Mitochondria play a crucial role in neuronal survival through efficient energy metabolism. In pathological conditions, mitochondrial stress leads to neuronal death, which is regulated by the anti-apoptotic BCL-2 family of proteins. MCL-1 is an anti-apoptotic BCL-2 protein localized to mitochondria either in the outer membrane (OM) or inner membrane (Matrix), which have distinct roles in inhibiting apoptosis and promoting bioenergetics, respectively. While the anti-apoptotic role for Mcl1 is well characterized, the protective function of MCL-1 ^Matrix remains poorly understood. Here, we show MCL-1^OM and MCL-1^Matrix prevent neuronal death through distinct mechanisms. We report that MCL-1^Matrix functions to preserve mitochondrial energy transduction and improves respiratory chain capacity by modulating mitochondrial oxygen consumption in response to mitochondrial stress. We show that MCL-1^Matrix protects neurons from stress by enhancing respiratory function, and by inhibiting mitochondrial permeability transition pore opening. Taken together, our results provide novel insight into how MCL-1^Matrix may confer neuroprotection under stress conditions involving loss of mitochondrial function.

Mitochondria share attributes of vesicular transport with their bacterial ancestors given their ability to form mitochondrial‐derived vesicles (MDVs). MDVs are involved in mitochondrial quality control and their formation is enhanced with stress and may, therefore, play a potential role in mitochondrial‐cellular communication. However, MDV proteomic cargo has remained mostly undefined. In this study, we strategically used an in vitro MDV budding/reconstitution assay on cardiac mitochondria, followed by graded oxidative stress, to identify and characterize the MDV proteome. Our results confirmed previously identified cardiac MDV markers, while also revealing a complete map of the MDV proteome, paving the way to a better understanding of the role of MDVs. The oxidative stress vulnerability of proteins directed the cargo loading of MDVs, which was enhanced by antimycin A (Ant‐A). Among OXPHOS complexes, complexes III and V were found to be Ant‐A‐sensitive. Proteins from metabolic pathways such as the TCA cycle and fatty acid metabolism, along with Fe‐S cluster, antioxidant response proteins, and autophagy were also found to be Ant‐A sensitive. Intriguingly, proteins containing hyper‐reactive cysteine residues, metabolic redox switches, including professional redox enzymes and those that mediate iron metabolism, were found to be components of MDV cargo with Ant‐A sensitivity. Last, we revealed a possible contribution of MDVs to the formation of extracellular vesicles, which may indicate mitochondrial stress. In conclusion, our study provides an MDV proteomics signature that delineates MDV cargo selectivity and hints at the potential for MDVs and their novel protein cargo to serve as vital biomarkers during mitochondrial stress and related pathologies.

The French-Canadian variant of Leigh Syndrome (LSFC) is an autosomal recessive oxidative phosphorylation (OXPHOS) disorder caused by a mutation in LRPPRC, coding for a protein involved in the stability of mitochondrially-encoded mRNAs. Low levels of LRPPRC are present in all patient tissues, but result in a disproportionately severe OXPHOS defect in the brain and liver, leading to unpredictable subacute metabolic crises. To investigate the impact of the OXPHOS defect in the liver, we analyzed the mitochondrial phenotype in mice harboring an hepatocyte-specific inactivation of Lrpprc. Loss of LRPPRC in the liver caused a generalized growth delay, and typical histological features of mitochondrial hepatopathy. At the molecular level, LRPPRC deficiency caused destabilization of polyadenylated mitochondrial mRNAs, altered mitochondrial ultrastructure, and a severe complex IV (CIV) and ATP synthase (CV) assembly defect. The impact of LRPPRC deficiency was not limited to OXPHOS, but also included impairment of long-chain fatty acid oxidation, a striking dysregulation of the mitochondrial permeability transition pore, and an unsuspected alteration of trans-membrane H₂O₂ diffusion, which was traced to the ATP synthase assembly defect, and to changes in the lipid composition of mitochondrial membranes. This study underscores the value of mitochondria phenotyping to uncover complex and unexpected mechanisms contributing to the pathophysiology of mitochondrial disorders.

Protein O-GlcNAcylation is increasingly recognized as an important cellular regulatory mechanism, in multiple organs including the heart. However, the mechanisms leading to O-GlcNAcylation in mitochondria and the consequences on their function remain poorly understood. In this study, we use an in vitro reconstitution assay to characterize the intra-mitochondrial O-GlcNAc system without potential cytoplasmic confounding effects. We compare the O-GlcNAcylome of isolated cardiac mitochondria with that of mitochondria acutely exposed to NButGT, a specific inhibitor of glycoside hydrolase. Amongst the 409 O-GlcNAcylated mitochondrial proteins identified, 191 display increased O-GlcNAcylation in response to NButGT. This is associated with enhanced Complex I (CI) activity, increased maximal respiration in presence of pyruvate-malate, and a striking reduction of mitochondrial ROS release, which could be related to O-GlcNAcylation of specific subunits of ETC complexes (CI, CIII) and TCA cycle enzymes. In conclusion, our work underlines the existence of a dynamic mitochondrial O-GlcNAcylation system capable of rapidly modifying mitochondrial function.

Mitochondrial dysfunction characterizes many rare and common age-associated diseases. The biochemical consequences, underlying clinical manifestations, and potential therapeutic targets, remain to be better understood. We tested the hypothesis that lipid dyshomeostasis in mitochondrial disorders goes beyond mitochondrial fatty acid β-oxidation, particularly in liver. This was achieved using comprehensive untargeted and targeted lipidomics in a case-control cohort of patients with Leigh syndrome French-Canadian variant (LSFC), a mitochondrial disease caused by mutations in LRPPRC , and in mice harboring liver-specific inactivation of Lrpprc (H- Lrpprc ^–/– ). We discovered a plasma lipid signature discriminating LSFC patients from controls encompassing lower levels of plasmalogens and conjugated bile acids, which suggest perturbations in peroxisomal lipid metabolism. This premise was reinforced in H- Lrpprc ^–/– mice, which compared with littermates recapitulated a similar, albeit stronger peroxisomal metabolic signature in plasma and liver including elevated levels of very-long-chain acylcarnitines. These mice also presented higher transcript levels for hepatic markers of peroxisome proliferation in addition to lipid remodeling reminiscent of nonalcoholic fatty liver diseases. Our study underscores the value of lipidomics to unveil unexpected mechanisms underlying lipid dyshomeostasis ensuing from mitochondrial dysfunction herein implying peroxisomes and liver, which likely contribute to the pathophysiology of LSFC, but also other rare and common mitochondrial diseases.