Genome-wide histone acetylation analysis reveals altered transcriptional regulation in the Parkinson's disease brain.

BACKGROUND: Parkinson's disease (PD) is a complex, age-related neurodegenerative disorder of largely unknown etiology. PD is strongly associated with mitochondrial respiratory dysfunction, which can lead to epigenetic dysregulation and specifically altered histone acetylation. Nevertheless, and despite the emerging role of epigenetics in age-related brain disorders, the question of whether aberrant histone acetylation is involved in PD remains unresolved. METHODS: We studied fresh-frozen brain tissue from two independent cohorts of individuals with idiopathic PD (n = 28) and neurologically healthy controls (n = 21). We performed comprehensive immunoblotting to identify histone sites with altered acetylation levels in PD, followed by chromatin immunoprecipitation sequencing (ChIP-seq). RNA sequencing data from the same individuals was used to assess the impact of altered histone acetylation on gene expression. RESULTS: Immunoblotting analyses revealed increased acetylation at several histone sites in PD, with the most prominent change observed for H3K27, a marker of active promoters and enhancers. ChIP-seq analysis further indicated that H3K27 hyperacetylation in the PD brain is a genome-wide phenomenon with a strong predilection for genes implicated in the disease, including SNCA, PARK7, PRKN and MAPT. Integration of the ChIP-seq with transcriptomic data from the same individuals revealed that the correlation between promoter H3K27 acetylation and gene expression is attenuated in PD patients, suggesting that H3K27 acetylation may be decoupled from transcription in the PD brain. Strikingly, this decoupling was most pronounced among nuclear-encoded mitochondrial genes, corroborating the notion that impaired crosstalk between the nucleus and mitochondria is involved in the pathogenesis of PD. Our findings independently replicated in the two cohorts. CONCLUSIONS: Our findings strongly suggest that aberrant histone acetylation and altered transcriptional regulation are involved in the pathophysiology of PD. We demonstrate that PD-associated genes are particularly prone to epigenetic dysregulation and identify novel epigenetic signatures associated with the disease.

Endurance Exercise-Induced Autophagy/Mitophagy Coincides with a Reinforced Anabolic State and Increased Mitochondrial Turnover in the Cortex of Young Male Mouse Brain.

Autophagy/mitophagy, a cellular catabolic process necessary for sustaining normal cellular function, has emerged as a potential therapeutic strategy against numerous obstinate diseases. In this regard, endurance exercise (EXE)-induced autophagy/mitophagy (EIAM) has been considered as a potential health-enriching factor in various tissues including the brain; however, underlying mechanisms of EIAM in the brain has not been fully defined yet. This study investigated the molecular signaling nexus of EIAM pathways in the cortex of the brain. C57BL/6 young male mice were randomly assigned to a control group (CON, n = 12) and an endurance exercise group (EXE, n = 12). Our data demonstrated that exercise-induced autophagy coincided with an enhanced anabolic state (p-AKT, p-mTOR, and p-p70S6K); furthermore, mitophagy concurred with enhanced mitochondrial turnover: increases in both fission (DRP1, BNIP3, and PINK1) and fusion (OPA1 and MFN2) proteins. In addition, neither oxidative stress nor sirtuins (SIRT) 1 and 3 were associated with EIAM; instead, the activation of AMPK as well as a JNK-BCL2 axis was linked to EIAM promotion. Collectively, our results demonstrated that EXE-induced anabolic enrichment did not hinder autophagy/mitophagy and that the concurrent augmentation of mitochondrial fusion and fusion process contributed to sustaining mitophagy in the cortex of the brain. Our findings suggest that the EXE-induced concomitant potentiation of the catabolic and anabolic state is a unique molecular mechanism that simultaneously contributes to recycling and rebuilding the cellular structure, leading to upholding healthy cellular environment. Thus, the current study provides a novel autophagy/mitophagy mechanism, from which groundbreaking pharmacological strategies of autophagy can be developed.

Effects of perinatal factors on sirtuin 3, 8-hydroxy-2'- deoxyguanosine, brain-derived neurotrophic factor and serotonin in cord blood and early breast milk: an observational study.

BACKGROUND: The profile of sirtuin 3 (SIRT3), 8-hydroxy-2'-deoxyguanosine (8-OHdG), brain-derived neurotrophic factor (BDNF) and serotonin (5-HT) in cord blood and in early breast milk was studied and it was related to perinatal factors. 5-HT and BDNF signalling systems have been claimed to play a critical role in intrauterine development, postnatal adaptation and lactation. Since prematurity and Caesarean birth are frequently associated with inflammation and related oxidative stress, an attempt was made to reveal the adaptive changes of the protective SIRT3 and the complex interplay among these bioactive components in cord blood and early breast milk. METHODS: Three groups each consisting of 30 mothers were included in the study: mothers who underwent spontaneous vaginal birth at term (group I), Caesarean section at term (group II) and preterm birth (group III). Venous cord blood and early breast milk samples were collected for measuring the biomarkers. SIRT3, 8-OHdG, BDNF and 5-HT levels were determined by using commercially available ELISA kits. RESULTS: It was demonstrated that cord blood levels of SIRT3, BDNF and 5-HT were markedly reduced whereas those of 8-OHdG were significantly elevated after preterm birth when compared with birth at term. The Caesarean section was associated with a moderate decrease in BDNF and 5-HT, however, both SIRT3 and 8-OHdG remained unaffected. Breast milk levels of all biomarkers studied proved to be independent of their corresponding cord blood concentrations. In response to preterm birth breast milk SIRT3, 8-OHdG and 5-HT increased significantly, while a drastic fall occurred in BDNF. A significant positive relationship was found of 5-HT with SIRT3 and 8-OHdG irrespective of the gestational age and the mode of delivery. CONCLUSIONS: It is suggested that the selected biomarkers in the breast milk mostly derive from local production by the mammary glands and 5-HT may have an essential role in the control of this process.

Developmental programming: intrauterine caloric restriction promotes upregulation of mitochondrial sirtuin with mild effects on oxidative parameters in the ovaries and testes of offspring.

According to the developmental origins of health and disease (DOHaD) hypothesis, changes in the maternal environment are known to reprogram the metabolic response of offspring. Known for its redox modulation, caloric restriction extends the lifespan of some species, which contributes to diminished cellular damage. Little is known about the effects of gestational caloric restriction, in terms of antioxidant parameters and molecular mechanisms of action, on the reproductive organs of offspring. This study assessed the effects of moderate (20%) caloric restriction on redox status parameters, molecular expression of sirtuin (SIRT) 1 and SIRT3 and histopathological markers in the ovaries and testes of adult rats that were subjected to gestational caloric restriction. Although enzyme activity was increased, ovaries from female pups contained high levels of oxidants, whereas testes from male pups had decreased antioxidant enzyme defences, as evidenced by diminished glyoxalase I activity and reduced glutathione content. Expression of SIRT3, a deacetylase enzyme related to cellular bioenergetics, was increased in both ovaries and testes. Previous studies have suggested that, in ovaries, diminished antioxidant metabolism can lead to premature ovarian failure. Unfortunately, there is little information regarding the redox profile in the testis. This study is the first to assess the redox network in both ovaries and testes, suggesting that, although intrauterine caloric restriction improves molecular mechanisms, it has a negative effect on the antioxidant network and redox status of reproductive organs of young adult rats.

Effect of ApoE isoforms on mitochondria in Alzheimer disease.

OBJECTIVE: To test the hypothesis that ApoE isoforms affect mitochondrial structure and function that are related to cognitive impairment in Alzheimer disease (AD), we systematically investigated the effects of ApoE isoforms on mitochondrial biogenesis and dynamics, oxidative stress, synapses, and cognitive performance in AD. METHODS: We obtained postmortem human brain tissues and measured proteins that are responsible for mitochondrial biogenesis (peroxisome proliferator-activated receptor-gamma coactivator-1α [PGC-1α] and sirtuin 3 [SIRT3]), for mitochondrial dynamics (mitofusin 1 [MFN1], mitofusin 2 [MFN2], and dynamin-like protein 1 [DLP1]), for oxidative stress (superoxide dismutase 2 [SOD2] and forkhead-box protein O3a [Foxo3a]), and for synapses (postsynaptic density protein 95 [PSD95] and synapsin1 [Syn1]). A total of 46 cases were enrolled, including ApoE-ɛ4 carriers (n = 21) and noncarriers (n = 25). RESULTS: Levels of these proteins were compared between ApoE-ɛ4 carriers and noncarriers. ApoE-ɛ4 was associated with impaired mitochondrial structure and function, oxidative stress, and synaptic integrity in the human brain. Correlation analysis revealed that mitochondrial proteins and the synaptic protein were strongly associated with cognitive performance. CONCLUSION: ApoE isoforms influence mitochondrial structure and function, which likely leads to alteration in oxidative stress, synapses, and cognitive function. These mitochondria-related proteins may be a harbinger of cognitive decline in ApoE-ɛ4 carriers and provide novel therapeutic targets for prevention and treatment of AD.

Aging-Related Overactivity of the Angiotensin/AT1 Axis Decreases Sirtuin 3 Levels in the Substantia Nigra, Which Induces Vulnerability to Oxidative Stress and Neurodegeneration.

Sirtuin 3 (SIRT3) and angiotensin play a major role in aging-related disorders. Both modulate oxidative stress and neurodegeneration. We investigated the interaction between SIRT3 and angiotensin II (AngII) in the dopaminergic system. Both in vivo and in vitro, treatment with AngII decreased SIRT3 expression, which was reversed by angiotensin type 1 receptor (AT1) antagonists. Aged animals showed enhanced pro-oxidative RAS activity and low nigral SIRT3 levels, which significantly increased with treatment with the AT1 antagonist candesartan or AT1 deletion. Consistent with this, AT2 knockout mice and cells treated with AT2 blockers showed downregulation of SIRT3. Treatment with the specific SIRT3 inhibitor AGK7 induced overexpression of AT1 and AT2 in substantia nigra (SN) of rats, and in dopaminergic neuronal MES23.5 and microglial N9 cell lines. The results suggest that SIRT3 may initially counteract low levels of oxidative stress as part of the antioxidant response. However, high or persistent oxidative stress induced by overactivation of the angiotensin/AT1 pro-oxidative axis induces a decrease in nigral SIRT3 levels. Furthermore, a decrease in SIRT3 levels further increases AT1 activity, which may lead to a feed-forward mechanism. This is observed in aged rats and can be counteracted by treatment with AT1 antagonists such as candesartan.

Development of Fluorogenic Probes for Rapid High-Contrast Imaging of Transient Nuclear Localization of Sirtuin†3.

Protein labeling using fluorogenic probes enables the facile visualization of proteins of interest. Herein, we report new fluorogenic probes consisting of a rationally designed coumarin ligand for the live-cell fluorogenic labeling of the photoactive yellow protein (PYP)-tag. On the basis of the photochemical mechanisms of coumarin and the probe-tag interactions, we introduced a hydroxy group into an environment-sensitive coumarin ligand to modulate its spectroscopic properties and increase the labeling reaction rate. The resulting probe had a higher labeling reaction rate constant and a greater fluorescence OFF-ON ratio than any previously developed PYP-tag labeling probe. The probe enabled the fluorogenic labeling of intracellular proteins within minutes. Furthermore, we used our probe to investigate the localization of sirtuin 3 (SIRT3), a mitochondrial deacetylase. Although the nuclear localization of SIRT3 has been controversial, this transient nuclear localization was clearly captured by the rapid, high-contrast imaging enabled by our probe.

Myricetin Exerts Anti-Obesity Effects through Upregulation of SIRT3 in Adipose Tissue.

Myricetin is a biologically active natural polyphenol with beneficial effects on metabolic health. This study aimed to examine the effects of myricetin on the expression levels of genes involved in lipolysis and mitochondrial respiration in adipocytes and the anti-obesity potential of myricetin. The results indicated that myricetin reduced triglyceride (TG) content and increased mitochondrial content and oxygen consumption rate (OCR) in adipocytes in vitro. To determine anti-obesity effect of myricetin, C57BL6/J mice were fed a high-fat diet (HFD) for eight weeks and then treated with myricetin (10 mg/kg) for 2 weeks. The in vivo treatment of myricetin reduced body weight by 11%. Furthermore, it improved the glucose tolerance, and increased fatty acid consumption of HFD-fed mice. Myricetin treatment increased Sirt3 expression and reduced the acetylation of mitochondrial proteins in adipose tissue. Finally, the knockdown of Sirt3 in adipocytes reduced the myricetin-induced increase in mitochondrial oxygen consumption rate by about 27% compared to controls. Our results indicated that myricetin exerted anti-obesity effects through the upregulation of Sirt3 expression and mitochondrial metabolism in adipose tissue.

Hepatic Sirt3 expression declines postpartum in dairy goats.

The experiments reported in this research communication aimed to plot the expression pattern of Sirt3, a master regulator of energy metabolism and antioxidation defence, in the liver of dairy goats during perinatal period. Ten healthy dairy goats in late pregnancy were chosen, and needle biopsy was applied to collect liver samples at 1-week intervals. Protein levels of hepatic Sirt3 were analysed by western-blotting. Serum enzyme activities of manganese superoxide dismutase (Mn-SOD) and non-esterified fatty acids (NEFA) levels were measured, and their correlation with Sirt3 mRNA levels was also estimated. Compared with >3-week before parturition (BP), Sirt3 proteins were significantly reduced at 1-week after parturition (AP) and 2-week AP (P < 0·05), but increased on the day of parturition (P < 0·01). Correlation analysis revealed a positive association between hepatic Sirt3 mRNA levels and serum enzyme activity of Mn-SOD (r = 0·46), but a negative association between that and serum NEFA levels (r = -0·41). These data indicate that the decreased hepatic expression of Sirt3 might be one of the reasons that dairy goats undergo oxidative stress after parturition.

Metformin and insulin impact on clinical outcome in patients with advanced hepatocellular carcinoma receiving sorafenib: Validation study and biological rationale.

PURPOSE: In 2015, we published a study on a small series of patients with hepatocellular carcinoma (HCC) treated chronically with metformin for type II diabetes mellitus (DM2) who showed a poorer response to sorafenib. The aim of the present study was to validate the prognostic significance of metformin in HCC patients treated with sorafenib, providing a biological rationale for the mechanism of resistance to sorafenib in patients on chronic metformin therapy, and to clarify the role of sirtuin-3 (SIRT-3), a protein involved in metabolic diseases and acknowledged as a tumour suppressor in HCC, in this resistance. PATIENTS AND METHODS: We analysed 279 patients consecutively treated with sorafenib for the clinical analysis. Of the 86 (30%) patients with DM2, 52 (19%) were on chronic treatment with metformin and 34 (12%) with insulin. We included 43 patients with HCC for the biological study: 19 (44.1%) were diabetic and 14 (73.7%) of these received metformin for DM2. SIRT-3 expression was investigated by immunohistochemistry (IHC) in formalin-fixed and paraffin-embedded (FFPE) samples. RESULTS: In HCC patients undergoing chronic treatment with metformin, the use of sorafenib was associated with poor progression-free survival (PFS) and overall survival (OS) (1.9 and 6.6 months, respectively) compared to 3.7 months and 10.8 months, respectively, for patients without DM2 and 8.4 months and 16.6 months, respectively, for patients on insulin (P < .0001). We also observed that SIRT-3 protein expression was significantly higher in patients treated with metformin than in those not taking this medication (65% versus 25%, respectively) (P = .013). CONCLUSIONS: Our findings could be attributed to increased tumour aggressiveness and resistance to sorafenib caused by chronic treatment with metformin.

The Prognostic and Clinicopathological Roles of Sirtuin-3 in Various Cancers.

Sirtuin-3 (SIRT3) is a major mitochondrial NAD(+)-dependent deacetylase and plays a key role in the progression and development of human cancers. Although the prognostic and clinicopathological features of SIRT3 expression in various cancers have been investigated by different research groups, however, inconsistent and opposing results can be observed. In this study, we therefore performed a meta-analysis to evaluate the significance of SIRT3 expression in various cancers. Systematic literature searching was performed in PubMed, Embase, China National Knowledge Infrastructure, and Wanfang Data up to November 2015. Total effect analyses and subgroup analyses were performed to evaluate the relationship between SIRT3 expression and overall survival, cancer/non-cancer tissues, lymph node metastasis, pathological differentiation, tumor node metastasis (TNM) stage, tumor size, and gender, in various cancer patients. Hazard ratios (HRs) or odds ratios (ORs) with 95% confidence intervals (CIs) were calculated to clarify the risk or hazard association. A total of 14 studies comprising 2165 cancer patients were included to assess the association between SIRT3 immunohistochemical expression and overall survival or clinicopathological characteristics. SIRT3 expression was significantly associated with overall survival in gastric cancer (HR = 0.62, 95% CI = 0.43-0.89, P = 0.009) and hepatocellular carcinoma patients (HR = 0.56, 95% CI = 0.42-0.74, P<0.0001), cancer/non-cancer tissues in hepatocellular carcinoma patients (OR = 0.04, 95% CI = 0.01-0.16, P<0.0001), lymph node metastasis in breast cancer patients (OR = 2.20, 95% CI = 1.49-3.26, P<0.0001), and also pathological differentiation in hepatocellular carcinoma patients (OR = 0.69, 95% CI = 0.48-0.98, P = 0.04) and gastric cancer patients (OR = 0.33, 95% CI = 0.21-0.50, P<0.00001), by subgroup analyses. Furthermore, SIRT3 expression was significantly associated with pathological differentiation in total effect analysis (OR = 0.46, 95% CI = 0.29-0.74, P = 0.001). No detectable relation between SIRT3 expression and other clinicopathological parameters were found. This meta-analysis indicates that SIRT3 expression level is associated with prognostic and clinical features in specific cancers.

Adipose tissue and liver expression of SIRT1, 3, and 6 increase after extensive weight loss in morbid obesity.

BACKGROUND & AIMS: Severe obesity is associated with a state of chronic inflammation. Sirtuins (SIRT) are a family of conserved enzymes which are able to affect many metabolic and inflammatory pathways thereby potentially improving health and increasing lifespan. METHODS: We investigated the effect of weight loss on subcutaneous adipose tissue and liver mRNA and immunohistochemical expression of SIRT1, SIRT3, and SIRT6. Twenty-nine severely obese patients undergoing laparoscopic adjustable gastric banding (LAGB) were studied. Tissue samples were collected before and 6months after LAGB surgery. Tissue mRNA expression levels of SIRT1, SIRT3, and SIRT6 were correlated with clinical, biochemical, and histological parameters. In vitro, we studied sirtuin expression in native and stimulated monocytes, adipocytes, and hepatocytes. RESULTS: SIRT1, SIRT3, and SIRT6 mRNA expression was higher in the subcutaneous adipose tissue than in the liver. Weight loss resulted in a significant induction of SIRT1, SIRT3, and SIRT6 expression in the subcutaneous adipose tissue. In the liver, a significant increase after weight loss was observed, particularly for SIRT3 and SIRT6 mRNA expression; immunohistochemically, SIRT1 and SIRT3 expression was upregulated. Endotoxin and tumor necrosis factor-alpha suppressed SIRT1, SIRT3, and SIRT6 expression in human monocytes. The same stimuli suppressed total sirtuin deacetylase activity again, mainly in monocytes and less in adipocytes and hepatocytes. CONCLUSIONS: The relative abundance of adipose tissue mRNA expression of certain sirtuins exceeds its expression in the liver. Extensive weight loss increases sirtuin expression significantly both in adipose tissue and liver, probably as a consequence of reduced inflammation.

Receptor-interacting protein (RIP) and Sirtuin-3 (SIRT3) are on opposite sides of anoikis and tumorigenesis.

BACKGROUND: Regulating cross-talk between anoikis and survival signaling pathways is crucial to regulating tissue processes and mitigating diseases like cancer. Previously, the authors demonstrated that anoikis activates a signaling pathway involving the CD95/Fas-mediated signaling pathway that is regulated by receptor-interacting protein (RIP), a kinase that shuttles between Fas-mediated cell death and integrin/focal adhesion kinase (FAK)-mediated survival pathways. Because it is known that sirtuin-3 (SIRT3), a nicotinamide adenine dinucleotide-dependent deacetylase, regulates cell survival, metabolism, and tumorigenesis, the authors hypothesized that SIRT3 may engage in cross-talk with Fas/RIP/integrin/FAK survival-death pathways in cancer cell systems. METHODS: Using immunohistochemical staining, immunoblotting, human tissue microarrays, and overexpression and suppression approaches in vitro and in vivo, the roles of RIP and SIRT3 were examined in oral squamous cell carcinoma (OSCC) anoikis resistance and tumorigenesis. RESULTS: RIP and SIRT3 had opposite expression profiles in OSCC cells and tissues. Stable suppression of RIP enhanced SIRT3 levels, whereas stable suppression of SIRT3 did not impact RIP levels in OSCC cells. The authors observed that, as OSCC cells became anoikis-resistant, they formed multicellular aggregates or oraspheres in suspension conditions, and their expression of SIRT3 increased as their RIP expression decreased. Also, anoikis-resistant OSCC cells with higher SIRT3 and low RIP expression induced an increased tumor burden and incidence in mice, unlike their adherent OSCC cell counterparts. Furthermore, stable suppression of SIRT3 inhibited anoikis resistance and reduced tumor incidence. CONCLUSIONS: The current results indicted that RIP is a likely upstream, negative regulator of SIRT3 in anoikis resistance, and an anoikis-resistant orasphere phenotype defined by higher SIRT3 and low RIP expression contributes to a more aggressive phenotype in OSCC development.

SIRT3 and cancer: tumor promoter or suppressor?

Sirtuins (SIRT1-7), the mammalian homologues of the Sir2 gene in yeast, have emerging roles in age-related diseases, such as cardiac hypertrophy, diabetes, obesity, and cancer. However, the role of several sirtuin family members, including SIRT1 and SIRT3, in cancer has been controversial. The aim of this review is to explore and discuss the seemingly dichotomous role of SIRT3 in cancer biology with particular emphasis on its potential role as a tumor promoter and tumor suppressor. This review will also discuss the potential role of SIRT3 as a novel therapeutic target to treat cancer.