Mitochondrial Metabolic Pathways Research Articles

Succinylation Links Metabolism to Protein Functions.

Post-translational modifications (PTMs) are important regulators of protein function, and integrate metabolism with physiological and pathological processes. Phosphorylation and acetylation are particularly well studied PTMs. A relatively recently discovered novel PTM is succinylation in which metabolically derived succinyl CoA modifies protein lysine groups. Succinylation causes a protein charge flip from positive to negative and a relatively large increase in mass compared to other PTMs. Hundreds of protein succinylation sites are present in proteins of multiple tissues and species, and the significance is being actively investigated. The few completed studies demonstrate that succinylation alters rates of enzymes and pathways, especially mitochondrial metabolic pathways. Thus, succinylation provides an elegant and efficient mechanism to coordinate metabolism and signaling by utilizing metabolic intermediates as sensors to regulate metabolism. Even though the brain is one of the most metabolically active organs, an understanding of the role succinylation in the nervous system is largely unknown. Data from other tissues and other PTMs suggest that succinylation provides a coupling between metabolism and protein function in the nervous system and in neurological diseases. This review provides a new insight into metabolism in neurological diseases and suggests that the drug development for these diseases requires a better understanding of succinylation and de-succinylation in the brain and other tissues.

The Role of Iron in Friedreich’s Ataxia: Insights From Studies in Human Tissues and Cellular and Animal Models

Friedreich’s ataxia (FRDA) is a rare early-onset degenerative disease that affects both the central and peripheral nervous systems, and other extraneural tissues, mainly the heart and endocrine pancreas. This disorder progresses as a mixed sensory and cerebellar ataxia, primarily disturbing the proprioceptive pathways in the spinal cord, peripheral nerves and nuclei of the cerebellum. FRDA is an inherited disease with an autosomal recessive pattern caused by an insufficient amount of the nuclear-encoded mitochondrial protein frataxin, which is an essential and highly evolutionary conserved protein whose deficit results in iron metabolism dysregulation and mitochondrial dysfunction. The first experimental evidence connecting frataxin with iron homeostasis came from Saccharomyces cerevisiae; iron accumulates in the mitochondria of yeast with deletion of the frataxin ortholog gene. This finding was soon linked to previous observations of iron deposits in the hearts of FRDA patients and was later reported in animal models of the disease. Despite advances made in the understanding of FRDA pathophysiology, the role of iron in this disease has not yet been completely clarified. Some of the questions still unresolved include the molecular mechanisms responsible for the iron accumulation and iron-mediated toxicity. Here, we review the contribution of the cellular and animal models of FRDA and relevance of the studies using FRDA patient samples to gain knowledge about these issues. Mechanisms of mitochondrial iron overload are discussed considering the potential roles of frataxin in the major mitochondrial metabolic pathways that use iron. We also analyzed the effect of iron toxicity on neuronal degeneration in FRDA by reactive oxygen species (ROS)-dependent and ROS-independent mechanisms. Finally, therapeutic strategies based on the control of iron toxicity are considered.

Chronic Inhibition of Mitochondrial Dihydrolipoamide Dehydrogenase (DLDH) as an Approach to Managing Diabetic Oxidative Stress.

Mitochondrial dihydrolipoamide dehydrogenase (DLDH) is a redox enzyme involved in decarboxylation of pyruvate to form acetyl-CoA during the cascade of glucose metabolism and mitochondrial adenine triphosphate (ATP) production. Depending on physiological or pathophysiological conditions, DLDH can either enhance or attenuate the production of reactive oxygen species (ROS) and reactive nitrogen species. Recent research in our laboratory has demonstrated that inhibition of DLDH induced antioxidative responses and could serve as a protective approach against oxidative stress in stroke injury. In this perspective article, we postulated that chronic inhibition of DLDH could also attenuate oxidative stress in type 2 diabetes. We discussed DLDH-involving mitochondrial metabolic pathways and metabolic intermediates that could accumulate upon DLDH inhibition and their corresponding roles in abrogating oxidative stress in diabetes. We also discussed a couple of DLDH inhibitors that could be tested in animal models of type 2 diabetes. It is our belief that DLDH inhibition could be a novel approach to fighting type 2 diabetes.

MiRNA-378a as a key regulator of cardiovascular health following engineered nanomaterial inhalation exposure

Nano-titanium dioxide (nano-TiO2), though one of the most utilized and produced engineered nanomaterials (ENMs), diminishes cardiovascular function through dysregulation of metabolism and mitochondrial bioenergetics following inhalation exposure. The molecular mechanisms governing this cardiac dysfunction remain largely unknown. The purpose of this study was to elucidate molecular mediators that connect nano-TiO2 exposure with impaired cardiac function. Specifically, we were interested in the role of microRNA (miRNA) expression in the resulting dysfunction. Not only are miRNA global regulators of gene expression, but also miRNA-based therapeutics provide a realistic treatment modality. Wild type and MiRNA-378a knockout mice were exposed to nano-TiO2 with an aerodynamic diameter of 182 ± 1.70 nm and a mass concentration of 11.09 mg/m3 for 4 h. Cardiac function, utilizing the Vevo 2100 Imaging System, electron transport chain complex activities, and mitochondrial respiration assessed cardiac and mitochondrial function. Immunoblotting and qPCR examined molecular targets of miRNA-378a. MiRNA-378a-3p expression was increased 48 h post inhalation exposure to nano-TiO2. Knockout of miRNA-378a preserved cardiac function following exposure as revealed by preserved E/A ratio and E/SR ratio. In knockout animals, complex I, III, and IV activities (∼2- to 6-fold) and fatty acid respiration (∼5-fold) were significantly increased. MiRNA-378a regulated proteins involved in mitochondrial fusion, transcription, and fatty acid metabolism. MiRNA-378a-3p acts as a negative regulator of mitochondrial metabolic and biogenesis pathways. MiRNA-378a knockout animals provide a protective effect against nano-TiO2 inhalation exposure by altering mitochondrial structure and function. This is the first study to manipulate a miRNA to attenuate the effects of ENM exposure.

Enterococcus faecalis sir2-like gene enhances aerobic metabolism of themselves and mitochondrial respiration of mammal cells to bring about improving metabolic syndrome through the PGC-1α pathway.

Recent studies showed that probiotics could improve metabolic syndrome, making the identification of factors affecting metabolic control more important than ever. The mammalian sirtuin protein family has received much attention for its regulatory role, especially in various mitochondrial ATP, glucose, and lipid metabolic pathways. However, compared with the mammalian sirtuin protein family, the function of prokaryotic sir2 protein is much less known. We studied the effects of probiotics sir2 protein on cell energy metabolize pathway, which showed that deletion of Enterococcus faecalis sir2 inhibited the aerobic oxidation of bacteria and increased the bacterial fermentation. The study of EF-sir2 (sir2 protein of E.faecalis) role of molecular targets demonstrated that deacetylation of EF-sir2 was via Rho upregulating in E.faecalis. When transfected into HEK293T cells, EF-sir2 could significantly facilitate aerobic oxidation of glucose, enhance the respiration to generate more ATP, and cause upregulation of NRF1 target gene. Then, we found EF-sir2 could increase activity of PGC-1α by deacetylation and PGC-1α inhibition decreased the expression of NRF1 target gene. Finally, we demonstrated that EF-sir2 could significantly improve the metabolic index of mammalian cells through insulin resistanced model in vitro and metabolic syndrome rat model in vivo. Our results first revealed that prokaryotic sir2 genes affect the molecular mechanism of cellular metabolism and the regulatory of cell homeostasis in prokaryotic and mammalian cells, suggesting that EF-sir2 has a positive regulatory effect on metabolic disturbance and may be used for the prevention and treatment of pathological processes related to metabolic syndrome.

Mitochondrial function modulates touch signalling in Arabidopsis thaliana.

Plants respond to short- and long-term mechanical stimuli, via altered transcript abundance and growth respectively. Jasmonate, gibberellic acid and calcium have been implicated in mediating responses to mechanical stimuli. Previously it has been shown that the transcript abundance for the outer mitochondrial membrane protein of 66 kDa (OM66), is induced several fold after 30min in response to touch. Therefore, the effect of mitochondrial function on the response to mechanical stimulation by touch at 30 min was investigated. Twenty-five mutants targeting mitochondrial function or regulators revealed that all affected the touch transcriptome. Double and triple mutants revealed synergistic or antagonistic effects following the observed responses in the single mutants. Changes in the touch-responsive transcriptome were localised, recurring with repeated rounds of stimulus. The gene expression kinetics after repeated touch were complex, displaying five distinct patterns. These transcriptomic responses were altered by some regulators of mitochondrial retrograde signalling, such as cyclic dependent protein kinase E1, a kinase protein in the mediator complex, and KIN10 (SnRK1 - sucrose non-fermenting related protein kinase 1), revealing an overlap between the touch response and mitochondrial stress signalling and alternative mitochondrial metabolic pathways. Regulatory network analyses revealed touch-induced stress responses and suppressed growth and biosynthetic processes. Interaction with the phytohormone signalling pathways indicated that ethylene and gibberellic acid had the greatest effect. Hormone measurements revealed that mutations of genes that encoded mitochondrial proteins altered hormone concentrations. Mitochondrial function modulates touch-induced changes in gene expression directly through altered regulatory networks, and indirectly via altering hormonal levels.

Quantifying Competition among Mitochondrial Protein Acylation Events Induced by Ethanol Metabolism.

Mitochondrial dysfunction is one of many key factors in the etiology of alcoholic liver disease (ALD). Lysine acetylation is known to regulate numerous mitochondrial metabolic pathways, and recent reports demonstrate that alcohol-induced protein acylation negatively impacts these processes. To identify regulatory mechanisms attributed to alcohol-induced protein post-translational modifications, we employed a model of alcohol consumption within the context of wild type (WT), sirtuin 3 knockout (SIRT3 KO), and sirtuin 5 knockout (SIRT5 KO) mice to manipulate hepatic mitochondrial protein acylation. Mitochondrial fractions were examined by label-free quantitative HPLC-MS/MS to reveal competition between lysine acetylation and succinylation. A class of proteins defined as "differential acyl switching proteins" demonstrate select sensitivity to alcohol-induced protein acylation. A number of these proteins reveal saturated lysine-site occupancy, suggesting a significant level of differential stoichiometry in the setting of ethanol consumption. We hypothesize that ethanol downregulates numerous mitochondrial metabolic pathways through differential acyl switching proteins. Data are available via ProteomeXchange with identifier PXD012089.

Biclustering Analysis of Co-regulation Patterns in Nuclear-Encoded Mitochondrial Genes and Metabolic Pathways.

Transcription of a large set of nuclear-encoded genes underlies biogenesis of mitochondria, regulated by a complex network of transcription factors and co-regulators. A remarkable heterogeneity can be detected in the expression of these genes in different cell types and tissues, and the recent availability of large gene expression compendiums allows the quantification of specific mitochondrial biogenesis patterns. We have developed a method to effectively perform this task. Massively correlated biclustering (MCbiclust) is a novel bioinformatics method that has been successfully applied to identify co-regulation patterns in large genesets, underlying essential cellular functions and determining cell types. The method has been recently evaluated and made available as a package in Bioconductor for R. One of the potential applications of the method is to compare expression of nuclear-encoded mitochondrial genes or larger sets of metabolism-related genes between different cell types or cellular metabolic states. Here we describe the essential steps to use MCbiclust as a tool to investigate co-regulation of mitochondrial genes and metabolic pathways.

CD4+ T Cell Fate in Multiple Myeloma

The average incidence of multiple myeloma (MM) is in the 7th decade that coincides with the development of immunosenescence and thymic atrophy, meaning that lymphocyte recovery after lymphopenia-inducing therapies (most notably autologous stem cell transplant, ASCT) is largely reliant on homeostatic proliferation of peripheral T cells rather than replenishing the T cell pool with new thymic emigrants. We have previously shown that there is a significant reduction in circulating naïve T cells with a reciprocal expansion of antigen-experienced cells from newly diagnosed MM (NDMM) to relapsed/refractory disease (RRMM). This results in a reduced TCR repertoire and the accumulation of senescence-associated secretory phenotype cytotoxic T cells, which maintain the ability to produce IFNγ but lose proliferative potential. A reduction in CD4:8 ratio is also a characteristic finding in MM with disease progression, which can be explained by high IL-15 levels in lymphopenic states that preferentially drive expansion of CD8+ memory T cells.We wanted to further evaluate what changes were occurring in the CD4+ T cell population with disease progression in MM. We analyzed paired peripheral blood (PB) samples from patients with NDMM and RRMM, and compared with age-matched normal donors (ND). In the NDMM cohort, we examined T cells from PB samples at baseline, after 4 cycles of lenalidomide and dexamethasone (len/dex), and after ASCT; and in the RRMM cohort samples from baseline and after 6 cycles of len/dex.We firstly confirmed in flow cytometric analysis of T cells at serial intervals in NDMM patients that the reduction in circulating naïve T cells and in CD4:8 ratio occurs post ASCT and does not recover by time of last follow-up.We next utilised RNA-seq to analyse differences in CD4+ T cells from NDMM, RRMM and ND. CD4+ T cells from RRMM showed downregulation of cytosolic ribosomal activity but maintenance of mitochondrial ribosomal activity and significant upregulation of pathways involved with calcium signalling. To this end, we evaluated mitochondrial biogenesis and metabolic pathways involved with mitochondrial respiration. Flow cytometric analysis of mitochondrial mass showed a marked increase in RRMM compared with ND, in keeping with a shift towards memory phenotype. Key rate-limiting enzymes in fatty acid β-oxidation (CPT1-A, ACAA2 and ACADVL) were all significantly increased in RRMM compared with ND.To analyse whether these cells were metabolically active, we also measured mitochondrial membrane potential and reactive oxygen species (ROS), gating on cells with high mitochondrial mass. Mitochondrial membrane potential was significantly increased in RRMM compared with ND, although ROS was reduced. The significance of this is not clear, as ROS are not only implicated in cell senescence and activation-induced cell death, but are also positively involved in tyrosine kinase and PI3K-signalling pathways.PD-1 has been shown to play a role in transitioning activated CD4+ T cells from glycolysis to FAO metabolism, and elevating ROS in activated CD8+ T cells. We analysed PD-1 expression on T cells in RRMM and at treatment intervals in NDMM (as described earlier). The proportion of CD4+ and CD8+ T cells expressing PD-1 was increased 4-6 months post-ASCT and remained elevated in CD4+ T cells 9-12 months post-ASCT, but normalised to baseline levels in CD8+ T cells. Increased PD-1 expressing CD4+ T cells was also evident in RRMM patient samples. This may suggest that in the lymphopenic state, PD-1 expression enhances longevity in a subset of CD4+ T cells by promoting reliance on mitochondrial respiration; however, their ability to undergo homeostatic proliferation is impaired. In CD8+ T cells, high PD-1 expression may lead to cell death via ROS accumulation, and these cells do not persist.ASCT remains a backbone of myeloma treatment in medically fit patients. However, this leads to significant permanent defects in the T cell repertoire, which may have unintended adverse outcomes. Additionally, T cells post-ASCT may not be metabolically adequate for the production of CAR-T cells, nor respond to checkpoint blockade therapies. DisclosuresQuach:Amgen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Sanofi Genzyme: Research Funding; Janssen Cilag: Consultancy. Harrison:Janssen-Cilag: Other: Scientific advisory board. Prince:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Integrated Pharmacodynamic Analysis Identifies Two Metabolic Adaption Pathways to Metformin in Breast Cancer

SummaryLate-phase clinical trials investigating metformin as a cancer therapy are underway. However, there remains controversy as to the mode of action of metformin in tumors at clinical doses. We conducted a clinical study integrating measurement of markers of systemic metabolism, dynamic FDG-PET-CT, transcriptomics, and metabolomics at paired time points to profile the bioactivity of metformin in primary breast cancer. We show metformin reduces the levels of mitochondrial metabolites, activates multiple mitochondrial metabolic pathways, and increases 18-FDG flux in tumors. Two tumor groups are identified with distinct metabolic responses, an OXPHOS transcriptional response (OTR) group for which there is an increase in OXPHOS gene transcription and an FDG response group with increased 18-FDG uptake. Increase in proliferation, as measured by a validated proliferation signature, suggested that patients in the OTR group were resistant to metformin treatment. We conclude that mitochondrial response to metformin in primary breast cancer may define anti-tumor effect.

Role of the malonyl-CoA synthetase ACSF3 in mitochondrial metabolism.

Malonyl-CoA is a central metabolite in fatty acid biochemistry. It is the rate-determining intermediate in fatty acid synthesis but is also an allosteric inhibitor of the rate-setting step in mitochondrial long-chain fatty acid oxidation. While these canonical cytoplasmic roles of malonyl-CoA have been well described, malonyl-CoA can also be generated within the mitochondrial matrix by an alternative pathway: the ATP-dependent ligation of malonate to Coenzyme A by the malonyl-CoA synthetase ACSF3. Malonate, a competitive inhibitor of succinate dehydrogenase of the TCA cycle, is a potent inhibitor of mitochondrial respiration. A major role for ACSF3 is to provide a metabolic pathway for the clearance of malonate by the generation of malonyl-CoA, which can then be decarboxylated to acetyl-CoA by malonyl-CoA decarboxylase. Additionally, ACSF3-derived malonyl-CoA can be used to malonylate lysine residues on proteins within the matrix of mitochondria, possibly adding another regulatory layer to post-translational control of mitochondrial metabolism. The discovery of ACSF3-mediated generation of malonyl-CoA defines a new mitochondrial metabolic pathway and raises new questions about how the metabolic fates of this multifunctional metabolite intersect with mitochondrial metabolism.

Matcha green tea (MGT) inhibits the propagation of cancer stem cells (CSCs), by targeting mitochondrial metabolism, glycolysis and multiple cell signalling pathways.

Matcha green tea (MGT) is a natural product that is currently used as a dietary supplement and may have significant anti-cancer properties. However, the molecular mechanism(s) underpinning its potential health benefits remain largely unknown. Here, we used MCF7 cells (an ER(+) human breast cancer cell line) as a model system, to systematically dissect the effects of MGT at the cellular level, via i) metabolic phenotyping and ii) unbiased proteomics analysis. Our results indicate that MGT is indeed sufficient to inhibit the propagation of breast cancer stem cells (CSCs), with an IC-50 of ~0.2 mg/ml, in tissue culture. Interestingly, metabolic phenotyping revealed that treatment with MGT is sufficient to suppress both oxidative mitochondrial metabolism (OXPHOS) and glycolytic flux, shifting cancer cells towards a more quiescent metabolic state. Unbiased label-free proteomics analysis identified the specific mitochondrial proteins and glycolytic enzymes that were down-regulated by MGT treatment. Moreover, to discover the underlying signalling pathways involved in this metabolic shift, we subjected our proteomics data sets to bio-informatics interrogation via Ingenuity Pathway Analysis (IPA) software. Our results indicate that MGT strongly affected mTOR signalling, specifically down-regulating many components of the 40S ribosome. This raises the intriguing possibility that MGT can be used as inhibitor of mTOR, instead of chemical compounds, such as rapamycin. In addition, other key pathways were affected, including the anti-oxidant response, cell cycle regulation, as well as interleukin signalling. Our results are consistent with the idea that MGT may have significant therapeutic potential, by mediating the metabolic reprogramming of cancer cells.

Abstract 232: NFkB1 Pathway is a Major Dysregulated Biological Pathway in Cardiac Myocytes in Myocyte-Specific Lamin A/C Deficient Mice

Lamin A/C (LMNA) is a major component of the nuclear membrane, involved in regulation of gene expression. Mutations in the LMNA gene cause a heterogeneous group of diseases, collectively referred to as laminopathies. Cardiac involvement is one of the most prominent feature of the laminopathies, presenting with dilated cardiomyopathy (DCM), conduction defects, and sudden death. Molecular mechanisms involved in the pathogenesis of cardiac phenotypes in laminopathies are not well known. To gain insights into molecular pathogenesis of DCM caused by LMNA deficiency, Lmna gene was deleted in cardiac myocytes ( Myh6-Cre:Lmna Flox ). Homozygous ( Myh6-Cre:Lmna F/F ) had a normal cardiac function and histology at 2 weeks of age. However, the mice showed progressive cardiac dilatation and dysfunction, interstitial fibrosis, increased apoptosis, and died by 4 weeks of age. To identify transcriptomic changes that precede cardiac dysfunction, cardiomyocytes were isolated from 2-week old wild type (WT) and Myh6-Cre:Lmna F/F mice and analyzed by RNA-Seq. A total of 1,420 genes were differentially expressed between WT and Myh6-Cre:Lmna F/F . Pathways analysis of differentially expressed genes (DEGs) showed enrichment of inflammatory, epithelial to mesenchymal transition, and cell death pathways, whereas mitochondrial oxidative function, adipogenesis, and fatty acid metabolism pathways were depressed in Myh6-Cre:Lmna F/F . Consistently, DEGs were enriched for the binding motif of RELA, the main transcription factor of the NFκB1 pathway. Activation of the NFκB1 pathway was validated by western blot showing increased nuclear localization of RELA. To determine therapeutic benefits of targeting the NFκB1 pathway, mice are being treated a specific inhibitor of NFκB (IKK-16). Effects on survival, cardiac function, apoptosis, and fibrosis are being analyzed. In conclusion, LMNA deficiency results in dysregulation of a large number genes, including NFκB1 pathway target genes that precede the onset of cardiac dysfunction and fibrosis. The findings point to the prominent role of inflammation in the development of cardiac dysfunction in myocyte-specific LMNA deficiency and identify the NFκB1 pathway as a therapeutic target in DCM caused by LMNA deficiency.

Abstract 418: Perm1 is a Novel Regulator of Mitochondrial Energetics in Cardiomyocytes

Mitochondrial oxidative metabolism and energy transduction pathways are critical for cardiac function. The expression of genes important for mitochondrial energetics is under the control of members of the peroxisome proliferator-activated receptor coactivator 1 (PGC-1) family and the estrogen-related receptor (ERR) subfamily. Perturbations in PGC-1 and/or ERR activities have been associated with impaired mitochondrial function and the development of heart failure. The mechanisms by which PGC-1 and ERR proteins regulate muscle specific transcriptional programs is not fully understood. P GC-1 and E RR-induced r egulator in m uscle 1 (Perm1) was recently identified in skeletal muscle as a muscle-specific key regulator of mitochondrial energetics. Although overexpression of Perm1 enhances oxidative capacity and fatigue resistance in mouse skeletal muscle, the underlying mechanisms remain to be elucidated. Perm1 is highly expressed in mouse and human hearts, and its expression level was reduced in pressure overload-induced heart failure in mice (49.9 ± 12.1% of sham, n=4, p<0.05) and in patients with heart failure (45.5 ± 12.1% of donors, n=5, p<0.05). Moreover, Perm1 is expressed in both the nucleus and the cytoplasm in primary cultured neonatal rat ventricular myocytes (NRVMs) and phenylephrine-induced cellular hypertrophy led to downregulation of Perm1 in both fractions (56.7 ± 1.7% of control). RNAseq followed by bioinformatic analysis revealed that siRNA-mediated Perm1 knockdown (KD) in NRVMs led to global downregulation of ERRα/PGC1α-target genes involved in oxidative phosphorylation, including the electron transport chain and TCA cycle ( Ndufs8; Ndufb8; Cox5a; Cox6a1; Coq2; Cox5a; Cox8a; Coq10b; Sdhb; Sdhd) . Basal respiration and ATP production rates were significantly reduced in Perm1-KD H9c2 rat ventricular myocyte-derived cells (60.4 ± 4.2% and 35.2 ± 8.8% of control cells that were transfected with scrambled-siRNA, both p<0.05). This study demonstrates a novel role of Perm1 in cardiomyocytes that provides an additional layer of mitochondrial energetics regulation, presumably in an ERRα- and PGC1α-mediated manner, and suggests that downregulation of Perm1 may contribute to mitochondrial impairment during heart failure.

Cerebrospinal fluid monoamines, pterins, and folate in patients with mitochondrial diseases: systematic review and hospital experience.

Mitochondrial diseases are a group of genetic disorders leading to the dysfunction of mitochondrial energy metabolism pathways. We aimed to assess the clinical phenotype and the biochemical cerebrospinal fluid (CSF) biogenic amine profiles of patients with different diagnoses of genetic mitochondrial diseases. We recruited 29 patients with genetically confirmed mitochondrial diseases harboring mutations in either nuclear or mitochondrial DNA (mtDNA) genes. Signs and symptoms of impaired neurotransmission and neuroradiological data were recorded. CSF monoamines, pterins, and 5-methyltetrahydrofolate (5MTHF) concentrations were analyzed using high-performance liquid chromatography with electrochemical and fluorescence detection procedures. The mtDNA mutations were studied by Sanger sequencing, Southern blot, and real-time PCR, and nuclear DNA was assessed either by Sanger or next-generation sequencing. Five out of 29 cases showed predominant dopaminergic signs not attributable to basal ganglia involvement, harboring mutations in different nuclear genes. A chi-square test showed a statistically significant association between high homovanillic acid (HVA) values and low CSF 5-MTHF values (chi-square = 10.916; p = 0.001). Seven out of the eight patients with high CSF HVA values showed cerebral folate deficiency. Five of them harbored mtDNA deletions associated with Kearns-Sayre syndrome (KSS), one had a mitochondrial point mutation at the mtDNA ATPase6 gene, and one had a POLG mutation. In conclusion, dopamine deficiency clinical signs were present in some patients with mitochondrial diseases with different genetic backgrounds. High CSF HVA values, together with a severe cerebral folate deficiency, were observed in KSS patients and in other mtDNA mutation syndromes.

Abstract 799: The novel histone deacetylase inhibitor pracinostat is an effective anti-lymphoma agent

Abstract Background. Currently, 3 histone deacetylase inhibitors (HDACi) are clinically approved for the treatment of T-cell lymphomas: vorinostat, romidepsin and belinostat. Pracinostat (SB939) is a class I, II and IV HDACi (Novotny-Diermayr et al, Mol Cancer Ther 2010), in phase 3 in combination with azacitidine for acute myeloid leukemia patients who are unfit to receive intensive remission induction chemotherapy (NCT03151408). Here, we tested it on a large panel of lymphoma cell lines alone or in combination with a BCL2 inhibitor, venetoclax and with 5-azacitidine. Methods. 60 cell lines derived from T-cell lymphoma (10), diffuse large B cell lymphoma (DLBCL, 25), mantle cell lymphoma (10), marginal zone lymphoma (5), Hodgkin lymphoma (4), chronic lymphocytic leukemia (2), primary mediastinal B-cell lymphoma (1), murine (2) and canine (1) lymphomas were exposed to increasing doses of pracinostat and vorinostat alone or to pracinostat in combination with venetoclax or 5-azacitidine for 72h. Cell proliferation was assessed using the MTT assay. Chou-Talalay index was used to determine synergism. For apoptosis and cell cycle analyses, cells were treated with 250 nM pracinostat for 72h then stained with Annexin V and 7-AAD (apoptosis) or fixed then stained with 7-AAD (cell cycle) before flow cytometry. Results. All lymphoma subtypes responded well to pracinostat (median IC50 250 nM; 95%C.I., 171-324 nM). Pracinostat compared favorably with vorinostat (306 nM; 95%C.I., 241-363 nM), and the anti-proliferative activity of the two compounds was correlated (R=0.8, P<0.0001). Comparison of consensus cluster classification DLBCL subgroups showed that BCR DLBCL were more sensitive to HDACi than OxPhos DLBCL (pracinostat median IC50 BCR: 171 nM, OxPhos: 865 nM, P 0.001; vorinostat median IC50 BCR: 246 nM, OxPhos: 829 nM, P 0.01. No differences were observed between the DLBCL subtypes based on the cell of origin. Pracinostat mostly provoked cell cycle arrest in the S and G2/M phases with S-phase arrest associated with OxPhos and G2/M arrest with BCR DLBCL. Pracinostat caused pronounced apoptosis in 2/8 DLBCL cell lines. Functional annotation analysis to compare DLBCL with IC50 values < 200 nM (n=12) and DLBCL with IC50 > 400 nM (n=7) revealed an enrichment of mitochondrial metabolic pathways, DNA repair and cell cycle regulation in DLBCL with IC50 >400 nM. Combination with venetoclax was beneficial in 3/3 cell lines, while the addition of 5-azacitidine benefited 2 out of 3 cell lines. Conclusions. Pracinostat robustly inhibits the proliferation of lymphoma cells with a similar IC50 range to vorinostat. Its anti-proliferative activity can be cytotoxic or cytostatic. Combination of pracinostat with other compounds further inhibits lymphoma cell proliferation. In DLBCL, the OxPhos phenotype and an enrichment of mitochondrial metabolism, DNA repair and cell cycle pathways are associated with a poorer response to pracinostat. Citation Format: Afua A. Mensah, Filippo Spriano, Eugenio Gaudio, Chiara Tarantelli, Luciano Cascione, Luca Aresu, Emanuela Lovati, Emanuele Zucca, Anastasios Stathis, Claudio Pietra, Francesco Bertoni. The novel histone deacetylase inhibitor pracinostat is an effective anti-lymphoma agent [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 799.

Zuo Gui Wan Alters Expression of Energy Metabolism Genes and Prevents Cell Death in High-Glucose Loaded Mouse Embryos.

Background Zuo Gui Wan (ZGW) is a classic formula in traditional chinese medicine (TCM). Previous studies have shown that it is beneficial for impaired glucose tolerance (IGT) of adults and the offspring as well. This study aimed to understand the molecular mechanisms of the efficacy of ZGW on IGT. Methods We used high-glucose loaded 2-cell stage mouse embryos as a model and took advantage of single-cell RNA sequencing technology to analyze the transcriptome of the model with or without ZGW. Differential gene expression analysis was performed with DESeq2. Results High glucose can downregulate genes in the ribosome pathway, while ZGW can reverse this inhibition and as a result prevent embryo cell death caused by high glucose. Furthermore, high glucose can affect sugar metabolism and influence mitochondrial function, but ZGW can promote sugar metabolism via the tricarboxylic acid cycle mainly through upregulating the genes in the respiratory chain and oxidative phosphorylation. Conclusions ZGW had a protective effect on embryonic cell death caused by glucose loading. The reversion of inhibition of ribosome pathway and regulation of mitochondrial energy metabolism are main effects of ZGW on high-glucose loaded embryos. This research not only revealed the global gene regulation changes of high glucose affecting 2-cell stage embryos but also provided insight into the potential molecular mechanisms of ZGW on the IGT model.

Panax ginseng Polysaccharide Protected H9c2 Cardiomyocyte From Hypoxia/Reoxygenation Injury Through Regulating Mitochondrial Metabolism and RISK Pathway.

Background and Objective: Ischemic heart disease (IHD) has been the major issue of public health. Panax ginseng (ginseng) has been verified as an effective traditional Chinese medicines and exerted cardioprotective effect. This study aimed to investigate the polysaccharide fraction of ginseng on hypoxia/reoxygenation (H/R) injury in cardiomyocytes and the underlying mechanisms.Methods: Ginseng was extracted by ethanol and fractionated by high-speed counter current chromatography (HSCCC) and column separation. The cardioprotective effect was evaluated in H9c2 cardiomyocytes underwent H/R treatment. The cell viability, apoptosis and mitochondrial respiration were examined.Results: An acid polysaccharides fraction of ginseng (AP1) was identified the most effective fraction in protecting cardiomyocytes from H/R injury. AP1 restored the mitochondrial function by maintaining mitochondrial membrane potential (MMP), blocking the release of cytochrome C, and increasing the ATP generation and oxygen consumption rate (OCR) of cardiomyocytes. Meanwhile, AP1 induced the expression of glucocorticoid receptor (GR) and estrogen receptor (ER) which further activated reperfusion injury salvage kinase (RISK) pathway. Finally, AP1 increased nitric oxide (NO) production and regulated endothelial function by increasing endothelial NO synthase (eNOS) expression and decreasing inducible NOS (iNOS) expression in H/R injury.Conclusion: The results suggested that AP1 exerted a protective effect in myocardial H/R injury mainly through maintaining myocardial mitochondrial function, thereby inhibiting myocardial H/R caused apoptosis and increasing the expressions of GR and ER, which in turn mediated the activation of RISK pathway and eNOS-dependent mechanism to resist the reperfusion injury.

A Sensitive Near-Infrared Fluorescent Sensor for Mitochondrial Hydrogen Sulfide.

Hydrogen sulfide (H2S) is an important gasotransmitter. Although a large number of fluorescent probes for cellular H2S have been reported, only a few can detect H2S in mitochondria, a cellular organelle connecting H2S with mitochondrial function and metabolic pathways. We hereby describe a novel near-infrared fluorescent probe, nimazide, by introducing sulfonyl azide to the core structure of a QSY-21 dark quencher. Nimazide responded quickly to H2S, resulting in robust fluorescence turn-off changes. This conversion displayed high specificity and fast kinetics. More impressively, we observed a robust fluorescence decrease in live cells loaded with mitochondrial nimazide in response to extracellular addition of nanomolar H2S, and successfully imaged biologically generated mitochondrial H2S in live mammalian cells. Nimazide is one of the most sensitive fluorescent probes for mitochondrial H2S.

H1/H2 histamine receptor blockade lowers substrate‐dependent mitochondrial H2O2 emission in deep gastrocnemius muscle following a bout of prolonged exercise

Prolonged exercise increases oxidative metabolism in skeletal muscle mitochondria, which is facilitated, in part, by increased blood flow and delivery of nutrients and oxygen. Histamine mediates many acute exercise responses, including sustained vasodilation and associated delivery of substrates to muscle. We tested the hypothesis that H1 and H2 histamine receptor blockade would attenuate exercise‐induced changes in skeletal muscle mitochondrial function. Adult male Wistar rats (75–94 days old; 441.2 ± 39.5 g) were randomly assigned to one of three groups (n = 12/group): exercise, in the form of one hour of continuous treadmill running at 20 cm/sec (Group 1); mass‐specific oral gavage with H1 antagonist fexofenadine (7.11 mg/kg) and H2 antagonist ranitidine (3.95 mg/kg) one hour prior to the same exercise protocol (Group 2); control, which received neither antihistamines nor exercise (Group 3). An hour post exercise, mitochondrial function was assessed in saponin‐permeabilized myofibers from deep gastrocnemius (mixed fiber types) and soleus (oxidative) skeletal muscle. A sequential substrate‐inhibitor titration protocol was used to examine multiple mitochondrial metabolic pathways (i.e., malate + octanoyl‐carnitine, ADP, lactate + NAD+, pyruvate, glutamate, succinate, inhibitors of antioxidant defense glutathione peroxidase and thioredoxin). Substrate‐dependent oxygen consumption (JO2; high‐resolution respirometry) was not different between treatments; however, H1 and H2 histamine receptor blockade significantly decreased hydrogen peroxide emission (mH2O2; fluorometric monitoring of Amplex Red oxidation) in deep gastrocnemius fibres (P < .05). Given the widespread and routine use of antihistamines, further research into the effects of these antagonists on mitochondrial function, in the context of exercise, is warranted.Support or Funding InformationThe Natural Sciences and Engineering Research Council of Canada (NSERC), The Nova Scotia Health Research Foundation (NSHRF), and Canada Foundation for Innovation (CFI)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.