Acute inhibition of OGA sex-dependently alters the networks associated with bioenergetics, autophagy, and neurodegeneration.

The accumulation of neurotoxic proteins characteristic of age-related neurodegenerative pathologies such as Alzheimer's and Parkinson's diseases is associated with the perturbation of metabolism, bioenergetics, and mitochondrial quality control. One approach to exploit these interactions therapeutically is to target the pathways that regulate metabolism. In this respect, the nutrient-sensing hexosamine biosynthesis pathway is of particular interest since it introduces a protein post-translational modification known as O-GlcNAcylation, which modifies different proteins in control versus neurodegenerative disease postmortem brains. A potent inhibitor of the O-GlcNAcase enzyme that removes the modification from proteins, Thiamet G (TG), has been proposed to have potential benefits in Alzheimer's disease. We tested whether key factors in the O-GlcNAcylation are correlated with mitochondrial electron transport and proteins related to the autophagy/lysosomal pathways in the cortex of male and female mice with and without exposure to TG (10 mg/kg i.p.). Mitochondrial complex activities were measured in the protein homogenates, and a panel of metabolic, autophagy/lysosomal proteins and O-GlcNAcylation enzymes were assessed by either enzyme activity assay or by western blot analysis. We found that the networks associated with O-GlcNAcylation enzymes and activities with mitochondrial parameters, autophagy-related proteins as well as neurodegenerative disease-related proteins exhibited sex and TG dependent differences. Taken together, these studies provide a framework of interconnectivity for multiple O-GlcNAc-dependent pathways in mouse brain of relevance to aging and sex/age-dependent neurodegenerative pathogenesis and response to potential therapies.

Bioenergetic maladaptation and release of HMGB1 in calcineurin inhibitor-mediated nephrotoxicity.

Calcineurin inhibitors (CNIs) are potent immunosuppressive agents, universally used following solid organ transplantation to prevent rejection. Although effective, the long-term use of CNIs is associated with nephrotoxicity. The etiology of this adverse effect is complex, and effective therapeutic interventions remain to be determined. Using a combination of in vitro techniques and a mouse model of CNI-mediated nephrotoxicity, we found that the CNIs, cyclosporine A (CsA), and tacrolimus (TAC) share a similar mechanism of tubular epithelial kidney cell injury, including mitochondrial dysfunction and release of High-Mobility Group Box I (HMGB1). CNIs promote bioenergetic reprogramming due to mitochondrial dysfunction and a shift toward glycolytic metabolism. These events were accompanied by diminished cell-to-cell adhesion, loss of the epithelial cell phenotype, and release of HMGB1. Notably, Erk1/2 inhibitors effectively diminished HMGB1 release, and similar inhibitor was observed on inclusion of pan-caspase inhibitor zVAD-FMK. In vivo, while CNIs activate tissue proremodeling signaling pathways, MAPK/Erk1/2 inhibitor prevented nephrotoxicity, including diminished HMGB1 release from kidney epithelial cells and accumulation in urine. In summary, HMGB1 is an early indicator and marker of progressive nephrotoxicity induced by CNIs. We suggest that proremodeling signaling pathway and loss of mitochondrial redox/bioenergetics homeostasis are crucial therapeutic targets to ameliorate CNI-mediated nephrotoxicity.

A precision medicine approach to defining the impact of doxorubicin on the bioenergetic-metabolite interactome in human platelets.

Non-invasive measures of the response of individual patients to cancer therapeutics is an emerging strategy in precision medicine. Platelets offer a potential dynamic marker for metabolism and bioenergetic responses in individual patients since they have active glycolysis and mitochondrial oxidative phosphorylation and can be easily isolated from a small blood sample. We have recently shown how the bioenergetic-metabolite interactome can be defined in platelets isolated from human subjects by measuring metabolites and bioenergetics in the same sample. In the present study, we used a model system to assess test the hypothesis that this interactome is modified by xenobiotics using exposure to the anti-cancer drug doxorubicin (Dox) in individual donors. We found that unsupervised analysis of the metabolome showed clear differentiation between the control and Dox treated group. Dox treatment resulted in a concentration-dependent decrease in bioenergetic parameters with maximal respiration being most sensitive and this was associated with significant changes in over 166 features. A metabolome-wide association study of Dox was also conducted, and Dox was found to have associations with metabolites in the glycolytic and TCA cycle pathways. Lastly, network analysis showed the impact of Dox on the bioenergetic-metabolite interactome and revealed profound changes in the regulation of reserve capacity. Taken together, these data support the conclusion that platelets are a suitable platform to predict and monitor therapeutic efficacy as well as anticipate susceptibility to toxicity in the context of precision medicine.

Regulation of autophagy, mitochondrial dynamics, and cellular bioenergetics by 4-hydroxynonenal in primary neurons.

The production of reactive species contributes to the age-dependent accumulation of dysfunctional mitochondria and protein aggregates, all of which are associated with neurodegeneration. A putative mediator of these effects is the lipid peroxidation product 4-hydroxynonenal (4-HNE), which has been shown to inhibit mitochondrial function, and accumulate in the postmortem brains of patients with neurodegenerative diseases. This deterioration in mitochondrial quality could be due to direct effects on mitochondrial proteins, or through perturbation of the macroautophagy/autophagy pathway, which plays an essential role in removing damaged mitochondria. Here, we use a click chemistry-based approach to demonstrate that alkyne-4-HNE can adduct to specific mitochondrial and autophagy-related proteins. Furthermore, we found that at lower concentrations (5-10 µM), 4-HNE activates autophagy, whereas at higher concentrations (15 µM), autophagic flux is inhibited, correlating with the modification of key autophagy proteins at higher concentrations of alkyne-4-HNE. Increasing concentrations of 4-HNE also cause mitochondrial dysfunction by targeting complex V (the ATP synthase) in the electron transport chain, and induce significant changes in mitochondrial fission and fusion protein levels, which results in alterations to mitochondrial network length. Finally, inhibition of autophagy initiation using 3-methyladenine (3MA) also results in a significant decrease in mitochondrial function and network length. These data show that both the mitochondria and autophagy are critical targets of 4-HNE, and that the proteins targeted by 4-HNE may change based on its concentration, persistently driving cellular dysfunction.

Inhibition of autophagy with bafilomycin and chloroquine decreases mitochondrial quality and bioenergetic function in primary neurons.

Autophagy is an important cell recycling program responsible for the clearance of damaged or long-lived proteins and organelles. Pharmacological modulators of this pathway have been extensively utilized in a wide range of basic research and pre-clinical studies. Bafilomycin A1 and chloroquine are commonly used compounds that inhibit autophagy by targeting the lysosomes but through distinct mechanisms. Since it is now clear that mitochondrial quality control, particularly in neurons, is dependent on autophagy, it is important to determine whether these compounds modify cellular bioenergetics. To address this, we cultured primary rat cortical neurons from E18 embryos and used the Seahorse XF96 analyzer and a targeted metabolomics approach to measure the effects of bafilomycin A1 and chloroquine on bioenergetics and metabolism. We found that both bafilomycin and chloroquine could significantly increase the autophagosome marker LC3-II and inhibit key parameters of mitochondrial function, and increase mtDNA damage. Furthermore, we observed significant alterations in TCA cycle intermediates, particularly those downstream of citrate synthase and those linked to glutaminolysis. Taken together, these data demonstrate a significant impact of bafilomycin and chloroquine on cellular bioenergetics and metabolism consistent with decreased mitochondrial quality associated with inhibition of autophagy.

The Bioenergetic Health Index: a new concept in mitochondrial translational research.

Bioenergetics has become central to our understanding of pathological mechanisms, the development of new therapeutic strategies and as a biomarker for disease progression in neurodegeneration, diabetes, cancer and cardiovascular disease. A key concept is that the mitochondrion can act as the 'canary in the coal mine' by serving as an early warning of bioenergetic crisis in patient populations. We propose that new clinical tests to monitor changes in bioenergetics in patient populations are needed to take advantage of the early and sensitive ability of bioenergetics to determine severity and progression in complex and multifactorial diseases. With the recent development of high-throughput assays to measure cellular energetic function in the small number of cells that can be isolated from human blood these clinical tests are now feasible. We have shown that the sequential addition of well-characterized inhibitors of oxidative phosphorylation allows a bioenergetic profile to be measured in cells isolated from normal or pathological samples. From these data we propose that a single value-the Bioenergetic Health Index (BHI)-can be calculated to represent the patient's composite mitochondrial profile for a selected cell type. In the present Hypothesis paper, we discuss how BHI could serve as a dynamic index of bioenergetic health and how it can be measured in platelets and leucocytes. We propose that, ultimately, BHI has the potential to be a new biomarker for assessing patient health with both prognostic and diagnostic value.

Inhibition of autophagy and glycolysis by nitric oxide during hypoxia-reoxygenation impairs cellular bioenergetics and promotes cell death in primary neurons.

Excessive nitric oxide (NO) production is known to damage mitochondrial proteins and the autophagy repair pathway and so can potentially contribute to neurotoxicity. Accordingly, we hypothesized that protection against protein damage from reactive oxygen and nitrogen species under conditions of low oxygen by the autophagy pathway in neurons would be impaired by NO and enhance bioenergetic dysfunction. Rat primary cortical neurons had the same basal cellular respiration in hypoxia as in normoxia, whereas NO-exposed cells exhibited a gradual decrease in mitochondrial respiration in hypoxia. Upon reoxygenation, the respiration in NO-treated cells did not recover to prehypoxic levels. Hypoxia-reoxygenation in the presence of NO was associated with inhibition of autophagy, and the inability to recover during reoxygenation was exacerbated by an inhibitor of autophagy, 3-methyladenine. The effects of hypoxia could be recapitulated by inhibiting glycolytic flux under normoxic conditions. Under both normoxic and hypoxic conditions NO exposure induced immediate stimulation of glycolysis, but prolonged NO exposure, associated with irreversible inhibition of mitochondrial respiration in hypoxia, inhibited glycolysis. Importantly, we found that NO inhibited basal respiration under normoxic conditions only when glucose was absent from the medium or glycolysis was inhibited by 2-deoxy-d-glucose, revealing a novel NO-dependent mechanism for the inhibition of mitochondrial respiration that is modulated by glycolysis. Taken together these data suggest an oxygen-dependent interaction between mitochondrial respiration, glycolysis, and autophagy in protecting neuronal cells exposed to NO. Importantly, they indicate that mitochondrial dysfunction is intimately linked to a failure of glycolytic flux induced by exposure to NO. In addition, these studies provide new insights into the understanding of how autophagy and NO may play interactive roles in neuroinflammation-induced cellular damage, which is pertinent to our understanding of the pathology of neurodegenerative diseases in which excessive NO is generated.

Bioenergetic and autophagic control by Sirt3 in response to nutrient deprivation in mouse embryonic fibroblasts.

Sirt3 (sirtuin 3) is an NAD-dependent deacetylase localized to mitochondria. Sirt3 expression is increased in mouse muscle and liver by starvation, which could protect against the starvation-dependent increase in oxidative stress and protein damage. Damaged proteins and organelles depend on autophagy for removal and this is critical for cell survival, but the role of Sirt3 is unclear. To examine this, we used Sirt3-KO (knockout) mouse embryonic fibroblast cells, and found that, under basal conditions, Sirt3-KO cells exhibited increased autophagy flux compared with WT (wild-type) cells. In response to nutrient deprivation, both WT and KO cells exhibited increased basal and ATP-linked mitochondrial respiration, indicating an increased energy demand. Both cells exhibited lower levels of phosphorylated mTOR (mammalian target of rapamycin) and higher autophagy flux, with KO cells exhibiting lower maximal mitochondrial respiration and reserve capacity, and higher levels of autophagy than WT cells. KO cells exhibit higher phospho-JNK (c-Jun N-terminal kinase) and phospho-c-Jun than WT cells under starvation conditions. However, inhibition of JNK activity in Sirt3-KO cells did not affect LC3-I (light chain 3-I) and LC3-II levels, indicating that Sirt3-regulated autophagy is independent of the JNK pathway. Caspase 3 activation and cell death are significantly higher in Sirt3-KO cells compared with WT cells in response to nutrient deprivation. Inhibition of autophagy by chloroquine exacerbated cell death in both WT and Sirt3-KO cells, and by 3-methyadenine exacerbated cell death in Sirt3-KO cells. These data suggest that nutrient deprivation-induced autophagy plays a protective role in cell survival, and Sirt3 decreases the requirement for enhanced autophagy and improves cellular bioenergetics.

Mitochondrially targeted compounds and their impact on cellular bioenergetics.

Mitochondria are recognized as critical sites of localized injury in a number of chronic pathologies which has led to the development of organelle directed therapeutics. One of the approaches employed to target molecules to the mitochondrion is to conjugate a delocalized cation such as triphenylphosphonium (TPP+) to various redox active compounds. Mitochondrially targeted antioxidants have also been used in numerous cell culture based studies as probes of the contribution of the mitochondrial generation of reactive oxygen species on cell signaling events. However, concentrations used in vitro are typically 10-100 times greater than those generated from oral dosing in a wide range of animal models and in humans. In the present study, we determined the effects of mitochondrial targeted antioxidants, MitoQ, MitoTempol, and MitoE on cellular bioenergetics of mesangial cells in culture and compared these to TPP+ conjugated compounds which lack the antioxidant functional group. We found that all TPP+ compounds inhibited oxidative phosphorylation to different extents independent of the antioxidant functional groups. These findings show that the TPP+ moiety can disrupt mitochondrial function at concentrations frequently observed in cell culture and this behavior is dependent on the linker group and independent of antioxidant properties. Moreover, TPP+ moiety alone is unlikely to achieve the concentrations needed to contribute to the protective mechanisms of the mitochondrially targeted compounds that have been reported in vivo.

Role of cellular bioenergetics in smooth muscle cell proliferation induced by platelet-derived growth factor.

Abnormal smooth muscle cell proliferation is a hallmark of vascular disease. Although growth factors are known to contribute to cell hyperplasia, the changes in metabolism associated with this response, particularly mitochondrial respiration, remain unclear. Given the increased energy requirements for proliferation, we hypothesized that PDGF (platelet-derived growth factor) would stimulate glycolysis and mitochondrial respiration and that this elevated bioenergetic capacity is required for smooth muscle cell hyperplasia. To test this hypothesis, cell proliferation, glycolytic flux and mitochondrial oxygen consumption were measured after treatment of primary rat aortic VSMCs (vascular smooth muscle cells) with PDGF. PDGF increased basal and maximal rates of glycolytic flux and mitochondrial oxygen consumption; enhancement of these bioenergetic pathways led to a substantial increase in the mitochondrial reserve capacity. Interventions with the PI3K (phosphoinositide 3-kinase) inhibitor LY-294002 or the glycolysis inhibitor 2-deoxy-D-glucose abrogated PDGF-stimulated proliferation and prevented augmentation of glycolysis and mitochondrial reserve capacity. Similarly, when L-glucose was substituted for D-glucose, PDGF-dependent proliferation was abolished, as were changes in glycolysis and mitochondrial respiration. Interestingly, LDH (lactate dehydrogenase) protein levels and activity were significantly increased after PDGF treatment. Moreover, substitution of L-lactate for D-glucose was sufficient to increase mitochondrial reserve capacity and cell proliferation after treatment with PDGF; these effects were inhibited by the LDH inhibitor oxamate. These results suggest that glycolysis, by providing substrates that enhance the mitochondrial reserve capacity, plays an essential role in PDGF-induced cell proliferation, underscoring the integrated metabolic response required for proliferation of VSMCs in the diseased vasculature.

Hydrogen sulfide mediates the vasoactivity of garlic.

The consumption of garlic is inversely correlated with the progression of cardiovascular disease, although the responsible mechanisms remain unclear. Here we show that human RBCs convert garlic-derived organic polysulfides into hydrogen sulfide (H(2)S), an endogenous cardioprotective vascular cell signaling molecule. This H(2)S production, measured in real time by a novel polarographic H(2)S sensor, is supported by glucose-maintained cytosolic glutathione levels and is to a large extent reliant on reduced thiols in or on the RBC membrane. H(2)S production from organic polysulfides is facilitated by allyl substituents and by increasing numbers of tethering sulfur atoms. Allyl-substituted polysulfides undergo nucleophilic substitution at the alpha carbon of the allyl substituent, thereby forming a hydropolysulfide (RS(n)H), a key intermediate during the formation of H(2)S. Organic polysulfides (R-S(n)-R'; n > 2) also undergo nucleophilic substitution at a sulfur atom, yielding RS(n)H and H(2)S. Intact aorta rings, under physiologically relevant oxygen levels, also metabolize garlic-derived organic polysulfides to liberate H(2)S. The vasoactivity of garlic compounds is synchronous with H(2)S production, and their potency to mediate relaxation increases with H(2)S yield, strongly supporting our hypothesis that H(2)S mediates the vasoactivity of garlic. Our results also suggest that the capacity to produce H(2)S can be used to standardize garlic dietary supplements.

Inhibition of NF-kappaB-mediated transcription and induction of apoptosis by melampolides and repandolides.

PURPOSE: Nuclear factor-kappaB (NF-kappaB) plays a crucial role in the regulation of inflammatory processes, cell proliferation, and apoptosis. Blocking NF-kappaB signaling may represent a therapeutic strategy in cancer and inflammation therapy. The aim of this study was to investigate the effects of sesquiterpenes isolated from Asteraceae, namely melampolides (enhydrin, tetraludin A) and repandolides (repandins A, B, D and E) on the activation of NF-kappaB, cell growth of cancer cells, cell cycle progression and apoptosis. In addition, their effects on the activity of cyclooxygenase-2 (COX-2) enzyme were also evaluated. METHODS: Cell-based reporter gene assay was conducted in SW1353 cells. COX-2 enzyme activity and cell growth inhibition was determined by enzyme immunoassay and MTT assay respectively. Cell cycle analysis was carried out by flow cytometry and apoptosis was observed by DAPI staining assay. RESULTS: In SW1353 cells, transcription mediated by NF-kappaB was inhibited by enhydrin, tetraludin A and repandins A, B, D and E, while Sp-1 mediated transcription was not affected. COX-2 enzyme activity was inhibited by enhydrin, repandin A and E, but not by tetraludin A, repandin B and D. These compounds were effective in inhibiting the growth of a panel of human tumor cell lines in a concentration-dependent manner. Cell cycle analysis and DAPI staining indicated cell cycle arrest in G(2)/M phase and induction of apoptosis. CONCLUSIONS: Enhydrin, tetraludin A and repandins A, B, D and E inhibited tumor cell growth and induced cell cycle arrest and apoptosis. These effects may be related to inhibition of NF-B activation.