Bi-allelic loss of function variants in COX20 gene cause autosomal recessive sensory neuronopathy.

Sensory neuronopathies are a rare and distinct subgroup of peripheral neuropathies, characterized by degeneration of the dorsal root ganglia neurons. About 50% of sensory neuronopathies are idiopathic and genetic causes remain to be clarified. Through a combination of homozygosity mapping and whole exome sequencing, we linked an autosomal recessive sensory neuronopathy to pathogenic variants in the COX20 gene. We identified eight unrelated families from the eastern Chinese population carrying a founder variant c.41A>G (p.Lys14Arg) within COX20 in either a homozygous or compound heterozygous state. All patients displayed sensory ataxia with a decrease in non-length-dependent sensory potentials. COX20 encodes a key transmembrane protein implicated in the assembly of mitochondrial complex IV. We showed that COX20 variants lead to reduction of COX20 protein in patient's fibroblasts and transfected cell lines, consistent with a loss-of-function mechanism. Knockdown of COX20 expression in ND7/23 sensory neuron cells resulted in complex IV deficiency and perturbed assembly of complex IV, which subsequently compromised cell spare respiratory capacity and reduced cell proliferation under metabolic stress. Consistent with mitochondrial dysfunction in knockdown cells, reduced complex IV assembly, enzyme activity and oxygen consumption rate were also found in patients' fibroblasts. We speculated that the mechanism of COX20 was similar to other causative genes (e.g. SURF1, COX6A1, COA3 and SCO2) for peripheral neuropathies, all of which are functionally important in the structure and assembly of complex IV. Our study identifies a novel causative gene for the autosomal recessive sensory neuronopathy, whose vital function in complex IV and high expression in the proprioceptive sensory neuron further underlines loss of COX20 contributing to mitochondrial bioenergetic dysfunction as a mechanism in peripheral sensory neuron disease.

Mitochondrial disease disrupts hepatic allostasis and lowers the threshold for immune-mediated liver toxicity.

OBJECTIVE: In individuals with mitochondrial disease, respiratory viral infection can result in metabolic decompensation with mitochondrial hepatopathy. Here, we used a mouse model of liver-specific Complex IV deficiency to study hepatic allostasis during respiratory viral infection. METHODS: Mice with hepatic cytochrome c oxidase deficiency (LivCox10-/-) were infected with aerosolized influenza, A/PR/8 (PR8), and euthanized on day five after infection following three days of symptoms. This time course is marked by a peak in inflammatory cytokines and mimics the timing of a common clinical scenario in which caregivers may first attempt to manage the illness at home before seeking medical attention. Metabolic decompensation and mitochondrial hepatopathy in mice were characterized by serum hepatic testing, histology, electron microscopy, biochemistry, metabolomics, and bioenergetic profiling. RESULTS: Following influenza infection, LivCox10-/- mice displayed marked liver disease including hepatitis, enlarged mitochondria with cristae loss, and hepatic steatosis. This pathophysiology was associated with viremia. Primary hepatocytes from LivCox10-/- mice cocultured with WT Kupffer cells in the presence of PR8 showed enhanced lipid accumulation. Treatment of hepatocytes with recombinant TNFα implicated Kupffer cell-derived TNFα as a precipitant of steatosis in LivCox10-/- mice. Eliminating Kupffer cells or blocking TNFα in vivo during influenza infection mitigated the steatosis and mitochondrial morphologic changes. CONCLUSIONS: Taken together, our data shift the narrative of metabolic decompensation in mitochondrial hepatopathy beyond the bioenergetic costs of infection to include an underlying susceptibility to immune-mediated damage. Moreover, our work suggests that immune modulation during metabolic decompensation in mitochondrial disease represents a future viable treatment strategy needing further exploration.

A Case of Reversible Infantile Respiratory Chain Deficiency Presenting With Hypotonia, Hyperammonemia, and Failure to Thrive.

Reversible infantile respiratory chain deficiency, previously termed reversible infantile cytochrome c oxidase (COX) deficiency myopathy, is a rare mitochondrial disorder that is characterized by severe hypotonia and generalized muscle weakness in infancy that is associated with lactic acidosis. Affected infants will spontaneously recover, if they survive the first months of life. Here, we present the case of a 4-week-old girl who initially presented with hyperammonemia, hypotonia, and failure to thrive, for which she was referred for genetic evaluation. After several tests, a distinct genetic syndrome could not be identified and she continued to deteriorate. A muscle biopsy was performed and demonstrated severe mitochondrial myopathy with abundant COX-negative fibers. Ultrastructural abnormalities of the mitochondria, diagnostic of mitochondrial myopathy, were identified on electron microscopy. Molecular studies revealed the classic homoplasmic disease causing mutation, m.14674 T>C in the MT-TE gene, associated with reversible COX deficiency. Although hyperammonemia is an unusual presentation for mitochondrial myopathies, specifically reversible infantile respiratory chain deficiency, it should be included in the list of possible presenting symptoms for this condition.

Low Cytochrome Oxidase 1 Links Mitochondrial Dysfunction to Atherosclerosis in Mice and Pigs.

BACKGROUND: Cytochrome oxidase IV complex regulates energy production in mitochondria. Therefore, we determined the relation of COX genes with atherosclerosis in mice and pigs. METHODS AND RESULTS: First, we compared atherosclerosis in the aortic arch of age-matched (24 weeks) C57BL/6J control (n = 10), LDL-receptor deficient (n = 8), leptin-deficient ob/ob (n = 10), and double knock-out (lacking LDL-receptor and leptin) mice (n = 12). Low aortic mitochondria-encoded cytochrome oxidase 1 in obese diabetic double knock-out mice was associated with a larger plaque area and higher propensity of M1 macrophages and oxidized LDL. Caloric restriction increased mitochondria-encoded cytochrome oxidase 1 and reduced plaque area and oxidized LDL. This was associated with a reduction of titer of anti-oxidized LDL antibodies, a proxy of systemic oxidative stress. Low of mitochondria-encoded cytochrome oxidase 1 was related to low expression of peroxisome proliferative activated receptors α, δ, and γ and of peroxisome proliferative activated receptor, gamma, co-activator 1 alpha reflecting mitochondrial dysfunction. Caloric restriction increased them. To investigate if there was a diabetic/obesity requirement for mitochondria-encoded cytochrome oxidase 1 to be down-regulated, we then studied atherosclerosis in LAD of hypercholesterolemic pigs (n = 37). Pigs at the end of the study were divided in three groups based on increasing LAD plaque complexity according to Stary (Stary I: n = 12; Stary II: n = 13; Stary III: n = 12). Low mitochondria-encoded cytochrome oxidase 1 in isolated plaque macrophages was associated with more complex coronary plaques and oxidized LDL. Nucleus-encoded cytochrome oxidase 4I1 and cytochrome oxidase 10 did not correlate with plaque complexity and oxidative stress. In mice and pigs, MT-COI was inversely related to insulin resistance. CONCLUSIONS: Low MT-COI is related to mitochondrial dysfunction, oxidative stress and atherosclerosis and plaque complexity.

Adult, isolated respiratory chain complex IV deficiency with minimal manifestations.

OBJECTIVES: Isolated complex IV (cytochrome c oxidase) deficiency is one of the most frequent respiratory chain defects in mitochondrial disorders (MIDs) and usually occurs together with severe pediatric or rarely adult multisystem disease. Here we report an adult with isolated complex IV deficiency with unusually mild clinical manifestations. CASE REPORT: A 50-year-old man had developed generalized muscle aches and occasional twitching and stiffness of the musculature since age 48 years. He had a previous history of diabetes, acute hearing loss, hyperlipidemia, hyperuricemia, arterial hypertension, polyarthrosis, hypogonadism, and hypothyroidism. The family history was positive for diabetes (mother), CK elevation (brother), myalgias (brother), and proximal weakness of the upper limbs (mother). Work-up revealed hypoacusis, postural tremor and reduced tendon reflexes, recurrent mild hyper-CK-emia, neurogenic needle electromyography, and a muscle biopsy with mild non-specific changes. Biochemical investigations of the muscle homogenate revealed an isolated complex IV defect and reduced amounts of coenzyme Q (CoQ). He profited from CoQ supplementation, low-carbohydrate diet, and gluten-free diet. CONCLUSIONS: Isolated complex IV deficiency may present with only mild muscular, endocrine, or cardiac manifestations in adults. Coenzyme Q supplementation, low-carbohydrate diet, and gluten-free diet may have a beneficial effect at least on some of the manifestations.

Cytochrome c oxidase deficit is associated with the seizure onset zone in young patients with focal cortical dysplasia Type II.

It has been postulated that mitochondrial dysfunction may be an important factor in epileptogenesis of intractable epilepsy. The current study tests the hypothesis that mitochondrial Complex IV (CIV) or cytochrome c oxidase dysfunction is associated with the seizure onset zone (SOZ) in patients with focal cortical dysplasia (FCD). Subjects were selected based on: age <19y; epilepsy surgery between May, 2010 and October, 2011; pathological diagnosis of isolated focal cortical dysplasia Type I (FCDI) or Type II (FCDII); and sufficient residual cortical tissue to conduct analysis of electron transport chain complex (ETC) activity in SOZ and adjacent cortical regions. In this retrospective study, patients were identified who had sufficient unfixed, frozen brain tissue for biochemical analysis in tissue homogenates. Specimens were subtyped using ILAE classification for FCD, and excluded if diagnosed with FCD Type III or dual pathology. Analysis of ETC activity in resected tissues was conducted independently and without knowledge of the identity, diagnosis, or clinical status of individual subjects. Seventeen patients met the inclusion criteria, including 6 FCDI and 11 FCDII. Comparison of adjacent cortical resections showed decreased CIV activity in the SOZ of the FCDII group (P = 0.003), but no significant CIV difference in adjacent tissues of the FCDI group. Because of the importance of CIV as the terminal and rate-limiting complex in the mitochondrial electron transport chain, these authors conclude that 1) a deficit of CIV is associated with the SOZ of patients with FCDII; 2) CIV deficiency may contribute to the spectrum of FCD neuropathology; and 3) further investigation of CIV in FCD may lead to the discovery of new targets for neuroprotective therapies for patients with intractable epilepsy.

Effect of resveratrol on cultured skin fibroblasts from patients with oxidative phosphorylation defects.

Few therapeutic options are available to patients with oxidative phosphorylation disorders. Administering pharmacological agents that are able to stimulate mitochondrial biogenesis have been put forward as a possible treatment, yet the approach remains in need of thorough testing. We investigated the effect of resveratrol in an in vitro setting. Mitochondrial enzymatic activities were tested in cultured skin fibroblasts from patients harboring a nuclear defect in either complex II or complex IV (n = 11), and in fibroblasts from healthy controls (n = 11). In the latter, preincubation with resveratrol resulted in a significant increase of citrate synthase, complex II and complex IV enzyme activity. In patients with complex II or complex IV deficiency, however, activity of the deficient complex could not be substantially augmented, and response was dependent upon the residual activity. We conclude that resveratrol is not capable of normalizing oxidative phosphorylation activities in deficient cell lines.

Molecular basis of infantile reversible cytochrome c oxidase deficiency myopathy.

Childhood-onset mitochondrial encephalomyopathies are usually severe, relentlessly progressive conditions that have a fatal outcome. However, a puzzling infantile disorder, long known as 'benign cytochrome c oxidase deficiency myopathy' is an exception because it shows spontaneous recovery if infants survive the first months of life. Current investigations cannot distinguish those with a good prognosis from those with terminal disease, making it very difficult to decide when to continue intensive supportive care. Here we define the principal molecular basis of the disorder by identifying a maternally inherited, homoplasmic m.14674T>C mt-tRNA(Glu) mutation in 17 patients from 12 families. Our results provide functional evidence for the pathogenicity of the mutation and show that tissue-specific mechanisms downstream of tRNA(Glu) may explain the spontaneous recovery. This study provides the rationale for a simple genetic test to identify infants with mitochondrial myopathy and good prognosis.

Cytochrome oxidase deficiency protects Escherichia coli from cell death but not from filamentation due to thymine deficiency or DNA polymerase inactivation.

Temperature-sensitive DNA polymerase mutants (dnaE) are protected from cell death on incubation at nonpermissive temperature by mutation in the cydA gene controlling cytochrome bd oxidase. Protection is observed in complex (Luria-Bertani [LB]) medium but not on minimal medium. The cydA mutation protects a thymine-deficient strain from death in the absence of thymine on LB but not on minimal medium. Both dnaE and Deltathy mutants filament under nonpermissive conditions. Filamentation per se is not the cause of cell death, because the dnaE cydA double mutant forms long filaments after 24 h of incubation in LB medium at nonpermissive temperature. These filaments have multiply dispersed nucleoids and produce colonies on return to permissive conditions. The protective effect of a deficiency of cydA at high temperature is itself suppressed by overexpression of cytochrome bo3, indicating that the phenomenon is related to energy metabolism rather than to a specific effect of the cydA protein. We propose that filamentation and cell death resulting from thymine deprivation or slowing of DNA synthesis are not sequential events but occur in response to the same or a similar signal which is modulated in complex medium by cytochrome bd oxidase. The events which follow inhibition of replication fork progression due to either polymerase inactivation, thymine deprivation, or hydroxyurea inhibition differ in detail from those following actual DNA damage.

Decreased affinity for oxygen of cytochrome-c oxidase in Leigh syndrome caused by SURF1 mutations.

Mutations in the gene SURF1 prevent synthesis of cytochrome-c oxidase (COX)-specific assembly protein and result in a fatal neurological disorder, Leigh syndrome. Because this severe COX deficiency presents with barely detectable changes of cellular respiratory rates under normoxic conditions, we analyzed the respiratory response to low oxygen in cultured fibroblasts harboring SURF1 mutations with high-resolution respirometry. The oxygen kinetics was quantified by the partial pressure of oxygen (PO2) at half-maximal respiration rate (P50) in intact coupled cells and in digitonin-permeabilized uncoupled cells. In both cases, the P50 in patients was elevated 2.1- and 3.3-fold, respectively, indicating decreased affinity of COX for oxygen. These results suggest that at physiologically low intracellular PO2, the depressed oxygen affinity may lead in vivo to limitations of respiration, resulting in impaired energy provision in Leigh syndrome patients.

Reversible fulminant lactic acidosis and liver failure in an infant with hepatic cytochrome-c oxidase deficiency.

Cytochrome-c oxidase (COX) is the most common respiratory chain complex involved in liver failure, either as a single enzyme deficiency or as part of multiple enzyme deficiencies. We describe an infant who presented with fulminant lactic acidosis in the neonatal period. The lactic acidosis resolved spontaneously but liver and pancreatic insufficiency ensued. Isolated cytochrome-c oxidase deficiency was found in liver but not in muscle and fibroblasts. mtDNA rearrangements or depletion were ruled out. By the age of one year, liver and pancreatic functions have normalized completely and neurodevelopment is normal.

Neuroprotective effects of bilobalide, a component of the Ginkgo biloba extract (EGb 761), in gerbil global brain ischemia.

The neuroprotective effect of Ginkgo biloba extract (EGb 761) against ischemic injury has been demonstrated in animal models. In this study, we compared the protective effect of bilobalide, a purified terpene lactone from EGb 761, and EGb 761 against ischemic injury. We measured neuronal loss and the levels of mitochondrial DNA (mtDNA)-encoded cytochrome oxidase (COX) subunit III mRNA in vulnerable hippocampal regions of gerbils. At 7 days of reperfusion after 5 min of transient global forebrain ischemia, a significant increase in neuronal death and a significant decrease in COX III mRNA were observed in the hippocampal CA1 neurons. Oral administration of EGb 761 at 25, 50 and 100 mg/kg/day and bilobalide at 3 and 6 mg/kg/day for 7 days before ischemia progressively protected CA1 neurons from death and from ischemia-induced reductions in COX III mRNA. In addition, both bilobalide and EGb 761 protected against ischemia-induced reductions in COX III mRNA in CA1 neurons prior to their death, at 1 day of reperfusion. These results suggest that oral administration of bilobalide and EGb 761 protect against ischemia-induced neuron death and reductions in mitochondrial gene expression.