Mitochondrial Gene Function Research Articles

Characterization of hydrogen sulfide toxicity to human corneal stromal fibroblasts.

Hydrogen sulfide gas (H2 S) is a chemical weapon and a common environmental pollutant. H2 S intoxication is lethal to humans and animals. H2 S contact to the eye can cause vision loss. However, the molecular mechanisms associated with H2 S toxicity to the cornea remain unclear, and no specific therapy exists to mitigate ocular damage from H2 S. Here, we report H2 S-induced cytotoxicity and the parameters contributing to the molecular mechanisms associated with corneal toxicity using primary human corneal stromal fibroblasts (hCSFs) in vitro. Sodium hydrosulfide (NaSH) was used as a source of H2 S, and the cytotoxicity of H2 S was determined by treating hCSF cells with varying concentrations of NaSH (0-10 mM) for 0-72 hours. Changes in cell proliferation, oxidative stress factors, and the expression of inflammatory and fibrotic genes were studied using standard commercial kits and qRT-PCR. NaSH exposure to hCSFs showed dose- and time-dependent cytotoxicity. The IC50 of NaSH was determined to be 5.35 mM. NaSH 5.35mM exposure led to significantly decreased cytochrome c oxidase activity, increased ROS production, and increased expression of inflammatory and fibrotic genes in hCSF cells. H2 S/NaSH exposure alters normal mitochondrial function, oxidative stress, and inflammatory and fibrotic gene responses in corneal stromal fibroblasts in vitro.

Mitochondrial-induced Epigenetic Modifications: From Biology to Clinical Translation.

Mitochondria are maternally inherited semi-autonomous organelles that play a central role in redox balance, energy metabolism, control of integrated stress responses, and cellular homeostasis. The molecular communication between mitochondria and the nucleus is intricate and bidirectional in nature. Though mitochondrial genome encodes for several key proteins involved in oxidative phosphorylation, several regulatory factors encoded by nuclear DNA are prominent contributors to mitochondrial biogenesis and function. The loss of synergy between this reciprocal control of anterograde (nuclear to mitochondrial) and retrograde (mitochondrial to nuclear) signaling, triggers epigenomic imbalance and affects mitochondrial function and global gene expressions. Recent expansions of our knowledge on mitochondrial epigenomics have offered novel perspectives for the study of several non-communicable diseases including cancer. As mitochondria are considered beacons for pharmacological interventions, new frontiers in targeted delivery approaches could provide opportunities for effective disease management and cure through reversible epigenetic reprogramming. This review focuses on recent progress in the area of mitochondrial-nuclear cross-talk and epigenetic regulation of mitochondrial DNA methylation, mitochondrial micro RNAs, and post-translational modification of mitochondrial nucleoid-associated proteins that hold major opportunities for targeted drug delivery and clinical translation.

Exposure to waterborne cadmium induce oxidative stress, autophagy and mitochondrial dysfunction in the liver of Procypris merus

The present study was performed to determine the effect of waterborne cadmium (Cd) exposure on oxidative stress, autophagy and mitochondrial dysfunction, and to explore the mechanism of Cd-induced liver damage in freshwater teleost Procypris merus. To this end, P. merus were exposed to waterborne 0, 0.25 and 0.5 mg/L Cd for 30 days (equal to 0, 2.22 and 4.45 μmol Cd/l). The waterborne Cd exposure significantly increased hepatic Cd accumulation and impaired histological structure of the liver of P. merus. both low and high-dose waterborne Cd exposure induced oxidative stress in the liver of P. merus, through increases Malondialdehyde (MDA) and reactive oxide species (ROS) accumulation in the liver. The Cd-induced oxidative stress in liver may result from reduction of enzyme activities (superoxide dismutases (SOD), catalases (CAT), GSH-S-transferases (GST)) and transcriptional expression of antioxidant related genes (gpx1, gpx2, cata, gsta1, sod1). Furthermore, the present study showed that waterborne Cd exposure decreased the transcriptional factor (nrf2) expression, which might lead to the down-regulation of antioxidant gene expression. Transmission electron microscopy (TEM) observations demonstrated that waterborne Cd exposure induced autophagy in the liver of P. merus. Gene expression analysis showed that waterborne Cd exposure also induced mRNA expression of a set of genes (beclin1, ulk1, atg5, lc3a, atg4b, atg9a, and p62) involved in the autophagy process, indicating that the influence of Cd on autophagy involved transcription regulation of autophagy gene expression. Waterborne Cd exposure induced a sharp decrease in ATP content in the liver of P. merus. In addition, the expression of mitochondrial function genes (sdha, cox4i1, cox1, atp5f1, and mt-cyb) are significantly decreased in the liver of P. merus in Cd treated groups, manifesting the suppression of Cd on mitochondrial energy metabolism. Taken together, our experiments demonstrate that waterborne Cd exposure induced oxidative stress, autophagy and mitochondrial dysfunction in the liver of P. merus. These results may contribute to the understanding of mechanisms that hepatotoxicity of Cd in teleost.

1945-P: Pioglitazone Promotes the Function of White and Brown Adipose Tissue in Diet-Induced Obese Mice

PPARγ agonist pioglitazone is an insulin sensitizer in the treatment of type 2 diabetes. Here, we investigate the effects of pioglitazone on white and brown fat in diet-induced obese mice. After 16-week diet intervention, C57BL/6 mice were divided randomly into three groups: normal chow diet group (NC group), high-fat diet group (HFD group) and high-fat diet + pioglitazone group (HFDP group). Pioglitazone (30-40mg/kg/day) was administered in drinking water for 6 weeks. After 6-week pioglitazone treatment, there was no significant difference in both body weight and fat mass between the HFDP group and the HFD group. However, fasting blood glucose, glucose tolerance and insulin sensitivity were significantly improved in HFDP group. What’s more, the weight of liver and epididymal adipose tissue (eWAT) were not reduced by pioglitazone treatment. However, the weight of brown adipose tissue (BAT) in the HFDP group was significantly higher than HFD group. Histological staining showed that the hepatic steatosis was ameliorated by pioglitazone. Quantitative real-time PCR revealed that mRNA levels of heat-producing genes (PRDM16, FGF-21, UCP1) and mitochondrial biosynthesis and functional genes (Tfam, cidea, cox7a1, Fabp4) were upregulated in eWAT by pioglitazone. Western blotting results also showed that the protein expression of UCP1 and PGC1α was upregulated by pioglitazone both in eWAT and in BAT when compared with the HFD group. To sum up, our study revealed that pioglitazone could promote the browning of WAT and activate the function of BAT with improved hepatic steatosis in diet-induced obese mice. Disclosure F. Xu: None. W. Wang: None. X. Zheng: None. W. Guo: None. Y. Shen: None.

41 Changes of Mitochondria-related Gene Expression Profile Associated with Burn-induced Cardiomyopathy

Abstract Introduction Burn-related cardiac mitochondria dysfunction (BRMD) is associated with negative health outcomes and decreased health-related quality of life; however, few studies of the molecular-genetic mechanisms of BRMD exist. Methods 60% of total body surface area (TBSA) burned rats was employed. O2K Respirometer system (Innsbruck, Austria) was utilized to measure cardiac mitochondrial function. OXPHOS complex activities were determined by using OXPHOS enzyme complex activity assays (Cayman Chemical, Ann Arbor, Michigan). The Rat Mitochondria RT2 Profiler PCR Array was used to identify differential regulation of genes involved in mitochondrial biogenesis and metabolism function. Results Burn injury induced cardiac mit dysfunction by decreasingOXPHOS oxygen consumption at State 3 energized by malate/pyruvate and succinate and declining mit ETC activity in complex I, III, IV and V. 84 rat mit-related gene profiles were measured. The mitochondrial gene profile showed that 30/84 genes related to mitochondrial function and structure were differentially expressed. Of these 30 genes, 17 (ATP12a, ATP4a, ATP6v0a2, ATP6v1e2, ATP6v1g3, COX8c, LHPP, NDUFA5, SLC25a10, SLC25a15, UCP1, UCP2, UCP3, UQCRFS1, LDHA and RGDC) were more than 2 fold up-regulated, and 13 (ATP5c1, ATP5i, ATP5L, COX17, COX6c, COX7a2, NDUFA8, NDUFB3, NDUFB7, NDUFB9, NDUFS4, NDUFS8, and UQCRB) were greater than 2-fold down-regulated. Furthermore, 8 genes (AIFM2, BCL2, FIS1, IMMP2L, MSTO1, SLC25A23, SLC25A37, SLC25A4) that had significant differentially expression were associated with heart dysfunction. Conclusions This study provides preliminary evidence that 30 mitochondrial function genes were significantly associated with burn-induced heart dysfunction in 24 hpb rats. Applicability of Research to Practice These findings elucidate possible pathways and early biomarkers for targeting novel interventions for burn-induced heart dysfunction.

Omega3-polyunsaturated fatty acid ameliorates metabolic disorders in adulthood rats caused by postnatal early overfeeding

Objective To explore the effect and mechanism of omega 3-polyunsaturated fatty acid(ω3-PUFA) dietary intervention on mitochondrial function of white adipose tissue in adult rats with postnatal early overfeeding. Methods An overfed animal model by adjusting litter size was developed for the study of neonatal overfeeding. The litter size was adjusted to 3 male rats per litter(small litter, SL group) and 10 pups per litter(normal litter, NL group). After weaning(week 3), the pups were fed standard chow or ω3-PUFA diet(SL-FO) until postnatal weeks 13. Food intake, body weight, and rectal temperature of rats were measured regularly, and energy metabolism of animals was monitored in week 13. During week 3 and 13, subcutaneous adipose tissue was collected. Inguinal preadipocytes of mice were isolated and induced to differentiate, and 50 μmol/L eicosapentaenoicacid(EPA) was administered for 48 h at the late stage of differentiation. The mRNA and protein expression levels of mitochondrial related genes, mitochondrial copy number, and oxygen consumption rate of adipocytes were detected in adipose tissue and adipocytes. Results By the 3rd week, the body weight, food intake, and fat cell area in SL group were higher than those in NL group while the body temperature was lower until to 13 weeks. By the 13th week, the O2 consumption, CO2 output, and heat production of rats in SL group were lower than those in NL group. Meanwhile, the expressions of mitochondrial function related genes such as uncoupling protein 1(UCP1), carnitine palmitoyltransferase 1(CPT1), SIRT1, and mitochondrial biosynthesis regulatory gene peroxisome proliferator-activated receptor coativator-1 (PGC1α) in adipose tissue by the 3rd and 13th week were significantly reduced(P<0.05). After weaning, ω3-PUFA diet significantly reduced weight gain in SL rats, increased UCP1 protein expression, restored energy metabolism level and mitochondrial function related gene expression. In vitro intervention of EPA increased the mitochondrial copy number, the mRNA and protein expression levels of mitochondrial biosynthesis and functional genes, as well as the mitochondrial basic oxygen consumption rate(P<0.05). Conclusion ω3-PUFA improves postnatal overfeeding-induced impairment of the mitochondrial function and biosynthesis of subcutaneous white adipose tissue in rats, which may be an important mechanism for fish oil diet to inhibit the early over-nutrition program and restore the thermogenic metabolism. Key words: Omega 3-PUFA; Postnatal overfeeding; White fat; Mitochondria

Mitoepigenetics and Its Emerging Roles in Cancer.

In human beings, there is a ∼16,569 bp circular mitochondrial DNA (mtDNA) encoding 22 tRNAs, 12S and 16S rRNAs, 13 polypeptides that constitute the central core of ETC/OxPhos complexes, and some non-coding RNAs. Recently, mtDNA has been shown to have some covalent modifications such as methylation or hydroxylmethylation, which play pivotal epigenetic roles in mtDNA replication and transcription. Post-translational modifications of proteins in mitochondrial nucleoids such as mitochondrial transcription factor A (TFAM) also emerge as essential epigenetic modulations in mtDNA replication and transcription. Post-transcriptional modifications of mitochondrial RNAs (mtRNAs) including mt-rRNAs, mt-tRNAs and mt-mRNAs are important epigenetic modulations. Besides, mtDNA or nuclear DNA (n-DNA)-derived non-coding RNAs also play important roles in the regulation of translation and function of mitochondrial genes. These evidences introduce a novel concept of mitoepigenetics that refers to the study of modulations in the mitochondria that alter heritable phenotype in mitochondria itself without changing the mtDNA sequence. Since mitochondrial dysfunction contributes to carcinogenesis and tumor development, mitoepigenetics is also essential for cancer. Understanding the mode of actions of mitoepigenetics in cancers may shade light on the clinical diagnosis and prevention of these diseases. In this review, we summarize the present study about modifications in mtDNA, mtRNA and nucleoids and modulations of mtDNA/nDNA-derived non-coding RNAs that affect mtDNA translation/function, and overview recent studies of mitoepigenetic alterations in cancer.

ACTL6A is a novel oncogene and prognostic biomarker for prostate cancer

Abstract ACTL6A (Actin-like 6A protein) is a member of ATP-dependent SWI/SNF-like BAF chromatin remodeling complexes encoding a family of actin-related proteins. Using 3D culture and Micro-Western Array analysis targeting for epigenetic regulatory proteins, we discovered that ACTL6A plays essential role in regulation of the prostate acinar morphogenesis, a differentiation procedure. Expression level of ACTL6A protein decreases dramatically during prostate acinar morphogenesis, suggesting that ACTL6A is a potential oncogene in prostate cancer (PCa). We analyzed Oncomine online database and found that ACTL6A is higher in prostate cancer vs. adjacent normal prostate tissues, while ACTL6A protein level is even higher in metastatic PCa. High ACTL6A gene expression level in prostate tumors correlates to worse PCa patient survival outcome. We knocked down ACTL6A in PC-3 PCa cells, and the reduction of ACTL6A suppressed proliferation, migration, and invasion of PC-3 cells. Knockdown of ACTL6A also reduced population of CD44+ and ALDH enzyme activity-positive PCa cells, which represents the population of cancer stem cell (CSC) in PCa. Decrease of ACTL6A reduced expression of a few oncogenic long non-coding RNA, including HIFCAR, MALAT1, and PCAT-1. Seahorse mito-stress and glycol-stress analysis revealed that knockdown of ACTL6A suppressed OCR and ECAR of mitochondria in PCa cells. ACTL6A knockdown up-regulates expression of glycolysis-related genes, suggesting that ACTL6A regulate cancer metabolism. Our study suggested that ACTL6A regulates expression and activity of YAP and c-Myc, which in turn regulates stemness factors Nanog and Sox2, thus enhances the cancer stemness of PCa cells. ACTL6A also regulates mitochondrial function and metabolic genes PKM2, LDHA, GLS1, partially through c-Myc, therefore ACTL6A causes metabolism rewiring in PCa. All these regulations contribute to the metastasis of PCa. In conclusion, ACTL6A is a novel oncogene and prognostic biomarker for PCa.

Propionic acid induces mitochondrial dysfunction and affects gene expression for mitochondria biogenesis and neuronal differentiation in SH-SY5Y cell line

Studies in animal models have shown that the short-chain fatty acid, propionic acid (PPA), interferes with mitochondrial metabolism leading to mitochondrial dysfunction and behavioral abnormalities. The aim of this study was to investigate the effects of PPA on mitochondrial function and gene expression in neuronal cells. SH-SY5Y cells and normal human neural progenitor (NHNP) cells were exposed to 1, 5 mM PPA for 4 or 24 h and we found that the mitochondrial potential measured in SH-SY5Y cells decreased in a dose-dependent manner after PPA treatment. Electron microscopy analysis revealed that the size of the mitochondria was significantly reduced following PPA treatment. A dose-dependent increase in the mitochondrial DNA copy number was observed in the PPA-treated cells. The expression of the mitochondrial biogenesis-related proteins PGC-1α, TFAM, SIRT3, and COX4 was significantly increased after PPA treatment. Transcriptome analysis revealed that mRNA expression in the notch signaling-related genes ASCL1 and LFNG changed after PPA treatment and the positive correlated protein expression changes were also observed. These results revealed that PPA treatment may affect neurodevelopment by altering mitochondrial function and notch signaling-related gene expression.

Mitochondrial genes associated with pyrethroid resistance revealed by mitochondrial genome and transcriptome analyses in the malaria vector Anopheles sinensis (Diptera: Culicidae)

Insecticides are still the main method of mosquito control, but mosquito resistance presents a large obstacle. The function of mitochondrial genes in the evolution of insecticide resistance is still poorly understood. Pyrethroid is the most commonly used insecticide, and Anopheles sinensis is an important malaria vector in China and Southeast Asia. In this study, we investigated the mitochondrial genes associated with pyrethroid resistance through their genetic and expression variation based on analyses of transcriptomes and 36 individuals with resequencing in three geographical populations in China. The nucleotide diversity (Pi) in 18 resistant individuals was much lower than that in 18 susceptible individuals, which suggests that some sites experienced purifying selection subject to pyrethroid stress. Ka/Ks and amino acid analyses showed that ND4 experienced positive selection and had 23 amino acid mutations due to pyrethroid stress. These mutations might change the ND4 structure and function and thus alter the efficiency of the respiratory chain. ND5 was significantly upregulated, and ATP8 was significantly downregulated in these three pyrethroid resistant populations, which suggests that these two genes function in the production and maintenance of pyrethroid resistance. There are differences in mitochondrial genes involved in pyrethroid resistance among these three populations. This is the first study to reveal the association of mitochondrial genes in the evolution of insecticide resistance through amino acid mutation and expression patterns and can help us further understand insecticide resistance mechanisms. © 2019 Society of Chemical Industry.

Regulation of nuclear epigenome by mitochondrial DNA heteroplasmy

Diseases associated with mitochondrial DNA (mtDNA) mutations are highly variable in phenotype, in large part because of differences in the percentage of normal and mutant mtDNAs (heteroplasmy) present within the cell. For example, increasing heteroplasmy levels of the mtDNA tRNALeu(UUR) nucleotide (nt) 3243A > G mutation result successively in diabetes, neuromuscular degenerative disease, and perinatal lethality. These phenotypes are associated with differences in mitochondrial function and nuclear DNA (nDNA) gene expression, which are recapitulated in cybrid cell lines with different percentages of m.3243G mutant mtDNAs. Using metabolic tracing, histone mass spectrometry, and NADH fluorescence lifetime imaging microscopy in these cells, we now show that increasing levels of this single mtDNA mutation cause profound changes in the nuclear epigenome. At high heteroplasmy, mitochondrially derived acetyl-CoA levels decrease causing decreased histone H4 acetylation, with glutamine-derived acetyl-CoA compensating when glucose-derived acetyl-CoA is limiting. In contrast, α-ketoglutarate levels increase at midlevel heteroplasmy and are inversely correlated with histone H3 methylation. Inhibition of mitochondrial protein synthesis induces acetylation and methylation changes, and restoration of mitochondrial function reverses these effects. mtDNA heteroplasmy also affects mitochondrial NAD+/NADH ratio, which correlates with nuclear histone acetylation, whereas nuclear NAD+/NADH ratio correlates with changes in nDNA and mtDNA transcription. Thus, mutations in the mtDNA cause distinct metabolic and epigenomic changes at different heteroplasmy levels, potentially explaining transcriptional and phenotypic variability of mitochondrial disease.

Adiponectin ameliorated pancreatic islet mitochondrial injury induced by chronic intermittent hypoxia

Objective: To explore the effect of adiponectin (APN) on islet injury induced by chronic intermittent hypoxia (CIH). Methods: Thirty-six SD rats were randomly divided into three groups: Normal control (NC), CIH, and CIH + APN groups. The rats in the CIH and CIH+APN groups received an intermittent hypoxia exposure while the rats in NC group received the room air only. The rats in CIH+APN group received the intravenous injection of APN. The intermittent hypoxia events persisted 8 hours a day and last for 35 days. The fasting blood glucose and fasting insulin were detected at the time of 0, 7, 14, 21, 28, and 35 day. After 35 days, the level of serum adiponectin, and adenosine triphosphate (ATP) level, superoxide dismutase (SOD), malondialdehyde (MDA), the mRNA levels of mitochondrial oxidative phosphorylation function and mitochondrial synthesis gene, and the protein level of mitochondrial and cytoplasmic cytochrome C of pancreatic islet were detected. Results: The glucose and insulin level had no statistically differences among three groups at different time points (all P>0.05). However, compared with NC and CIH+APN groups, CIH reduced the serum adiponectin [(7 265±2 209) ng/ml, (6 536±1 678) ng/ml vs (4 923±1 742) ng/ml, both P<0.05], ATP levels [(30.92±1.12) nmol/mg, (26.55±0.72) nmol/mg vs (20.22±1.47) nmol/mg, both P<0.05], mRNA levels of mitochondria oxidative phosphorylation function and mitochondrial synthesis gene, the activity of SOD, and the rate of mitochondrial/cytoplasmic cytochrome C protein level while increased the MDA level in pancreatic islet. Compared with NC group, the MDA level increased (P<0.05) and the APN level had no statistically difference, while the level of other indicators decreased in CIH+APN group (all P<0.05). Conclusion: APN ameliorates the pancreatic islet injury induced by CIH through inhibition of oxidative stress.

Lycopene supplementation attenuates western diet-induced body weight gain through increasing the expressions of thermogenic/mitochondrial functional genes and improving insulin resistance in the adipose tissue of obese mice

This passive overconsumption of western diet has precipitated a steep rise in obesity and its comorbidities, and obesity has become one of the main threats to health worldwide. Thus, deciphering the molecular mechanisms leading to obesity is therefore of utmost importance to guide the search for novel therapeutic and preventive strategies. Lycopene (LYC), a major carotenoid present in tomato, has been regarded as a nutraceutical that has powerful anti-oxidant and anti-obesity bioactivities. Even though substantial progress has been made in deciphering the mechanism of how LYC affects obesity in recent years, whether thermogenic genes, mitochondrial function and insulin resistance are involved in the anti-obesity effect of LYC is yet to be elucidated. In the current study, we demonstrated that LYC remarkably suppressed HFFD-elevated mice body weight gain. LYC blocked lipid accumulation in adipose tissue by decreasing the expressions of lipogenesis genes and increasing the expressions of lipidolysis related genes, including thermogenic and mitochondrial functional genes. Moreover, LYC improved HFFD-induced insulin resistance in WATs via inhibiting the inflammation responses in WATs, decreasing circulating proinflammatory cytokines, suppressing gut leak and intestinal inflammation. Our study indicating that the supplementation of LYC might be a nutritional preventive strategy to combat obesity.

Imsnc761 and DDX6 synergistically suppress cell proliferation and promote apoptosis via p53 in testicular embryonal carcinoma cells.

Intermediate-sized non-coding RNAs (imsncRNAs) have been shown to play important regulatory roles in the development of several eukaryotic organisms. In the present research, we selected imsncRNA 761 (imsnc761) as a research target. Expression analyses in a previous study showed that imsnc761 was down-regulated in maturation-arrested testis tissues as compared with the level in normal controls. In the present study, we found that imsnc761 could interact with DEAD-box helicase 6 (DDX6) to induce NTERA-2 (NT2 (testicular embryonal carcinoma cell)) cell apoptosis and proliferation inhibition via the p53 pathway. This interaction between imsnc761 and DDX6 also inhibited mitochondrial function and specific gene transcription and translation. To facilitate further research, we used label-free quantitation method to analyze the associated differences in Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathways and biological processes. This confirmed the changes in several specific pathways, which matched our molecular experimental results.

Cellular and molecular approaches to enhance myocardial recovery after myocardial infarction.

Reperfusion therapy has resulted in significant improvement in post-myocardial infarction morbidity and mortality in over the last 4 decades. Nonetheless, it is well recognized that simply restoring patency of the epicardial artery may not stop or reverse damage at microvascular level, and myocardial salvage is often suboptimal. Numerous efforts have been undertaken to elucidate the mechanisms underlying extensive myonecrosis to facilitate the discovery of therapies to provide additional and incremental benefits over current therapeutic pathways. To date, conclusively effective strategies to promote myocardial recovery have not yet been established. Novel approaches are investigating the foundational cellular and molecular bases of myocardial ischemia and irreversible injury. Herein, we review the emerging concepts and proposed therapies that may improve myocardial protection and reduce infarct size. We examine the preclinical and clinical evidence for reduced infarct size with these strategies, including anti-inflammatory agents, intracellular ion channel modulators, agents affecting the reperfusion injury salvage kinase (RISK) and nitric oxide signaling pathways, modulators of mitochondrial function, anti-apoptotic agents, and stem cell and gene therapy. We review the potential reasons of failures to date and the potential for new strategies to further promote myocardial recovery and improve prognosis.

Early burst of glycolysis in microglia regulates mitochondrial dysfunction in oligodendrocytes under neuro-inflammation

Abstract Brain resident microglia plays a pathological role in the loss of oligodendrocyte’s function under neuroinflammation. The precise signaling mechanism by which microglial driven factor attenuates the myelin gene expression under such condition is poorly defined. Here we used bioenergetic approach to understand the microglia-oligodendrocyte crosstalk. We found that mixed glial cells under inflammatory stimulation using lipopolysaccharide plus interferon gamma (LI) had significantly enhanced glycolysis with drastic attenuation of mitochondrial respiration and myelin gene expression. Dissecting the mixed glial culture into pure microglia, astrocyte and oligodendrocytes (OLs) revealed that primary microglia was the main source of higher glycolysis, inflammatory cytokines, and nitric oxide (NO). An early burst of glycolysis in microglia in response to LI treatment caused the production of inflammatory cytokines and NO, which is mediated by PDK1-Akt signaling. Culturing of OLs with LI-conditioned microglial media (mCM) resulted in complete failure of mitochondrial respiration in OLsalong with decreased expression of myelin genes. We identified that nitric oxide produced by microglia is the key player in the loss of mitochondrial respiration in OLs and targeting either glycolysis, PDK1 or Akt in microglia resulted in significant recovery of mitochondrial function and myelin genes expression in OLs. In summary, we identified a potential therapeutic signaling cascade in microglia; an early burst of glycolysis mediated by PDK1 and Akt, responsible for hyper-production of NO, ensuing in the failure of mitochondrial respiration in oligodendrocytes, and leading in reduction of myelin genes expression.

Mechanisms Underpinning the Polypharmacy Effects of Medications in Psychiatry.

BackgroundBipolar disorder is a mental health condition with progressive social and cognitive function disturbances. Most patients’ treatments are based on polypharmacy, but with no biological basis and little is known of the drugs’ interactions. The aim of this study was to analyze the effects of lithium, valproate, quetiapine, and lamotrigine, and the interactions between them, on markers of inflammation, bioenergetics, mitochondrial function, and oxidative stress in neuron-like cells and microglial cells.MethodsNeuron-like cells and lipopolysaccharide-stimulated C8-B4 cells were treated with lithium (2.5 mM), valproate (0.5 mM), quetiapine (0.05 mM), and lamotrigine (0.05 mM) individually and in all possible combinations for 24 h. Twenty cytokines were measured in the media from lipopolysaccharide-stimulated C8-B4 cells. Metabolic flux analysis was used to measure bioenergetics, and real-time PCR was used to measure the expression of mitochondrial function genes in neuron-like cells. The production of superoxide in treated cells was also assessed.ResultsThe results suggest major inhibitory effects on proinflammatory cytokine release as a therapeutic mechanism of these medications when used in combination. The various combinations of medications also caused overexpression of PGC1α and ATP5A1 in neuron-like cells. Quetiapine appears to have a proinflammatory effect in microglial cells, but this was reversed by the addition of lamotrigine independent of the drug combination.ConclusionPolypharmacy in bipolar disorder may have antiinflammatory effects on microglial cells as well as effects on mitochondrial biogenesis in neuronal cells.

Increased expression of mitochondrial transcription factor A and nuclear respiratory factor-1 predicts a poor clinical outcome of breast cancer.

Nuclear respiratory factor-1 (Nrf1) and mitochondrial transcription factor A (TFAM) are involved in the regulation of a variety of mitochondrial functional genes, which are associated with decreased sensitivity of tumor cells to chemotherapy. However, the expression status of Nrf1 and TFAM, as well as their clinical significance in breast cancer, is unknown. In the present study, tumor tissues and corresponding adjacent normal tissues were collected from 336 patients with breast cancer, and Nrf1 and TFAM expression was analyzed by immunohistochemistry using a tissue microarray. Expression of Nrf1 and TFAM was significantly increased in breast cancer tissue compared with adjacent normal tissues. In addition, patients positive for Nrf1 or TFAM had a poorer clinical prognosis than patients who were negative, and those positive for Nrf1 and TFAM had the shortest survival time. These results suggest that Nrf1 and TFAM are potential biomarkers for the determination of individualized therapy and the prognosis of breast cancer, and molecular targeting of Nrf1 and TFAM is a promising strategy for the sensitization of breast cancer cells to chemotherapeutics.

Quantifying engineered nanomaterial toxicity: comparison of common cytotoxicity and gene expression measurements

BackgroundWhen evaluating the toxicity of engineered nanomaterials (ENMS) it is important to use multiple bioassays based on different mechanisms of action. In this regard we evaluated the use of gene expression and common cytotoxicity measurements using as test materials, two selected nanoparticles with known differences in toxicity, 5 nm mercaptoundecanoic acid (MUA)-capped InP and CdSe quantum dots (QDs). We tested the effects of these QDs at concentrations ranging from 0.5 to 160 µg/mL on cultured normal human bronchial epithelial (NHBE) cells using four common cytotoxicity assays: the dichlorofluorescein assay for reactive oxygen species (ROS), the lactate dehydrogenase assay for membrane viability (LDH), the mitochondrial dehydrogenase assay for mitochondrial function, and the Comet assay for DNA strand breaks.ResultsThe cytotoxicity assays showed similar trends when exposed to nanoparticles for 24 h at 80 µg/mL with a threefold increase in ROS with exposure to CdSe QDs compared to an insignificant change in ROS levels after exposure to InP QDs, a twofold increase in the LDH necrosis assay in NHBE cells with exposure to CdSe QDs compared to a 50% decrease for InP QDs, a 60% decrease in the mitochondrial function assay upon exposure to CdSe QDs compared to a minimal increase in the case of InP and significant DNA strand breaks after exposure to CdSe QDs compared to no significant DNA strand breaks with InP. High-throughput quantitative real-time polymerase chain reaction (qRT-PCR) data for cells exposed for 6 h at a concentration of 80 µg/mL were consistent with the cytotoxicity assays showing major differences in DNA damage, DNA repair and mitochondrial function gene regulatory responses to the CdSe and InP QDs. The BRCA2, CYP1A1, CYP1B1, CDK1, SFN and VEGFA genes were observed to be upregulated specifically from increased CdSe exposure and suggests their possible utility as biomarkers for toxicity.ConclusionsThis study can serve as a model for comparing traditional cytotoxicity assays and gene expression measurements and to determine candidate biomarkers for assessing the biocompatibility of ENMs.

Neurochemical effects of the R form of α-lipoic acid and its neuroprotective mechanism in cellular models of Parkinson’s disease

ObjectiveParkinson’s disease (PD) is a common neurodegenerative disease. This study aimed to investigate the effects of the R form of α-lipoic acid (RLA) in cellular models of PD induced by 6-hydroxydopamine (6-OHDA) or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MethodsCell viability and apoptosis were detected using CCK8 and Annexin V-FITC assays, respectively. Intracellular reactive oxygen species (ROS) were detected by fluorescence staining. ELISA assays were performed to detect the levels of dopamine and α-synuclein. To evaluate the effects of RLA on mitochondrial function, cytotoxicity, ATP levels, and mitochondrial gene expression were assayed. Additionally, the expression levels of autophagy-related proteins, including Parkin, PINK1, p62, ATG12, and LC3, were analyzed by western blot, and cell autophagy was visualized by immunofluorescence. ResultsRLA increased cell viability and decreased apoptosis, intracellular ROS, and cytotoxicity, and induced cell autophagy in PD models induced by 6-OHDA and MPTP. RLA also reversed the decreased dopamine and increased α-synuclein expression induced by 6-OHDA and MPTP. The mitochondrial regulatory protein PGC-1α was significantly up-regulated by RLA. The expression levels of autophagy-related proteins, including Parkin, PINK1, p62, and ATG12, were down-regulated after RLA treatment, while LC3 expression was up-regulated. ConclusionsRLA has a protective effect against cellular damage induced by 6-OHDA and MPTP. The neuroprotective mechanism of RLA may be associated with improvement of mitochondrial function and autophagy. Therefore, RLA may serve as a promising potential adjuvant for PD treatment.