Mitochondrial Respiratory Chain Deficiency Research Articles

The complexities of investigating mitochondria dynamics in multiple sclerosis and mouse models of MS.

Multiple sclerosis (MS) is a demyelinating, degenerating disorder of the central nervous system (CNS) that is accompanied by mitochondria energy production failure. A loss of myelin paired with a deficit in energy production can contribute to further neurodegeneration and disability in patients in MS. Mitochondria are essential organelles that produce adenosine triphosphate (ATP) via oxidative phosphorylation in all cells in the CNS, including neurons, oligodendrocytes, astrocytes, and immune cells. In the context of demyelinating diseases, mitochondria have been shown to alter their morphology and undergo an initial increase in metabolic demand. This is followed by mitochondrial respiratory chain deficiency and abnormalities in mitochondrial transport that contribute to progressive neurodegeneration and irreversible disability. The current methodologies to study mitochondria are limiting and are capable of providing only a partial snapshot of the true mitochondria activity at a particular timepoint during disease. Mitochondrial functional studies are mostly performed in cell culture or whole brain tissue, which prevents understanding of mitochondrial pathology in distinct cell types in vivo. A true understanding of cell-specific mitochondrial pathophysiology of MS in mouse models is required. Cell-specific mitochondria morphology, mitochondria motility, and ATP production studies in animal models of MS will help us understand the role of mitochondria in the normal and diseased CNS. In this review, we present currently used methods to investigate mitochondria function in MS mouse models and discuss the current advantages and caveats with using each technique. In addition, we present recently developed mitochondria transgenic mouse lines expressing Cre under the control of CNS specific promoters to relate mitochondria to disease in vivo.

TEFM variants impair mitochondrial transcription causing childhood-onset neurological disease

Mutations in the mitochondrial or nuclear genomes are associated with a diverse group of human disorders characterized by impaired mitochondrial respiration. Within this group, an increasing number of mutations have been identified in nuclear genes involved in mitochondrial RNA biology. The TEFM gene encodes the mitochondrial transcription elongation factor responsible for enhancing the processivity of mitochondrial RNA polymerase, POLRMT. We report for the first time that TEFM variants are associated with mitochondrial respiratory chain deficiency and a wide range of clinical presentations including mitochondrial myopathy with a treatable neuromuscular transmission defect. Mechanistically, we show muscle and primary fibroblasts from the affected individuals have reduced levels of promoter distal mitochondrial RNA transcripts. Finally, tefm knockdown in zebrafish embryos resulted in neuromuscular junction abnormalities and abnormal mitochondrial function, strengthening the genotype-phenotype correlation. Our study highlights that TEFM regulates mitochondrial transcription elongation and its defect results in variable, tissue-specific neurological and neuromuscular symptoms.

Mitochondrial DNA involvement in patients with autism spectrum disorders and intellectual disability

BackgroundAutism spectrum disorders (ASD) are neurodevelopmental disorders, with heterogeneous etiology, characterized by deficiencies in social interaction, communication, and by repetitive and stereotyped behaviors.Mitochondrial encephalomyopathies are inherited disorders of oxidative metabolism, with a wide clinical, biochemical and genetic heterogeneity, requiring a complex diagnostic flow-chart. Infantile forms often occur with defects in development, intellectual disabilities and dysmorphisms.ASD has been associated with mitochondrial respiratory chain deficiency and/or mitochondrial DNA (mtDNA) mutations. However, few studies carried out detailed clinical, instrumental, morphological, biochemical investigations, and mtDNA sequencing in ASD patients. MethodThe purpose of this study was the identification of mitochondrial dysfunction due to mtDNA variants in 19 selected subjects presenting ASD and clinical features of mitochondrial disease. For these patients we adopted a multidisciplinary approach combining clinical and laboratory investigations on muscle biopsy, with biochemical, histological and genetic techniques. ResultsThe histological examination showed myogenic or neurogenic changes in 79 % of patients; furthermore, 58 % of the patients had lipid accumulation, mitochondrial proliferation and COX deficient fibers. The biochemical investigations reported in three patients impairments involving one or more of the respiratory chain complexes. In addition, genetic studies revealed in one patient with normal histology and biochemistry multiple mtDNA deletions, and in four patients different mtDNA point mutations. ConclusionsThe present study confirms the hypothesis of an association between ASD and mitochondrial dysfunction. However, further studies in a larger group of subjects are needed.

ITCH deficiency clinical phenotype expansion and mitochondrial dysfunction

Autoimmune Disease, Multisystem, with Facial Dysmorphism (ADMFD) is an autosomal recessive disorder due to pathogenic variants in the ITCH gene. It is characterized by failure to thrive, dysmorphic facial features, developmental delay, and systemic autoimmunity that can manifest variably with autoimmune hepatitis, thyroiditis, and enteropathy, among other organ manifestations. It was originally described in 10 consanguineous Old Order Amish patients, and more recently in two patients of White British and Black German ethnicities. While the role of ITCH protein in apoptosis and inflammation has previously been characterized, a defect in cellular bioenergetics has not yet been reported in ITCH deficiency. Here we present a Caucasian female originally evaluated for possible mitochondrial respiratory chain deficiency, who ultimately was found to have two novel variants in ITCH with absence of ITCH protein in patient derived fibroblasts. Clinical studies of patient muscle showed mitochondrial DNA copy number of 57% compared to controls. Functional studies in skin fibroblasts revealed decreased activity of mitochondrial fatty acid oxidation and oxidative phosphorylation, and decreased overall ATP production. Our findings confirm mitochondrial energy dysfunction in a patient with ITCH deficiency offering the opportunity to assess alternative therapeutic options.

Two independent respiratory chains adapt OXPHOS performance to glycolytic switch

The structural and functional organization of the mitochondrial respiratory chain (MRC) remains intensely debated. Here, we show the co-existence of two separate MRC organizations in human cells and postmitotic tissues, C-MRC and S-MRC, defined by the preferential expression of three COX7A subunit isoforms, COX7A1/2 and SCAFI (COX7A2L). COX7A isoforms promote the functional reorganization of distinct co-existing MRC structures to prevent metabolic exhaustion and MRC deficiency. Notably, prevalence of each MRC organization is reversibly regulated by the activation state of the pyruvate dehydrogenase complex (PDC). Under oxidative conditions, the C-MRC is bioenergetically more efficient, whereas the S-MRC preferentially maintains oxidative phosphorylation (OXPHOS) upon metabolic rewiring toward glycolysis. We show a link between the metabolic signatures converging at the PDC and the structural and functional organization of the MRC, challenging the widespread notion of the MRC as a single functional unit and concluding that its structural heterogeneity warrants optimal adaptation to metabolic function.

I.01Congenital myasthenic syndromes

Neuromuscular junction disorders are a heterogeneous group of acquired (Myasthenia Gravis, MG) and inherited (Congenital Myasthenic Syndromes, CMS) disorders associated with distinctive clinical, electrophysiological, laboratory and ultrastructural abnormalities. The genetic defects in CMS either impair neuromuscular transmission directly or result in secondary impairments, which eventually compromise the safety margin of neuromuscular transmission. More recently, we have identified two genes (<i>DOK7, GFPT1</i>) that cause fatigable weakness of muscles in a limb-girdle distribution, but rarely affecting facial or eye muscles. Next-generation sequencing and deep phenotyping, in combination with international data sharing, reveals new genetic causes of CMS, but also unusual, overlapping clinical phenotypes, which blur the boundaries with primary myopathies and motor neuropathies. Mitochondrial dysfunction has been implicated with both myopathies and neuromuscular transmission defects. TEFM is a nuclear gene that codes for a mitochondrial transcription elongation factor – the protein responsible for prolonging activity of RNA polymerase to ensure distal transcripts of the mitochondrial genome are transcribed. We identified the first human <i>TEFM</i> variants associated with mitochondrial respiratory chain deficiency in seven patients with varying clinical presentations. While symptoms included classical features of a mitochondrial disease, some patients also presented with a treatable neuromuscular transmission defect. We will cover the significant progress made in understanding the molecular pathogenesis of CMS, which is important for both patients and clinicians in terms of reaching a definite diagnosis and selecting the most appropriate treatment.

Detecting respiratory chain defects in osteoblasts from osteoarthritic patients using imaging mass cytometry.

Osteoporosis is a skeletal disease which is characterised by reduced bone mass and microarchitecture, with a subsequent loss of strength that predisposes to fragility and risk of fractures. The pathogenesis of falling bone mineral density, ultimately leading to a diagnosis of osteoporosis is incompletely understood but the disease is currently thought to be multifactorial. Humans are known to accumulate mitochondrial mutations and respiratory chain deficiency with age and mounting evidence suggests that this may indeed be the overarching cause intrinsic to the changing phenotype in advancing age and age-related disease. Mitochondrial mutations are detectable from the age of about 30years onwards. Mitochondria contain their own genome which encodes 13 essential mitochondrial proteins and accumulates somatic variants at up to 10 times the rate of the nuclear genome. Once the concentration of any pathogenic mitochondrial genome variant exceeds a threshold, respiratory chain deficiency and cellular dysfunction occur. The PolgD257A/D257A mouse model is a knock-in mutant that expresses a proof-reading-deficient version of PolgA, a nuclear encoded subunit of mtDNA polymerase. These mice are a useful model of age-related accumulation of mtDNA mutations in humans since their defective proof-reading mechanism leads to a mitochondrial DNA mutation rate 3-5 times higher than in wild-type mice. These mice showed enhanced levels of age-related osteoporosis along with respiratory chain deficiency in osteoblasts. To explore whether respiratory chain deficiency is also seen in human osteoblasts, we developed a protocol and analysis framework for imaging mass cytometry in bone tissue sections to analyse osteoblasts in situ. By comparing bone tissue sampled at one timepoint from femoral neck of 10 older healthy volunteers aged 40-85 with samples from young patients aged 1-19, we have identified complex I defect in osteoblasts from 6 out of 10 older volunteers, complex II defect in 2 out of 10 older volunteers, complex IV defect in 1 out of 10 older volunteers and complex V defect in 4 out of 10 older volunteers. These observations are consistent with findings from the PolgD257A/D257A mouse model and suggest that respiratory chain deficiency, as a consequence of the accumulation of age-related pathogenic mitochondrial DNA mutations, may play a significant role in the pathogenesis of human age-related osteoporosis.

Research progress in mitochondrial transfer mediated by tunneling nanotube in the field of tumor

Mitochondria, as the main site of cell metabolism and energy generation, contains the genome encoding the respiratory chain-associated complexes. Deletions or mutations of mitochondria will lead to mitochondrial respiratory chain deficiencies and these deficiencies play an important role in metabolic reprogramming which is considered as one of the important features of tumorigenesis and development. Many studies have found that tunneling nanotube (TNT), a well-established mitochondrial transfer pathway, is able to restore mitochondrial respiratory deficiencies. This review article focuses on the occurrence of mitochondrial transfer, the mechanism of TNT formation and the promising therapeutic targets acting on mitochondrial transfer.

New subtype of PCH1C caused by novel EXOSC8 variants in a 16-year-old Spanish patient

We report the case of a 16-year-old Spanish boy with cerebellar and spinal muscular atrophy, spasticity, psychomotor retardation, nystagmus, ophthalmoparesis, epilepsy, and mitochondrial respiratory chain (MRC) deficiency. Whole exome sequencing (WES) uncovered three variants (two of them novel) in a compound heterozygous in EXOSC8 gene (NM_181503.3:c.[390+1delG];[628C>T;815G>C]) that encodes the exosome complex component RRP43 protein (EXOSC8). In order to assess the pathogenicity of these variants, expression experiments of RNA and protein for EXOSC8 were carried out. The c.[390+1delG] variant produces the elimination of exon 7 (r.[345_390del]; p.[Ser116LysfsTer27]) and a decrease of the RNA expression in relation to the other allele (p.[Pro210Ser;Ser272Thr]). Furthermore, total mRNA expression is reduced by 30% and the protein level by 65%. EXOSC8 is an essential protein of the exosome core, a ubiquitously expressed complex responsible for RNA processing and degradation. Recessive mutations in EXOSC8 cause pontocerebellar hypoplasia type 1C (PCH1C), and currently, only two homozygous variants in this gene have been described. However, unlike PCH1C-affected individuals with EXOSC8 variants, our patient presents a normal supratentorial cerebral tissue (neither corpus callosum hypoplasia nor hypomyelination) with a less severe phenotype and longer survival. In conclusion, our data expand both genetic and phenotypic spectrum associated with EXOSC8 variants.

Mitochondrial dysfunction impairs osteogenesis, increases osteoclast activity, and accelerates age related bone loss

The pathogenesis of declining bone mineral density, a universal feature of ageing, is not fully understood. Somatic mitochondrial DNA (mtDNA) mutations accumulate with age in human tissues and mounting evidence suggests that they may be integral to the ageing process. To explore the potential effects of mtDNA mutations on bone biology, we compared bone microarchitecture and turnover in an ageing series of wild type mice with that of the PolgAmut/mut mitochondrial DNA ‘mutator’ mouse. In vivo analyses showed an age-related loss of bone in both groups of mice; however, it was significantly accelerated in the PolgAmut/mut mice. This accelerated rate of bone loss is associated with significantly reduced bone formation rate, reduced osteoblast population densities, increased osteoclast population densities, and mitochondrial respiratory chain deficiency in osteoblasts and osteoclasts in PolgAmut/mut mice compared with wild-type mice. In vitro assays demonstrated severely impaired mineralised matrix formation and increased osteoclast resorption by PolgAmut/mut cells. Finally, application of an exercise intervention to a subset of PolgAmut/mut mice showed no effect on bone mass or mineralised matrix formation in vitro. Our data demonstrate that mitochondrial dysfunction, a universal feature of human ageing, impairs osteogenesis and is associated with accelerated bone loss.

Evidence of diaphragmatic dysfunction with severe alveolar hypoventilation syndrome in mitochondrial respiratory chain deficiency

Diaphragmatic dysfunction has been reported in congenital myopathies, muscular dystrophies, and occasionally, mitochondrial respiratory chain deficiency. Using a minimally invasive procedure in 3 young girls, 1 with a heteroplasmic MT-CYB mutation and 2 with biallelic pathogenic TK2 variants, we provided functional evidence of diaphragmatic dysfunction with global respiratory muscle weakness in mitochondrial respiratory chain deficiency. Analysis of respiratory muscle performance using esogastric pressures revealed paradoxical breathing and severe global inspiratory and expiratory muscle weakness with a sniff esophageal inspiratory pressure and a gastric pressure during cough averaging 50% and 40% of predicted values, respectively. This diaphragmatic dysfunction was responsible for severe undiagnosed nocturnal hypoventilation, requiring noninvasive ventilation. Our results underline the interest of this minimally invasive procedure for the evaluation of respiratory muscle performance and its potential value for the monitoring of future clinical trials in respiratory chain deficiency.

Early-onset coenzyme Q10 deficiency associated with ataxia and respiratory chain dysfunction due to novel pathogenic COQ8A variants, including a large intragenic deletion.

Coenzyme Q10 (CoQ10) deficiency is a clinically and genetically heterogeneous subtype of mitochondrial disease. We report two girls with ataxia and mitochondrial respiratory chain deficiency who were shown to have primary CoQ10 deficiency. Muscle histochemistry displayed signs of mitochondrial dysfunction—ragged red fibers, mitochondrial paracrystalline inclusions, and lipid deposits while biochemical analyses revealed complex II+III respiratory chain deficiencies. MRI brain demonstrated cerebral and cerebellar atrophy. Targeted molecular analysis identified a homozygous c.1015G>A, p.(Ala339Thr) COQ8A variant in subject 1, while subject 2 was found to harbor a single heterozygous c.1029_1030delinsCA variant predicting a p.Gln343_Val344delinsHisMet amino acid substitution. Subsequent investigations identified a large‐scale COQ8A deletion in trans to the c.1029_1030delinsCA allele. A skin biopsy facilitated cDNA studies that confirmed exon skipping in the fibroblast derived COQ8A mRNA transcript. This report expands the molecular genetic spectrum associated with COQ8A‐related mitochondrial disease and highlights the importance of thorough investigation of candidate pathogenic variants to establish phase. Rapid diagnosis is of the utmost importance as patients may benefit from therapeutic CoQ10 supplementation.

Mitochondrial respiratory chain deficiency correlates with the severity of neuropathology in sporadic Creutzfeldt-Jakob disease

Mitochondrial dysfunction has been implicated in multiple neurodegenerative diseases but remains largely unexplored in Creutzfeldt-Jakob disease. Here, we characterize the mitochondrial respiratory chain at the individual neuron level in the MM1 and VV2 common molecular subtypes of sporadic Creutzfeldt-Jakob disease. Moreover, we investigate the associations between the mitochondrial respiratory chain and neuropathological markers of the disease.Brain tissue from individuals with sporadic Creutzfeldt-Jakob disease and age-matched controls were obtained from the brain collection of the Austrian Creutzfeldt-Jakob Surveillance. The mitochondrial respiratory chain was studied through a dichotomous approach of immunoreactivities in the temporal cortex and the hippocampal subregions of CA4 and CA3.We show that profound deficiency of all mitochondrial respiratory complexes (I-V) occurs in neurons of the severely affected temporal cortex of patients with Creutzfeldt-Jakob disease. This deficiency correlates strongly with the severity of neuropathological changes, including vacuolation of the neuropil, gliosis and disease associated prion protein load. Respiratory chain deficiency is less pronounced in hippocampal CA4 and CA3 regions compared to the temporal cortex. In both areas respiratory chain deficiency shows a predilection for the MM1 molecular subtype of Creutzfeldt-Jakob disease.Our findings indicate that aberrant mitochondrial respiration could be involved early in the pathogenesis of sporadic Creutzfeldt-Jakob disease and contributes to neuronal death, most likely via ATP depletion. Based on these results, we propose that the restricted MRI diffusion profile seen in the brain of patients with sporadic Creutzfeldt-Jakob disease might reflect cytotoxic changes due to neuronal respiratory chain failure and ATP loss.

Mapping mitochondrial respiratory chain deficiencies by respirometry: Beyond the Mito Stress Test

Cell-based respirometers, such as the Seahorse Extracellular Flux Analyzer, are valuable tools to assess the functionality of mitochondria within adherent neurons, as well as other cell types. The Mito Stress Test is the most frequently employed protocol of drug additions to evaluate mitochondrial bioenergetic function. Sequential exposure of cells to an ATP synthase inhibitor such as oligomycin and an uncoupler such as FCCP cause changes in oxygen consumption rate that allow estimation of the cellular efficiency and capacity for mitochondrial ATP synthesis. While a useful first step in assessing whether an experimental treatment or genetic manipulation affects mitochondrial energetics, the Mito Stress Test does not identify specific sites of altered respiratory chain function. This article discusses limitations of the Mito Stress Test, proposes a refined protocol for comparing cell populations that requires independent drug titrations at multiple cell densities, and describes a stepwise series of respirometry-based assays that “map” locations of electron transport deficiency. These include strategies to test for cytochrome c release, to probe the functionality of specific electron transport chain complexes within intact or permeabilized cells, and to measure NADH oxidation by the linked activity of Complexes I, III, and IV. To illustrate utility, we show that although UK5099 and ABT-737 each decrease the spare respiratory capacity of cortical neurons, the stepwise assays reveal different underlying mechanisms consistent with their established drug targets: deficient Complex I substrate supply induced by the mitochondrial pyruvate carrier inhibitor UK5099 and cytochrome c release induced by the anti-apoptotic BCL-2 family protein inhibitor ABT-737.

Investigation of mitochondrial biogenesis defects in single substantia nigra neurons using post-mortem human tissues

Mitochondrial respiratory chain deficiency and mitochondrial DNA deletions are reported in substantia nigra neurons from healthy aged and Parkinson's disease cases, with extensive neuronal loss only seen in the latter. This study aimed to understand the pathological relevance of mitochondrial defects for neuronal survival. Using post-mortem human midbrain, substantia nigra neurons exposed to different types of mitochondrial defects (including mitochondrial DNA point mutations, single and multiple deletions) were compared to neurons from healthy aged and Parkinson's disease cases (either sex) at a single neuronal level. We identified mitochondrial deficiencies in all cases, though these deficiencies were more severe in the mitochondrial disease patients with multiple deletions. A significant reduction in TFAM expression was detected in Parkinson's disease compared to cases with other mitochondrial defects. Higher mitochondrial DNA copy number was detected in healthy aged neurons, despite a deletion level equivalent to Parkinson's disease. Our data support that in individuals with pathogenic mitochondrial defects, neurons respond to mitochondrial defect to survive and such an adaptation may involve TFAM.

Mutations in ELAC2 associated with hypertrophic cardiomyopathy impair mitochondrial tRNA 3'-end processing.

Mutations in either the mitochondrial or nuclear genomes are associated with a diverse group of human disorders characterized by impaired mitochondrial respiration. Within this group, an increasing number of mutations have been identified in nuclear genes involved in mitochondrial RNA metabolism, including ELAC2. The ELAC2 gene codes for the mitochondrial RNase Z, responsible for endonucleolytic cleavage of the 3′ ends of mitochondrial pre‐tRNAs. Here, we report the identification of 16 novel ELAC2 variants in individuals presenting with mitochondrial respiratory chain deficiency, hypertrophic cardiomyopathy (HCM), and lactic acidosis. We provide evidence for the pathogenicity of the novel missense variants by studying the RNase Z activity in an in vitro system. We also modeled the residues affected by a missense mutation in solved RNase Z structures, providing insight into enzyme structure and function. Finally, we show that primary fibroblasts from the affected individuals have elevated levels of unprocessed mitochondrial RNA precursors. Our study thus broadly confirms the correlation of ELAC2 variants with severe infantile‐onset forms of HCM and mitochondrial respiratory chain dysfunction. One rare missense variant associated with the occurrence of prostate cancer (p.Arg781His) impairs the mitochondrial RNase Z activity of ELAC2, suggesting a functional link between tumorigenesis and mitochondrial RNA metabolism

Understanding mitochondrial DNA maintenance disorders at the single muscle fibre level.

Clonal expansion of mitochondrial DNA (mtDNA) deletions is an important pathological mechanism in adults with mtDNA maintenance disorders, leading to a mosaic mitochondrial respiratory chain deficiency in skeletal muscle. This study had two aims: (i) to determine if different Mendelian mtDNA maintenance disorders showed similar pattern of mtDNA deletions and respiratory chain deficiency and (ii) to investigate the correlation between the mitochondrial genetic defect and corresponding respiratory chain deficiency. We performed a quantitative analysis of respiratory chain deficiency, at a single cell level, in a cohort of patients with mutations in mtDNA maintenance genes. Using the same tissue section, we performed laser microdissection and single cell genetic analysis to investigate the relationship between mtDNA deletion characteristics and the respiratory chain deficiency. The pattern of respiratory chain deficiency is similar with different genetic defects. We demonstrate a clear correlation between the level of mtDNA deletion and extent of respiratory chain deficiency within a single cell. Long-range and single molecule PCR shows the presence of multiple mtDNA deletions in approximately one-third of all muscle fibres. We did not detect evidence of a replicative advantage for smaller mtDNA molecules in the majority of fibres, but further analysis is needed to provide conclusive evidence.

Mitochondrial Dysfunction and Multiple Sclerosis.

In recent years, several studies have examined the potential associations between mitochondrial dysfunction and neurodegenerative diseases such as multiple sclerosis (MS), Parkinson’s disease and Alzheimer’s disease. In MS, neurological disability results from inflammation, demyelination, and ultimately, axonal damage within the central nervous system. The sustained inflammatory phase of the disease leads to ion channel changes and chronic oxidative stress. Several independent investigations have demonstrated mitochondrial respiratory chain deficiency in MS, as well as abnormalities in mitochondrial transport. These processes create an energy imbalance and contribute to a parallel process of progressive neurodegeneration and irreversible disability. The potential roles of mitochondria in neurodegeneration are reviewed. An overview of mitochondrial diseases that may overlap with MS are also discussed, as well as possible therapeutic targets for the treatment of MS and other neurodegenerative conditions.

Mitochondrial respiratory chain deficiency inhibits lysosomal hydrolysis

ABSTRACTMitochondria are key organelles for cellular metabolism, and regulate several processes including cell death and macroautophagy/autophagy. Here, we show that mitochondrial respiratory chain (RC) deficiency deactivates AMP-activated protein kinase (AMPK, a key regulator of energy homeostasis) signaling in tissue and in cultured cells. The deactivation of AMPK in RC-deficiency is due to increased expression of the AMPK-inhibiting protein FLCN (folliculin). AMPK is found to be necessary for basal lysosomal function, and AMPK deactivation in RC-deficiency inhibits lysosomal function by decreasing the activity of the lysosomal Ca2+ channel MCOLN1 (mucolipin 1). MCOLN1 is regulated by phosphoinositide kinase PIKFYVE and its product PtdIns(3,5)P2, which is also decreased in RC-deficiency. Notably, reactivation of AMPK, in a PIKFYVE-dependent manner, or of MCOLN1 in RC-deficient cells, restores lysosomal hydrolytic capacity. Building on these data and the literature, we propose that downregulation of the AMPK-PIKFYVE-PtdIns(3,5)P2-MCOLN1 pathway causes lysosomal Ca2+ accumulation and impaired lysosomal catabolism. Besides unveiling a novel role of AMPK in lysosomal function, this study points to the mechanism that links mitochondrial malfunction to impaired lysosomal catabolism, underscoring the importance of AMPK and the complexity of organelle cross-talk in the regulation of cellular homeostasis.Abbreviation: ΔΨm: mitochondrial transmembrane potential; AMP: adenosine monophosphate; AMPK: AMP-activated protein kinase; ATG5: autophagy related 5; ATP: adenosine triphosphate; ATP6V0A1: ATPase, H+ transporting, lysosomal, V0 subbunit A1; ATP6V1A: ATPase, H+ transporting, lysosomal, V0 subbunit A; BSA: bovine serum albumin; CCCP: carbonyl cyanide-m-chlorophenylhydrazone; CREB1: cAMP response element binding protein 1; CTSD: cathepsin D; CTSF: cathepsin F; DMEM: Dulbecco’s modified Eagle’s medium; DMSO: dimethyl sulfoxide; EBSS: Earl’s balanced salt solution; ER: endoplasmic reticulum; FBS: fetal bovine serum; FCCP: carbonyl cyanide-p-trifluoromethoxyphenolhydrazone; GFP: green fluorescent protein; GPN: glycyl-L-phenylalanine 2-naphthylamide; LAMP1: lysosomal associated membrane protein 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MCOLN1/TRPML1: mucolipin 1; MEF: mouse embryonic fibroblast; MITF: melanocyte inducing transcription factor; ML1N*2-GFP: probe used to detect PtdIns(3,5)P2 based on the transmembrane domain of MCOLN1; MTORC1: mechanistic target of rapamycin kinase complex 1; NDUFS4: NADH:ubiquinone oxidoreductase subunit S4; OCR: oxygen consumption rate; PBS: phosphate-buffered saline; pcDNA: plasmid cytomegalovirus promoter DNA; PCR: polymerase chain reaction; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns(3,5)P2: phosphatidylinositol-3,5-bisphosphate; PIKFYVE: phosphoinositide kinase, FYVE-type zinc finger containing; P/S: penicillin-streptomycin; PVDF: polyvinylidene fluoride; qPCR: quantitative real time polymerase chain reaction; RFP: red fluorescent protein; RNA: ribonucleic acid; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis; shRNA: short hairpin RNA; siRNA: small interfering RNA; TFEB: transcription factor EB; TFE3: transcription factor binding to IGHM enhancer 3; TMRM: tetramethylrhodamine, methyl ester, perchlorate; ULK1: unc-51 like autophagy activating kinase 1; ULK2: unc-51 like autophagy activating kinase 2; UQCRC1: ubiquinol-cytochrome c reductase core protein 1; v-ATPase: vacuolar-type H+-translocating ATPase; WT: wild-type

Leigh syndrome caused by mutations in MTFMT is associated with a better prognosis.

ObjectivesMitochondrial methionyl‐tRNA formyltransferase (MTFMT) is required for the initiation of translation and elongation of mitochondrial protein synthesis. Pathogenic variants in MTFMT have been associated with Leigh syndrome (LS) and mitochondrial multiple respiratory chain deficiencies. We sought to elucidate the spectrum of clinical, neuroradiological and molecular genetic findings of patients with bi‐allelic pathogenic variants in MTFMT.MethodsRetrospective cohort study combining new cases and previously published cases.ResultsThirty‐eight patients with pathogenic variants in MTFMT were identified, including eight new cases. The median age of presentation was 14 months (range: birth to 17 years, interquartile range [IQR] 4.5 years), with developmental delay and motor symptoms being the most frequent initial manifestation. Twenty‐nine percent of the patients survived into adulthood. MRI headings in MTFMT pathogenic variants included symmetrical basal ganglia changes (62%), periventricular and subcortical white matter abnormalities (55%), and brainstem lesions (48%). Isolated complex I and combined respiratory chain deficiencies were identified in 31% and 59% of the cases, respectively. Reduction of the mitochondrial complex I and complex IV subunits was identified in the fibroblasts (13/13). Sixteen pathogenic variants were identified, of which c.626C>T was the most common. Seventy‐four percent of the patients were alive at their last clinical review (median 6.8 years, range: 14 months to 31 years, IQR 14.5 years).InterpretationPatients that harbour pathogenic variants in MTFMT have a milder clinical phenotype and disease progression compared to LS caused by other nuclear defects. Fibroblasts may preclude the need for muscle biopsy, to prove causality of any novel variant.