Scaling of smaller pyramidal neuron size and lower energy production in schizophrenia.

BACKGROUND: Dorsolateral prefrontal cortex (DLPFC) dysfunction in schizophrenia appears to reflect alterations in layer 3 pyramidal neurons (L3PNs), including smaller cell bodies and lower expression of mitochondrial energy production genes. However, prior somal size studies used biased strategies for identifying L3PNs, and somal size and levels of energy production markers have not been assessed in individual L3PNs. STUDY DESIGN: We combined fluorescent in situ hybridization (FISH) of vesicular glutamate transporter 1 (VGLUT1) mRNA and immunohistochemical-labeling of NeuN to determine if the cytoplasmic distribution of VGLUT1 mRNA permits the unbiased identification and somal size quantification of L3PNs. Dual-label FISH for VGLUT1 mRNA and cytochrome C oxidase subunit 4I1 (COX4I1) mRNA, a marker of energy production, was used to assess somal size and COX4I1 transcript levels in individual DLPFC L3PNs from schizophrenia (12 males; 2 females) and unaffected comparison (13 males; 1 female) subjects. STUDY RESULTS: Measures of L3PN somal size with NeuN immunohistochemistry or VGLUT1 mRNA provided nearly identical results (ICC = 0.96, p < 0.0001). Mean somal size of VGLUT1-identified L3PNs was 8.7% smaller (p = 0.004) and mean COX4I1 mRNA levels per L3PN were 16.7% lower (p = 0.01) in schizophrenia. These measures were correlated across individual L3PNs in both subject groups (rrm = 0.81-0.86). CONCLUSIONS: This preliminary study presents a novel method for combining unbiased neuronal identification with quantitative assessments of somal size and mRNA levels. We replicated findings of smaller somal size and lower COX4I1 mRNA levels in DLPFC L3PNs in schizophrenia. The normal scaling of COX4I1 mRNA levels with somal size in schizophrenia suggests that lower markers of energy production are secondary to L3PN morphological alterations in the illness.

HIF1A transcriptional regulation of COX4I2 impacts angiogenesis in pheochromocytoma.

BACKGROUND: Pheochromocytoma (PCC) is a rare neuroendocrine tumor. Angiogenesis is primary contributing factor for tumorigenesis. Cytochrome c oxidase 4I2 (COX4I2) has been confirmed to take part in the progression of cancer. Hypoxia-inducible factor 1A (HIF1A) is the main regulatory factor for the steady-state response of hypoxia, involved in metabolism and angiogenesis. In this study, we intended to explore the functions of COX4I2 in PCC and the effect mechanism between HIF1A and COX4I2. MATERIALS AND METHODS: The RNA-sequencing and immunohistochemistry tested COX4I2 expression in highly vascular PCC. Small interfering RNA (siRNA) was used to reduce the mRNA expression of COX4I2, and a small molecule inhibitor was utilized to reduce the protein expression of HIF1A. Culturing cells in 1% O2environment was performed to activate HIF1A. Western blot was applied to quantify the expression of target genes at the protein levels. The supernatant from PCC cells and fibroblasts acted as the conditioned medium. We conducted the tube formation and transwell assays in human vascular endothelial cells (HUVECs) to determine angiogenesis, the binding of COX4I2 promoter and HIF1A was evaluated by the dual luciferase reporter assay. RESULTS: COX4I2 had been rigorously shown to be overexpressed in highly vascular PCC. Knockdown of COX4I2 in PCC cells (MPC) did not significantly impact angiogenesis, while knockdown of COX4I2 in fibroblast (3T3) notably inhibited angiogenesis. RNA sequencing suggested that the expression of 11 vascular markers, such as CD34 and angiogenesis associated pathways in 3T3, decreased with knockdown of COX4I2. HIF1A had been shown to enhance the mRNA expression of COX4I2 through transcriptional regulation. Activation and inhibition of HIF1A resulted in upregulation and downregulation of COX4I2, respectively. The HIF1A inhibitor demonstrated a reduction in angiogenesis. CONCLUSION: COX4I2 is overexpressed in highly vascular PCC and contributes to angiogenesis in fibroblasts. Mechanistically, HIF1A transcriptional regulation enhances COX4I2 and its effects on angiogenesis in PCC. COX4I2 might serve as a vascular marker and represent a potential target for vascular therapy.

PTENα is responsible for protection of brain against oxidative stress during aging.

Neural cells are continuously subjected to oxidative stress arising from electrochemical activity, and cellular protection systems can turn on the oxidative stress response to detect and alleviate adverse conditions. However, the function and mechanism of the protective systems are complicated and remain largely elusive. We report that PTENα, an isoform of the PTEN family, mediates defense signaling in response to oxidative stress during brain aging. We show that genetic ablation of Ptenα in mice increases oxidative stress and results in neuronal cell death, culminating in accelerated decline of cognition and motor coordination as age increases. PTENα maintains COX activity and promotes energy metabolism through abrogating NEDD4L-mediated degradation of COX4 in response to oxidative stress. In the presence of Parkinson's disease-associated mutation, PTENα loses the capability to protect COX4 and ameliorate defects caused by Ptenα deletion. Our study reveals an important role of PTENα in response to oxidative stress. We propose that dysregulation of PTENα signaling may accelerate the rate of brain aging and promote the development of neurodegenerative disorders.

Identification of COX4I2 as a hypoxia-associated gene acting through FGF1 to promote EMT and angiogenesis in CRC.

BACKGROUND: Current evidence suggests that the hypoxic tumor microenvironment further aggravates tumor progression, leading to poor therapeutic outcomes. There is as yet no biomarker capable of evaluating the hypoxic state of the tumor. The cytochrome c oxidase (COX) subunit is crucial to the mitochondrial respiratory chain. METHODS: We investigated the potential oncogenic role of COX subunit 4 isoform 2 gene (COX4I2) in colorectal cancer (CRC) by least absolute shrinkage and selection operator (LASSO) and COX regression analysis to examine whether COX4I2 overexpression can predict colorectal cancer (CRC) prognosis. The association of COX4I2 levels with clinical features and its biological actions were evaluated both in vitro and in vivo. RESULTS: Our analysis showed that elevated COX4I2 levels were correlated with poor clinical outcomes. We also observed that that COX4I2 may be involved in epithelial-mesenchymal transition, activation of cancer-related fibroblasts and angiogenesis in relation to fibroblast growth factor 1. CONCLUSIONS: The COX4I2 level may be a predictor of outcome in CRC and may represent a novel target for treatment development.

Seasonal changes in mitochondrial bioenergetics and physiological performance of the bluegill sunfish, Lepomis macrochirus, from a shallow, Midwest river.

As global temperature shifts due to anthropogenic impacts, seasonal temperatures in shallow aquatic ecosystems are expected to increase. Previous studies on freshwater fishes that experience significant temperature changes during the annual seasons found pronounced physiological restructuring not observed in animals inhabiting more thermally stable environments. Studies evaluating mitochondrial bioenergetics in fish are often performed on animals acclimated to constant temperatures in the laboratory. However, natural habitats are much more complex. Fishes may experience substantial daily and seasonal variation in temperature, energy requirements and resource availability, which are impossible to emulate on acclimation studies. Our study explores the effects of these more complex natural environments on whole-organism thermal tolerance and mitochondrial bioenergetics in bluegill sunfish (Lepomis macrochirus), a native fish to the temperate zone of North America. Compensatory mechanisms and variations in physiological thresholds were observed in specimens acclimatized to the fall season compared to specimens acclimatized to spring and summer seasons. Somatic indices, such as relative weights and hepatosomatic indices, showed significant differences across seasons and critical thermal maxima significantly decreased in the cold acclimatized specimens. Liver mitochondria from L. macrochirus also showed significantly higher uncoupled proton conductance, cytochrome c oxidase (COX) activity, and reduced respiratory control ratios in individuals sampled in the colder season. These findings suggest that mechanisms regulating proton conductance and COX activity modulate mitochondrial function across seasons to sustain physiological fitness in ectotherms inhabiting shallow, inland aquatic habitats.

Replicative Stress Coincides with Impaired Nuclear DNA Damage Response in COX4-1 Deficiency.

Cytochrome c oxidase (COX), a multimeric protein complex, is the final electron acceptor in the mitochondrial electron transfer chain. Primary COX deficiency, caused by mutations in either mitochondrial DNA or nuclear-encoded genes, is a heterogenous group of mitochondrial diseases with a wide range of presentations, ranging from fatal infantile to subtler. We previously reported a patient with primary COX deficiency due to a pathogenic variant in COX4I1 (encoding the common isoform of COX subunit 4, COX4-1), who presented with bone marrow failure, genomic instability, and short stature, mimicking Fanconi anemia (FA). In the present study, we demonstrated that accumulative DNA damage coincided primarily with proliferative cells in the patient's fibroblasts and in COX4i1 knockdown cells. Expression analysis implicated a reduction in DNA damage response pathways, which was verified by demonstrating impaired recovery from genotoxic insult and decreased DNA repair. The premature senescence of the COX4-1-deficient cells prevented us from undertaking additional studies; nevertheless, taken together, our results indicate replicative stress and impaired nuclear DNA damage response in COX4-1 deficiency. Interestingly, our in vitro findings recapitulated the patient's presentation and present status.

The Dynll1-Cox4i1 Complex Regulates Intracellular Pathogen Clearance via Release of Mitochondrial Reactive Oxygen Species.

Cellular membrane proteins are a critical part of the host defense mechanisms against infection and intracellular survival of Listeria monocytogenes The complex spatiotemporal regulation of bacterial infection by various membrane proteins has been challenging to study. Here, using mass spectrometry analyses, we depicted the dynamic expression landscape of membrane proteins upon L. monocytogenes infection in dendritic cells. We showed that Dynein light chain 1 (Dynll1) formed a persistent complex with the mitochondrial cytochrome oxidase Cox4i1, which is disturbed by pathogen insult. We discovered that the dissociation of the Dynll1-Cox4i1 complex is required for the release of mitochondrial reactive oxygen species and serves as a regulator of intracellular proliferation of Listeria monocytogenes Our study shows that Dynll1 is an inhibitor of mitochondrial reactive oxygen species and can serve as a potential molecular drug target for antibacterial treatment.

Biallelic variants in COX4I1 associated with a novel phenotype resembling Leigh syndrome with developmental regression, intellectual disability, and seizures.

Autosomal recessive COX4I1 deficiency has been previously reported in a single individual with a homozygous pathogenic variant in COX4I1, who presented with short stature, poor weight gain, dysmorphic features, and features of Fanconi anemia. COX4I1 encodes subunit 4, isoform 1 of cytochrome c oxidase. Cytochrome c oxidase is a respiratory chain enzyme that plays an important role in mitochondrial electron transport and reduces molecular oxygen to water leading to the formation of ATP. Defective production of cytochrome c oxidase leads to a variable phenotypic spectrum ranging from isolated myopathy to Leigh syndrome. Here, we describe two siblings, born to consanguineous parents, who presented with encephalopathy, developmental regression, hypotonia, pathognomonic brain imaging findings resembling Leigh-syndrome, and a novel homozygous variant on COX4I1, expanding the known clinical phenotype associated with pathogenic variants in COX4I1.

Long-term HIF-1α transcriptional activation is essential for heat-acclimation-mediated cross tolerance: mitochondrial target genes.

An important adaptive feature of heat acclimation (HA) is the induction of cross tolerance against novel stressors (HACT) Reprogramming of gene expression leading to enhanced innate cytoprotective features by attenuating damage and/or enhancing the response of "help" signals plays a pivotal role. Hypoxia-inducible factor-1α (HIF-1α), constitutively upregulated by HA (1 mo, 34°C), is a crucial transcription factor in this program, although its specific role is as yet unknown. By using a rat HA model, we studied the impact of disrupting HIF-1α transcriptional activation [HIF-1α:HIF-1ß dimerization blockade by intraperitoneal acriflavine (4 mg/kg)] on its mitochondrial gene targets [phosphoinositide-dependent kinase-1 (PDK1), LON, and cyclooxygenase 4 (COX4) isoforms] in the HA rat heart. Physiological measures of cardiac HACT were infarct size after ischemia-reperfusion and time to rigor contracture during hypoxia in cardiomyocytes. We show that HACT requires transcriptional activation of HIF-1α throughout the course of HA and that this activation is accompanied by two metabolic switches: 1) profound upregulation of PDK1, which reduces pyruvate entry into the mitochondria, consequently increasing glycolytic lactate production; 2) remodeling of the COX4 isoform ratio, inducing hypoxic-tolerant COX4.2 dominance, and optimizing electron transfer and possibly ATP production during the ischemic and hypoxic insults. LON and COX4.2 transcript upregulation accompanied this shift. Loss of HACT despite elevated expression of the cytoprotective protein heat shock protein-72 concomitantly with disrupted HIF-1α dimerization suggests that HIF-1α is essential for HACT. The role of a PDK1 metabolic switch is well known in hypoxia acclimation but not in the HA model and its ischemic setting. Remodeling of COX4 isoforms by environmental acclimation is a novel finding.

[Effect of Ligustri Lucidi Fructus on hepatic COX activity in normal rats and preliminary investigation of its DNA methylation mechanism].

The study aims to investigate the effect of Ligustri Lucidi Fructus on cytochrome c oxidase(COX) activity in rat hepatic tissues and explore its possible mechanism of DNA methylation. Male SD rats received aqueous extract of Ligustri Lucidi Fructus (2.0，6.0 g•kg⁻¹) by intragastric administration for 30 d. After the rats were sacrificed, hepatic tissues of rats were taken to detect COX activity, protein concentration of COX4I1, TET1 and DNMT3A protein levels, mRNA expression levels of Cox4i1, Dnmt3a and Tet1, and determine the DNA methylation frequency of Cox4i1.Results showed that both low and high doses of Ligustri Lucidi Fructus could significantly increase COX activity and concentration of COX4I1(P<0.05), with a decreasing tendency of both TET1 protein and DNMT3a protein expression； however, mRNA expression levels of Cox4i1, Dnmt3a and Tet1 were not significantly changed by Ligustri Lucidi Fructus. In addition, DNA methylation frequency of Cox4i1 in high dose group showed a declining tendency as compared with the blank control group, but without significant difference.These results indicated that Ligustri Lucidi Fructus had promotive effect on hepatic COX activity in rats, which may be achieved by increasing protein content of COX4I1. Moreover, a decreased tendency of DNMT3A protein could be one of the reasons for the lower trend of Cox4i1 methylation rate. In addition, Ligustri Lucidi Fructus may regulate the expression levels of DNMT3A and TET1 in the same direction and its mechanism is not clear.

Evolution of the oxygen sensitivity of cytochrome c oxidase subunit 4.

Vertebrates possess two paralogs of cytochrome c oxidase (COX) subunit 4: a ubiquitous COX4-1 and a hypoxia-linked COX4-2. Mammalian COX4-2 is thought to have a role in relation to fine-tuning metabolism in low oxygen levels, conferred through both structural differences in the subunit protein structure and regulatory differences in the gene. We sought to elucidate the pervasiveness of this feature across vertebrates. The ratio of COX4-2/4-1 mRNA is generally low in mammals, but this ratio was higher in fish and reptiles, particularly turtles. The COX4-2 gene appeared unresponsive to low oxygen in nonmammalian models (zebrafish, goldfish, tilapia, anoles, and turtles) and fish cell lines. Reporter genes constructed from the amphibian and reptile homologues of the mammalian oxygen-responsive elements and hypoxia-responsive elements did not respond to low oxygen. Unlike the rodent ortholog, the promoter of goldfish COX4-2 did not respond to hypoxia or anoxia. The protein sequences of the COX4-2 peptide showed that the disulfide bridge seen in human and rodent orthologs would be precluded in other mammalian lineages and lower vertebrates, all of which lack the requisite pair of cysteines. The coordinating ligands of the ATP-binding site are largely conserved across mammals and reptiles, but in Xenopus and fish, sequence variations may disrupt the ability of the protein to bind ATP at this site. Collectively, these results suggest that many of the genetic and structural features of COX4-2 that impart responsiveness and benefits in hypoxia may be restricted to the Euarchontoglires lineage that includes primates, lagomorphs, and rodents.

An algorithm-based technique for counting mitochondria in cells using immunohistochemical staining of formalin-fixed and paraffin-embedded sections.

PURPOSE: Visualizing mitochondria in cancer cells from human pathological specimens may improve our understanding of cancer biology. However, using immunohistochemistry to evaluate mitochondria remains difficult because almost all cells contain mitochondria and the number of mitochondria per cell may have important effects on mitochondrial function. Herein, we established an objective system (Mito-score) for evaluating mitochondria using machine-based processing of hue, saturation, and value color spaces. METHODS: The Mito-score was defined as the number of COX4 (mitochondrial inner membrane) immunohistochemistry-positive pixels divided by the number of nuclei per cell. The system was validated using four lung cancer cell lines, normal tissues, and lung cancer tissues (199 cases). RESULTS: The Mito-score correlated with MitoTracker, a fluorescent dye used to selectively label and visualize mitochondria within cells under a microscope (R2 = 0.68) and with the number of mitochondria counted using electron microscopy (R2 = 0.79). Histologically, the Mito-score of small cell carcinoma (57.25) was significantly lower than that of adenocarcinoma (147.5, p < 0.0001), squamous cell carcinoma (120.6, p = 0.0004), and large cell neuroendocrine carcinoma (111.8, p = 0.002). CONCLUSION: The Mito-score method enables the analysis of the mitochondrial status of human formalin-fixed paraffin-embedded specimens and may provide insights into the metabolic status of cancer.

EBF1-COX4I2 signaling axis promotes a myofibroblast-like phenotype in cancer-associated fibroblasts (CAFs) and is associated with an immunosuppressive microenvironment.

Immunotherapy has limited response rates in colorectal cancer (CRC) due to an immunosuppressive tumor microenvironment (TME). Combining transcriptome sequencing, clinical specimens, and functional experiments, we identified a unique group of CAF subpopulations (COX4I2 + ) with inhibited mitochondrial respiration and enhanced glycolysis. Through bioinformatics predictions and luciferase reporter assays, we determined that EBF1 can upstreamly regulate COX4I2 transcription. COX4I2 + CAFs functionally and phenotypically resemble myofibroblasts, are important for the formation of the fibrotic TME, and are capable of activating the M2 phenotype of macrophages. In vitro experiments demonstrated that COX4I2 + CAFs promote immunosuppressive TME by blocking CD8 + T cell infiltration and inducing CD8 + T cell dysfunction. Using multiple independent cohorts, we also found a strong correlation between the immunotherapy response rate of CRC patients and COX4I2 expression in their tumors. Our results identify a CAF subpopulation characterized by activation of the EBF1-COX4I2 axis, and this group of CAFs can be targeted to improve cancer immunotherapy outcomes.

Cytochrome c oxidase IV isoform 1 (COX4-1) regulates the proliferation, migration and invasion of trophoblast cells via modulating mitochondrial function.

INTRODUCTION: Spontaneous miscarriage is a common complication of early pregnancy. Previous studies have shown that mitochondrial function plays an important role in establishment of a successful pregnancy. Cytochrome c oxidase subunit 4 isoform 1 (COX4I1), a component of electron transport chain complex Ⅳ, is required for coupling the rate of ATP production to energetic requirements. However, there is very limited research on its role in trophoblast biology and how its dysfunction may contribute to spontaneous miscarriage. METHODS: Placental villi (7-10 weeks gestational age) collected from either induced termination of pregnancy or after spontaneous miscarriage were examined for expression of COX4I1. COX4I1 was knocked down by siRNA transfection of primary isolates of EVT cells. Real-time cell analysis (RTCA) and 5-Ethynyl-2'-deoxyuridine (EdU) were used to detect changes in proliferation ability after COX4I1 knockdown of EVT cells. Migration and invasion indices were determined by RTCA. Mitochondrial morphology was observed via MitoTracker staining. Oxidative phosphorylation, ATP production, and glycolysis in COX4I1-deficient cells and controls were assessed by a cellular energy metabolism analyzer (Seahorse). RESULTS: In placental villous tissue, COX4I1 expression was significantly decreased in the spontaneous miscarriage group. Knockdown of COX4I1 inhibited EVT cell proliferation, increased the migration and invasion ability and mitochondrial fusion of EVT cells. Mitochondrial respiration and glycolysis were impaired in COX4I1-deficient EVT cells. Knockdown of MMP1 could rescue the increased migration and invasion induced by COX4I1 silencing. DISCUSSION: Low expression of COX4I1 leads to mitochondrial dysfunction in EVT, resulting in altered trophoblast function, and ultimately to pregnancy loss.

Reprogramming of Mitochondrial Respiratory Chain Complex by Targeting SIRT3-COX4I2 Axis Attenuates Osteoarthritis Progression.

Mitochondrial homeostasis is of great importance for cartilage integrity and associated with the progression of osteoarthritis (OA); however, the underlying mechanisms are unknown. This study aims to investigate the role of mitochondrial deacetylation reaction and investigate the mechanistic relationship OA development. Silent mating type information regulation 2 homolog 3 (SIRT3) expression has a negative correlation with the severity of OA in both human arthritic cartilage and mice inflammatory chondrocytes. Global SIRT3 deletion accelerates pathological phenotype in post-traumatic OA mice, as evidenced by cartilage extracellular matrix collapse, osteophyte formation, and synovial macrophage M1 polarization. Mechanistically, SIRT3 prevents OA progression by targeting and deacetylating cytochrome c oxidase subunit 4 isoform 2 (COX4I2) to maintain mitochondrial homeostasis at the post-translational level. The activation of SIRT3 by honokiol restores cartilage metabolic equilibrium and protects mice from the development of post-traumatic OA. Collectively, the loss of mitochondrial SIRT3 is essential for the development of OA, whereas SIRT3-mediated proteins deacetylation of COX4I2 rescues OA-impaired mitochondrial respiratory chain functions to improve the OA phenotype. Herein, the induction of SIRT3 provides a novel therapeutic candidate for OA treatment.

A SNP of the COX4I2 gene associated with environmental adaptation in Chinese cattle.

COX4I2 is an isoform of cytochrome C oxidase subunit IV (COX4), which plays an important role in mitochondrial oxidative phosphorylation. This gene affects heat production and thus affects thermoregulatory capacity in mammals. A splice region variant (rs109072064, NC_037340.1:g.61202988C > T) was identified in COX4I2 by using Ensembl, which transforms the amino acid arginine into cysteine in XP_005214921.1. In this study, we sought to determine the relationship between the mutant locus and the environment in which the cattle are located. We verified that mRNA (XM_005214864.4), which translated XP_005214921.1, is expressed in bovine muscle, fat, heart, liver, kidney, lung and testis tissues. The g.61202988C > T variant was then genotyped in 569 individuals of 34 cattle breeds. Compared with the CC genotype, southern cattle carried more the CT and TT genotypes. Furthermore, the association results carried out that the frequencies of genotypes (CC, CT, TT) and the value of climate parameters (mean annual temperature (T), relative humidity (RH) and temperature humidity index (THI)) were significantly correlated (P < 0.01). Hence, we speculated that the g.61202988C > T variant of COX4I2 gene was associated with the environmental adaptation trait in Chinese cattle and the locus may be considered as a molecular marker for Chinese cattle breeding.

COX4-1 promotes mitochondrial supercomplex assembly and limits reactive oxide species production in radioresistant GBM.

Glioblastoma (GBM) is a fatal disease with recurrences often associated with radioresistance. Although often effective at treating newly diagnosed GBM, increasing evidence suggests that radiotherapy-induced alterations in tumor metabolism promote GBM recurrence and aggressiveness. Using isogenic radiosensitive and radioresistant GBM cell lines and patient-derived xenolines, we found that acquired radioresistance is associated with a shift from a glycolytic metabolism to a more oxidative metabolism marked by a substantial increase in the activity of the mitochondrial respiratory chain complex cytochrome c oxidase (CcO). This elevated CcO activity was associated with a switch in the isoform expression of the CcO regulatory subunit COX4, from COX4-2 to COX4-1, assembly of CcO-containing mitochondrial supercomplexes (SCs), and reduced superoxide (O2 •-) production. Overexpression of COX4-1 in the radiosensitive cells was sufficient to promote the switch from glycolytic to oxidative metabolism and the incorporation of CcO into SCs, with a concomitant reduction in O2 •- production. Conversely, silencing of COX4-1 expression in normally radioresistant cells reduced CcO activity, promoted the disassembly of mitochondrial SCs, and increased O2 •- production. Additionally, gain or loss of COX4-1 expression was sufficient to induce the radioresistant or radiosensitive phenotype, respectively. Our results demonstrate that COX4-1 promotes SC assembly in GBM cells, and SC assembly may in turn regulate the production of reactive oxygen species and thus the acquisition of radioresistance in GBM.

Fibroblasts mediate the angiogenesis of pheochromocytoma by increasing COX4I2 expression.

Objective: Our previous work found COX4I2 was associated with angiogenesis in pheochromocytoma. The purpose of this study was to explore the role of COX4I2 in regulating angiogenesis in pheochromocytoma. Methods: Distribution of COX4I2 was evaluated by scRNA-seq in one case of pheochromocytoma and the findings were verified by immunostaining. COX4I2 was further knocked down in target cells. Changes of angiogenesis-related genes were evaluated by qPCR in target cells. Results: The scRNA-seq revealed high mRNA expression of COX4I2 in fibroblasts rather than tumor cells. Immunostaining of COX4I2 confirmed its distribution in fibroblasts. Knocking down COX4I2 in NIH3T3 cell line led to significant reduction of angiogenesis-related genes, especially ANG1 and HGF. Conclusions: Fibroblasts mediate the angiogenesis of pheochromocytoma by increasing COX4I2 expression, possibly by affecting ANG1 and HGF.

Effect of Expression of Nuclear-Encoded Cytochrome C Oxidase Subunit 4 Isoforms on Metabolic Profiles of Glioma Cells.

Although often effective at treating newly diagnosed glioblastoma (GBM), increasing evidence suggests that chemo- and radiotherapy-induced alterations in tumor metabolism promote GBM recurrence and aggressiveness, as well as treatment resistance. Recent studies have demonstrated that alterations in glioma cell metabolism, induced by a switch in the isoform expression of cytochrome c oxidase subunit 4 (COX4), a key regulatory subunit of mammalian cytochrome c oxidase, could promote these effects. To understand how the two COX4 isoforms (COX4-1 and COX4-2) differentially affect glioma metabolism, glioma samples harvested from COX4-1- or COX4-2-overexpressing U251 cells were profiled using Gas chromatography-mass spectrometry GC-MS and Liquid Chromatography - Tandem Mass Spectrometry LC-MS/MS metabolomics platforms. The concentration of 362 metabolites differed significantly in the two cell types. The two most significantly upregulated pathways associated with COX4-1 overexpression were purine and glutathione metabolism; the two most significantly downregulated metabolic pathways associated with COX4-1 expression were glycolysis and fatty acid metabolism. Our study provides new insights into how Cytochrome c oxidase (CcO) regulatory subunits affect cellular metabolic networks in GBM and identifies potential targets that may be exploited for therapeutic benefit.

Mitochondrial oxygen sensing of acute hypoxia in specialized cells - Is there a unifying mechanism?

Acclimation to acute hypoxia through cardiorespiratory responses is mediated by specialized cells in the carotid body and pulmonary vasculature to optimize systemic arterial oxygenation and thus oxygen supply to the tissues. Acute oxygen sensing by these cells triggers hyperventilation and hypoxic pulmonary vasoconstriction which limits pulmonary blood flow through areas of low alveolar oxygen content. Oxygen sensing of acute hypoxia by specialized cells thus is a fundamental pre-requisite for aerobic life and maintains systemic oxygen supply. However, the primary oxygen sensing mechanism and the question of a common mechanism in different specialized oxygen sensing cells remains unresolved. Recent studies unraveled basic oxygen sensing mechanisms involving the mitochondrial cytochrome c oxidase subunit 4 isoform 2 that is essential for the hypoxia-induced release of mitochondrial reactive oxygen species and subsequent acute hypoxic responses in both, the carotid body and pulmonary vasculature. This review compares basic mitochondrial oxygen sensing mechanisms in the pulmonary vasculature and the carotid body.