Opposite Modulation of RAC1 by Mutations in TRIO Is Associated with Distinct, Domain-Specific Neurodevelopmental Disorders

The Rho-guanine nucleotide exchange factor (RhoGEF) TRIO acts as a key regulator of neuronal migration, axonal outgrowth, axon guidance, and synaptogenesis by activating the GTPase RAC1 and modulating actin cytoskeleton remodeling. Pathogenic variants in TRIO are associated with neurodevelopmental diseases, including intellectual disability (ID) and autism spectrum disorders (ASD). Here, we report the largest international cohort of 24 individuals with confirmed pathogenic missense or nonsense variants in TRIO. The nonsense mutations are spread along the TRIO sequence, and affected individuals show variable neurodevelopmental phenotypes. In contrast, missense variants cluster into two mutational hotspots in the TRIO sequence, one in the seventh spectrin repeat and one in the RAC1-activating GEFD1. Although all individuals in this cohort present with developmental delay and a neuro-behavioral phenotype, individuals with a pathogenic variant in the seventh spectrin repeat have a more severe ID associated with macrocephaly than do most individuals with GEFD1 variants, who display milder ID and microcephaly. Functional studies show that the spectrin and GEFD1 variants cause a TRIO-mediated hyper- or hypo-activation of RAC1, respectively, and we observe a striking correlation between RAC1 activation levels and the head size of the affected individuals. In addition, truncations in TRIO GEFD1 in the vertebrate model X. tropicalis induce defects that are concordant with the human phenotype. This work demonstrates distinct clinical and molecular disorders clustering in the GEFD1 and seventh spectrin repeat domains and highlights the importance of tight control of TRIO-RAC1 signaling in neuronal development.<br/

Circadian clock nuclear receptor REV-ERBα is a novel regulator of beta-cell function, survival and autophagy under diabetogenic conditions

International audienceBackground and aims:The circadian clock regulates diverse cellular and molecular rhythms employing CLOCK-BMAL1 transcriptional heterodimer with nuclear receptor REV-ERBα (encoded by gene Nr1d1) playing an important role as a clock repressor through modulation of Bmal1 transcription. Importantly, in addition to its core circadian clock function, recent studies have identified REV-ERBα as a potent transcriptional repressor of autophagy. Therefore, in the current study we set out to address whether impaired beta-cell function and survival associated with exposure to diabetogenic stressors (e.g. glucotoxicty and inflammation) is attributed in part to REV-ERBα-mediated inhibition of autophagy. Materials and methods: Experiments were performed with the rat pancreatic beta-cell line (INS-1E). p62 (also known as sequestosome-1) levels were used to monitor autophagic degradation and evaluated by western blot. Because p62 aggregated forms were reported to be largely insoluble, we also evaluated the detergent-solubility of p62 by fractionation and western blot analysis. Apoptosis was evidenced by cleaved caspase-3 emergence. Glucose-induced insulin secretion was assessed by Homogeneous Time Resolved Fluorescence (HTRF) technology.Results: Exposure of beta-cells to either glucotoxicity (30 mM glucose for 48h) or cytokines (cytomix of IL-1β, TNFα and IFNγ for 24h) resulted in robust induction of REV-ERBα expression (1.5-2 fold, p<0.05) and corresponded with impaired autophagy flux characterized by increased protein levels of p62 (1.5-2 fold, p<0.05). Consistent with these data, exposure of beta-cells to a REV-ERBα agonist (SR9011) was characterized by impaired autophagy (increased p62 levels, aggregated and insoluble forms, p<0.05), defective glucose-stimulated insulin secretion (70 % decrease, p<0.05) and increased beta-cell apoptosis (increased cleaved caspase-3, p<0.01 vs. vehicle). In contrast, REV-ERBα specific antagonist (SR8278) protected beta-cells from deleterious effects of glucotoxicity or cytokines-induced inflammation by enhancing autophagy flux and attenuating beta-cell apoptosis (~30%). Conclusion:Taken together, these data reveal for the first time an underexplored link between the core circadian clock nuclear receptor REV-ERBα, autophagy and beta-cell failure under diabetogenic conditions. These data also suggest a potential therapeutic potential of modulating REV-ERBα levels in beta-cells to enhance function and survival in diabetes

The nuclear receptor REV-ERBα is implicated in the alteration of β-cell autophagy and survival under diabetogenic conditions

International audiencePancreatic β-cell failure in type 2 diabetes mellitus (T2DM) is associated with impaired regulation of autophagy which controls β-cell development, function, and survival through clearance of misfolded proteins and damaged organelles. However, the mechanisms responsible for defective autophagy in T2DM β-cells remain unknown. Since recent studies identified circadian clock transcriptional repressor REV-ERBα as a novel regulator of autophagy in cancer, in this study we set out to test whether REV-ERBα-mediated inhibition of autophagy contributes to the β-cell failure in T2DM. Our study provides evidence that common diabetogenic stressors (e.g., glucotoxicity and cytokine-mediated inflammation) augment β-cell REV-ERBα expression and impair β-cell autophagy and survival. Notably, pharmacological activation of REV-ERBα was shown to phenocopy effects of diabetogenic stressors on the β-cell through inhibition of autophagic flux, survival, and insulin secretion. In contrast, negative modulation of REV-ERBα was shown to provide partial protection from inflammation and glucotoxicity-induced β-cell failure. Finally, using bioinformatic approaches, we provide further supporting evidence for augmented REV-ERBα activity in T2DM human islets associated with impaired transcriptional regulation of autophagy and protein degradation pathways. In conclusion, our study reveals a previously unexplored causative relationship between REV-ERBα expression, inhibition of autophagy, and β-cell failure in T2DM

Le récepteur nucléaire Rev-erbα est un nouveau régulateur de l’autophagie et de la survie des cellules β pancréatiques en conditions diabétogènes

International audienceIntroductionLe diabète de type 2 (DT2) est caractérisé par une hyperglycémie liée à un déficit en cellules β. L’altération du rythme circadien est un facteur de risque du DT2. Au niveau moléculaire, les rythmes circadiens sont contrôlés par Clock-Bmal1 et le répresseur Rev-erbα. En dehors de sa fonction circadienne, Rev-erbα bloque l’autophagie, cette dernière étant cruciale pour l’intégrité des cellules β. Nous émettons l’hypothèse selon laquelle l’inhibition de l’autophagie par Rev-erbα en conditions diabétogènes est impliquée dans le déficit β-cellulaire. Matériel et MéthodesLes expériences sont réalisées avec des cellules ß (INS-1E de rat, EndoC-ßH1 humaines) et des îlots humains. Les niveaux protéiques de Rev-erbα sont évalués par western-blot. L’efficacité de l’autophagie est évaluée grâce aux marqueurs LC3-II (nombre d’autophagosomes) et p62 (efficacité de dégradation lysosomale). L’apoptose est évaluée par le clivage de la caspase-3. La sécrétion d’insuline est mesurée par HTRF (Homogeneous Time Resolved Fluorescence). RésultatsL’exposition à des concentrations élevées en glucose mimant l'hyperglycémie chronique ou à des cytokines pro-inflammatoires entraine une augmentation de Rev-erbα corrélée à une altération de l’autophagie dans les cellules INS-1E. Dans les îlots humains exposés à des conditions de glucotoxicité ou lipotoxicité, l’augmentation de l’expression protéique de Rev-erbα est aussi observée. L’activation pharmacologique de Rev-erbα (agoniste SR9009) altère l’autophagie dans les cellules INS-1E et les ilots humains. En conséquence, cela entraine un déficit insulinosécrétoire et l’apoptose des cellules INS-1E. A l’inverse, l’inhibition pharmacologique de Rev-erbα (antagoniste SR8278) ou son invalidation par siRNA protège les cellules ß de l’apoptose induite par la glucotoxicité (INS-1E et EndoC-ßH1), ou par les cytokines (INS-1E et ilots humains).Conclusion-discussionNotre étude révèle pour la première fois un lien entre le gène de l’horloge Rev-erba, l’autophagie et l’altération des cellules ß en conditions diabétogènes, identifiant ainsi une nouvelle cible potentielle