Distinct fission signatures predict mitochondrial degradation or biogenesis.

Mitochondrial fission is a highly regulated process that, when disrupted, can alter metabolism, proliferation and apoptosis1-3. Dysregulation has been linked to neurodegeneration3,4, cardiovascular disease3 and cancer5. Key components of the fission machinery include the endoplasmic reticulum6 and actin7, which initiate constriction before dynamin-related protein 1 (DRP1)8 binds to the outer mitochondrial membrane via adaptor proteins9-11, to drive scission12. In the mitochondrial life cycle, fission enables both biogenesis of new mitochondria and clearance of dysfunctional mitochondria through mitophagy1,13. Current models of fission regulation cannot explain how those dual fates are decided. However, uncovering fate determinants is challenging, as fission is unpredictable, and mitochondrial morphology is heterogeneous, with ultrastructural features that are below the diffraction limit. Here, we used live-cell structured illumination microscopy to capture mitochondrial dynamics. By analysing hundreds of fissions in African green monkey Cos-7 cells and mouse cardiomyocytes, we discovered two functionally and mechanistically distinct types of fission. Division at the periphery enables damaged material to be shed into smaller mitochondria destined for mitophagy, whereas division at the midzone leads to the proliferation of mitochondria. Both types are mediated by DRP1, but endoplasmic reticulum- and actin-mediated pre-constriction and the adaptor MFF govern only midzone fission. Peripheral fission is preceded by lysosomal contact and is regulated by the mitochondrial outer membrane protein FIS1. These distinct molecular mechanisms explain how cells independently regulate fission, leading to distinct mitochondrial fates.

MPC2 variants disrupt mitochondrial pyruvate metabolism and cause an early-onset mitochondriopathy.

Pyruvate is an essential metabolite produced by glycolysis in the cytosol and must be transported across the inner mitochondrial membrane into the mitochondrial matrix, where it is oxidized to fuel mitochondrial respiration. Pyruvate import is performed by the mitochondrial pyruvate carrier (MPC), a hetero-oligomeric complex composed by interdependent subunits MPC1 and MPC2. Pathogenic variants in the MPC1 gene disrupt mitochondrial pyruvate uptake and oxidation and cause autosomal-recessive early-onset neurological dysfunction in humans. The present work describes the first pathogenic variants in MPC2 associated with human disease in four patients from two unrelated families. In the first family, patients presented with antenatal developmental abnormalities and harboured a homozygous c.148T>C (p.Trp50Arg) variant. In the second family, patients that presented with infantile encephalopathy carried a missense c.2T>G (p.Met1?) variant disrupting the initiation codon. Patient-derived skin fibroblasts exhibit decreased pyruvate-driven oxygen consumption rates with normal activities of the pyruvate dehydrogenase complex and mitochondrial respiratory chain and no defects in mitochondrial content or morphology. Re-expression of wild-type MPC2 restored pyruvate-dependent respiration rates in patient-derived fibroblasts. The discovery of pathogenic variants in MPC2 therefore broadens the clinical and genetic landscape associated with inborn errors in pyruvate metabolism.

CHCHD10 and SLP2 control the stability of the PHB complex: a key factor for motor neuron viability.

CHCHD10 is an amyotrophic lateral sclerosis/frontotemporal dementia gene that encodes a mitochondrial protein whose precise function is unclear. Here we show that Coiled-Coil-Helix-Coiled-Coil-Helix Domain Containing protein 10 interacts with the Stomatin-Like Protein 2 and participates in the stability of the prohibitin complex in the inner mitochondrial membrane. By using patient fibroblasts and mouse models expressing the same CHCHD10 variant (p.Ser59Leu), we show that Stomatin-Like Protein 2 forms aggregates with prohibitins, found in vivo in the hippocampus and as aggresome-like inclusions in spinal motor neurons of Chchd10S59L/+ mice. Affected cells and tissues display instability of the prohibitin complex, which participates at least in part in the activation of the OMA1 cascade with OPA1 processing leading to mitochondrial fragmentation, abnormal mitochondrial cristae morphogenesis and neuronal death found in spinal cord and the hippocampus of Chchd10S59L/+ animals. Destabilization of the prohibitin complex leads to the instability of the mitochondrial contact site and cristae organizing the system complex, probably by the disruption of OPA1-mitofilin interaction. Thus, Stomatin-Like Protein 2/prohibitin aggregates and destabilization of the prohibitin complex are critical in the sequence of events leading to motor neuron death in CHCHD10S59L-related disease.

Mitochondrial dysfunction caused by novel ATAD3A mutations.

Mitochondrial respiratory chain integrity depends on a number of proteins encoded by nuclear and mitochondrial genomes. Mutations of such factors can result in isolated or combined respiratory chain deficits, some of which can induce abnormal morphology of the mitochondrial network or accumulation of intermediary metabolites. Consequently, affected patients are clinically heterogeneous, presenting with central nervous system, muscular, or neurodegenerative disorders. ATAD3A is a nuclear-encoded ATPase protein of the AAA+ family and has been localized to the inner mitochondrial membrane. Recently reported mutations or large deletions in the ATDA3A gene in patients have been shown to induce altered mitochondrial structure and function and abnormal cholesterol metabolism in a recessive or dominant manner. Here, we report two siblings presenting axonal sensory-motor neuropathy associated with neonatal cataract. Genetic analyses identified two novel mutations in ATAD3A; a point mutation and an intronic 15 bp deletion affecting splicing and leading to exon skipping. Biochemical analysis in patient cells and tissues showed abnormal function of the mitochondrial respiratory chain in muscle and abnormal mitochondrial cristae structure. These new cases underline the large spectrum of biochemical and clinical presentations of ATAD3A deficiency and the different modes of inheritance, making it an atypical mitochondrial disorder.

The membrane scaffold SLP2 anchors a proteolytic hub in mitochondria containing PARL and the i-AAA protease YME1L.

The SPFH (stomatin, prohibitin, flotillin, HflC/K) superfamily is composed of scaffold proteins that form ring-like structures and locally specify the protein-lipid composition in a variety of cellular membranes. Stomatin-like protein 2 (SLP2) is a member of this superfamily that localizes to the mitochondrial inner membrane (IM) where it acts as a membrane organizer. Here, we report that SLP2 anchors a large protease complex composed of the rhomboid protease PARL and the i-AAA protease YME1L, which we term the SPY complex (for SLP2-PARL-YME1L). Association with SLP2 in the SPY complex regulates PARL-mediated processing of PTEN-induced kinase PINK1 and the phosphatase PGAM5 in mitochondria. Moreover, SLP2 inhibits the stress-activated peptidase OMA1, which can bind to SLP2 and cleaves PGAM5 in depolarized mitochondria. SLP2 restricts OMA1-mediated processing of the dynamin-like GTPase OPA1 allowing stress-induced mitochondrial hyperfusion under starvation conditions. Together, our results reveal an important role of SLP2 membrane scaffolds for the spatial organization of IM proteases regulating mitochondrial dynamics, quality control, and cell survival.

SLIRP Regulates the Rate of Mitochondrial Protein Synthesis and Protects LRPPRC from Degradation.

We have studied the in vivo role of SLIRP in regulation of mitochondrial DNA (mtDNA) gene expression and show here that it stabilizes its interacting partner protein LRPPRC by protecting it from degradation. Although SLIRP is completely dependent on LRPPRC for its stability, reduced levels of LRPPRC persist in the absence of SLIRP in vivo. Surprisingly, Slirp knockout mice are apparently healthy and only display a minor weight loss, despite a 50-70% reduction in the steady-state levels of mtDNA-encoded mRNAs. In contrast to LRPPRC, SLIRP is dispensable for polyadenylation of mtDNA-encoded mRNAs. Instead, deep RNA sequencing (RNAseq) of mitochondrial ribosomal fractions and additional molecular analyses show that SLIRP is required for proper association of mRNAs to the mitochondrial ribosome and efficient translation. Our findings thus establish distinct functions for SLIRP and LRPPRC within the LRPPRC-SLIRP complex, with a novel role for SLIRP in mitochondrial translation. Very surprisingly, our results also demonstrate that mammalian mitochondria have a great excess of transcripts under basal physiological conditions in vivo.

Is mitochondrial morphology important for cellular physiology?

Mitochondria are double membrane-bound organelles the network morphology of which in cells is shaped by opposing events of fusion and fission executed by dynamin-like GTPases. Mutations in these genes can perturb the form and functions of mitochondria in cell and animal models of mitochondrial diseases. An expanding array of chemical, mechanical, and genetic stressors can converge on mitochondrial-shaping proteins and disrupt mitochondrial morphology. In recent years, studies aimed at disentangling the multiple roles of mitochondrial-shaping proteins beyond fission or fusion have provided insights into the homeostatic relevance of mitochondrial morphology. Here, I review the pleiotropy of mitochondrial fusion and fission proteins with the aim of understanding whether mitochondrial morphology is important for cell and tissue physiology.

Dynamic label-free analysis of SARS-CoV-2 infection reveals virus-induced subcellular remodeling.

Assessing the impact of SARS-CoV-2 on organelle dynamics allows a better understanding of the mechanisms of viral replication. We combine label-free holotomographic microscopy with Artificial Intelligence to visualize and quantify the subcellular changes triggered by SARS-CoV-2 infection. We study the dynamics of shape, position and dry mass of nucleoli, nuclei, lipid droplets and mitochondria within hundreds of single cells from early infection to syncytia formation and death. SARS-CoV-2 infection enlarges nucleoli, perturbs lipid droplets, changes mitochondrial shape and dry mass, and separates lipid droplets from mitochondria. We then used Bayesian network modeling on organelle dry mass states to define organelle cross-regulation networks and report modifications of organelle cross-regulation that are triggered by infection and syncytia formation. Our work highlights the subcellular remodeling induced by SARS-CoV-2 infection and provides an Artificial Intelligence-enhanced, label-free methodology to study in real-time the dynamics of cell populations and their content.

Gimap3 regulates tissue-specific mitochondrial DNA segregation.

Mitochondrial DNA (mtDNA) sequence variants segregate in mutation and tissue-specific manners, but the mechanisms remain unknown. The segregation pattern of pathogenic mtDNA mutations is a major determinant of the onset and severity of disease. Using a heteroplasmic mouse model, we demonstrate that Gimap3, an outer mitochondrial membrane GTPase, is a critical regulator of this process in leukocytes. Gimap3 is important for T cell development and survival, suggesting that leukocyte survival may be a key factor in the genetic regulation of mtDNA sequence variants and in modulating human mitochondrial diseases.

Mitochondrial dynamics and metabolic regulation control T cell fate in the thymus.

Several studies demonstrated that mitochondrial dynamics and metabolic pathways control T cell fate in the periphery. However, little is known about their implication in thymocyte development. Our results showed that thymic progenitors (CD3-CD4-CD8- triple negative, TN), in active division, have essentially a fused mitochondrial morphology and rely on high glycolysis and mitochondrial oxidative phosphorylation (OXPHOS). As TN cells differentiate to double positive (DP, CD4+CD8+) and single positive (SP, CD4+ and CD8+) stages, they became more quiescent, their mitochondria fragment and they downregulate glycolysis and OXPHOS. Accordingly, in vitro inhibition of the mitochondrial fission during progenitor differentiation on OP9-DL4 stroma, affected the TN to DP thymocyte transition by enhancing the percentage of TN and reducing that of DP, leading to a decrease in the total number of thymic cells including SP T cells. We demonstrated that the stage 3 triple negative pre-T (TN3) and the stage 4 triple negative pre-T (TN4) have different metabolic and functional behaviors. While their mitochondrial morphologies are both essentially fused, the LC-MS based analysis of their metabolome showed that they are distinct: TN3 rely more on OXPHOS whereas TN4 are more glycolytic. In line with this, TN4 display an increased Hexokinase II expression in comparison to TN3, associated with high proliferation and glycolysis. The in vivo inhibition of glycolysis using 2-deoxyglucose (2-DG) and the absence of IL-7 signaling, led to a decline in glucose metabolism and mitochondrial membrane potential. In addition, the glucose/IL-7R connection affects the TN3 to TN4 transition (also called ß-selection transition), by enhancing the percentage of TN3, leading to a decrease in the total number of thymocytes. Thus, we identified additional components, essential during ß-selection transition and playing a major role in thymic development.

Mtfp1 ablation enhances mitochondrial respiration and protects against hepatic steatosis.

Hepatic steatosis is the result of imbalanced nutrient delivery and metabolism in the liver and is the first hallmark of Metabolic dysfunction-associated steatotic liver disease (MASLD). MASLD is the most common chronic liver disease and involves the accumulation of excess lipids in hepatocytes, inflammation, and cancer. Mitochondria play central roles in liver metabolism yet the specific mitochondrial functions causally linked to MASLD remain unclear. Here, we identify Mitochondrial Fission Process 1 protein (MTFP1) as a key regulator of mitochondrial and metabolic activity in the liver. Deletion of Mtfp1 in hepatocytes is physiologically benign in mice yet leads to the upregulation of oxidative phosphorylation (OXPHOS) activity and mitochondrial respiration, independently of mitochondrial biogenesis. Consequently, liver-specific knockout mice are protected against high fat diet-induced steatosis and metabolic dysregulation. Additionally, Mtfp1 deletion inhibits mitochondrial permeability transition pore opening in hepatocytes, conferring protection against apoptotic liver damage in vivo and ex vivo. Our work uncovers additional functions of MTFP1 in the liver, positioning this gene as an unexpected regulator of OXPHOS and a therapeutic candidate for MASLD.

Mitochondrial Fission Process 1 controls inner membrane integrity and protects against heart failure.

Mitochondria are paramount to the metabolism and survival of cardiomyocytes. Here we show that Mitochondrial Fission Process 1 (MTFP1) is an inner mitochondrial membrane (IMM) protein that is dispensable for mitochondrial division yet essential for cardiac structure and function. Constitutive knockout of cardiomyocyte MTFP1 in mice resulted in a fatal, adult-onset dilated cardiomyopathy accompanied by extensive mitochondrial and cardiac remodeling during the transition to heart failure. Prior to the onset of disease, knockout cardiac mitochondria displayed specific IMM defects: futile proton leak dependent upon the adenine nucleotide translocase and an increased sensitivity to the opening of the mitochondrial permeability transition pore, with which MTFP1 physically and genetically interacts. Collectively, our data reveal new functions of MTFP1 in the control of bioenergetic efficiency and cell death sensitivity and define its importance in preventing pathogenic cardiac remodeling.

Mitochondrial Nucleic Acid as a Driver of Pathogenic Type I Interferon Induction in Mendelian Disease.

The immune response to viral infection involves the recognition of pathogen-derived nucleic acids by intracellular sensors, leading to type I interferon (IFN), and downstream IFN-stimulated gene, induction. Ineffective discrimination of self from non-self nucleic acid can lead to autoinflammation, a phenomenon implicated in an increasing number of disease states, and well highlighted by the group of rare genetic disorders referred to as the type I interferonopathies. To understand the pathogenesis of these monogenic disorders, and polyfactorial diseases associated with pathogenic IFN upregulation, such as systemic lupus erythematosus and dermatomyositis, it is important to define the self-derived nucleic acid species responsible for such abnormal IFN induction. Recently, attention has focused on mitochondria as a novel source of immunogenic self nucleic acid. Best appreciated for their function in oxidative phosphorylation, metabolism and apoptosis, mitochondria are double membrane-bound organelles that represent vestigial bacteria in the cytosol of eukaryotic cells, containing their own DNA and RNA enclosed within the inner mitochondrial membrane. There is increasing recognition that a loss of mitochondrial integrity and compartmentalization can allow the release of mitochondrial nucleic acid into the cytosol, leading to IFN induction. Here, we provide recent insights into the potential of mitochondrial-derived DNA and RNA to drive IFN production in Mendelian disease. Specifically, we summarize current understanding of how nucleic acids are detected as foreign when released into the cytosol, and then consider the findings implicating mitochondrial nucleic acid in type I interferonopathy disease states. Finally, we discuss the potential for IFN-driven pathology in primary mitochondrial disorders.

Quality control of the mitochondrion.

Mitochondria are essential organelles that execute and coordinate various metabolic processes in the cell. Mitochondrial dysfunction severely affects cell fitness and contributes to disease. Proper organellar function depends on the biogenesis and maintenance of mitochondria and its >1,000 proteins. As a result, the cell has evolved mechanisms to coordinate protein and organellar quality control, such as the turnover of proteins via mitochondria-associated degradation, the ubiquitin-proteasome system, and mitoproteases, as well as the elimination of mitochondria through mitophagy. Specific quality control mechanisms are engaged depending upon the nature and severity of mitochondrial dysfunction, which can also feed back to elicit transcriptional or proteomic remodeling by the cell. Here, we will discuss the current understanding of how these different quality control mechanisms are integrated and overlap to maintain protein and organellar quality and how they may be relevant for cellular and organismal health.

Mitochondrial respiration restricts Listeria monocytogenes infection by slowing down host cell receptor recycling.

Mutations in mitochondrial genes impairing energy production cause mitochondrial diseases (MDs), and clinical studies have shown that MD patients are prone to bacterial infections. However, the relationship between mitochondrial (dys)function and infection remains largely unexplored, especially in epithelial cells, the first barrier to many pathogens. Here, we generate an epithelial cell model for one of the most common mitochondrial diseases, Leigh syndrome, by deleting surfeit locus protein 1 (SURF1), an assembly factor for respiratory chain complex IV. We use this genetic model and a complementary, nutrient-based approach to modulate mitochondrial respiration rates and show that impaired mitochondrial respiration favors entry of the human pathogen Listeria monocytogenes, a well-established bacterial infection model. Reversely, enhanced mitochondrial energy metabolism decreases infection efficiency. We further demonstrate that endocytic recycling is reduced in mitochondrial respiration-dependent cells, dampening L. monocytogenes infection by slowing the recycling of its host cell receptor c-Met, highlighting a previously undescribed role of mitochondrial respiration during infection.

High-throughput screening identifies suppressors of mitochondrial fragmentation in OPA1 fibroblasts.

Mutations in OPA1 cause autosomal dominant optic atrophy (DOA) as well as DOA+, a phenotype characterized by more severe neurological deficits. OPA1 deficiency causes mitochondrial fragmentation and also disrupts cristae, respiration, mitochondrial DNA (mtDNA) maintenance, and cell viability. It has not yet been established whether phenotypic severity can be modulated by genetic modifiers of OPA1. We screened the entire known mitochondrial proteome (1,531 genes) to identify genes that control mitochondrial morphology using a first-in-kind imaging pipeline. We identified 145 known and novel candidate genes whose depletion promoted elongation or fragmentation of the mitochondrial network in control fibroblasts and 91 in DOA+ patient fibroblasts that prevented mitochondrial fragmentation, including phosphatidyl glycerophosphate synthase (PGS1). PGS1 depletion reduces CL content in mitochondria and rebalances mitochondrial dynamics in OPA1-deficient fibroblasts by inhibiting mitochondrial fission, which improves defective respiration, but does not rescue mtDNA depletion, cristae dysmorphology, or apoptotic sensitivity. Our data reveal that the multifaceted roles of OPA1 in mitochondria can be functionally uncoupled by modulating mitochondrial lipid metabolism, providing novel insights into the cellular relevance of mitochondrial fragmentation.

Enhanced cGAS-STING-dependent interferon signaling associated with mutations in ATAD3A.

Mitochondrial DNA (mtDNA) has been suggested to drive immune system activation, but the induction of interferon signaling by mtDNA has not been demonstrated in a Mendelian mitochondrial disease. We initially ascertained two patients, one with a purely neurological phenotype and one with features suggestive of systemic sclerosis in a syndromic context, and found them both to demonstrate enhanced interferon-stimulated gene (ISG) expression in blood. We determined each to harbor a previously described de novo dominant-negative heterozygous mutation in ATAD3A, encoding ATPase family AAA domain-containing protein 3A (ATAD3A). We identified five further patients with mutations in ATAD3A and recorded up-regulated ISG expression and interferon α protein in four of them. Knockdown of ATAD3A in THP-1 cells resulted in increased interferon signaling, mediated by cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING). Enhanced interferon signaling was abrogated in THP-1 cells and patient fibroblasts depleted of mtDNA. Thus, mutations in the mitochondrial membrane protein ATAD3A define a novel type I interferonopathy.

The role of mitochondrial DNA copy number in mammalian fertility.

Mammalian mitochondrial DNA (mtDNA) is a small, maternally inherited genome that codes for 13 essential proteins in the respiratory chain. Mature oocytes contain more than 150 000 copies of mtDNA, at least an order of magnitude greater than the number in most somatic cells, but sperm contain only approximately 100 copies. Mitochondrial oxidative phosphorylation has been suggested to be an important determinant of oocyte quality and sperm motility; however, the functional significance of the high mtDNA copy number in oocytes, and of the low copy number in sperm, remains unclear. To investigate the effects of mtDNA copy number on fertility, we genetically manipulated mtDNA copy number in the mouse by deleting one copy of Tfam, an essential component of the mitochondrial nucleoid, at different stages of germline development. We show that males can tolerate at least a threefold reduction in mtDNA copy number in their sperm without impaired fertility, and in fact, they preferentially transmit a deleted Tfam allele. Surprisingly, oocytes with as few as 4000 copies of mtDNA can be fertilized and progress normally through preimplantation development to the blastocyst stage. The mature oocyte, however, has a critical postimplantation developmental threshold of 40 000-50 000 copies of mtDNA in the mature oocyte. These observations suggest that the high mtDNA copy number in the mature oocyte is a genetic device designed to distribute mitochondria and mtDNAs to the cells of the early postimplantation embryo before mitochondrial biogenesis and mtDNA replication resumes, whereas down-regulation of mtDNA copy number is important for normal sperm function.

Loss of the mitochondrial i-AAA protease YME1L leads to ocular dysfunction and spinal axonopathy.

Disturbances in the morphology and function of mitochondria cause neurological diseases, which can affect the central and peripheral nervous system. The i-AAA protease YME1L ensures mitochondrial proteostasis and regulates mitochondrial dynamics by processing of the dynamin-like GTPase OPA1. Mutations in YME1L cause a multi-systemic mitochondriopathy associated with neurological dysfunction and mitochondrial fragmentation but pathogenic mechanisms remained enigmatic. Here, we report on striking cell-type-specific defects in mice lacking YME1L in the nervous system. YME1L-deficient mice manifest ocular dysfunction with microphthalmia and cataracts and develop deficiencies in locomotor activity due to specific degeneration of spinal cord axons, which relay proprioceptive signals from the hind limbs to the cerebellum. Mitochondrial fragmentation occurs throughout the nervous system and does not correlate with the degenerative phenotype. Deletion of Oma1 restores tubular mitochondria but deteriorates axonal degeneration in the absence of YME1L, demonstrating that impaired mitochondrial proteostasis rather than mitochondrial fragmentation causes the observed neurological defects.

Lung ultrasound as a translational approach for non-invasive assessment of heart failure with reduced or preserved ejection fraction in mice.

AIMS: Heart failure (HF) has become an epidemic and constitutes a major medical, social, and economic problem worldwide. Despite advances in medical treatment, HF prognosis remains poor. The development of efficient therapies is hampered by the lack of appropriate animal models in which HF can be reliably determined, particularly in mice. The development of HF in mice is often assumed based on the presence of cardiac dysfunction, but HF itself is seldom proved. Lung ultrasound (LUS) has become a helpful tool for lung congestion assessment in patients at all stages of HF. We aimed to apply this non-invasive imaging tool to evaluate HF in mouse models of both systolic and diastolic dysfunction. METHODS AND RESULTS: We used LUS to study HF in a mouse model of systolic dysfunction, dilated cardiomyopathy, and in a mouse model of diastolic dysfunction, diabetic cardiomyopathy. LUS proved to be a reliable and reproducible tool to detect pulmonary congestion in mice. The combination of LUS and echocardiography allowed discriminating those mice that develop HF from those that do not, even in the presence of evident cardiac dysfunction. The study showed that LUS can be used to identify the onset of HF decompensation and to evaluate the efficacy of therapies for this syndrome. CONCLUSIONS: This novel approach in mouse models of cardiac disease enables for the first time to adequately diagnose HF non-invasively in mice with preserved or reduced ejection fraction, and will pave the way to a better understanding of HF and to the development of new therapeutic approaches.