A Drosophila model of mitochondrial disease phenotypic heterogeneity.

Mutations in genes that affect mitochondrial function cause primary mitochondrial diseases. Mitochondrial diseases are highly heterogeneous and even patients with the same mitochondrial disease can exhibit broad phenotypic heterogeneity, which is poorly understood. Mutations in subunits of mitochondrial respiratory complex I cause complex I deficiency, which can result in severe neurological symptoms and death in infancy. However, some complex I deficiency patients present with much milder symptoms. The most common nuclear gene mutated in complex I deficiency is the highly conserved core subunit NDUFS1. To model the phenotypic heterogeneity in complex I deficiency, we used RNAi lines targeting the Drosophila NDUFS1 homolog ND-75 with different efficiencies. Strong knockdown of ND-75 in Drosophila neurons resulted in severe behavioural phenotypes, reduced lifespan, altered mitochondrial morphology, reduced endoplasmic reticulum (ER)-mitochondria contacts and activation of the unfolded protein response (UPR). By contrast, weak ND-75 knockdown caused much milder behavioural phenotypes and changes in mitochondrial morphology. Moreover, weak ND-75 did not alter ER-mitochondria contacts or activate the UPR. Weak and strong ND-75 knockdown resulted in overlapping but distinct transcriptional responses in the brain, with weak knockdown specifically affecting proteosome activity and immune response genes. Metabolism was also differentially affected by weak and strong ND-75 knockdown including gamma-aminobutyric acid (GABA) levels, which may contribute to neuronal dysfunction in ND-75 knockdown flies. Several metabolic processes were only affected by strong ND-75 knockdown including the pentose phosphate pathway and the metabolite 2-hydroxyglutarate (2-HG), suggesting 2-HG as a candidate biomarker of severe neurological mitochondrial disease. Thus, our Drosophila model provides the means to dissect the mechanisms underlying phenotypic heterogeneity in mitochondrial disease.

Unique prenatal manifestations of biallelic NDUFAF5 variants: expansion of phenotype.

OBJECTIVE: Mitochondrial complex-I deficiency, nuclear type 16, is a rare autosomal recessive disorder caused by biallelic pathogenic variants in NDUFAF5 (C20orf7) (OMIM 618238). The aim of this study was to describe a severe early prenatal manifestation of this disorder, which was previously considered to occur only postnatally. METHODS: This was a multicenter retrospective case series including five fetuses from three non-related families, which shared common sonographic abnormalities, including brain cysts, corpus callosal malformations, non-immune hydrops fetalis and growth restriction. Genetic evaluation included chromosomal microarray analysis and exome sequencing. Two fetuses from the same family were also available for pathology examination, including electron microscopy. RESULTS: Chromosomal microarray analysis revealed no chromosomal abnormality in any of the tested cases. Trio exome sequencing demonstrated that three affected fetuses from three unrelated families were compound heterozygous or homozygous for likely pathogenic variants in NDUFAF5. No other causative variants were detected. The association between NDUFAF5 variants and fetal malformations was further confirmed by segregation analysis. Histological evaluation of fetal tissues and electron microscopy of the skeletal muscle, liver, proximal tubules and heart demonstrated changes that resembled postmortem findings in patients with mitochondrial depletion disorders as well as previously undescribed findings. CONCLUSIONS: Mitochondrial complex-I deficiency and specifically biallelic mutations in NDUFAF5 have a role in abnormal fetal development, presenting with severe congenital malformations. Mitochondrial complex-I disorders should be considered in the differential diagnosis of corpus callosal malformations and brain cysts, especially when associated with extracranial abnormalities, such as fetal growth restriction and non-immune hydrops fetalis. © 2023 International Society of Ultrasound in Obstetrics and Gynecology.

Transgenic NADH dehydrogenase restores oxygen regulation of breathing in mitochondrial complex I-deficient mice.

The hypoxic ventilatory response (HVR) is a life-saving reflex, triggered by the activation of chemoreceptor glomus cells in the carotid body (CB) connected with the brainstem respiratory center. The molecular mechanisms underlying glomus cell acute oxygen (O2) sensing are unclear. Genetic disruption of mitochondrial complex I (MCI) selectively abolishes the HVR and glomus cell responsiveness to hypoxia. However, it is unknown what functions of MCI (metabolic, proton transport, or signaling) are essential for O2 sensing. Here we show that transgenic mitochondrial expression of NDI1, a single-molecule yeast NADH/quinone oxidoreductase that does not directly contribute to proton pumping, fully recovers the HVR and glomus cell sensitivity to hypoxia in MCI-deficient mice. Therefore, maintenance of mitochondrial NADH dehydrogenase activity and the electron transport chain are absolutely necessary for O2-dependent regulation of breathing. NDI1 expression also rescues other systemic defects caused by MCI deficiency. These data explain the role of MCI in acute O2 sensing by arterial chemoreceptors and demonstrate the optimal recovery of complex organismal functions by gene therapy.

Noninvasive Ophthalmic Imaging Measures Retinal Degeneration and Vision Deficits in Ndufs4-/- Mouse Model of Mitochondrial Complex I Deficiency.

Purpose: To characterize postnatal ocular pathology in a Ndufs4-/- mouse model of complex I deficiency using noninvasive retinal imaging and visual testing. Methods: Ndufs4-/- mice and wild-type (WT) littermates were analyzed at 3, 5, and 7 weeks postnatal. Retinal morphology was visualized by optical coherence tomography (OCT). OCT images were analyzed for changes in retinal thickness and reflectivity profiles. Visual function was assessed by electroretinogram (ERG) and optomotor reflex (OMR). Results: Ndufs4-/- animals have normal OCT morphology at weaning and develop inner plexiform layer atrophy over weeks 5 to 7. Outer retinal layers show hyporeflectivity of the external limiting membrane (ELM) and photoreceptor ellipsoid zone (EZ). Retinal function is impaired at 3 weeks, with profound deficits in b-wave, a-wave, and oscillatory potential amplitudes. The b-wave and oscillatory potential implicit times are delayed, but the a-wave implicit time is unaffected. Ndufs4-/- animals have normal OMR at 3 weeks and present with increasing acuity and contrast OMR deficits at 5 and 7 weeks. Physiological thinning of inner retinal layers, attenuation of ELM reflectivity, and attenuation of ERG b- and a-wave amplitudes occur in WT C57BL/6 littermates between weeks 3 and 7. Conclusions: Noninvasive ocular imaging captures early-onset retinal degeneration in Ndufs4-/- mice and is a tractable approach for investigating retinal pathology subsequent to complex I deficiency. Translational Relevance: Ophthalmic imaging captures clinically relevant measures of retinal disease in a fast-progressing mouse model of complex I deficiency consistent with human Leigh syndrome.

Lip cyanosis as the first symptom of Leigh syndrome associated with mitochondrial complex I deficiency due to a compound heterozygous NDUFS1 mutation: A case report.

BACKGROUND: Leigh syndrome (LS) is a rare, progressive, and fatal neurodegenerative disease that occurs mainly in infants and children. Neonatal LS has not yet been fully described. METHODS: The study design was approved by the ethics review board of Shenzhen Children's Hospital. RESULTS: A 24-day-old full-term male infant presented with a 2-day history of lip cyanosis when crying in September 2021. He was born to nonconsanguineous Asian parents. After birth, the patient was fed poorly. A recurrent decrease in peripheral oxygen saturation and difficulty in weaning from mechanical ventilation during hospitalization were observed. There were no abnormalities on brain magnetic resonance imaging (MRI) or blood and urine organic acid analyses on admission. His lactic acid level increased markedly, and repeat MRI showed symmetrical abnormal signal areas in the bilateral basal ganglia and brainstem with disease progression. Trio whole-exome sequencing revealed 2 heterozygous mutations (c.64C > T [p.R22X] and c.584T > C [p.L195S]) in NDUFS1. Based on these findings, mitochondrial respiratory chain complex I deficiency-related LS was diagnosed. The patient underwent tracheal intubation and mechanical ventilation for respiratory failure. His oxygen saturation levels were maintained at normal levels with partially assisted ventilation. He was administered broad-spectrum antibiotics, oral coenzyme Q10, multivitamins, and idebenone. During hospitalization, the patient developed progressive consciousness impairment and respiratory and circulatory failure. He died on day 30. CONCLUSION: Lip cyanosis is an important initial symptom in LS. Mild upper respiratory tract infections can induce LS and aggravate the disease. No abnormal changes in the brain MRI were observed in the early LS stages in this patient. Multiple MRIs and blood lactic acid tests during disease progression and genetic testing are important for prompt and accurate diagnosis of LS.

Metabolic rescue ameliorates mitochondrial encephalo-cardiomyopathy in murine and human iPSC models of Leigh syndrome.

BACKGROUND: Mice with deletion of complex I subunit Ndufs4 develop mitochondrial encephalomyopathy resembling Leigh syndrome (LS). The metabolic derangement and underlying mechanisms of cardio-encephalomyopathy in LS remains incompletely understood. METHODS: We performed echocardiography, electrophysiology, confocal microscopy, metabolic and molecular/morphometric analysis of the mice lacking Ndufs4. HEK293 cells, human iPS cells-derived cardiomyocytes and neurons were used to determine the mechanistic role of mitochondrial complex I deficiency. RESULTS: LS mice develop severe cardiac bradyarrhythmia and diastolic dysfunction. Human-induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) with Ndufs4 deletion recapitulate LS cardiomyopathy. Mechanistically, we demonstrate a direct link between complex I deficiency, decreased intracellular (nicotinamide adenine dinucleotide) NAD+ /NADH and bradyarrhythmia, mediated by hyperacetylation of the cardiac sodium channel NaV 1.5, particularly at K1479 site. Neuronal apoptosis in the cerebellar and midbrain regions in LS mice was associated with hyperacetylation of p53 and activation of microglia. Targeted metabolomics revealed increases in several amino acids and citric acid cycle intermediates, likely due to impairment of NAD+ -dependent dehydrogenases, and a substantial decrease in reduced Glutathione (GSH). Metabolic rescue by nicotinamide riboside (NR) supplementation increased intracellular NAD+ / NADH, restored metabolic derangement, reversed protein hyperacetylation through NAD+ -dependent Sirtuin deacetylase, and ameliorated cardiomyopathic phenotypes, concomitant with improvement of NaV 1.5 current and SERCA2a function measured by Ca2+ -transients. NR also attenuated neuronal apoptosis and microglial activation in the LS brain and human iPS-derived neurons with Ndufs4 deletion. CONCLUSIONS: Our study reveals direct mechanistic explanations of the observed cardiac bradyarrhythmia, diastolic dysfunction and neuronal apoptosis in mouse and human induced pluripotent stem cells (iPSC) models of LS.

Case report: novel mutations of NDUFS6 and NHLRC2 genes potentially cause the quick postnatal death of a Chinese Hani minority neonate with mitochondrial complex I deficiency and FINCA syndrome.

INTRODUCTION: Mitochondrial complex I deficiency (MCID) and abbFINCA syndrome are lethal congenital diseases and cases in the neonatal period are rarely reported. Here, we identified a Chinese Hani minority neonate with rare MCID and FINCA syndrome. This study was to analyze the clinical manifestations and pathogenic gene variations, and to investigate causes of quick postnatal death of patient and possible molecular pathogenic mechanisms. PATIENT CONCERNS: A 17-day-old patient had reduced muscle tension, diminished primitive reflexes, significantly abnormal blood gas analysis, and progressively increased blood lactate and blood glucose. Imaging studies revealed pneumonia, pulmonary hypertension, and brain abnormalities. DIAGNOSIS: Whole-exome sequencing revealed that the NDUFS6 gene of the patient carried c. 344G > T (p.C115F) novel homozygous variation, and the NHLRC2 gene carried c. 1749C > G (p.F583L) and c. 2129C > T (p.T710M) novel compound heterozygous variation. INTERVENTIONS AND OUTCOMES: The patient was given endotracheal intubation, respiratory support, high-frequency ventilation, antishock therapy, as well as iNO and Alprostadil to reduce pulmonary hypertension and maintain homeostatic equilibrium. However, the patient was critically ill and died in 27 days. CONCLUSION: The patient has MCID due to a novel mutation in NDUFS6 and FINCA syndrome due to novel mutations in NHLRC2, which is the main reason for the rapid onset and quick death of the patient.

Early Forms of α-Synuclein Pathology Are Associated with Neuronal Complex I Deficiency in the Substantia Nigra of Individuals with Parkinson's Disease.

Idiopathic Parkinson's disease (iPD) is characterized by degeneration of the dopaminergic substantia nigra pars compacta (SNc), typically in the presence of Lewy pathology (LP) and mitochondrial respiratory complex I (CI) deficiency. LP is driven by α-synuclein aggregation, morphologically evolving from early punctate inclusions to Lewy bodies (LBs). The relationship between α-synuclein aggregation and CI deficiency in iPD is poorly understood. While studies in models suggest they are causally linked, observations in human SNc show that LBs preferentially occur in CI intact neurons. Since LBs are end-results of α-synuclein aggregation, we hypothesized that the relationship between LP and CI deficiency may be better reflected in neurons with early-stage α-synuclein pathology. Using quadruple immunofluorescence in SNc tissue from eight iPD subjects, we assessed the relationship between neuronal CI or CIV deficiency and early or late forms of LP. In agreement with previous findings, we did not observe CI-negative neurons with late LP. In contrast, early LP showed a significant predilection for CI-negative neurons (p = 6.3 × 10-5). CIV deficiency was not associated with LP. Our findings indicate that early α-syn aggregation is associated with CI deficiency in iPD, and suggest a double-hit mechanism, where neurons exhibiting both these pathologies are selectively lost.

The decylTPP mitochondria-targeting moiety lowers electron transport chain supercomplex levels in primary human skin fibroblasts.

Attachment of cargo molecules to lipophilic triphenylphosphonium (TPP+) cations is a widely applied strategy for mitochondrial targeting. We previously demonstrated that the vitamin E-derived antioxidant Trolox increases the levels of active mitochondrial complex I (CI), the first complex of the electron transport chain (ETC), in primary human skin fibroblasts (PHSFs) of Leigh Syndrome (LS) patients with isolated CI deficiency. Primed by this finding, we here studied the cellular effects of mitochondria-targeted Trolox (MitoE10), mitochondria-targeted ubiquinone (MitoQ10) and their mitochondria-targeting moiety decylTPP (C10-TPP+). Chronic treatment (96 h) with these molecules of PHSFs from a healthy subject and an LS patient with isolated CI deficiency (NDUFS7-V122M mutation) did not greatly affect cell number. Unexpectedly, this treatment reduced CI levels/activity, lowered the amount of ETC supercomplexes, inhibited mitochondrial oxygen consumption, increased extracellular acidification, altered mitochondrial morphology and stimulated hydroethidine oxidation. We conclude that the mitochondria-targeting decylTPP moiety is responsible for the observed effects and advocate that every study employing alkylTPP-mediated mitochondrial targeting should routinely include control experiments with the corresponding alkylTPP moiety.

Mesenchymal stem cells improve redox homeostasis and mitochondrial respiration in fibroblast cell lines with pathogenic MT-ND3 and MT-ND6 variants.

The most frequent biochemical defect of inherited mitochondrial disease is isolated complex I deficiency. There is no cure for this disorder, and treatment is mainly supportive. In this study, we investigated the effects of human mesenchymal stem cells (MSCs) on skin fibroblast derived from three individuals with complex I deficiency carrying different pathogenic variants in mitochondrial DNA-encoded subunits (MT-ND3, MT-ND6). Complex I-deficient fibroblasts were transiently co-cultured with bone marrow-derived MSCs. Mitochondrial transfer was analysed by fluorescence labelling and validated by Sanger sequencing. Levels of reactive oxygen species (ROS) were measured using MitoSOX Red. Moreover, mitochondrial respiration was analysed by Seahorse XFe96 Extracellular Flux Analyzer. Levels of antioxidant proteins were investigated via immunoblotting. Co-culturing of complex I-deficient fibroblast with MSCs lowered cellular ROS levels. The effect on ROS production was more sustained compared to treatment of patient fibroblasts with culture medium derived from MSC cultures. Investigation of cellular antioxidant defence systems revealed an upregulation of SOD2 (superoxide dismutase 2, mitochondrial) and HO-1 (heme oxygenase 1) in patient-derived cell lines. This adaptive response was normalised upon MSC treatment. Moreover, Seahorse experiments revealed a significant improvement of mitochondrial respiration, indicating a mitigation of the oxidative phosphorylation defect. Experiments with repetitive MSC co-culture at two consecutive time points enhanced this effect. Our study indicates that MSC-based treatment approaches might constitute an interesting option for patients with mitochondrial DNA-encoded mitochondrial diseases. We suggest that this strategy may prove more promising for defects caused by mitochondrial DNA variants compared to nuclear-encoded defects.

Compound heterozygous mutations of NDUFV1 identified in a child with mitochondrial complex I deficiency.

BACKGROUND: Mitochondrial complex I deficiency (MCID) is the most common biochemical defect identified in childhood with mitochondrial diseases, mainly including Leigh syndrome, encephalopathy, macrocephaly with progressive leukodystrophy, hypertrophic cardiomyopathy and myopathy. OBJECTIVE: To identify genetic cause in a patient with early onset autosomal recessive MCID. METHODS: Trio whole-exome sequencing was performed and phenotype-related data analyses were conducted. All candidate mutations were confirmed by Sanger sequencing. RESULTS: Here we report a child of Leigh syndrome presented with global developmental delay, progressive muscular hypotonia and myocardial damage. A missense mutation c.118C > T (p.Arg40Trp) and a previously reported mutation c.1157G > A (p.Arg386His) in NDUFV1 have been identified as compound heterozygous in the patient. The mutation p.Arg386His is closely associated with the impairment of 4Fe-4S domain and this mutation has been reported pathogenic. The c.118C > T mutation has not been reported in ClinVar and HGMD database. In silico protein analyses showed that p.Arg40 is highly conserved in a wide range of species, and the amino acid substitution p.Trp40 largely decreases the stability of NDUFV1. In addition, the mutation has not been detected in the Asian populations and it was predicted to be deleterious by numerous prediction tools. CONCLUSION: This research expands the mutation spectrum of NDUFV1 and substantially provides an early and accurate diagnosis basis of MCID, which would benefit subsequently effective genetic counseling and prenatal diagnosis for future reproduction of the family.

Restoring cellular NAD(P)H levels by PPARα and LXRα stimulation to improve mitochondrial complex I deficiency.

Mitochondrial complex I (CI), the first multiprotein enzyme complex of the oxidative phosphorylation system, plays a crucial role in cellular energy production. CI deficiency is associated with a variety of clinical phenotypes, including Leigh syndrome. At the cellular level, an increased NAD(P)H concentration is one of the hallmarks in CI-deficiency. AIMS: Here, we aimed to attenuate increased NAD(P)H levels by stimulation of ATP-dependent cassette (ABC)A1 and ABCG1-mediated cellular cholesterol efflux with various PPARα and LXRα agonists. MAIN METHODS: Mitochondrial CI-deficient fibroblasts and chemically-induced CI-deficient HeLa cells were used to study the dose-dependent effects of various PPARα and LXRα agonists on cellular NAD(P)H levels and cholesterol efflux. KEY FINDINGS: In patient-derived mitochondrial CI-deficient fibroblasts, GW590735, astaxanthin, oleoylethanolamide, and GW3965 significantly reduced the enhanced NAD(P)H levels in CI-deficient fibroblasts. Similar effects were observed in chemically-induced CI-impaired HeLa cells, in which BMS-687453, Wy14643, GW7647, T0901317, DMHCA also demonstrated a beneficial effect. Surprisingly, no effect on ABCA1- and ABCG1-mediated cholesterol efflux in HeLa cells and fibroblasts was found after treatment with these compounds. The reduction in NAD(P)H levels by GW590735 could be partially reversed by inhibition of fatty acid synthase and ß-oxidation, which suggests that its beneficial effects are possibly mediated via stimulation of fatty acid metabolism rather than cholesterol efflux. SIGNIFICANCE: Collectively, PPARα and LXRα stimulation resulted in attenuated cellular NAD(P)H levels in CI-impaired HeLa cells and patient-derived fibroblasts and could eventually have a therapeutic potential in CI deficiency.

Mitochondrial complex I subunit deficiency promotes pancreatic α-cell proliferation.

OBJECTIVE: There is strong evidence that mitochondrial DNA mutations and mitochondrial dysfunction play a role in diabetes pathogenesis. The homozygous knock-in mtDNA mutator mouse is a model of premature aging due to the accumulation of mitochondrial DNA mutations. We used this mouse model to investigate the relationship between mitochondrial subunit expression and pancreatic islet cell composition. METHODS: Quadruple immunofluorescence was used to quantify mitochondrial subunit expression (complex I and IV) and cell composition in pancreatic islets from mitochondrial DNA mutator mice (PolgAmut/mut) and control C57BL/6 mice at 12 and 44 weeks of age. RESULTS: Mitochondrial complex I subunit expression was decreased in islets from 12 week PolgAmut/mut mice. This complex I deficiency persisted with age and was associated with decreased insulin staining intensity at 44 weeks. Complex I deficiency was greater in α-cells compared with ß-cells in islets from 44 week PolgAmut/mut mice. Islet cell composition was normal in 12 week PolgAmut/mut mice, but the ß: α cell ratio was decreased in islets from 44 week PolgAmut/mut mice. This was due to an increase in α-cell number linked to an increase in α-cell proliferation. CONCLUSION: Complex I deficiency promotes α-cell proliferation and alters islet cell composition.

NDUFA1 p.Gly32Arg variant in early-onset dementia.

Early-onset dementia (EOD) is highly heritable. However, in many EOD cases the genetic etiology remains unknown. Mitochondrial dysfunction is associated with neurodegeneration and the complex I (CI) deficiency is the most common enzyme deficiency in diseases related to oxidative phosphorylation. The X-chromosomal NDUFA1 gene is essential for the activity of CI. Mutations in NDUFA1 are associated with mitochondrial diseases especially with Leigh syndrome. CI deficiency is also associated with neurodegenerative diseases, such as Alzheimer's disease (AD). The aim of this study was to evaluate the role of NDUFA1 variants in EOD patients. Next-generation sequencing panel was used to screen NDUFA1 variants in a cohort of 37 EOD patients with a family history of dementia or an atypical or rapidly progressive course of disease. We identified a hemizygous p.Gly32Arg variant in two brothers with AD. Subsequent screening of the variant in a larger cohort of EOD patients (n = 279) revealed three additional variant carriers (one male and two heterozygote females), suggesting that NDUFA1 variant p.Gly32Arg may play a role in neurodegenerative dementia.

Adipose Mitochondrial Complex I Deficiency Modulates Inflammation and Glucose Homeostasis in a Sex-Dependent Manner.

Mitochondrial dysfunction in adipose tissue has been associated with type 2 diabetes, but it is unclear whether it is a cause or the consequence. Mitochondrial complex I is a major site of reactive oxygen species generation and a therapeutic target. Here we report that genetic deletion of the complex I subunit Ndufs4 specifically in adipose tissue results in an increased propensity to develop diet-induced weight gain, glucose intolerance, and elevated levels of fat inflammatory genes. This outcome is apparent in young males but not in young females, suggesting that females are relatively protected from the adverse consequences of adipose mitochondrial dysfunction for metabolic health. Mutant mice of both sexes exhibit defects in brown adipose tissue thermogenesis. Fibroblast growth factor 21 (FGF21) signaling in adipose tissue is selectively blunted in male mutant mice relative to wild-type littermates, consistent with sex-dependent regulation of its autocrine/paracrine action in adipocytes. Together, these findings support that adipocyte-specific mitochondrial dysfunction is sufficient to induce tissue inflammation and can cause systemic glucose abnormalities in male mice.

Genome sequencing and RNA-seq analyses of mitochondrial complex I deficiency revealed Alu insertion-mediated deletion in NDUFV2.

Isolated complex I deficiency is the most common cause of pediatric mitochondrial disease. Exome sequencing (ES) has revealed many complex I causative genes. However, there are limitations associated with identifying causative genes by ES analysis. In this study, we performed multiomics analysis to reveal the causal variants. We here report two cases with mitochondrial complex I deficiency. In both cases, ES identified a novel c.580G>A (p.Glu194Lys) variant in NDUFV2. One case additionally harbored c.427C>T (p.Arg143*), but no other variants were observed in the other case. RNA sequencing showed aberrant exon splicing of NDUFV2 in the unsolved case. Genome sequencing revealed a novel heterozygous deletion in NDUFV2, which included one exon and resulted in exon skipping. Detailed examination of the breakpoint revealed that an Alu insertion-mediated rearrangement caused the deletion. Our report reveals that combined use of transcriptome sequencing and GS was effective for diagnosing cases that were unresolved by ES.

TAT-Conjugated NDUFS8 Can Be Transduced into Mitochondria in a Membrane-Potential-Independent Manner and Rescue Complex I Deficiency.

NADH dehydrogenase (ubiquinone) Fe-S protein 8 (NDUFS8) is a nuclear-encoded core subunit of human mitochondrial complex I. Defects in NDUFS8 are associated with Leigh syndrome and encephalomyopathy. Cell-penetrating peptide derived from the HIV-1 transactivator of transcription protein (TAT) has been successfully applied as a carrier to bring fusion proteins into cells without compromising the biological function of the cargoes. In this study, we developed a TAT-mediated protein transduction system to rescue complex I deficiency caused by NDUFS8 defects. Two fusion proteins (TAT-NDUFS8 and NDUFS8-TAT) were exogenously expressed and purified from Escherichia coli for transduction of human cells. In addition, similar constructs were generated and used in transfection studies for comparison. The results showed that both exogenous TAT-NDUFS8 and NDUFS8-TAT were delivered into mitochondria and correctly processed. Interestingly, the mitochondrial import of TAT-containing NDUFS8 was independent of mitochondrial membrane potential. Treatment with TAT-NDUFS8 not only significantly improved the assembly of complex I in an NDUFS8-deficient cell line, but also partially rescued complex I functions both in the in-gel activity assay and the oxygen consumption assay. Our current findings suggest the considerable potential of applying the TAT-mediated protein transduction system for treatment of complex I deficiency.

Biallelic P4HTM variants associated with HIDEA syndrome and mitochondrial respiratory chain complex I deficiency.

We report a patient with profound congenital hypotonia, central hypoventilation, poor visual behaviour with retinal hypopigmentation, and significantly decreased mitochondrial respiratory chain complex I activity in muscle, who died at 7 months of age having made minimal developmental progress. Biallelic predicted truncating P4HTM variants were identified following trio whole-genome sequencing, consistent with a diagnosis of hypotonia, hypoventilation, intellectual disability, dysautonomia, epilepsy and eye abnormalities (HIDEA) syndrome. Very few patients with HIDEA syndrome have been reported previously and mitochondrial abnormalities were observed in three of four previous cases who had a muscle biopsy, suggesting the possibility that HIDEA syndrome represents a primary mitochondrial disorder. P4HTM encodes a transmembrane prolyl 4-hydroxylase with putative targets including hypoxia inducible factors, RNA polymerase II and activating transcription factor 4, which has been implicated in the integrated stress response observed in cell and animal models of mitochondrial disease, and may explain the mitochondrial dysfunction observed in HIDEA syndrome.

Berberine alleviates lipid metabolism disorders via inhibition of mitochondrial complex I in gut and liver.

This study is to investigate the relationship between berberine (BBR) and mitochondrial complex I in lipid metabolism. BBR reversed high-fat diet-induced obesity, hepatic steatosis, hyperlipidemia and insulin resistance in mice. Fatty acid consumption, ß-oxidation and lipogenesis were attenuated in liver after BBR treatment which may be through reduction in SCD1, FABP1, CD36 and CPT1A. BBR promoted fecal lipid excretion, which may result from the reduction in intestinal CD36 and SCD1. Moreover, BBR inhibited mitochondrial complex I-dependent oxygen consumption and ATP synthesis of liver and gut, but no impact on activities of complex II, III and IV. BBR ameliorated mitochondrial swelling, facilitated mitochondrial fusion, and reduced mtDNA and citrate synthase activity. BBR decreased the abundance and diversity of gut microbiome. However, no change in metabolism of recipient mice was observed after fecal microbiota transplantation from BBR treated mice. In primary hepatocytes, BBR and AMPK activator A769662 normalized oleic acid-induced lipid deposition. Although both the agents activated AMPK, BBR decreased oxygen consumption whereas A769662 increased it. Collectively, these findings indicated that BBR repressed complex I in gut and liver and consequently inhibited lipid metabolism which led to alleviation of obesity and fatty liver. This process was independent of intestinal bacteria.