Mitochondrial F0F1-ATP synthase governs the induction of mitochondrial fission.

Mitochondrial dynamics is a process that balances fusion and fission events, the latter providing a mechanism for segregating dysfunctional mitochondria. Fission is controlled by the mitochondrial membrane potential (ΔΨm), optic atrophy 1 (OPA1) cleavage, and DRP1 recruitment. It is thought that this process is closely linked to the activity of the mitochondrial respiratory chain (MRC). However, we report here that MRC inhibition does not decrease ΔΨm nor increase fission, as evidenced by hyperconnected mitochondria. Conversely, blocking F0F1-ATP synthase activity induces fragmentation. We show that the F0F1-ATP synthase is sensing the inhibition of MRC activity by immediately promoting its reverse mode of action to hydrolyze matrix ATP and restoring ΔΨm, thus preventing fission. While this reverse mode is expected to be inhibited by the ATPase inhibitor ATPIF1, we show that this sensing is independent of this factor. We have unraveled an unexpected role of F0F1-ATP synthase in controlling the induction of fission by sensing and maintaining ΔΨm.

Primary mitochondrial disorders and mimics: Insights from a large French cohort.

OBJECTIVE: The objective of this study was to evaluate the implementation of NGS within the French mitochondrial network, MitoDiag, from targeted gene panels to whole exome sequencing (WES) or whole genome sequencing (WGS) focusing on mitochondrial nuclear-encoded genes. METHODS: Over 2000 patients suspected of Primary Mitochondrial Diseases (PMD) were sequenced by either targeted gene panels, WES or WGS within MitoDiag. We described the clinical, biochemical, and molecular data of 397 genetically confirmed patients, comprising 294 children and 103 adults, carrying pathogenic or likely pathogenic variants in nuclear-encoded genes. RESULTS: The cohort exhibited a large genetic heterogeneity, with the identification of 172 distinct genes and 253 novel variants. Among children, a notable prevalence of pathogenic variants in genes associated with oxidative phosphorylation (OXPHOS) functions and mitochondrial translation was observed. In adults, pathogenic variants were primarily identified in genes linked to mtDNA maintenance. Additionally, a substantial proportion of patients (54% (42/78) and 48% (13/27) in children and adults, respectively), undergoing WES or WGS testing displayed PMD mimics, representing pathologies that clinically resemble mitochondrial diseases. INTERPRETATION: We reported the largest French cohort of patients suspected of PMD with pathogenic variants in nuclear genes. We have emphasized the clinical complexity of PMD and the challenges associated with recognizing and distinguishing them from other pathologies, particularly neuromuscular disorders. We confirmed that WES/WGS, instead of panel approach, was more valuable to identify the genetic basis in patients with "possible" PMD and we provided a genetic testing flowchart to guide physicians in their diagnostic strategy.

Homozygous MFN2 variants causing severe antenatal encephalopathy with clumped mitochondria.

Pathogenic variants in the MFN2 gene are commonly associated with autosomal dominant (CMT2A2A) or recessive (CMT2A2B) Charcot-Marie-Tooth disease, with possible involvement of the CNS. Here, we present a case of severe antenatal encephalopathy with lissencephaly, polymicrogyria and cerebellar atrophy. Whole genome analysis revealed a homozygous deletion c.1717-274_1734 del (NM_014874.4) in the MFN2 gene, leading to exon 16 skipping and in-frame loss of 50 amino acids (p.Gln574_Val624del), removing the proline-rich domain and the transmembrane domain 1 (TM1). MFN2 is a transmembrane GTPase located on the mitochondrial outer membrane that contributes to mitochondrial fusion, shaping large mitochondrial networks within cells. In silico modelling showed that the loss of the TM1 domain resulted in a drastically altered topological insertion of the protein in the mitochondrial outer membrane. Fetus fibroblasts, investigated by fluorescent cell imaging, electron microscopy and time-lapse recording, showed a sharp alteration of the mitochondrial network, with clumped mitochondria and clusters of tethered mitochondria unable to fuse. Multiple deficiencies of respiratory chain complexes with severe impairment of complex I were also evidenced in patient fibroblasts, without involvement of mitochondrial DNA instability. This is the first reported case of a severe developmental defect due to MFN2 deficiency with clumped mitochondria.

Kynurenic Acid: A Novel Player in Cardioprotection against Myocardial Ischemia/Reperfusion Injuries.

BACKGROUND: Myocardial infarction is one of the leading causes of mortality worldwide; hence, there is an urgent need to discover novel cardioprotective strategies. Kynurenic acid (KYNA), a metabolite of the kynurenine pathway, has been previously reported to have cardioprotective effects. However, the mechanisms by which KYNA may be protective are still unclear. The current study addressed this issue by investigating KYNA's cardioprotective effect in the context of myocardial ischemia/reperfusion. METHODS: H9C2 cells and rats were exposed to hypoxia/reoxygenation or myocardial infarction, respectively, in the presence or absence of KYNA. In vitro, cell death was quantified using flow cytometry analysis of propidium iodide staining. In vivo, TTC-Evans Blue staining was performed to evaluate infarct size. Mitochondrial respiratory chain complex activities were measured using spectrophotometry. Protein expression was evaluated by Western blot, and mRNA levels by RT-qPCR. RESULTS: KYNA treatment significantly reduced H9C2-relative cell death as well as infarct size. KYNA did not exhibit any effect on the mitochondrial respiratory chain complex activity. SOD2 mRNA levels were increased by KYNA. A decrease in p62 protein levels together with a trend of increase in PARK2 may mark a stimulation of mitophagy. Additionally, ERK1/2, Akt, and FOXO3α phosphorylation levels were significantly reduced after the KYNA treatment. Altogether, KYNA significantly reduced myocardial ischemia/reperfusion injuries in both in vitro and in vivo models. CONCLUSION: Here we show that KYNA-mediated cardioprotection was associated with enhanced mitophagy and antioxidant defense. A deeper understanding of KYNA's cardioprotective mechanisms is necessary to identify promising novel therapeutic targets and their translation into the clinical arena.

Case report: Thirty-year progression of an EMPF1 encephalopathy due to defective mitochondrial and peroxisomal fission caused by a novel de novo heterozygous DNM1L variant.

Mutations in DNM1L (DRP1), which encode a key player of mitochondrial and peroxisomal fission, have been reported in patients with the variable phenotypic spectrum, ranging from non-syndromic optic atrophy to lethal infantile encephalopathy. Here, we report a case of an adult female patient presenting with a complex neurological phenotype that associates axonal sensory neuropathy, spasticity, optic atrophy, dysarthria, dysphasia, dystonia, and ataxia, worsening with aging. Whole-exome sequencing revealed a heterozygous de novo variant in the GTPase domain of DNM1L [NM_001278464.1: c.176C>A p.(Thr59Asn)] making her the oldest patient suffering from encephalopathy due to defective mitochondrial and peroxisomal fission-1. In silico analysis suggested a protein destabilization effect of the variant Thr59Asn. Unexpectedly, Western blotting disclosed profound decrease of DNM1L expression, probably related to the degradation of DNM1L complexes. A detailed description of mitochondrial and peroxisomal anomalies in transmission electron and 3D fluorescence microscopy studies confirmed the exceptional phenotype of this patient.

The multiple facets of mitochondrial regulations controlling cellular thermogenesis.

Understanding temperature production and regulation in endotherm organisms becomes a crucial challenge facing the increased frequency and intensity of heat strokes related to global warming. Mitochondria, located at the crossroad of metabolism, respiration, Ca2+ homeostasis, and apoptosis, were recently proposed to further act as cellular radiators, with an estimated inner temperature reaching 50 °C in common cell lines. This inner thermogenesis might be further exacerbated in organs devoted to produce consistent efforts as muscles, or heat as brown adipose tissue, in response to acute solicitations. Consequently, pathways promoting respiratory chain uncoupling and mitochondrial activity, such as Ca2+ fluxes, uncoupling proteins, futile cycling, and substrate supplies, provide the main processes controlling heat production and cell temperature. The mitochondrial thermogenesis might be further amplified by cytoplasmic mechanisms promoting the over-consumption of ATP pools. Considering these new thermic paradigms, we discuss here all conventional wisdoms linking mitochondrial functions to cellular thermogenesis in different physiological conditions.

Glutamate-Induced Deregulation of Krebs Cycle in Mitochondrial Encephalopathy Lactic Acidosis Syndrome Stroke-Like Episodes (MELAS) Syndrome Is Alleviated by Ketone Body Exposure.

(1) Background: The development of mitochondrial medicine has been severely impeded by a lack of effective therapies. (2) Methods: To better understand Mitochondrial Encephalopathy Lactic Acidosis Syndrome Stroke-like episodes (MELAS) syndrome, neuronal cybrid cells carrying different mutation loads of the m.3243A > G mitochondrial DNA variant were analysed using a multi-omic approach. (3) Results: Specific metabolomic signatures revealed that the glutamate pathway was significantly increased in MELAS cells with a direct correlation between glutamate concentration and the m.3243A > G heteroplasmy level. Transcriptomic analysis in mutant cells further revealed alterations in specific gene clusters, including those of the glutamate, gamma-aminobutyric acid pathways, and tricarboxylic acid (TCA) cycle. These results were supported by post-mortem brain tissue analysis from a MELAS patient, confirming the glutamate dysregulation. Exposure of MELAS cells to ketone bodies significantly reduced the glutamate level and improved mitochondrial functions, reducing the accumulation of several intermediate metabolites of the TCA cycle and alleviating the NADH-redox imbalance. (4) Conclusions: Thus, a multi-omic integrated approach to MELAS cells revealed glutamate as a promising disease biomarker, while also indicating that a ketogenic diet should be tested in MELAS patients.

Next-Generation Sequencing Identifies Novel PMPCA Variants in Patients with Late-Onset Dominant Optic Atrophy.

Dominant Optic Atrophy (DOA) is one of the most common inherited mitochondrial diseases, leading to blindness. It is caused by the chronic degeneration of the retinal ganglion cells (RGCs) and their axons forming the optic nerve. Until now, DOA has been mainly associated with genes encoding proteins involved in mitochondrial network dynamics. Using next-generation and exome sequencing, we identified for the first time heterozygous PMPCA variants having a causative role in the pathology of late-onset primary DOA in five patients. PMPCA encodes an α subunit of the mitochondrial peptidase (MPP), responsible for the cleavage and maturation of the mitochondrial precursor proteins imported from the cytoplasm into mitochondria. Recently, PMPCA has been identified as the gene responsible for Autosomal Recessive Cerebellar Ataxia type 2 (SCAR2) and another severe recessive mitochondrial disease. In this study, four PMPCA variants were identified, two are frameshifts (c.309delA and c.820delG) classified as pathogenic and two are missenses (c.1363G>A and c.1547G>A) classified with uncertain pathological significance. Functional assays on patients' fibroblasts show a hyperconnection of the mitochondrial network and revealed that frameshift variants reduced α-MPP levels, while not significantly affecting the respiratory machinery. These results suggest that alterations in mitochondrial peptidase function can affect the fusion-fission balance, a key element in maintaining the physiology of retinal ganglion cells, and consequently lead to their progressive degeneration.

Expanding the phenotype of DNAJC30-associated Leigh syndrome.

Leigh syndrome (LS) is a progressive neurodegenerative disease, characterized by extensive clinical, biochemical, and genetic heterogeneity. Recently, biallelic variants in DNAJC30 gene, encoding a protein crucial for the repair of mitochondrial complex I subunits, have been associated with Leber hereditary optic neuropathy and LS. It was suggested that clinical heterogeneity of DNAJC30-associated mitochondrial disease may be attributed to digenic inheritance. We describe three Polish patients, a 9-year-old boy, and female and male siblings, aged 17 and 11 years, with clinical and biochemical manifestations of LS. Exome sequencing (ES) identified a homozygous pathogenic variant in DNAJC30 c.152A>G, p.(Tyr51Cys) in the 9-year-old boy. In the siblings, ES identified two DNAJC30 variants: c.152A>G, p.(Tyr51Cys) and c.130_131del, p.(Ser44ValfsTer8) in a compound heterozygous state. In addition, both siblings carried a novel heterozygous c.484G>T, p.(Val162Leu) variant in NDUFS8 gene. This report provides further evidence for the association of DNAJC30 variants with LS. DNAJC30-associated LS is characterized by variable age at onset, movement disorder phenotype and normal or moderately elevated blood lactate level. Identification of a candidate heterozygous variant in NDUFS8 supports the hypothesis of digenic inheritance. Importantly, DNAJC30 pathogenic variants should be suspected in patients with LS irrespective of optic nerve involvement.

A homozygous splice variant in ATP5PO, disrupts mitochondrial complex V function and causes Leigh syndrome in two unrelated families.

Mitochondrial complex V plays an important role in oxidative phosphorylation by catalyzing the generation of ATP. Most complex V subunits are nuclear encoded and not yet associated with recognized Mendelian disorders. Using exome sequencing, we identified a rare homozygous splice variant (c.87+3A>G) in ATP5PO, the complex V subunit which encodes the oligomycin sensitivity conferring protein, in three individuals from two unrelated families, with clinical suspicion of a mitochondrial disorder. These individuals had a similar, severe infantile and often lethal multi-systemic disorder that included hypotonia, developmental delay, hypertrophic cardiomyopathy, progressive epileptic encephalopathy, progressive cerebral atrophy, and white matter abnormalities on brain MRI consistent with Leigh syndrome. cDNA studies showed a predominant shortened transcript with skipping of exon 2 and low levels of the normal full-length transcript. Fibroblasts from the affected individuals demonstrated decreased ATP5PO protein, defective assembly of complex V with markedly reduced amounts of peripheral stalk proteins, and complex V hydrolytic activity. Further, expression of human ATP5PO cDNA without exon 2 (hATP5PO-∆ex2) in yeast cells deleted for yATP5 (ATP5PO homolog) was unable to rescue growth on media which requires oxidative phosphorylation when compared to the wild type construct (hATP5PO-WT), indicating that exon 2 deletion leads to a non-functional protein. Collectively, our findings support the pathogenicity of the ATP5PO c.87+3A>G variant, which significantly reduces but does not eliminate complex V activity. These data along with the recent report of an affected individual with ATP5PO variants, add to the evidence that rare biallelic variants in ATP5PO result in defective complex V assembly, function and are associated with Leigh syndrome.

Cancer/Testis Antigen 55 is required for cancer cell proliferation and mitochondrial DNA maintenance.

Cancer/Testis Antigens (CTAs) represent a group of proteins whose expression under physiological conditions is restricted to testis but activated in many human cancers. Also, it was observed that co-expression of multiple CTAs worsens the patient prognosis. Five CTAs were reported acting in mitochondria and we recently reported 147 transcripts encoded by 67 CTAs encoding for proteins potentially targeted to mitochondria. Among them, we identified the two isoforms encoded by CT55 for whom the function is poorly understood. First, we found that patients with tumors expressing wild-type CT55 are associated with poor survival. Moreover, CT55 silencing decreases dramatically cell proliferation. Second, to investigate the role of CT55 on mitochondria, we first show that CT55 is localized to both mitochondria and endoplasmic reticulum (ER) due to the presence of an ambiguous N-terminal targeting signal. Then, we show that CT55 silencing decreases mtDNA copy number and delays mtDNA recovery after an acute depletion. Moreover, demethylation of CT55 promotor increases its expression, which in turn increases mtDNA copy number. Finally, we measured the mtDNA copy number in NCI-60 cell lines and screened for genes whose expression is strongly correlated to mtDNA amount. We identified CT55 as the second highest correlated hit. Also, we show that compared to siRNA scrambled control (siCtrl) treatment, CT55 specific siRNA (siCT55) treatment down-regulates aerobic respiration, indicating that CT55 sustains mitochondrial respiration. Altogether, these data show for first time that CT55 acts on mtDNA copy number, modulates mitochondrial activity to sustain cancer cell proliferation.

Variants in Mitochondrial ATP Synthase Cause Variable Neurologic Phenotypes.

OBJECTIVE: ATP synthase (ATPase) is responsible for the majority of ATP production. Nevertheless, disease phenotypes associated with mutations in ATPase subunits are extremely rare. We aimed at expanding the spectrum of ATPase-related diseases. METHODS: Whole-exome sequencing in cohorts with 2,962 patients diagnosed with mitochondrial disease and/or dystonia and international collaboration were used to identify deleterious variants in ATPase-encoding genes. Findings were complemented by transcriptional and proteomic profiling of patient fibroblasts. ATPase integrity and activity were assayed using cells and tissues from 5 patients. RESULTS: We present 10 total individuals with biallelic or de novo monoallelic variants in nuclear ATPase subunit genes. Three unrelated patients showed the same homozygous missense ATP5F1E mutation (including one published case). An intronic splice-disrupting alteration in compound heterozygosity with a nonsense variant in ATP5PO was found in one patient. Three patients had de novo heterozygous missense variants in ATP5F1A, whereas another 3 were heterozygous for ATP5MC3 de novo missense changes. Bioinformatics methods and populational data supported the variants' pathogenicity. Immunohistochemistry, proteomics, and/or immunoblotting revealed significantly reduced ATPase amounts in association to ATP5F1E and ATP5PO mutations. Diminished activity and/or defective assembly of ATPase was demonstrated by enzymatic assays and/or immunoblotting in patient samples bearing ATP5F1A-p.Arg207His, ATP5MC3-p.Gly79Val, and ATP5MC3-p.Asn106Lys. The associated clinical profiles were heterogeneous, ranging from hypotonia with spontaneous resolution (1/10) to epilepsy with early death (1/10) or variable persistent abnormalities, including movement disorders, developmental delay, intellectual disability, hyperlactatemia, and other neurologic and systemic features. Although potentially reflecting an ascertainment bias, dystonia was common (7/10). INTERPRETATION: Our results establish evidence for a previously unrecognized role of ATPase nuclear-gene defects in phenotypes characterized by neurodevelopmental and neurodegenerative features. ANN NEUROL 2022;91:225-237.

Acetoacetate protects macrophages from lactic acidosis-induced mitochondrial dysfunction by metabolic reprograming.

Lactic acidosis, the extracellular accumulation of lactate and protons, is a consequence of increased glycolysis triggered by insufficient oxygen supply to tissues. Macrophages are able to differentiate from monocytes under such acidotic conditions, and remain active in order to resolve the underlying injury. Here we show that, in lactic acidosis, human monocytes differentiating into macrophages are characterized by depolarized mitochondria, transient reduction of mitochondrial mass due to mitophagy, and a significant decrease in nutrient absorption. These metabolic changes, resembling pseudostarvation, result from the low extracellular pH rather than from the lactosis component, and render these cells dependent on autophagy for survival. Meanwhile, acetoacetate, a natural metabolite produced by the liver, is utilized by monocytes/macrophages as an alternative fuel to mitigate lactic acidosis-induced pseudostarvation, as evidenced by retained mitochondrial integrity and function, retained nutrient uptake, and survival without the need of autophagy. Our results thus show that acetoacetate may increase tissue tolerance to sustained lactic acidosis.

The Long Non-Coding RNA SAMMSON Is a Regulator of Chemosensitivity and Metabolic Orientation in MCF-7 Doxorubicin-Resistant Breast Cancer Cells.

Despite improvements in therapeutic strategies for treating breast cancers, tumor relapse and chemoresistance remain major issues in patient outcomes. Indeed, cancer cells display a metabolic plasticity allowing a quick adaptation to the tumoral microenvironment and to cellular stresses induced by chemotherapy. Recently, long non-coding RNA molecules (lncRNAs) have emerged as important regulators of cellular metabolic orientation. In the present study, we addressed the role of the long non-coding RNA molecule (lncRNA) SAMMSON on the metabolic reprogramming and chemoresistance of MCF-7 breast cancer cells resistant to doxorubicin (MCF-7dox). Our results showed an overexpression of SAMMSON in MCF-7dox compared to doxorubicin-sensitive cells (MCF-7). Silencing of SAMMSON expression by siRNA in MCF-7dox cells resulted in a metabolic rewiring with improvement of oxidative metabolism, decreased mitochondrial ROS production, increased mitochondrial replication, transcription and translation and an attenuation of chemoresistance. These results highlight the role of SAMMSON in the metabolic adaptations leading to the development of chemoresistance in breast cancer cells. Thus, targeting SAMMSON expression levels represents a promising therapeutic route to circumvent doxorubicin resistance in breast cancers.

Doxorubicin-Induced Autophagolysosome Formation Is Partly Prevented by Mitochondrial ROS Elimination in DOX-Resistant Breast Cancer Cells.

Since its discovery, mitophagy has been viewed as a protective mechanism used by cancer cells to prevent the induction of mitochondrial apoptosis. Most cancer treatments directly or indirectly cause mitochondrial dysfunction in order to trigger signals for cell death. Elimination of these dysfunctional mitochondria by mitophagy could thus prevent the initiation of the apoptotic cascade. In breast cancer patients, resistance to doxorubicin (DOX), one of the most widely used cancer drugs, is an important cause of poor clinical outcomes. However, the role played by mitophagy in the context of DOX resistance in breast cancer cells is not well understood. We therefore tried to determine whether an increase in mitophagic flux was associated with the resistance of breast cancer cells to DOX. Our first objective was to explore whether DOX-resistant breast cancer cells were characterized by conditions that favor mitophagy induction. We next tried to determine whether mitophagic flux was increased in DOX-resistant cells in response to DOX treatment. For this purpose, the parental (MCF-7) and DOX-resistant (MCF-7dox) breast cancer cell lines were used. Our results show that mitochondrial reactive oxygen species (ROS) production and hypoxia-inducible factor-1 alpha (HIF-1 alpha) expression are higher in MCF-7dox in a basal condition compared to MCF-7, suggesting DOX-resistant breast cancer cells are prone to stimuli to induce a mitophagy-related event. Our results also showed that, in response to DOX, autophagolysosome formation is induced in DOX-resistant breast cancer cells. This mitophagic step following DOX treatment seems to be partly due to mitochondrial ROS production as autophagolysosome formation is moderately decreased by the mitochondrial antioxidant mitoTEMPO.

Mitochondrial Dysfunction in Mitochondrial Medicine: Current Limitations, Pitfalls, and Tomorrow.

Until recently restricted to hereditary mitochondrial diseases, mitochondrial dysfunction is now recognized as a key player and strategic factor in the pathophysiological of many human diseases, ranging from the metabolism, vascular, cardiac, and neurodegenerative diseases to cancer. Because of their participation in a myriad of cellular functions and signaling pathways, precisely identifying the cause of mitochondrial "dysfunctions" can be challenging and requires robust and controlled techniques. Initially limited to the analysis of the respiratory chain functioning, these analytical techniques now enlarge to the analyses of mitochondrial and cellular metabolism, based on metabolomic approaches.Here, we address the methods used to assay mitochondrial dysfunction, with a highlight on the techniques used in diagnosis on tissues and cells derived from patients, the information they provide, and their strength and weakness.Targeting mitochondrial dysfunction by various strategies is a huge challenge, requires robust methods of evaluation, and should be able to take into consideration the mitochondria dynamics and localization. The future of mitochondrial medicine is strongly related to a perfect comprehension of its dysfunction.

Dominant ACO2 mutations are a frequent cause of isolated optic atrophy.

Biallelic mutations in ACO2, encoding the mitochondrial aconitase 2, have been identified in individuals with neurodegenerative syndromes, including infantile cerebellar retinal degeneration and recessive optic neuropathies (locus OPA9). By screening European cohorts of individuals with genetically unsolved inherited optic neuropathies, we identified 61 cases harbouring variants in ACO2, among whom 50 carried dominant mutations, emphasizing for the first time the important contribution of ACO2 monoallelic pathogenic variants to dominant optic atrophy. Analysis of the ophthalmological and clinical data revealed that recessive cases are affected more severely than dominant cases, while not significantly earlier. In addition, 27% of the recessive cases and 11% of the dominant cases manifested with extraocular features in addition to optic atrophy. In silico analyses of ACO2 variants predicted their deleterious impacts on ACO2 biophysical properties. Skin derived fibroblasts from patients harbouring dominant and recessive ACO2 mutations revealed a reduction of ACO2 abundance and enzymatic activity, and the impairment of the mitochondrial respiration using citrate and pyruvate as substrates, while the addition of other Krebs cycle intermediates restored a normal respiration, suggesting a possible short-cut adaptation of the tricarboxylic citric acid cycle. Analysis of the mitochondrial genome abundance disclosed a significant reduction of the mitochondrial DNA amount in all ACO2 fibroblasts. Overall, our data position ACO2 as the third most frequently mutated gene in autosomal inherited optic neuropathies, after OPA1 and WFS1, and emphasize the crucial involvement of the first steps of the Krebs cycle in the maintenance and survival of retinal ganglion cells.

Improved detection of mitochondrial DNA instability in mitochondrial genome maintenance disorders.

PURPOSE: Diseases caused by defects in mitochondrial DNA (mtDNA) maintenance machinery, leading to mtDNA deletions, form a specific group of disorders. However, mtDNA deletions also appear during aging, interfering with those resulting from mitochondrial disorders. METHODS: Here, using next-generation sequencing (NGS) data processed by eKLIPse and data mining, we established criteria distinguishing age-related mtDNA rearrangements from those due to mtDNA maintenance defects. MtDNA deletion profiles from muscle and urine patient samples carrying pathogenic variants in nuclear genes involved in mtDNA maintenance (n = 40) were compared with age-matched controls (n = 90). Seventeen additional patient samples were used to validate the data mining model. RESULTS: Overall, deletion number, heteroplasmy level, deletion locations, and the presence of repeats at deletion breakpoints were significantly different between patients and controls, especially in muscle samples. The deletion number was significantly relevant in adults, while breakpoint repeat lengths surrounding deletions were discriminant in young subjects. CONCLUSION: Altogether, eKLIPse analysis is a powerful tool for measuring the accumulation of mtDNA deletions between patients of different ages, as well as in prioritizing novel variants in genes involved in mtDNA stability.

Optic neuropathy linked to ACAD9 pathogenic variants: A potentially riboflavin-responsive disorder?

Mitochondrial complex I (CI) deficiencies (OMIM 252010) are the commonest inherited mitochondrial disorders in children. Acyl-CoA dehydrogenase 9 (ACAD9) is a flavoenzyme involved chiefly in CI assembly and possibly in fatty acid oxidation. Biallelic pathogenic variants result in CI dysfunction, with a phenotype ranging from early onset and sometimes fatal mitochondrial encephalopathy with lactic acidosis to late-onset exercise intolerance. Cardiomyopathy is often associated. We report a patient with childhood-onset optic and peripheral neuropathy without cardiac involvement, related to CI deficiency. Genetic analysis revealed compound heterozygous pathogenic variants in ACAD9, expanding the clinical spectrum associated to ACAD9 mutations. Importantly, riboflavin treatment (15 mg/kg/day) improved long-distance visual acuity and demonstrated significant rescue of CI activity in vitro.

Iron Deficiency without Anemia Decreases Physical Endurance and Mitochondrial Complex I Activity of Oxidative Skeletal Muscle in the Mouse.

Iron deficiency (ID), with or without anemia, is responsible for physical fatigue. This effect may be linked to an alteration of mitochondrial metabolism. Our aim was to assess the impact of ID on skeletal striated muscle mitochondrial metabolism. Iron-deficient non-anemic mice, obtained using a bloodletting followed by a low-iron diet for three weeks, were compared to control mice. Endurance was assessed using a one-hour submaximal exercise on a Rotarod device and activities of mitochondrial complexes I and IV were measured by spectrophotometry on two types of skeletal striated muscles, the soleus and the quadriceps. As expected, ID mice displayed hematologic markers of ID and reduced iron stores, although none of them were anemic. In ID mice, endurance was significantly reduced and activity of the respiratory chain complex I, normalized to citrate synthase activity, was significantly reduced in the soleus muscle but not in the quadriceps. Complex IV activities were not significantly different, neither in the soleus nor in the quadriceps. We conclude that ID without anemia is responsible for impaired mitochondrial complex I activity in skeletal muscles with predominant oxidative metabolism. These results bring pathophysiological support to explain the improved physical activity observed when correcting ID in human. Further studies are needed to explore the mechanisms underlying this decrease in complex I activity and to assess the role of iron therapy on muscle mitochondrial metabolism.