COX5A Alleviates Doxorubicin-Induced Cardiotoxicity by Suppressing Oxidative Stress, Mitochondrial Dysfunction and Cardiomyocyte Apoptosis.

Doxorubicin (DOX) as a chemotherapeutic agent can cause mitochondrial dysfunction and heart failure. COX5A has been described as an important regulator of mitochondrial energy metabolism. We investigate the roles of COX5A in DOX-induced cardiomyopathy and explore the underlying mechanisms. C57BL/6J mice and H9c2 cardiomyoblasts were treated with DOX, and the COX5A expression was assessed. An adeno-associated virus serum type 9 (AAV9) and lenti-virus system were used to upregulate COX5A expression. Echocardiographic parameters, morphological and histological analyses, transmission electron microscope and immunofluorescence assays were used to assess cardiac and mitochondrial function. In a human study, we found that cardiac COX5A expression was dramatically decreased in patients with end-stage dilated cardiomyopathy (DCM) compared to the control group. COX5A was significantly downregulated following DOX stimulation in the heart of mice and H9c2 cells. Reduced cardiac function, decreased myocardium glucose uptake, mitochondrial morphology disturbance, reduced activity of mitochondrial cytochrome c oxidase (COX) and lowered ATP content were detected after DOX stimulation in mice, which could be significantly improved by overexpression of COX5A. Overexpression of COX5A effectively protected against DOX-induced oxidative stress, mitochondrial dysfunction and cardiomyocyte apoptosis in vivo and in vitro. Mechanistically, the phosphorylation of Akt (Thr308) and Akt (Ser473) were also decreased following DOX treatment, which could be reserved by the upregulation of COX5A. Furthermore, PI3K inhibitors abrogated the protection effects of COX5A against DOX-induced cardiotoxicity in H9c2 cells. Thus, we identified that PI3K/Akt signaling was responsible for the COX5A-mediated protective role in DOX-induced cardiomyopathy. These results demonstrated the protective effect of COX5A in mitochondrial dysfunction, oxidative stress, and cardiomyocyte apoptosis, providing a potential therapeutic target in DOX-induced cardiomyopathy.

Mitochondrial electron transport chain defects modify Parkinson's disease phenotypes in a Drosophila model.

INTRODUCTION: Mitochondrial defects have been implicated in Parkinson's disease (PD) since complex I poisons were found to cause accelerated parkinsonism in young people in the early 1980s. More evidence of mitochondrial involvement arose when many of the genes whose mutations caused inherited PD were discovered to be subcellularly localized to mitochondria or have mitochondrial functions. However, the details of how mitochondrial dysfunction might impact or cause PD remain unclear. The aim of our study was to better understand mitochondrial dysfunction in PD by evaluating mitochondrial respiratory complex mutations in a Drosophila melanogaster (fruit fly) model of PD. METHODS: We have conducted a targeted heterozygous enhancer/suppressor screen using Drosophila mutations within mitochondrial electron transport chain (ETC) genes against a null PD mutation in parkin. The interactions were assessed by climbing assays at 2-5 days as an indicator of motor function. A strong enhancer mutation in COX5A was examined further for L-dopa rescue, oxygen consumption, mitochondrial content, and reactive oxygen species. A later timepoint of 16-20 days was also investigated for both COX5A and a suppressor mutation in cyclope. Generalized Linear Models and similar statistical tests were used to verify significance of the findings. RESULTS: We have discovered that mutations in individual genes for subunits within the mitochondrial respiratory complexes have interactions with parkin, while others do not, irrespective of complex. One intriguing mutation in a complex IV subunit (cyclope) shows a suppressor rescue effect at early time points, improving the gross motor defects caused by the PD mutation, providing a strong candidate for drug discovery. Most mutations, however, show varying degrees of enhancement or slight suppression of the PD phenotypes. Thus, individual mitochondrial mutations within different oxidative phosphorylation complexes have different interactions with PD with regard to degree and direction. Upon further investigation of the strongest enhancer (COX5A), the mechanism by which these interactions occur initially does not appear to be based on defects in ATP production, but rather may be related to increased levels of reactive oxygen species. CONCLUSIONS: Our work highlights some key subunits potentially involved in mechanisms underlying PD pathogenesis, implicating ETC complexes other than complex I in PD.

A novel homozygous variant in COX5A causes an attenuated phenotype with failure to thrive, lactic acidosis, hypoglycemia, and short stature.

Genetic defect in the nuclear encoded subunits of cytochrome c oxidase are very rare. To date, most deleterious variants affect the mitochondrially encoded subunits of complex IV and the nuclear genes encoded for assembly factors. A biallelic pathogenic variant in the mitochondrial complex IV subunit COX5A was previously reported in a couple of sibs with failure to thrive, lactic acidosis and pulmonary hypertension and a lethal phenotype. Here, we describe a second family with a 11-year-old girl presenting with failure to thrive, lactic acidosis, hypoglycemia and short stature. Clinical exome revealed the homozygous missense variant c.266 T > G in COX5A, which produces a drop of the corresponding protein and a reduction of the COX activity. Compared to the previous observation, this girl showed an attenuated metabolic derangement without involvement of the cardiovascular system and neurodevelopment. Our observation confirms that COX5A recessive variants may cause mitochondrial disease and expands the associated phenotype to less severe presentations.

Overexpression of COX5A protects H9c2 cells against doxorubicin-induced cardiotoxicity.

Mitochondrial dysfunction plays a pivotal role in doxorubicin (DOX)-induced cardiomyopathy. Cytochrome c oxidase subunit 5A (COX5A) is a nuclear-encoded subunit of the terminal oxidase involved in mitochondrial electron transport. Although COX5A appears to play a key role in modulating the physiological activity of COX and involve in energy metabolism, the involvement of COX5A in DOX-induced cardiotoxicity remains unclear. In this study, we showed that COX5A was significantly downregulated by DOX treatment of H9c2 cells. Overexpression of COX5A in H9c2 cells effectively attenuated DOX-induced apoptosis. Meanwhile, DOX-induced decrease in mitochondrial membrane potential could be reserved by COX5A overexpression. Furthermore, COX5A overexpression relieved the DOX-induced suppression of mitochondrial respiration, due an increase in basal respiration, maximal respiration, ATP production, and spare respiratory capacity. These findings indicate that up-regulation of COX5A may inhibit the apoptosis and alleviate the mitochondrial dysfunction of DOX-treated H9c2 cells. Thus, COX5A may have potential for clinical use as a therapeutic target in DOX-induced cardiotoxicity.

COX5A over-expression protects cortical neurons from hypoxic ischemic injury in neonatal rats associated with TPI up-regulation.

BACKGROUND: Neonatal hypoxic-ischemic encephalopathy (HIE) represents as a major cause of neonatal morbidity and mortality. However, the underlying molecular mechanisms in brain damage are still not fully elucidated. This study was conducted to determine the specific potential molecular mechanism in the hypoxic-ischemic induced cerebral injury. METHODS: Here, hypoxic-ischemic (HI) animal models were established and primary cortical neurons were subjected to oxygen-glucose deprivation (OGD) to mimic HIE model in vivo and in vitro. The HI-induced neurological injury was evaluated by Zea-longa scores, Triphenyte-trazoliumchloride (TTC) staining the Terminal Deoxynucleotidyl Transferased Utp Nick End Labeling (TUNEL) and immunofluorescent staining. Then the expression of Cytochrome c oxidase subunit 5a (COX5A) was determined by immunohistochemistry, western blotting (WB) and quantitative real time Polymerase Chain Reaction (qRT-PCR) techniques. Moreover, HSV-mediated COX5A over-expression virus was transducted into OGD neurons to explore the role of COX5A in vitro, and the underlying mechanism was predicted by GeneMANIA, then verified by WB and qRT-PCR. RESULTS: HI induced a severe neurological dysfunction, brain infarction, and cell apoptosis as well as obvious neuron loss in neonatal rats, in corresponding to the decrease on the expression of COX5A in both sides of the brain. What's more, COX5A over-expression significantly promoted the neuronal survival, reduced the apoptosis rate, and markedly increased the neurites length after OGD. Moreover, Triosephosephate isomerase (TPI) was predicted as physical interactions with COX5A, and COX5A over-expression largely increased the expressional level of TPI. CONCLUSIONS: The present findings suggest that COX5A plays an important role in promoting neurological recovery after HI, and this process is related to TPI up-regulation.

Mutation in mitochondrial complex IV subunit COX5A causes pulmonary arterial hypertension, lactic acidemia, and failure to thrive.

COX5A is a nuclear-encoded subunit of mitochondrial respiratory chain complex IV (cytochrome c oxidase). We present patients with a homozygous pathogenic variant in the COX5A gene. Clinical details of two affected siblings suffering from early-onset pulmonary arterial hypertension, lactic acidemia, failure to thrive, and isolated complex IV deficiency are presented. We show that the variant lies within the evolutionarily conserved COX5A/COX4 interface domain, suggesting that it alters the interaction between these two subunits during complex IV biogenesis. In patient skin fibroblasts, the enzymatic activity and protein levels of complex IV and several of its subunits are reduced. Lentiviral complementation rescues complex IV deficiency. The monomeric COX1 assembly intermediate accumulates demonstrating a function of COX5A in complex IV biogenesis. A potential therapeutic lead is demonstrated by showing that copper supplementation leads to partial rescue of complex IV deficiency in patient fibroblasts.

A mitochondrial CO2-adenylyl cyclase-cAMP signalosome controls yeast normoxic cytochrome c oxidase activity.

Mitochondria, the major source of cellular energy in the form of ATP, respond to changes in substrate availability and bioenergetic demands by employing rapid, short-term, metabolic adaptation mechanisms, such as phosphorylation-dependent protein regulation. In mammalian cells, an intramitochondrial CO2-adenylyl cyclase (AC)-cyclic AMP (cAMP)-protein kinase A (PKA) pathway regulates aerobic energy production. One target of this pathway involves phosphorylation of cytochrome c oxidase (COX) subunit 4-isoform 1 (COX4i1), which modulates COX allosteric regulation by ATP. However, the role of the CO2-sAC-cAMP-PKA signalosome in regulating COX activity and mitochondrial metabolism and its evolutionary conservation remain to be fully established. We show that in Saccharomyces cerevisiae, normoxic COX activity measured in the presence of ATP is 55% lower than in the presence of ADP. Moreover, the adenylyl cyclase Cyr1 activity is present in mitochondria, and it contributes to the ATP-mediated regulation of COX through the normoxic subunit Cox5a, homologue of human COX4i1, in a bicarbonate-sensitive manner. Furthermore, we have identified 2 phosphorylation targets in Cox5a (T65 and S43) that modulate its allosteric regulation by ATP. These residues are not conserved in the Cox5b-containing hypoxic enzyme, which is not regulated by ATP. We conclude that across evolution, a CO2-sAC-cAMP-PKA axis regulates normoxic COX activity.

CLPP inhibition triggers apoptosis in human ovarian granulosa cells via COX5A abnormality-Mediated mitochondrial dysfunction.

Premature ovarian insufficiency (POI) is characterized by early loss of ovarian function before the age of 40 years. It is confirmed to have a strong and indispensable genetic component. Caseinolytic mitochondrial matrix peptidase proteolytic subunit (CLPP) is a key inducer of mitochondrial protein quality control for the clearance of misfolded or damaged proteins, which is necessary to maintain mitochondrial function. Previous findings have shown that the variation in CLPP is closely related to the occurrence of POI, which is consistent with our findings. This study identified a novel CLPP missense variant (c.628G > A) in a woman with POI who presented with secondary amenorrhea, ovarian dysfunction, and primary infertility. The variant was located in exon 5 and resulted in a change from alanine to threonine (p.Ala210Thr). Importantly, Clpp was mainly localized in the cytoplasm of mouse ovarian granulosa cells and oocytes, and was relatively highly expressed in granulosa cells. Moreover, the overexpression of c.628G > A variant in human ovarian granulosa cells decreased the proliferative capacity. Functional experiments revealed that the inhibition of CLPP decreased the content and activity of oxidative respiratory chain complex IV by affecting the degradation of aggregated or misfolded COX5A, leading to the accumulation of reactive oxygen species and reduction of mitochondrial membrane potential, ultimately activating the intrinsic apoptotic pathways. The present study demonstrated that CLPP affected the apoptosis of granulosa cells, which might be one of the mechanisms by which CLPP aberrations led to the development of POI.

COX5A as a potential biomarker for disease activity and organ damage in lupus.

Systemic Lupus Erythematosus (SLE) is a complex autoimmune disease with limited therapeutic targets or clinical outcome predictors. This study aimed to gain more insights into the underlying immunological pathways and prognostic biomarkers of SLE. Integrated analyses of RNA-seq data from 64 SLE and 62 healthy controls, examining 27 immune cell types to explore the key pathways and driver genes in SLE pathogenesis. Single-cell RNA sequencing data from the skin and kidney were used to determine the association of COX5A expression with organ damage. The associations of COX5A with SLE phenotypes were further evaluated in two independent cohorts, and receiver operating characteristic (ROC) curves were constructed to assess the value of COX5A as a biomarker for disease activity and organ damage in SLE. We found that oxidative phosphorylation (OXPHOS) is the most significantly altered metabolic pathway in SLE, especially in effector T cells. Notably, we identified an OXPHOS-related enzyme, COX5A, whose expression was significantly higher in effector T cells than in naïve T cells and showed associations with disease activity, organ damage, and steroid treatment of SLE. Furthermore, ROC curves showed that COX5A is a robust biomarker for disease activity, kidney involvement, and new-onset skin lesions, with the area under the curve (AUC) values of 0.880, 0.801, and 0.805, respectively. Our results identified the OXPHOS signature as a prominent feature in SLE T cells, and COX5A as a potential candidate biomarker for disease activity and organ damage in SLE.

Protective Role of Cytochrome C Oxidase 5A (COX5A) against Mitochondrial Disorder and Oxidative Stress in VSMC Phenotypic Modulation and Neointima Formation.

BACKGROUND: The pathological role of cytochrome c oxidase 5A (COX5A) in vascular neointima formation remains unknown. AIM: This study aims to investigate the role of COX5A on platelet-derived growth factor BB (PDGFBB)- mediated smooth muscle phenotypic modulation and neointima formation and clarify the molecular mechanisms behind this effect. METHODS: For in vitro assays, human aortic vascular smooth muscle cells (HA-VSMCs) were transfected with pcDNA3.1-COX5A and COX5A siRNA to overexpress and knockdown COX5A, respectively. Mitochondrial complex IV activity, oxygen consumption rate (OCR), H2O2 and ATP production, reactive oxygen species (ROS) generation, cell proliferation, and migration were measured. For in vivo assays, rats after balloon injury (BI) were injected with recombinant lentivirus carrying the COX5A gene. Mitochondrial COX5A expression, carotid arterial morphology, mitochondrial ultrastructure, and ROS were measured. RESULTS: The results showed that PDGF-BB reduced the level and altered the distribution of COX5A in mitochondria, as well as reduced complex IV activity, ATP synthesis, and OCR while increasing H2O2 synthesis, ROS production, and cell proliferation and migration. These effects were reversed by overexpression of COX5A and aggravated by COX5A knockdown. In addition, COX5A overexpression attenuated BI-induced neointima formation, muscle fiber area ratio, VSMC migration to the intima, mitochondrial ultrastructural damage, and vascular ROS generation. CONCLUSION: The present study demonstrated that COX5A protects VSMCs against phenotypic modulation by improving mitochondrial respiratory function and attenuating mitochondrial damage, as well as reducing oxidative stress, thereby preventing neointima formation.

COX5A Plays a Vital Role in Memory Impairment Associated With Brain Aging via the BDNF/ERK1/2 Signaling Pathway.

Cytochrome c oxidase subunit Va (COX5A) is involved in maintaining normal mitochondrial function. However, little is known on the role of COX5A in the development and progress of Alzheimer's disease (Martinez-Losa et al., 2018). In this study, we established and characterized the genomic profiles of genes expressed in the hippocampus of Senescence-Accelerated Mouse-prone 8 (SAMP8) mice, and revealed differential expression of COX5A among 12-month-aged SAMP8 mice and 2-month-aged SAMP8 mice. Newly established transgenic mice with systemic COX5A overexpression (51% increase) resulted in the improvement of spatial recognition memory and hippocampal synaptic plasticity, recovery of hippocampal CA1 dendrites, and activation of the BDNF/ERK1/2 signaling pathway in vivo. Moreover, mice with both COX5A overexpression and BDNF knockdown showed a poor recovery in spatial recognition memory as well as a decrease in spine density and branching of dendrites in CA1, when compared to mice that only overexpressed COX5A. In vitro studies supported that COX5A affected neuronal growth via BDNF. In summary, this study was the first to show that COX5A in the hippocampus plays a vital role in aging-related cognitive deterioration via BDNF/ERK1/2 regulation, and suggested that COX5A may be a potential target for anti-senescence drugs.

The Dual Role of AQP4 in Cytotoxic and Vasogenic Edema Following Spinal Cord Contusion and Its Possible Association With Energy Metabolism via COX5A.

Spinal cord edema, mainly including vasogenic and cytotoxic edema, influences neurological outcome after spinal cord contusion (SCC). Aquaporin 4 (AQP4) is the most ubiquitous water channel in the central nervous system (CNS), which is a rate-limiting factor in vasogenic edema expressing in brain injury, and it contributes to the formation of cytotoxic edema locating in astrocytes. However, little is known about the regulatory mechanism of AQP4 within vasogenic and cytotoxic edema in SCC, and whether the regulation mechanism of AQP4 is related to Cytochrome coxidase (COX5A) affecting energy metabolism. Therefore, the SCC model is established by Allen's method, and the degree of edema and neuronal area is measured. The motor function of rats is evaluated by the Basso, Beattie, and Bresnahan (BBB) scoring system. Meanwhile, AQP4 and COX5A are detected by real-time quantitative PCR (qRT-PCR) and western blot (WB). The localization of targeted protein is exhibited by immunohistochemical staining (IHC) and immunofluorescence (IF). Additionally, the methodology of AQP4 lentivirus-mediated RNA interference (AQP4-RNAi) is used to reveal the effect on edema of SCC and the regulating molecular mechanism. Firstly, we observe that the tissue water content increases after SCC and decreases after the peak value of tissue water content at 3 days (P < 0.05) with abundant expression of AQP4 protein locating around vascular endothelial cells (VECs), which suggests that the increasing AQP4 promotes water reabsorption and improves vasogenic edema in the early stage of SCC. However, the neuronal area is larger than in the sham group in the 7 days (P < 0.05) with the total water content of spinal cord decrease. Meanwhile, AQP4 migrates from VECs to neuronal cytomembrane, which indicates that AQP4 plays a crucial role in aggravating the formation and development of cytotoxic edema in the middle stages of SCC. Secondly, AQP4-RNAi is used to elucidate the mechanism of AQP4 to edema of SCC. The neuronal area shrinks and the area of cytotoxic edema reduces after AQP4 downregulation. The BBB scores are significantly higher than in the vector group after AQP4-RNAi at 5, 7, and 14 (P < 0.05). There is a relationship between AQP4 and COX5A shown by bioinformatics analysis. After AQP4 inhibition, the expression of COX5A is significantly upregulated in the swelling astrocytes. Therefore, the inhibition of AQP4 expression reduces cytotoxic edema in SCC and improves motor function, which may be associated with upregulation of COX5A via affecting energy metabolism. Moreover, it is not clear how the inhibition of AQP4 directly causes the upregulation of COX5A.