Test di nefrotossicità in vitro: l'uso delle "renal cortical slices".

Studi di tossicità eseguiti in vitro mediante il modello sperimentale delle fettine di corteccia renale.ope

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

Test di nefrotossicità in vitro: l'uso delle "renal cortical slices".

Studi di tossicità eseguiti in vitro mediante il modello sperimentale delle fettine di corteccia renale

La SUMOylation de l'ataxine-7 et ses conséquences fonctionnelles dans la physiopathologie de l'ataxie spinocérébelleuse de type 7 (SCA7)

Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disease caused by a CAG expansion (polyQ) in the protein ataxin-7. SUMOylation, a post-translational modification that we identified to modulate mutant protein aggregation in a SCA7 cellular model, is facilitated by a SUMO E3 ligase. Here, we identified RanBP2 (Nup358), a nucleoporin belonging to the nuclear pore complex, as the major E3 enzyme implicated in ataxin-7 modification by SUMO-1. Indeed, RanBP2 silencing renders mutant ataxin-7 more prone to aggregation, thus demonstrating the implication of RanBP2 in SCA7 pathophysiology. We also show that endogenous ataxin-7 is a target for both SUMO-1 and -2 modification. Poly-SUMOylated ataxin-7 presents a docking site composed of SUMO2/3 chains for the recruitment of RNF4: this protein is a SUMO E3 ubiquitin ligase that mediates degradation of mutant ataxin-7 by the proteasome pathway. The degradation is abolished in presence of a mutant form of RNF4. In a SCA7 knock-in mouse model we quantified expression of SUMO-pathway related genes in cerebellum and retina, the most affected regions using quantitative RT-PCR. SUMO-related genes show expanded repeat-dependent alterations in expression patterns. At 6 months (before onset), deregulations begin to occur; by 12 months (late stage of disease), there is a statistically significant impairment in Sumo-1 levels in Atxn7100Q/5Q cerebellum. These results, together with the observation that SUMO-1 and RanBP2 protein accumulate abnormally in the cerebellum of a SCA7 patient, suggest that in vivo SUMO-modifying pathways may be perturbed in SCA7.L'ataxie spinocérébelleuse de type 7 (SCA7) est une maladie neurodégénerative due à une expansion de CAG traduit en polyQ dans la protéine ataxine-7. La SUMOylation, modification post-traductionnelle que nous avons identifiée moduler l'agrégation de la protéine mutante, est facilitée par une SUMO E3 ligase.Nous avons identifié RanBP2, une nucléoporine appartenant au complexe du pore nucléaire en tant que SUMO E3 ligase, via SUMO-1 de l'ataxine-7. En effet, le silencing de RanBP2 induit l'agrégation de l'ataxine-7 mutante, ce qui démontre l'implication de RanBP2 dans la physiopathologie de SCA7. Nous montrons également que l'ataxine-7 endogène est une cible modifiée par SUMO-1 et -2. L'ataxine-7 poly-SUMOylée, grâce à la présence de chaines SUMO2/3, est capable de recruter RNF4. Cette protéine conduit à la dégradation de l'ataxine-7 mutante par la voie du protéasome. La dégradation est abolie en présence d'un mutant de RNF4.Dans un modèle murin KI SCA7, nous avons quantifié l'expression des gènes impliqués dans la voie de la SUMOylation au niveau des régions les plus touchées du cerveau. Le niveau d'expression des ARNs messagers montre des altérations dépendantes des répétitions CAG du gène SCA7. A 6 mois (avant le début de la pathologie), les premières dérégulations sont observées; à 12 mois (à un stade avancé de la maladie), on note une diminution statistiquement significative de Sumo-1 dans le cervelet des souris Atxn7100Q/5Q. Ces résultats, alliés à l'observation de l'accumulation anormale des protéines SUMO-1 et RanBP2 dans le cervelet d'un patient SCA7, suggèrent que les voies de la SUMOylation in vivo peuvent être perturbées dans SCA7

Characterization of Neuromuscular Junctions in Mice by Combined Confocal and Super-Resolution Microscopy

International audienc

Characterization of Neuromuscular Junctions in Mice by Combined Confocal and Super-Resolution Microscopy

International audienc