Selenium Drives a Transcriptional Adaptive Program to Block Ferroptosis and Treat Stroke.

Ferroptosis, a non-apoptotic form of programmed cell death, is triggered by oxidative stress in cancer, heat stress in plants, and hemorrhagic stroke. A homeostatic transcriptional response to ferroptotic stimuli is unknown. We show that neurons respond to ferroptotic stimuli by induction of selenoproteins, including antioxidant glutathione peroxidase 4 (GPX4). Pharmacological selenium (Se) augments GPX4 and other genes in this transcriptional program, the selenome, via coordinated activation of the transcription factors TFAP2c and Sp1 to protect neurons. Remarkably, a single dose of Se delivered into the brain drives antioxidant GPX4 expression, protects neurons, and improves behavior in a hemorrhagic stroke model. Altogether, we show that pharmacological Se supplementation effectively inhibits GPX4-dependent ferroptotic death as well as cell death induced by excitotoxicity or ER stress, which are GPX4 independent. Systemic administration of a brain-penetrant selenopeptide activates homeostatic transcription to inhibit cell death and improves function when delivered after hemorrhagic or ischemic stroke.

Hemin-Induced Death Models Hemorrhagic Stroke and Is a Variant of Classical Neuronal Ferroptosis.

Ferroptosis is a caspase-independent, iron-dependent form of regulated necrosis extant in traumatic brain injury, Huntington disease, and hemorrhagic stroke. It can be activated by cystine deprivation leading to glutathione depletion, the insufficiency of the antioxidant glutathione peroxidase-4, and the hemolysis products hemoglobin and hemin. A cardinal feature of ferroptosis is extracellular signal-regulated kinase (ERK)1/2 activation culminating in its translocation to the nucleus. We have previously confirmed that the mitogen-activated protein (MAP) kinase kinase (MEK) inhibitor U0126 inhibits persistent ERK1/2 phosphorylation and ferroptosis. Here, we show that hemin exposure, a model of secondary injury in brain hemorrhage and ferroptosis, activated ERK1/2 in mouse neurons. Accordingly, MEK inhibitor U0126 protected against hemin-induced ferroptosis. Unexpectedly, U0126 prevented hemin-induced ferroptosis independent of its ability to inhibit ERK1/2 signaling. In contrast to classical ferroptosis in neurons or cancer cells, chemically diverse inhibitors of MEK did not block hemin-induced ferroptosis, nor did the forced expression of the ERK-selective MAP kinase phosphatase (MKP)3. We conclude that hemin or hemoglobin-induced ferroptosis, unlike glutathione depletion, is ERK1/2-independent. Together with recent studies, our findings suggest the existence of a novel subtype of neuronal ferroptosis relevant to bleeding in the brain that is 5-lipoxygenase-dependent, ERK-independent, and transcription-independent. Remarkably, our unbiased phosphoproteome analysis revealed dramatic differences in phosphorylation induced by two ferroptosis subtypes. As U0126 also reduced cell death and improved functional recovery after hemorrhagic stroke in male mice, our analysis also provides a template on which to build a search for U0126's effects in a variant of neuronal ferroptosis.SIGNIFICANCE STATEMENT Ferroptosis is an iron-dependent mechanism of regulated necrosis that has been linked to hemorrhagic stroke. Common features of ferroptotic death induced by diverse stimuli are the depletion of the antioxidant glutathione, production of lipoxygenase-dependent reactive lipids, sensitivity to iron chelation, and persistent activation of extracellular signal-regulated kinase (ERK) signaling. Unlike classical ferroptosis induced in neurons or cancer cells, here we show that ferroptosis induced by hemin is ERK-independent. Paradoxically, the canonical MAP kinase kinase (MEK) inhibitor U0126 blocks brain hemorrhage-induced death. Altogether, these data suggest that a variant of ferroptosis is unleashed in hemorrhagic stroke. We present the first, unbiased phosphoproteomic analysis of ferroptosis as a template on which to understand distinct paths to cell death that meet the definition of ferroptosis.

Temporary induction of hypoxic adaptations by preconditioning fails to enhance Schwann cell transplant survival after spinal cord injury.

Hypoxic preconditioning is protective in multiple models of injury and disease, but whether it is beneficial for cells transplanted into sites of spinal cord injury (SCI) is largely unexplored. In this study, we analyzed whether hypoxia-related preconditioning protected Schwann cells (SCs) transplanted into the contused thoracic rat spinal cord. Hypoxic preconditioning was induced in SCs prior to transplantation by exposure to either low oxygen (1% O2 ) or pharmacological agents (deferoxamine or adaptaquin). All preconditioning approaches induced hypoxic adaptations, including increased expression of HIF-1α and its target genes. These adaptations, however, were transient and resolved within 24 h of transplantation. Pharmacological preconditioning attenuated spinal cord oxidative stress and enhanced transplant vascularization, but it did not improve either transplanted cell survival or recovery of sensory or motor function. Together, these experiments show that hypoxia-related preconditioning is ineffective at augmenting either cell survival or the functional outcomes of SC-SCI transplants. They also reveal that the benefits of hypoxia-related adaptations induced by preconditioning for cell transplant therapies are not universal.

Epigenetic regulators of neuronal ferroptosis identify novel therapeutics for neurological diseases: HDACs, transglutaminases, and HIF prolyl hydroxylases.

A major thrust of our laboratory has been to identify how physiological stress is transduced into transcriptional responses that feed back to overcome the inciting stress or its consequences, thereby fostering survival and repair. To this end, we have adopted the use of an in vitro model of ferroptosis, a caspase-independent, but iron-dependent form of cell death (Dixon et al., 2012; Ratan, 2020). In this review, we highlight three distinct epigenetic targets that have evolved from our studies and which have been validated in vivo studies. In the first section, we discuss our studies of broad, pan-selective histone deacetylase (HDAC) inhibitors in ferroptosis and how these studies led to the validation of HDAC inhibitors as candidate therapeutics in a host of disease models. In the second section, we discuss our studies that revealed a role for transglutaminase as an epigenetic modulator of proferroptotic pathways and how these studies set the stage for recent elucidation of monoamines as post-translation modifiers of histone function. In the final section, we discuss our studies of iron-, 2-oxoglutarate-, and oxygen-dependent dioxygenases and the role of one family of these enzymes, the HIF prolyl hydroxylases, in mediating transcriptional events necessary for ferroptosis in vitro and for dysfunction in a host of neurological conditions. Overall, our studies highlight the importance of epigenetic proteins in mediating prodeath and prosurvival responses to ferroptosis. Pharmacological agents that target these epigenetic proteins are showing robust beneficial effects in diverse rodent models of stroke, Parkinson's disease, Huntington's disease, and Alzheimer's disease.

The drug adaptaquin blocks ATF4/CHOP-dependent pro-death Trib3 induction and protects in cellular and mouse models of Parkinson's disease.

Identifying disease-causing pathways and drugs that target them in Parkinson's disease (PD) has remained challenging. We uncovered a PD-relevant pathway in which the stress-regulated heterodimeric transcription complex CHOP/ATF4 induces the neuron prodeath protein Trib3 that in turn depletes the neuronal survival protein Parkin. Here we sought to determine whether the drug adaptaquin, which inhibits ATF4-dependent transcription, could suppress Trib3 induction and neuronal death in cellular and animal models of PD. Neuronal PC12 cells and ventral midbrain dopaminergic neurons were assessed in vitro for survival, transcription factor levels and Trib3 or Parkin expression after exposure to 6-hydroxydopamine or 1-methyl-4-phenylpyridinium with or without adaptaquin co-treatment. 6-hydroxydopamine injection into the medial forebrain bundle was used to examine the effects of systemic adaptaquin on signaling, substantia nigra dopaminergic neuron survival and striatal projections as well as motor behavior. In both culture and animal models, adaptaquin suppressed elevation of ATF4 and/or CHOP and induction of Trib3 in response to 1-methyl-4-phenylpyridinium and/or 6-hydroxydopamine. In culture, adaptaquin preserved Parkin levels, provided neuroprotection and preserved morphology. In the mouse model, adaptaquin treatment enhanced survival of dopaminergic neurons and substantially protected their striatal projections. It also significantly enhanced retention of nigrostriatal function. These findings define a novel pharmacological approach involving the drug adaptaquin, a selective modulator of hypoxic adaptation, for suppressing Parkin loss and neurodegeneration in toxin models of PD. As adaptaquin possesses an oxyquinoline backbone with known safety in humans, these findings provide a firm rationale for advancing it towards clinical evaluation in PD.

N-acetylcysteine targets 5 lipoxygenase-derived, toxic lipids and can synergize with prostaglandin E2 to inhibit ferroptosis and improve outcomes following hemorrhagic stroke in mice.

OBJECTIVES: N-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. METHODS: Hemin 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. RESULTS: NAC 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 ). INTERPRETATION: NAC 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.

Neuronal Death After Hemorrhagic Stroke In Vitro and In Vivo Shares Features of Ferroptosis and Necroptosis.

BACKGROUND AND PURPOSE: Intracerebral hemorrhage leads to disability or death with few established treatments. Adverse outcomes after intracerebral hemorrhage result from irreversible damage to neurons resulting from primary and secondary injury. Secondary injury has been attributed to hemoglobin and its oxidized product hemin from lysed red blood cells. The aim of this study was to identify the underlying cell death mechanisms attributable to secondary injury by hemoglobin and hemin to broaden treatment options. METHODS: We investigated cell death mechanisms in cultured neurons exposed to hemoglobin or hemin. Chemical inhibitors implicated in all known cell death pathways were used. Identified cell death mechanisms were confirmed using molecular markers and electron microscopy. RESULTS: Chemical inhibitors of ferroptosis and necroptosis protected against hemoglobin- and hemin-induced toxicity. By contrast, inhibitors of caspase-dependent apoptosis, protein or mRNA synthesis, autophagy, mitophagy, or parthanatos had no effect. Accordingly, molecular markers of ferroptosis and necroptosis were increased after intracerebral hemorrhage in vitro and in vivo. Electron microscopy showed that hemin induced a necrotic phenotype. Necroptosis and ferroptosis inhibitors each abrogated death by >80% and had similar therapeutic windows in vitro. CONCLUSIONS: Experimental intracerebral hemorrhage shares features of ferroptotic and necroptotic cell death, but not caspase-dependent apoptosis or autophagy. We propose that ferroptosis or necroptotic signaling induced by lysed blood is sufficient to reach a threshold of death that leads to neuronal necrosis and that inhibition of either of these pathways can bring cells below that threshold to survival.

Hydroxamic acid-based histone deacetylase (HDAC) inhibitors can mediate neuroprotection independent of HDAC inhibition.

Histone deacetylase (HDAC) inhibition improves function and extends survival in rodent models of a host of neurological conditions, including stroke, and neurodegenerative diseases. Our understanding, however, of the contribution of individual HDAC isoforms to neuronal death is limited. In this study, we used selective chemical probes to assess the individual roles of the Class I HDAC isoforms in protecting Mus musculus primary cortical neurons from oxidative death. We demonstrated that the selective HDAC8 inhibitor PCI-34051 is a potent neuroprotective agent; and by taking advantage of both pharmacological and genetic tools, we established that HDAC8 is not critically involved in PCI-34051's mechanism of action. We used BRD3811, an inactive ortholog of PCI-34051, and showed that, despite its inability to inhibit HDAC8, it exhibits robust neuroprotective properties. Furthermore, molecular deletion of HDAC8 proved insufficient to protect neurons from oxidative death, whereas both PCI-34051 and BRD3811 were able to protect neurons derived from HDAC8 knock-out mice. Finally, we designed and synthesized two new, orthogonal negative control compounds, BRD9715 and BRD8461, which lack the hydroxamic acid motif and showed that they stably penetrate cell membranes but are not neuroprotective. These results indicate that the protective effects of these hydroxamic acid-containing small molecules are likely unrelated to direct epigenetic regulation via HDAC inhibition, but rather due to their ability to bind metals. Our results suggest that hydroxamic acid-based HDAC inhibitors may mediate neuroprotection via HDAC-independent mechanisms and affirm the need for careful structure-activity relationship studies when using pharmacological approaches.

In vitro ischemia suppresses hypoxic induction of hypoxia-inducible factor-1α by inhibition of synthesis and not enhanced degradation.

Hypoxia-inducible factor (HIF) mediates a broad, conserved adaptive response to hypoxia, and the HIF pathway is a potential therapeutic target in cerebral ischemia. This study investigated the mechanism by which in vitro ischemia (oxygen-glucose deprivation; OGD) affects canonical hypoxic HIF-1α stabilization. We validated the use of a reporter containing the oxygen-dependent degradation domain of HIF-1α fused to firefly luciferase (ODD-luc) to monitor quantitatively distinct biochemical events leading to hypoxic HIF-1α expression or stabilization in a human neuroblastoma cell line (SH-SY5Y). When OGD was imposed following a 2-hr hypoxic stabilization of ODD-luc, the levels of the reporter were reduced, consistent with prior models proposing that OGD enhances HIF prolylhydroxylase (PHD) activity. Surprisingly, PHD inhibitors and proteasome inhibitors do not stabilize ODD-luc in OGD. Furthermore, OGD does not affect the half-life of ODD-luc protein following hypoxia, suggesting that OGD abrogates hypoxic HIF-1α induction by reducing HIF-1α synthesis rather than by enhancing its degradation. We observed ATP depletion under OGD vs. hypoxia and propose that ATP depletion enhances translational suppression, overcoming the selective synthesis of HIF concurrent with global decreases in protein synthesis in hypoxia. Taken together, these findings biochemically characterize a practical reporter for monitoring HIF-1α levels and support a novel model for HIF regulation in an in vitro model of human ischemia.

Sodium salicylate protects against rotenone-induced parkinsonism in rats.

Complex I deficiency culminating in oxidative stress is proposed as one of the upstream mechanisms of nigral neuronal death in Parkinson's disease. We investigated whether sodium salicylate, an active metabolite of aspirin, could afford protection against rotenone-induced oxidative stress, neuronal degeneration, and behavioral dysfunction in rats, because it has the potential to accept a molecule each of hydroxyl radical (•OH) at the third or fifth position of its benzyl ring. Rotenone caused dose-dependent increase in •OH in isolated mitochondria from the cerebral cortex and time- (24-48 h) and dose-dependent (0.1-100 µM) increase in the substantia nigra and the striatum, ipsilateral to the side of rotenone infusion. Administration of sodium salicylate at 12-h intervals for 4 days showed dose-dependent (50-100 mg/kg, i.p) reductions in the levels of •OH in the nigra on the fifth day. These animals showed significant attenuation in rotenone-induced loss in striatal dopamine levels, number of nigral dopaminergic neurons, reduced and oxidized glutathione levels, and complex I activity loss, but superoxide dismutase activity was increased further. Amphetamine- or apomorphine-induced ipsilateral rotations in rotenone-treated rats were significantly reduced in rats treated with sodium salicylate. Our results indicate a direct role of •OH in mediating nigral neuronal death by rotenone and confirm the neuroprotective potential of salicylate in a rodent model of parkinsonism.

2-Deoxyglucose drives plasticity via an adaptive ER stress-ATF4 pathway and elicits stroke recovery and Alzheimer's resilience.

Intermittent fasting (IF) is a diet with salutary effects on cognitive aging, Alzheimer's disease (AD), and stroke. IF restricts a number of nutrient components, including glucose. 2-deoxyglucose (2-DG), a glucose analog, can be used to mimic glucose restriction. 2-DG induced transcription of the pro-plasticity factor, Bdnf, in the brain without ketosis. Accordingly, 2-DG enhanced memory in an AD model (5xFAD) and functional recovery in an ischemic stroke model. 2-DG increased Bdnf transcription via reduced N-linked glycosylation, consequent ER stress, and activity of ATF4 at an enhancer of the Bdnf gene, as well as other regulatory regions of plasticity/regeneration (e.g., Creb5, Cdc42bpa, Ppp3cc, and Atf3) genes. These findings demonstrate an unrecognized role for N-linked glycosylation as an adaptive sensor to reduced glucose availability. They further demonstrate that ER stress induced by 2-DG can, in the absence of ketosis, lead to the transcription of genes involved in plasticity and cognitive resilience as well as proteostasis.

A STING agonist preconditions against ischaemic stroke via an adaptive antiviral Type 1 interferon response.

Converging lines of inquiry have highlighted the importance of the Type I antiviral response not only in defending against viruses but also in preconditioning the brain against ischaemic stroke. Despite this understanding, treatments that foster brain resilience by driving antiviral interferon responses have yet to be developed for human use. Studies from our laboratory showed that tilorone, the first human antiviral immunomodulatory agent to be developed, robustly preconditioned against stroke in mice and rats. Tilorone is a DNA intercalator; therefore, we hypothesized that it stabilizes cytosolic DNA (released from the mitochondria or the nucleus), thereby activating cyclic GMP-AMP synthase, a homeostatic DNA sensor, and its downstream pathway. This pathway involves st imulator of in terferon g enes (STING), tank-binding kinase 1 (TBK1), and i nterferon r egulatory p rotein-3 and culminates in a protective Type I interferon response. We tested this hypothesis by examining the ability of structurally diverse small-molecule agonists of STING to protect against oxygen/glucose deprivation in vitro in mouse cortical cultures and in vivo against transient ischaemia in mice. The STING agonists significantly reduced cell death both in vitro and in vivo but failed to do so in STING knockout mice. As expected, STING agonist-induced protection was associated with the induction of interferon related genes and the effects could be abrogated in vitro by a TBK1 inhibitor. Taken together, these findings in mice identify STING as a therapeutic target for preconditioning the brain against ischaemic stroke in vitro and in vivo. Moreover, they suggest that clinically approved STING agonists such as Ganciclovir or α-Mangostin are candidate drugs that could be tested in humans as a prophylactic treatment to alleviate brain injury associated with ischaemic stroke.

HIF1α stabilization in hypoxia is not oxidant-initiated.

Hypoxic adaptation mediated by HIF transcription factors requires mitochondria, which have been implicated in regulating HIF1α stability in hypoxia by distinct models that involve consuming oxygen or alternatively converting oxygen into the second messenger peroxide. Here, we use a ratiometric, peroxide reporter, HyPer to evaluate the role of peroxide in regulating HIF1α stability. We show that antioxidant enzymes are neither homeostatically induced nor are peroxide levels increased in hypoxia. Additionally, forced expression of diverse antioxidant enzymes, all of which diminish peroxide, had disparate effects on HIF1α protein stability. Moreover, decrease in lipid peroxides by glutathione peroxidase-4 or superoxide by mitochondrial SOD, failed to influence HIF1α protein stability. These data show that mitochondrial, cytosolic or lipid ROS were not necessary for HIF1α stability, and favor a model where mitochondria contribute to hypoxic adaptation as oxygen consumers.

Hypoxia-inducible factor prolyl hydroxylase domain (PHD) inhibition after contusive spinal cord injury does not improve locomotor recovery.

Traumatic spinal cord injury (SCI) is a devastating neurological condition that involves both primary and secondary tissue loss. Various cytotoxic events including hypoxia, hemorrhage and blood lysis, bioenergetic failure, oxidative stress, endoplasmic reticulum (ER) stress, and neuroinflammation contribute to secondary injury. The HIF prolyl hydroxylase domain (PHD/EGLN) family of proteins are iron-dependent, oxygen-sensing enzymes that regulate the stability of hypoxia inducible factor-1α (HIF-1α) and also mediate oxidative stress caused by free iron liberated from the lysis of blood. PHD inhibition improves outcome after experimental intracerebral hemorrhage (ICH) by reducing activating transcription factor 4 (ATF4)-driven neuronal death. As the ATF4-CHOP (CCAAT-enhancer-binding protein homologous protein) pathway plays a role in the pathogenesis of contusive SCI, we examined the effects of PHD inhibition in a mouse model of moderate T9 contusive SCI in which white matter damage is the primary driver of locomotor dysfunction. Pharmacological inhibition of PHDs using adaptaquin (AQ) moderately lowers acute induction of Atf4 and Chop mRNAs and prevents the acute decline of oligodendrocyte (OL) lineage mRNAs, but does not improve long-term recovery of hindlimb locomotion or increase chronic white matter sparing. Conditional genetic ablation of all three PHD isoenzymes in OLs did not affect Atf4, Chop or OL mRNAs expression levels, locomotor recovery, and white matter sparing after SCI. Hence, PHDs may not be suitable targets to improve outcomes in traumatic CNS pathologies that involve acute white matter injury.

Longitudinal transcriptomics define the stages of myeloid activation in the living human brain after intracerebral hemorrhage.

Opportunities to interrogate the immune responses in the injured tissue of living patients suffering from acute sterile injuries such as stroke and heart attack are limited. We leveraged a clinical trial of minimally invasive neurosurgery for patients with intracerebral hemorrhage (ICH), a severely disabling subtype of stroke, to investigate the dynamics of inflammation at the site of brain injury over time. Longitudinal transcriptional profiling of CD14+ monocytes/macrophages and neutrophils from hematomas of patients with ICH revealed that the myeloid response to ICH within the hematoma is distinct from that in the blood and occurs in stages conserved across the patient cohort. Initially, hematoma myeloid cells expressed a robust anabolic proinflammatory profile characterized by activation of hypoxia-inducible factors (HIFs) and expression of genes encoding immune factors and glycolysis. Subsequently, inflammatory gene expression decreased over time, whereas anti-inflammatory circuits were maintained and phagocytic and antioxidative pathways up-regulated. During this transition to immune resolution, glycolysis gene expression and levels of the potent proresolution lipid mediator prostaglandin E2 remained elevated in the hematoma, and unexpectedly, these elevations correlated with positive patient outcomes. Ex vivo activation of human macrophages by ICH-associated stimuli highlighted an important role for HIFs in production of both inflammatory and anti-inflammatory factors, including PGE2, which, in turn, augmented VEGF production. Our findings define the time course of myeloid activation in the human brain after ICH, revealing a conserved progression of immune responses from proinflammatory to proresolution states in humans after brain injury and identifying transcriptional programs associated with neurological recovery.

New Mechanistic Insights, Novel Treatment Paradigms, and Clinical Progress in Cerebrovascular Diseases.

The past decade has brought tremendous progress in diagnostic and therapeutic options for cerebrovascular diseases as exemplified by the advent of thrombectomy in ischemic stroke, benefitting a steeply increasing number of stroke patients and potentially paving the way for a renaissance of neuroprotectants. Progress in basic science has been equally impressive. Based on a deeper understanding of pathomechanisms underlying cerebrovascular diseases, new therapeutic targets have been identified and novel treatment strategies such as pre- and post-conditioning methods were developed. Moreover, translationally relevant aspects are increasingly recognized in basic science studies, which is believed to increase their predictive value and the relevance of obtained findings for clinical application.This review reports key results from some of the most remarkable and encouraging achievements in neurovascular research that have been reported at the 10th International Symposium on Neuroprotection and Neurorepair. Basic science topics discussed herein focus on aspects such as neuroinflammation, extracellular vesicles, and the role of sex and age on stroke recovery. Translational reports highlighted endovascular techniques and targeted delivery methods, neurorehabilitation, advanced functional testing approaches for experimental studies, pre-and post-conditioning approaches as well as novel imaging and treatment strategies. Beyond ischemic stroke, particular emphasis was given on activities in the fields of traumatic brain injury and cerebral hemorrhage in which promising preclinical and clinical results have been reported. Although the number of neutral outcomes in clinical trials is still remarkably high when targeting cerebrovascular diseases, we begin to evidence stepwise but continuous progress towards novel treatment options. Advances in preclinical and translational research as reported herein are believed to have formed a solid foundation for this progress.

Dietary supplementation with resveratrol reduces plaque pathology in a transgenic model of Alzheimer's disease.

Resveratrol, a polyphenol found in red wine, peanuts, soy beans, and pomegranates, possesses a wide range of biological effects. Since resveratrol's properties seem ideal for treating neurodegenerative diseases, its ability to diminish amyloid plaques was tested. Mice were fed clinically feasible dosages of resveratrol for forty-five days. Neither resveratrol nor its conjugated metabolites were detectable in brain. Nevertheless, resveratrol diminished plaque formation in a region specific manner. The largest reductions in the percent area occupied by plaques were observed in medial cortex (-48%), striatum (-89%) and hypothalamus (-90%). The changes occurred without detectable activation of SIRT-1 or alterations in APP processing. However, brain glutathione declined 21% and brain cysteine increased 54%. The increased cysteine and decreased glutathione may be linked to the diminished plaque formation. This study supports the concept that onset of neurodegenerative disease may be delayed or mitigated with use of dietary chemo-preventive agents that protect against beta-amyloid plaque formation and oxidative stress.

Ferroptosis in Neurons and Cancer Cells Is Similar But Differentially Regulated by Histone Deacetylase Inhibitors.

Ferroptotic death is a mechanism for tumor suppression by pharmacological inhibitors that target the Xc- transporter (cystine/glutamate antiporter) in a host of non-CNS and CNS tumors. Inhibition of this transporter leads to reduction of cystine uptake, cyst(e)ine deprivation, subsequent depletion of the versatile antioxidant glutathione, and reactive lipid species-dependent death. Accordingly, pharmacological inhibitors of the Xc- transporter can also induce neuronal cell death raising concerns about toxicity in the CNS and PNS if these agents are used for chemotherapy. Here, we show that ferroptotic death induced by the canonical ferroptosis inducer erastin is similar in HT1080 fibrosarcoma cells and primary cortical neurons although cell death is mediated more potently in cancer cells. Reducing the toxicity of ferroptosis inducers will require, among other things, the identification of agents that protect neurons from ferroptosis but exacerbate it in tumor cells. Although we show that a number of agents known to block ferroptosis in primary mouse neurons also inhibit ferroptosis in fibrosarcoma cells, class I histone deacetylase (HDAC) inhibitors selectively protect neurons while augmenting ferroptosis in cancer cells. Our results further suggest that cell death pathways induced by erastin in these two cell types are statistically identical to each other and identical to oxidative glutamate toxicity in neurons, where death is also mediated via inhibition of Xc- cystine transport. Together, these studies identify HDACs inhibitors as a novel class of agents to augment tumor suppression by ferroptosis induction and to minimize neuronal toxicity that could manifest as peripheral neuropathy or chemo brain.

The interferon response as a common final pathway for many preconditioning stimuli: unexpected crosstalk between hypoxic adaptation and antiviral defense.

Despite major advances in understanding how the brain goes awry in disease, identification of therapeutics for neuroprotection in stroke remains an unsolved challenge. A promising strategy to delineate endogenous mechanisms of neuroprotection is to understand adaptive homeostatic transcription induced by sublethal ischemia. Homeostatic adaptation is defined as the body's restorative responses to stress. Activating adaptive homeostatic pathways can lead to transcription of a panoply of genes involved in cell survival and repair, can suppress pro-death signaling, and can stimulate metabolic changes congruent with survival. All of these mechanisms have been shown to be operative in protection induced by sublethal stress. In this context, central mediators of cellular adaptation to hypoxic and viral stress have been implicated in preconditioning. Here we present data that suggest an unexpected convergence in the pathways triggering adaptation to hypoxia and viral infection leading to preconditioning neuroprotection in the CNS.

Exercise promotes the expression of brain derived neurotrophic factor (BDNF) through the action of the ketone body ß-hydroxybutyrate.

Exercise induces beneficial responses in the brain, which is accompanied by an increase in BDNF, a trophic factor associated with cognitive improvement and the alleviation of depression and anxiety. However, the exact mechanisms whereby physical exercise produces an induction in brain Bdnf gene expression are not well understood. While pharmacological doses of HDAC inhibitors exert positive effects on Bdnf gene transcription, the inhibitors represent small molecules that do not occur in vivo. Here, we report that an endogenous molecule released after exercise is capable of inducing key promoters of the Mus musculus Bdnf gene. The metabolite ß-hydroxybutyrate, which increases after prolonged exercise, induces the activities of Bdnf promoters, particularly promoter I, which is activity-dependent. We have discovered that the action of ß-hydroxybutyrate is specifically upon HDAC2 and HDAC3, which act upon selective Bdnf promoters. Moreover, the effects upon hippocampal Bdnf expression were observed after direct ventricular application of ß-hydroxybutyrate. Electrophysiological measurements indicate that ß-hydroxybutyrate causes an increase in neurotransmitter release, which is dependent upon the TrkB receptor. These results reveal an endogenous mechanism to explain how physical exercise leads to the induction of BDNF.