Heme oxygenase-1 drives metaflammation and insulin resistance in mouse and man.

Obesity and diabetes affect more than half a billion individuals worldwide. Interestingly, the two conditions do not always coincide and the molecular determinants of "healthy" versus "unhealthy" obesity remain ill-defined. Chronic metabolic inflammation (metaflammation) is believed to be pivotal. Here, we tested a hypothesized anti-inflammatory role for heme oxygenase-1 (HO-1) in the development of metabolic disease. Surprisingly, in matched biopsies from "healthy" versus insulin-resistant obese subjects we find HO-1 to be among the strongest positive predictors of metabolic disease in humans. We find that hepatocyte and macrophage conditional HO-1 deletion in mice evokes resistance to diet-induced insulin resistance and inflammation, dramatically reducing secondary disease such as steatosis and liver toxicity. Intriguingly, cellular assays show that HO-1 defines prestimulation thresholds for inflammatory skewing and NF-κB amplification in macrophages and for insulin signaling in hepatocytes. These findings identify HO-1 inhibition as a potential therapeutic strategy for metabolic disease.

Publisher Correction: The metabolite BH4 controls T cell proliferation in autoimmunity and cancer.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

The metabolite BH4 controls T cell proliferation in autoimmunity and cancer.

Genetic regulators and environmental stimuli modulate T cell activation in autoimmunity and cancer. The enzyme co-factor tetrahydrobiopterin (BH4) is involved in the production of monoamine neurotransmitters, the generation of nitric oxide, and pain1,2. Here we uncover a link between these processes, identifying a fundamental role for BH4 in T cell biology. We find that genetic inactivation of GTP cyclohydrolase 1 (GCH1, the rate-limiting enzyme in the synthesis of BH4) and inhibition of sepiapterin reductase (the terminal enzyme in the synthetic pathway for BH4) severely impair the proliferation of mature mouse and human T cells. BH4 production in activated T cells is linked to alterations in iron metabolism and mitochondrial bioenergetics. In vivo blockade of BH4 synthesis abrogates T-cell-mediated autoimmunity and allergic inflammation, and enhancing BH4 levels through GCH1 overexpression augments responses by CD4- and CD8-expressing T cells, increasing their antitumour activity in vivo. Administration of BH4 to mice markedly reduces tumour growth and expands the population of intratumoral effector T cells. Kynurenine-a tryptophan metabolite that blocks antitumour immunity-inhibits T cell proliferation in a manner that can be rescued by BH4. Finally, we report the development of a potent SPR antagonist for possible clinical use. Our data uncover GCH1, SPR and their downstream metabolite BH4 as critical regulators of T cell biology that can be readily manipulated to either block autoimmunity or enhance anticancer immunity.

Pathological Interplay between Inflammation and Mitochondria Aggravates Glutamate Toxicity.

Mitochondrial dysfunction and glutamate toxicity are associated with neural disorders, including brain trauma. A review of the literature suggests that toxic and transmission actions of neuronal glutamate are spatially and functionally separated. The transmission pathway utilizes synaptic GluN2A receptors, rapidly released pool of glutamate, evoked release of glutamate mediated by Synaptotagmin 1 and the amount of extracellular glutamate regulated by astrocytes. The toxic pathway utilizes extrasynaptic GluN2B receptors and a cytoplasmic pool of glutamate, which results from the spontaneous release of glutamate mediated by Synaptotagmin 7 and the neuronal 2-oxoglutarate dehydrogenase complex (OGDHC), a tricarboxylic acid (TCA) cycle enzyme. Additionally, the inhibition of OGDHC observed upon neuro-inflammation is due to an excessive release of reactive oxygen/nitrogen species by immune cells. The loss of OGDHC inhibits uptake of glutamate by mitochondria, thus facilitating its extracellular accumulation and stimulating toxic glutamate pathway without affecting transmission. High levels of extracellular glutamate lead to dysregulation of intracellular redox homeostasis and cause ferroptosis, excitotoxicity, and mitochondrial dysfunction. The latter affects the transmission pathway demanding high-energy supply and leading to cell death. Mitochondria aggravate glutamate toxicity due to impairments in the TCA cycle and become a victim of glutamate toxicity, which disrupts oxidative phosphorylation. Thus, therapies targeting the TCA cycle in neurological disorders may be more efficient than attempting to preserve mitochondrial oxidative phosphorylation.

Endoplasmic Reticulum Stress Induces Vasodilation in Liver Vessels That Is Not Mediated by Unfolded Protein Response.

There is a growing body of evidence that ER stress and the unfolded protein response (UPR) play a key role in numerous diseases. Impaired liver perfusion and ER stress often accompany each other in liver diseases. However, the exact impact of ER stress and UPR on the hepatic perfusion is not fully understood. The aim of this study was to disclose the effect of ER stress and UPR on the size of liver vessels and on the levels of Ca2+ and nitric oxide (NO), critical regulators of vascular tonus. This study was carried out in precisely cut liver tissue slices. Confocal microscopy was used to create 3D images of vessels. NO levels were determined either using either laser scan microscopy (LSM) in cells or by NO-analyser in medium. Ca2+ levels were analysed by LSM. We show that tunicamycin, an inducer of ER stress, acts similarly with vasodilator acetylcholine. Both exert a similar effect on the NO and Ca2+ levels; both induce significant vasodilation. Notably, this vasodilative effect persisted despite individual inhibition of UPR pathways-ATF-6, PERK, and IRE1-despite confirming the activation of UPR. Experiments with HUVEC cells showed that elevated NO levels did not result from endothelial NO synthase (eNOS) activation. Our study suggests that tunicamycin-mediated ER stress induces liver vessel vasodilation in an NO-dependent manner, which is mediated by intracellular nitrodilator-activatable NO store (NANOS) in smooth muscle cells rather than by eNOS.

Efficacy and safety of nivolumab combined with brentuximab vedotin after nivolumab monotherapy failure in patients with relapsed and refractory classic Hodgkin lymphoma.

OBJECTIVES: Therapy of patients with relapsed and refractory classic Hodgkin lymphoma (r/r cHL) after PD-1 inhibitors failure remains an unresolved issue. The aim of this study was to evaluate the efficacy and safety of the combination of nivolumab with brentuximab vedotin (Nivo + BV) after nivolumab monotherapy failure. METHODS: This study retrospectively analyzed 21 patients with r/r cHL who were treated with the combination of Nivo + BV after Nivo failure. The response was evaluated by PET-CT scan according to the LYRIC criteria. Adverse events (AEs) were assessed according to NCI CTCAE v.4.03. RESULTS: Median follow-up was 19 (9-47) months. The ORR was 57%. The median OS was not reached, 24 month OS was 80% (95% CI 50-93%). Median PFS was 12 months with 24 month PFS of 31% (95% CI 12-53%). Any grade AEs were observed in 12 patients (63%), 3-4 grade AEs in 2 patients (10%). Allogeneic hematopoietic stem cell transplantation (allo-HSCT) after Nivo + BV was performed in 8 (38%) patients. The median time between Nivo + BV and allo-HSCT was 8 (5-21) months. CONCLUSIONS: Combination of Nivo + BV in r/r cHL after nivolumab monotherapy failure is potentially an effective and safe approach.

Nivolumab discontinuation and retreatment in patients with relapsed or refractory Hodgkin lymphoma.

Immune checkpoint inhibitors (ICI) have demonstrated high therapeutic efficacy in relapsed or refractory classical Hodgkin lymphoma (r/r cHL). Nevertheless, despite the accumulated data, the question of the ICI therapy duration and efficacy of nivolumab retreatment remains unresolved. In this retrospective study, in a cohort of 23 adult patients with r/r cHL who discontinued nivolumab in complete response (CR), the possibility of durable remission achievement (2-year PFS was 55.1%) was demonstrated. Retreatment with nivolumab has demonstrated efficacy with high overall response rate (ORR) and CR (67% and 33.3% respectively). At the final analysis, all patients were alive with median PFS of 16.5 months. Grade 3-4 adverse events (AEs) were reported in 36% of patients, and there was no deterioration in terms of nivolumab retreatment-associated complications.

Cerebral nitric oxide and mitochondrial function in patients suffering aneurysmal subarachnoid hemorrhage-a translational approach.

BACKGROUND: Cerebral ischemia and neuroinflammation following aneurysmal subarachnoid hemorrhage (SAH) are major contributors to poor neurological outcome. Our study set out to investigate in an exploratory approach the interaction between NO and energy metabolism following SAH as both hypoxia and inflammation are known to affect nitric oxide (NO) metabolism and NO in turn affects mitochondria. METHODS: In seven patients under continuous multimodality neuromonitoring suffering poor-grade aneurysmal SAH, cerebral metabolism and NO levels (determined as a sum of nitrite plus nitrate) were determined in cerebral microdialysate for 14 days following SAH. In additional ex vivo experiments, rat cortex homogenate was subjected to the NO concentrations determined in SAH patients to test whether these NO concentrations impair mitochondrial function (determined by means of high-resolution respirometry). RESULTS: NO levels showed biphasic kinetics with drastically increased levels during the first 7 days (74.5 ± 29.9 µM) and significantly lower levels thereafter (47.5 ± 18.7 µM; p = 0.02). Only during the first 7 days, NO levels showed a strong negative correlation with brain tissue oxygen tension (r = - 0.78; p < 0.001) and a positive correlation with cerebral lactate (r = 0.79; p < 0.001), pyruvate (r = 0.68; p < 0.001), glutamate (r = 0.65; p < 0.001), as well as the lactate-pyruvate ratio (r = 0.48; p = 0.01), suggesting mitochondrial dysfunction. Ex vivo experiments confirmed that the increase in NO levels determined in patients during the acute phase is sufficient to impair mitochondrial function (p < 0.001). Mitochondrial respiration was inhibited irrespectively of whether glutamate (substrate of complex I) or succinate (substrate of complex II) was used as mitochondrial substrate suggesting the inhibition of mitochondrial complex IV. The latter was confirmed by direct determination of complex IV activity. CONCLUSIONS: Exploratory analysis of our data suggests that during the acute phase of SAH, NO plays a key role in the neuronal damage impairing mitochondrial function and facilitating accumulation of mitochondrial substrate; further studies are required to understand mechanisms underlying this observation.

Crosstalk between inflammatory mediators and endoplasmic reticulum stress in liver diseases.

An excessive inflammatory response is frequently associated with cellular dysfunction and cell death. The latter may cause single and multiple organ failure. The most susceptible organs are liver, lung, kidney, heart and intestine. This review will focus on the liver as a target organ for an excessive inflammatory response. It is commonly accepted that organ failure is caused by the action of inflammatory cytokines released in excess during the inflammatory response. It has been suggested that inflammation mediated liver failure is not due to an increased death rate of parenchymal cells, but due to an intracellular metabolic disorder. This metabolic disorder is associated with mitochondrial and endoplasmic reticulum (ER) dysfunction during the acute phase response elicited by systemic inflammation. An overproduction of acute phase proteins in the liver as well as elevated reactive oxygen species (ROS) generation induce ER stress, triggering the unfolded protein response (UPR), which may initiate or aggravate inflammation. It is known that certain inflammatory mediators, such as the pro-inflammatory cytokines IL-1ß, IL-6 and TNF-α induce ER stress. These findings suggest that ER stress and the subsequent UPR on the one hand, and the inflammatory response on the other create a kind of feed forward loop, which can be either beneficial (e.g., elimination of the pathogen and restoration of tissue homeostasis) or deleterious (e.g., excessive cell dysfunction and cell death). This review aims to unfurl the different pathways contributing to this loop and to highlight the relevance of UPR signaling (IRE1α, ATF6, and PERK) and mediators of the inflammatory response (NF-κB, STAT3, IL-1ß, IL-6, TLR) which have a particular role as pathophysiological triggers in the liver.

Thiamine preserves mitochondrial function in a rat model of traumatic brain injury, preventing inactivation of the 2-oxoglutarate dehydrogenase complex.

BACKGROUND AND PURPOSE: Based on the fact that traumatic brain injury is associated with mitochondrial dysfunction we aimed at localization of mitochondrial defect and attempted to correct it by thiamine. EXPERIMENTAL APPROACH: Interventional controlled experimental animal study was used. Adult male Sprague-Dawley rats were subjected to lateral fluid percussion traumatic brain injury. Thiamine was administered 1 h prior to trauma; cortex was extracted for analysis 4 h and 3 d after trauma. KEY RESULTS: Increased expression of inducible nitric oxide synthase (iNOS) and tumor necrosis factor receptor 1 (TNF-R1) by 4 h was accompanied by a decrease in mitochondrial respiration with glutamate but neither with pyruvate nor succinate. Assays of TCA cycle flux-limiting 2-oxoglutarate dehydrogenase complex (OGDHC) and functionally linked enzymes (glutamate dehydrogenase, glutamine synthetase, pyruvate dehydrogenase, malate dehydrogenase and malic enzyme) indicated that only OGDHC activity was decreased. Application of the OGDHC coenzyme precursor thiamine rescued the activity of OGDHC and restored mitochondrial respiration. These effects were not mediated by changes in the expression of the OGDHC sub-units (E1k and E3), suggesting post-translational mechanism of thiamine effects. By the third day after TBI, thiamine treatment also decreased expression of TNF-R1. Specific markers of unfolded protein response did not change in response to thiamine. CONCLUSION AND IMPLICATIONS: Our data point to OGDHC as a major site of damage in mitochondria upon traumatic brain injury, which is associated with neuroinflammation and can be corrected by thiamine. Further studies are required to evaluate the pathological impact of these findings in clinical settings.

Minimum Quality Threshold in Pre-Clinical Sepsis Studies (MQTiPSS): an international expert consensus initiative for improvement of animal modeling in sepsis.

PURPOSE: Pre-clinical animal studies precede the majority of clinical trials. While the clinical sepsis definitions and recommended treatments are regularly updated, a systematic review of pre-clinical models of sepsis has not been done and clear modeling guidelines are lacking. To address this deficit, a Wiggers-Bernard Conference on pre-clinical sepsis modeling was held in Vienna in May, 2017. The conference goal was to identify limitations of pre-clinical sepsis models and to propose a set of guidelines, defined as the "Minimum Quality Threshold in Pre-Clinical Sepsis Studies" (MQTiPSS), to enhance translational value of these models. METHODS: 31 experts from 13 countries participated and were divided into 6 thematic Working Groups (WG): (1) Study Design, (2) Humane modeling, (3) Infection types, (4) Organ failure/dysfunction, (5) Fluid resuscitation and (6) Antimicrobial therapy endpoints. As basis for the MQTiPSS discussions, the participants conducted a literature review of the 260 most highly cited scientific articles on sepsis models (2002-2013). RESULTS: Overall, the participants reached consensus on 29 points; 20 at "recommendation" (R) and 9 at "consideration" (C) strength. This Executive Summary provides a synopsis of the MQTiPSS consensus (Tables 1, 2 and 3). CONCLUSIONS: We believe that these recommendations and considerations will serve to bring a level of standardization to pre-clinical models of sepsis and ultimately improve translation of pre-clinical findings. These guideline points are proposed as "best practices" that should be implemented for animal sepsis models. In order to encourage its wide dissemination, this article is freely accessible in Shock, Infection and Intensive Care Medicine Experimental.

Correction to: Minimum Quality Threshold in Pre-Clinical Sepsis Studies (MQTiPSS): an international expert consensus initiative for improvement of animal modeling in sepsis.

The original version of this article unfortunately contained mistakes.

Alterations in nitric oxide homeostasis during traumatic brain injury.

Changes in nitric oxide (NO) levels have been often associated with various forms of trauma, including secondary damage after traumatic brain injury (TBI). Several studies demonstrate the upregulation of NO synthase (NOS) enzymes, and concomitant increases in brain NO levels, which contribute to the TBI-associated glutamate cytotoxicity, including the pathogenesis of mitochondrial dysfunction. TBI is also associated with elevated NO levels in remote organs, indicating that TBI can induce systemic changes in NO regulation, which can be either beneficial or detrimental. Here we review the possible mechanisms responsible for changes in NO metabolism during TBI. Better understanding of the changes in NO homeostasis in TBI will be necessary to design rational therapeutic approaches for TBI. This article is part of a Special Issue entitled: Immune and Metabolic Alterations in Trauma and Sepsis edited by Dr. Raghavan Raju.

EPR analysis of extra- and intracellular nitric oxide in liver biopsies.

PURPOSE: To develop an assay that can enable the quantification of intra- and extracellular nitric oxide (NO) levels in liver biopsies without application of potentially harmful exogenous NO traps. THEORY: Electron paramagnetic resonance (EPR) spectroscopy is currently the most appropriate method of measuring NO in biological samples due to the outstanding specificity resulting from the interaction of NO with exogenous NO traps. Because such traps are not allowed in clinical settings, we tested the reliability of endogenous NO traps for the determination of NO levels in blood and liver compartments. METHODS: Rats were injected with 0-8 mg/kg lipopolysaccharide (LPS) to gradually induce a systemic inflammatory response. Specific features of NO-hemoglobin and NO-Fe EPR signals were quantified using a specifically developed calibration procedure. RESULTS: Whereas both NO-hemoglobin (NO-HbLIVER BLOOD ) and NO-Fe (NO-FeLIVER ) complexes were detected in nonperfused liver tissue, only NO-Fe complexes were detected in perfused tissue and only NO-Hb complexes were detected in blood (NO-HbBLOOD ). The NO concentrations increased in the sequence NO-HbBLOOD < NO-FeLIVER < NO-HbLIVER BLOOD (9.4, 18.5, 27.9 nmol/cm3 , respectively at 2.5 mg/kg LPS). The detection limit of the method was 0.61 nmol/cm3 for NO-Hb and 0.52 nmol/cm3 for NO-Fe. CONCLUSION: The assay reported here does not influence natural NO pathways and enables the quantification of NO distribution in two liver compartments using a single liver biopsy. Magn Reson Med 77:2372-2380, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

Cellular ROS and Antioxidants: Physiological and Pathological Role.

Reactive oxygen species (ROS) are highly reactive oxygen derivatives that include free radicals such as superoxide anion radical (O2•-) and hydroxyl radical (HO•), as well as non-radical molecules hydrogen peroxide (H2O2), peroxynitrite (ONOO-), and hypochlorous acid (HOCl) [...].

Interplay between Energy Supply and Glutamate Toxicity in the Primary Cortical Culture.

Limited substrate availability because of the blood-brain barrier (BBB) has made the brain develop specific molecular mechanisms to survive, using lactate synthesized by astrocytes as a source of energy in neurons. To understand if lactate improves cellular viability and susceptibility to glutamate toxicity, primary cortical cells were incubated in glucose- or lactate-containing media and toxic concentrations of glutamate for 24 h. Cell death was determined by immunostaining and lactate dehydrogenase (LDH) release. Mitochondrial membrane potential and nitric oxide (NO) levels were measured using Tetramethylrhodamine, methyl ester (TMRM) and 4-Amino-5-Methylamino-2',7'-Difluorofluorescein Diacetate (DAF-FM) live staining, respectively. LDH activity was quantified in single cells in the presence of lactate (LDH substrate) and oxamate (LDH inhibitor). Nuclei of cells were stained with DAPI and neurons with MAP2. Based on the distance between neurons and glial cells, they were classified as linked (<10 µm) and non-linked (>10 µm) neurons. Lactate increased cell death rate and the mean value of endogenous NO levels compared to glucose incubations. Mitochondrial membrane potential was lower in the cells cultured with lactate, but this effect was reversed when glutamate was added to the lactate medium. LDH activity was higher in linked neurons compared to non-linked neurons, supporting the hypothesis of the existence of the lactate shuttle between astrocytes and at least a portion of neurons. In conclusion, glucose or lactate can equally preserve primary cortical neurons, but those neurons having a low level of LDH activity and incubated with lactate cannot cover high energetic demand solely with lactate and become more susceptible to glutamate toxicity.

Growth-hormone-induced signal transducer and activator of transcription 5 signaling causes gigantism, inflammation, and premature death but protects mice from aggressive liver cancer.

UNLABELLED: Persistently high levels of growth hormone (GH) can cause liver cancer. GH activates multiple signal-transduction pathways, among them janus kinase (JAK) 2-signal transducer and activator of transcription (STAT) 5 (signal transducer and activator of transcription 5). Both hyperactivation and deletion of STAT5 in hepatocytes have been implicated in the development of hepatocellular carcinoma (HCC); nevertheless, the role of STAT5 in the development of HCC as a result of high GH levels remains enigmatic. Thus, we crossed a mouse model of gigantism and inflammatory liver cancer caused by hyperactivated GH signaling (GH(tg) ) to mice with hepatic deletion of STAT5 (STAT5(Δhep) ). Unlike GH(tg) mice, GH(tg) STAT5(Δhep) animals did not display gigantism. Moreover, the premature mortality, which was associated with chronic inflammation, as well as the pathologic alterations of hepatocytes observed in GH(tg) mice, were not observed in GH(tg) animals lacking STAT5. Strikingly, loss of hepatic STAT5 proteins led to enhanced HCC development in GH(tg) mice. Despite reduced chronic inflammation, GH(tg) STAT5(Δhep) mice displayed earlier and more advanced HCC than GH(tg) animals. This may be attributed to the combination of increased peripheral lipolysis, hepatic lipid synthesis, loss of hepatoprotective mediators accompanied by aberrant activation of tumor-promoting c-JUN and STAT3 signaling cascades, and accumulation of DNA damage secondary to loss of cell-cycle control. Thus, HCC was never observed in STAT5(Δhep) mice. CONCLUSION: As a result of their hepatoprotective functions, STAT5 proteins prevent progressive fatty liver disease and the formation of aggressive HCC in the setting of hyperactivated GH signaling. At the same time, they play a key role in controlling systemic inflammation and regulating organ and body size.

Effect of mitoTEMPO on Redox Reactions in Different Body Compartments upon Endotoxemia in Rats.

Mitochondrial ROS (mitoROS) control many reactions in cells. Biological effects of mitoROS in vivo can be investigated by modulation via mitochondria-targeted antioxidants (mtAOX, mitoTEMPO). The aim of this study was to determine how mitoROS influence redox reactions in different body compartments in a rat model of endotoxemia. We induced inflammatory response by lipopolysaccharide (LPS) injection and analyzed effects of mitoTEMPO in blood, abdominal cavity, bronchoalveolar space, and liver tissue. MitoTEMPO decreased the liver damage marker aspartate aminotransferase; however, it neither influenced the release of cytokines (e.g., tumor necrosis factor, IL-4) nor decreased ROS generation by immune cells in the compartments examined. In contrast, ex vivo mitoTEMPO treatment substantially reduced ROS generation. Examination of liver tissue revealed several redox paramagnetic centers sensitive to in vivo LPS and mitoTEMPO treatment and high levels of nitric oxide (NO) in response to LPS. NO levels in blood were lower than in liver, and were decreased by in vivo mitoTEMPO treatment. Our data suggest that (i) inflammatory mediators are not likely to directly contribute to ROS-mediated liver damage and (ii) mitoTEMPO is more likely to affect the redox status of liver cells reflected in a redox change of paramagnetic molecules. Further studies are necessary to understand these mechanisms.

Bacterial Communities of Lamiacea L. Medicinal Plants: Structural Features and Rhizosphere Effect.

Bacterial communities associated with medicinal plants are an essential part of ecosystems. The rhizosphere effect is rather important in the cultivation process. The purpose of the study was to analyze the rhizosphere effect of oregano (Origanum vulgare L.), peppermint (Mentha piperita L.), thyme (Thymus vulgaris L.), creeping thyme (Thymus serpillum L.) and sage (Salvia officinalis L.). To estimate the quantity of 16S bacteria ribosomal genes, qPCR assays were used. To compare bacterial communities' structure of medicinal plants rhizosphere with bulk soil high-throughput sequencing of the 16S rRNA targeting variable regions V3-V4 of bacteria was carried out. The highest bacterial abundance was associated with T. vulgaris L., M. piperita L. and S. officinalis L., and the lowest was associated with the O. vulgare L. rhizosphere. Phylum Actinobacteriota was predominant in all rhizosphere samples. The maximum bacterial α-diversity was found in S. officinalis L. rhizosphere. According to bacterial ß-diversity calculated by the Bray-Curtis metric, T. vulgaris L. root zone significantly differed from bulk soil. The rhizosphere effect was positive to the Myxococcota, Bacteroidota, Verrucomicrobiota, Proteobacteria and Gemmatimonadota.

Oxoglutarate dehydrogenase complex controls glutamate-mediated neuronal death.

Brain injury is accompanied by neuroinflammation, accumulation of extracellular glutamate and mitochondrial dysfunction, all of which cause neuronal death. The aim of this study was to investigate the impact of these mechanisms on neuronal death. Patients from the neurosurgical intensive care unit suffering aneurysmal subarachnoid hemorrhage (SAH) were recruited retrospectively from a respective database. In vitro experiments were performed in rat cortex homogenate, primary dissociated neuronal cultures, B35 and NG108-15 cell lines. We employed methods including high resolution respirometry, electron spin resonance, fluorescent microscopy, kinetic determination of enzymatic activities and immunocytochemistry. We found that elevated levels of extracellular glutamate and nitric oxide (NO) metabolites correlated with poor clinical outcome in patients with SAH. In experiments using neuronal cultures we showed that the 2-oxoglutarate dehydrogenase complex (OGDHC), a key enzyme of the glutamate-dependent segment of the tricarboxylic acid (TCA) cycle, is more susceptible to the inhibition by NO than mitochondrial respiration. Inhibition of OGDHC by NO or by succinyl phosphonate (SP), a highly specific OGDHC inhibitor, caused accumulation of extracellular glutamate and neuronal death. Extracellular nitrite did not substantially contribute to this NO action. Reactivation of OGDHC by its cofactor thiamine (TH) reduced extracellular glutamate levels, Ca2+ influx into neurons and cell death rate. Salutary effect of TH against glutamate toxicity was confirmed in three different cell lines. Our data suggest that the loss of control over extracellular glutamate, as described here, rather than commonly assumed impaired energy metabolism, is the critical pathological manifestation of insufficient OGDHC activity, leading to neuronal death.