NRF-2α and mitophagy underlie enhanced mitochondrial functions and biogenesis induced by T-2 toxin in GH3 cells.

T-2 toxin is a natural contaminant in grain cereals produced by species of Fusarium. Studies indicate that T-2 toxin can positively affect mitochondrial function, but the underlying mechanism is unclear. In this study, we examined the role of nuclear respiratory factor 2α (NRF-2α) in T-2 toxin-activated mitochondrial biogenesis and the direct target genes of NRF-2α. Furthermore, we investigated T-2 toxin-induced autophagy and mitophagy, and the role of mitophagy in changes in mitochondrial function and apoptosis. It was found that T-2 toxin significantly increased NRF-2α levels and nuclear localization of NRF-2α was induced. NRF-2α deletion significantly increased the production of reactive oxygen species (ROS), abrogated T-2 toxin-induced increases in ATP and mitochondrial complex I activity, and inhibited the mitochondrial DNA copy number. Meanwhile, With chromatin immunoprecipitation sequencing (ChIP-Seq), various novel NRF-2α target genes were identified, such as mitochondrial iron-sulphur subunits (Ndufs 3,7) and mitochondrial transcription factors (Tfam, Tfb1m, and Tfb2m). Some target genes were also involved in mitochondrial fusion and fission (Drp1), mitochondrial translation (Yars2) and splicing (Ddx55), and mitophagy. Further studies showed that T-2 toxin induced Atg5 dependent autophagy and Atg5/PINK1-dependent mitophagy. In addition, mitophagy defects increase ROS production, inhibit ATP levels and the expression of genes related to mitochondrial dynamics, and promote apoptosis in the presence of T-2 toxins. Altogether, these results suggest that NRF-2α plays a critical role in promoting mitochondrial function and biogenesis through regulation of mitochondrial genes, and, interestingly, mitophagy caused by T-2 toxin positively affected mitochondrial function and protected cell survival against T-2 toxin.

Platycodin D Protects Human Fibroblast Cells from Premature Senescence Induced by H2O2 through Improving Mitochondrial Biogenesis.

BACKGROUND: Although Platycodin D (PLD) is the main active saponin of Platycodon grandiflorum (PG) and responsible for multiple therapeutic benefits, including antioxidant and antiaging, only few direct demonstrations have been reported on the role of PLD in antiaging process. The present investigation was carried out to elucidate the protection of PLD against aging in vitro and associated molecular mechanisms on H2O2-induced premature senescence model in human -fetal lung diploid fibroblasts 2BS cells. METHODS: The cellular morphology, cell cycle, and senescence-associated ß-galactosidase activity assays were used for senescence-like phenotypes determination in the oxidant challenged model. The oxygen-free radicals reactive oxygen species (ROS), 4-hydroxynonenal (4-HNE), and malondialdehyde (MDA) determinations were estimated by enzyme-linked immunosorbent assay assay. The potential of the mitochondria mass and the mitochondrial membrane were used to observe the alteration of mitochondria. Western blot analysis was performed to determine the protein expression. RESULTS: The results showed that PLD significantly reversed senescence-like phenotypes in the oxidant challenged model, as well as related molecules expression such as p53, p21, and p16. Moreover, PLD treatment significantly decreased the levels of ROS, 4-HNE, and MDA in H2O2-treated 2BS cells. The mechanisms responsible for the antioxidant and antiaging effects of PLD were investigated, we found that mitochondria under PLD conditions show increase membrane potential ratio and stimulate the proliferation of mitochondria mass. In addition, the protein expression of peroxisome proliferator activated receptor gamma coactivator 1α and its downstream targets, that is, nuclear respiratory factor and mitochondrial transcription factor A were also increased in mitochondrial biogenesis. CONCLUSION: These results indicated that PLD prevented H2O2-induced premature senescence in vitro by improving mitochondrial biogenesis to attenuate age-dependent endogenous oxidative damage. Key Message: The study revealed the antioxidant and antiaging potential of PLD against H2O2-induced premature senescence.

Effects of low-dose ALA-PDT on fibroblast photoaging induced by UVA irradiation and the underlying mechanisms.

OBJECTIVES: To investigate the effects of low-dose aminolevulinic acid photodynamic therapy (ALA-PDT) on photoaging in human dermal fibroblasts (HDFs) and to explore the mechanism of Nuclear factor erythroid 2-related factor 2(Nrf2)-mediated photorejuvenation in vitro. METHODS: A photoaging model was established through repeated exposure of HDFs to UVA. Total superoxide dismutase (SOD) expression was detected by a SOD activity assay. Nrf2 was knocked down through adenovirus infection, and successful knockdown was confirmed by Western blot analysis and quantitative polymerase chain reaction. RESULTS: Sustained exposure to UVA induced photoaging in HDFs. Total SOD activity was significantly increased by low-dose aminolevulinic acid (ALA)-PDT. Upon application of low doses of ALA-PDT to photoaging HDFs, Nrf2 was translocated to the nucleus; in addition, the expression of Nrf2, transforming growth factor-ß1 (TGF-ß1), type I and III collagen (COL1 and COL3), heme oxygenase 1 (HO-1), and p-ERK was increased, while the expression of matrix metalloproteinase 9 (MMP-9) was decreased. However, after Nrf2 was knocked down in HDFs, the expression of TGF-ß1, COL1, COL3, and HO-1 was significantly decreased, while the expression of MMP-9 was increased. CONCLUSION: This study revealed that low-dose ALA-PDT decreases UVA-mediated photoaging through an Nrf2-mediated antioxidant effect.

Mitochondrial biogenesis is impaired in osteoarthritis chondrocytes but reversible via peroxisome proliferator-activated receptor γ coactivator 1α.

OBJECTIVE: The etiology of chondrocyte mitochondrial dysfunction in osteoarthritis (OA) is not completely understood. OA chondrocytes are deficient in the metabolic biosensors active AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT-1), which modulate the mitochondrial biogenesis "master regulator" peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α). Moreover, PGC-1α critically mediates AMPK anticatabolic activity in chondrocytes. The aim of this study was to test the hypothesis that mitochondrial biogenesis is deficient in human OA chondrocytes and that this deficiency functionally increases chondrocyte procatabolic responses, which are reversed by activation of the AMPK/SIRT-1/PGC-1α pathway. METHODS: We assessed the expression and activity (phosphorylation) of AMPKα, SIRT-1, and PGC-1α in human knee chondrocytes and human and mouse knee cartilage, and we defined and compared the content and function of mitochondria, including oxidative phosphorylation and expression of mitochondrial biogenesis factors (mitochondrial transcriptional factor A [TFAM] and nuclear respiratory factors [NRFs]). RESULTS: Human knee OA chondrocytes had a decreased mitochondrial biogenesis capacity, which was linked to reduced AMPKα activity and decreased expression of SIRT-1, PGC-1α, TFAM, NRF-1, and NRF-2. Human knee OA and aging mouse knee cartilage had decreased expression of TFAM and ubiquinol-cytochrome c reductase core protein, a subunit of mitochondrial complex III, in situ. Chondrocyte TFAM knockdown inhibited mitochondrial biogenesis and enhanced procatabolic responses to interleukin-1ß. Finally, activation of AMPK by A-769662 increased PGC-1α expression via SIRT-1 and reversed impairments in mitochondrial biogenesis, oxidative phosphorylation, and intracellular ATP in human knee OA chondrocytes. CONCLUSION: Mitochondrial biogenesis is deficient in human OA chondrocytes, and this deficiency promotes chondrocyte procatabolic responses. TFAM-mediated activation of the AMPK/SIRT-1/PGC-1α pathway reverses these effects, suggesting translational potential of pharmacologic AMPK activators to limit OA progression.

Lipid exposure elicits differential responses in gene expression and DNA methylation in primary human skeletal muscle cells from severely obese women.

The skeletal muscle of obese individuals exhibits an impaired ability to increase the expression of genes linked with fatty acid oxidation (FAO) upon lipid exposure. The present study determined if this response could be attributed to differential DNA methylation signatures. RNA and DNA were isolated from primary human skeletal muscle cells (HSkMC) from lean and severely obese women following lipid incubation. mRNA expression and DNA methylation were quantified for genes that globally regulate FAO [PPARγ coactivator (PGC-1α), peroxisome proliferator-activated receptors (PPARs), nuclear respiratory factors (NRFs)]. With lipid oversupply, increases in NRF-1, NRF-2, PPARα, and PPARδ expression were dampened in skeletal muscle from severely obese compared with lean women. The expression of genes downstream of the PPARs and NRFs also exhibited a pattern of not increasing as robustly upon lipid exposure with obesity. Increases in CpG methylation near the transcription start site with lipid oversupply were positively related to PPARδ expression; increases in methylation with lipid were depressed in HSkMC from severely obese women. With severe obesity, there is an impaired ability to upregulate global transcriptional regulators of FAO in response to lipid exposure. Transient changes in DNA methylation patterns and differences in the methylation signature with severe obesity may play a role in the transcriptional regulation of PPARδ in response to lipid. The persistence of differential responses to lipid in HSkMC derived from lean and obese subjects supports the possibility of stable epigenetic programming of skeletal muscle cells by the respective environments.

Eating habits modulate short term memory and epigenetical regulation of brain derived neurotrophic factor in hippocampus of low- and high running capacity rats.

Exercise capacity and dietary restriction (DR) are linked to improved quality of life, including enhanced brain function and neuro-protection. Brain derived neurotrophic factor (BDNF) is one of the key proteins involved in the beneficial effects of exercise on brain. Low capacity runner (LCR) and high capacity runner (HCR) rats were subjected to DR in order to investigate the regulation of BDNF. HCR-DR rats out-performed other groups in a passive avoidance test. BDNF content increased significantly in the hippocampus of HCR-DR groups compared to control groups (p<0.05). The acetylation of H3 increased significantly only in the LCR-DR group. However, chip-assay revealed that the specific binding between acetylated histone H3 and BNDF promoter was increased in both LCR-DR and HCR-DR groups. In spite of these increases in binding, at the transcriptional level only, the LCR-DR group showed an increase in BDNF mRNA content. Additionally, DR also induced the activity of cAMP response element-binding protein (CREB), while the content of SIRT1 was not altered. Peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) was elevated in HCR-DR groups. But, based on the levels of nuclear respiratory factor-1 and cytocrome c oxidase, it appears that DR did not cause mitochondrial biogenesis. The data suggest that DR-mediated induction of BDNF levels includes chromatin remodeling. Moreover, DR does not induce mitochondrial biogenesis in the hippocampus of LCR/HCR rats. DR results in different responses to a passive avoidance test, and BDNF regulation in LCR and HCR rats.

Regulatory elements associated with paternally-expressed genes in the imprinted murine Angelman/Prader-Willi syndrome domain.

The Angelman/Prader-Willi syndrome (AS/PWS) domain contains at least 8 imprinted genes regulated by a bipartite imprinting center (IC) associated with the SNRPN gene. One component of the IC, the PWS-IC, governs the paternal epigenotype and expression of paternal genes. The mechanisms by which imprinting and expression of paternal genes within the AS/PWS domain - such as MKRN3 and NDN - are regulated by the PWS-IC are unclear. The syntenic region in the mouse is organized and imprinted similarly to the human domain with the murine PWS-IC defined by a 6 kb interval within the Snrpn locus that includes the promoter. To identify regulatory elements that may mediate PWS-IC function, we mapped the location and allele-specificity of DNase I hypersensitive (DH) sites within the PWS-IC in brain cells, then identified transcription factor binding sites within a subset of these DH sites. Six major paternal-specific DH sites were detected in the Snrpn gene, five of which map within the 6 kb PWS-IC. We postulate these five DH sites represent functional components of the murine PWS-IC. Analysis of transcription factor binding within multiple DH sites detected nuclear respiratory factors (NRF's) and YY1 specifically on the paternal allele. NRF's and YY1 were also detected in the paternal promoter region of the murine Mrkn3 and Ndn genes. These results suggest that NRF's and YY1 may facilitate PWS-IC function and coordinately regulate expression of paternal genes. The presence of NRF's also suggests a link between transcriptional regulation within the AS/PWS domain and regulation of respiration. 3C analyses indicated Mkrn3 lies in close proximity to the PWS-IC on the paternal chromosome, evidence that the PWS-IC functions by allele-specific interaction with its distal target genes. This could occur by allele-specific co-localization of the PWS-IC and its target genes to transcription factories containing NRF's and YY1.

Decreased mRNA expression of PGC-1α and PGC-1α-regulated factors in the SOD1G93A ALS mouse model and in human sporadic ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective motoneuron loss. Although the cause of ALS is unknown, oxidative stress, inflammation, and mitochondrial dysfunction have been identified as important components of its pathogenesis. Peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) plays a central role in the regulation of mitochondrial metabolism and biogenesis via activation of transcription factors, such as nuclear respiratory factors 1 and 2 and mitochondrial transcription factor A (Tfam). Alterations in PGC-1α expression and function have previously been described in models of Huntington and Alzheimer diseases. Moreover, the protective effects of PGC-1α have been shown in animal models of ALS. Levels of PGC-1α correlate with the number of acetylcholine receptor clusters in muscle. This is of particular interest because neurodegeneration in ALS may be a dying-back process. We investigated mRNA and protein expressions of PGC-1α and PGC-1α-regulated factors in the spinal cord and muscle tissues of SOD1 ALS mice and in ALS patients. We detected significant alterations in mRNA expression of PGC-1α and downstream factors with their earliest occurrence in muscle tissue. Our data provide evidence for a role of PGC-1α in mitochondrial dysfunction both in the ALS mouse model and in human sporadic ALS that is probably most relevant in the skeletal muscle.

Impaired mitochondrial biogenesis contributes to mitochondrial dysfunction in Alzheimer's disease.

Mitochondrial dysfunction is a prominent feature of Alzheimer's disease (AD) brain. Our prior studies demonstrated reduced mitochondrial number in susceptible hippocampal neurons in the brain from AD patients and in M17 cells over-expressing familial AD-causing amyloid precursor protein (APP) mutant (APPswe). In the current study, we investigated whether alterations in mitochondrial biogenesis contribute to mitochondrial abnormalities in AD. Mitochondrial biogenesis is regulated by the peroxisome proliferator activator receptor gamma-coactivator 1α (PGC-1α)-nuclear respiratory factor (NRF)-mitochondrial transcription factor A pathway. Expression levels of PGC-1α, NRF 1, NRF 2, and mitochondrial transcription factor A were significantly decreased in both AD hippocampal tissues and APPswe M17 cells, suggesting a reduced mitochondrial biogenesis. Indeed, APPswe M17 cells demonstrated decreased mitochondrial DNA/nuclear DNA ratio, correlated with reduced ATP content, and decreased cytochrome C oxidase activity. Importantly, over-expression of PGC-1α could completely rescue while knockdown of PGC-1α could exacerbate impaired mitochondrial biogenesis and mitochondrial deficits in APPswe M17 cells, suggesting reduced mitochondrial biogenesis is likely involved in APPswe-induced mitochondrial deficits. We further demonstrated that reduced expression of p-CREB and PGC-1α in APPswe M17 cells could be rescued by cAMP in a dose-dependent manner, which could be inhibited by PKA inhibitor H89, suggesting that the PKA/CREB pathway plays a critical role in the regulation of PGC-1α expression in APPswe M17 cells. Overall, this study demonstrated that impaired mitochondrial biogenesis likely contributes to mitochondrial dysfunction in AD.

Hydroxytyrosol promotes mitochondrial biogenesis and mitochondrial function in 3T3-L1 adipocytes.

Hydroxytyrosol (HT) in extra-virgin olive oil is considered one of the most important polyphenolic compounds responsible for the health benefits of the Mediterranean diet for lowering incidence of cardiovascular disease, the most common and most serious complication of diabetes. We propose that HT may prevent these diseases by a stimulation of mitochondrial biogenesis that leads to enhancement of mitochondrial function and cellular defense systems. In the present study, we investigated effects of HT that stimulate mitochondrial biogenesis and promote mitochondrial function in 3T3-L1 adipocytes. HT over the concentration range of 0.1-10 micromol/L stimulated the promoter transcriptional activation and protein expression of peroxisome proliferator-activated receptor (PPAR) coactivator 1 alpha (PPARGC1 alpha, the central factor for mitochondrial biogenesis) and its downstream targets; these included nuclear respiration factors 1 and 2 and mitochondrial transcription factor A, which leads to an increase in mitochondrial DNA (mtDNA) and in the number of mitochondria. Knockdown of Ppargc1 alpha by siRNA blocked HT's stimulating effect on Complex I expression and mtDNA copy number. The HT treatment resulted in an enhancement of mitochondrial function, including an increase in activity and protein expression of Mitochondrial Complexes I, II, III and V; increased oxygen consumption; and a decrease in free fatty acid contents in the adipocytes. The mechanistic study of the PPARGC1 alpha activation signaling pathway demonstrated that HT is an activator of 5'AMP-activated protein kinase and also up-regulates gene expression of PPAR alpha, CPT-1 and PPAR gamma. These data suggest that HT is able to promote mitochondrial function by stimulating mitochondrial biogenesis.

Pyrroloquinoline quinone stimulates mitochondrial biogenesis through cAMP response element-binding protein phosphorylation and increased PGC-1alpha expression.

Bioactive compounds reported to stimulate mitochondrial biogenesis are linked to many health benefits such increased longevity, improved energy utilization, and protection from reactive oxygen species. Previously studies have shown that mice and rats fed diets lacking in pyrroloquinoline quinone (PQQ) have reduced mitochondrial content. Therefore, we hypothesized that PQQ can induce mitochondrial biogenesis in mouse hepatocytes. Exposure of mouse Hepa1-6 cells to 10-30 microm PQQ for 24-48 h resulted in increased citrate synthase and cytochrome c oxidase activity, Mitotracker staining, mitochondrial DNA content, and cellular oxygen respiration. The induction of this process occurred through the activation of cAMP response element-binding protein (CREB) and peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha), a pathway known to regulate mitochondrial biogenesis. PQQ exposure stimulated phosphorylation of CREB at serine 133, activated the promoter of PGC-1alpha, and increased PGC-1alpha mRNA and protein expression. PQQ did not stimulate mitochondrial biogenesis after small interfering RNA-mediated reduction in either PGC-1alpha or CREB expression. Consistent with activation of the PGC-1alpha pathway, PQQ increased nuclear respiratory factor activation (NRF-1 and NRF-2) and Tfam, TFB1M, and TFB2M mRNA expression. Moreover, PQQ protected cells from mitochondrial inhibition by rotenone, 3-nitropropionic acid, antimycin A, and sodium azide. The ability of PQQ to stimulate mitochondrial biogenesis accounts in part for action of this compound and suggests that PQQ may be beneficial in diseases associated with mitochondrial dysfunction.

Thioredoxin1 upregulates mitochondrial proteins related to oxidative phosphorylation and TCA cycle in the heart.

Thioredoxin1 (Trx1) inhibits hypertrophy and exhibits protective functions in the heart. To elucidate further the cardiac functions of Trx1, we used a DNA microarray analysis, with hearts from transgenic mice with cardiac- specific overexpression of Trx1 (Tg-Trx1, n = 4) and nontransgenic controls (n = 4). Expression of a large number of genes is regulated in Tg-Trx1, with a greater number of genes downregulated, versus upregulated, at high-fold changes. The peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1gamma) gene was among the top 50 significantly upregulated genes. By pathway analyses, we found that genes involved in both mitochondrial oxidative phosphorylation and the TCA cycle were upregulated in Tg-Trx1. We confirmed upregulation of cytochrome c oxidase (COX) components and mitochondrial transcription factor A in Tg-Trx1. The activity of citrate synthase and COX and the cardiac ATP content were significantly higher in Tg-Trx1. A transcription factor binding-site analysis showed that upregulated genes frequently contained binding sites for nuclear respiratory factor 1 (NRF1). Expression of NRF1 and PGC-1gamma was upregulated in Tg-Trx1, and Trx1 stimulated the transcriptional activity of NRF1 and NRF2 in cardiac myocytes. These results suggest that, in cardiac myocytes, Trx1 upregulates mitochondrial proteins and enhances mitochondrial functions, possibly through PGC-1alpha and NRFs.

Control of mitochondrial gene expression in the aging rat myocardium.

Aging induces complex changes in myocardium bioenergetic and contractile properties. Using F344BNF(1) rats, we examined age-dependent changes in myocardial bioenergetic enzymes (catalytic activities and transcript levels) and mRNA levels of putative transcriptional regulators of bioenergetic genes. Very old rats (35 months) showed a 22% increase in ventricular mass with no changes in DNA or RNA per gram. Age-dependent cardiac hypertrophy was accompanied by complex changes in mitochondrial enzymes. Enzymes of the Krebs cycle and electron transport system remained within 15% of the values measured in adult heart, significant decreases occurring in citrate synthase (10%) and aconitase (15%). Transcripts for these enzymes were largely unaffected by aging, although mRNA levels of putative transcriptional regulators of the enzymes (nuclear respiratory factor (NRF) 1 and 2 alpha subunit) increased by about 30%-50%. In contrast, enzymes of fatty acid oxidation exhibited a more diverse pattern, with a 50% decrease in beta-hydroxyacyl-CoA dehydrogenase (HOAD) and no change in long-chain acyl-CoA dehydrogenase or carnitine palmitoyltransferase. Transcript levels for fatty acid oxidizing enzymes covaried with HOAD, which declined significantly by 30%. There were no significant changes in the relative transcript levels of regulators of genes for fatty acid oxidizing enzymes: peroxisome proliferator-activated receptor-alpha (PPARalpha), PPARbeta, or PPARgamma coactivator-1alpha (PGC-1alpha). There were no changes in the mRNA levels of Sirt1, a histone-modifying enzyme that interacts with PGC-1alpha. Collectively, these data suggest that aging causes complex changes in the enzymes of myocardial energy metabolism, triggered in part by NRF-independent pathways as well as post-transcriptional regulation.

Nuclear respiratory factor 2 senses changing cellular energy demands and its silencing down-regulates cytochrome oxidase and other target gene mRNAs.

Cytochrome c oxidase (COX), the terminal enzyme of the electron transport chain, is a bigenomic enzyme with 13 subunits. The mechanism coordinating the transcription of these subunits is poorly understood. We investigated the role of nuclear respiratory factor-2 (NRF-2) in intragenomic regulation of nuclear COX genes. Vector-mediated short-hairpin RNA interference against NRF-2alpha reduced all 10 COX nuclear subunit mRNAs and mRNAs of other genes involved in mitochondrial function/biogenesis. NRF-2 binding site was necessary for the rat COX 4i1 promoter to down-regulate in response to decreased energy demands in primary neurons. Over-expression of NRF-2 protein prevented the down-regulation of transcriptional activity by TTX. Finally, NRF-2 binding sites in isolation were sufficient for modulating COX subunit 4i1 and 6A1 promoters' activity in response to decreased energy demand. These results indicate that NRF-2 is a vital part of a molecular mechanism that senses upstream energy signals and modulates COX transcriptional levels in mammalian cells.