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Mitochondria critically rely on protein import and its tight regulation. Here, we found that the complex I assembly factor NDUFAF8 follows a two-step import pathway linking IMS and matrix import systems. A weak targeting sequence drives TIM23-dependent NDUFAF8 matrix import, and en route, allows exposure to the IMS disulfide relay, which oxidizes NDUFAF8. Import is closely surveyed by proteases: YME1L prevents accumulation of excess NDUFAF8 in the IMS, while CLPP degrades reduced NDUFAF8 in the matrix. Therefore, NDUFAF8 can only fulfil its function in complex I biogenesis if both oxidation in the IMS and subsequent matrix import work efficiently. We propose that the two-step import pathway for NDUFAF8 allows integration of the activity of matrix complex I biogenesis pathways with the activity of the mitochondrial disulfide relay system in the IMS. Such coordination might not be limited to NDUFAF8 as we identified further proteins that can follow such a two-step import pathway.

Hereditary spastic paraplegia is a neurological condition characterized by predominant axonal degeneration in long spinal tracts, leading to weakness and spasticity in the lower limbs. The NAD + -consuming enzyme SARM1 has emerged as a key executioner of axonal degeneration upon nerve transection and in some neuropathies. An increase in the nicotinamide mononucleotide/NAD+ ratio activates SARM1, causing catastrophic NAD+ depletion and axonal degeneration. However, the role of SARM1 in the pathogenesis of hereditary spastic paraplegia has not been investigated. Here, we report an enhanced mouse model for hereditary spastic paraplegia caused by mutations in SPG7. eSpg7 knock-out mice carry a deletion in both Spg7 and Afg3l1, a redundant homologue expressed in mice but not in humans. eSpg7 knock-out mice recapitulate the phenotypic features of human patients, showing progressive symptoms of spastic-ataxia and degeneration of axons in the spinal cord as well as the cerebellum. We show that the lack of SPG7 rewires the mitochondrial proteome in both tissues, leading to an early onset decrease in mitoribosomal subunits and a remodelling of mitochondrial solute carriers and transporters. To interrogate mechanisms leading to axonal degeneration in this mouse model, we explored the involvement of SARM1. Deletion of SARM1 delays the appearance of ataxic signs, rescues mitochondrial swelling and axonal degeneration of cerebellar granule cells and dampens neuroinflammation in the cerebellum. The loss of SARM1 also prevents endoplasmic reticulum abnormalities in long spinal cord axons, but does not halt the degeneration of these axons. Our data thus reveal a neuron-specific interplay between SARM1 and mitochondrial dysfunction caused by lack of SPG7 in hereditary spastic paraplegia.

Dilated cardiomyopathy (DCM), an incurable disease of the cardiomyocyte terminating in systolic heart failure (HFrEF), is prevalent, causes hospitalization and is associated with increased mortality. Despite evidence of immune activation in DCM, anti-inflammatory interventions so far did not prove to alter the course of this disease. Here we show that myeloperoxidase (MPO), the principal heme peroxidase expressed by polymorphonuclear neutrophils (PMN) and monocytes, critically contributes to HFrEF in DCM.Muscle LIM protein (MLP) deficient mice, which spontaneously develop DCM, display increased circulating PMN counts and augmented levels of vessel-immobilized MPO. Genetic ablation and pharmacological inhibition of MPO resulted in enhanced nitric oxide (NO) bioavailability of systemic conductance and resistance vessels, and subsequently restoration of systolic left ventricular (LV) function, whereas infusion of MPO worsened systolic LV function. When patients diagnosed for DCM were treated with an orally available MPO inhibitor, systolic LV function increased, natriuretic peptides declined, and functional status improved.Impairment of endothelial NO bioavailability by release of leukocyte-derived MPO evolves as a disease-aggravating mechanism in DCM. MPO inhibition profoundly improved ventricular function by lowering systemic vascular resistance and thus holds promise as a novel and complementary treatment strategy for patients with DCM.

Branched-chain amino acid (BCAA) metabolism is a central hub for energy production and regulation of numerous physiological processes. Controversially, both increased and decreased levels of BCAAs are associated with longevity. Using genetics and multi-omics analyses in Caenorhabditis elegans, we identified adaptive regulation of the ubiquitin-proteasome system (UPS) in response to defective BCAA catabolic reactions after the initial transamination step. Worms with impaired BCAA metabolism show a slower turnover of a GFP-based proteasome substrate, which is suppressed by loss-of-function of the first BCAA catabolic enzyme, the branched-chain aminotransferase BCAT-1. The exogenous supply of BCAA-derived carboxylic acids, which are known to accumulate in the body fluid of patients with BCAA metabolic disorders, is sufficient to regulate the UPS. The link between BCAA intermediates and UPS function presented here sheds light on the unexplained role of BCAAs in the aging process and opens future possibilities for therapeutic interventions.

To ensure correct function, mitochondria have developed several mechanisms of protein quality control (QC). Protein homeostasis highly relies on chaperones and proteases to maintain proper folding and remove damaged proteins that might otherwise form cell-toxic aggregates. Besides quality control, mitochondrial proteases modulate and regulate many essential functions, such as trafficking, processing and activation of mitochondrial proteins, mitochondrial dynamics, mitophagy and apoptosis. Therefore, the impaired function of mitochondrial proteases is associated with various pathological conditions, including cancer, metabolic syndromes and neurodegenerative disorders. This review recapitulates and discusses the emerging roles of two major proteases of the mitochondrial matrix, LON and ClpXP. Although commonly acknowledge for their protein quality control role, recent advances have uncovered several highly regulated processes controlled by the LON and ClpXP connected to mitochondrial gene expression and respiratory chain function maintenance. Furthermore, both proteases have been lately recognized as potent targets for anticancer therapies, and we summarize those findings.

The mitochondrial intermembrane space protein AIFM1 has been reported to mediate the import of MIA40/CHCHD4, which forms the import receptor in the mitochondrial disulfide relay. Here, we demonstrate that AIFM1 and MIA40/CHCHD4 cooperate beyond this MIA40/CHCHD4 import. We show that AIFM1 and MIA40/CHCHD4 form a stable long-lived complex in vitro, in different cell lines, and in tissues. In HEK293 cells lacking AIFM1, levels of MIA40 are unchanged, but the protein is present in the monomeric form. Monomeric MIA40 neither efficiently interacts with nor mediates the import of specific substrates. The import defect is especially severe for NDUFS5, a subunit of complex I of the respiratory chain. As a consequence, NDUFS5 accumulates in the cytosol and undergoes rapid proteasomal degradation. Lack of mitochondrial NDUFS5 in turn results in stalling of complex I assembly. Collectively, we demonstrate that AIFM1 serves two overlapping functions: importing MIA40/CHCHD4 and constituting an integral part of the disulfide relay that ensures efficient interaction of MIA40/CHCHD4 with specific substrates.

Gut homeostasis is maintained by the close interaction between commensal intestinal microbiota and the host, affecting the most complex physiological processes, such as aging. Some commensal bacteria with the potential to promote healthy aging arise as attractive candidates for the development of pro-longevity probiotics. Here, we showed that heat-inactivated human commensal Lactobacillus fermentum BGHV110 (BGHV110) extends the lifespan of Caenorhabditis elegans and improves age-related physiological features, including locomotor function and lipid metabolism. Mechanistically, we found that BGHV110 promotes HLH-30/TFEB-dependent autophagy to delay aging, as longevity assurance was completely abolished in the mutant lacking HLH-30, a major autophagy regulator in C. elegans. Moreover, we observed that BGHV110 partially decreased the content of lipid droplets in an HLH-30-dependent manner and, at the same time, slightly increased mitochondrial activity. In summary, this study demonstrates that specific factors from commensal bacteria can be used to exploit HLH-30/TFEB-mediated autophagy in order to promote longevity and fitness of the host.

International audience; The mitochondrial integrated stress response (mitoISR) has emerged as a major adaptive pathway to respiratory chain deficiency, but both the tissue specificity of its regulation, and how mitoISR adapts to different levels of mitochondrial dysfunction are largely unknown. Here, we report that diverse levels of mitochondrial cardiomyopathy activate mitoISR, including high production of FGF21, a cytokine with both paracrine and endocrine function, shown to be induced by respiratory chain dysfunction. Although being fully dispensable for the cell-autonomous and systemic responses to severe mitochondrial cardiomyopathy, in the conditions of mild-to-moderate cardiac OXPHOS dysfunction, FGF21 regulates a portion of mitoISR. In the absence of FGF21, a large part of the metabolic adaptation to mitochondrial dysfunction (one-carbon metabolism, transsulfuration, and serine and proline biosynthesis) is strongly blunted, independent of the primary mitoISR activator ATF4. Collectively, our work highlights the complexity of mitochondrial stress responses by revealing the importance of the tissue specificity and dose dependency of mitoISR.

Cockayne syndrome (CS) is a congenital syndrome characterized by growth and mental retardation, and premature ageing. The complexity of CS and mammalian models warrants simpler metazoan models that display CS-like phenotypes that could be studied in the context of a live organism. Here, we provide a characterization of neuronal and mitochondrial aberrations caused by a mutation in the csb-1 gene in Caenorhabditis elegans. We report a progressive neurodegeneration in adult animals that is enhanced upon UV-induced DNA damage. The csb-1 mutants show dysfunctional hyperfused mitochondria that degrade upon DNA damage, resulting in diminished respiratory activity. Our data support the role of endogenous DNA damage as a driving factor of CS-related neuropathology and underline the role of mitochondrial dysfunction in the disease.

We combined quantitative proteomics with N terminome profiling and substrate trapping AP-MS to identify new ClpXP substrates. Mitochondrial protein N termini isolated from mouse hearts revealed frequent aminopeptidase processing after MTS cleavage and many internal protease-generated neo-N termini. CLPP-deficient mice showed perturbed processing patterns with overall accumulation of protease-generated neo-N termini. New high-confidence candidate ClpXP substrates were identified by strong changes in N termini abundance or pull-downs with inactive CLPP and validated using immunoblotting and cycloheximide-chase experiments., Graphical Abstract Highlights • Mitochondrial heart N terminome shows aminopeptidase processing after MTS cleavage. • CLPP-deficiency alters protein processing patterns in mouse heart mitochondria. • Candidate substrates identified by N termini accumulation and interaction with inactive ClpXP. • UQCRC1, HSPA9 and OAT validated biochemically as high confidence ClpXP substrates., The mammalian mitochondrial proteome consists of more than 1100 annotated proteins and their proteostasis is regulated by only a few ATP-dependent protease complexes. Technical advances in protein mass spectrometry allowed for detailed description of the mitoproteome from different species and tissues and their changes under specific conditions. However, protease-substrate relations within mitochondria are still poorly understood. Here, we combined Terminal Amine Isotope Labeling of Substrates (TAILS) N termini profiling of heart mitochondria proteomes isolated from wild type and Clpp−/− mice with a classical substrate-trapping screen using FLAG-tagged proteolytically active and inactive CLPP variants to identify new ClpXP substrates in mammalian mitochondria. Using TAILS, we identified N termini of more than 200 mitochondrial proteins. Expected N termini confirmed sequence determinants for mitochondrial targeting signal (MTS) cleavage and subsequent N-terminal processing after import, but the majority were protease-generated neo-N termini mapping to positions within the proteins. Quantitative comparison revealed widespread changes in protein processing patterns, including both strong increases or decreases in the abundance of specific neo-N termini, as well as an overall increase in the abundance of protease-generated neo-N termini in CLPP-deficient mitochondria that indicated altered mitochondrial proteostasis. Based on the combination of altered processing patterns, protein accumulation and stabilization in CLPP-deficient mice and interaction with CLPP, we identified OAT, HSPA9 and POLDIP2 and as novel bona fide ClpXP substrates. Finally, we propose that ClpXP participates in the cooperative degradation of UQCRC1. Together, our data provide the first landscape of the heart mitochondria N terminome and give further insights into regulatory and assisted proteolysis mediated by ClpXP.

Proliferating cells undergo metabolic changes in synchrony with cell cycle progression and cell division. Mitochondria provide fuel, metabolites, and ATP during different phases of the cell cycle, however it is not completely understood how mitochondrial function and the cell cycle are coordinated. CLUH is a post-transcriptional regulator of mRNAs encoding mitochondrial proteins involved in oxidative phosphorylation and several metabolic pathways. Here, we show a role of CLUH in regulating the expression of astrin, which is involved in metaphase to anaphase progression, centrosome integrity, and mTORC1 inhibition. We find that CLUH binds both the SPAG5 mRNA and its product astrin, and controls the synthesis and the stability of the full-length astrin-1 isoform. We show that CLUH interacts with astrin-1 specifically during interphase. Astrin-depleted cells show mTORC1 hyperactivation and enhanced anabolism. On the other hand, cells lacking CLUH show decreased astrin levels and increased mTORC1 signaling, but cannot sustain anaplerotic and anabolic pathways. In absence of CLUH, cells fail to grow during G1, and progress faster through the cell cycle, indicating dysregulated matching of growth, metabolism and cell cycling. Our data reveal a role of CLUH in coupling growth signaling pathways and mitochondrial metabolism with cell cycle progression.

Proliferating cells undergo metabolic changes in synchrony with cell cycle progression and cell division. Mitochondria provide fuel, metabolites, and ATP during different phases of the cell cycle, however it is not completely understood how mitochondrial function and the cell cycle are coordinated. CLUH (clustered mitochondria homolog) is a post-transcriptional regulator of mRNAs encoding mitochondrial proteins involved in oxidative phosphorylation and several metabolic pathways. Here, we show a role of CLUH in regulating the expression of astrin, which is involved in metaphase to anaphase progression, centrosome integrity, and mTORC1 inhibition. We find that CLUH binds both the SPAG5 mRNA and its product astrin, and controls the synthesis and the stability of the full-length astrin-1 isoform. We show that CLUH interacts with astrin-1 specifically during interphase. Astrin-depleted cells show mTORC1 hyperactivation and enhanced anabolism. On the other hand, cells lacking CLUH show decreased astrin levels and increased mTORC1 signaling, but cannot sustain anaplerotic and anabolic pathways. In absence of CLUH, cells fail to grow during G1, and progress faster through the cell cycle, indicating dysregulated matching of growth, metabolism, and cell cycling. Our data reveal a role of CLUH in coupling growth signaling pathways and mitochondrial metabolism with cell cycle progression.

Skeletal muscle subsarcolemmal mitochondria (SSM) and intermyofibrillar mitochondria subpopulations have distinct metabolic activity and sensitivity, though the mechanisms that localize SSM to peripheral areas of muscle fibers are poorly understood. A protein interaction study and complexome profiling identifies PERM1 interacts with the MICOS-MIB complex. Ablation of Perm1 in mice reduces muscle force, decreases mitochondrial membrane potential and complex I activity, and reduces the numbers of SSM in skeletal muscle. We demonstrate PERM1 interacts with the intracellular adaptor protein ankyrin B (ANKB) that connects the cytoskeleton to the plasma membrane. Moreover, we identify a C-terminal transmembrane helix that anchors PERM1 into the outer mitochondrial membrane. We conclude PERM1 functions in the MICOS-MIB complex and acts as an adapter to connect the mitochondria with the sarcolemma via ANKB., Mitochondria in skeletal muscle have distinct localization and properties through unclear mechanisms. Here, the authors use complexome profiling and immunoprecipitations to identify PERM1 as a MICOS-MIB complex interactor that also binds ankyrin B, suggesting PERM1 directs the mitochondria to the membrane.

Germline proteostasis coordinates mitochondrial networks and aggregation of disease-related proteins across the organism., Protein aggregation causes intracellular changes in neurons, which elicit signals to modulate proteostasis in the periphery. Beyond the nervous system, a fundamental question is whether other organs also communicate their proteostasis status to distal tissues. Here, we examine whether proteostasis of the germ line influences somatic tissues. To this end, we induce aggregation of germline-specific PGL-1 protein in germline stem cells of Caenorhabditis elegans. Besides altering the intracellular mitochondrial network of germline cells, PGL-1 aggregation also reduces the mitochondrial content of somatic tissues through long-range Wnt signaling pathway. This process induces the unfolded protein response of the mitochondria in the soma, promoting somatic mitochondrial fragmentation and aggregation of proteins linked with neurodegenerative diseases such as Huntington’s and amyotrophic lateral sclerosis. Thus, the proteostasis status of germline stem cells coordinates mitochondrial networks and protein aggregation through the organism.

Most manipulations that extend lifespan also increase resistance to various stress factors and environmental cues in a range of animals from yeast to mammals. However, the underlying molecular mechanisms regulating stress resistance during aging are still largely unknown. Here we identify Krüppel-like factor 1 (KLF-1) as a mediator of a cytoprotective response that dictates longevity induced by reduced mitochondrial function. A redox-regulated KLF-1 activation and transfer to the nucleus coincides with the peak of somatic mitochondrial biogenesis that occurs around a transition from larval stage L3 to D1. We further show that KLF-1 activates genes involved in the xenobiotic detoxification programme and identified cytochrome P450 oxidases, the KLF-1 main effectors, as longevity-assurance factors of mitochondrial mutants. Collectively, these findings underline the importance of the xenobiotic detoxification in the mitohormetic, longevity assurance pathway and identify KLF-1 as a central factor in orchestrating this response., Cytochrome P450 oxidases (CYPs) are enzymes that participate in the xenobiotic detoxification and their expression is enhanced in long-lived model organisms. Here the authors show that KLF-1 promotes cyp expression and ensures lifespan extension in C. elegans mitomutants by activating mitohormesis.

Mitochondria are essential organelles found in every eukaryotic cell, required to convert food into usable energy. Therefore, it is not surprising that mutations in either mtDNA or nuclear DNA-encoded genes of mitochondrial proteins cause diseases affecting the oxidative phosphorylation system, which are heterogeneous from a clinical, genetic, biochemical and molecular perspective and can affect patients at any age. Despite all this, it is surprising that our understanding of the mechanisms governing mitochondrial gene expression and its associated pathologies remain superficial and therapeutic interventions largely unexplored. We recently showed that loss of the mitochondrial matrix protease caseinolytic protease proteolytic subunit (CLPP) ameliorates phenotypes in cells characterized by defects in oxidative phosphorylation maintenance. Here, we build upon this finding by showing that CLPP depletion is indeed beneficial in vivo for various types of neuronal populations, including Purkinje cells in the cerebellum and cortical and hippocampal neurons in the forebrain, as it strongly improves distinct phenotypes of mitochondria encephalopathy, driven by the deficiency of the mitochondrial aspartyl tRNA synthase DARS2. In the absence of CLPP, neurodegeneration of DARS2-deficient neurons is delayed as they present milder oxidative phosphorylation dysfunction. This in turn leads to a decreased neuroinflammatory response and significantly improved motor functions in both double-deficient models (Purkinje cell-specific or forebrain neuron-specific Dars2/Clpp double knockout mice). We propose that diminished turnover of respiratory complex I caused by the loss of CLPP is behind the improved phenotype in Dars2/Clpp double knockout animals, even though this intervention might not restore respiratory complex I activity but rather improve mitochondrial cristae morphology or help maintain the NAD+/NADH ratio inside mitochondria. These results also open the possibility of targeting CLPP activity in many other mitochondrial encephalopathies characterized by respiratory complex I instability.

Mutations in POLG, encoding POLγA, the catalytic subunit of the mitochondrial DNA polymerase, cause a spectrum of disorders characterized by mtDNA instability. However, the molecular pathogenesis of POLG-related diseases is poorly understood and efficient treatments are missing. Here, we generate the PolgA449T/A449T mouse model, which reproduces the A467T change, the most common human recessive mutation of POLG. We show that the mouse A449T mutation impairs DNA binding and mtDNA synthesis activities of POLγ, leading to a stalling phenotype. Most importantly, the A449T mutation also strongly impairs interactions with POLγB, the accessory subunit of the POLγ holoenzyme. This allows the free POLγA to become a substrate for LONP1 protease degradation, leading to dramatically reduced levels of POLγA in A449T mouse tissues. Therefore, in addition to its role as a processivity factor, POLγB acts to stabilize POLγA and to prevent LONP1-dependent degradation. Notably, we validated this mechanism for other disease-associated mutations affecting the interaction between the two POLγ subunits. We suggest that targeting POLγA turnover can be exploited as a target for the development of future therapies., Graphical Abstract Graphical AbstractThe A449T mutation in mouse POLγA, corresponding to the common A467T in patients, impairs the interaction between POLγA and B subunits, making POLγA amenable to degradation by LONP1 protease.

International audience; Hereditary spastic paraplegias are heterogeneous neurodegenerative disorders. Understanding of their pathogenicmechanisms remains sparse, and therapeutic options are lacking. We characterized a mouse model lacking the Cyp2u1 gene, lossof which is known to be involved in a complex form of these diseases in humans. We showed that this model partially recapitulated the clinical and biochemical phenotypes of patients. Using electron microscopy, lipidomic, and proteomicstudies, we identified vitamin B2 as a substrate of the CYP2U1 enzyme, as well as coenzyme Q, neopterin, and IFN-α levels asputative biomarkers in mice and fluids obtained from the largest series of CYP2U1-mutated patients reported so far. We alsoconfirmed brain calcifications as a potential biomarker in patients. Our results suggest that CYP2U1 deficiency disruptsmitochondrial function and impacts proper neurodevelopment, which could be prevented by folate supplementation in ourmouse model, followed by a neurodegenerative process altering multiple neuronal and extraneuronal tissues.

Wound healing is a coordinated process that initially relies on pro-inflammatory macrophages, followed by a pro-resolution function of these cells. Changes in cellular metabolism likely dictate these distinct activities, but the nature of these changes has been unclear. Here, we profiled early- versus late-stage skin wound macrophages in mice at both the transcriptional and functional levels. We found that glycolytic metabolism in the early phase is not sufficient to ensure productive repair. Instead, by combining conditional disruption of the electron transport chain with deletion of tgcqmitochondrial aspartyl-tRNA synthetase, followed by single-cell sequencing analysis, we found that a subpopulation of early-stage wound macrophages are marked by mitochondrial ROS (mtROS) production and HIF1alpha stabilization, which ultimately drives a pro-angiogenic program essential for timely healing. In contrast, late-phase, pro-resolving wound macrophages are marked by IL-4Ralpha-mediated mitochondrial respiration and mitohormesis. Collectively, we identify changes in mitochondrial metabolism as a critical control mechanism for macrophage effector functions during wound healing.

Mitochondrial proteases constitute a fundamental part of the organellar protein quality control system to ensure the timely removal of damaged or obsolete proteins. The analysis of proteases is often limited to the identification of bona fide substrates that are degraded in the presence and become more abundant in the absence of the respective protease. However, proteases in numerous organisms from bacteria to humans can process specific substrates to release shortened proteins with potentially altered activities. Here, we describe an adaptation of the substrate-trapping approach, as well as the N-terminal profiling protocol Terminal Amine Isotope Labeling of Substrates (TAILS) for the identification of bona fide substrates and mitochondrial proteins that undergo complete or partial proteolysis.

Mitochondrial proteases constitute a fundamental part of the organellar protein quality control system to ensure the timely removal of damaged or obsolete proteins. The analysis of proteases is often limited to the identification of bona fide substrates that are degraded in the presence and become more abundant in the absence of the respective protease. However, proteases in numerous organisms from bacteria to humans can process specific substrates to release shortened proteins with potentially altered activities. Here, we describe an adaptation of the substrate-trapping approach, as well as the N-terminal profiling protocol Terminal Amine Isotope Labeling of Substrates (TAILS) for the identification of bona fide substrates and mitochondrial proteins that undergo complete or partial proteolysis.

Upon mitochondrial dysfunction, CHOP acts as a rheostat that attenuates prolonged stress and delays the onset of cardiomyopathy., In response to disturbed mitochondrial gene expression and protein synthesis, an adaptive transcriptional response sharing a signature of the integrated stress response (ISR) is activated. We report an intricate interplay between three transcription factors regulating the mitochondrial stress response: CHOP, C/EBPβ, and ATF4. We show that CHOP acts as a rheostat that attenuates prolonged ISR, prevents unfavorable metabolic alterations, and postpones the onset of mitochondrial cardiomyopathy. Upon mitochondrial dysfunction, CHOP interaction with C/EBPβ is needed to adjust ATF4 levels, thus preventing overactivation of the ATF4-regulated transcriptional program. Failure of this interaction switches ISR from an acute to a chronic state, leading to early respiratory chain deficiency, energy crisis, and premature death. Therefore, contrary to its previously proposed role as a transcriptional activator of mitochondrial unfolded protein response, our results highlight a role of CHOP in the fine-tuning of mitochondrial ISR in mammals.

Mutations in POLG, encoding POLγA, the catalytic subunit of the mitochondrial DNA polymerase, cause a spectrum of disorders characterized by mtDNA instability. However, the molecular pathogenesis of POLG-related diseases is poorly understood and efficient treatments are missing. Here, we generated a POLGA449T/A449T mouse model, which reproduces the most common human recessive mutation of POLG, encoding the A467T change, and dissected the mechanisms underlying pathogenicity. We show that the A449T mutation impairs DNA binding and mtDNA synthesis activities of POLγ in vivo and in vitro. Interestingly, the A467T mutation also strongly impairs interactions with POLγB, the homodimeric accessory subunit of holo-POLγ. This allows the free POLγA to become a substrate for LONP1 protease degradation, leading to dramatically reduced levels of POLγA, which in turn exacerbates the molecular phenotypes of PolgA449T/A449T mice. Importantly, we validated this mechanism for other mutations affecting the interaction between the two POLγ subunits. We suggest that LONP1 dependent degradation of POLγA can be exploited as a target for the development of future therapies.

Heart development relies on PTMs that control cardiomyocyte proliferation, differentiation and cardiac morphogenesis. We generated a map of phosphorylation sites during the early stages of cardiac postnatal development in mice; we quantified over 10,000 phosphorylation sites and 5000 proteins that were assigned to different pathways. Analysis of mitochondrial proteins led to the identification of PGC-1- and ERR-induced regulator in muscle 1 (PERM1), which is specifically expressed in skeletal muscle and heart tissue and associates with the outer mitochondrial membrane. We demonstrate PERM1 is subject to rapid changes mediated by the UPS through phosphorylation of its PEST motif by casein kinase 2. Ablation of Perm1 in mice results in reduced protein expression of lipin-1 accompanied by accumulation of specific phospholipid species. Isolation of Perm1-deficient mitochondria revealed significant downregulation of mitochondrial transport proteins for amino acids and carnitines, including SLC25A12/13/29/34 and CPT2. Consistently, we observed altered levels of various lipid species, amino acids, and acylcarnitines in Perm1(-/-) mitochondria. We conclude that the outer mitochondrial membrane protein PERM1 regulates homeostasis of lipid and amino acid metabolites in mitochondria.

Leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation disorder (LBSL) arises from mutations in mitochondrial aspartyl-tRNA synthetase (DARS2) gene. The disease has a childhood or juvenile-onset and is clinically characterized by cerebellar ataxia, cognitive decline and distinct morphological abnormalities upon magnetic resonance imaging. We previously demonstrated that neurons and not adult myelin-producing cells are specifically sensitive to DARS2 loss, hence likely the primary culprit in LBSL disorder. We used conditional Purkinje cell (PCs)-specific Dars2 deletion to elucidate further the cell-type-specific contribution of this class of neurons to the cerebellar impairment observed in LBSL. We show that DARS2 depletion causes a severe mitochondrial dysfunction concomitant with a massive loss of PCs by the age of 15 weeks, thereby rapidly deteriorating motor skills. Our findings conclusively show that DARS2 is indispensable for PC survival and highlights the central role of neuroinflammation in DARS2-related PC degeneration.

Cellular proteostasis describes all processes, which are involved in synthesis, folding and degradation of proteins. The balance between protein translation and degradation is strictly regulated in the cell to ensure its viability. For this purpose, the activity of the protein synthesis and degradation machinery can be adapted according to cellular needs. Disturbance of proteostasis leads for example to accumulation of misfolded or damaged proteins in the cell and is linked to aging and conformational diseases (proteinopathies) such as neurodegenerative, cardiovascular and pulmonary diseases. The ubiquitin-proteasome system plays a central role for the balanced protein turnover in the cell as it is responsible for the degradation of up to 80 % of all cellular proteins. The proteasome is a large protein complex, with catalytically active cleavage sites located within the 20S core proteasome. Ubiquitin-dependent degradation of folded proteins is mainly performed by the 26S proteasome, which is formed by the assembly of 20S core particle and 19S regulatory particles. Assembly and activity of the 26S proteasome are fine-tuned according to cellular needs such as growth and differentiation. Regulation of protein synthesis via the mammalian target of rapamycin (mTOR) has for example direct effects on 26S proteasome function. As 26S proteasome function is strictly dependent on energy in form of ATP, mitochondria – the powerhouses of the cell – are also involved in the regulation of protein degradation by the proteasome. Additionally, reactive oxygen species (ROS), which are mainly produced by dysfunctional mitochondria, negatively influence 26S proteasome assembly and activity. However, mitochondria are not only the main source of cellular ATP and ROS but also provide important metabolites and precursors generated by the tricarboxylic acid cycle (TCA), which are involved in central cellular processes such as proliferation. While a variety of regulatory mechanisms for protein translation and 26S proteasome mediated protein degradation have already been described, a metabolic regulation of cellular proteostasis mediated by mitochondria has not been demonstrated so far. Therefore, the main focus of the present study was to dissect a possible connection between mitochondrial metabolism and cellular proteostasis. For that three different models for mitochondrial respiratory chain dysfunction were used: mouse embryonic fibroblasts (MEFs) derived from the so-called mtDNA mutator mouse model, primary human skin fibroblasts with a mutation in the ND5 subunit of respiratory chain complex I and primary human skin and lung fibroblasts treated with the complex I inhibitor and anti-diabetic drug metformin. The different models are all characterized by respiratory chain complex I deficiency in the absence of increased ROS production. Cells maintained cellular viability and did not show signs of severe stress despite respiratory chain dysfunction. Mitochondrial complex I deficiency in mutator MEFs caused metabolic reprogramming of the TCA cycle resulting in diminished aspartate biosynthesis. Reduced aspartate levels caused downregulated proteostasis as both protein translation and 26S proteasome assembly and activity was decreased in respiration deficient cells but could be rescued by supplementation of aspartate. Furthermore, aspartate supplementation induced mTORC1 mediated protein synthesis and mTORC1-dependent transcriptional activation of defined proteasome assembly factors, which were involved in activation of 26S proteasome assembly and activity in cells with complex I deficiency. Similar data were obtained in ND5 mutant skin fibroblasts and upon metformin treatment. In contrast to diminished proteasome function, chronic respiratory chain impairment in mutator MEFs led to strongly induced immunoproteasome expression and activity. Upregulation of the immunoproteasome was accompanied by increased MHC class I antigen presentation during chronic mitochondrial dysfunction representing a so far unknown stress response which may probably serve to alert the immune system. This finding requires further analysis. These results thus uncover a novel concept of how mitochondrial metabolism adaptively adjusts protein synthesis and degradation by the proteasome to the metabolic condition of the cell. These data extend the knowledge about proteasomal regulation in the cell and have therapeutic implications for diseases and drug-targeted mitochondrial reprogramming.

Protein homeostasis involving protein synthesis and degradation by the proteasome system is intricately balanced in the cell and fine-tuned according to cellular needs and upon stress. Metabolic regulation of protein synthesis and proteasome activity by respiratory chain dysfunction has not been investigated so far. In our study, we used mouse embryonic fibroblasts (MEFs) isolated from the mitochondrial (mt)DNA mutator mouse model that is characterized by genetic respiratory chain dysfunction. Cellular proteostasis including protein synthesis and proteasome activity was significantly decreased in mutator MEFs. In particular, mTOR signaling was downregulated and the formation of 26S proteasome complexes was impaired. Electron microscopy and proteomic analysis of isolated mitochondria revealed massive respiratory chain complex I deficiency in mutator mitochondria resulting in the accumulation of NADH and altered Krebs cycle metabolism. Targeted metabolomics of mutator MEFs identified impaired biosynthesis of aspartate as a consequence of disturbed Krebs cycle function. Notably, supplementation with aspartate restored both mTOR mediated protein translation and proteasome activity in mutator cells. Furthermore, we delineated impairment of respiratory chain complex I activity as the driving factor of this metabolic regulation in primary human lung fibroblasts treated with the complex I inhibitor Metformin. Metformin downregulated both protein synthesis and proteasome activity, a phenotype that was rescued by aspartate supplementation. Our data thus unravel a previously unrecognized adaptation of proteostasis to metabolic alterations of the cell, which might be of therapeutic relevance for the treatment of lung diseases.

Mitochondrial dysfunction is critically involved in Parkinson's disease, characterized by loss of dopaminergic neurons (DaNs) in the substantia nigra (SNc), whereas DaNs in the neighboring ventral tegmental area (VTA) are much less affected. In contrast to VTA, SNc DaNs engage calcium channels to generate action potentials, which lead to oxidant stress by yet unknown pathways. To determine the molecular mechanisms linking calcium load with selective cell death in the presence of mitochondrial deficiency, we analyzed the mitochondrial redox state and the mitochondrial membrane potential in mice of both sexes with genetically induced, severe mitochondrial dysfunction in DaNs (MitoPark mice), at the same time expressing a redox-sensitive GFP targeted to the mitochondrial matrix. Despite mitochondrial insufficiency in all DaNs, exclusively SNc neurons showed an oxidized redox-system, i.e., a low reduced/oxidized glutathione (GSH-GSSG) ratio. This was mimicked by cyanide, but not by rotenone or antimycin A, making the involvement of reactive oxygen species rather unlikely. Surprisingly, a high mitochondrial inner membrane potential was maintained in MitoPark SNc DaNs. Antagonizing calcium influx into the cell and into mitochondria, respectively, rescued the disturbed redox ratio and induced further hyperpolarization of the inner mitochondrial membrane. Our data therefore show that the constant calcium load in SNc DaNs is counterbalanced by a high mitochondrial inner membrane potential, even under conditions of severe mitochondrial dysfunction, but triggers a detrimental imbalance in the mitochondrial redox system, which will lead to neuron death. Our findings thus reveal a new mechanism, redox imbalance, which underlies the differential vulnerability of DaNs to mitochondrial defects.SIGNIFICANCE STATEMENTParkinson's disease is characterized by the preferential degeneration of dopaminergic neurons (DaNs) of the substantia nigra pars compacta (SNc), resulting in the characteristic hypokinesia in patients. Ubiquitous pathological triggers cannot be responsible for the selective neuron loss. Here we show that mitochondrial impairment together with elevated calcium burden destabilize the mitochondrial antioxidant defense only in SNc DaNs, and thus promote the increased vulnerability of this neuron population.

HIV‐1 persists in a latent form during antiretroviral therapy, mainly in CD4+ T cells, thus hampering efforts for a cure. HIV‐1 infection is accompanied by metabolic alterations, such as oxidative stress, but the effect of cellular antioxidant responses on viral replication and latency is unknown. Here, we show that cells survive retroviral replication, both in vitro and in vivo in SIVmac‐infected macaques, by upregulating antioxidant pathways and the intertwined iron import pathway. These changes are associated with remodeling of promyelocytic leukemia protein nuclear bodies (PML NBs), an important constituent of nuclear architecture and a marker of HIV‐1 latency. We found that PML NBs are hyper‐SUMOylated and that PML protein is degraded via the ubiquitin–proteasome pathway in productively infected cells, before latency establishment and after reactivation. Conversely, normal numbers of PML NBs were restored upon transition to latency or by decreasing oxidative stress or iron content. Our results highlight antioxidant and iron import pathways as determinants of HIV‐1 latency and support their pharmacologic inhibition as tools to regulate PML stability and impair latency establishment., Activation of cellular oxidative stress responses and iron import upon HIV‐1 infection triggers latency establishment via remodeling of PML nuclear bodies.

Corrupted integrity of the mitochondrial genome (mtDNA) accompanied by a gradual decline of mitochondrial function is a hallmark of aging. The majority of people carry the detectable levels of mtDNA mutations in somatic tissues, and the burden of those mutations builds up considerably with age. Hence, the causative nature of the relationship between the mtDNA mutations and aging emerges, awaiting further rigorous testing. Although over several decades numerous studies have been conducted and many experimental models developed, the origin and a precise time of occurrence of mtDNA mutations in the course of aging remain controversial. Here, we review the possible sources of somatic mtDNA mutations in our cells, and the mechanism underlying their expansion during aging. We also discuss whether in light of currently available evidence the link between mtDNA mutations and aging is credible.