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.
Synopsis
In vivo expression in Drosophila motor neurons reveals that the two most prevalent forms of mitofusin alleles associated with CMT2A neuropathy (R94Q and R364W) have opposite effects on mitochondrial fusion.
Mutations near/within the GTP‐binding domain of mitofusin (R94Q and T105M) inhibit fusion and trigger aggregation.
Mutations within Helix‐Bundle 1 (R364W and L76P) enhance mitochondrial fusion.
Aggregation and excess fusion both impact on mitochondrial distribution and turn over and induce locomotor defects in Drosophila.
In vivo expression in Drosophila motor neurons reveals that the two most prevalent forms of mitofusin alleles associated with CMT2A neuropathy (R94Q and R364W) have opposite effects on mitochondrial fusion.
The RNA helicase SUV3 and the polynucleotide phosphorylase PNPase are involved in the degradation of mitochondrial mRNAs but their roles in vivo are not fully understood. Additionally, upstream ...processes, such as transcript maturation, have been linked to some of these factors, suggesting either dual roles or tightly interconnected mechanisms of mitochondrial RNA metabolism. To get a better understanding of the turn-over of mitochondrial RNAs in vivo, we manipulated the mitochondrial mRNA degrading complex in Drosophila melanogaster models and studied the molecular consequences. Additionally, we investigated if and how these factors interact with the mitochondrial poly(A) polymerase, MTPAP, as well as with the mitochondrial mRNA stabilising factor, LRPPRC. Our results demonstrate a tight interdependency of mitochondrial mRNA stability, polyadenylation and the removal of antisense RNA. Furthermore, disruption of degradation, as well as polyadenylation, leads to the accumulation of double-stranded RNAs, and their escape out into the cytoplasm is associated with an altered immune-response in flies. Together our results suggest a highly organised and inter-dependable regulation of mitochondrial RNA metabolism with far reaching consequences on cellular physiology.
Induction of the one-carbon cycle is an early hallmark of mitochondrial dysfunction and cancer metabolism. Vital intermediary steps are localized to mitochondria, but it remains unclear how ...one-carbon availability connects to mitochondrial function. Here, we show that the one-carbon metabolite and methyl group donor
-adenosylmethionine (SAM) is pivotal for energy metabolism. A gradual decline in mitochondrial SAM (mitoSAM) causes hierarchical defects in fly and mouse, comprising loss of mitoSAM-dependent metabolites and impaired assembly of the oxidative phosphorylation system. Complex I stability and iron-sulfur cluster biosynthesis are directly controlled by mitoSAM levels, while other protein targets are predominantly methylated outside of the organelle before import. The mitoSAM pool follows its cytosolic production, establishing mitochondria as responsive receivers of one-carbon units. Thus, we demonstrate that cellular methylation potential is required for energy metabolism, with direct relevance for pathophysiology, aging, and cancer.
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.
Les mitochondries forment un réseau très dynamique remodelé par deux processus antagonistes appelés : fusion et fission mitochondriales. Chez l’homme, une altération de ces processus, sont à ...l’origine de nombreuses maladies qui affectent essentiellement le système nerveux. L'objectif principal des travaux de ma thèse était de caractériser l'impact d'un déséquilibre entre la fusion et la fission mitochondriale dans le contexte d'une neuropathie héréditaire : la maladie de Charcot-Marie-Tooth de type 2A (CMT2A), qui est causée par des mutations dominantes dans la mitofusine MFN2. Dans le but d’étudier les mécanismes à l’origine de cette maladie, j’ai développé le premier modèle drosophile de CMT2A en exprimant dans les neurones de mouches quatre allèles de mitofusine retrouvés fréquemment chez les patients. De manière surprenante, les différents allèles altèrent très différemment la morphologie mitochondriale. En effet, alors que les mutations associées au domaine GTPase inhibent la fusion et agrègent les mitochondries, les mutations du domaine dit HB1 induisent au contraire un excès de fusion. J’ai pu ensuite déterminer que l’agrégation des mitochondries et l’excès de fusion, conduisent de manière commune à un défaut de transport des mitochondries au niveau des synapses et à une altération du métabolisme oxydatif associée à une accumulation de mutation dans l’ADN mitochondrial. Chez les drosophiles exprimant des allèles dominants actifs de mitofusine, tous ces dysfonctionnements disparaissent lorsqu’on augmente la fission suggérant que la pathogénicité des allèles du domaine HB1 résulte d’un déséquilibre de la balance entre fusion et fission en faveur de la fusion.
Mitochondria form a dynamic network remodeled by two antagonistic processes called mitochondrial fusion and fission. While mitochondrial fusion creates interconnections between mitochondria, mitochondrial fission result in fragmentation. These processes are mediated by Dynamin-related GTPases, the outer-membrane fusion protein mitofusin, and the fission factor DPR1.The main aim of my resaearch was to characterize the impact of an imbalance between mitochondrial fusion and fission in neurons in the context of a severe hereditary neuropathy called Charcot-Marie-Tooth type 2A (CMT2A). Indeed, this disease is caused by dominant mutations in the mitofusinMFN2.In order to dissect the mechanisms by which these mutations alter mitofusin properties and neuronal function, we developed four drosophila models of CMT2A expressing the two most frequent substitutions (R94Q, R364W) and two others localizing to similar domains (T105M, L76P). The four alleles resulted in mitochondrial depletion at neuromuscular junctions, decreased oxidative metabolism, increased mtDNA mutations, and impaired locomotion that were associated with aberrant mitochondrial morphology. Interestingly, while GTPase domain-associated mutations (R94Q, T105M) aggregate unfused mitochondria, mutations within helix bundle 1 (R364W, L76P) unexpectedly enhance mitochondrial fusion, as demonstrated by rescue of mitochondrial morphology and locomotion provided by the DRP1 fission factor. In conclusion, we show that both dominant negative and dominant active forms of mitofusin can cause CMT2A, and propose for the first time that excessive mitochondrial fusion drives CMT2A pathogenesis in a large number of patients.
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