COX Deficiency Research Articles

Cytochrome c oxidase deficiency detection in human fibroblasts using scanning electrochemical microscopy.

Cytochrome c oxidase deficiency (COXD) is an inherited disorder characterized by the absence or mutation in the genes encoding for the cytochrome c oxidase protein (COX). COX deficiency results in severe muscle weakness, heart, liver, and kidney disorders, as well as brain damage in infants and adolescents, leading to death in many cases. With no cure for this disorder, finding an efficient, inexpensive, and early means of diagnosis is essential to minimize symptoms and long-term disabilities. Furthermore, muscle biopsy, the traditional detection method, is invasive, expensive, and time-consuming. This study demonstrates the applicability of scanning electrochemical microscopy to quantify COX activity in living human fibroblast cells. Taking advantage of the interaction between the redox mediator N, N, N', N'-tetramethyl-para-phenylene-diamine, and COX, the enzymatic activity was successfully quantified by monitoring current changes using a platinum microelectrode and determining the apparent heterogeneous rate constant k0 using numerical modeling. This study provides a foundation for developing a diagnostic method for detecting COXD in infants, which has the potential to increase treatment effectiveness and improve the quality of life of affected individuals.

Prominent muscle involvement in a familial form of mitochondrial disease due to a COA8 variant

Isolated mitochondrial respiratory chain Complex IV (Cytochrome c Oxidase or COX) deficiency is the second most frequent isolated respiratory chain defect. Causative mutations are mainly identified in structural COX subunits or in proteins involved in the maturation and assembly of the COX holocomplex. We describe an Italian familial case of mitochondrial myopathy due to a variant in the COX assembly factor 8 gene (COA8). Patient 1 is a 52-year-old woman who presented generalized epilepsy and retinitis pigmentosa at 10 years of age. From her early adulthood she complained about cramps and myalgia after exercise, and bilateral hearing loss emerged. Last neurological examination (52 years of age) showed bilateral ptosis, muscle weakness, peripheral neuropathy, mild dysarthria and dysphonia, cognitive impairment. Muscle biopsy had shown the presence of ragged-red fibers. Patient 2 (Patient 1’s sister) is a 53-year-old woman presenting fatigability, myalgia, and hearing loss. Neurological examination showed ptosis and muscle weakness. Muscle biopsy displayed a diffuse reduction of COX activity staining and ragged-red fibers. Both sisters presented secondary amenorrhea. After ruling out mtDNA mutations, Whole Exome Sequencing analysis identified the novel homozygous COA8 defect c.170_173dupGACC, p.(Pro59fs) in the probands. Loss-of-function COA8 mutations have been associated with cavitating leukoencephalopathy with COX deficiency in 9 reported individuals. Disease course shows an early-onset rapid clinical deterioration, affecting both cognitive and motor functions over months, followed by stabilization and slow improvement over several years. Our findings expand the clinical spectrum of COA8-related disease. We confirm the benign course of this rare disorder, highlighting its (intrafamilial) clinical variability.

The Mitochondrial tRNASer(UCN) Gene: A Novel m.7484A>G Mutation Associated with Mitochondrial Encephalomyopathy and Literature Review

Mitochondrial tRNASer(UCN) is considered a hot-spot for non-syndromic and aminoglycoside-induced hearing loss. However, many patients have been described with more extensive neurological diseases, mainly including epilepsy, myoclonus, ataxia, and myopathy. We describe a novel homoplasmic m.7484A>G mutation in the tRNASer(UCN) gene affecting the third base of the anticodon triplet in a girl with profound intellectual disability, spastic tetraplegia, sensorineural hearing loss, a clinical history of epilepsia partialis continua and vomiting, typical of MELAS syndrome, leading to a myoclonic epilepticus status, and myopathy with severe COX deficiency at muscle biopsy. The mutation was also found in the homoplasmic condition in the mother who presented with mild cognitive deficit, cerebellar ataxia, myoclonic epilepsy, sensorineural hearing loss and myopathy with COX deficient ragged-red fibers consistent with MERRF syndrome. This is the first anticodon mutation in the tRNASer(UCN) and the second homoplasmic mutation in the anticodon triplet reported to date.

Human mtRF1 terminates COX1 translation and its ablation induces mitochondrial ribosome-associated quality control

Translation termination requires release factors that read a STOP codon in the decoding center and subsequently facilitate the hydrolysis of the nascent peptide chain from the peptidyl tRNA within the ribosome. In human mitochondria eleven open reading frames terminate in the standard UAA or UAG STOP codon, which can be recognized by mtRF1a, the proposed major mitochondrial release factor. However, two transcripts encoding for COX1 and ND6 terminate in the non-conventional AGA or AGG codon, respectively. How translation termination is achieved in these two cases is not known. We address this long-standing open question by showing that the non-canonical release factor mtRF1 is a specialized release factor that triggers COX1 translation termination, while mtRF1a terminates the majority of other mitochondrial translation events including the non-canonical ND6. Loss of mtRF1 leads to isolated COX deficiency and activates the mitochondrial ribosome-associated quality control accompanied by the degradation of COX1 mRNA to prevent an overload of the ribosome rescue system. Taken together, these results establish the role of mtRF1 in mitochondrial translation, which had been a mystery for decades, and lead to a comprehensive picture of translation termination in human mitochondria.

Zebrafish cox17 modulates primitive erythropoiesis via regulation of mitochondrial metabolism to facilitate hypoxia tolerance.

Cox17 is required in the assembly of mitochondrial intermembrane space (IMS) and Cu metallization of cytochrome C oxidase (CcO) in mitochondria as well as Cu homeostasis in cells. Cox deficiency is associated with hematopoietic diseases such as tubulopathy and leukodystrophy, but whether and how cox17 functions in hematopoiesis are still unknown. Here, we report the effects of zebrafish cox17 deficiency on primitive erythropoiesis, mitochondrial metabolism, and hypoxia tolerance. Cox17-/- larvae were sensitive to hypoxia stress, with reduced primitive erythropoiesis. Meanwhile, cox17-/- mutants showed a significant reduction in the expression of pivotal transcriptional regulators in erythropoiesis, such as scl, lmo2, and gata1a at 14 h post fertilization (hpf), with expression remaining downregulated for scl but upregulated for lmo2 and gata1a at 24 hpf. Mechanistically, cox17-/- mutants showed impaired mitochondrial metabolism, coupled with a significant decrease in the mitochondrial membrane potential, ATP and SAM content, and the ratio of SAM and SAH. Additionally, disrupting mitochondrial metabolism in wild type (WT) larvae treated with carbonyl cyanide 3-chlorophenylhydrazone (CCCP) could mimic the primitive erythropoiesis defects observed in cox17-/- mutants. Moreover, cox17-/- mutants exhibited significantly downregulated WNT signaling and upregulated ER stress, with a significant reduction of beta-Catenin in gata1a+ cells and of binding enrichment in both scl and lmo2 promoters of the WNT transcriptional factor TCF4. This is the first report on the novel linkage of cox17 deficiency with defective primitive erythropoiesis and reduced hypoxia tolerance. This study has shed light on the potential mechanism by which Cox deficiency, especially cox17 deficiency, induces Cu homeostasis imbalance, leading to hematopoietic diseases.

Generation of a homozygous COX6A2 knockout human embryonic stem cell line (WAe009-A-47) via an epiCRISPR/Cas9 system.

COX6A2 protein is a structural subunit of Complex IV (CIV/Cytochrome c oxidase/COX) in the mitochondrial respiratory chain. It is mainly expressed in the heart and skeletal muscle, also in some interneurons, regulating the assembly and catalytic activity of CIV. Its mutations can lead to COX deficiency, causing human myopathies, and maybe a potential cause of neurological abnormalities. Here, we used the CRISPR/Cas9 editing system to establish a homozygous COX6A2 knockout (COX6A2-KO) human embryonic stem cell (hESC) line. This COX6A2-KO hESC has normal morphology, pluripotency, and karyotype, which can differentiate into three germ layers in vivo.

A Novel Pathogenic Variant in MT-CO2 Causes an Isolated Mitochondrial Complex IV Deficiency and Late-Onset Cerebellar Ataxia.

Both nuclear and mitochondrial DNA defects can cause isolated cytochrome c oxidase (COX; complex IV) deficiency, leading to the development of the mitochondrial disease. We report a 52-year-old female patient who presented with a late-onset, progressive cerebellar ataxia, tremor and axonal neuropathy. No family history of neurological disorder was reported. Although her muscle biopsy demonstrated a significant COX deficiency, there was no clinical and electromyographical evidence of myopathy. Electrophysiological studies identified low frequency sinusoidal postural tremor at 3 Hz, corroborating the clinical finding of cerebellar dysfunction. Complete sequencing of the mitochondrial DNA genome in muscle identified a novel MT-CO2 variant, m.8163A>G predicting p.(Tyr193Cys). We present several lines of evidence, in proving the pathogenicity of this heteroplasmic mitochondrial DNA variant, as the cause of her clinical presentation. Our findings serve as an important reminder that full mitochondrial DNA analysis should be included in the diagnostic pipeline for investigating individuals with spinocerebellar ataxia.

MITOCHONDRIAL DISEASES I (Oral): I.5Perturbed mitochondrial homeostasis in the pathogenesis of mitochondrial disorders

Mitochondrial disorders come with an impressive variability of symptoms, organ involvement, and clinical course, which considerably impact the quality of life and quite often shorten the lifespan expectancy. The last 20 years have witnessed an exponential increase in understanding the genetic and biochemical mechanisms leading to disease. More recently, several homeostatic pathways, including the quality control and adaptive mechanisms controlling the bioenergetics proficiency and biogenesis of the mitochondrial network, have become a central issue to elucidate pathogenic mechanisms and develop rational therapies in mitochondrial disease. Indeed, new experimental approaches have recently been emerging, some of which have shown potential efficacy at the preclinical level: for instance, activators of mitochondrial biogenesis, modulators of autophagy and mTORC1 pathway, and regulators of mitochondrial dynamics and mitochondrial architecture. We and others have shown that activation of the PGC1a-dependent mitochondriogenic axis partially rescues the phenotype of COX-deficiency mouse models. Conversely, suppression of ROS production by expressing the alternative oxidase (AOX) worsens the phenotype and anticipates the fatal outcome of a mouse model of COX deficiency restricted to skeletal muscle (Cox15sm/sm). Autophagy is an intracellular recycling process that delivers misfolded proteins and dysfunctional organelles to lysosomes. This bulk degradation process may be impaired in several pathological conditions including mitochondrial disorders, where its activation can be exploited for the clearance of dysfunctional mitochondria and the restoration of the normal energy homeostasis and metabolism. Rapamycin is an inhibitor of mTOR complex 1 (mTORC1) that can both induce autophagy, through new autophagosome formation, and increase the autophagic flux by enhancing lysosomal biogenesis. Rapamycin improves the clinical conditions of the Cox15sm/sm mouse model, rescuing the myopathy caused by COX deficiency. This is the result of a coordinated activation of autophagy together with increased lysosomes, possibly through the action of TFEB, a master regulator of lysosomal biogenesis.

The pathomechanism of COX deficiency includes nuclear DNA damage and replicative stress

The pathomechanism of COX deficiency includes nuclear DNA damage and replicative stress

The pathomechanism of cytochrome c oxidase deficiency includes nuclear DNA damage

Mitochondrial cytochrome c oxidase (COX, respiratory chain complex IV), contributes to ATP production via oxidative phosphorylation (OXPHOS). Clinical presentation of COX deficiency is heterogeneous ranging from mild to severe neuromuscular diseases. Anemia is among the symptoms and we have previously reported Fanconi anemia like features in COX4-1 deficiency, suggesting genomic instability and our preliminary results detected nuclear double stranded DNA breaks (DSB). We now quantified the DSB by phospho histone H2AX Ser139 staining of COX4-1 and COX6B1 deficient fibroblasts (225% and 215% of normal, respectively) and confirmed their occurrence by neutral comet assay. We further explored the mechanism of DNA damage by studying normal fibroblasts treated with micromolar concentrations of cyanide (KCN). Present results demonstrate elevated nuclear DSB in cells treated with 50 μM KCN for 24 h (170% of normal) in high-glucose medium conditions where ROS and ATP remain normal, although Glutathione content was partially decreased. In glucose-free and serum-free medium, where growth is hampered, DSB were not elevated. Additionally we demonstrate the benefit of nicotinamide riboside (NR) which ameliorated DSB in COX4-1, COX6B1 and KCN treated cells (130%, 154% and 87% of normal cells, respectively). Conversely a negative effect of a poly[ADP-ribose] polymerase (PARP) inhibitor was found. Although additional investigation is needed, our findings raise the possibility that the pathomechanism of COX deficiency and possibly also in other OXPHOS defects, include nuclear DNA damage resulting from nicotinamide adenine dinucleotide (NAD+) deficit combined with a replicative state, rather than oxidative stress and energy depletion.

A novel histochemistry assay to assess and quantify focal cytochrome c oxidase deficiency

Defects in the respiratory chain, interfering with energy production in the cell, are major underlying causes of mitochondrial diseases. In spite of this, the surprising variety of clinical symptoms, disparity between ages of onset, as well as the involvement of mitochondrial impairment in ageing and age‐related diseases continue to challenge our understanding of the pathogenic processes. This complexity can be in part attributed to the unique metabolic needs of organs or of various cell types. In this view, it remains essential to investigate mitochondrial dysfunction at the cellular level. For this purpose, we developed a novel enzyme histochemical method that enables precise quantification in fresh‐frozen tissues using competing redox reactions which ultimately lead to the reduction of tetrazolium salts and formazan deposition in cytochrome c oxidase‐deficient mitochondria. We demonstrate that the loss of oxidative activity is detected at very low levels – this achievement is unequalled by previous techniques and opens up new opportunities for the study of early disease processes or comparative investigations. Moreover, human biopsy samples of mitochondrial disease patients of diverse genotypic origins were used and the successful detection of COX‐deficient cells suggests a broad application for this new method. Lastly, the assay can be adapted to a wide range of tissues in the mouse and extends to other animal models, which we show here with the fruit fly, Drosophila melanogaster. Overall, the new assay provides the means to quantify and map, on a cell‐by‐cell basis, the full extent of COX deficiency in tissues, thereby expending new possibilities for future investigation. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Quantitation of muscle pathology abnormalities in 18 patients with mitochondrial disorders

ABSTRACT INTRODUCTION: Mitochondrial disorders can lead to the accumulation of mitochondria in muscle fibers, as indicated by ragged red (RRF) or ragged blue fibers when stained with modified Gomori trichrome or succinate dehydrogenase (SDH+), respectively, and, absence of activity of cytochrome c oxidase, COX negative fibers (COX-). The combined COX-SDH stain (COMBO+) can reveal even more COX-deficient fibers. OBJECTIVE: To quantify RRFs, SDH+, COX-, and COMBO+ fibers in muscle biopsies with mitochondrial findings. MATERIAL AND METHODS: We retrospectively selected 18 muscle biopsies with mitochondrial abnormalities based on the Walker criteria (percentage of RRFs/COX- fibers, and clinical picture), and/or the Sleigh criteria (percentage of RRFs, SDH+, and COX- fibers). RESULTS: Females represented 83.3%, with a mean age of 38.6 years (5 months-70 years). Patients were diagnosed with chronic progressive external ophthalmoplegia (CPEO, 66.7%), proximal myopathy (22.2%), idiopathic hyperCKemia (11.1%), Kearns-Sayre syndrome (5.6%), mitochondrial encephalomyopathy with ragged red fibers and stroke-like episodes (5.6%), and a dystrophic pattern (5.6%). Some cases of CPEO were combined with proximal myopathy. The quantitative pathologic findings were: RRFs, 3.95% ± 3.17%; SDH+, 7.55% ± 6.1%; COX-, 10.9% ± 7.2%; COMBO+, 14.22% ± 12.79%. We found a slight variation in the diameter of muscle fibers, no necrosis or proliferative connective tissue, few fibers with internal nuclei, and some cases with fiber type grouping. CONCLUSION: Pathologic events, grouped in ascending order of frequency, were RRFs, SDH+ fibers, COX- fibers, and COMBO+ fibers. These data emphasize the importance of the COMBO technique in revealing occult COX deficiency in muscle fibers.

Investigating the cardiac pathology of SCO2-mediated hypertrophic cardiomyopathy using patients induced pluripotent stem cell-derived cardiomyocytes.

Mutations in SCO2 are among the most common causes of COX deficiency, resulting in reduced mitochondrial oxidative ATP production capacity, often leading to hypertrophic cardiomyopathy (HCM). To date, none of the recent pertaining reports provide deep understanding of the SCO2 disease pathophysiology. To investigate the cardiac pathology of the disease, we were the first to generate induced pluripotent stem cell (iPSC)‐derived cardiomyocytes (iPSC‐CMs) from SCO2‐mutated patients. For iPSC generation, we reprogrammed skin fibroblasts from two SCO2 patients and healthy controls. The first patient was a compound heterozygote to the common E140K mutation, and the second was homozygote for the less common G193S mutation. iPSC were differentiated into cardiomyocytes through embryoid body (EB) formation. To test the hypothesis that the SCO2 mutation is associated with mitochondrial abnormalities, and intracellular Ca2+‐overload resulting in functional derangements and arrhythmias, we investigated in SCO2‐mutated iPSC‐CMs (compared to control cardiomyocytes): (i) the ultrastructural changes; (ii) the inotropic responsiveness to β‐adrenergic stimulation, increased [Ca2+]o and angiotensin‐II (AT‐II); and (iii) the Beat Rate Variability (BRV) characteristics. In support of the hypothesis, we found in the mutated iPSC‐CMs major ultrastructural abnormalities and markedly attenuated response to the inotropic interventions and caffeine, as well as delayed afterdepolarizations (DADs) and increased BRV, suggesting impaired SR Ca2+ handling due to attenuated SERCA activity caused by ATP shortage. Our novel results show that iPSC‐CMs are useful for investigating the pathophysiological mechanisms underlying the SCO2 mutation syndrome.

P.67 - Integrated genome analysis of COX deficiency in Japan

P.67 - Integrated genome analysis of COX deficiency in Japan

Abstract IA29: Manipulating inflammation to raise cancer immunogenicity

Abstract Cyclooxygenase (COX)-2, one of the rate-limiting enzymes for production of prostanoids, is commonly upregulated in numerous cancers, and together with prostaglandin E2 (PGE2), has been implicated in several aspects of malignant growth including proliferation, angiogenesis, and invasion. We found that COX-dependent production of PGE2 by melanoma cells dominantly and primarily enables progressive tumor growth via immune escape. Melanoma cells, unable to produce PGE2 by means of genetic ablation of COX or prostaglandin E synthase, formed spontaneously regressing tumors in immune-competent mice but progressive tumors in immune-deficient hosts. By contrast, their PGE2-competent parental lines grew progressively and irrespectively of the presence of a fully competent immune system, demonstrating an essential role for PGE2 in immune evasion. Importantly, PGE2-dependent immune escape was crucial not only in melanoma but also in colorectal and breast cancer models, arguing that this is a general mechanism for subversion of anticancer immunity. COX deficiency in cancer cells was associated with a marked shift in the inflammatory signature at the tumor site characterized by lower expression of cancer-promoting factors and concomitant increase in several mediators typically associated with antitumor immunity. These drastic changes in the inflammatory nature of the tumor microenvironment due to reduced PGE2 levels suggested that COX inhibitors could be used to potentiate immune-dependent control. Indeed, we found that combining COX-inhibitors, such as aspirin, with anti-PD-1 blockade synergistically promoted growth control of tumors poorly responsive to either monotherapy. Finally, analysis of publicly available human melanoma datasets revealed a remarkable conservation of the COX-driven mouse inflammatory signature suggesting that COX activity might be a driver of immune suppression across species. Citation Format: Santiago Zelenay. Manipulating inflammation to raise cancer immunogenicity [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr IA29.

Identification of a novel deletion in SURF1 gene: Heterogeneity in Leigh syndrome with COX deficiency

Leigh syndrome (LS) is a rare, progressive neurodegenerative mitochondrial disorder of infancy. It is a genetically heterogeneous disease. The mutations in SURF1 gene are the most frequently known cause. Here two cases of LS likely caused by SURF1 gene variants are reported: a 39-year-old male patient with a novel homozygous deletion (c.-11_13del), and a case of a 6-year-old boy with the same deletion and a nonsense mutation (c.868dupT), both in heterozygosity. Blue native PAGE showed absence of assembled complex IV. This is the first report of a variant that may abolish the SURF1 gene initiation codon in two LS patients.

The Arabidopsis COX11 Homolog is Essential for Cytochrome c Oxidase Activity.

Members of the ubiquitous COX11 (cytochrome c oxidase 11) protein family are involved in copper delivery to the COX complex. In this work, we characterize the Arabidopsis thaliana COX11 homolog (encoded by locus At1g02410). Western blot analyses and confocal microscopy identified Arabidopsis COX11 as an integral mitochondrial protein. Despite sharing high sequence and structural similarities, the Arabidopsis COX11 is not able to functionally replace the Saccharomyces cerevisiae COX11 homolog. Nevertheless, further analysis confirmed the hypothesis that Arabidopsis COX11 is essential for COX activity. Disturbance of COX11 expression through knockdown (KD) or overexpression (OE) affected COX activity. In KD lines, the activity was reduced by ~50%, resulting in root growth inhibition, smaller rosettes and leaf curling. In OE lines, the reduction was less pronounced (~80% of the wild type), still resulting in root growth inhibition. Additionally, pollen germination was impaired in COX11 KD and OE plants. This effect on pollen germination can only partially be attributed to COX deficiency and may indicate a possible auxiliary role of COX11 in ROS metabolism. In agreement with its role in energy production, the COX11 promoter is highly active in cells and tissues with high-energy demand for example shoot and root meristems, or vascular tissues of source and sink organs. In COX11 KD lines, the expression of the plasma-membrane copper transporter COPT2 and of several copper chaperones was altered, indicative of a retrograde signaling pathway pertinent to copper homeostasis. Based on our data, we postulate that COX11 is a mitochondrial chaperone, which plays an important role for plant growth and pollen germination as an essential COX complex assembly factor.

P39. Assigning pathogenicity to three novel mt-tRNA mutations associated with different mitochondrial disease presentations

Mitochondrial transfer RNA (mt-tRNA) mutations are the commonest mitochondrial (mtDNA) mutations associated with human disease. Assigning pathogenicity to novel mt-tRNA variants is very important particularly regarding the polymorphic nature of mtDNA. We report three patients in whom we have identified novel mt-tRNA point mutations. Clinical presentation of patients 1 and 2 was isolated myopathy. Patient 3 showed multisytemic disease with myopathy, spinal ataxia, deafness, cataract and cognitive impairment. Muscle biopsies revealed extensive histopathological findings (up to 40% COX-deficient fibres). mtDNA sequencing in muscle identified variants in the mt-tRNA Ala gene (m.5631G>A and m.5610G>A) in patients 1 and 2, and a m.7539C>T mt-tRNA Asp transition in patient 3. Quantitative restriction fragment length polymorphism analysis and pyrosequencing confirmed mtDNA heteroplasmy for all mutations, with highest levels being demonstrated in muscle and lower levels observed in blood, buccal epithelium, urinary sediments and hair shafts. Family analysis in patients 1 and 2 showed maternal transmission of the mutations. Single muscle fibre segregation studies in individual, laser-microcaptured COX-positive and COX-deficient fibres detected statistically-significant higher mutation loads in COX-deficient fibres than in COX-positive fibres (patient 1: 95.1% + 0.45 vs. 83.8% + 3.38; patient 2: 98.0% + 0.46 vs. 78.9% + 4.81; patient 3: 96.1% + 0.38 vs. 69.1% + 2.98). Absence from control databases, presence of mtDNA heteroplasmy, hierarchical mt-tRNA mutation segregation within tissues, occurrence at conserved sequence positions, and single-fibre studies confirm these novel mt-tRNA mutations to be pathogenic. Interestingly, patients 1 and 2 showed very high mutation levels in all tissues, as described for other mt-tRNA mutations (e.g. m.14709T>C); moreover, the threshold for impairment of oxidative phosphorylation, as evidenced by COX deficiency, appears to be extremely high for both novel mt-tRNA Ala mutations. Previously-described cases of mt-tRNA Ala mutations (m.5591G>A, m.5650G>A) are also associated with a pure myopathic phenotype and demonstrate very high mutation thresholds, inferring at least some genotype:phenotype correlation for mutations within this tRNA gene.

Human mitochondrial COX1 assembly into cytochrome c oxidase at a glance.

Mitochondria provide the main portion of cellular energy in form of ATP produced by the F1Fo ATP synthase, which uses the electrochemical gradient, generated by the mitochondrial respiratory chain (MRC). In human mitochondria, the MRC is composed of four multisubunit enzyme complexes, with the cytochrome c oxidase (COX, also known as complex IV) as the terminal enzyme. COX comprises 14 structural subunits, of nuclear or mitochondrial origin. Hence, mitochondria are faced with the predicament of organizing and controlling COX assembly with subunits that are synthesized by different translation machineries and that reach the inner membrane by alternative transport routes. An increasing number of COX assembly factors have been identified in recent years. Interestingly, mutations in several of these factors have been associated with human disorders leading to COX deficiency. Recently, studies have provided mechanistic insights into crosstalk between assembly intermediates, import processes and the synthesis of COX subunits in mitochondria, thus linking conceptually separated functions. This Cell Science at a Glance article and the accompanying poster will focus on COX assembly and discuss recent discoveries in the field, the molecular functions of known factors, as well as new players and control mechanisms. Furthermore, these findings will be discussed in the context of human COX-related disorders.

Loss of LRPPRC causes ATP synthase deficiency

Defects of the oxidative phosphorylation system, in particular of cytochrome-c oxidase (COX, respiratory chain complex IV), are common causes of Leigh syndrome (LS), which is a rare neurodegenerative disorder with severe progressive neurological symptoms that usually present during infancy or early childhood. The COX-deficient form of LS is commonly caused by mutations in genes encoding COX assembly factors, e.g. SURF1, SCO1, SCO2 or COX10. However, other mutations affecting genes that encode proteins not directly involved in COX assembly can also cause LS. The leucine-rich pentatricopeptide repeat containing protein (LRPPRC) regulates mRNA stability, polyadenylation and coordinates mitochondrial translation. In humans, mutations in Lrpprc cause the French Canadian type of LS. Despite the finding that LRPPRC deficiency affects the stability of most mitochondrial mRNAs, its pathophysiological effect has mainly been attributed to COX deficiency. Surprisingly, we show here that the impaired mitochondrial respiration and reduced ATP production observed in Lrpprc conditional knockout mouse hearts is caused by an ATP synthase deficiency. Furthermore, the appearance of inactive subassembled ATP synthase complexes causes hyperpolarization and increases mitochondrial reactive oxygen species production. Our findings shed important new light on the bioenergetic consequences of the loss of LRPPRC in cardiac mitochondria.