Alzheimer's disease-linked risk alleles elevate microglial cGAS-associated senescence and neurodegeneration in a tauopathy model.

The strongest risk factors for late-onset sporadic Alzheimer's disease (AD) include the ε4 allele of apolipoprotein E (APOE), the R47H variant of triggering receptor expressed on myeloid cells 2 (TREM2), and female sex. Here, we combine APOE4 and TREM2R47H (R47H) in female P301S tauopathy mice to identify the pathways activated when AD risk is the strongest, thereby highlighting detrimental disease mechanisms. We find that R47H induces neurodegeneration in 9- to 10-month-old female APOE4 tauopathy mice. The combination of APOE4 and R47H (APOE4-R47H) worsened hyperphosphorylated tau pathology in the frontal cortex and amplified tauopathy-induced microglial cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling and downstream interferon response. APOE4-R47H microglia displayed cGAS- and BAX-dependent upregulation of senescence, showing association between neurotoxic signatures and implicating mitochondrial permeabilization in pathogenesis. By uncovering pathways enhanced by the strongest AD risk factors, our study points to cGAS-STING signaling and associated microglial senescence as potential drivers of AD risk.

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.

Tau activation of microglial cGAS-IFN reduces MEF2C-mediated cognitive resilience.

Pathological hallmarks of Alzheimer's disease (AD) precede clinical symptoms by years, indicating a period of cognitive resilience before the onset of dementia. Here, we report that activation of cyclic GMP-AMP synthase (cGAS) diminishes cognitive resilience by decreasing the neuronal transcriptional network of myocyte enhancer factor 2c (MEF2C) through type I interferon (IFN-I) signaling. Pathogenic tau activates cGAS and IFN-I responses in microglia, in part mediated by cytosolic leakage of mitochondrial DNA. Genetic ablation of Cgas in mice with tauopathy diminished the microglial IFN-I response, preserved synapse integrity and plasticity and protected against cognitive impairment without affecting the pathogenic tau load. cGAS ablation increased, while activation of IFN-I decreased, the neuronal MEF2C expression network linked to cognitive resilience in AD. Pharmacological inhibition of cGAS in mice with tauopathy enhanced the neuronal MEF2C transcriptional network and restored synaptic integrity, plasticity and memory, supporting the therapeutic potential of targeting the cGAS-IFN-MEF2C axis to improve resilience against AD-related pathological insults.

Parkin coregulates glutathione metabolism in adult mammalian brain.

We recently discovered that the expression of PRKN, a young-onset Parkinson disease-linked gene, confers redox homeostasis. To further examine the protective effects of parkin in an oxidative stress model, we first combined the loss of prkn with Sod2 haploinsufficiency in mice. Although adult prkn-/-//Sod2± animals did not develop dopamine cell loss in the S. nigra, they had more reactive oxidative species and a higher concentration of carbonylated proteins in the brain; bi-genic mice also showed a trend for more nitrotyrosinated proteins. Because these redox changes were seen in the cytosol rather than mitochondria, we next explored the thiol network in the context of PRKN expression. We detected a parkin deficiency-associated increase in the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) in murine brain, PRKN-linked human cortex and several cell models. This shift resulted from enhanced recycling of GSSG back to GSH via upregulated glutathione reductase activity; it also correlated with altered activities of redox-sensitive enzymes in mitochondria isolated from mouse brain (e.g., aconitase-2; creatine kinase). Intriguingly, human parkin itself showed glutathione-recycling activity in vitro and in cells: For each GSSG dipeptide encountered, parkin regenerated one GSH molecule and was S-glutathionylated by the other (GSSG + P-SH [Formula: see text] GSH + P-S-SG), including at cysteines 59, 95 and 377. Moreover, parkin's S-glutathionylation was reversible by glutaredoxin activity. In summary, we found that PRKN gene expression contributes to the network of available thiols in the cell, including by parkin's participation in glutathione recycling, which involves a reversible, posttranslational modification at select cysteines. Further, parkin's impact on redox homeostasis in the cytosol can affect enzyme activities elsewhere, such as in mitochondria. We posit that antioxidant functions of parkin may explain many of its previously described, protective effects in vertebrates and invertebrates that are unrelated to E3 ligase activity.

Targeting oxidative distress to treat chemobrain: Go with the choroid plexus-cerebrospinal fluid flow.

Chemobrain is a variable but debilitating consequence of lifesaving cancer treatments. In this issue of Neuron, Jang and colleagues provide a new conceptual framework based on choroid plexus-CSF antioxidant status that advances understanding of chemobrain's nervous system pathobiology and identifies novel therapeutic opportunities.

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.

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.

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.

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 Chemical Biology of Ferroptosis in the Central Nervous System.

Over the past five decades, thanatology has come to include the study of how individual cells in our bodies die appropriately and inappropriately in response to physiological and pathological stimuli. Morphological and biochemical criteria have been painstakingly established to create clarity around definitions of distinct types of cell death and mechanisms for their activation. Among these, ferroptosis has emerged as a unique, oxidative stress-induced cell death pathway with implications for diseases as diverse as traumatic brain injury, hemorrhagic stroke, Alzheimer's disease, cancer, renal ischemia, and heat stress in plants. In this review, I highlight some of the formative studies that fostered its recognition in the nervous system and describe how chemical biological tools have been essential in defining events necessary for its execution. Finally, I discuss emerging opportunities for antiferroptotic agents as therapeutic agents in neurological diseases.

Manipulating Adult Neural Stem and Progenitor Cells with G-Quadruplex Ligands.

G-quadruplexes are pervasive nucleic acid secondary structures in mammalian genomes and transcriptomes that regulate gene expression and genome duplication. Small molecule ligands that modify the stability of G-quadruplexes are widely studied in cancer, but whether G-quadruplex ligands can also be used to manipulate cell function under normal development and homeostatic conditions is largely unexplored. Here we show that two related G-quadruplex ligands (pyridostatin and carboxypyridostatin) can reduce proliferation of adult neural stem cell and progenitor cells derived from the adult mouse subventricular zone both in vitro and in vivo. Studies with neurosphere cultures show that pyridostatin reduces proliferation by a mechanism associated with DNA damage and cell death. By contrast, selectively targeting RNA G-quadruplex stability with carboxypyridostatin diminishes proliferation through a mechanism that promotes cell cycle exit and the production of oligodendrocyte progenitors. The ability to generate oligodendrocyte progenitors by targeting RNA G-quadruplex stability, however, is dependent on the cellular environment. Together, these findings show that ligands that can selectively stabilize RNA G-quadruplexes are an important, new class of molecular tool for neural stem and progenitor cell engineering, whereas ligands that target DNA G-quadruplexes have limited utility due to their toxicity.

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.

Paired Associative Stimulation as a Tool to Assess Plasticity Enhancers in Chronic Stroke.

BACKGROUND AND PURPOSE: The potential for adaptive plasticity in the post-stroke brain is difficult to estimate, as is the demonstration of central nervous system (CNS) target engagement of drugs that show promise in facilitating stroke recovery. We set out to determine if paired associative stimulation (PAS) can be used (a) as an assay of CNS plasticity in patients with chronic stroke, and (b) to demonstrate CNS engagement by memantine, a drug which has potential plasticity-modulating effects for use in motor recovery following stroke. METHODS: We examined the effect of PAS in fourteen participants with chronic hemiparetic stroke at five time-points in a within-subjects repeated measures design study: baseline off-drug, and following a week of orally administered memantine at doses of 5, 10, 15, and 20 mg, comprising a total of seventy sessions. Each week, MEP amplitude pre and post-PAS was assessed in the contralesional hemisphere as a marker of enhanced or diminished plasticity. Strength and dexterity were recorded each week to monitor motor-specific clinical status across the study period. RESULTS: We found that MEP amplitude was significantly larger after PAS in baseline sessions off-drug, and responsiveness to PAS in these sessions was associated with increased clinical severity. There was no observed increase in MEP amplitude after PAS with memantine at any dose. Motor threshold (MT), strength, and dexterity remained unchanged during the study. CONCLUSION: Paired associative stimulation successfully induced corticospinal excitability enhancement in chronic stroke subjects at the group level. However, this response did not occur in all participants, and was associated with increased clinical severity. This could be an important way to stratify patients for future PAS-drug studies. PAS was suppressed by memantine at all doses, regardless of responsiveness to PAS off-drug, indicating CNS engagement.

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.

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.