Mitochondrial-encoded Genes Research Articles

Plumbagin-induced anticancer effects are associated with mitochondrial-encoded respiratory gene downregulation in oral squamous cell carcinoma

Plumbagin (PL) is a known quinoid (5-hydroxyl-2-methyl-1,4-napthoquinone) initially isolated from the roots of Plumbago zeylanica L. It is reported to exert anti-proliferative effects in oral squamous cell carcinoma (OSCC) cells, suggesting that PL would be a promising therapeutic anti-cancer drug for OSCC; however, the molecular basis by which PL suppresses cell survival signaling is poorly understood. In this study, we conducted a comprehensive gene expression analysis to identify the molecular basis by which PL suppresses cell survival signaling. Human OSCC cell lines HSC-3 and SAS were used in this study. Comprehensive gene expression analysis was performed with an Agilent Whole Human Genome DNA microarray 4 × 44 format. Gene Set Enrichment Analysis was conducted to investigate the effect of PL on the oncogenic signaling pathway in the OSCC cells. cDNA microarray and subsequent gene set enrichment analyses showed that PL readily downregulates oxidative phosphorylation, MYC-target genes, and MTORC signaling at 4 h after treatment in HSC-3 and SAS cells. Semi-quantitative PCR analysis showed that gene expression levels of mitochondrial (Mt)-encoded COX-2, COX-3, and ND1 genes are significantly lower in the PL-treated OSCC cells than those in the untreated cells. In addition, an ROS scavenger, NAC, reversed the PL-induced downregulation of expression of Mt-encoded genes. Accordingly, intracellular ATP levels significantly decreased after PL treatment. Collectively, PL downregulates both oxidative phosphorylation and oncogenic signatures and decreases the survival of OSCC cells, thus highlighting its application as a potent treatment for patients with OSCC.

Abstract 3829: Microenvironmental stiffness induces metabolic reprogramming in glioblastoma

Abstract Glioblastoma (GBM) is the most common and lethal type of brain cancer with 5-year survival of <15%. The unique microenvironment of GBM is a key factor in tumor aggression. Similar to other solid tumors, GBM cells experience different biomechanical forces than do cells in healthy brain tissue. Here, we use atomic force microscopy to perform micro-compression mechanical analysis of xenografted GBM tumors. We show that tumors have a relatively stiff core (5-6 kPa Young’s modulus) and a softer edge (1-2 kPa Young’s modulus), yet all tumor tissue was stiffer than adjacent brain tissue (0.5-0.8 kPa Young’s modulus). To understand the functional implications of this biomechanical landscape, GBM spheroids were encapsulated in 3D culture matrices tuned to mimic the measured stiffnesses of a tumor regions and neighboring, non-cancerous tissue. These 3D matrices are fabricated from hyaluronic acid to promote GBM/matrix interaction through CD44 and RHAMM. RNA-sequencing analysis revealed overexpression of mitochondrial-encoded genes in stiff conditions, specifically genes encoding proteins involved in oxidation phosphorylation (OXPHOS), when compared to soft hydrogels. Using fluorescence-life time imaging microscopy to assess metabolic states at a single-cell level, we found that metabolism in GBM cells cultured in stiff matrices was dominated by OXPHOS while those in softer matrices was dominated by glycolysis (GLY). Furthermore, we show that this stiffness-induced shift towards OXPHOS in stiff matrices was associated with increased proliferation while the shift towards GLY in soft matrices was associated with increased migratory activity. Finally, inhibition of ezrin, an adaptor molecule linking the HA receptor CD44 to the actin cytoskeleton, eliminated the shift towards GLY induced in soft matrices, indicating that HA-CD44 interactions are necessary for these matrix-induced metabolic shifts. Citation Format: Alireza Sohrabi, Austin Lefebvre, Michael Condro, Harley Kornblum, Michelle Digman, Stephanie Seidlits. Microenvironmental stiffness induces metabolic reprogramming in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3829.

Maternal Western‐style diet and obesity during pregnancy reduces mitochondrial Complex I‐linked oxidative capacity in tandem with decreased oxidative stress in skeletal muscle of adolescent Japanese macaque offspring

BackgroundExposure to an obesogenic gestational environment during early development increases offspring risk of developing obesity and cardiometabolic diseases. Skeletal muscle of obese and insulin resistant individuals show signs of mitochondrial dysfunction including reduced oxidative capacity and increased oxidative stress. Subsequent oxidative damage is caused by reactive oxygen species (ROS) generated primarily by mitochondrial Complex I (CI). Previous work in our non‐human primate model shows decreased oxidative capacity in fetal skeletal muscle from obesogenic pregnancies, a phenotype we have confirmed is retained into adolescence. The purpose of this study was to evaluate how this reduced oxidative capacity correspond with ROS production in skeletal muscle of lean adolescent offspring.Experimental DesignLean (body fat <25%) adult female Japanese macaques were maintained on a control diet (CD) or Western‐style diet (WD) prior to and throughout pregnancy and lactation. Dams chronically consuming WD with body fat >30% were classified as obese and included in this study. Male and female offspring were weaned to CD and moved to independent housing at 7 months of age. Muscle samples were collected from offspring gastrocnemius (gastroc) and soleus at 40 months. Offspring data were analyzed by Student’s t‐test.ResultsExposure to maternal WD during pregnancy showed moderate but significant reductions in oxidative damage and a modest (but insignificant) decrease in ROS generation in gastroc of offspring from obese pregnancies. In the soleus, ROS production was significantly reduced in these offspring. Both nuclear‐ and mitochondrial‐encoded genes for CI were decreased with maternal obesity/WD pregnancy, indicating transcriptional regulation of CI in exposed offspring. Finally, CI protein abundance was also decreased in primary myotubes from these offspring in lieu of additional metabolic challenges, providing further evidence of lasting metabolic reprogramming in skeletal muscle.ConclusionsWe find that early life exposure to maternal WD‐induced obesity leaves a lasting impression on offspring skeletal muscle metabolism that persists into young adulthood. It is possible that transcriptional suppression of CI activity is a protective adaptation to mitigate oxidative damage caused by mitochondrial ROS. While long‐term consequences of altered oxidation patterns relating to ROS flux require further investigation, the loss of CI content and diminished oxidative capacity in these lean offspring likely drive their increased susceptibility for cardiometabolic diseases – particularly when faced with metabolic stress and environmental insults throughout the lifespan.

NRF2 Alters Mitochondrial Gene Expression in Neonate Mice Exposed to Hyperoxia.

Approximately 1 in 10 newborns are born preterm and require supplemental oxygen (O2) in an extrauterine environment following birth. Supplemental O2 can induce oxidative stress that can impair mitochondrial function, resulting in lung injury and increased risk in early life pulmonary diseases. The nuclear factor-erythroid 2 related factor 2 (NRF2) protects the cells from oxidative stress by regulating the expression of genes containing antioxidant response elements and many mitochondrial-associated genes. In this study, we compared Nrf2-deficient (Nrf2−/−) and wild-type (Nrf2+/+) mice to define the role of NRF2 in lung mitochondrial genomic features in late embryonic development in mice (embryonic days, E13.5 and E18.5) versus birth (postnatal day 0, PND0). We also determined whether NRF2 protects lung mitochondrial genome parameters in postnatal mice exposed to a 72 h hyperoxia environment. We found Nrf2−/− embryonic lungs were characterized by decreases in mtDNA copies from E13.5 to E18.5. Interestingly, Nrf2−/− heteroplasmy frequency was significantly higher than Nrf2+/+ at E18.5, though this effect reversed at PND0. In postnatal mice exposed to hyperoxia, we identified three- to four-fold increases in mitochondria-encoded mitochondrial genes, which regulate oxidative phosphorylation. Overall, our findings demonstrate a potentially critical role of NRF2 in mediating long-term effects of hyperoxia on mitochondrial function.

Functional Role of Mitochondrial DNA in Cancer Progression.

Mitochondrial DNA (mtDNA) has been identified as a significant genetic biomarker in disease, cancer and evolution. Mitochondria function as modulators for regulating cellular metabolism. In the clinic, mtDNA variations (mutations/single nucleotide polymorphisms) and dysregulation of mitochondria-encoded genes are associated with survival outcomes among cancer patients. On the other hand, nuclear-encoded genes have been found to regulate mitochondria-encoded gene expression, in turn regulating mitochondrial homeostasis. These observations suggest that the crosstalk between the nuclear genome and mitochondrial genome is important for cellular function. Therefore, this review summarizes the significant mechanisms and functional roles of mtDNA variations (DNA level) and mtDNA-encoded genes (RNA and protein levels) in cancers and discusses new mechanisms of crosstalk between mtDNA and the nuclear genome.

P107 mediated mitochondrial function controls muscle stem cell proliferative fates

Muscle diseases and aging are associated with impaired myogenic stem cell self-renewal and fewer proliferating progenitors (MPs). Importantly, distinct metabolic states induced by glycolysis or oxidative phosphorylation have been connected to MP proliferation and differentiation. However, how these energy-provisioning mechanisms cooperate remain obscure. Herein, we describe a mechanism by which mitochondrial-localized transcriptional co-repressor p107 regulates MP proliferation. We show p107 directly interacts with the mitochondrial DNA, repressing mitochondrial-encoded gene transcription. This reduces ATP production by limiting electron transport chain complex formation. ATP output, controlled by the mitochondrial function of p107, is directly associated with the cell cycle rate. Sirt1 activity, dependent on the cytoplasmic glycolysis product NAD+, directly interacts with p107, impeding its mitochondrial localization. The metabolic control of MP proliferation, driven by p107 mitochondrial function, establishes a cell cycle paradigm that might extend to other dividing cell types.

Mitonuclear interactions and introgression genomics of macaque monkeys (Macaca) highlight the influence of behaviour on genome evolution.

In most macaques, females are philopatric and males migrate from their natal ranges, which results in pronounced divergence of mitochondrial genomes within and among species. We therefore predicted that some nuclear genes would have to acquire compensatory mutations to preserve compatibility with diverged interaction partners from the mitochondria. We additionally expected that these sex-differences would have distinctive effects on gene flow in the X and autosomes. Using new genomic data from 29 individuals from eight species of Southeast Asian macaque, we identified evidence of natural selection associated with mitonuclear interactions, including extreme outliers of interspecies differentiation and metrics of positive selection, low intraspecies polymorphism and atypically long runs of homozygosity associated with nuclear-encoded genes that interact with mitochondria-encoded genes. In one individual with introgressed mitochondria, we detected a small but significant enrichment of autosomal introgression blocks from the source species of her mitochondria that contained genes which interact with mitochondria-encoded loci. Our analyses also demonstrate that sex-specific demography sculpts genetic exchange across multiple species boundaries. These findings show that behaviour can have profound but indirect effects on genome evolution by influencing how interacting components of different genomic compartments (mitochondria, the autosomes and the sex chromosomes) move through time and space.

Targeting adaptive cellular responses to mitochondrial bioenergetic deficiencies in human disease.

Mitochondrial dysfunction is increasingly appreciated as a central contributor to human disease. Oxidative metabolism at the mitochondrial respiratory chain produces ATP and is intricately tied to redox homeostasis and biosynthetic pathways. Metabolic stress arising from genetic mutations in mitochondrial genes and environmental factors such as malnutrition or overnutrition is perceived by the cell and leads to adaptive and maladaptive responses that can underlie pathology. Here, we will outline cellular sensors that react to alterations in energy production, organellar redox, and metabolites stemming from mitochondrial disease (MD) mutations. MD is a heterogeneous group of disorders primarily defined by defects in mitochondrial oxidative phosphorylation from nuclear or mitochondrial-encoded gene mutations. Preclinical therapies that improve fitness of MD mouse models have been recently identified. Targeting metabolic/energetic deficiencies, maladaptive signaling processes, and hyper-oxygenation of tissues are all strategies aside from direct genetic approaches that hold therapeutic promise. A further mechanistic understanding of these curative processes as well as the identification of novel targets will significantly impact mitochondrial biology and disease research.

DMEP induces mitochondrial damage regulated by inhibiting Nrf2 and SIRT1/PGC-1α signaling pathways in HepG2 cells

Dimethoxyethyl phthalate (DMEP) is an environmental endocrine disruptor. However, research into the underlying mechanisms of DMEP mitochondrial toxicity is still in its infancy. We therefore expect to understand whether DMEP induced mitochondrial damage in HepG2 cells and the associated signaling pathways. DMEP (0.125, 0.25, 0.5, 1 and 2 mM) exposure for 48 h induced a notable increment in reactive oxygen species (ROS), malondialdehyde (MDA), alanine aminotransferase (ALT), aspartate transaminase (AST) and 8-hydroxydeoxyguanosine (8-OHdG) in hepG2 cells, resulting in cellular oxidative stress. Low doses of DMEP upregulated nuclear factor E2-related factor 2 (Nrf2) and downstream protein haeme oxygenase-1 (HO-1) levels and high doses down-regulated their levels. Nrf2 levels increased after ROS scavenging by N-acetyl-L-cysteine (NAC), which indicated that the Nrf2 pathway may be affected by oxidative stress. We also found that DMEP decreased ATP content, mitochondrial copy number (mtDNA), translocase of the outer membrane subunit 20 (TOM20) expression, mitochondria-encoded genes CO1, CO2, CO3, ATP6, ATP8 expression, inhibited mitochondrial biogenesis pathway, down-regulated sirtuin 1(SIRT1), PPAR gamma co-activator 1 alpha (PGC-1α), Nuclear respiratory factor 1(Nrf1), Mitochondrial transcription factor A (TFAM) content and activated PINK1/Parkin autophagy pathway. DMEP also activated the mitochondrial apoptotic pathway, causing cytochrome c cytoplasmic translocation and caspase 3 cleavage. What’s more, DMEP activated the Nuclear factor-κB (NF-κB) pathway and levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) were significantly upregulated, causing an inflammatory response. In summary, DMEP can cause inflammatory response and oxidative stress in HepG2 cells, inhibited the Nrf2 pathway and mitochondrial biogenesis, and induced autophagy and apoptosis. And oxidative stress at least partially affected the Nrf2 pathway and mitochondrial biogenesis SIRT1/PGC-1α pathway.

Gender specific airway gene expression in COPD sub-phenotypes supports a role of mitochondria and of different types of leukocytes

Chronic obstructive pulmonary disease (COPD) is a destructive inflammatory disease and the genes expressed within the lung are crucial to its pathophysiology. We have determined the RNAseq transcriptome of bronchial brush cells from 312 stringently defined ex-smoker patients. Compared to healthy controls there were for males 40 differentially expressed genes (DEGs) and 73 DEGs for females with only 26 genes shared. The gene ontology (GO) term “response to bacterium” was shared, with several different DEGs contributing in males and females. Strongly upregulated genes TCN1 and CYP1B1 were unique to males and females, respectively. For male emphysema (E)-dominant and airway disease (A)-dominant COPD (defined by computed tomography) the term “response to stress” was found for both sub-phenotypes, but this included distinct up-regulated genes for the E-sub-phenotype (neutrophil-related CSF3R, CXCL1, MNDA) and for the A-sub-phenotype (macrophage-related KLF4, F3, CD36). In E-dominant disease, a cluster of mitochondria-encoded (MT) genes forms a signature, able to identify patients with emphysema features in a confirmation cohort. The MT-CO2 gene is upregulated transcriptionally in bronchial epithelial cells with the copy number essentially unchanged. Both MT-CO2 and the neutrophil chemoattractant CXCL1 are induced by reactive oxygen in bronchial epithelial cells. Of the female DEGs unique for E- and A-dominant COPD, 88% were detected in females only. In E-dominant disease we found a pronounced expression of mast cell-associated DEGs TPSB2, TPSAB1 and CPA3. The differential genes discovered in this study point towards involvement of different types of leukocytes in the E- and A-dominant COPD sub-phenotypes in males and females.

Mitochondria and the Frozen Frog.

The wood frog, Rana sylvatica, is the best-studied of a small group of amphibian species that survive whole body freezing during the winter months. These frogs endure the freezing of 65–70% of their total body water in extracellular ice masses. They have implemented multiple adaptations that manage ice formation, deal with freeze-induced ischemia/reperfusion stress, limit cell volume reduction with the production of small molecule cryoprotectants (glucose, urea) and adjust a wide variety of metabolic pathways for prolonged life in a frozen state. All organs, tissues, cells and intracellular organelles are affected by freeze/thaw and its consequences. This article explores mitochondria in the frozen frog with a focus on both the consequences of freezing (e.g., anoxia/ischemia, cell volume reduction) and mitigating defenses (e.g., antioxidants, chaperone proteins, upregulation of mitochondria-encoded genes, enzyme regulation, etc.) in order to identify adaptive strategies that defend and adapt mitochondria in animals that can be frozen for six months or more every year. A particular focus is placed on freeze-responsive genes in wood frogs that are encoded on the mitochondrial genome including ATP6/8, ND4 and 16S RNA. These were strongly up-regulated during whole body freezing (24 h at −2.5 °C) in the liver and brain but showed opposing responses to two component stresses: strong upregulation in response to anoxia but no response to dehydration stress. This indicates that freeze-responsive upregulation of mitochondria-encoded genes is triggered by declining oxygen and likely has an adaptive function in supporting cellular energetics under indeterminate lengths of whole body freezing.

Cyclophilin D-dependent mitochondrial permeability transition amplifies inflammatory reprogramming in endotoxemia.

Microorganisms or LPS (lipopolysaccharide), an outer membrane component of Gram‐negative bacteria, can induce a systemic inflammatory response that leads to sepsis, multiple organ dysfunction, and mortality. Here, we investigated the role of cyclophilin D (CypD)‐dependent mitochondrial permeability transition (mPT) in the immunosuppressive phase of LPS‐induced endotoxic shock. The liver plays an important role in immunity and organ dysfunction; therefore, we used liver RNA sequencing (RNA‐seq) data, Ingenuity® Pathway Analysis (IPA ®) to investigate the complex role of mPT formation in inflammatory reprogramming and disease progression. LPS induced significant changes in the expression of 2844 genes, affecting 179 pathways related to mitochondrial dysfunction, defective oxidative phosphorylation, nitric oxide (NO) and reactive oxygen species (ROS) accumulation, nuclear factor, erythroid 2 like 2 (Nrf2), Toll‐like receptors (TLRs), and tumor necrosis factor α receptor (TNFR)‐mediated processes in wild‐type mice. The disruption of CypD reduced LPS‐induced alterations in gene expression and pathways involving TNFRs and TLRs, in addition to improving survival and attenuating oxidative liver damage and the related NO‐ and ROS‐producing pathways. CypD deficiency diminished the suppressive effect of LPS on mitochondrial function, nuclear‐ and mitochondrial‐encoded genes, and mitochondrial DNA (mtDNA) quantity, which could be critical in improving survival. Our data propose that CypD‐dependent mPT is an amplifier in inflammatory reprogramming and promotes disease progression. The mortality in human sepsis and shock is associated with mitochondrial dysfunction. Prevention of mPT by CypD disruption reduces inflammatory reprogramming, mitochondrial dysfunction, and lethality; therefore, CypD can be a novel drug target in endotoxic shock and related inflammatory diseases.

Abstract PO-022: The NKX3.1 homeoprotein functions in the mitochondria to protect against oxidative stress-dependent prostate tumorigenesis

Abstract The NKX3.1 homeobox gene is a key regulator of prostate differentiation, whose loss-of-function predisposes to prostate cancer. Here, we uncover an unexpected role for NKX3.1 in mitochondrial function and bioenergetics that regulates cancer initiation under conditions of oxidative stress. To investigate whether NKX3.1 plays a direct functional role in protection against oxidative stress, we studied the consequences of its loss- or gain-of-function in tumorigenic and non-tumorigenic human prostate cells, mutant mice, and primary human prostate cultures. Utilizing redox compounds to exogenously induce and study oxidative stress responses in our models, we show that NKX3.1 localizes to the mitochondria to protect against oxidative stress, while its loss-of-function leads to deregulation of mitochondrial-encoded oxidative phosphorylation genes, elevated reactive oxygen species, and accelerated prostate cancer initiation. Importantly, our studies reveal that cells lacking NKX3.1 have impaired intracellular energetics under oxidative stress, warranting further investigation into the role of NKX3.1 loss in cancer metabolism. These observations uncover a novel function for a tissue-specific differentiation regulator in mitochondria and demonstrate its functional relevance for suppressing prostate cancer initiation. Citation Format: Alexandros Papachristodoulou, Sukanya Panja, Antonina Mitrofanova, Aditya Dutta, Cory Abate-Shen. The NKX3.1 homeoprotein functions in the mitochondria to protect against oxidative stress-dependent prostate tumorigenesis [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-022.

Mitochondrial delivery of microRNA mimic let-7b to NSCLC cells by PAMAM-based nanoparticles

Many biological mechanisms including cellular metabolism and cell death are regulated by mitochondria known as powerhouse of the cell. Recently, let-7b, a tumour-suppressor microRNA has been detected in mitochondria of human cells targeting several mitochondrial-encoded respiratory chain genes. Triphenylphosphonium cation (TPP) is one of the major classes of mitochondriotropics that possess the ability of specifically targeting the mitochondria. PAMAM dendrimers are one of the most available agents in gene delivery due to their well-defined and beneficial features such as large density of surface functional groups. Hyaluronic acid (HA), a natural polysaccharide has been demonstrated to have the abilities such as good biocompatibility and targeting CD44 overexpressed receptors on non-small cell lung cancer (NSCLC) cells. In this research, let-7b-PAMAM (G5)-TPP and let-7b-PAMAM (G5)-TPP-HA nano-carriers were designed to deliver let-7b miRNA mimic to NSCLC cells’ mitochondria as a novel way of cancer cells inhibition. Nano-carriers were capable of being successfully taken up by A549 cells and localised in mitochondria environment. Let-7b loaded nanoparticles reduced cell viability and induced apoptosis significantly. Expression of genes involved in mitochondrial oxidative function was decreased resulting in nanoparticles effect on mitochondria. Application of mitochondria targeted-miRNA delivery systems could regulate cellular functions to inhibit lung cancer.

Evaluation of Mitochondrial Energy Metabolism and Translation in Clear Cell Renal Cell Carcinoma

Clear cell renal cell carcinoma (ccRCC) is one of the most common renal cell carcinomas. The defining morphological hallmark of ccRCC is the accumulation of glycogen and lipid droplets in the cytoplasm of the cells due to the reprogramming of glucose and fatty acid metabolism and oxidative phosphorylation (OXPHOS). Energy metabolism by OXPHOS is supported by both nuclear and mitochondrial‐encoded genes. Alterations in the expression of mitochondrial genes involved in OXPHOS have been well documented in ccRCC.We proposed that the mitochondrial translation machinery that exists solely for the synthesis of 13 mitochondrially‐encoded subunits of OXPHOS complexes is also implicated in the progression of ccRCC.To test this hypothesis, immunoblotting analyses of 14 ccRCC biopsies and their matched normal tissues were performed to investigate the role of mitochondrial translation machinery in the remodeling of OXPHOS complexes. Along with changes in the expression of nuclear and mitochondrial encoded subunits of OXPHOS complexes, we discovered that the expression of several mitochondrial translation factors such as mtEF‐Tu (TUFM), mtIF2 (MTIF2), and mtEF‐G (GFM1) and ribosomal proteins were also significantly reduced in ccRCC biopsies. To identify the key factors involved in mitochondrial translation further, genomics and proteomics data mining analyses of publicly available ccRCC databases at the Cancer Genome Atlas (TCGA) and The Clinical Proteomic Tumor Analysis Consortium (CPTAC) were also performed.Based on the evidence provided in our studies, we propose that changes in the expression of mitochondrial translation components, specifically translation factors and mitochondrial ribosomal proteins, are part of the remodeling of mitochondrial energy metabolism and resistance to apoptosis in ccRCC.

Aerobic Exercise Training Induces the Mitonuclear Imbalance and UPRmt in the Skeletal Muscle of Aged Mice.

The impairment of the mitochondrial functions is a hallmark of aging. During aging, there is a downregulation of two mechanisms strictly associated with mitochondrial integrity, including the mitonuclear imbalance (eg, imbalance in mitochondrial- versus nuclear-encoded mitochondrial proteins) and the mitochondrial unfolded protein response (UPRmt). Here, we evaluated the effects of aerobic exercise in the mitonuclear imbalance and UPRmt markers in the skeletal muscle of old mice. We combined the physiological tests, molecular and bioinformatic analyzes to evaluate the effects of 4 weeks of aerobic exercise training on mitonuclear imbalance and UPRmt markers in the skeletal muscle of young (2 months) and aged (24 months) C57BL/6J mice. Initially, we found that aging reduced several mitochondrial genes in the gastrocnemius muscle, and it was accompanied by the low levels of UPRmt markers, including Yme1l1 and Clpp mRNA. As expected, physical training improved the whole-body metabolism and physical performance of aged mice. The aerobic exercise increased key proteins involved in the mitochondrial biogenesis/functions (VDAC and SIRT1) along with mitochondrial-encoded genes (mtNd1, mtCytB, and mtD-Loop) in the skeletal muscle of old mice. Interestingly, aerobic exercise induced the mitonuclear imbalance, increasing MTCO1/ATP5a ratio and UPRmt markers in the skeletal muscle, including HSP60, Lonp1, and Yme1L1 protein levels in the gastrocnemius muscle of aged mice. These data demonstrate that aerobic exercise training induced mitonuclear imbalance and UPRmt in the skeletal muscle during aging. These phenomena could be involved in the improvement of the mitochondrial metabolism and oxidative capacity in aged individuals.

STAT3 inhibition induced temozolomide-resistant glioblastoma apoptosis via triggering mitochondrial STAT3 translocation and respiratory chain dysfunction

Recent evidence has demonstrated that the signal transducer and activator of transcription 3 (STAT3) gene are abnormally active in glioblastoma multiforme (GBM), and this change is crucial for the tumor survival and chemotherapy-resistant. Certain preclinical pharmacology studies have focused on STAT3 phosphorylation and homodimerization, and have developed a class of salicylic acid-based inhibitors, which blocks the nuclear translocation-dependent canonical STAT3 signaling. In the present study, we demonstrated that the salicylic acid-based compound SH-4-54 was quite toxic to temozolomide (TMZ)-resistant GBM cells and could trigger apoptosis in these cells via enhancing mitochondrial translocation–dependent non-canonical STAT3 pathway. We demonstrated that incubation of TMZ-resistant GBM cells with SH-4-54 led to mitochondrial STAT3 (mitoSTAT3) activation and respiratory dysfunction reflected by disrupted (or suppressed) activities of oxidative phosphorylation complexes and oxygen consumption rate. Mechanistically, we proved that SH-4-54 could increase mitoSTAT3 transmembrane import via GRIM-19 and reinforce the association between mitoSTAT3 and mitochondrial transcription factor A (TFAM), indicating that SH-4-54 could facilitate the binding of mitoSTAT3 to mitochondria DNA (mtDNA) and negatively regulate mitochondrial-encoded genes, thus leading to the abnormal oxidation respiratory. Lastly, using GRIM-19 knockout cell line and subcutaneous xenotransplanted tumor model, we elaborately showed the enrichment of SH-4-54 in mitochondria by LC-MS/MS analysis. In conclusion, our data demonstrate thatthe salicylic acid-based compound SH-4-54 is quite effective in killing TMZ-resistant GBM cells and this cytotoxicity is attributed to mitoSTAT3 activation.

Relaxed sequence constraints favor mutational freedom in idiosyncratic metazoan mitochondrial tRNAs

Metazoan complexity and life-style depend on the bioenergetic potential of mitochondria. However, higher aerobic activity and genetic drift impose strong mutation pressure and risk of irreversible fitness decline in mitochondrial (mt)DNA-encoded genes. Bilaterian mitochondria-encoded tRNA genes, key players in mitochondrial activity, have accumulated mutations at significantly higher rates than their cytoplasmic counterparts, resulting in foreshortened and fragile structures. Here we show that fragility of mt tRNAs coincided with the evolution of bilaterian animals. We demonstrate that bilaterians compensated for this reduced structural complexity in mt tRNAs by sequence-independent induced-fit adaption to the cognate mitochondrial aminoacyl-tRNA synthetase (aaRS). Structural readout by nuclear-encoded aaRS partners relaxed the sequence constraints on mt tRNAs and facilitated accommodation of functionally disruptive mutational insults by cis-acting epistatic compensations. Our results thus suggest that mutational freedom in mt tRNA genes is an adaptation to increased mutation pressure that was associated with the evolution of animal complexity.

Arabinoxylan rice bran (MGN-3/Biobran) alleviates radiation-induced intestinal barrier dysfunction of mice in a mitochondrion-dependent manner

MGN-3 is an arabinoxylan from rice bran that has been shown to be an excellent antioxidant and radioprotector. This study examined the protective effects of MGN-3 on radiation-induced intestinal injury. Mice were treated with MGN-3 prior to irradiation, then continued to receive MGN-3 for 4 weeks thereafter. MGN-3 increased the activity of mitochondrial respiratory chain complexes Ⅰ, Ⅲ, Ⅳ and Ⅴ, the intercellular ATP content, the mitochondria-encoded gene expression and mitochondrial copy numbers in the jejunal and colonic mucosa. MGN-3 reduced the oxidative stress levels and inflammatory response indicators in the serum and jejunal and colonic mucosa. Antioxidant indicators such as superoxide dismutase, glutathione peroxidase, catalase and total antioxidant capacity were significantly increased in the serum and jejunal and colonic mucosa in the MGN-3 group. Moreover, MGN-3 decreased the gene abundances and enzymatic activities of caspase-3, 8, 9 and 10 in the jejunal and colonic mucosa. The endotoxin, diamine peroxidase, d-lactate and zonulin levels were significantly reduced in the serum and jejunal and colonic mucosa in the MGN-3 group. MGN-3 also markedly upregulated the gene abundances of ZO-1, occludin, claudin-1 and mucin 2. MGN-3 effectively attenuated radiation-induced changes in the intestinal epithelial mitochondrial function, oxidative stress, inflammatory response, apoptosis, intestinal permeability and barrier function in mice. These findings add to our understanding of the potential mechanisms by which MGN-3 alleviates radioactive intestinal injury.

Hyperandrogenism and insulin resistance modulate gravid uterine and placental ferroptosis in PCOS-like rats.

Women with polycystic ovary syndrome (PCOS) have hyperandrogenism and insulin resistance and a high risk of miscarriage during pregnancy. Similarly, in rats, maternal exposure to 5α-dihydrotestosterone (DHT) and insulin from gestational day 7.5 to 13.5 leads to hyperandrogenism and insulin resistance and subsequently increased fetal loss. A variety of hormonal and metabolic stimuli are able to trigger different types of regulated cell death under physiological and pathological conditions. These include ferroptosis, apoptosis and necroptosis. We hypothesized that, in rats, maternal hyperandrogenism and insulin-resistance-induced fetal loss is mediated, at least in part, by changes in the ferroptosis, apoptosis and necroptosis pathways in the gravid uterus and placenta. Compared with controls, we found that co-exposure to DHT and insulin led to decreased levels of glutathione peroxidase 4 (GPX4) and glutathione, increased glutathione + glutathione disulfide and malondialdehyde, aberrant expression of ferroptosis-associated genes (Acsl4, Tfrc, Slc7a11, and Gclc), increased iron deposition and activated ERK/p38/JNK phosphorylation in the gravid uterus. In addition, we observed shrunken mitochondria with electron-dense cristae, which are key features of ferroptosis-related mitochondrial morphology, as well as increased expression of Dpp4, a mitochondria-encoded gene responsible for ferroptosis induction in the uteri of rats co-exposed to DHT and insulin. However, in the placenta, DHT and insulin exposure only partially altered the expression of ferroptosis-related markers (e.g. region-dependent GPX4, glutathione + glutathione disulfide, malondialdehyde, Gls2 and Slc7a11 mRNAs, and phosphorylated p38 levels). Moreover, we found decreased expression of Dpp4 mRNA and increased expression of Cisd1 mRNA in placentas of rats co-exposed to DHT and insulin. Further, DHT + insulin-exposed pregnant rats exhibited decreased apoptosis in the uterus and increased necroptosis in the placenta. Our findings suggest that maternal hyperandrogenism and insulin resistance causes the activation of ferroptosis in the gravid uterus and placenta, although this is mediated via different mechanisms operating at the molecular and cellular levels. Our data also suggest that apoptosis and necroptosis may play a role in coordinating or compensating for hyperandrogenism and insulin-resistance-induced ferroptosis when the gravid uterus and placenta are dysfunctional.