Oxidative Phosphorylation Genes Research Articles

Pathomic and bioinformatics analysis of clinical-pathological and genomic factors for pancreatic cancer prognosis.

Pancreatic cancer exhibits a high degree of malignancy with a poor prognosis, lacking effective prognostic targets. Utilizing histopathological methodologies, this study endeavors to predict the expression of pathological features in pancreatic ductal adenocarcinoma (PAAD) and investigate their underlying molecular mechanisms. Pathological images, transcriptomic, and clinical data from TCGA-PAAD were collected for survival analysis. Image segmentation using unsupervised machine learning was employed to extract features, perform clustering, and establish models. The prognostic value of pathological features and associated clinical risk factors were evaluated; the correlation between pathological features and molecular mechanisms, gene mutations, and immune infiltration was analyzed. By clustering 45 effective pathological features, we divided PAAD patients into two groups: cluster 1 and cluster 2. Significant associations with poor prognosis were found for cluster 2 in both the training group (n = 113) and validation group (n = 75) (p = 0.006), with pathological stages II-IV identified as potential synergistic risk factors (HR = 2.421, 95% CI = 1.263-4.639, p = 0.008). Subsequently, through multi-omics correlation analysis, we further revealed a close association between cluster 2 and the oxidative phosphorylation mechanism. Within the cluster 2 group, 28 oxidative phosphorylation genes exhibited reduced expression, CDKN2A gene mutations were upregulated, and there was significant downregulation of Tregs infiltration and related immune gene expression. The pathomic model constructed using machine learning serves as a valuable prognostic target for PAAD. The histopathological features cluster 2 are closely associated with the downregulation of oxidative phosphorylation levels and Tregs immune infiltration.

CNSC-46. GLIOBLASTOMA INITIATION, MIGRATION, AND CELL TYPES ARE REGULATED BY CORE BHLH TRANSCRIPTION FACTORS ASCL1 AND OLIG2

Abstract Glioblastomas (GBMs) are highly aggressive, infiltrative, and heterogeneous brain tumors driven by complex genetic alterations. The neurodevelopmental transcription factors ASCL1 and OLIG2 are highly co-expressed in GBMs. However, their combinatorial roles in regulating the hierarchy and heterogeneity of GBM cells are unknown. Here, we show that induction of somatic mutations in neural progenitor cells lead to the dysregulation of ASCL1 and OLIG2, which then function redundantly and are required for brain tumor formation in a mouse model of GBM. Subsequently, the binding of ASCL1 and OLIG2 to each other’s loci and to downstream target genes then determine the cell types and degree of migration of tumor cells. Notably, single-cell RNA sequencing (scRNA-seq) reveals that a high level of ASCL1 is key in promoting neural stem cell (NSC)/astrocyte-like tumor cell types, which are highly proliferative, migratory, and are marked by upregulation of ribosomal protein, oxidative phosphorylation, cancer metastasis, and therapeutic resistance genes.

EIF5A controls mitoprotein import by relieving ribosome stalling at TIM50 translocase mRNA.

Efficient import of nuclear-encoded proteins into mitochondria is crucial for proper mitochondrial function. The conserved translation factor eIF5A binds ribosomes, alleviating stalling at polyproline-encoding sequences. eIF5A impacts mitochondrial function across species, though the precise molecular mechanism is unclear. We found that eIF5A depletion in yeast reduces the translation and levels of the TCA cycle and oxidative phosphorylation proteins. Loss of eIF5A causes mitoprotein precursors to accumulate in the cytosol and triggers a mitochondrial import stress response. We identify an essential polyproline protein as a direct target of eIF5A: the mitochondrial inner membrane protein and translocase component Tim50. Thus, eIF5A controls mitochondrial protein import by alleviating ribosome stalling along Tim50 mRNA at the mitochondrial surface. Removal of polyprolines from Tim50 partially rescues the mitochondrial import stress response and translation of oxidative phosphorylation genes. Overall, our findings elucidate how eIF5A impacts the mitochondrial function by promoting efficient translation and reducing ribosome stalling of co-translationally imported proteins, thereby positively impacting the mitochondrial import process.

Modified mRNA-based gene editing reveals sarcomere-based regulation of gene expression in human induced-pluripotent stem cell-derived cardiomyocytes

Mutations in genes coding sarcomere components are the major causes of human inherited cardiomyopathy. Genome editing is widely applied to genetic modification of human pluripotent stem cells (hPSCs) before hPSCs were differentiated into cardiomyocytes to model cardiomyopathy. Whether genetic mutations influence the early hPSC differentiation process or solely the terminally differentiated cardiomyocytes during cardiac pathogenesis remains challenging to distinguish. To solve this problem, here we harnessed chemically modified mRNA (modRNA) and synthetic single-guide RNA to develop an efficient genome editing approach in hPSC-derived cardiomyocytes (hPSC-CMs). We showed that modRNA-based CRISPR/Cas9 mutagenesis of TNNT2, the coding gene for cardiac troponin T, results in sarcomere disassembly and contractile dysfunction in hPSC-CMs. These structural and functional phenotypes were associated with profound downregulation of oxidative phosphorylation genes and upregulation of cardiac stress markers NPPA and NPPB. These data confirmed that sarcomeres regulate gene expression in hPSC-CMs and highlighted the RNA technology as a powerful tool to achieve stage-specific genome editing during hPSC differentiation.

Dichloroacetate reduces cisplatin-induced apoptosis by inhibiting the JNK/14-3-3/Bax/caspase-9 pathway and suppressing caspase-8 activation via cFLIP in murine tubular cells

Cisplatin-induced injury to renal proximal tubular cells stems from mitochondrial damage-induced apoptosis and inflammation. Dichloroacetate (DCA), a pyruvate dehydrogenase kinase (PDK) inhibitor, a potential generator of ROS and ATP, protects against cisplatin-induced nephrotoxicity by promoting the TCA cycle. However, its effects on apoptotic pathways and ROS production in renal tubular cells remain unclear. Here, we investigated the detailed molecular mechanisms of the DCA’s effects by immunoblot, RT-PCR, RNA-sequencing, and RNA-silencing in a murine renal proximal tubular (mProx) cell line and mouse kidneys. In mProx cells, DCA suppressed cisplatin-induced apoptosis by attenuating the JNK/14-3-3/Bax/caspase-9 and death receptor/ligand/caspase-8 pathways without impeding inflammatory signaling. RNA-sequencing demonstrated that DCA increased the cisplatin-reduced expression of cFLIP, a caspase-8 inactivator, and decreased the expression of almost all oxidative phosphorylation (OXPHOS) genes. DCA also increased NF-kB activation and ROS production, probably enhancing the cFLIP induction and OXPHOS gene reduction, respectively. Furthermore, cFLIP silencing weakened the DCA’s anti-apoptotic effects. Finally, in mouse kidneys, DCA attenuated cisplatin-caused injuries such as functional and histological damages, caspase activation, JNK/14-3-3 activation, and cFLIP reduction. Conclusively, DCA mitigates cisplatin-induced nephrotoxicity by attenuating the JNK/14-3-3/Bax/caspase-9 pathway and inhibiting the caspase-8 pathways via cFLIP induction, probably outweighing the cisplatin plus DCA-derived cytotoxic effects including ROS.

Alterations in cellular metabolic pathway and epithelial cell maturation induced by MYO5B defects are partially reversible by LPAR5 activation.

Functional loss of the motor protein, Myosin Vb (MYO5B), induces various defects in intestinal epithelial function and causes a congenital diarrheal disorder, microvillus inclusion disease (MVID). Utilizing the MVID model mice, Vil1-CreERT2;Myo5bflox/flox (MYO5B∆IEC) and Vil1-CreERT2;Myo5bflox/G519R (MYO5B(G519R)), we previously reported that functional MYO5B loss disrupts progenitor cell differentiation and enterocyte maturation that result in villus blunting and deadly malabsorption symptoms. In this study, we determined that both absence and a point mutation of MYO5B impair lipid metabolism and alter mitochondrial structure, which may underlie the progenitor cell malfunction observed in MVID intestine. Along with a decrease in fatty acid oxidation, the lipogenesis pathway was enhanced in the MYO5B∆IEC small intestine. Consistent with these observations in vivo, RNA-sequencing of enteroids generated from the two MVID mouse strains showed similar downregulation of energy metabolic enzymes, including mitochondrial oxidative phosphorylation genes. In our previous studies, lysophosphatidic acid (LPA) signaling ameliorates epithelial cell defects in MYO5B∆IEC tissues and enteroids. The present study demonstrated that the highly soluble LPAR5-preferred agonist, Compound-1, improved sodium transporter localization and absorptive function, and tuft cell differentiation in patient-modeled MVID animals that carry independent mutations in MYO5B. Body weight loss in male MYO5B(G519R) mice was ameliorated by Compound-1. These observations suggest that Compound-1 treatment has a trophic effect on intestine with MYO5B functional loss through epithelial cell-autonomous pathways that can accelerate the differentiation of progenitor cells and the maturation of enterocytes. Targeting LPAR5 may represent an effective therapeutic approach for treatment of MVID symptoms induced by different point mutations in MYO5B.

Rapid evolution of mitochondrion-related genes in haplodiploid arthropods

BackgroundMitochondrial genes and nuclear genes cooperate closely to maintain the functions of mitochondria, especially in the oxidative phosphorylation (OXPHOS) pathway. However, mitochondrial genes among arthropod lineages have dramatic evolutionary rate differences. Haplodiploid arthropods often show fast-evolving mitochondrial genes. One hypothesis predicts that the small effective population size of haplodiploid species could enhance the effect of genetic drift leading to higher substitution rates in mitochondrial and nuclear genes. Alternatively, positive selection or compensatory changes in nuclear OXPHOS genes could lead to the fast-evolving mitochondrial genes. However, due to the limited number of arthropod genomes, the rates of evolution for nuclear genes in haplodiploid species, besides hymenopterans, are largely unknown. To test these hypotheses, we used data from 76 arthropod genomes, including 5 independently evolved haplodiploid lineages, to estimate the evolutionary rates and patterns of gene family turnover of mitochondrial and nuclear genes.ResultsWe show that five haplodiploid lineages tested here have fast-evolving mitochondrial genes and fast-evolving nuclear genes related to mitochondrial functions, while nuclear genes not related to mitochondrion showed no significant evolutionary rate differences. Among hymenopterans, bees and ants show faster rates of molecular evolution in mitochondrial genes and mitochondrion-related nuclear genes than sawflies and wasps. With genome data, we also find gene family expansions and contractions in mitochondrion-related genes of bees and ants.ConclusionsOur results reject the small population size hypothesis in haplodiploid species. A combination of positive selection and compensatory changes could lead to the observed patterns in haplodiploid species. The elevated evolutionary rates in OXPHOS complex 2 genes of bees and ants suggest a unique evolutionary history of social hymenopterans.

Inflammatory Gene Expression in Livers Undergoing Ex Situ Normothermic Perfusion Is Attenuated by Leukocyte Removal From the Perfusate.

Ex situ normothermic perfusion (ESNP) is a method to evaluate and potentially recondition organs before transplantation. However, increased expression of inflammatory molecules, including by tissue-resident immune cells, may occur during the perfusion process, potentially negating the beneficial effects of perfusion. We used RNA sequencing to assess gene expression in 31 livers undergoing ESNP, including 23 donated after circulatory death (DCD) and 8 donated after brain death. In 7 DCD livers, a leucocyte filter was added to the circuit during perfusion. Biopsies were available for transcriptomic assessment in all cases at the start of perfusion and at varying time points postperfusion. During ESNP in DCD livers, we observed an increase in proinflammatory, profibrinolytic, and prorepair pathway genes. SERPINE1, encoding plasminogen activator inhibitor-1, was among the genes most significantly upregulated during perfusion in DCD livers, potentially promoting fibrin clot persistence in vasculature. We also found increased expression of monocyte and neutrophil recruiting chemokine and proinflammatory cytokine transcripts during ESNP, but several prorepair molecules, including thymic stromal lymphopoietin, were also upregulated. In both DCD and donation after brain death livers, interferon-gamma response genes were enriched, whereas oxidative phosphorylation genes decreased in organs with high perfusate alanine transaminase, a biomarker associated with adverse clinical outcomes. The inclusion of a leukocyte filter in the perfusion circuit mitigated the induction of inflammation/immune pathway genes during perfusion and was associated with enrichment in oxidative phosphorylation genes. Leukocyte removal during ESNP abrogates transcriptional changes that are associated with unfavorable clinical outcomes, potentially benefiting human livers undergoing ESNP.

Calorie restriction exacerbates folic acid-induced kidney fibrosis by altering mitochondria metabolism

Calorie restriction (CR) is known to confer health benefits, including longevity and disease prevention. Although CR is promising in preventing chronic kidney disease (CKD), its potential impact on the progression of kidney fibrosis from acute kidney injury (AKI) to CKD remains unclear. Here, we present evidence that CR exacerbates renal damage in a mouse model of folic acid (FA)-induced renal fibrosis by altering mitochondrial metabolism and inflammation. Mice subjected to CR (60% of ad libitum) for three days were subjected to high dose of FA (250 mg/kg) injection and maintained under CR for an additional week before being sacrificed. Biochemical analyses showed that CR mice exhibited increased kidney injury and fibrosis. RNA sequencing analysis demonstrated decreased electron transport and oxidative phosphorylation (OXPHOS) in CR kidneys with injury, heightened inflammatory, and fibrotic responses. CR significantly decreased OXPHOS gene and protein levels and reduced β-oxidation-associated proteins in the kidney. To determine whether defects in mitochondrial metabolism is associated with inflammation in the kidney, further in vitro experiments were performed. NRK52E kidney epithelial cells were treated with antimycin A to induce mitochondrial damage. Antimycin A treatment significantly increased chemokine expression via a STING-dependent pathway. Serum restriction in NRK49F kidney fibroblasts was observed to enhance the fibrotic response induced by TGFβ under in vitro conditions. In summary, our results indicate that CR exacerbates fibrosis and inflammatory responses in the kidney by altering mitochondrial metabolism, highlighting the importance of adequate energy supply for an effective response to AKI and fibrosis development.

Age-related increase of CD38 directs osteoclastogenic potential of monocytic myeloid-derived suppressor cells through mitochondrial dysfunction in male mice.

An aged immune system undergoes substantial changes where myelopoiesis dominates within the bone marrow. Monocytic-MDSCs (M-MDSCs) have been found to play an important role in osteoclastogenesis and bone resorption. In this study, we sought to provide a more comprehensive understanding of the osteoclastogenic potential of bone marrow M-MDSCs during normal aging through transcriptomic and metabolic changes. Using young mature and aged mice, detailed immunophenotypic analyses of myeloid cells revealed that the M-MDSCs were not increased in bone marrow, however M-MDSCS were significantly expanded in peripheral tissues. Although aged mice exhibited a similar number of M-MDSCs in bone marrow, these M-MDSCs had significantly higher osteoclastogenic potential and greater demineralization activity. Intriguingly, osteoclast progenitors from aged bone marrow M-MDSCs exhibited greater mitochondrial respiration rate and glucose metabolism. Further, transcriptomic analyses revealed the upregulation of mitochondrial oxidative phosphorylation and glucose metabolism genes. Interestingly, there was 8-fold increase in Cd38 mRNA gene expression, consistent with the Mouse Aging Cell Atlas transcriptomic database, and confirmed by qRT-PCR. CD38 regulates NAD+ availability, and 78c, a small molecule inhibitor of CD38, reduced the mitochondrial oxygen consumption rate and glucose metabolism and inhibited the osteoclastogenic potential of aged mice bone marrow-derived M-MDSCs. These results indicate that the age-related increase in Cd38 expression in M-MDSCs bias the transcriptome of M-MDSCs towards osteoclastogenesis. This enhanced understanding of the mechanistic underpinnings of M-MDSCs and their osteoclastogenesis during aging could lead to new therapeutic approaches for age-related bone loss and promote healthy aging.

Mitochondrial Pathogenic Mutations and Expression Pattern of Oxidative Phosphorylation Genes in COVID-19 Patients.

Mitochondrial missense mutations and pathogenic variants have been implicated in the pathogenesis of COVID-19. This study evaluated the role of mitochondrial DNA (mtDNA) mutations and changes in gene expression in the progression of COVID-19 and their correlation with clinical characteristics. Next-generation sequencing with high throughput was used to identify mtDNA mutations in 30 COVID-19 patients compared to 20 healthy controls. The potential impact of identified mutations on protein structure and stability was predicted using bioinformatic tools. Quantitative real-time polymerase chain reaction was employed to assess the expression levels of mtDNA-encoded genes involved in oxidative phosphorylation in COVID-19 patients and healthy controls. Correlations between gene expression levels, clinical parameters, including leukocyte, lymphocyte, neutrophil, and platelet count, as well as creatinine, alanine transaminase (ALT), aspartate transaminase (AST), and blood urea nitrogen (BUN) levels, and disease severity were analyzed. We found 8 different mtDNA mutations in ND1, ND5, CO3, ATP6, and CYB genes, which were predicted to alter amino acids and decrease protein stability. Two missense unique mutations, C9555T in CO3 and A12418T in ND5 were identified and correlated with Complexes I and IV, respectively. This downregulation was correlated with age, elevated levels of leukocytes, lymphocytes, neutrophils, platelets, creatinine, ALT, AST, and BUN, as well as disease severity. These findings suggest that mtDNA mutations and altered expression of oxidative phosphorylation genes contribute to mitochondrial dysfunction in COVID-19. Targeting mitochondrial dysfunction may represent a promising therapeutic strategy for COVID-19 treatment.

Insights into gemcitabine resistance in pancreatic cancer: association with metabolic reprogramming and TP53 pathogenicity in patient derived xenografts

BackgroundWith poor prognosis and high mortality, pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies. Standard of care therapies for PDAC have included gemcitabine for the past three decades, although resistance often develops within weeks of chemotherapy initiation through an array of possible mechanisms.MethodsWe reanalyzed publicly available RNA-seq gene expression profiles of 28 PDAC patient-derived xenograft (PDX) models before and after a 21-day gemcitabine treatment using our validated analysis pipeline to identify molecular markers of intrinsic and acquired resistance.ResultsUsing normalized RNA-seq quantification measurements, we first identified oxidative phosphorylation and interferon alpha pathways as the two most enriched cancer hallmark gene sets in the baseline gene expression profile associated with intrinsic gemcitabine resistance and sensitivity, respectively. Furthermore, we discovered strong correlations between drug-induced expression changes in glycolysis and oxidative phosphorylation genes and response to gemcitabine, which suggests that these pathways may be associated with acquired gemcitabine resistance mechanisms. Thus, we developed prediction models using baseline gene expression profiles in those pathways and validated them in another dataset of 12 PDAC models from Novartis. We also developed prediction models based on drug-induced expression changes in genes from the Molecular Signatures Database (MSigDB)’s curated 50 cancer hallmark gene sets. Finally, pathogenic TP53 mutations correlated with treatment resistance.ConclusionOur results demonstrate that concurrent upregulation of both glycolysis and oxidative phosphorylation pathways occurs in vivo in PDAC PDXs following gemcitabine treatment and that pathogenic TP53 status had association with gemcitabine resistance in these models. Our findings may elucidate the molecular basis for gemcitabine resistance and provide insights for effective drug combination in PDAC chemotherapy.

Abstract Tu124: The Transcriptional Regulator FOG2S Represses GATA4-ERRα-PGC1α Complex to Decrease Mitochondrial Metabolism

Background: Cardiac transcription factors (CTFs) and coregulators drive the developmental transition from immature to mature cardiomyocytes (CMs). The nuclear receptor estrogen-related receptor (ERR), with coregulator PGC-1α and CTF GATA4, has been shown to serve as a critical role in CM maturation. Friend of GATA 2 (FOG2), a transcriptional coregulator, is crucial for normal cardiac development and exists in 2 isoforms: FOG2 and FOG2S, a shortened isoform lacking the N-terminal repressor domain and more prevalent in developing heart. Little is known about the role of FOG2S. We sought to investigate the mechanisms whereby FOG2S serves as a transcriptional regulator and assess its potential interaction with maturation CTFs. Methods: FOG2S was overexpressed in 8-week-old male C57/BL6 mice and human iPSC-derived cardiomyocytes (iPSC-CMs) using cardiac-specific AAV (cTNT.AAV9). Hearts were processed for RNA-seq and electron microscopy. iPSC-CMs were also treated with 0.1 mM DMOG for 16 h to induce hypoxia, essential for metabolic flexibility, and assessed with Seahorse Bioanalyzer. Hearts and iPSC-CMs were also analyzed for oxidative phosphorylation genes by qRTPCR. Co-transfection studies using a 3XERR-RE upstream of a minimal promoter driving luciferase reporter was performed in HEK293 cells with 3xFLAG-ERRα, PGC-1α-Myc/His-, GATA4-HA-, FOG2-FLAG- and/or FOG2S-FLAG-tagged plasmids. Results: FOG2S mice showed 678 differentially expressed genes (p<0.001, 424 genes up and 254 genes down). Gene set enrichment analysis identified oxidative phosphorylation as the top hallmark pathway, driven by decreased Nduf’s and Cox’s and increased Bnip3, a mitophagy gene. FOG2S hearts had smaller and rounder mitochondria with less cristae. In vitro , DMOG-treated FOG2S iPSC-CMs exhibited decreased basal and maximal respiration and reduction in mitochondrial network. There was a significant 40-80% decrease in mitochondrial gene expression, MFN1, COX10, TIMM21, TOMM22, NDUFA11, NDUFAB1. GSEA for transcription factor targets identified ERRα as the top hit. Luciferase reporter assay for 3XERR-RE activity showed FOG2S inhibited ERRα-PGC1α-GATA4 mediated activation in a dose-dependent manner. Inhibition was also mediated through GATA4 and independent of PGC1α. Conclusion: FOG2S overexpression suppresses oxidative phosphorylation and mitochondrial metabolism by inhibiting GATA4 in the context of ERRα-PGC1α-GATA4 complex. These results suggest that FOG2S modulates CM metabolic maturation.

Deciphering tissue-specific expression profiles of mitochondrial genome-encoded tRNAs and rRNAs through transcriptomic profiling in buffalo.

Mitochondria, essential for cellular energy production through oxidative phosphorylation (OXPHOS), integrate mt-DNA and nuclear-encoded genes. This cooperation extends to the mitochondrial translation machinery, involving crucial mtDNA-encoded RNAs: 22 tRNAs (mt-tRNAs) as adapters and two rRNAs (mt-rRNAs) for ribosomal assembly, enabling mitochondrial-encoded mRNA translation. Disruptions in mitochondrial gene expression can strongly impact energy generation and overall animal health. Our study investigates the tissue-specific expression patterns of mt-tRNAs and mt-rRNAs in buffalo. To investigate the expression patterns of mt-tRNAs and mt-rRNAs in different tissues and gain a better understanding of tissue-specific variations, RNA-seq was performed on various tissues, such as the kidney, heart, brain, and ovary, from post-pubertal female buffaloes. Subsequently, we identified transcripts that were differentially expressed in various tissue comparisons. The findings reveal distinct expression patterns among specific mt-tRNA and mt-rRNA genes across various tissues, with some exhibiting significant upregulation and others demonstrating marked downregulation in specific tissue contexts. These identified variations reflect tissue-specific physiological roles, underscoring their significance in meeting the unique energy demands of each tissue. Notably, the brain exhibits the highest mtDNA copy numbers and an abundance of mitochondrial mRNAs of our earlier findings, potentially linked to the significant upregulation of mt-tRNAs in brain. This suggests a plausible association between mtDNA replication and the regulation of mtDNA gene expression. Overall, our study unveils the tissue-specific expression of mitochondrial-encoded non-coding RNAs in buffalo. As we proceed, our further investigations into tissue-specific mitochondrial proteomics and microRNA studies aim to elucidate the intricate mechanisms within mitochondria, contributing to tissue-specific mitochondrial attributes. This research holds promise to elucidate the critical role of mitochondria in animal health and disease.

Pleiotropic effects of an eQTL in the CELSR2/PSRC1/SORT1 cluster that associates with LDL-C and resting metabolic rate.

The locus CELSR2-PSRC1-SORT1, a primary genetic signal for lipids, has recently been implicated in different metabolic processes. Our investigation identified its association with energy metabolism. To determine biological mechanisms that govern diverse functions of this locus. Genotypes for 491,265 variants in 7,000 clinically characterized American Indians were previously determined using a custom-designed array specific for this longitudinally studied American Indian population. Among the genotyped individuals, 5,205 had measures of fasting lipid levels and 509 had measures of resting metabolic rate (RMR) and substrate oxidation rate assessed through indirect calorimetry. A genome-wide association study (GWAS) for LDL-C levels identified a variant in CELSR2 and the molecular impact of this variant on gene expression was assessed in skeletal muscle biopsies from 207 participants, followed by functional validation in mouse myoblasts using a luciferase assay. A GWAS in American Indians identified rs12740374 in CELSR2 as the top signal for LDL-C levels (P = 1 × 10-22); further analysis of this variant identified an unexpected correlation with reduced RMR (effect = -44.3 kcal/day/minor-allele) and carbohydrate oxidation rate (effect = -5.21 mg/hour/kg-EMBS). Tagged variants showed a distinct linkage disequilibrium architecture in American Indians, highlighting a potential functional variant, rs6670347 (minor-allele frequency = 0.20). Positioned in the glucocorticoid receptor's core binding motif, rs6670347 is part of a skeletal muscle-specific enhancer. Human skeletal muscle transcriptome analysis showed CELSR2 as the most differentially expressed gene (P = 1.9 × 10-7), with the RMR-lowering minor allele elevating gene expression. Experiments in mouse myoblasts confirmed enhancer-based regulation of CELSR2 expression, dependent on glucocorticoids. Rs6670347 also associated with increased oxidative phosphorylation gene expression; CELSR2 as a regulator of these genes, suggests potential influence on energy metabolism through muscle oxidative capacity. Variants in the CELSR2/PSRC1/SORT1 locus exhibit tissue-specific effects on metabolic traits, with an independent role in muscle metabolism through glucocorticoid signaling.

Mitochondrial antioxidants abate SARS-COV-2 pathology in mice

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection inhibits mitochondrial oxidative phosphorylation (OXPHOS) and elevates mitochondrial reactive oxygen species (ROS, mROS) which activates hypoxia-inducible factor-1alpha (HIF-1α), shifting metabolism toward glycolysis to drive viral biogenesis but also causing the release of mitochondrial DNA (mtDNA) and activation of innate immunity. To determine whether mitochondrially targeted antioxidants could mitigate these viral effects, we challenged mice expressing human angiotensin-converting enzyme 2 (ACE2) with SARS-CoV-2 and intervened using transgenic and pharmacological mitochondrially targeted catalytic antioxidants. Transgenic expression of mitochondrially targeted catalase (mCAT) or systemic treatment with EUK8 decreased weight loss, clinical severity, and circulating levels of mtDNA; as well as reduced lung levels of HIF-1α, viral proteins, and inflammatory cytokines. RNA-sequencing of infected lungs revealed that mCAT and Eukarion 8 (EUK8) up-regulated OXPHOS gene expression and down-regulated HIF-1α and its target genes as well as innate immune gene expression. These data demonstrate that SARS-CoV-2 pathology can be mitigated by catalytically reducing mROS, potentially providing a unique host-directed pharmacological therapy for COVID-19 which is not subject to viral mutational resistance.

Accumulation and growth toxicity mechanisms of fluxapyroxad revealed by physiological, hepatopancreas transcriptome, and gut microbiome analysis in Pacific white shrimp (Litopenaeus vannamei)

Fluxapyroxad (FX), a typical succinate dehydrogenase inhibitor fungicide, is causing increased global concerns due to its fungicide effects. However, the accumulation and grow toxicity of FX to Litopenaeus vannamei (L. vannamei) is poorly understand. Therefore, the accumulation pattern of FX in L. vannamei was investigated for the first time in environmental concentrations. FX accumulated rapidly in shrimp muscle. Meanwhile, growth inhibition was observed and the mechanism derived by primarily accelerated glycolipid metabolism and reduced glycolipid content. Moreover, exposure to environmental concentrations of FX induced significant growth inhibition and oxidative stress and inhibited oxidative phosphorylation and TCA cycle in L. vannamei. The endocytosis signaling pathway genes were activated, thereby driving growth toxicity. Oxidative phosphorylation and cytosolic gene expression were further rescued in elimination experiments, demonstrating the mechanism of growth toxicity by FX exposure. The results revealed that FX persistently altered the gut microbiome of L. vannamei using gut microbiome sequencing, particularly with increased Garcinia Purple Pseudoalteromonas luteoviolacea for organic pollutant degradation. This study provided new insights into the potential toxicity of FX to marine organisms, emphasizing the need for further investigation and potential regulatory considerations.

Mitonuclear compatibility is maintained despite relaxed selection on male mitochondrial DNA in bivalves with doubly uniparental inheritance.

Mitonuclear coevolution is common in eukaryotes, but bivalve lineages that have doubly uniparental inheritance (DUI) of mitochondria may be an interesting example. In this system, females transmit mtDNA (F mtDNA) to all offspring, while males transmit a different mtDNA (M mtDNA) solely to their sons. Molecular evolution and functional data suggest oxidative phosphorylation (OXPHOS) genes encoded in M mtDNA evolve under relaxed selection due to their function being limited to sperm only (vs. all other tissues for F mtDNA). This has led to the hypothesis that mitonuclear coevolution is less important for M mtDNA. Here, we use comparative phylogenetics, transcriptomics, and proteomics to understand mitonuclear interactions in DUI bivalves. We found nuclear OXPHOS proteins coevolve and maintain compatibility similarly with both F and M mtDNA OXPHOS proteins. Mitochondrial recombination did not influence mitonuclear compatibility and nuclear-encoded OXPHOS genes were not upregulated in tissues with M mtDNA to offset dysfunction. Our results support that selection maintains mitonuclear compatibility with F and M mtDNA despite relaxed selection on M mtDNA. Strict sperm transmission, lower effective population size, and higher mutation rates may explain the evolution of M mtDNA. Our study highlights that mitonuclear coevolution and compatibility may be broad features of eukaryotes.

Identification of a longevity gene through evolutionary rate covariation of insect mito-nuclear genomes.

Oxidative phosphorylation, essential for energy metabolism and linked to the regulation of longevity, involves mitochondrial and nuclear genes. The functions of these genes and their evolutionary rate covariation (ERC) have been extensively studied, but little is known about whether other nuclear genes not targeted to mitochondria evolutionarily and functionally interact with mitochondrial genes. Here we systematically examined the ERC of mitochondrial and nuclear benchmarking universal single-copy ortholog (BUSCO) genes from 472 insects, identifying 75 non-mitochondria-targeted nuclear genes. We found that the uncharacterized gene CG11837-a putative ortholog of human DIMT1-regulates insect lifespan, as its knockdown reduces median lifespan in five diverse insect species and Caenorhabditis elegans, whereas its overexpression extends median lifespans in fruit flies and C. elegans and enhances oxidative phosphorylation gene activity. Additionally, DIMT1 overexpression protects human cells from cellular senescence. Together, these data provide insights into the ERC of mito-nuclear genes and suggest that CG11837 may regulate longevity across animals.

Abstract 133: Trem2 Agonist Promotes Atherosclerotic Plaque Stability

Atherosclerosis is an inflammatory disease of the arteries that is a major cause of cardiovascular disease (CVD). Atherosclerotic plaque progression is driven by the deposition of low-density lipoprotein (LDL) within the intima, which is internalized by macrophages, forming lipid-engorged foamy cells. Importantly, the accumulation of excess LDL within foamy macrophages is cytotoxic, driving apoptosis, which ultimately promotes necrotic core formation and increased potential for plaque rupture. Recently, our lab identified the lipid receptor Trem2 to be highly expressed by foamy macrophages and regulate cell survival, proliferation, and cholesterol efflux. Thus, we tested whether agonizing Trem2 could drive macrophage survival to promote plaque stability. Using a Trem2 agonist antibody (AL002a, Alector Inc.), we treated Ldlr -/- mice that had preexisting atherosclerotic plaque with either isotype control or AL002a antibody for 8 weeks. AL002a treated mice showed an increase in plaque size in the aortic arch and aortic sinus. Plaque analysis determined that this was primarily due to an expansion of macrophage cellularity, through promotion of cell survival and proliferation. Furthermore, we found that plaques from AL002a treated mice had decreased necrotic core formation, increased fibrous cap size, and increased collagen deposition, consistent with improved stability. Single cell RNA-sequencing of atherosclerotic aortae from isotype and AL002a treated mice found that Trem2-agonism dramatically reshaped transcriptomes of both foamy macrophages and smooth muscle cells, including upregulation of oxidative phosphorylation (OXPHOS), cholesterol efflux, collagen, and pro-survival genes while downregulating inflammatory pathways. In vitro studies using bone marrow derived macrophages confirmed AL002a’s ability to drive cell survival, proliferation, OXPHOS, and cholesterol efflux in culture. Finally, we determined that AL002a’s ability to reshape aspects of foamy macrophage function was dependent on its ability to activate the kinase SYK downstream of Trem2. Overall, this data identifies Trem2 as a viable therapeutic target for promoting atherosclerotic plaque stability and improving outcomes in CVD.