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Author(s): Karuppagounder, Saravanan S; Alin, Lauren; Chen, Yingxin; Brand, David; Bourassa, Megan W; Dietrich, Kristen; Wilkinson, Cassandra M; Nadeau, Colby A; Kumar, Amit; Perry, Steve; Pinto, John T; Darley-Usmar, Victor; Sanchez, Stephanie; Milne, Ginger L; Pratico, Domenico; Holman, Theodore R; Carmichael, S Thomas; Coppola, Giovanni; Colbourne, Frederick; Ratan, Rajiv R | Abstract: ObjectivesN-acetylcysteine (NAC) is a clinically approved thiol-containing redox modulatory compound currently in trials for many neurological and psychiatric disorders. Although generically labeled as an "antioxidant," poor understanding of its site(s) of action is a barrier to its use in neurological practice. Here, we examined the efficacy and mechanism of action of NAC in rodent models of hemorrhagic stroke.MethodsHemin was used to model ferroptosis and hemorrhagic stroke in cultured neurons. Striatal infusion of collagenase was used to model intracerebral hemorrhage (ICH) in mice and rats. Chemical biology, targeted lipidomics, arachidonate 5-lipoxygenase (ALOX5) knockout mice, and viral-gene transfer were used to gain insight into the pharmacological targets and mechanism of action of NAC.ResultsNAC prevented hemin-induced ferroptosis by neutralizing toxic lipids generated by arachidonate-dependent ALOX5 activity. NAC efficacy required increases in glutathione and is correlated with suppression of reactive lipids by glutathione-dependent enzymes such as glutathione S-transferase. Accordingly, its protective effects were mimicked by chemical or molecular lipid peroxidation inhibitors. NAC delivered postinjury reduced neuronal death and improved functional recovery at least 7 days following ICH in mice and can synergize with clinically approved prostaglandin E2 (PGE2 ).InterpretationNAC is a promising, protective therapy for ICH, which acted to inhibit toxic arachidonic acid products of nuclear ALOX5 that synergized with exogenously delivered protective PGE2 in vitro and in vivo. The findings provide novel insight into a target for NAC, beyond the generic characterization as an antioxidant, resulting in neuroprotection and offer a feasible combinatorial strategy to optimize efficacy and safety in dosing of NAC for treatment of neurological disorders involving ferroptosis such as ICH. Ann Neurol 2018;84:854-872.

Parkinson's disease (PD) is a movement disorder with widespread neurodegeneration in the brain. Significant oxidative, reductive, metabolic, and proteotoxic alterations have been observed in PD postmortem brains. The alterations of mitochondrial function resulting in decreased bioenergetic health is important and needs to be further examined to help develop biomarkers for PD severity and prognosis. It is now becoming clear that multiple hits on metabolic and signaling pathways are likely to exacerbate PD pathogenesis. Indeed, data obtained from genetic and genome association studies have implicated interactive contributions of genes controlling protein quality control and metabolism. For example, loss of key proteins that are responsible for clearance of dysfunctional mitochondria through a process called mitophagy has been found to cause PD, and a significant proportion of genes associated with PD encode proteins involved in the autophagy-lysosomal pathway. In this review, we highlight the evidence for the targeting of mitochondria by proteotoxic, redox and metabolic stress, and the role autophagic surveillance in maintenance of mitochondrial quality. Furthermore, we summarize the role of α-synuclein, leucine-rich repeat kinase 2, and tau in modulating mitochondrial function and autophagy. Among the stressors that can overwhelm the mitochondrial quality control mechanisms, we will discuss 4-hydroxynonenal and nitric oxide. The impact of autophagy is context depend and as such can have both beneficial and detrimental effects. Furthermore, we highlight the potential of targeting mitochondria and autophagic function as an integrated therapeutic strategy and the emerging contribution of the microbiome to PD susceptibility.

Alcoholic hepatitis occurs in about one third of individuals reporting long term heavy alcohol use. It is associated with high short-term mortality, economic burden, and hospital resources utilization. We performed this systematic review to a) describe clinical characteristics and genomics associated with the risk of alcoholic hepatitis; b) discuss role and limitations of liver biopsy and prognostic scoring systems; c) summarize evidence regarding the currently available therapies including liver transplantation; and d) outline emerging therapies with areas of unmet need. Literature search was performed for studies published in English language (January 1971 through - March 2016). Following search engines were used: Pubmed, Elsevier Embase, PsychINFO, and Cochrane Library. For the treatment section, only randomized controlled studies were included for this review. A total of 138 studies (59 randomized, 22 systematic reviews or meta-analyses, 7 surveys or guidelines, 7 population based, and 43 prospective cohort) were cited. There are over 325,000 annual admissions with alcoholic hepatitis contributing to about 0.8% of all hospitalizations in the US. Liver biopsy may be required in about 25–30% cases for uncertain clinical diagnosis. Corticosteroids with or without N-acetylcysteine remains the only available therapy for severe episode. Data are emerging on the role of liver transplantation as salvage therapy for select patients. Abstinence remains the most important factor impacting long-term prognosis. Results from the ongoing clinical trials within the National institute on Alcohol Abuse and Alcoholism funded consortia are awaited for more effective and safer therapies. Alcoholic hepatitis is a potentially lethal condition with a significant short-term mortality. A high index of suspicion is required. There remains an unmet need for non-invasive biomarkers for diagnosis, and predicting prognosis and response to therapy.

Summary In summary, the studies reported during this session ofthe symposium emphasize the diverse and complex mech-anisms by which ethanol acutely or chronically can affectcell functions involving mitochondria. These functions in-volve cellular energy metabolism, the regulation of Ca 2 homeostasis, and the response to oxidative stress and hyp-oxia. Session Three: Fig. 1 summarizes some of the mech-anisms by which the actions of ethanol on mitochondrialfunction may be mediated. Current evidence suggests thatthese involve a variety of cell signaling mechanisms, includ-ing PKC , nitric oxide radicals and reactive oxygen species,Ca 2 , and probably stress signaling pathways that regulatecell survival. The mitochondrial targets of these signalingpathways include components of the electron transportchain and the energy conservation system, the complexes ofthe mitochondrial permeability transition (in which theBzrp protein is thought to be involved) and, remarkably,ALDH2. As a result of these interactions, ethanol alsoimpacts on the signaling pathways that control mitochon-drial biogenesis and the regulation of cell death, with im-plications for fetal development, cardiac and liver function,and other actions. Much more work will be required tounderstand the detailed mechanisms by which these pro-cesses are interconnected, providing novel perspectives onthe underlying cellular and molecular mechanisms by whichacute and chronic ethanol intake exerts damaging, or some-times protective effects.Brookes et al., 2002 Shiva et al., 2003 Clerk et al., 2003

Xanthine oxidase (XO)-derived superoxide contributes to endothelial dysfunction in humans and animal models of hypercholesterolemia (HC). Since L-arginine supplementation prevents defects in NO signaling, we tested the hypothesis that L-arginine blunts the inhibitory effect of XO on vascular function. Acetylcholine-mediated relaxation was significantly impaired in ring segments of HC rabbits, a response that was associated with an increase in plasma XO activity. L-Arginine treatment of HC rabbits reduced plasma XO and improved endothelial function. L-Arginine also modestly prolonged the lag time for oxidation in isolated lipoprotein samples. These results reveal that the principal action of L-arginine is to protect against the XO-dependent inactivation of NO in arteries of HC rabbits.

Oxidant formation in the vasculature contributes to vascular disease and dysfunction associated with obesity. In contrast, exercise-dependent production of oxidants may stimulate adaptive responses that protect against the development of such diseases. In this review, we discuss current concepts in the biology of reactive oxygen and nitrogen species and how their function is modulated in the context of vascular disease, obesity, and aerobic exercise.

The role of glutathione (GSH) in inflammation is largely discussed from the context of providing reducing equivalents to detoxify reactive oxygen and nitrogen species. Inflammation is now recognized to be an underlying cause of many vascular diseases including atherosclerosis, a disease in which endothelial GSH concentrations are decreased. However, mechanisms that control GSH levels are poorly understood. Key players in the inflammatory process are endothelial adhesion molecules, including intercellular adhesion molecule-1 (ICAM-1). This adhesion molecule is present constitutively and can be induced by a variety of inflammatory stimuli. In this study, using mouse aortic endothelial cells (MAEC) deficient in ICAM-1, we demonstrate a novel interplay between constitutive ICAM-1 and cellular GSH. Deficiency of ICAM-1 was associated with an approximately twofold increase in total GSH content. Inhibiting glutamate-cysteine ligase (GCL), the enzyme that catalyses the rate-limiting step in GSH biosynthesis, prevented the increase in GSH. In addition, the catalytic subunit of GCL was increased (approximately 1.6-fold) in ICAM-1 deficient relative to wild-type cells, suggesting that constitutive ICAM-1 represses GCL expression. Furthermore, the ratio of reduced (GSH) to oxidized (GSSG) glutathione was also increased suggesting a role for ICAM-1 in modulating cellular redox status. Interestingly, increasing cytosolic GSH in wild-type mouse endothelial cells decreased constitutive ICAM-1, suggesting the presence of an inverse and reciprocal pathway. To test the effects of inducible ICAM-1 on GSH, cells were stimulated with the proinflammatory cytokine TNF-alpha. TNF-alpha stimulated production of ICAM-1, which was however not associated with induction of GSH. In contrast, supplementation of endothelial cells with GSH before TNF-alpha addition, inhibited induction of ICAM-1. These data suggest a novel regulatory pathway between constitutive ICAM-1 and GSH synthesis in the endothelium and are discussed in the context of modulating the inflammatory response.

1. Administration of peroxynitrite (ONOO-, 30-300 microM) caused relaxation of rabbit aortic strips superfused in series in a cascade. The compound responsible for this effect had a half-life greater than 20 s and could not therefore be either nitric oxide (NO) or ONOO- which have half-lives in the order of 1-2 s under these conditions. However the relaxation was inhibited by oxyhaemoglobin, suggesting the compound could be converted to NO in the vascular tissues or in the superfusate. 2. The products of the reactions between ONOO- and Krebs buffer containing 11 mM glucose, but not glucose-free Krebs buffer, caused relaxation of the bioassay tissues. These data suggest that stable NO donor(s) were formed from the reaction of ONOO- with glucose. We therefore prepared these NO donor(s) by the reaction of glucose solutions with ONOO- in order to characterize their ability to release NO. 3. These reaction product(s) caused relaxation in the cascade and inhibition of platelet aggregation. Both effects were dependent on the concentration of D-glucose, were equally effective if L-glucose was used as a reactant and were reversed by oxyhaemoglobin. 3. The products of the reaction between ONOO- and glucose or other biological molecules containing an alcohol functional group, such as fructose, glycerol, or glyceraldehyde, released NO in the presence of Cu2+and L-cysteine. 5. These results indicate that ONOO- reacts with sugars or other compounds containing an alcohol functional group(s) to form NO donors with the characteristics of organic nitrate/nitrites. This may represent a further detoxification pathway for ONOO- in vivo.

Incubation of rat skeletal muscle mitochondria with the nitric oxide generator, S-nitrosoglutathione (GSNO) reversibly inhibited oxygen utilisation with all substrates tested. The visible absorption spectra of the inhibited mitochondria showed that cytochromes c+c1, b and a+a3 were reduced, indicating a block at the distal end of the respiratory chain. Analysis of the respiratory chain enzyme activities in the presence of GSNO localised the site of inhibition to cytochrome c oxidase alone. These results indicate that nitric oxide is capable of rapidly and reversibly inhibiting the mitochondrial respiratory chain and may be implicated in the cytotoxic effects of nitric oxide in the CNS and other tissues.

Peroxynitrite may be formed in the vasculature by the reaction of puperoxide with nitric oxide. When the blue copper-containing protein, caeruloplasmin, is incubated with peroxynitrite, copper is released, and ferroxidase activity and the blue colouration are lost. When plasma from normal subjects is incubated with peroxynitrite, the oxidant reacts with numerous plasma constituents but is still able to release copper from caeruloplasmin. As the ferroxidase activity of caeruloplasmin is lost in plasma in the presence of peroxynitrite, a second ferroxidase activity associated with peroxidised lipids, and not inhibited by azide, is formed.

Peroxynitrite is an oxidant which could be formed in the vasculature by the reaction of superoxide with nitric oxide. It is capable of modifying amino acid residues and of initiating lipid peroxidation. In the present study we have shown that peroxynitrite converts low density lipoprotein to a form recognized by the macrophage scavenger receptor and that this process is associated with modification of the protein and lipid, and with the oxidation of alpha-tocopherol to alpha-tocopherol quinone.

The recent upsurge in proteomics research has been facilitated largely by streamlining of two-dimensional (2-D) gel technology and the parallel development of facile mass spectrometry for analysis of peptides and proteins. However, application of these technologies to the mitochondrial proteome has been limited due to the considerable complement of hydrophobic membrane proteins in mitochondria, which precipitate during first dimension isoelectric focusing of standard 2-D gels. In addition, functional information regarding protein:protein interactions is lost during 2-D gel separation due to denaturing conditions in both gel dimensions. To resolve these issues, 2-D blue-native gel electrophoresis was applied to the mitochondrial proteome. In this technique, membrane protein complexes such as those of the respiratory chain are solubilized and resolved in native form in the first dimension. A second dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel then denatures the complexes and resolves them into their component subunits. Refinements to this technique have yielded the levels of throughput and reproducibility required for proteomics. By coupling to tryptic peptide fingerprinting using matrix-assisted laser desorption/ionization-time of flight mass spectrometry, a partial mitochondrial proteome map has been assembled. Applications of this functional mitochondrial proteomics method are discussed.

Since the discovery of nitric oxide (NO) as a vasodilatory messenger, in particular after its identification as an endothelial-derived relaxing factor (EDRF), there has been a mushroom effect in the research of NO in biological systems. Monitoring of NO in biological samples in vivo and in real time is desirable in many fields of NO research. Although several techniques are available for measurement of NO, the electrochemical method is most advantageous because of its speed and sensitivity. Since the first commercially available electrochemical NO detection system, many NO electrodes have been developed with dimensions from μm to cm, and with detection limits in the low nanomolar range. However, there is still a continuing demand for new NO sensors with lower detection limits. An electrochemical sensor to detect nitric oxide gas dissolved in solution as well as in gas phase is described as well as a fabrication method for the electrode. The sensor is based on the selective oxidation of nitric oxide by a multielectrode array of microelectrodes created on activated carbon, which has been deposited on a silicon chip substrate. The array, in turn, is further modified with several layers of cationic ion exchanger then the subsequent addition of NO selective membranes. The microchip NO sensor, is characterized by a linear response to concentrations of NO up to 100 μM, a response time of a few seconds, and a detection limit of less than 0.3 nM. In biological samples the sensor discriminates against substances such as nitrite, dopamine and ascorbic acid. Moreover, compared with present NO sensors, this sensor is significantly less temperature sensitive, resulting in significantly improved sensor detectivity as compared with present electrochemical sensors. Major applications for measurement of NO concentration are in chemical media, biological tissue, cell cultures, or in blood.