PML clastosomes prevent nuclear accumulation of mutant ataxin-7 and other polyglutamine proteins

The pathogenesis of spinocerebellar ataxia type 7 and other neurodegenerative polyglutamine (polyQ) disorders correlates with the aberrant accumulation of toxic polyQ-expanded proteins in the nucleus. Promyelocytic leukemia protein (PML) nuclear bodies are often present in polyQ aggregates, but their relation to pathogenesis is unclear. We show that expression of PML isoform IV leads to the formation of distinct nuclear bodies enriched in components of the ubiquitin-proteasome system. These bodies recruit soluble mutant ataxin-7 and promote its degradation by proteasome-dependent proteolysis, thus preventing the aggregate formation. Inversely, disruption of the endogenous nuclear bodies with cadmium increases the nuclear accumulation and aggregation of mutant ataxin-7, demonstrating their role in ataxin-7 turnover. Interestingly, β-interferon treatment, which induces the expression of endogenous PML IV, prevents the accumulation of transiently expressed mutant ataxin-7 without affecting the level of the endogenous wild-type protein. Therefore, clastosomes represent a potential therapeutic target for preventing polyQ disorders

Lentiviral vector-mediated overexpression of mutant ataxin-7 recapitulates SCA7 pathology and promotes accumulation of the FUS/TLS and MBNL1 RNA-binding proteins

International audienceBackground: We used lentiviral vectors (LVs) to generate a new SCA7 animal model overexpressing a truncated mutant ataxin-7 (MUT ATXN7) fragment in the mouse cerebellum, in order to characterize the specific neuropathological and behavioral consequences of the genetic defect in this brain structure. Results: LV-mediated overexpression of MUT ATXN7 into the cerebellum of C57/BL6 adult mice induced neuropathological features similar to that observed in patients, such as intranuclear aggregates in Purkinje cells (PC), loss of synaptic markers, neuroinflammation, and neuronal death. No neuropathological changes were observed when truncated wild-type ataxin-7 (WT ATXN7) was injected. Interestingly, the local delivery of LV-expressing mutant ataxin-7 (LV-MUT-ATXN7) into the cerebellum of wild-type mice also mediated the development of an ataxic phenotype at 8 to 12 weeks post-injection. Importantly, our data revealed abnormal levels of the FUS/TLS, MBNL1, and TDP-43 RNA-binding proteins in the cerebellum of the LV-MUT-ATXN7 injected mice. MUT ATXN7 overexpression induced an increase in the levels of the pathological phosphorylated TDP-43, and a decrease in the levels of soluble FUS/TLS, with both proteins accumulating within ATXN7-positive intranuclear inclusions. MBNL1 also co-aggregated with MUT ATXN7 in most PC nuclear inclusions. Interestingly, no MBNL2 aggregation was observed in cerebellar MUT ATXN7 aggregates. Immunohistochemical studies in postmortem tissue from SCA7 patients and SCA7 knock-in mice confirmed SCA7-induced nuclear accumulation of FUS/TLS and MBNL1, strongly suggesting that these proteins play a physiopathological role in SCA7. Conclusions: This study validates a novel SCA7 mouse model based on lentiviral vectors, in which strong and sustained expression of MUT ATXN7 in the cerebellum was found sufficient to generate motor defects

Epissage in vivo des RNAs premessagers de l'unite E3 d'adenovirus-2

SIGLECNRS T Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Ataxie spinocérébelleuse de type 7 (approches physiopathologique et thérapeutique)

L ataxie spinocérébelleuse de type 7 (SCA7) est une affection neurodégénérative héréditaire autosomique dominante causée par une expansion de polyQ dans l ataxine7 (ATXN7).Dans un premier temps nous avons testé le potentiel thérapeutique de l ifn-b dans le traitement de SCA7 dans un modèle murin Knock-In exprimant l ATXN7 mutée. Nous avons montré que ce traitement permettait d améliorer la coordination motrice des animaux traités, évaluée grâce à 2 tests : le Locotronic et le Beam Walking.Ensuite, je me suis intéressée aux modifications transcriptionnelles induites par la mutation dans le cervelet des souris KI SCA7. Par une approche transcriptomique réalisée durant le développement post-natal du cervelet et à un stade post symptomatique, nous avons identifié trois voies dérégulées précocement dans le cervelet des souris KI SCA7 : la myélinisation, le développement du système nerveux central et le métabolisme des monoamines. Dans chacune de ces voies nous avons validé les résultats obtenus par PCR quantitative et par des approches fonctionnelles.Ainsi, mon travail de thèse montre que l ifn-b possède un réel potentiel thérapeutique chez la souris KI SCA7. Après une étude approfondie des mécanismes moléculaires sous tendant cet effet, une étude clinique sera alors envisageable. De plus, l analyse transcriptomique montre que la majorité des dérégulations de la transcription ont lieu durant le développement post-natal du cervelet et ont donc probablement des conséquences sur son bon déroulement. Cette étude met donc en évidence pour la première fois l existence d une composante développementale dans la physiopathologie de l'ataxie SCA7.PARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Rôle des corps nucléaires PML dans la physiopathologie des maladies à expansion de polyglutamine

L ataxie spino-cérébelleuse de type 7 (SCA7) est une affection neurodégénérative héréditaire de la famille des maladies à polyglutamine (polyQ). La mutation déstabilise l ataxine7 (ATXN7) qui acquière des propriétés toxiques. L accumulation de la protéine mutée dans le noyau des neurones est une étape clef dans la physiopathologie. Notre travail démontre le rôle des corps nucléaires PML (CN) dans la dégradation de l ATXN7 mutée. Les CN sont des complexes multiprotéiques dont la fonction n est pas encore élucidée. La protéine PML (ProMyelocytic Leukaemia protein) est essentielle à la formation des CN et peut exister sous plusieurs isoformes. Notre étude montre que la surexpression spécifique de l isoforme IV de PML conduit à la formation de CN fortement enrichis en acteurs de la dégradation protéique (protéasome, ubiquitine, chaperones) appelés clastosomes. Ces clastosomes sont capables de recruter l ATXN7 mutée et de la dégrader via le protéasome, diminuant ainsi la quantité d ATXN7 mutée. De même, l interféron (INF), qui induit l expression de PML et la formation des CN, augmente la dégradation et l élimination de l ATXN7 mutée. L INF diminue ainsi la toxicité de l ATXN7 mutée dans un modèle cellulaire de SCA7 et améliore le phénotype locomoteur dans un modèle murin. L INF, en augmentant les capacités de dégradation protéique du noyau, permet donc de mieux lutter contre l accumulation de la protéine mutée et représente une nouvelle voie thérapeutique potentielle pour le traitement des maladies à polyQ.PARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Self-assembly of polyglutamine-containing huntingtin fragments into amyloid-like fibrils: Implications for Huntington’s disease pathology

Huntington’s disease is a progressive neurodegenerative disorder caused by a polyglutamine [poly(Q)] repeat expansion in the first exon of the huntingtin protein. Previously, we showed that N-terminal huntingtin peptides with poly(Q) tracts in the pathological range (51–122 glutamines), but not with poly(Q) tracts in the normal range (20 and 30 glutamines), form high molecular weight protein aggregates with a fibrillar or ribbon-like morphology, reminiscent of scrapie prion rods and β-amyloid fibrils in Alzheimer’s disease. Here we report that the formation of amyloid-like huntingtin aggregates in vitro not only depends on poly(Q) repeat length but also critically depends on protein concentration and time. Furthermore, the in vitro aggregation of huntingtin can be seeded by preformed fibrils. Together, these results suggest that amyloid fibrillogenesis in Huntington’s disease, like in Alzheimer’s disease, is a nucleation-dependent polymerization

SUMOylation attenuates the aggregation propensity and cellular toxicity of the polyglutamine expanded ataxin-7

International audiencePost-translational modification by SUMO (small ubiquitin-like modifier) was proposed to modulate the pathogenesis of several neurodegenerative diseases. Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disorder, whose pathology is caused by an expansion of a polyglutamine stretch in the protein ataxin-7 (ATXN7). Here, we identified ATXN7 as new target for SUMOylation in vitro and in vivo. The major SUMO acceptor site was mapped to lysine 257, which is part of an evolutionarily conserved consensus SUMOylation motif. SUMOylation did not influence the subcellular localization of ATXN7 nor its interaction with components of the TFTC/STAGA complex. Expansion of the polyglutamine stretch did not impair the SUMOylation of ATXN7. Furthermore, SUMO1 and SUMO2 colocalized with ATXN7 in a subset of neuronal intranuclear inclusions in the brain of SCA7 patients and SCA7 knock-in mice. In a COS-7 cellular model of SCA7, in addition to diffuse nucleoplasmic staining we identified two populations of nuclear inclusions: homogenous or non-homogenous. Non-homogenous inclusions showed significantly reduced colocalization with SUMO1 and SUMO2, but were highly enriched in Hsp70, 19S proteasome and ubiquitin. Interestingly, they were characterized by increased staining with the apoptotic marker caspase-3 and by disruption of PML nuclear bodies. Importantly, preventing the SUMOylation of expanded ATXN7 by mutating the SUMO site increased both the amount of SDS-insoluble aggregates and of caspase-3 positive non-homogenous inclusions, which act toxic to the cells. Our results demonstrate an influence of SUMOylation on the multistep aggregation process of ATXN7 and implicate a role for ATXN7 SUMOylation in SCA7 pathogenesis