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.

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.

Arginase overexpression in neurons and its effect on traumatic brain injury.

Arginine is a semi-essential amino acid which serves as a substrate for nitric oxide (NO) production by nitric oxide synthase (NOS) and a precursor for various metabolites including ornithine, creatine, polyamines, and agmatine. Arginase competes with nitric oxide synthase for substrate arginine to produce orthinine and urea. There is contradictory evidence in the literature on the role of nitric oxide in the pathophysiology of traumatic brain injury (TBI). These contradictory perspectives are likely due to different NOS isoforms - endothelial (eNOS), inducible (iNOS) and neuronal (nNOS) which are expressed in the central nervous system. Of these, the role of nNOS in acute injury remains less clear. This study aimed to employ a genetic approach by overexpressing arginase isoforms specifically in neurons using a Thy-1 promoter to manipulate cell autonomous NO production in the context of TBI. The hypothesis was that increased arginase would divert arginine from pathological NO production. We generated 2 mouse lines that overexpress arginase I (a cytoplasmic enzyme) or arginase II (a mitochondrial enzyme) in neurons of FVB mice. We found that two-weeks after induction of controlled cortical injury, overexpressing arginase I but not arginase II in neurons significantly reduced contusion size and contusion index compared to wild-type (WT) mice. This study establishes enhanced neuronal arginase levels as a strategy to affect the course of TBI and provides support for the potential role of neuronal NO production in this condition.

Distinct Nrf2 Signaling Mechanisms of Fumaric Acid Esters and Their Role in Neuroprotection against 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine-Induced Experimental Parkinson's-Like Disease.

UNLABELLED: A promising approach to neurotherapeutics involves activating the nuclear-factor-E2-related factor 2 (Nrf2)/antioxidant response element signaling, which regulates expression of antioxidant, anti-inflammatory, and cytoprotective genes. Tecfidera, a putative Nrf2 activator, is an oral formulation of dimethylfumarate (DMF) used to treat multiple sclerosis. We compared the effects of DMF and its bioactive metabolite monomethylfumarate (MMF) on Nrf2 signaling and their ability to block 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced experimental Parkinson's disease (PD). We show that in vitro DMF and MMF activate the Nrf2 pathway via S-alkylation of the Nrf2 inhibitor Keap1 and by causing nuclear exit of the Nrf2 repressor Bach1. Nrf2 activation by DMF but not MMF was associated with depletion of glutathione, decreased cell viability, and inhibition of mitochondrial oxygen consumption and glycolysis rates in a dose-dependent manner, whereas MMF increased these activities in vitro However, both DMF and MMF upregulated mitochondrial biogenesis in vitro in an Nrf2-dependent manner. Despite the in vitro differences, both DMF and MMF exerted similar neuroprotective effects and blocked MPTP neurotoxicity in wild-type but not in Nrf2 null mice. Our data suggest that DMF and MMF exhibit neuroprotective effects against MPTP neurotoxicity because of their distinct Nrf2-mediated antioxidant, anti-inflammatory, and mitochondrial functional/biogenetic effects, but MMF does so without depleting glutathione and inhibiting mitochondrial and glycolytic functions. Given that oxidative damage, neuroinflammation, and mitochondrial dysfunction are all implicated in PD pathogenesis, our results provide preclinical evidence for the development of MMF rather than DMF as a novel PD therapeutic. SIGNIFICANCE STATEMENT: Almost two centuries since its first description by James Parkinson, Parkinson's disease (PD) remains an incurable disease with limited symptomatic treatment. The current study provides preclinical evidence that a Food and Drug Administration-approved drug, dimethylfumarate (DMF), and its metabolite monomethylfumarate (MMF) can block nigrostriatal dopaminergic neurodegeneration in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of PD. We elucidated mechanisms by which DMF and its active metabolite MMF activates the redox-sensitive transcription factor nuclear-factor-E2-related factor 2 (Nrf2) to upregulate antioxidant, anti-inflammatory, mitochondrial biosynthetic and cytoprotective genes to render neuroprotection via distinct S-alkylating properties and depletion of glutathione. Our data suggest that targeting Nrf2-mediated gene transcription using MMF rather than DMF is a promising approach to block oxidative stress, neuroinflammation, and mitochondrial dysfunction for therapeutic intervention in PD while minimizing side effects.

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.

Daidzein Augments Cholesterol Homeostasis via ApoE to Promote Functional Recovery in Chronic Stroke.

Stroke is the world's leading cause of physiological disability, but there are currently no available agents that can be delivered early after stroke to enhance recovery. Daidzein, a soy isoflavone, is a clinically approved agent that has a neuroprotective effect in vitro, and it promotes axon growth in an animal model of optic nerve crush. The current study investigates the efficacy of daidzein on neuroprotection and functional recovery in a clinically relevant mouse model of stroke recovery. In light of the fact that cholesterols are essential lipid substrates in injury-induced synaptic remodeling, we found that daidzein enhanced the cholesterol homeostasis genetic program, including Lxr and downstream transporters, Apoe, Abca1, and Abcg1 genes in vitro. Daidzein also elevated the cholesterol homeostasis genes in the poststroke brain with Apoe, the highest expressing transporter, but did not affect infarct volume or hemispheric swelling. Despite the absence of neuroprotection, daidzein improved motor/gait function in chronic stroke and elevated synaptophysin expression. However, the daidzein-enhanced functional benefits and synaptophysin expression were abolished in Apoe-knock-out mice, suggesting the importance of daidzein-induced ApoE upregulation in fostering stroke recovery. Dissociation between daidzein-induced functional benefits and the absence of neuroprotection further suggest the presence of nonoverlapping mechanisms underlying recovery processes versus acute pathology. With its known safety in humans, early and chronic use of daidzein aimed at augmenting ApoE may serve as a novel, translatable strategy to promote functional recovery in stroke patients without adverse acute effect. SIGNIFICANCE STATEMENT: There have been recurring translational failures in treatment strategies for stroke. One underlying issue is the disparity in outcome analysis between animal and clinical studies. The former mainly depends on acute infarct size, whereas long-term functional recovery is an important outcome in patients. In an attempt to identify agents that promote functional recovery, we discovered that an FDA-approved soy isoflavone, daidzein, improved stroke-induced behavioral deficits via enhancing cholesterol homeostasis in chronic stroke, and this occurs without causing adverse effects in the acute phase. With its known safety in humans, the study suggests that the early and chronic use of daidzein serves as a potential strategy to promote functional recovery in stroke patients.

Hypoxic Adaptation in the Nervous System: Promise for Novel Therapeutics for Acute and Chronic Neurodegeneration.

Homeostasis is the process by which cells adapt to stress and prevent or repair injury. Unique programs have evolved to sense and activate these homeostatic mechanisms and as such, homeostatic sensors may be potent therapeutic targets. The hypoxic response mediated by hypoxia inducible factor (HIF) downstream of oxygen sensing by HIF prolyl 4-hydroxylases (PHDs) has been well-studied, revealing cell-type specific regulation of HIF stability, activity, and transcriptional targets. HIF's paradoxical roles in nervous system development, physiology, and pathology arise from its complex roles in hypoxic adaptation and normoxic biology. Understanding how to engage the hypoxic response so as to recapitulate the protective mechanism of ischemic preconditioning is a high priority. Indeed, small molecules that activate the hypoxic response provide broad neuroprotection in several clinically relevant injury models. Screens for PHD inhibitors have identified novel therapeutics for neuroprotection that are ready to proceed to clinical trials for ischemic stroke. Better understanding the mechanisms of how to engage hypoxic adaption without altering development or physiology may identify additional novel therapeutic targets for diverse acute and chronic neuropathologies.

An adaptive role for BDNF Val66Met polymorphism in motor recovery in chronic stroke.

Little is known about the influence of genetic diversity on stroke recovery. One exception is the polymorphism in brain derived neurotrophic factor (BDNF), a critical neurotrophin for brain repair and plasticity. Humans have a high-frequency single nucleotide polymorphism (SNP) in the prodomain of the BDNF gene. Previous studies show that the BDNF Val66Met variant negatively affects motor learning and severity of acute stroke. To investigate the impact of this common BDNF SNP on stroke recovery, we used a mouse model that contains the human BDNF Val66Met variant in both alleles (BDNF(M/M)). Male BDNF(+/+) and BDNF(M/M) littermates received sham or transient middle cerebral artery occlusion. We assessed motor function regularly for 6 months after stroke and then performed anatomical analyses. Despite reported negative association of the SNP with motor learning and acute deficits, we unexpectedly found that BDNF(M/M) mice displayed significantly enhanced motor/kinematic performance in the chronic phase of motor recovery, especially in ipsilesional hindlimb. The enhanced recovery was associated with significant increases in striatum volume, dendritic arbor, and elevated excitatory synaptic markers in the contralesional striatum. Transient inactivation of the contralateral striatum during recovery transiently abolished the enhanced function. This study showed an unexpected benefit of the BDNFVal66Met carriers for functional recovery, involving structural and molecular plasticity in the nonstroked hemisphere. Clinically, this study suggests a role for BDNF genotype in predicting stroke recovery and identifies a novel systems-level mechanism for enhanced motor recovery.

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.

cAMP-responsive element-binding protein (CREB) and cAMP co-regulate activator protein 1 (AP1)-dependent regeneration-associated gene expression and neurite growth.

To regenerate damaged axons, neurons must express a cassette of regeneration-associated genes (RAGs) that increases intrinsic growth capacity and confers resistance to extrinsic inhibitory cues. Here we show that dibutyrl-cAMP or forskolin combined with constitutive-active CREB are superior to either agent alone in driving neurite growth on permissive and inhibitory substrates. Of the RAGs examined, only arginase 1 (Arg1) expression correlated with the increased neurite growth induced by the cAMP/CREB combination, both of which were AP1-dependent. This suggests that cAMP-induced AP1 activity is necessary and interacts with CREB to drive expression of RAGs relevant for regeneration and demonstrates that combining a small molecule (cAMP) with an activated transcription factor (CREB) stimulates the gene expression necessary to enhance axonal regeneration.

Specific acetylation of p53 by HDAC inhibition prevents DNA damage-induced apoptosis in neurons.

Histone deacetylase (HDAC) inhibitors have been used to promote neuronal survival and ameliorate neurological dysfunction in a host of neurodegenerative disease models. The precise molecular mechanisms whereby HDAC inhibitors prevent neuronal death are currently the focus of intensive research. Here we demonstrate that HDAC inhibition prevents DNA damage-induced neurodegeneration by modifying the acetylation pattern of the tumor suppressor p53, which decreases its DNA-binding and transcriptional activation of target genes. Specifically, we identify that acetylation at K382 and K381 prevents p53 from associating with the pro-apoptotic PUMA gene promoter, activating transcription, and inducing apoptosis in mouse primary cortical neurons. Paradoxically, acetylation of p53 at the same lysines in various cancer cell lines leads to the induction of PUMA expression and death. Together, our data provide a molecular understanding of the specific outcomes of HDAC inhibition and suggest that strategies aimed at enhancing p53 acetylation at K381 and K382 might be therapeutically viable for capturing the beneficial effects in the CNS, without compromising tumor suppression.

Inhibition of 12/15-lipoxygenase as therapeutic strategy to treat stroke.

Targeting newly identified damage pathways in the ischemic brain can help to circumvent the currently severe limitations of acute stroke therapy. Here we show that the activity of 12/15-lipoxygenase was increased in the ischemic mouse brain, and 12/15-lipoxygenase colocalized with a marker for oxidized lipids, MDA2. This colocalization was also detected in the brain of 2 human stroke patients, where it also coincided with increased apoptosis-inducing factor. A novel inhibitor of 12/15-lipoxygenase, LOXBlock-1, protected neuronal HT22 cells against oxidative stress. In a mouse model of transient focal ischemia, the inhibitor reduced infarct sizes both 24 hours and 14 days poststroke, with improved behavioral parameters. Even when treatment was delayed until at least 4 hours after onset of ischemia, LOXBlock-1 was protective. Furthermore, it reduced tissue plasminogen activator-associated hemorrhage in a clot model of ischemia/reperfusion. This study establishes inhibition of 12/15-lipoxygenase as a viable strategy for first-line stroke treatment.

miR-886-3p levels are elevated in Friedreich ataxia.

Friedreich ataxia (FRDA) is the most common inherited ataxia caused primarily by an intronic GAA.TTC triplet repeat expansion in the frataxin (FXN) gene. FXN RNA and protein levels are reduced in patients leading to progressive gait and limb ataxia, sensory loss, reduced tendon reflexes, dysarthria, absent lower limb reflexes, and loss of position and vibration sense. Neurological manifestations ensue from primary loss of dorsal root ganglia neurons and their associated axons ascending centrally in the spinal cord and peripherally in large myelinated nerves. Small noncoding RNAs such as microRNAs have been shown to be dysregulated in neurodegenerative diseases such as Alzheimer's and Huntington's disease. Here we report that hsa-miR-886-3p (miR-886-3p) was increased in patient cells as well as peripheral patient blood samples. Selective reduction in miR-886-3p by an anti-miR led to elevation of FXN message and protein levels without associated changes in histone marks at the FXN locus. Nevertheless, derepression of frataxin by a histone deacetylase inhibitor leads to a decrease in miR-886-3p. These results outline involvement of a small RNA, miR-886-3p in FRDA and a novel therapeutic approach to this disease using an anti-miR-886-3p.

Transglutaminase inhibition protects against oxidative stress-induced neuronal death downstream of pathological ERK activation.

Molecular deletion of transglutaminase 2 (TG2) has been shown to improve function and survival in a host of neurological conditions including stroke, Huntington's disease, and Parkinson's disease. However, unifying schemes by which these cross-linking or polyaminating enzymes participate broadly in neuronal death have yet to be presented. Unexpectedly, we found that in addition to TG2, TG1 gene expression level is significantly induced following stroke in vivo or due to oxidative stress in vitro. Forced expression of TG1 or TG2 proteins is sufficient to induce neuronal death in Rattus norvegicus cortical neurons in vitro. Accordingly, molecular deletion of TG2 alone is insufficient to protect Mus musculus neurons from oxidative death. By contrast, structurally diverse inhibitors used at concentrations that inhibit TG1 and TG2 simultaneously are neuroprotective. These small molecules inhibit increases in neuronal transamidating activity induced by oxidative stress; they also protect neurons downstream of pathological ERK activation when added well after the onset of the death stimulus. Together, these studies suggest that multiple TG isoforms, not only TG2, participate in oxidative stress-induced cell death signaling; and that isoform nonselective inhibitors of TG will be most efficacious in combating oxidative death in neurological disorders.

15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) protects neurons from oxidative death via an Nrf2 astrocyte-specific mechanism independent of PPARγ.

Astrocytes are critical for the antioxidant support of neurons. Recently, we demonstrated that low level hydrogen peroxide (H(2) O(2) ) facilitates astrocyte-dependent neuroprotection independent of the antioxidant transcription factor Nrf2, leaving the identity of the endogenous astrocytic Nrf2 activator to question. In this study, we show that an endogenous electrophile, 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), non-cell autonomously protects neurons from death induced by depletion of the major antioxidant glutathione. Nrf2 knockdown in astrocytes abrogated 15d-PGJ2's neuroprotective effect as well as 15d-PGJ2 facilitated Nrf2-target gene induction. In contrast, knockdown of the transcription factor peroxisome proliferator activated-receptor gamma (PPARγ), a well-characterized 15d-PGJ2 target, did not alter 15d-PGJ2 non-cell autonomous neuroprotection. In addition, several PPARγ agonists of the thiazolidinedione (TZD) family failed to induce neuroprotection. Unexpectedly, however, the TZD troglitazone (which contains a chromanol moiety found on vitamin E) induced astrocyte-mediated neuroprotection, an effect which was mimicked by the vitamin E analogs alpha-tocopherol or alpha-tocotrienol. Our findings lead to two important conclusions: (i) 15d-PGJ2 induces astrocyte-mediated neuroprotection via an Nrf2 but not PPARγ mediated pathway, suggesting that 15d-PGJ2 is a candidate endogenous modulator of Nrf2 protective pathways in astrocytes; (ii) selective astrocyte treatment with analogs or compounds containing the chromanol moiety of vitamin E facilitates non-cell autonomous neuroprotection.