Structural insights into respiratory complex I deficiency and assembly from the mitochondrial disease-related ndufs4-/- mouse.

Respiratory complex I (NADH:ubiquinone oxidoreductase) is essential for cellular energy production and NAD+ homeostasis. Complex I mutations cause neuromuscular, mitochondrial diseases, such as Leigh Syndrome, but their molecular-level consequences remain poorly understood. Here, we use a popular complex I-linked mitochondrial disease model, the ndufs4-/- mouse, to define the structural, biochemical, and functional consequences of the absence of subunit NDUFS4. Cryo-EM analyses of the complex I from ndufs4-/- mouse hearts revealed a loose association of the NADH-dehydrogenase module, and discrete classes containing either assembly factor NDUFAF2 or subunit NDUFS6. Subunit NDUFA12, which replaces its paralogue NDUFAF2 in mature complex I, is absent from all classes, compounding the deletion of NDUFS4 and preventing maturation of an NDUFS4-free enzyme. We propose that NDUFAF2 recruits the NADH-dehydrogenase module during assembly of the complex. Taken together, the findings provide new molecular-level understanding of the ndufs4-/- mouse model and complex I-linked mitochondrial disease.

Disruption of mitochondrial complex I induces progressive parkinsonism.

Loss of functional mitochondrial complex I (MCI) in the dopaminergic neurons of the substantia nigra is a hallmark of Parkinson's disease1. Yet, whether this change contributes to Parkinson's disease pathogenesis is unclear2. Here we used intersectional genetics to disrupt the function of MCI in mouse dopaminergic neurons. Disruption of MCI induced a Warburg-like shift in metabolism that enabled neuronal survival, but triggered a progressive loss of the dopaminergic phenotype that was first evident in nigrostriatal axons. This axonal deficit was accompanied by motor learning and fine motor deficits, but not by clear levodopa-responsive parkinsonism-which emerged only after the later loss of dopamine release in the substantia nigra. Thus, MCI dysfunction alone is sufficient to cause progressive, human-like parkinsonism in which the loss of nigral dopamine release makes a critical contribution to motor dysfunction, contrary to the current Parkinson's disease paradigm3,4.

MDM2 Integrates Cellular Respiration and Apoptotic Signaling through NDUFS1 and the Mitochondrial Network.

Signaling diversity and subsequent complexity in higher eukaryotes is partially explained by one gene encoding a polypeptide with multiple biochemical functions in different cellular contexts. For example, mouse double minute 2 (MDM2) is functionally characterized as both an oncogene and a tumor suppressor, yet this dual classification confounds the cell biology and clinical literatures. Identified via complementary biochemical, organellar, and cellular approaches, we report that MDM2 negatively regulates NADH:ubiquinone oxidoreductase 75 kDa Fe-S protein 1 (NDUFS1), leading to decreased mitochondrial respiration, marked oxidative stress, and commitment to the mitochondrial pathway of apoptosis. MDM2 directly binds and sequesters NDUFS1, preventing its mitochondrial localization and ultimately causing complex I and supercomplex destabilization and inefficiency of oxidative phosphorylation. The MDM2 amino-terminal region is sufficient to bind NDUFS1, alter supercomplex assembly, and induce apoptosis. Finally, this pathway is independent of p53, and several mitochondrial phenotypes are observed in Drosophila and murine models expressing transgenic Mdm2.

Yeast NDI1 reconfigures neuronal metabolism and prevents the unfolded protein response in mitochondrial complex I deficiency.

Mutations in subunits of the mitochondrial NADH dehydrogenase cause mitochondrial complex I deficiency, a group of severe neurological diseases that can result in death in infancy. The pathogenesis of complex I deficiency remain poorly understood, and as a result there are currently no available treatments. To better understand the underlying mechanisms, we modelled complex I deficiency in Drosophila using knockdown of the mitochondrial complex I subunit ND-75 (NDUFS1) specifically in neurons. Neuronal complex I deficiency causes locomotor defects, seizures and reduced lifespan. At the cellular level, complex I deficiency does not affect ATP levels but leads to mitochondrial morphology defects, reduced endoplasmic reticulum-mitochondria contacts and activation of the endoplasmic reticulum unfolded protein response (UPR) in neurons. Multi-omic analysis shows that complex I deficiency dramatically perturbs mitochondrial metabolism in the brain. We find that expression of the yeast non-proton translocating NADH dehydrogenase NDI1, which reinstates mitochondrial NADH oxidation but not ATP production, restores levels of several key metabolites in the brain in complex I deficiency. Remarkably, NDI1 expression also reinstates endoplasmic reticulum-mitochondria contacts, prevents UPR activation and rescues the behavioural and lifespan phenotypes caused by complex I deficiency. Together, these data show that metabolic disruption due to loss of neuronal NADH dehydrogenase activity cause UPR activation and drive pathogenesis in complex I deficiency.

Abnormal morphology and function in retinal ganglion cells derived from patients-specific iPSCs generated from individuals with Leber's hereditary optic neuropathy.

Leber's hereditary optic neuropathy (LHON) is a maternally inherited eye disease that results from degeneration of retinal ganglion cells (RGC). Mitochondrial ND4 11778G > A mutation, which affects structural components of complex I, is the most prevalent LHON-associated mitochondrial DNA (mtDNA) mutation worldwide. The m.11778G > A mutation is the primary contributor underlying the development of LHON and X-linked PRICKLE3 allele (c.157C > T, p.Arg53Trp) linked to biogenesis of ATPase interacts with m.11778G > A mutation to cause LHON. However, the lack of appropriate cell and animal models of LHON has been significant obstacles for deep elucidation of disease pathophysiology, specifically the tissue-specific effects. Using RGC-like cells differentiated from induced pluripotent stem cells (iPSCs) from members of one Chinese family (asymptomatic subjects carrying only m.11778G > A mutation or PRICKLE3 p.Arg53Trp mutation, symptomatic individuals bearing both m.11778G > A and PRICKLE3 p.Arg53Trp mutations and control lacking these mutations), we demonstrated the deleterious effects of mitochondrial dysfunctions on the morphology and functions of RGCs. Notably, iPSCs bearing only m.11778G > A or p.Arg53Trp mutation exhibited mild defects in differentiation to RGC-like cells. The RGC-like cells carrying only m.11778G > A or p.Arg53Trp mutation displayed mild defects in RGC morphology, including the area of soma and numbers of neurites, electrophysiological properties, ATP contents and apoptosis. Strikingly, those RGC-like cells derived from symptomatic individuals harboring both m.11778G > A and p.Arg53Trp mutations displayed greater defects in the development, morphology and functions than those in cells bearing single mutation. These findings provide new insights into pathophysiology of LHON arising from RGC deficiencies caused by synergy between m.11778G > A and PRICKLE3 p.Arg53Trp mutation.

Digenic Leigh syndrome on the background of the m.11778G>A Leber hereditary optic neuropathy variant.

Leigh syndrome spectrum (LSS) is a primary mitochondrial disorder defined neuropathologically by a subacute necrotizing encephalomyelopathy and characterized by bilateral basal ganglia and/or brainstem lesions. LSS is associated with variants in several mitochondrial DNA genes and more than 100 nuclear genes, most often related to mitochondrial complex I (CI) dysfunction. Rarely, LSS has been reported in association with primary Leber hereditary optic neuropathy (LHON) variants of the mitochondrial DNA, coding for CI subunits (m.3460G>A in MT-ND1, m.11778G>A in MT-ND4 and m.14484T>C in MT-ND6). The underlying mechanism by which these variants manifest as LSS, a severe neurodegenerative disease, as opposed to the LHON phenotype of isolated optic neuropathy, remains an open question. Here, we analyse the exome sequencing of six probands with LSS carrying primary LHON variants, and report digenic co-occurrence of the m.11778G > A variant with damaging heterozygous variants in nuclear disease genes encoding CI subunits as a plausible explanation. Our findings suggest a digenic mechanism of disease for m.11778G>A-associated LSS, consistent with recent reports of digenic disease in individuals manifesting with LSS due to biallelic variants in the recessive LHON-associated disease gene DNAJC30 in combination with heterozygous variants in CI subunits.

Respiratory complex I with charge symmetry in the membrane arm pumps protons.

Energy-converting NADH:ubiquinone oxidoreductase, respiratory complex I, is essential for cellular energy metabolism coupling NADH oxidation to proton translocation. The mechanism of proton translocation by complex I is still under debate. Its membrane arm contains an unusual central axis of polar and charged amino acid residues connecting the quinone binding site with the antiporter-type subunits NuoL, NuoM, and NuoN, proposed to catalyze proton translocation. Quinone chemistry probably causes conformational changes and electrostatic interactions that are propagated through these subunits by a conserved pattern of predominantly lysine, histidine, and glutamate residues. These conserved residues are thought to transfer protons along and across the membrane arm. The distinct charge distribution in the membrane arm is a prerequisite for proton translocation. Remarkably, the central subunit NuoM contains a conserved glutamate residue in a position that is taken by a lysine residue in the two other antiporter-type subunits. It was proposed that this charge asymmetry is essential for proton translocation, as it should enable NuoM to operate asynchronously with NuoL and NuoN. Accordingly, we exchanged the conserved glutamate in NuoM for a lysine residue, introducing charge symmetry in the membrane arm. The stably assembled variant pumps protons across the membrane, but with a diminished H+/e- stoichiometry of 1.5. Thus, charge asymmetry is not essential for proton translocation by complex I, casting doubts on the suggestion of an asynchronous operation of NuoL, NuoM, and NuoN. Furthermore, our data emphasize the importance of a balanced charge distribution in the protein for directional proton transfer.

NDUFA8 is transcriptionally regulated by EP300/H3K27ac and promotes mitochondrial respiration to support proliferation and inhibit apoptosis in cervical cancer.

Cervical cancer, a common malignancy in women, poses a significant health burden worldwide. In this study, we aimed to investigate the expression, function, and potential mechanisms of NADH: ubiquinone oxidoreductase subunit A8 (NDUFA8) in cervical cancer. The Gene Expression Profiling Interactive Analysis (GEPIA) database and immunohistochemical scoring were used to analyze NDUFA8 expression in cervical cancer tissues and normal tissues. Quantitative real-time PCR and Western blot analyses were performed to assess the expression level of NDUFA8 in cervical cancer cell lines. NDUFA8 knockdown or overexpression experiments were conducted to evaluate its impact on cell proliferation and apoptosis. The mitochondrial respiratory status was analyzed by measuring cellular oxygen consumption, adenosine triphosphate (ATP) levels, and the expression levels of Mitochondrial Complex I activity, and Mitochondrial Complex IV-associated proteins Cytochrome C Oxidase Subunit 5B (COX5B) and COX6C. NDUFA8 exhibited high expression levels in cervical cancer tissues, and these levels were correlated with reduced survival rates. A significant upregulation of NDUFA8 expression was observed in cervical cancer cell lines compared to normal cells. Silencing NDUFA8 hindered cell proliferation, promoted apoptosis, and concurrently suppressed cellular mitochondrial respiration, resulting in decreased levels of available ATP. Conversely, NDUFA8 overexpression induced the opposite effects. Herein, we also found that E1A Binding Protein P300 (EP300) overexpression facilitated Histone H3 Lysine 27 (H3K27) acetylation enrichment, enhancing the activity of the NDUFA8 promoter region. NDUFA8, which is highly expressed in cervical cancer, is regulated by transcriptional control via EP300/H3K27 acetylation. By promoting mitochondrial respiration, NDUFA8 contributes to cervical cancer cell proliferation and apoptosis. These findings provide novel insights into NDUFA8 as a therapeutic target in cervical cancer.

Alternative splicing expands the clinical spectrum of NDUFS6-related mitochondrial disorders.

PURPOSE: We describe 3 families with Charcot-Marie-Tooth neuropathy (CMT), harboring a homozygous NDUFS6 NM_004553.6:c.309+5G>A variant previously linked to fatal Leigh syndrome. We aimed to characterize clinically and molecularly the newly identified patients and understand the mechanism underlying their milder phenotype. METHODS: The patients underwent extensive clinical examinations. Exome sequencing was done in 4 affected individuals. The functional effect of the c.309+5G>A variant was investigated in patient-derived EBV-transformed lymphoblasts at the complementary DNA, protein, and mitochondrial level. Alternative splicing was evaluated using complementary DNA long-read sequencing. RESULTS: All patients presented with early-onset, slowly progressive axonal CMT, and nystagmus; some exhibited additional central nervous system symptoms. The c.309+5G>A substitution caused the expression of aberrantly spliced transcripts and negligible levels of the canonical transcript. Immunoblotting showed reduced levels of mutant isoforms. No detectable defects in mitochondrial complex stability or bioenergetics were found. CONCLUSION: We expand the clinical spectrum of NDUFS6-related mitochondrial disorders to include axonal CMT, emphasizing the clinical and pathophysiologic overlap between these 2 clinical entities. This work demonstrates the critical role that alternative splicing may play in modulating the severity of a genetic disorder, emphasizing the need for careful consideration when interpreting splice variants and their implications on disease prognosis.

Scrutinizing a Lactococcus lactis mutant with enhanced capacity for extracellular electron transfer reveals a unique role for a novel type-II NADH dehydrogenase.

Lactococcus lactis, a lactic acid bacterium used in food fermentations and commonly found in the human gut, is known to possess a fermentative metabolism. L. lactis, however, has been demonstrated to transfer metabolically generated electrons to external electron acceptors, a process termed extracellular electron transfer (EET). Here, we investigated an L. lactis mutant with an unusually high capacity for EET that was obtained in an adaptive laboratory evolution (ALE) experiment. First, we investigated how global gene expression had changed, and found that amino acid metabolism and nucleotide metabolism had been affected significantly. One of the most significantly upregulated genes encoded the NADH dehydrogenase NoxB. We found that this upregulation was due to a mutation in the promoter region of NoxB, which abolished carbon catabolite repression. A unique role of NoxB in EET could be attributed and it was directly verified, for the first time, that NoxB could support respiration in L. lactis. NoxB, was shown to be a novel type-II NADH dehydrogenase that is widely distributed among gut microorganisms. This work expands our understanding of EET in Gram-positive electroactive microorganisms and the special significance of a novel type-II NADH dehydrogenase in EET.IMPORTANCEElectroactive microorganisms with extracellular electron transfer (EET) ability play important roles in biotechnology and ecosystems. To date, there have been many investigations aiming at elucidating the mechanisms behind EET, and determining the relevance of EET for microorganisms in different niches. However, how EET can be enhanced and harnessed for biotechnological applications has been less explored. Here, we compare the transcriptomes of an EET-enhanced L. lactis mutant with its parent and elucidate the underlying reason for its superior performance. We find that one of the most significantly upregulated genes is the gene encoding the NADH dehydrogenase NoxB, and that upregulation is due to a mutation in the catabolite-responsive element that abolishes carbon catabolite repression. We demonstrate that NoxB has a special role in EET, and furthermore show that it supports respiration to oxygen, which has never been done previously. In addition, a search reveals that this novel NoxB-type NADH dehydrogenase is widely distributed among gut microorganisms.

Mitochondrial gene defects in Arabidopsis can broadly affect mitochondrial gene expression through copy number.

How mitochondria regulate the expression of their genes is poorly understood, partly because methods have not been developed for stably transforming mitochondrial genomes. In recent years, the disruption of mitochondrial genes has been achieved in several plant species using mitochondria-localized TALEN (mitoTALEN). In this study, we attempted to disrupt the NADH dehydrogenase subunit7 (NAD7) gene, a subunit of respiratory chain complex I, in Arabidopsis (Arabidopsis thaliana) using the mitoTALEN method. In some of the transformants, disruption of NAD7 was accompanied by severe growth inhibition and lethality, suggesting that NAD7 has an essential function in Arabidopsis. In addition, the mitochondrial genome copy number and overall expression of genes encoding mitochondrial proteins were generally increased by nad7 knockout. Similar increases were also observed in mutants with decreased NAD7 transcripts and with dysfunctions of other mitochondrial respiratory complexes. In these mutants, the expression of nuclear genes involved in mitochondrial translation or protein transport was induced in sync with mitochondrial genes. Mitochondrial genome copy number was also partly regulated by the nuclear stress-responsive factors NAC domain containing protein 17 and Radical cell death 1. These findings suggest the existence of overall gene-expression control through mitochondrial genome copy number in Arabidopsis and that disruption of single mitochondrial genes can have additional broad consequences in both the nuclear and mitochondrial genomes.

Evolutionary characteristics of the mitochondrial NADH dehydrogenase subunit 6 gene in some populations of four sympatric Mustela species (Mustelidae, Mammalia) from central Europe.

BACKGROUND: Selection on or reticulate evolution of mtDNA is documented in various mammalian taxa and could lead to misleading phylogenetic conclusions if not recognized. We sequenced the MT-ND6 gene of four sympatric Mustelid species of the genus Mustela from some central European populations. We hypothesised positive selection on MT-ND6, given its functional importance and the different body sizes and life histories of the species, even though climatic differences may be unimportant for adaptation in sympatry. METHODS AND RESULTS: MT-ND6 genes were sequenced in 187 sympatric specimens of weasels, Mustela nivalis, stoats, M. erminea, polecats, M. putorius, and steppe polecats, M. eversmannii, from eastern Austria and of fourteen allopatric polecats from eastern-central Germany. Median joining networks, neighbour joining and maximum likelihood analyses as well as Bayesian inference grouped all species according to earlier published phylogenetic models. However, polecats and steppe polecats, two very closely related species, shared the same two haplotypes. We found only negative selection within the Mustela sequences, including 131 downloaded ones covering thirteen species. Positive selection was observed on three MT-ND6 codons of other mustelid genera retrieved from GenBank. CONCLUSIONS: Negative selection for MT-ND6 within the genus Mustela suggests absence of both environmental and species-specific effects of cellular energy metabolism despite large species-specific differences in body size. The presently found shared polymorphism in European polecats and steppe polecats may result from ancestral polymorphism before speciation and historical or recent introgressive hybridization; it may indicate mtDNA capture of steppe polecats by M. putorius in Europe.

Ocular stress enhances contralateral transfer of lenadogene nolparvovec gene therapy through astrocyte networks.

Lenadogene nolparvovec (GS010) was developed to treat a point mutation in mitochondrial ND4 that causes Leber hereditary optic neuropathy. GS010 delivers human cDNA encoding wild-type ND4 packaged into an rAAV2/2 vector that transduces retinal ganglion cells, to induce allotopic expression of hybrid mitochondrial ND4. GS010 clinical trials improved best-corrected visual acuity (BCVA) up to 5 years after treatment. Interestingly, unilateral treatment improved BCVA bilaterally. Subsequent studies revealed GS010 DNA in visual tissues contralateral to the injected eye, suggesting migration. Here we tested whether unilateral intraocular pressure (IOP) elevation could influence the transfer of viral ND4 RNA in contralateral tissues after GS010 delivery to the IOP-elevated eye and probed a potential mechanism mediating translocation in mice. We found IOP elevation enhanced viral ND4 RNA transcripts in contralateral visual tissues, including retinas. Using conditional transgenic mice, we depleted astrocytic gap junction connexin 43 (Cx43), required for distant redistribution of metabolic resources between astrocytes during stress. After unilateral IOP elevation and GS010 injection, Cx43 knockdown eradicated ND4 RNA transcript detection in contralateral retinal tissues, while transcript was still detectable in optic nerves. Overall, our study indicates long-range migration of GS010 product to contralateral visual tissues is enhanced by Cx43-linked astrocyte networks.

Leishmania type II dehydrogenase is essential for parasite viability irrespective of the presence of an active complex I.

Type II NADH dehydrogenases (NDH2) are monotopic enzymes present in the external or internal face of the mitochondrial inner membrane that contribute to NADH/NAD+ balance by conveying electrons from NADH to ubiquinone without coupled proton translocation. Herein, we characterize the product of a gene present in all species of the human protozoan parasite Leishmania as a bona fide, matrix-oriented, type II NADH dehydrogenase. Within mitochondria, this respiratory activity concurs with that of type I NADH dehydrogenase (complex I) in some Leishmania species but not others. To query the significance of NDH2 in parasite physiology, we attempted its genetic disruption in two parasite species, exhibiting a silent (Leishmania infantum, Li) and a fully operational (Leishmania major, Lm) complex I. Strikingly, this analysis revealed that NDH2 abrogation is not tolerated by Leishmania, not even by complex I-expressing Lm species. Conversely, complex I is dispensable in both species, provided that NDH2 is sufficiently expressed. That a type II dehydrogenase is essential even in the presence of an active complex I places Leishmania NADH metabolism into an entirely unique perspective and suggests unexplored functions for NDH2 that span beyond its complex I-overlapping activities. Notably, by showing that the essential character of NDH2 extends to the disease-causing stage of Leishmania, we genetically validate NDH2-an enzyme without a counterpart in mammals-as a candidate target for leishmanicidal drugs.

Phylogenetic relationship of Prohemistomum vivax to other trematodes based on the internal transcribed spacer region and mitochondrial genes.

Prohemistomum vivax is a zoonotic small cyathocotylid trematode that inhabits the intestines of fish-eating birds and mammals. Here, we amplified the internal transcribed spacer (ITS) sequence and six mitochondrial protein-coding genes (PCGs) from P. vivax. The ITS region was 1389 base pairs long and had a partial 18S ribosomal RNA gene, a full ITS1, 5.8S rRNA, and ITS2 sequence, and a partial 28S rRNA gene. The ITS region of P. vivax showed a minimum pairwise distance (0.3-0.6%) from the ITS sequences of Cyathocotylidae sp. 1 and 2 metacercariae from Clarias gariepinus. This result suggests that these metacercariae belong to P. vivax metacercariae. We first amplified mitochondrial genes from P. vivax, including cytochrome c oxidase subunit III (cox3) partial sequence; tRNA-His, cytochrome b (cytb), and NADH dehydrogenase subunit 4L (nad4L) complete sequences; and NADH dehydrogenase subunit 4 (nad4), cytochrome c oxidase I (cox1), and NADH dehydrogenase subunit 5 (nad5) partial sequences. P. vivax was most closely related to Cyathocotyle prussica (NC_039780) and Holostephanus sp. (OP082179), with cox1, cox3, and cytb genes conserved among the three trematodes. The ML phylogenetic tree of ITS sequences supports the order Diplostomida, divided into two main clades (the superfamily Diplostomoidea and Schistosomatoidea). The phylogeny of concatenated amino acid sequences of P. vivax six PCGs revealed that diplostomoids and Clinostomum sp. evolved in a clade with Plagiorchiida members, away from Schistosoma species. These results may yield ribosomal and mitochondrial genetic markers for molecular epidemiological investigations of cyathocotylid intestinal flukes.

Intra- and interspecies variation and population dynamics of Fasciola gigantica among ruminants in Sudan.

Fasciola gigantica is a widespread parasite that causes neglected disease in livestock worldwide. Its high transmissibility and dispersion are attributed to its ability to infect intermediate snail hosts and adapt to various mammalian definitive hosts. This study investigated the variation and population dynamics of F. gigantica in cattle, sheep, and goats from three states in Sudan. Mitochondrial cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit 1 (ND1) genes were sequenced successfully to examine intra and interspecific differences. ND1 exhibited higher diversity than COI, with 15 haplotypes and 10 haplotypes, respectively. Both genes had high haplotype diversity but low nucleotide diversity, with 21 and 11 polymorphic sites for ND1 and COI, respectively. Mismatch distribution analysis and neutrality tests revealed that F. gigantica from different host species was in a state of population expansion. Maximum likelihood phylogenetic trees and median networks revealed that F. gigantica in Sudan and other African countries had host-specific and country-specific lineages for both genes. The study also indicated that F. gigantica-infected small ruminants were evolutionarily distant, suggesting deep and historical interspecies adaptation.

A small protein coded within the mitochondrial canonical gene nd4 regulates mitochondrial bioenergetics.

BACKGROUND: Mitochondria have a central role in cellular functions, aging, and in certain diseases. They possess their own genome, a vestige of their bacterial ancestor. Over the course of evolution, most of the genes of the ancestor have been lost or transferred to the nucleus. In humans, the mtDNA is a very small circular molecule with a functional repertoire limited to only 37 genes. Its extremely compact nature with genes arranged one after the other and separated by short non-coding regions suggests that there is little room for evolutionary novelties. This is radically different from bacterial genomes, which are also circular but much larger, and in which we can find genes inside other genes. These sequences, different from the reference coding sequences, are called alternatives open reading frames or altORFs, and they are involved in key biological functions. However, whether altORFs exist in mitochondrial protein-coding genes or elsewhere in the human mitogenome has not been fully addressed. RESULTS: We found a downstream alternative ATG initiation codon in the + 3 reading frame of the human mitochondrial nd4 gene. This newly characterized altORF encodes a 99-amino-acid-long polypeptide, MTALTND4, which is conserved in primates. Our custom antibody, but not the pre-immune serum, was able to immunoprecipitate MTALTND4 from HeLa cell lysates, confirming the existence of an endogenous MTALTND4 peptide. The protein is localized in mitochondria and cytoplasm and is also found in the plasma, and it impacts cell and mitochondrial physiology. CONCLUSIONS: Many human mitochondrial translated ORFs might have so far gone unnoticed. By ignoring mtaltORFs, we have underestimated the coding potential of the mitogenome. Alternative mitochondrial peptides such as MTALTND4 may offer a new framework for the investigation of mitochondrial functions and diseases.

Pathogenic Bi-allelic Mutations in NDUFAF8 Cause Leigh Syndrome with an Isolated Complex I Deficiency.

Leigh syndrome is one of the most common neurological phenotypes observed in pediatric mitochondrial disease presentations. It is characterized by symmetrical lesions found on neuroimaging in the basal ganglia, thalamus, and brainstem and by a loss of motor skills and delayed developmental milestones. Genetic diagnosis of Leigh syndrome is complicated on account of the vast genetic heterogeneity with >75 candidate disease-associated genes having been reported to date. Candidate genes are still emerging, being identified when "omics" tools (genomics, proteomics, and transcriptomics) are applied to manipulated cell lines and cohorts of clinically characterized individuals who lack a genetic diagnosis. NDUFAF8 is one such protein; it has been found to interact with the well-characterized complex I (CI) assembly factor NDUFAF5 in a large-scale protein-protein interaction screen. Diagnostic next-generation sequencing has identified three unrelated pediatric subjects, each with a clinical diagnosis of Leigh syndrome, who harbor bi-allelic pathogenic variants in NDUFAF8. These variants include a recurrent splicing variant that was initially overlooked due to its deep-intronic location. Subject fibroblasts were found to express a complex I deficiency, and lentiviral transduction with wild-type NDUFAF8-cDNA ameliorated both the assembly defect and the biochemical deficiency. Complexome profiling of subject fibroblasts demonstrated a complex I assembly defect, and the stalled assembly intermediates corroborate the role of NDUFAF8 in early complex I assembly. This report serves to expand the genetic heterogeneity associated with Leigh syndrome and to validate the clinical utility of orphan protein characterization. We also highlight the importance of evaluating intronic sequence when a single, definitively pathogenic variant is identified during diagnostic testing.

Optimized allotopic expression of mitochondrial ND6 transgene restored complex I and apoptosis deficiencies caused by LHON-linked ND6 14484T > C mutation.

BACKGROUND: Leber's hereditary optic neuropathy (LHON) is a maternally inherited eye disease due to mutations in mitochondrial DNA. However, there is no effective treatment for this disease. LHON-linked ND6 14484T > C (p.M64V) mutation caused complex I deficiency, diminished ATP production, increased production of reactive oxygen species (ROS), elevated apoptosis, and impaired mitophagy. Here, we investigated if the allotopic expression of human mitochondrial ND6 transgene corrected the mitochondrial dysfunctions due to LHON-associated m.14484T > C mutation. METHODS: Nucleus-versions of ND6 was generated by changing 6 non-universal codons with universal codons and added to mitochondrial targeting sequence of COX8. Stable transfectants were generated by transferring human ND6 cDNA expressed in a pCDH-puro vector into mutant cybrids carrying the m.14484T > C mutation and control cybrids. The effect of allotopic expression of ND6 on oxidative phosphorylation (OXPHOS) was evaluated using Blue Native gel electrophoresis and extracellular flux analyzer. Assessment of ROS production in cell lines was performed by flow cytometry with MitoSOX Red reagent. Analyses for apoptosis and mitophagy were undertaken via flow cytometry, TUNEL and immunofluorescence assays. RESULTS: The transfer of human ND6 into the cybrids carrying the m.14484T > C mutation raised the levels of ND6, ND1 and ND4L but did not change the levels of other mitochondrial proteins. The overexpression of ND6 led to 20~23% increases in the assembly and activity of complex I, and ~ 53% and ~ 33% increases in the levels of mitochondrial ATP and ΔΨm in the mutant cybrids bearing m.14484T > C mutation. Furthermore, mutant cybrids with overexpression of ND6 exhibited marked reductions in the levels of mitochondrial ROS. Strikingly, ND6 overexpression markedly inhibited the apoptosis process and restored impaired mitophagy in the cells carrying m.14484T > C mutation. However, overexpression of ND6 did not affect the ND6 level and mitochondrial functions in the wild-type cybrids, indicating that this ND6 level appeared to be the maximum threshold level to maintain the normal cell function. CONCLUSION: We demonstrated that allotopic expression of nucleus-versions of ND6 restored complex I, apoptosis and mitophagy deficiencies caused by the m.14484T > C mutation. The restoration of m.14484T > C mutation-induced mitochondrial dysfunctions by overexpression of ND6 is a step toward therapeutic interventions for LHON and mitochondrial diseases.

Whole genome and exome sequencing identify NDUFV2 mutations as a new cause of progressive cavitating leukoencephalopathy.

BACKGROUND: Progressive cavitating leukoencephalopathy (PCL) is thought to result from mutations in nuclear genes affecting mitochondrial function and energy metabolism. To date, mutations in two subunits of complex I, NDUFS1 and NDUFV1, have been reported to be related to PCL. METHODS: Patients underwent clinical examinations, brain MRI, skin biopsy and muscle biopsy. Whole-genome or whole-exome sequencing was performed on the index patients from two unrelated families with PCL. The effects of the mutations were examined through complementation of the NDUFV2 mutation by cDNA expression. RESULTS: The common clinical features of the patients in this study were recurring episodes of acute or subacute developmental regression that appeared in the first years of life, followed by gradual remissions and prolonged periods of stability. MRI showed leukoencephalopathy with multiple cavities. Three novel NDUFV2 missense mutations were identified in these families. Complex I deficiency was confirmed in affected individuals' fibroblasts and a muscle biopsy. Functional and structural analyses revealed that these mutations affect the structural stability and function of the NDUFV2 protein, indicating that defective NDUFV2 function is responsible for the phenotypes in these individuals. CONCLUSIONS: Here, we report the clinical presentations, neuroimaging and molecular and functional analyses of novel mutations in NDUFV2 in two sibling pairs of two Chinese families presenting with PCL. We hereby expand the knowledge on the clinical phenotypes associated with mutations in NDUFV2 and the genotypes causative for PCL.