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Charcot-Marie-Tooth disease type 2A (CMT2A) is caused by dominant alleles of the mitochondrial pro-fusion factor Mitofusin 2 (MFN2). To address the consequences of these mutations on mitofusin activity and neuronal function, we generate Drosophila models expressing in neurons the two most frequent substitutions (R94Q and R364W, the latter never studied before) and two others localizing to similar domains (T105M and L76P). All alleles trigger locomotor deficits associated with mitochondrial depletion at neuromuscular junctions, decreased oxidative metabolism and increased mtDNA mutations, but they differently alter mitochondrial morphology and organization. Substitutions near or within the GTPase domain (R94Q, T105M) result in loss of function and provoke aggregation of unfused mitochondria. In contrast, mutations within helix bundle 1 (R364W, L76P) enhance mitochondrial fusion, as demonstrated by the rescue of mitochondrial alterations and locomotor deficits by over-expression of the fission factor DRP1. In conclusion, we show that both dominant negative and dominant active forms of mitofusin can cause CMT2A-associated defects and propose for the first time that excessive mitochondrial fusion drives CMT2A pathogenesis in a large number of patients.

Long-term exposure to environmental oxidative stressors, like the herbicide paraquat (PQ), has been linked to the development of Parkinson's disease (PD), the most frequent neurodegenerative movement disorder. Paraquat is thus frequently used in the fruit fly Drosophila melanogaster and other animal models to study PD and the degeneration of dopaminergic neurons (DNs) that characterizes this disease. Here, we show that a D1-like dopamine (DA) receptor, DAMB, actively contributes to the fast central nervous system (CNS) failure induced by PQ in the fly. First, we found that a long-term increase in neuronal DA synthesis reduced DAMB expression and protected against PQ neurotoxicity. Secondly, a striking age-related decrease in PQ resistance in young adult flies correlated with an augmentation of DAMB expression. This aging-associated increase in oxidative stress vulnerability was not observed in a DAMB-deficient mutant. Thirdly, targeted inactivation of this receptor in glutamatergic neurons (GNs) markedly enhanced the survival of Drosophila exposed to either PQ or neurotoxic levels of DA, whereas, conversely, DAMB overexpression in these cells made the flies more vulnerable to both compounds. Fourthly, a mutation in the Drosophila ryanodine receptor (RyR), which inhibits activity-induced increase in cytosolic Ca(2+), also strongly enhanced PQ resistance. Finally, we found that DAMB overexpression in specific neuronal populations arrested development of the fly and that in vivo stimulation of either DNs or GNs increased PQ susceptibility. This suggests a model for DA receptor-mediated potentiation of PQ-induced neurotoxicity. Further studies of DAMB signaling in Drosophila could have implications for better understanding DA-related neurodegenerative disorders in humans.

International audience; L-glutamate is the major excitatory neurotransmitter in the mammalian brain. Specific proteins, the Na+/K+-dependent high affinity excitatory amino acid transporters (EAATs), are involved in the extracellular clearance and recycling of this amino acid. Type I synapses of the Drosophila neuromuscular junction (NMJ) similarly use L-glutamate as an excitatory transmitter. However, the localization and function of the only high-affinity glutamate reuptake transporter in Drosophila, dEAAT1, at the NMJ was unknown. Using a specific antibody and transgenic strains, we observed that dEAAT1 is present at the adult, but surprisingly not at embryonic and larval NMJ, suggesting a physiological maturation of the junction during metamorphosis. We found that dEAAT1 is not localized in motor neurons but in glial extensions that closely follow motor axons to the adult NMJ. Inactivation of the dEAAT1 gene by RNA interference generated viable adult flies that were able to walk but were flight-defective. Electrophysiological recordings of the thoracic dorso-lateral NMJ were performed in adult dEAAT1-deficient flies. The lack of dEAAT1 prolonged the duration of the individual responses to motor nerve stimulation and this effect was progressively increased during physiological trains of stimulations. Therefore, glutamate reuptake by glial cells is required to ensure normal activity of the Drosophila NMJ, but only in adult flies.

Huntington's disease (HD) is a late onset heritable neurodegenerative disorder caused by expansion of a polyglutamine (polyQ) sequence in the protein huntingtin (Htt). Transgenic models in mice have suggested that the motor and cognitive deficits associated to this disease are triggered by extended neuronal and possibly glial dysfunction, whereas neuronal death occurs late and selectively. Here, we provide in vivo evidence that expanded polyQ peptides antagonize epidermal growth factor receptor (EGFR) signaling in Drosophila glia. We targeted the expression of the polyQ-containing domain of Htt or an extended polyQ peptide alone in a subset of Drosophila glial cells, where the only fly glutamate transporter, dEAAT1, is detected. This resulted in formation of nuclear inclusions, progressive decrease in dEAAT1 transcription and shortened adult lifespan, but no significant glial cell death. We observed that brain expression of dEAAT1 is normally sustained by the EGFR-Ras-extracellular signal-regulated kinase (ERK) signaling pathway, suggesting that polyQ could act by antagonizing this pathway. We found that the presence of polyQ peptides indeed abolished dEAAT1 upregulation by constitutively active EGFR and potently inhibited EGFR-mediated ERK activation in fly glial cells. Long polyQ also limited the effect of activated EGFR on Drosophila eye development. Our results further indicate that the polyQ acts at an upstream step in the pathway, situated between EGFR and ERK activation. This suggests that disruption of EGFR signaling and ensuing glial cell dysfunction could play a direct role in the pathogenesis of HD and other polyQ diseases in humans.

International audience; Cristae are mitochondrial inner-membrane structures that concentrate respiratory chain complexes and hence regulate ATP production. Mechanisms controlling crista morphogenesis are poorly understood and few crista determinants have been identified. Among them are the Mitofilins that are required to establish crista junctions and ATP-synthase subunits that bend the membrane at the tips of the cristae. We report here the phenotypic consequences associated with the in vivo inactivation of the inner-membrane protein Pantagruelian Mitochondrion I (PMI) both at the scale of the whole organism, and at the level of mitochondrial ultrastructure and function. We show that flies in which PMI is genetically inactivated experience synaptic defects and have a reduced life span. Electron microscopy analysis of the inner-membrane morphology demonstrates that loss of PMI function increases the average length of mitochondrial cristae in embryonic cells. This phenotype is exacerbated in adult neurons in which cristae form a dense tangle of elongated membranes. Conversely, we show that PMI overexpression is sufficient to reduce crista length in vivo. Finally, these crista defects are associated with impaired respiratory chain activity and increases in the level of reactive oxygen species. Since PMI and its human orthologue TMEM11 are regulators of mitochondrial morphology, our data suggest that, by controlling crista length, PMI influences mitochondrial diameter and tubular shape.

Huntington’s disease (HD) is a fatal neurodegenerative disorder caused by aberrant expansion of CAG repeat in the huntingtin gene. Mutant Huntingtin (mHtt) alters multiple cellular processes, leading to neuronal dysfunction and death. Among those alterations, impaired mitochondrial metabolism seems to have a major role in HD pathogenesis. In this study, we used the Drosophila model system to further investigate the role of mitochondrial damages in HD. We first analyzed the impact of mHtt on mitochondrial morphology, and surprisingly, we revealed the formation of abnormal ring-shaped mitochondria in photoreceptor neurons. Because such mitochondrial spheroids were previously detected in cells where mitophagy is blocked, we analyzed the effect of PTEN-induced putative kinase 1 (PINK1), which controls Parkin-mediated mitophagy. Consistently, we found that PINK1 overexpression alleviated mitochondrial spheroid formation in HD flies. More importantly, PINK1 ameliorated ATP levels, neuronal integrity and adult fly survival, demonstrating that PINK1 counteracts the neurotoxicity of mHtt. This neuroprotection was Parkin-dependent and required mitochondrial outer membrane proteins, mitofusin and the voltage-dependent anion channel. Consistent with our observations in flies, we demonstrated that the removal of defective mitochondria was impaired in HD striatal cells derived from HdhQ111 knock-in mice, and that overexpressing PINK1 in these cells partially restored mitophagy. The presence of mHtt did not affect Parkin-mediated mitochondrial ubiquitination but decreased the targeting of mitochondria to autophagosomes. Altogether, our findings suggest that mitophagy is altered in the presence of mHtt and that increasing PINK1/Parkin mitochondrial quality control pathway may improve mitochondrial integrity and neuroprotection in HD.