Glutamine-Expanded Ataxin-7 Alters TFTC/STAGA Recruitment and Chromatin Structure Leading to Photoreceptor Dysfunction

Spinocerebellar ataxia type 7 (SCA7) is one of several inherited neurodegenerative disorders caused by a polyglutamine (polyQ) expansion, but it is the only one in which the retina is affected. Increasing evidence suggests that transcriptional alterations contribute to polyQ pathogenesis, although the mechanism is unclear. We previously demonstrated that theSCA7 gene product, ataxin-7 (ATXN7), is a subunit of the GCN5 histone acetyltransferase–containing coactivator complexes TFTC/STAGA. We show here that TFTC/STAGA complexes purified from SCA7 mice have normal TRRAP, GCN5, TAF12, and SPT3 levels and that their histone or nucleosomal acetylation activities are unaffected. However, rod photoreceptors from SCA7 mouse models showed severe chromatin decondensation. In agreement, polyQ-expanded ataxin-7 induced histone H3 hyperacetylation, resulting from an increased recruitment of TFTC/STAGA to specific promoters. Surprisingly, hyperacetylated genes were transcriptionally down-regulated, and expression analysis revealed that nearly all rod-specific genes were affected, leading to visual impairment in SCA7 mice. In conclusion, we describe here a set of events accounting for SCA7 pathogenesis in the retina, in which polyQ-expanded ATXN7 deregulated TFTC/STAGA recruitment to a subset of genes specifically expressed in rod photoreceptors, leading to chromatin alterations and consequent progressive loss of rod photoreceptor function

Maladies par expansion de polygluatamine (Dérégulations transcriptionnelles, altération du programme de différenciation neuronale et dégénérescence)

STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Les maladies neurodégénératives par expansion de polyglutamine : physiopathologie et stratégies thérapeutiques

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Disease Progression Despite Early Loss of Polyglutamine Protein Expression in SCA7 Mouse Model

International audienceNine neurodegenerative diseases including Huntington's disease (HD) and spinocerebellar ataxia type 7 (SCA7) are caused by an expansion of a polyglutamine (polyQ) stretch in the respective proteins. Aggregation of expanded polyQ-containing proteins into the nucleus is a hallmark of these diseases. Recent evidence indicates that transcriptional dysregulation may contribute to the molecular pathogen-esis of these diseases. Using SCA7 and HD mouse models in which we recently described a retinal phenotype, we investigated whether altered gene expression underlies photoreceptor dysfunction. In both models, rhodopsin promoter activity was early and dramatically repressed, suggesting that downregulation of photoreceptor-specific genes plays a major role in polyQ-induced retinal dysfunction. Because the rhodopsin promoter drives mutant ataxin-7 expression in our SCA7 mice, we also assessed whether downregulation of mutant SCA7 transgene would reverse retinopathy progression and aggregate formation. Although residual expression of mutant ataxin-7 was found negligible from 9 weeks of age, SCA7 transgenic mice showed a progressive decline of photoreceptor activity leading to a complete loss of electroretinographic responses from 1 year of age. At this age, aggregates were cleared in only half of the photore-ceptors, indicating that their formation is not fully reversible in this model. We demonstrate here that abolishing full-length mutant ataxin-7 expression did not reverse retinopathy progression in SCA7 mice, raising the possibility that some polyQ-induced pathological events might be irreversible

COQ8A and MED25 Mutations in a Child with Intellectual Disability, Microcephaly, Seizures, and Spastic Ataxia: Synergistic Effect of Digenic Variants?

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Polyglutamine expansion causes neurodegeneration by altering the neuronal differentiation program

International audienceHuntington's disease (HD) and spinocerebellar ataxia type 7 (SCA7) belong to a group of inherited neurodegenerative diseases caused by polyglutamine (polyQ) expansion in corresponding proteins. Transcriptional alteration is a unifying feature of polyQ disorders; however, the relationship between polyQ-induced gene expression deregulation and degenerative processes remains unclear. R6/2 and R7E mouse models of HD and SCA7, respectively, present a comparable retinal degeneration characterized by progressive reduction of electroretinograph activity and important morphological changes of rod photoreceptors. The retina, which is a simple central nervous system tissue, allows correlating functional, morphological and molecular defects. Taking advantage of comparing polyQ-induced degeneration in two retina models, we combined gene expression profiling and molecular biology techniques to decipher the molecular pathways underlying polyQ expansion toxicity. We show that R7E and R6/2 retinal phenotype strongly correlates with loss of expression of a large cohort of genes specifically involved in phototransduction function and morphogenesis of differentiated rod photoreceptors. Accordingly, three key transcription factors (Nrl, Crx and Nr2e3) controlling rod differentiation genes, hence expression of photoreceptor specific traits, are down-regulated. Interestingly, other transcription factors known to cause inhibitory effects on photoreceptor differentiation when mis-expressed, such as Stat3, are aberrantly re-activated. Thus, our results suggest that independently from the protein context, polyQ expansion overrides the control of neuronal differentiation and maintenance, thereby causing dysfunction and degeneration

Technical tools and guidance for the detection and identification of Living Modified Organisms

The TECHNICAL TOOLS AND GUIDANCE FOR THE DETECTION AND IDENTIFICATION OF LIVING MODIFIED ORGANISMS has been compiled by members of the NETWORK OF LABORATORIES FOR THE DETECTION AND IDENTIFICATION OF LIVING MODIFIED ORGANISMS as a tool toward the implementation of the Strategic Plan for the Cartagena Protocol on Biosafety for the period 2011-2020 and in particular, in line with Operational Objectives 1.6, 1.8 and 2.3 of the Strategic Plan, addressing the issue of LMO detection and identification, as a tool to assist Parties in the detection and identification of LMOs in the context of the Protocol. The TECHNICAL TOOLS AND GUIDANCE addresses the following topics: (a) Overview of available detection methods, including validated methods; (b) Overview of available databases for methods and sequence information, and available screening matrixes; (c) Minimum performance criteria for sample handling, extraction, detection and identification methodology; (d) Experience and case-studies on detection and identification. Each of the topics addressed is enriched with online resources, representing a wide array of tools and guidance on the detection and identification of LMOs, proposed by the members of the Network during the online discussions organised by the CBD executive Secretariat. The introductory texts produced by the moderators and the resources submitted by the Network during the online discussions were compiled by the Secretariat of the Convention on Biological Diversity UNEP as “Technical Tools and Guidance for the Detection and Identification of LMOs” available through the BCH at http://bch.cbd.int/protocol/cpb_detection/toolsandguidance.shtml. The outcome was presented at the Seventh meeting of the CONFERENCE OF THE PARTIES TO THE CONVENTION ON BIOLOGICAL DIVERSITY SERVING AS THE MEETING OF THE PARTIES TO THE CARTAGENA PROTOCOL ON BIOSAFETY (COP7) held in Pyeongchang, Republic of Korea, 29 September-3 October 2014.JRC.I.3-Molecular Biology and Genomic

TFTC Subunit Composition and HAT Activity Are Not Altered by PolyQ-Expanded ATXN7 in R7E Retina

<div><p>(A) Western blot analysis of TFTC-type complexes immunopurified from 4-wk-old R7N or R7E retinal homogenates using an anti-ATXN7 antibody. ATXN7 IPs revealed comparable levels of Trrap, Gcn5, and Taf12 in complexes purified from R7N or R7E retina.</p> <p>(B and C) Whole retinal extracts from 4-wk-old R7N and R7E animals were immunoprecipitated with an anti-SPT3 (B) and anti-TRRAP antibody (C). Using the same antibodies, complexes were also immunoprecipitated from HeLa cell nuclear extract as positive controls. The retinal extracts (Input) and the immunopurified complexes were analyzed by immunoblotting with an anti-ATXN7 antibody. Complexes contained similar amounts of normal or mutant ATXN7.</p> <p>(D) Western blot analysis using anti-TRRAP and anti-GCN5 antibodies on complexes immunoprecipitated with an anti-TRRAP antibody from 10-wk-old R7N and R7E retinal homogenates. Using the same antibody, complexes were also immunoprecipitated from HeLa cell nuclear extract as positive controls. Comparable levels of Gcn5 and Trrap were detected in purified complexes from R7N and R7E retina.</p> <p>(E–G) HAT activities of immunopurified complexes bound to either the anti-ATXN7 pAb (E), anti-SPT3 mAb (F), or anti-TRRAP mAb beads (G) from R7N and R7E retina were measured by an in vitro HAT assay performed on free core histones. Histones were separated by SDS-PAGE and stained by Coomassie Brilliant Blue (CBB) and acetylated histones were visualized by fluorography (Fluorogr). The position of each histone is indicated. The pattern of histone acetylation was identical to that of a highly purified TFTC fraction used as a positive control. No histone acetylation could be detected when the antibody was omitted. The age of the mice are indicated in weeks in brackets. These results are representative of three independent experiments. Quantification of all HAT assays performed on R7N and R7E retina is provided in<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040067#sg006" target="_blank">Figure S6</a>A.</p></div

Loss of a Specific Gene Compartmentalization in Rods from SCA7 Mice

<div><p>(A–F) BAC probes containing<i>Rho</i> (A–C) or<i>Acta2</i> (D–F) genes were hybridized to retinal cryosections from 2-mo-old WT (A), (B), (D), and (E) or R7E (C) and (F) animals. FISH signals appear in green whereas DAPI-stained photoreceptor nuclei were pseudo-colored in red for better visualization. Merged images were collected by confocal imaging analysis and showed co-localization of<i>Acta2</i> within the densely DAPI-stained heterochromatin region in WT rod nuclei (arrowheads in [D] and [E]). By contrast,<i>Rho</i> was excluded from this compact heterochromatin region. In R7E rod nuclei,<i>Rho</i> and<i>Acta2</i> showed a comparable random pattern of intranuclear distribution. Both could be found co-localizing within the central densely DAPI-stained region (arrowheads in [C] and [F]). Scale bars represent 2 μm.</p> <p>(G) Distribution of<i>Rho</i> and<i>Acta2</i> between heterochromatin and euchromatin territories in WT and R7E rod nuclei was estimated by counting the number of spots detected in the densely DAPI-stained region. Counting was performed on the projection of four consecutive<i>z</i> stacks (1.2 μm between each stack) taken through the retinal section such that only entire rod nuclei were included in the counting. More than 500 nuclei were analyzed, and each bar represents the mean value ± SEM of three independent experiments performed on two different animals.</p></div