly helps support this idea that we’ve all tried to understand about why Alzheimer’s is age-associated and why, while amyloid is necessary for the development of Alzheimer’s disease, it certainly is not sufficient.”

He added, “There have to be some other processes and triggers that result in Alzheimer’s.”

Correction: March 19, 2014 
Because of an editing error, an earlier version of this article misstated the gender of Dr. Li-Huei Tsai. Dr. Tsai is a woman.

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TyrNovo’s Novel and Unique Compound, named NT219, selectively Inhibits the process of Aging and Neurodegenerative Diseases, without affecting Lifespan

Reporter: Aviva Lev-Ari, PhD, RN

A step toward development of drugs for diseases such as Alzheimer’s, Parkinson’s and Huntington’s

December 3, 2013


Jerusalem – A successful joint collaboration between researchers at The Hebrew university of Jerusalem and the startup company TyrNovo may lead to a potential treatment of brain diseases. The researchers found that TyrNovo’s novel and unique compound, named NT219, selectively inhibits the process of aging in order to protect the brain from neurodegenerative diseases, without affecting lifespan. This is a first and important step towards the development of future drugs for the treatment of various neurodegenerative maladies.
Human neurodegenerative diseases such as Alzheimer’s, Parkinson’s andHuntington’s diseases share two key features: they stem from toxic proteinaggregation and emerge late in life. The common temporal emergence pattern exhibited by these maladies proposes that the aging process negatively regulates protective mechanisms that prevent their manifestation early in life, exposing the elderly to disease. This idea has been the major focus of the work in the laboratory of Dr. Ehud Cohen of the Department of Biochemistry and Molecular Biology, at The Hebrew University of Jerusalem‘s Faculty of Medicine.
Dr. Cohen’s first breakthrough in this area occurred when he discovered, working with Dr. Ehud Cohenworms, that reducing the activity of the signaling mechanism conveyed through insulin and the growth hormone IGF1, a major aging regulating pathway, constituted a defense against the aggregation of the Aβ protein which is mechanistically-linked with Alzheimer’s disease. Later, he found that the inhibition of this signaling route also protected Alzheimer’s-model mice from behavioral impairments and pathological phenomena typical to the disease. In these studies, the path was reduced through genetic manipulation, a method not applicable in humans.
Dr. Hadas Reuveni, the CEO of TyrNovo, a startup company formed for the clinical development of NT219, and Professor Alexander Levitzki from the Department of Biological Chemistry at The Hebrew University, with their research teams, discovered a new set of compounds that inhibit the activity of the IGF1 signaling cascade in a unique and efficient mechanism, primarily for cancer treatment, and defined NT219 as the leading compound for further development.
Now, in a fruitful collaboration Dr. Cohen and Dr. Reuveni, together with Dr. Cohen’s associates Tayir El-Ami and Lorna Moll, have demonstrated that NT219 efficiently inhibits IGF1 signaling, in both worms and human cells. The inhibition of this signaling pathway by NT219 protected worms from toxic protein aggregation that in humans is associated with the development of Alzheimer’s or Huntington’s disease.
The discoveries achieved during this project, which was funded by the Rosetrees Trust of Britain, were published this week in the journal Aging Cell (“A novel inhibitor of the insulin/IGF signaling pathway protects from age-onset, neurodegeneration-linked proteotoxicity”). The findings strengthen the notion that the inhibition of the IGF1 signaling pathway has a therapeutic potential as a treatment for neurodegenerative disorders. They also point at NT219 as the first compound that provides protection from neurodegeneration-associated toxic protein aggregation through a selective manipulation of aging.
Cohen, Reuveni and Levitzki have filed a patent application that protects the use of NT219 as a treatment for neurodegenerative maladies through Yissum, the technology transfer company of The Hebrew University. Dr. Gil Pogozelich, chairman of Goldman Hirsh Partners Ltd., which holds the controlling interest in TyrNovo, says that he sees great importance in the cooperation on this project with The Hebrew University, and that TyrNovo represents a good example of how scientific and research initiatives can further health care together with economic benefits.
Recently, Dr. Cohen’s laboratory obtained an ethical approval to test the therapeutic efficiency of NT219 as a treatment in Alzheimer’s-model mice, hoping to develop a future treatment for hitherto incurable neurodegenerative disorders.


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Ubiquitin Pathway Involved in Neurodegenerative Diseases

Larry H Bernstein, MD,  FCAP


PINK1 and Parkin and Parkinson’s Disease

Studies of the familial Parkinson disease-related proteins PINK1 and Parkin have demonstrated that these factors promote the fragmentation and turnover of mitochondria following treatment of cultured cells with mitochondrial depolarizing agents. Whether PINK1 or Parkin influence mitochondrial quality control under normal physiological conditions in dopaminergic neurons, a principal cell type that degenerates in Parkinson disease, remains unclear. To address this matter, we developed a method to purify and characterize neural subtypes of interest from the adult Drosophila brain.

Using this method, we find that dopaminergic neurons from Drosophila parkin mutants accumulate enlarged, depolarized mitochondria, and that genetic perturbations that promote mitochondrial fragmentation and turnover rescue the mitochondrial depolarization and neurodegenerative phenotypes of parkin mutants. In contrast, cholinergic neurons from parkin mutants accumulate enlarged depolarized mitochondria to a lesser extent than dopaminergic neurons, suggesting that a higher rate of mitochondrial damage, or a deficiency in alternative mechanisms to repair or eliminate damaged mitochondria explains the selective vulnerability of dopaminergic neurons in Parkinson disease.

Our study validates key tenets of the model that PINK1 and Parkin promote the fragmentation and turnover of depolarized mitochondria in dopaminergic neurons. Moreover, our neural purification method provides a foundation to further explore the pathogenesis of Parkinson disease, and to address other neurobiological questions requiring the analysis of defined neural cell types.

Burmana JL, Yua S, Poole AC, Decala RB , Pallanck L. Analysis of neural subtypes reveals selective mitochondrial dysfunction in dopaminergic neurons from parkin mutants.

Autophagy in Parkinson’s Disease.

Parkinson’s disease is a common neurodegenerative disease in the elderly. To explore the specific role of autophagy and the ubiquitin-proteasome pathway in apoptosis, a specific proteasome inhibitor and macroautophagy inhibitor and stimulator were selected to investigate pheochromocytoma (PC12) cell lines transfected with human mutant (A30P) and wildtype (WT) -synuclein.

The apoptosis ratio was assessed by flow cytometry. LC3, heat shock protein 70 (hsp70) and caspase-3 expression in cell culture were determined by Western blot. The hallmarks of apoptosis and autophagy were assessed with transmission electron microscopy. Compared to the control group or the rapamycin (autophagy stimulator) group, the apoptosis ratio in A30P and WT cells was significantly higher after treatment with inhibitors of the proteasome and macroautophagy. The results of Western blots for caspase-3 expression were similar to those of flow cytometry; hsp70 protein was significantly higher in the proteasome inhibitor group than in control, but in the autophagy inhibitor and stimulator groups, hsp70 was similar to control. These findings show that inhibition of the proteasome and autophagy promotes apoptosis, and the macroautophagy stimulator rapamycin reduces the apoptosis ratio. And inhibiting or stimulating autophagy has less impact on hsp70 than the proteasome pathway.

In conclusion, either stimulation or inhibition of macroautophagy, has less impact on hsp70 than on the proteasome pathway. This study found that rapamycin decreased apoptotic cells in A30P cells independent of caspase-3 activity. Although several lines of evidence recently demonstrated crosstalk between autophagy and caspase-independent apoptosis, we could not confirm that autophagy activation protects cells from caspase-independent cell death. Undoubtedly, there are multiple connections between the apoptotic and autophagic processes.

Inhibition of autophagy may subvert the capacity of cells to remove damaged organelles or to remove misfolded proteins, which would favor apoptosis. However, proteasome inhibition activated macroautophagy and accelerated apoptosis. A likely explanation is inhibition of the proteasome favors oxidative reactions that trigger apoptosis, presumably through

  • a direct effect on mitochondria, and
  • the absence of NADPH2 and ATP

which may deinhibit the activation of caspase-2 or MOMP. Another possibility is that aggregated proteins induced by proteasome inhibition increase apoptosis.

Yang F, Yanga YP, Maoa CJ, Caoa BY, et al. Role of autophagy and proteasome degradation pathways in apoptosis of PC12 cells overexpressing human -synuclein. Neuroscience Letters 2009; 454:203–208. doi:10.1016/j.neulet.2009.03.027. www.elsevier.com/locate/neulet   http://neurosciletters.com/ Role_of_autophagy_and_proteasome_degradation_pathways_in_apoptosis_of_PC12_cells_
overexpressing_human –synuclein/

Parkin-dependent Ubiquitination of Endogenous Bax

Autosomal recessive loss-of-function mutations within the PARK2 gene functionally inactivate the E3 ubiquitin ligase parkin, resulting in neurodegeneration of catecholaminergic neurons and a familial form of Parkinson disease. Current evidence suggests both a mitochondrial function for parkin and a neuroprotective role, which may in fact be interrelated. The antiapoptotic effects of Parkin have been widely reported, and may involve fundamental changes in the threshold for apoptotic cytochrome c release, but the substrate(s) involved in Parkin dependent protection had not been identified. Here, we demonstrate the Parkin-dependent ubiquitination of endogenous Bax comparing primary cultured neurons from WT and Parkin KO mice and using multiple Parkin-overexpressing cell culture systems. The direct ubiquitination of purified Bax was also observed in vitro following incubation with recombinant parkin. The authors found that Parkin prevented basal and apoptotic stress induced translocation of Bax to the mitochondria. Moreover, an engineered ubiquitination-resistant form of Bax retained its apoptotic function, but Bax KO cells complemented with lysine-mutant Bax did not manifest the antiapoptotic effects of Parkin that were observed in cells expressing WT Bax. These data suggest that Bax is the primary substrate responsible for the antiapoptotic effects of Parkin, and provide mechanistic insight into at least a subset of the mitochondrial effects of Parkin.

Johnson BN, Berger AK, Cortese GP, and LaVoie MJ. The ubiquitin E3 ligase Parkin regulates the proapoptotic function of Bax. PNAS 2012, pp 6. www.pnas.org/cgi/doi/10.1073/pnas.1113248109
PNAS.org/ The_ubiquitin_E3_ligase_Parkin_regulates_the_proapoptotic_function_of_Bax

Parkin Promotes Mitochondrial Loss in Autophagy

Parkin, an E3 ubiquitin ligase implicated in Parkinson’s disease, promotes degradation of dysfunctional mitochondria by autophagy. Using proteomic and cellular approaches, we show that upon translocation to mitochondria, Parkin activates the ubiquitin–proteasome system (UPS) for widespread degradation of outer membrane proteins. This is evidenced by an increase in K48-linked polyubiquitin on mitochondria, recruitment of the 26S proteasome and rapid degradation of multiple outer membrane proteins. The degradation of proteins by the UPS occurs independently of the autophagy pathway, and inhibition of the 26S proteasome completely abrogates Parkin-mediated mitophagy in HeLa, SH-SY5Y and mouse cells. Although the mitofusins Mfn1 and Mfn2 are rapid degradation targets of Parkin, degradation of additional targets is essential for mitophagy. These results indicate that remodeling of the mitochondrial outer membrane proteome is important for mitophagy, and reveal a causal link between the UPS and autophagy, the major pathways for degradation of intracellular substrates.

Chan NC, Salazar AM, Pham AH, Sweredoski MJ, et al. Broad activation of the ubiquitin–proteasome system by Parkin is critical for mitophagy. Human Molecular Genetics 2011; 20(9): 1726–1737. doi:10.1093/hmg/ddr048.  http://HumMolecGenetics.com/ Broad_activation_of_the_ubiquitin–proteasome_system_by_Parkin_is_critical_for_mitophagy/

Interactome Networks and Protein Expression

Aloy P. Shaping the future of interactome networks. (A report of the third Interactome Networks Conference, Hinxton, UK, 29 August-1 September 2007). Genome Biology 2007; 8:316 (doi:10.1186/gb-2007-8-10-316)

Complex systems are often networked, and biology is no exception. Following on from the genome sequencing projects, experiments show that proteins in living organisms are highly connected, which helps to explain how such great complexity can be achieved by a comparatively small set of gene products. At a recent conference on interactome networks held outside Cambridge, UK, the most recent advances in research on cellular networks were discussed. This year’s conference focused on identifying the strengths and weaknesses of currently resolved interaction networks and the techniques used to determine them – reflecting the fact that the field of mapping interaction networks is maturing.

Peroutka RJ, Orcutt SJ, Strickler JE, and Butt TR. SUMO Fusion Technology for Enhanced Protein Expression and Purification in Prokaryotes and Eukaryotes. Chapter 2. in T.C. Evans, M.-Q. Xu (eds.), Heterologous Gene Expression in E. coli, Methods in Molecular Biology 705:15-29. DOI 10.1007/978-1-61737-967-3_2, © Springer Science+Business Media, LLC 2011

The preparation of sufficient amounts of high-quality protein samples is the major bottleneck for structural proteomics. The use of recombinant proteins has increased significantly during the past decades. The most commonly used host, Escherichia coli, presents many challenges including protein misfolding, protein degradation, and low solubility. A novel SUMO fusion technology appears to enhance protein expression and solubility (www.lifesensors.com). Efficient removal of the SUMO tag by SUMO protease in vitro facilitates the generation of target protein with a native N-terminus. In addition to its physiological relevance in eukaryotes, SUMO can be used as a powerful biotechnology tool forenhanced functional protein expression in prokaryotes and eukaryotes.

IL-6 regulation on mitochondrial remodeling/dysfunction

Muscle protein turnover regulation during cancer cachexia is being rapidly defined, and skeletal muscle mitochondria function appears coupled to processes regulating muscle wasting. Skeletal muscle oxidative capacity and the expression of proteins regulating mitochondrial biogenesis and dynamics are disrupted in severely cachectic ApcMin/+ mice. It has not been determined if these changes occur at the onset of cachexia and are necessary for the progression of muscle wasting. Exercise and anti-cytokine therapies have proven effective in preventing cachexia development in tumor bearing mice, while their effect on mitochondrial content, biogenesis and dynamics is not well understood.

The purposes of this study were to

1) determine IL-6 regulation on mitochondrial remodeling/dysfunction during the progression of cancer cachexia and

2) to determine if exercise training can attenuate mitochondrial dysfunction and the induction of proteolytic pathways during IL-6 induced cancer cachexia.

ApcMin/+ mice were examined during the progression of cachexia, after systemic interleukin (IL)-6r antibody treatment, or after IL-6 over-expression with or without exercise. Direct effects of IL-6 on mitochondrial remodeling were examined in cultured C2C12 myoblasts.

  • Mitochondrial content was not reduced during the initial development of cachexia, while muscle PGC-1α and fusion (Mfn1, Mfn2) protein expression was repressed.
  • With progressive weight loss mitochondrial content decreased, PGC-1α and fusion proteins were further suppressed, and fission protein (FIS1) was induced.

IL-6 receptor antibody administration after the onset of cachexia improved mitochondrial content,

  • PGC-1α,
  • Mfn1/Mfn2 and
  • FIS1 protein expression.

IL-6 over-expression in pre-cachectic mice accelerated body weight loss and muscle wasting, without reducing mitochondrial content, while PGC-1α and Mfn1/Mfn2 protein expression was suppressed and FIS1 protein expression induced. Exercise normalized these IL-6 induced effects. C2C12 myotubes administered IL-6 had

  • increased FIS1 protein expression,
  • increased oxidative stress, and
  • reduced PGC-1α gene expression
  • without altered mitochondrial protein expression.

Altered expression of proteins regulating mitochondrial biogenesis and fusion are early events in the initiation of cachexia regulated by IL-6, which precede the loss of muscle mitochondrial content. Furthermore, IL-6 induced mitochondrial remodeling and proteolysis can be rescued with moderate exercise training even in the presence of high circulating IL-6 levels.

White JP, Puppa MJ, Sato S, Gao S. IL-6 regulation on skeletal muscle mitochondrial remodeling during cancer cachexia in the ApcMin/+ mouse. Skeletal Muscle 2012; 2:14-30.

Starvation-induced Autophagy

Upon starvation cells undergo autophagy, a cellular degradation pathway important in the turnover of whole organelles and long lived proteins. Starvation-induced protein degradation has been regarded as an unspecific bulk degradation process. We studied global protein dynamics during amino acid starvation-induced autophagy by quantitative mass spectrometry and were able to record nearly 1500 protein profiles during 36 h of starvation. Cluster analysis of the recorded protein profiles revealed that cytosolic proteins were degraded rapidly, whereas proteins annotated to various complexes and organelles were degraded later at different time periods. Inhibition of protein degradation pathways identified the lysosomal/autophagosomal system as the main degradative route.

Thus, starvation induces degradation via autophagy, which appears to be selective and to degrade proteins in an ordered fashion and not completely arbitrarily as anticipated so far.

Kristensen AR, Schandorff S, Høyer-Hansen M, Nielsen MO, et al. Ordered Organelle Degradation during Starvation-induced Autophagy. Molecular & Cellular Proteomics 2008; 7:2419–2428.

Skeletal Muscle Macroautophagy

Skeletal muscles are the agent of motion and one of the most important tissues responsible for the control of metabolism. Coordinated movements are allowed by the highly organized structure of the cytosol of muscle fibers (or myofibers), the multinucleated and highly specialized cells of skeletal muscles involved in contraction. Contractile proteins are assembled into repetitive structures, the basal unit of which is the sarcomere, that are well packed into the myofiber cytosol. Myonuclei are located at the edge of the myofibers, whereas the various organelles such as mitochondria and sarcoplasmic reticulum are embedded among the myofibrils. Many different changes take place in the cytosol of myofibers during catabolic conditions:

  • proteins are mobilized
  • organelles networks are reorganized for energy needs
  • the setting of myonuclei can be modified.


  • strenuous physical activity,
  • improper dietary regimens and
  • aging

lead to mechanical and metabolic damages of

  • myofiber organelles,
  • especially mitochondria, and
  • contractile proteins.

During aging the protein turnover is slowed down, therefore it is easier to accumulate aggregates of dysfunctional proteins. Therefore, a highly dynamic tissue such as skeletal muscle requires a rapid and efficient system for the removal of altered organelles, the elimination of protein aggregates, and the disposal of toxic products.

The two major proteolytic systems in muscle are the ubiquitin-proteasome and the autophagy-lysosome pathways. The proteasome system requires

  • the transcription of the two ubiquitin ligases (atrogin-1 and MuRF1) and
  • the ubiquitination of the substrates.

Therefore, the ubiquitin-proteasome system can provide the rapid elimination of single proteins or small aggregates. Conversely, the autophagic system is able to degrade entire organelles and large proteins aggregates. In the autophagy-lysosome system, double-membrane vesicles named autophagosomes are able to engulf a portion of the cytosol and fuse with lysosomes, where their content is completely degraded by lytic enzymes.

The autophagy flux can be biochemicaly monitored following LC3 lipidation and p62 degradation. LC3 is the mammalian homolog of the yeast Atg8 gene, which is lipidated when recruited for the double-membrane commitment and growth. p62 (SQSTM-1) is a polyubiquitin-binding protein involved in the proteasome system and that can either reside free in the cytosol and nucleus or occur within autophagosomes and lysosomes. The GFP-LC3 transgenic mouse model allows easy detection of autophagosomes by simply monitoring the presence of bright GFP-positive puncta inside the myofibrils and beneath the plasma membrane of the myofibers, thus investigate the activation of autophagy in skeletal muscles with different contents of slow and fast-twitching myofibers and in response to stimuli such as fasting. For example, in the fast-twiching extensor digitorum longus muscle few GFP-LC3 dots were observed before starvation, while many small GFP-LC3 puncta appeared between myofibrils and in the perinuclear regions after 24 h starvation. Conversely, in the slow-twitching soleus muscle, autophagic puncta were almost absent in standard condition and scarcely induced after 24 h starvation.

Autophagy in Muscle Homeostasis

The autophagic flux was found to be increased during certain catabolic conditions, such as fasting, atrophy , and denervation , thus contributing to protein breakdown. Food deprivation is one of the strongest stimuli known to induce autophagy in muscle. Indeed skeletal muscle, after the liver, is the most responsive tissue to autophagy activation during food deprivation. Since muscles are the biggest reserve of amino acids in the body, during fasting autophagy has the vital role to maintain the amino acid pool by digesting muscular protein and organelles. In mammalian cells, mTORC1, which consists of

  • mTOR and
  • Raptor,

is the nutrient sensor that negatively regulates autophagy.

During atrophy, protein breakdown is mediated by atrogenes, which are under the forkhead box O (FoxO) transcription factors control, and activation of autophagy seems to aggravate muscle loss during atrophy. In vivo and in vitro studies demonstrated that several genes coding for components of the autophagic machinery, such as

  • LC3,
  • Vps34,
  • Atg12 and
  • Bnip3,

are controlled by FoxO3 transcription factor. FoxO3 is able to regulate independently

  1. the ubiquitin-proteasome system and
  2. the autophagy-lysosome machinery in vivo and in vitro.

Denervation is also able to induce autophagy in skeletal muscle, although at a slower rate than fasting. This effect is mediated by RUNX1, a transcription factor upregulated during autophagy; the lack of RUNX1 results in

  • excessive autophagic flux in denervated muscle and leads to atrophy.

The generation of Atg5 and Atg7 muscle-specific knockout mice have shown that

  • with suppression of autophagy both models display muscle weakness and atrophy and
  • a significant reduction of weight, which is
  • correlated with the important loss of muscle tissue due to an atrophic condition.

An unbalanced autophagy flux is highly detrimental for muscle, as too much induces atrophy whereas too little leads to muscle weakness and degeneration. Muscle wasting associated with autophagy inhibition becomes evident and symptomatic only after a number of altered proteins and dysfunctional organelles are accumulated, a condition that becomes evident after months or even years. On the other hand, the excessive increase of autophagy flux is able to induce a rapid loss of muscle mass (within days or weeks).  Alterations of autophagy are involved in the pathogenesis of several myopathies and dystrophies.

The maintenance of muscle homeostasis is finely regulated by the balance between catabolic and anabolic process. Macroautophagy (or autophagy) is a catabolic process that provides the degradation of protein aggregation and damaged organelles through the fusion between autophagosomes and lysosomes. Proper regulation of the autophagy flux is fundamental for

  • the homeostasis of skeletal muscles during physiological situations and
  • in response to stress.

Defective as well as excessive autophagy is harmful for muscle health and has a pathogenic role in several forms of muscle diseases.

Grumati P, Bonaldo P. Autophagy in Skeletal Muscle Homeostasis and in Muscular Dystrophies. Cells 2012, 1, 325-345; doi:10.3390/cells1030325. ISSN 2073-4409. www.mdpi.com/journal/cells
http://cell.com/ Autophagy in Skeletal Muscle Homeostasis and in Muscular Dystrophies/

Parkinson’s Disease Mutations

Mutations in parkin, a ubiquitin ligase, cause early-onset familial Parkinson’s disease (AR-JP). How Parkin suppresses Parkinsonism remains unknown. Parkin was recently shown to promote the clearance of impaired mitochondria by autophagy, termed mitophagy. Here, we show that Parkin promotes mitophagy by catalyzing mitochondrial ubiquitination, which in turn recruits ubiquitin-binding autophagic components, HDAC6 and p62, leading to mitochondrial clearance.

During the process, juxtanuclear mitochondrial aggregates resembling a protein aggregate-induced aggresome are formed. The formation of these “mito-aggresome” structures requires microtubule motor-dependent transport and is essential for efficient mitophagy. Importantly, we show that AR-JP–causing Parkin mutations are defective in supporting mitophagy due to distinct defects at

  • recognition,
  • transportation, or
  • ubiquitination of impaired mitochondria,

thereby implicating mitophagy defects in the development of Parkinsonism. Our results show that impaired mitochondria and protein aggregates are processed by common ubiquitin-selective autophagy machinery connected to the aggresomal pathway, thus identifying a mechanistic basis for the prevalence of these toxic entities in Parkinson’s disease.

Lee JY,Nagano Y, Taylor JP,Lim KL, and Yao TP. Disease-causing mutations in Parkin impair mitochondrial ubiquitination, aggregation, and HDAC6-dependent mitophagy. J Cell Biol 2010; 189(4):671-679. www.jcb.org/cgi/doi/10.1083/jcb.201001039
http://JCellBiol.com/Disease-causing_mutations_in_Parkin_impair_mitochondrial_ubiquitination_ aggregation_and_HDAC6-dependent_mitophagy/

Drosophila Parkin Requires PINK1

Loss of the E3 ubiquitin ligase Parkin causes early onset Parkinson’s disease, a neurodegenerative disorder of unknown etiology.  Parkin has been linked to multiple cellular processes including

  • protein degradation,
  • mitochondrial homeostasis, and
  • autophagy;

however, its precise role in pathogenesis is unclear. Recent evidence suggests that Parkin is recruited to damaged mitochondria, possibly affecting

  • mitochondrial fission and/or fusion,
  • to mediate their autophagic turnover.

The precise mechanism of recruitment and the ubiquitination target are unclear. Here we show in Drosophila cells that PINK1 is required to recruit Parkin to dysfunctional mitochondria and promote their degradation. Furthermore, PINK1 and Parkin mediate the ubiquitination of the profusion factor Mfn on the outer surface of mitochondria. Loss of Drosophila PINK1 or parkin causes an increase in Mfn abundance in vivo and concomitant elongation of mitochondria. These findings provide a molecular mechanism by which the PINK1/Parkin pathway affects mitochondrial fission/fusion as suggested by previous genetic interaction studies. We hypothesize that Mfn ubiquitination may provide a mechanism by which terminally damaged mitochondria are labeled and sequestered for degradation by autophagy.

Ziviani E, Tao RN, and Whitworth AJ. Drosophila Parkin requires PINK1 for mitochondrial translocation and ubiquitinates Mitofusin. PNAS 2010. Pp6 www.pnas.org/cgi/doi/10.1073/pnas.0913485107
http://PNAS.org/ Drosophila_Parkin_requires_PINK1_for_ mitochondrial_translocation_and_ubiquitinates_Mitofusin

Dynamin-related protein 1 (Drp1) in Parkinson’s

Mutations in Parkin, an E3 ubiquitin ligase that regulates protein turnover, represent one of the major causes of familial Parkinson’s disease (PD), a neurodegenerative disorder characterized by the loss of dopaminergic neurons and impaired mitochondrial functions. The underlying mechanism by which pathogenic parkin mutations induce mitochondrial abnormality is not fully understood. Here we demonstrate that Parkin interacts with and subsequently ubiquitinates dynamin-related protein 1 (Drp1), for promoting its proteasome-dependent degradation. Pathogenic mutation or knockdown of Parkin inhibits the ubiquitination and degradation of Drp1, leading to an increased level of Drp1 for mitochondrial fragmentation. These results identify Drp1 as a novel substrate of Parkin and suggest a potential mechanism linking abnormal Parkin expression to mitochondrial dysfunction in the pathogenesis of PD.

Wang H, Song P, Du L, Tian W. Parkin ubiquitinates Drp1 for proteasome-dependent degradation: implication of dysregulated mitochondrial dynamics in Parkinson’s disease.

JBC Papers in Press. Published on February 3, 2011 as Manuscript M110.144238. http://www.jbc.org/cgi/doi/10.1074/jbc.M110.144238
http://JBC.org/ Parkin_ubiquitinates_Drp1_for_proteasome-dependent_degradation_implication_of_ dysregulated_mitochondrial_dynamics_in_Parkinson’s_disease

Pink1, Parkin, and DJ-1 Form a Complex

Mutations in the genes PTEN-induced putative kinase 1 (PINK1), PARKIN, and DJ-1 cause autosomal recessive forms of Parkinson disease (PD), and the Pink1/Parkin pathway regulates mitochondrial integrity and function. An important question is whether the proteins encoded by these genes function to regulate activities of other cellular compartments. A study in mice, reported by Xiong et al. in this issue of the JCI, demonstrates that Pink1, Parkin, and DJ-1 can form a complex in the cytoplasm, with Pink1 and DJ-1 promoting the E3 ubiquitin ligase activity of Parkin to degrade substrates via the proteasome (see the related article, doi:10.1172/ JCI37617).

This protein complex in the cytosol may or may not be related to the role of these proteins in regulating mitochondrial function or oxidative stress in vivo. Three models for the role of the PPD complex. In this issue of the JCI, Xiong et al. report that Pink1, Parkin, and DJ-1 bind to each other and form a PPD E3 ligase complex in which Pink1 and DJ-1 modulate Parkin-dependent ubiquitination and subsequent degradation of substrates via the proteasome. Previous work suggests that the Pink1/Parkin pathway regulates mitochondrial integrity and promotes mitochondrial fission in Drosophila.

(A) Parkin and DJ-1 may be recruited to the mitochondrial outer membrane during stress and interact with Pink1. These interactions may facilitate the ligase activity of Parkin, thereby facilitating the turnover of molecules that regulate mitochondrial dynamics and mitophagy. The PPD complex may have other roles in the cytosol that result in degradative ubiquitination and/or relay information from mitochondria to other cellular compartments.

(B) Alternatively, Pink1 may be released from mitochondria after cleavage to interact with DJ-1 and Parkin in the cytosol.

A and B differ in the site of action of the PPD complex and the cleavage status of Pink1.

The complex forms on the mitochondrial outer membrane potentially containing full-length Pink1 in A, and in the cytosol with cleaved Pink1 in B.

Lack of DJ-1 function results in phenotypes that are distinct from the mitochondrial phenotypes observed in null mutants of Pink1 or Parkin in Drosophila. Thus, although the PPD complex is illustrated here as regulating mitochondrial fission, the role of DJ-1 in vivo remains to be clarified.

(C) It is also possible that the action occurs in the cytosol and is independent of the function of Pink1/Parkin in regulating mitochondrial integrity and function.

The Xiong et al. study offers an entry point for explorations of the role of Pink1, Parkin, and DJ-1 in the cytoplasm. It remains to be shown whether Parkin, in complex with Pink1 and DJ-1, carries out protein degradation in vivo.

Li H, and Guo M. Protein degradation in Parkinson disease revisited: it’s complex. commentaries. J Clin Invest.  doi:10.1172/JCI38619. http://www.jci.org
http://JCI.org/ Protein_degradation_in_Parkinson_disease_revisited_it’s_complex/

Xiong, H., et al. Parkin, PINK1, and DJ-1 form a ubiquitin E3 ligase complex promoting unfolded protein degradation. J. Clin. Invest. 2009; 119:650–660.
http://JCI.org/ Parkin_PINK1_DJ1_form_ubiquitin_E3_ligase_complex_promoting_unfolded_protein_degradation/

Mitochondrial Ubiquitin Ligase, MITOL, protects neuronal cells

Nitric oxide (NO) is implicated in neuronal cell survival. However, excessive NO production mediates neuronal cell death, in part via mitochondrial dysfunction. Here, we report that the mitochondrial ubiquitin ligase, MITOL, protects neuronal cells from mitochondrial damage caused by accumulation of S-nitrosylated microtubule associated protein 1B-light chain 1 (LC1). S-nitrosylation of LC1 induces a conformational change that serves both to activate LC1 and to promote its ubiquination by MITOL, indicating that microtubule stabilization by LC1 is regulated through its interaction with MITOL. Excessive NO production can inhibit MITOL, and MITOL inhibition resulted in accumulation of S-nitrosylated LC1 following stimulation of NO production by calcimycin and N-methyl-D-aspartate. LC1 accumulation under these conditions resulted in mitochondrial dysfunction and neuronal cell death. Thus, the balance between LC1 activation by S-nitrosylation and down-regulation by MITOL is critical for neuronal cell survival. Our findings may contribute significantly to an understanding of the mechanisms of neurological diseases caused by nitrosative stress-mediated mitochondrial dysfunction.

Yonashiro R, Kimijima Y, Shimura T, Kawaguchi K, et al. Mitochondrial ubiquitin ligase MITOL blocks S-nitrosylated MAP1B-light chain 1-mediated mitochondrial dysfunction and neuronal cell death. PNAS; 2012. pp 6. www.pnas.org/cgi/doi/10.1073/pnas.1114985109

Ubiquitin–Proteasome System in Neurodegeneration

A common histopathological hallmark of most neurodegenerative diseases is the presence of aberrant proteinaceous inclusions inside affected neurons. Because these protein aggregates are detected using antibodies against components of the ubiquitin–proteasome system (UPS), impairment of this machinery for regulated proteolysis has been suggested to be at the root of neurodegeneration. This hypothesis has been difficult to prove in vivo owing to the lack of appropriate tools. The recent report of transgenic mice with ubiquitous expression of a UPS-reporter protein should finally make it possible to test in vivo the role of the UPS in neurodegeneration.

Hernandez F, Dıaz-Hernandez M, Avila J and Lucas JJ. Testing the ubiquitin–proteasome hypothesis of neurodegeneration in vivo. TRENDS in Neurosciences 2004; 27(2): 66-68.

ALP in Parkinson’s

The ubiquitin-proteasome system (UPS) and autophagy-lysosome pathway (ALP) are the two most important mechanisms that normally repair or remove abnormal proteins. Alterations in the function of these systems to degrade misfolded and aggregated proteins are being increasingly recognized as playing a pivotal role in the pathogenesis of many neurodegenerative disorders such as Parkinson’s disease. Dysfunction of the UPS has been already strongly implicated in the pathogenesis of this disease and, more recently, growing interest has been shown in identifying the role of ALP in neurodegeneration. Mutations of a-synuclein and the increase of intracellular concentrations of non-mutant a-synuclein have been associated with Parkinson’s disease phenotype.

The demonstration that a-synuclein is degraded by both proteasome and autophagy indicates a possible linkage between the dysfunction of the UPS or ALP and the occurrence of this disorder.The fact that mutant a-synucleins inhibit ALP functioning by tightly binding to the receptor on the lysosomal membrane for autophagy pathway further supports the assumption that impairment of the ALP may be related to the development of Parkinson’s disease.  In this review, we summarize the recent findings related to this topic and discuss the unique role of the ALP in this neurogenerative disorder and the putative therapeutic potential through ALP enhancement.

Pan Y, Kondo S, Le W, Jankovic J. The role of autophagy-lysosome pathway in neurodegeneration associated with Parkinson’s disease. Brain 2008; 131: 1969-1978. doi:10.1093/brain/awm318.

Ubiquitin-Proteasome System in Parkinson’s

There is growing evidence that dysfunction of the mitochondrial respiratory chain and failure of the cellular protein degradation machinery, specifically the ubiquitin-proteasome system, play an important role in the pathogenesis of Parkinson’s disease. We now show that the corresponding pathways of these two systems are linked at the transcriptomic level in Parkinsonian substantia nigra. We examined gene expression in medial and lateral substantia nigra (SN) as well as in frontal cortex using whole genome DNA oligonucleotide microarrays. In this study, we use a hypothesis-driven approach in analysing microarray data to describe the expression of mitochondrial and ubiquitin-proteasomal system (UPS) genes in Parkinson’s disease (PD).

Although a number of genes showed up-regulation, we found an overall decrease in expression affecting the majority of mitochondrial and UPS sequences. The down-regulated genes include genes that encode subunits of complex I and the Parkinson’s-disease-linked UCHL1. The observed changes in expression were very similar for both medial and lateral SN and also affected the PD cerebral cortex. As revealed by “gene shaving” clustering analysis, there was a very significant correlation between the transcriptomic profiles of both systems including in control brains.

Therefore, the mitochondria and the proteasome form a higher-order gene regulatory network that is severely perturbed in Parkinson’s disease. Our quantitative results also suggest that Parkinson’s disease is a disease of more than one cell class, i.e. that it goes beyond the catecholaminergic neuron and involves glia as well.

Duke DC, Moran LB, Kalaitzakis ME, Deprez M, et al. Transcriptome analysis reveals link between proteasomal and mitochondrial pathways in Parkinson’s disease. Neurogenetics 2006; 7:139-148.

The causes of various neurodegenerative diseases, particularly sporadic cases, remain unknown, but increasing evidence suggests that these diseases may share similar molecular and cellular mechanisms of pathogenesis. One prominent feature common to most neurodegenerative diseases is the accumulation of misfolded proteins in the form of insoluble protein aggregates or inclusion bodies. Although these aggregates have different protein compositions, they all contain ubiquitin and proteasome subunits, implying a failure of the ubiquitin-proteasome system (UPS) in the removal of misfolded proteins.

A direct link between UPS dysfunction and neurodegeneration has been provided by recent findings that genetic mutations in UPS components cause several rare, familial forms of neurodegenerative diseases. Furthermore, it is becoming increasingly clear that oxidative stress, which results from aging or exposure to environmental toxins, can directly damage UPS components, thereby contributing to the pathogenesis of sporadic forms of neurodegenerative diseases.

Aberrations in the UPS often result in defective proteasome-mediated protein degradation, leading to accumulation of toxic proteins and eventually to neuronal cell death. Interestingly, emerging evidence has begun to suggest that impairment in substrate-specific components of the UPS, such as E3 ubiquitin-protein ligases, may cause aberrant ubiquitination and neurodegeneration in a proteasome-independent manner. This provides an overview of the molecular components of the UPS and their impairment in familial and sporadic forms of neurodegenerative diseases, and summarizes present knowledge about the pathogenic mechanisms of UPS dysfunction in neurodegeneration.

Molecular mechanisms of protein ubiquitination and degradation by the UPS. Ubiquitination involves a highly specific enzyme cascade in which

  • ubiquitin (Ub) is first activated by the ubiquitinactivating enzyme (E1),
  • then transferred to an ubiquitin-conjugating enzyme (E2), and
  • finally covalently attached to the substrate by an ubiquitin-protein ligase (E3).

Ubiquitination is a reversible posttranslational modification in which the removal of Ub is mediated by a deubiquitinating enzyme (DUB).

  • Substrate proteins can be either monoubiquitinated or polyubiquitinated through successive conjugation of Ub moieties to an internal lysine residue in Ub.
  • K48-linked poly-Ub chains are recognized by the 26S proteasome, resulting in degradation of the substrate and recycling of Ub.

Monoubiquitination or K63-linked polyubiquitination plays a number of regulatory roles in cells that are proteasome-independent.


Loss-of-function mutations in parkin, a 465-amino-acid RING-type E3 ligase, were first identified as the cause for autosomal recessive juvenile Parkinsonism (AR-JP) and subsequently found to account for ~50% of all recessively transmitted early-onset PD cases. Interestingly, patients with parkin mutations do not exhibit Lewy body pathology.

Possible pathogenic mechanisms by which impaired UPS components cause neurodegeneration. Genetic mutations or oxidative stress from aging and/or exposure to environmental toxins have been shown to impair the ubiquitination machinery (particularly E3 ubiquitin-protein ligases) and deubiquitinating enzymes (DUBs), resulting in abnormal ubiquitination. Depending on the type of ubiquitination affected, the impairment could cause neurodegeneration through two different mechanisms.

  1. aberrant K48-linked polyubiquitination resulting from impaired E3s or DUBs alters protein degradation by the proteasome, leading to accumulation of toxic proteins and subsequent neurodegeneration. The proteasomes could be directly damaged by oxidative stress or might be inhibited by protein aggregation, which exacerbates the neurotoxicity.
  2. aberrant monoubiquitination or K63-linked polyubiquitination resulting from impaired E3s or DUBs alters crucial non-proteasomal functions, such as gene transcription and protein trafficking, thereby causing neurodegeneration without protein aggregation.

These two models are not mutually exclusive because a single E3 or DUB enzyme, such as parkin or UCH-L1, could regulate more than one type of ubiquitination. In addition, abnormal ubiquitination and neurodegeneration could also result from mutation or oxidative stress-induced structural changes in the protein substrates that alter their recognition and degradation by the UPS.

Lian Li and Chin LS. IMPAIRMENT OF THE UBIQUITIN-PROTEASOME SYSTEM: A COMMON PATHOGENIC MECHANISM IN NEURODEGENERATIVE DISORDERS. In The Ubiquitin Proteasome System…Chapter 23. (Eds: Eds: Mario Di Napoli and Cezary Wojcik) 553-577 © 2007 Nova Science Publishers, Inc. ISBN 978-1-60021-749-4.

filedesc Schematic diagram of the ubiquitylation system. Created by Roger B. Dodd (Photo credit: Wikipedia)

Current Noteworthy Work

Statins inhibit HMG-CoA reductase, a key enzyme in cholesterol synthesis, and are widely used to treat hypercholesterolemia.

These drugs can lead to a number of side effects in muscle, including muscle fiber breakdown; however, the mechanisms of muscle injury by statins are poorly understood. We report that lovastatin induced the expression of atrogin-1, a key gene involved in skeletal muscle atrophy, in humans with statin myopathy, in zebrafish embryos, and in vitro in murine skeletal muscle cells. In cultured mouse myotubes, atrogin-1 induction following lovastatin treatment was accompanied by distinct morphological changes, largely absent in atrogin-1 null cells. In zebrafish embryos, lovastatin promoted muscle fiber damage, an effect that was closely mimicked by knockdown of zebrafish HMG-CoA reductase. Moreover, atrogin-1 knockdown in zebrafish embryos prevented lovastatin-induced muscle injury. Finally, overexpression of PGC-1α, a transcriptional coactivator that induces mitochondrial biogenesis and protects against the development of muscle atrophy, dramatically prevented lovastatin-induced muscle damage and abrogated atrogin-1 induction both in fish and in cultured mouse myotubes. Collectively, our human, animal, and in vitro findings shed light on the molecular mechanism of statin-induced myopathy and suggest that atrogin-1 may be a critical mediator of the muscle damage induced by statins.

Inami Y, Waguri S, Sakamoto A, Kouno T, et al.  Persistent activation of Nrf2 through p62 in hepatocellular carcinoma cells. J. Cell Biol. 2011; 193(2): 275–284. www.jcb.org/cgi/doi/10.1083/jcb.201102031

Macroautophagy (hereafter referred to as autophagy) is a cellular degradation system in which cytoplasmic components, including organelles, are sequestered by double membrane structures called autophagosomes and the sequestered materials are degraded by lysosomal hydrolases for supply of amino acids and for cellular homeostasis. Although autophagy has generally been considered nonselective, recent studies have shed light on another indispensable role for basal autophagy in cellular homeostasis, which is mediated by selective degradation of a specific substrate(s).  p62 is a ubiquitously expressed cellular protein that is conserved in metazoa but not in plants and fungi, and recently it has been known as one of the selective substrates for autophagy.

This protein is localized at the autophagosome formation site and directly interacts with LC3, an autophagosome localizing protein . Subsequently, the p62 is incorporated into the autophagosome and then degraded. Therefore, impaired autophagy is accompanied by accumulation of p62 followed by the formation of p62 and ubiquitinated protein aggregates because of the nature of both self- oligomerization and ubiquitin binding of p62.


This extensive review leaves little left unopened. We have seen the central role that the UPS system plays in normal organelle proteolysis in concert with autophagy. Impaired ubiquitination occurs from aging, and/or toxins, under oxidative stress involving E3s or DUBs.

This leads to altered gene transcripton, altered protein trafficking, and plays a role in neurodegenative disease, and muscle malfunction.

English: A cartoon representation of a lysine 48-linked diubiquitin molecule. The two ubiquitin chains are shown as green cartoons with each chain labelled. The components of the linkage are indicated and shown as orange sticks. Image was created using PyMOL from PDB id 1aar. (Photo credit: Wikipedia)

Different forms of protein ubiquitylation (Photo credit: Wikipedia)


                              nature10774-f6.2 (1)  tetra-ubiquitin chain conjugated to the undtructured initiation region of a substrate and bound to the ubiquitin receptor Rpn13. substrate poised for deubiquination by Rpn11

filedesc Schematic diagram of the ubiquitylati...

filedesc Schematic diagram of the ubiquitylation system. Created by Roger B. Dodd (Photo credit: Wikipedia)


Autophagy (Photo credit: Wikipedia)

English: Structure of the PARK2 protein. Based...

English: Structure of the PARK2 protein. Based on PyMOL rendering of PDB 1iyf. (Photo credit: Wikipedia)

Comparison of the process of macroautophagy ve...

Comparison of the process of macroautophagy versus microautophagy. (Photo credit: Wikipedia)

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Reporter: Aviva Lev-Ari, PhD, RN


Press Release

13 January, 2013

Hebrew University study finds key mechanism in calcium regulation.  The finding is important element in road towards development of new drugs for neurodegenerative diseases


All living cells keep their cellular calcium concentration at a very low level. Since a small increase in calcium can affect many critical cellular functions (an elevated calcium concentration over an extended period can induce cell death), powerful cellular mechanisms ensure that calcium concentration quickly returns to its low level.

It is known that impairments of cellular calcium regulation underlie almost all neurodegenerative diseases. For example, age-related loss of calcium regulation was shown to promote cell vulnerability in Alzheimer’s disease.

In a study recently published in the Journal of Neuroscience, Hebrew University of Jerusalem researchers, along with others from Israel and the US, presented their findings of a previously undescribed cellular mechanism which is essential for keeping cellular calcium concentration low. 
This mechanism operates together with other already characterized mechanisms.

Dr. Shirley Weiss and Prof. Baruch Minke of the Hebrew University’s Institute of Medical Research Israel-Canada (IMRIC) and the Edmond and Lily Safra Center for Brain Sciences (ELSC) characterized this mechanism using photoreceptor cells of the fruit fly, which is a powerful model for studying basic biological processes. 

They found that a protein-designated calphotin (a calcium buffer) operates by sequestering elevated calcium concentration. Genetic elimination of calphotin led to a light-induced rise in cellular calcium for an abnormally extended time, leading to retinal photoreceptor degeneration in the fruit flies.

The researchers stress that this kind of research, leading to a better understanding of the fundamental mechanisms underlying cellular calcium regulation, is critical for the development of new drugs and treatments for neurodegenerative diseases. 




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Mitochondrial Dynamics and Cardiovascular Diseases

Author and Curator: Ritu Saxena, Ph.D.


Morphological changes in mitochondria have been observed in several human diseases including myopathies, diabetes mellitus, liver diseases, neurodegeneration, aging, and cancer. Ong et al (2010) studied neonatal rat ventricular myocytes as an experimental model of aging and concluded that the interplay between mitochondrial fission and autophagy controls the rate of mitochondrial turnover. A disturbance in the balance is observed in aging heart cells resulting in giant mitochondria. This observation is an indication that mitochondrial morphology is connected to pathogenesis of cardiac disease. http://www.ncbi.nlm.nih.gov/pubmed/20631158 Thus, it is important to understand the mechanism of mitochondrial dynamics in order to correlate it with the development of cardiovascular diseases.

Mitochondrial dynamics

The shape of mitochondria is very dynamic in living cells, constantly interchanging between thread-like and grain-like morphology through what we know now as the fusion and fission processes, respectively. The fusion and fission processes together with the mitochondrial movement have been termed “mitochondrial dynamics”.  Nucleoids, the assemblies of mitochondrial DNA (mtDNA) with its associated proteins, are distributed during fission in such a way that each mitochondrion contains at least one nucleoid.

Mitochondrial fusion is a complex process that involves the fusing together of four lipid bilayers. Proteins involved in the mitochondrial fission and fusion have been discussed in an earlier post published on October 31, 2012. Mitochondrial fusion requires two 85kD-GTPase isoforms mitofusin1 (Mfn1) and mitofusin2 (Mfn2). Mfn1 and Mfn1 are both anchored to the outer mitochondrial membrane. They contain – two transmembrane domains connected by a small intermembrane-space loop, a cytosolic N-terminal GTPase domain and two cytosolic hydrophobic heptad-repeat coiled-coil domains. The coiled-coil domains of Mfn1 and Mfn2 help in tethering adjacent mitochondria in both homo-oligomeric and hetero-oligomeic fashion. The fusion process requires GTP hydrolysis and the cells where Mfn2 had a GTPase mutation; mitochondria were not able to undergo fusion even after tethering. Mitochondrial fission and fusion have been illustrated in Figure 1.

Mitochondrial fission is opposite of the fusion process. Mammalian mitochondria undergo fission by the interaction of two proteins: dynamin-like protein 1 or dynamin-related protein 1 (DLP1/Drp1), an 80–85-kD cytosolic GTPase, and human fission protein 1 (hFis1), a 17-kD outer mitochondrial membrane anchored protein. Mitochondrial fission too requires GTP hydrolysis. DLP1 mainly localizes in the cytosol and with the help of hFis1, DLP1 is recruited to the constriction sites of the membrane. DLP1 translocation depends on actin and microtubules and once inside, DLP1 oligomerizes into a ring around the mitochondrion. The self-assembly of DLP1 stimulates the final step of fission which is disassembly and it requires GTP hydrolysis.

Figure 1: Model of mammalian mitochondrial fission and fusion (Hom et al, J Mol Cell Cardiol, 2009)


Additional information on different aspects of mitochondria could be found articles published earlier in the Pharmaceutical Intelligence webpage.

Mitochondrial dynamics in the heart

In cultured cardiovascular cell line the mitochondria are arranged in a filamentous network and are highly dynamic, constantly undergoing fusion and fission. Similar mitochondrial network is observed in vascular smooth muscle cells, cardiac stem cells, and neonatal cardiomyocytes. Thus, these cell types have been used to study mitochondrial dynamics.

However, in the adult cardiomyocyte, there are three distinct populations of mitochondria:

(i)           peri-nuclear mitochondria,

(ii)         subsarcolemmal (SSC) mitochondria, and

(iii)       interfibrillar (IF) mitochondria

Electron micrographs of adult cardiac muscle cells, especially ventricular myocytes, show that mitochondria are numerous, making up about 35% of the cell volume, and that mitochondria are highly organized and compacted between contractile filaments and next to T-tubules. This crystal-like pattern of mitochondria in adult ventricular myocytes raises an interesting question- Do the mitochondria in these cells also undergo physiological fission, fusion, and movement just like other cell types? Whether the crystal-like lattice arrangement restricts their movements and prevents them from undergoing fusion or fission is unclear. It has been speculated that the fission and fusion processes might occur at a slower rate because of the tight packing. A four-dimensional (x, y, z axis and time) live-cell imaging is needed to detect possible movements like mitochondria winding slowly through the myofibrils in the third dimension.

Figure 2. Representative electron micrograph of adult murine heart depicting the three subpopulations of mitochondria: perinuclear (PN) mitochondria; interfibrillar (IF) mitochondria; and subsarcolemmal mitochondria (SSM). Photo credit: Ong et al, Cardiovascular Research (2010).

Expression of fission/fusion proteins in adult heart: Interestingly, it has been observed that proteins required for mitochondrial dynamics including fission and fusion proteins is abundantly present in the adult heart and would have been active during cardiomyocyte differentiation to ensure the unique spatial organization of the three different subpopulations of cardiac mitochondria.

Several studies suggest the existence of fission and fusion proteins in the adult heart.

  • Mfn1 and Mfn2 fusion proteins have been found to be expressed in highest amounts in the heart compared to that in human tissues of pancreas, skeletal muscle, brain, liver, placenta, lung, and kidney using both Northern and Western blot analysis. Infact, Mfn2 mRNA was found to be abundantly expressed in heart and muscle tissue but expressed only at low levels in other tissues. Mfn1 and Mfn2 expression has also been confirmed in heart tissue of rat and mouse by RT-PCR.
  • hFis1, a fission protein, has been shown to be ubiquitously expressed in isolated rat mitochondria in heart tissue apart from several other tissues.
  • DLP1 mRNA, coding for a fusion protein, have been detected in high levels in several adult tissues including heart, skeletal muscle, kidney and brain.
  • OPA1 codes for another fusion protein and four transcripts of OPA1 have been detected in adult mouse hearts.

Mitochondria in cardiac diseases:

Morphological changes in mitochondria have been observed in several human diseases including myopathies, diabetes mellitus, liver diseases, neurodegeneration, aging, and cancer. Ong et al (2010) studied neonatal rat ventricular myocytes as an experimental model of aging and concluded that the interplay between mitochondrial fission and autophagy controls the rate of mitochondrial turnover. A disturbance in the balance is observed in aging heart cells resulting in giant mitochondria. This observation is an indication that mitochondrial morphology is connected to pathogenesis of cardiac disease. http://www.ncbi.nlm.nih.gov/pubmed/20631158

Abnormal mitochondrial morphology corresponding to various cardiac diseases has been listed as follows:

  • Abnormally small and disorganized mitochondria – observed in endstage dilated cardiomyopathy, myocardial hibernation, cardiac rhabdomyoma, and ventricular-associated congenital heart diseases.
  • Disorganized clusters of fragmented mitochondria – observed in Tetralogy of Fallot and are located away from contractile filaments, along with having a very small diameter measured to be 0.1 μm as observed in the electron micrographs.
  • Big and defective mitochondria – observed in senescent cardiomyocytes.



Condition Cell type Change in mitochondrial morphology Other findings Study
Ischemia-perfusion injury HL-1 cells Fission P38 inhibition at reperfusion allows mitochondrial re-fusion Brady et al
β – Adrenergic stimulation by isoproterenol or exercise Adult murine heart Not investigated Phosphorylation and inhibiton of Drp1 at Ser656 Cribbs and Strack et al
Cardiac differentiation Embryonic stem cells Fusion Fusion is required to support Oxidative phosphorylation Chung et al
Hyperglycemia H9C2 rat myoblast Fission Yu et al
Post-MI heart failure and dilated cardiomyopathy Adult rat and human heart Fragmentation Decrease in OPA1 Chen et al
Diabetes Murine coronary endothelial cell Fission Decreased OPA1, increased Drp1 Makino et al
Diabetes Adult murine diabetic heart Fission Lower mitochondrial membrane potential Williamson et al
Ischaemia-reperfusion injury and cardioprotection HL-1 cells, adult heart Fission Inhibiting fission cardioprotective Ong et al
Cytosolic calcium overload Neonatal cardiomyocytes and adult heart Fission Hom et al

Table 1: Studies implicating changes in mitochondrial morphology in cardiovascular diseases, Adapted from Ong et al, Cardiovascular Research (2010).

Mitochondrial dynamics in heart failure

Fission and Fusion in Heart Failure

Mutation or abnormal expression of fission and fusion proteins have been implicated in several diseases including neuropathies, Parkinson’s disease, type 2 diabetes and so on. However, few studies have addressed the involvement of mitochondrial dynamics in heart failure. Research groups have used cardiac-like cell lines, neonatal and adult cardiomyocytes, and animal models to demonstrate the importance of fission and fusion proteins. Observations from some studies have been listed below:

  • Mitochondria are highly organized and compacted between contractile filaments (interfibrillar) or adjacent to the sarcolemma (subsarcolemmal) in adult mammalian cardiomyocytes. However, during heart failure, interfibrillar mitochondria may lose their normal organization.
  • There is also a reduction in size and density of interfibrillar mitochondria in rodent models of heart failure.
  • It was recently reported that OPA1 is decreased in both human and rat heart failure.
  • Electron microscopic data showed an increase in the number and decrease in the size of the mitochondria in a coronary artery ligation rat heart failure model.
  • Inhibition of fission in cultured neonatal ventricular myocytes by overexpression of dominant negative mutant form of Drp1, Drp1-K38A, prevents overproduction of ROS, mitochondrial permeability transient pore formation and ultimately cell death under high glucose conditions.
  • In cultured neonatal and adult cardiomyocytes, cytosolic Ca2+ overload induced by thapsigargin (Tg) or potassium chloride (KCl) resulted in rapid mitochondrial fragmentation. Calcium overload is a common feature in heart failure, which might lead to increase in fission contributing to decrease in energy production in the failing heart.
  • In H9c2 cells, reduction in OPA1 increased apoptosis both at baseline and after simulated ischemia, via cytochrome c release from mitochondria.
  • Drosophila heart tube-specific silencing of OPA1 and mitochondrial assembly regulatory factor (MARF) increased mitochondrial morphometric heterogeneity and induced heart tube dilation with profound contractile impairment. In this model, human MFN1/2 was rescued MARF RNAi induced cardiomyopathy.
  • MFN-2-deficient mice have mild cardiac hypertrophy and mild depression of cardiac function. Also, mitochondria of cardiac myocytes lacking MFN-2 are pleiotropic and larger.
  • In rat hearts, decreased MFN2, increased Fis1 and no change in OPA1 expression was observed 12–18 weeks after myocardial infarction. http://www.ncbi.nlm.nih.gov/pubmed/22848903

However, further research is needed to accurately and fully define the role of abnormal mitochondrial morphology in heart failure. Those researches might lead to developing new interventions for treating abnormal mitochondrial function based diseases.


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