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Glycosylation of lipids and proteins significantly increases the molecular diversity in the brain. Membrane-localized glycoconjugates facilitate critical neuro-immune interactions. Therefore, glycodysregulation is increasingly recognized as a novel hallmark of various acute and chronic neurological diseases. Although RNAs are heavily modified, they are never thought to be substrates for glycosylation due to their inaccessibility to the glycosylation machinery in the Golgi apparatus. The astonishing discovery of cell surface glycoRNAs opened new avenues for glycomedicine. This review highlighted the key features of GlycoRNAs and further discussed their potential immunomodulatory role in the brain, particularly focusing on post-stroke neuroinflammation.

Background: FTO (fat mass and obesity-associated protein) demethylates N 6 -methyladenosine (m 6 A), which is a critical epitranscriptomic regulator of neuronal function. We previously reported that ischemic stroke induces m 6 A hypermethylation with a simultaneous decrease in FTO expression in neurons. Currently, we evaluated the functional significance of restoring FTO with an adeno-associated virus 9, and thus reducing m 6 A methylation in poststroke brain damage. Methods: Adult male and female C57BL/6J mice were injected with FTO adeno-associated virus 9 (intracerebral) at 21 days prior to inducing transient middle cerebral artery occlusion. Poststroke brain damage (infarction, atrophy, and white matter integrity) and neurobehavioral deficits (motor function, cognition, depression, and anxiety-like behaviors) were evaluated between days 1 and 28 of reperfusion. Results: FTO overexpression significantly decreased the poststroke m 6 A hypermethylation. More importantly, exogenous FTO substantially decreased poststroke gray and white matter damage and improved motor function recovery, cognition, and depression-like behavior in both sexes. Conclusions: These results demonstrate that FTO-dependent m 6 A demethylation minimizes long-term sequelae of stroke independent of sex.

Accumulating evidence indicates a central role for epigenetic modifications in the progression of stroke pathology. These epigenetic mechanisms are involved in complex and dynamic processes that modulate post-stroke gene expression, cellular injury response, motor function, and cognitive ability. Despite decades of research, stroke continues to be classified as a leading cause of death and disability worldwide with limited clinical interventions. Thus, technological advances in the field of epigenetics may provide innovative targets to develop new stroke therapies. This review presents the evidence on the impact of epigenomic readers, writers, and erasers in both ischemic and hemorrhagic stroke pathophysiology. We specifically explore the role of DNA methylation, DNA hydroxymethylation, histone modifications, and epigenomic regulation by long non-coding RNAs in modulating gene expression and functional outcome after stroke. Furthermore, we highlight promising pharmacological approaches and biomarkers in relation to epigenetics for translational therapeutic applications.

Stroke leads to gut bacterial dysbiosis that impacts the post-stroke outcome. The gut microbiome also contains a high abundance of viruses which might play a crucial role in disease progression and recovery by modulating the metabolism of both host and host’s gut bacteria. We presently analyzed the virome composition (viruses and phages) by shotgun metagenomics in the fecal samples obtained at 1 day of reperfusion following transient focal ischemia in adult mice. Viral genomes, viral auxiliary metabolic genes, and viral protein networks were compared between stroke and sham conditions (stroke vs sham, exclusive to sham and exclusive to stroke). Following focal ischemia, abundances of 2 viral taxa decreased, and 5 viral taxa increased compared with the sham. Furthermore, the abundance of Clostridia-like phages and Erysipelatoclostridiaceae-like phages were altered in the stroke compared with the sham cohorts. This is the first report to show that the gut virome responds acutely to stroke.

Vitamin C has recently been identified as an epigenetic regulator by activating ten-eleven translocases (TETs), enzymes involved in generating DNA hydroxymethylcytosine (5hmC). Currently, we investigated whether high-dose vitamin C promotes neuroprotection through epigenetic modulation of 5hmC, if there are sex-specific differences in outcome, and the therapeutic potential of vitamin C in stroke-related comorbidities in adult mice. Post-stroke treatment with ascorbate (reduced form), but not dehydroascorbate (oxidized form), increased TET3 activity and 5hmC levels and reduced infarct following focal ischemia. Hydroxymethylation DNA immunoprecipitation sequencing showed that ascorbate increased 5hmC across the genome and specifically in promoters of several stroke pathophysiology-related genes, particularly anti-inflammatory genes. Ascorbate also decreased markers of oxidative stress, mitochondrial fragmentation, and apoptosis in cortical peri-infarct neurons and promoted motor and cognitive functional recovery in both sexes via TET3. Furthermore, post-stroke ascorbate treatment reduced infarct volume and improved motor function recovery in aged, hypertensive and diabetic male and female mice. Delayed ascorbate treatment at 6 h of reperfusion was still effective at reducing infarct volume and motor impairments in adult mice. Collectively, this study shows that post-stroke treatment with high-dose ascorbate protects the brain through epigenetic reprogramming and may function as a robust therapeutic against stroke injury.

Epigenetic mechanisms have been shown to play a major role in the progression of stroke pathophysiology. The ten eleven translocases (TETs) are enzymes involved in generating DNA hydroxymethylcytosine (5hmC), a brain enriched epigenetic modification that is associated with transcriptional activation and neuroprotection. We have previously shown that the TET3 isoform may be involved in promoting endogenous neuroprotective pathways following brain injury. In the current study, we investigated the role of TET3 activity after stroke by examining functional recovery, neuroprotection, and sex-specific differences after TET3 modulation. Adult C57BL6/J mice were subjected to transient middle cerebral artery occlusion (MCAO) to induce focal cerebral ischemia. Dot blotting analysis revealed robust induction of 5hmC levels from 1 hour to 24 hours of reperfusion in the mouse cortex. Intracerebral injection of a neuronal-specific TET3 adenovirus further enhanced TET activity and 5hmC levels in both male and female mice. Overexpression of TET3 led to decreased infarct volume and edema and improved neurological scores at 24 hours of reperfusion. Furthermore, both male and female mice subjected to a battery of motor function assessments from 3 days to 14 days of reperfusion displayed enhanced motor function recovery with increased TET3. These results indicate that TET3-mediated epigenetic regulation may hold therapeutic potential following cerebral ischemia.

We currently evaluated the effect of focal cerebral ischemia on gut virome and bacteriome in adult mice. Adult male C57BL/6 mice were subjected to transient middle cerebral artery occlusion or sham surgery. Virome and bacteriome were analyzed using shotgun metagenomics in the fecal samples collected from each mouse before and at 24h of reperfusion. Bioinformatics tools including VIBRANT (v1.2.1), DIAMOND Blastp (v0.9.14.115), VConTACT2 (v0.9.5), Cytoscape (v3.7.2), mash (v2.0), MUMmer (v3.1), Samtools (v1.11), DESeq2 (v1.28.1), MetaWRAP, and CRISPR Recognition Tool (v1.2) were used to assess viral networks, viral auxiliary metabolic genes, and viral protein network changes. Bacteriome was analyzed by kneaddata v0.7.2, MetaPhlAn version 2.7.7, and humann2 v2.8.1. Focal ischemia induced significant differences in fecal viral and bacterial taxa at the strain levels compared with sham. Furthermore, viral protein networks altered significantly after stroke. In particular, the clusters of Clostridia-like phages and Erysipelatoclostridiaceae phages showed a differential association between stroke and sham. In addition, we identified a significant reduction in the phages and bacteria of Lactobacillus after stroke. These studies indicate a possible viral-bacterial correlative change in the gut after stroke.

Fat mass and obesity-associated protein (FTO) demethylates N 6 -methyladenosine (m 6 A), which is a critical epitranscriptomic regulator of neuronal function. We previously reported that ischemic stroke induces m 6 A hypermethylation with a simultaneous decrease in FTO expression in the neurons. Currently, we evaluated the functional significance of restoring FTO with an adeno-associated virus 9 (AAV9), and thus reducing m 6 A methylation in the post-stroke brain damage. Adult male and female C57BL/6J mice were injected with FTO AAV9 (intracerebral) at 21 days prior to inducing transient middle cerebral artery occlusion. Post-stroke brain damage (infarction, atrophy and white matter integrity) and neurobehavioral deficits (motor function, cognition, depression and anxiety-like behaviors) were evaluated between days 1 and 28 of reperfusion. FTO overexpression significantly decreased the post-stroke m 6 A hypermethylation. More importantly, exogenous FTO substantially decreased post-stroke grey and white matter damage and improved motor function recovery, cognition and depression-like behavior in both sexes. These results demonstrate that FTO-dependent m 6 A demethylation minimizes long-term sequelae of stroke independent of sex.

Due to their role in controlling translation, microRNAs emerged as novel therapeutic targets to modulate post-stroke outcomes. We previously reported that miR-21 is the most abundantly induced microRNA in the brain of rodents subjected to preconditioning-induced cerebral ischemic tolerance. We currently show that intracerebral administration of miR-21 mimic decreased the infarct volume and promoted better motor function recovery in adult male and female C57BL/6 mice subjected to transient middle cerebral artery occlusion. The miR-21 mimic treatment is also efficacious in aged mice of both sexes subjected to focal ischemia. Mechanistically, miR-21 mimic treatment decreased the post-ischemic levels of several pro-apoptotic and pro-inflammatory RNAs, which might be responsible for the observed neuroprotection. We further observed post-ischemic neuroprotection in adult mice administered with miR-21 mimic intravenously. Overall, the results of this study implicate miR-21 as a promising candidate for therapeutic translation after stroke.

The role of tenascin-C (TNC) in ischemic stroke pathology is not known despite its prognostic association with cerebrovascular diseases. Here, we investigated the effect of TNC knockdown on post-stroke brain damage and its putative mechanism of action in adult mice of both sexes. Male and female C57BL/6 mice were subjected to transient middle cerebral artery occlusion and injected (i.v.) with either TNC siRNA or a negative (non-targeting) siRNA at 5 min after reperfusion. Motor function (beam walk and rotarod tests) was assessed between days 1 and 14 of reperfusion. Infarct volume (T2-MRI), BBB damage (T1-MRI with contrast), and inflammatory markers were measured at 3 days of reperfusion. The TNC siRNA treated cohort showed significantly curtailed post-stroke TNC protein expression, motor dysfunction, infarction, BBB damage, and inflammation compared to the sex-matched negative siRNA treated cohort. These results demonstrate that the induction of TNC during the acute period after stroke might be a mediator of post-ischemic inflammation and secondary brain damage independent of sex.

FTO (fat mass and obesity-associated protein) demethylatesAdult male and female C57BL/6J mice were injected with FTO adeno-associated virus 9 (intracerebral) at 21 days prior to inducing transient middle cerebral artery occlusion. Poststroke brain damage (infarction, atrophy, and white matter integrity) and neurobehavioral deficits (motor function, cognition, depression, and anxiety-like behaviors) were evaluated between days 1 and 28 of reperfusion.FTO overexpression significantly decreased the poststroke mThese results demonstrate that FTO-dependent m

Background and Purpose: Stroke induces the expression of several long noncoding RNAs in the brain. However, their functional significance in poststroke outcome is poorly understood. We recently observed that a brain-specific long noncoding RNA called Fos downstream transcript (FosDT) is induced rapidly in the rodent brain following focal ischemia. Using FosDT knockout rats, we presently evaluated the role of FosDT in poststroke brain damage. Methods: FosDT knockout rats were generated using CRISPR-Cas9 genome editing on a Sprague-Dawley background. Male and female FosDT −/− and FosDT +/+ cohorts were subjected to transient middle cerebral artery occlusion. Postischemic sensorimotor deficits were evaluated between days 1 and 7 and lesion volume on day 7 of reperfusion. The developmental expression profile of FosDT was determined with real-time polymerase chain reaction and mechanistic implications of FosDT in the ischemic brain were conducted with RNA-sequencing analysis and immunostaining of pathological markers. Results: FosDT expression is developmentally regulated, with the adult cerebral cortex showing significantly higher FosDT expression than neonates. FosDT −/− rats did not show any anomalies in growth and development, fertility, brain cytoarchitecture, and cerebral vasculature. However, when subjected to transient focal ischemia, FosDT −/− rats of both sexes showed enhanced sensorimotor recovery and reduced brain damage. RNA-sequencing analysis showed that improved poststroke functional outcome in FosDT −/− rats is partially associated with curtailed induction of inflammatory genes, reduced apoptosis, mitochondrial dysfunction, and oxidative stress. Conclusions: Our study shows that FosDT is developmentally dispensable, mechanistically important, and a functionally promising target to reduce ischemic brain damage and facilitate neurological recovery.

The complex pathophysiology of post-traumatic brain damage might need a polypharmacological strategy with a combination of drugs that target multiple, synergistic mechanisms. We currently tested a combination of apocynin (curtails formation of reactive oxygen species), tert-butylhydroquinone (promotes disposal of reactive oxygen species), and salubrinal (prevents endoplasmic reticulum stress) following a moderate traumatic brain injury (TBI) induced by controlled cortical impact in adult mice. Adult mice of both sexes treated with the above tri-combo showed alleviated motor and cognitive deficits, attenuated secondary lesion volume, and decreased oxidative DNA damage. Concomitantly, tri-combo treatment regulated post-TBI inflammatory response by decreasing the infiltration of T cells and neutrophils and activation of microglia in both sexes. Interestingly, sexual dimorphism was seen in the case of TBI-induced microgliosis and infiltration of macrophages in the tri-combo–treated mice. Moreover, the tri-combo treatment prevented TBI-induced white matter volume loss in both sexes. The beneficial effects of tri-combo treatment were long-lasting and were also seen in aged mice. Thus, the present study supports the tri-combo treatment to curtail oxidative stress and endoplasmic reticulum stress concomitantly as a therapeutic strategy to improve TBI outcomes. SIGNIFICANCE STATEMENT Of the several mechanisms that contribute to TBI pathophysiology, oxidative stress, endoplasmic reticulum stress, and inflammation play a major role. The present study shows the therapeutic potential of a combination of apocynin, tert-butylhydroquinone, and salubrinal to prevent oxidative stress and endoplasmic reticulum stress and the interrelated inflammatory response in mice subjected to TBI. The beneficial effects of the tri-combo include alleviation of TBI-induced motor and cognitive deficits and lesion volume. The neuroprotective effects of the tri-combo are also linked to its ability to prevent TBI-induced white matter damage. Importantly, neuroprotection by the tri-combo treatment was observed to be not dependent on sex or age. Our data demonstrate that a polypharmacological strategy is efficacious after TBI.

Transient focal ischemia decreased microRNA-7 (miR-7) levels, leading to derepression of its major target α-synuclein (α-Syn) that promotes secondary brain damage. Circular RNA CDR1as is known to regulate miR-7 abundance and function. Hence, we currently evaluated its functional significance after focal ischemia. Transient middle cerebral artery occlusion (MCAO) in adult mice significantly downregulated both CDR1as and miR-7 levels in the peri-infarct cortex between 3 and 72 h of reperfusion. Interestingly, neither pri-miR-7a nor 7b was altered in the ischemic brain. Intracerebral injection of an AAV9 vector containing a CDR1as gene significantly increased CDR1as levels by 21 days that persisted up to 4 months without inducing any observable toxicity in both sham and MCAO groups. Following transient MCAO, there was a significant increase in miR-7 levels and CDR1as binding to Ago2/miR-7 in the peri-infarct cortex of AAV9-CDR1as cohort compared with AAV9-Control cohort at 1 day of reperfusion. CDR1as overexpression significantly suppressed post-stroke α-Syn protein induction, promoted motor function recovery, decreased infarct size, and curtailed the markers of apoptosis, autophagy mitochondrial fragmentation, and inflammation in the post-stroke brain compared with AAV9-Control-treated cohort. Overall, our findings imply that CDR1as reconstitution is neuroprotective after stroke, probably by protecting miR-7 and preventing α-Syn-mediated neuronal death.

Following traumatic brain injury (TBI), increased production of reactive oxygen species (ROS) and the ensuing oxidative stress promotes the secondary brain damage that encompasses both grey matter and white matter. As this contributes to the long-term neurological deficits, decreasing oxidative stress during the acute period of TBI is beneficial. While NADPH oxidase (NOX2) is the major producer of ROS, transcription factor Nrf2 that induces antioxidant enzymes promotes efficient ROS disposal. We recently showed that treatment with an antioxidant drug combo of apocynin (NOX2 inhibitor) and TBHQ (Nrf2 activator) protects the grey matter in adult mice subjected to TBI. We currently show that this antioxidant combo therapy given at 2 h and 24 h after TBI also protects white matter in mouse brain. Thus, the better functional outcomes after TBI in the combo therapy treated mice might be due to a combination of sparing both grey matter and white matter. Hence, the antioxidant combo we tested is a potent therapeutic option for translation in future.

Cellular RNAs are pervasively tagged with diverse chemical moieties, collectively called epitranscriptomic modifications. The methylation of adenosine at N6 position generates N6-methyladenosine (m6A), which is the most abundant and reversible epitranscriptomic modification in mammals. The m6A signaling is mediated by a dedicated set of proteins comprised of writers, erasers, and readers. Contrary to the activation–repression binary view of gene regulation, emerging evidence suggests that the m6A methylation controls multiple aspects of mRNA metabolism, such as splicing, export, stability, translation, and degradation, culminating in the fine-tuning of gene expression. Brain shows the highest abundance of m6A methylation in the body, which is developmentally altered. Within the brain, m6A methylation is biased toward neuronal transcripts and sensitive to neuronal activity. In a healthy brain, m6A maintains several developmental and physiological processes such as neurogenesis, axonal growth, synaptic plasticity, circadian rhythm, cognitive function, and stress response. The m6A imbalance contributes to the pathogenesis of acute and chronic CNS insults, brain cancer, and neuropsychiatric disorders. This review discussed the molecular mechanisms of m6A regulation and its implication in the developmental, physiological, and pathological processes of the brain.

A robust innate immune activation leads to downstream expression of inflammatory mediators that amplify tissue damage and consequently increase the morbidity after stroke. The Toll-like receptor 4 (TLR4) pathway is a major innate immune pathway activated acutely and chronically after stroke. Hence, understanding the intricacies of the temporal profile, specific control points, and cellular specificity of TLR4 activation is crucial for the development of any novel therapeutics targeting the endogenous innate immune response after focal cerebral ischemia. The goal of this review is to summarize the current findings related to TLR4 signaling after stroke with a specific focus on the components of the neurovascular unit such as astrocytes, neurons, endothelial cells, and pericytes. In addition, this review will examine the effects of focal cerebral ischemia on interaction of these neurovascular unit components.

Autophagy is an evolutionarily conserved intracellular system that routes distinct cytoplasmic cargo to lysosomes for degradation and recycling. Accumulating evidence highlight the mechanisms of autophagy, such as clearance of proteins, carbohydrates, lipids and damaged organelles. The critical role of autophagy in selective degradation of the transcriptome is still emerging and could shape the total proteome of the cell, and thus can regulate the homeostasis under stressful conditions. Unregulated autophagy that potentiates secondary brain damage is a key pathological features of acute CNS injuries such as stroke and traumatic brain injury. This review discussed the mutual modulation of autophagy and RNA and its significance in mediating the functional consequences of acute CNS injuries.

A proper diet is important for health and longevity. Controlling the amount of food consumed is immensely beneficial as it promotes multiple cellular and molecular protective mechanisms and simultaneously prevents toxic mechanisms. Intermittent fasting (IF) is a flexible and easy-to-adopt dietary modification that helps to mitigate metabolic disorders like diabetes and hypertension, and thus the devastating age-related diseases like heart attack, stroke and dementia. The benefits of IF seem to be mediated by altered epigenetic and transcriptional programming leading to reduced oxidative stress, inflammation, mitochondrial damage and cell death.

Intermittent fasting (IF) remains the most effective intervention to achieve robust anti-aging effects and attenuation of age-related diseases in various species. Epigenetic modifications mediate the biological effects of several environmental factors on gene expression; however, no information is available on the effects of IF on the epigenome. Here, we first found that IF for 3 months caused modulation of H3K9 trimethylation (H3K9me(3)) in the cerebellum, which in turn orchestrated a plethora of transcriptomic changes involved in robust metabolic switching processes commonly observed during IF. Second, a portion of both the epigenomic and transcriptomic modulations induced by IF was remarkably preserved for at least 3 months post-IF refeeding, indicating that memory of IF-induced epigenetic changes was maintained. Notably, though, we found that termination of IF resulted in a loss of H3K9me(3) regulation of the transcriptome. Collectively, our study characterizes the novel effects of IF on the epigenetic-transcriptomic axis, which controls myriad metabolic processes. The comprehensive analyses undertaken in this study reveal a molecular framework for understanding how IF impacts the metabolo-epigenetic axis of the brain and will serve as a valuable resource for future research. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11357-022-00537-z.

Background: Ischemic stroke is a leading cause of mortality worldwide, largely due to the inflammatory response to brain ischemia during post-stroke reperfusion. Despite ongoing intensive research, there have not been any clinically approved drugs targeting the inflammatory component to stroke. Preclinical studies have identified T cells as pro-inflammatory mediators of ischemic brain damage, yet mechanisms that regulate the infiltration and phenotype of these cells are lacking. Further understanding of how T cells migrate to the ischemic brain and facilitate neuronal death during brain ischemia can reveal novel targets for post-stroke intervention.Methods: To identify the population of T cells that produce IL-21 and contribute to stroke, we performed transient middle cerebral artery occlusion (tMCAO) in mice and performed flow cytometry on brain tissue. We also utilized immunohistochemistry in both mouse and human brain sections to identify cell types and inflammatory mediators related to stroke-induced IL-21 signaling. To mechanistically demonstrate our findings, we employed pharmacological inhibitor anti-CXCL13 and performed histological analyses with Cresyl violet to evaluate its effects on brain infarct damage. Finally, to evaluate cellular mechanisms of stroke, we exposed mouse primary neurons to oxygen glucose deprivation (OGD) conditions with or without IL-21 and measured cell viability, caspase activity and JAK/STAT signaling.Results: Flow cytometry on brains from mice following tMCAO identified a novel population of cells IL-21 producing CXCR5+ CD4+ ICOS-1+ T follicular helper cells (TFH) in the ischemic brain early after injury. We observed augmented expression of CXCL13 on inflamed brain vascular cells and demonstrated that inhibition of CXCL13 protects mice from tMCAO by restricting the migration and influence of IL-21 producing TFH cells in the ischemic brain. We also illustrate that neurons express IL-21R in the peri-infarct regions of both mice and human stroke tissue in vivo. Lastly, we found that IL-21 acts on mouse primary ischemic neurons to activate the JAK/STAT pathway and induce Caspase 3/7 mediated apoptosis in vitro. Conclusion: These findings identify a novel mechanism for how pro-inflammatory T cells are recruited to the ischemic brain to propagate stroke damage and provide a potential novel therapeutic target for stroke.

We previously reported that increased expression of matrix metalloproteinase-12 (MMP-12) mediates blood-brain barrier disruption via tight junction protein degradation after focal cerebral ischemia in rats. Currently, we evaluated whether MMP-12 knockdown protects the post-stroke mouse brain and promotes better functional recovery. Adult male mice were injected with negative siRNA or MMP-12 siRNA (intravenous) at 5 min of reperfusion following 1 h transient middle cerebral artery occlusion. MMP-12 knockdown significantly reduced the post-ischemic infarct volume and improved motor and cognitive functional recovery. Mechanistically, MMP-12 knockdown ameliorated degradation of tight junction proteins zonula occludens-1, claudin-5, and occludin after focal ischemia. MMP-12 knockdown also decreased the expression of inflammatory mediators, including monocyte chemoattractant protein-1, tumor necrosis factor-α, and interleukin-6, and the expression of apoptosis marker cleaved caspase-3 after ischemia. Overall, the present study indicates that MMP-12 promotes secondary brain damage after stroke and hence is a promising stroke therapeutic target.

Transient focal ischemia induces a sustained downregulation of miR-7 leading to derepression of its target α-synuclein (α-Syn), which promotes neuronal death. We previously showed that treatment with miR-7 mimic prevents α-Syn induction and protects brain after stroke in rodents irrespective of age and sex. To further decipher the role of miR-7, we currently studied infarction and motor function in miR-7 double knockout mice (lack both miR-7a and miR-7b) subjected to focal ischemia. Adult miR-7(−/−) mice showed similar motor and cognitive functions to miR-7(+/+) mice. However, when subjected to even a mild focal ischemia, the miR-7(−/−) mice showed exacerbated brain damage and worsened motor function compared with the miR-7(+/+) mice. Replenishing miR-7 in miR-7(−/−) mice (IV injection of miR-7 mimic) restored miR-7 mediated neuroprotection and motor recovery, potentially by preventing α-Syn protein induction. Thus, we show that miR-7 is an essential miRNA in the brain that prevents α-Syn translation and the ensuing brain damage after stroke.

Due to their role in controlling translation, microRNAs emerged as novel therapeutic targets to modulate post-stroke outcomes. We previously reported that miR-21 is the most abundantly induced microRNA in the brain of rodents subjected to preconditioning-induced cerebral ischemic tolerance. We currently show that intracerebral administration of miR-21 mimic decreased the infarct volume and promoted better motor function recovery in adult male and female C57BL/6 mice subjected to transient middle cerebral artery occlusion. The miR-21 mimic treatment is also efficacious in aged mice of both sexes subjected to focal ischemia. Mechanistically, miR-21 mimic treatment decreased the post-ischemic levels of several pro-apoptotic and pro-inflammatory RNAs, which might be responsible for the observed neuroprotection. We further observed post-ischemic neuroprotection in adult mice administered with miR-21 mimic intravenously. Overall, the results of this study implicate miR-21 as a promising candidate for therapeutic translation after stroke.

Diet is a significant factor in determining human well-being. Excessive eating and/or diets with higher than needed amounts of carbohydrates, salt, and fat are known to cause metabolic disorders and functional changes in the body. To compensate the ill effects, many designer diets including the Mediterranean diet, the Okinawa diet, vegetarian/vegan diets, keto diet, anti-inflammatory diet, and the anti-oxidant diet have been introduced in the past 2 decades. While these diets are either enriched or devoid of one or more specific components, a better way to control diet is to limit the amount of food consumed. Caloric restriction (CR), which involves limiting the amount of food consumed rather than eliminating any specific type of food, as well as intermittent fasting (IF), which entails limiting the time during which food can be consumed on a given day, have gained popularity because of their positive effects on human health. While the molecular mechanisms of these 2 dietary regimens have not been fully deciphered, they are known to prolong the life span, control blood pressure, and blood glucose levels. Furthermore, CR and IF were both shown to decrease the incidence of heart attack and stroke, as well as their ill effects. In particular, IF is thought to promote metabolic switching by altering gene expression profiles leading to reduced inflammation and oxidative stress, while increasing plasticity and regeneration.

Background and Purpose— Adenosine in many types of RNAs can be converted to m 6 A (N 6 -methyladenosine) which is a highly dynamic epitranscriptomic modification that regulates RNA metabolism and function. Of all organs, the brain shows the highest abundance of m 6 A methylation of RNAs. As recent studies showed that m 6 A modification promotes cell survival after adverse conditions, we currently evaluated the effect of stroke on cerebral m 6 A methylation in mRNAs and lncRNAs. Methods— Adult C57BL/6J mice were subjected to transient middle cerebral artery occlusion. In the peri-infarct cortex, m 6 A levels were measured by dot blot analysis, and transcriptome-wide m 6 A changes were profiled using immunoprecipitated methylated RNAs with microarrays (44 122 mRNAs and 12 496 lncRNAs). Gene ontology analysis was conducted to understand the functional implications of m 6 A changes after stroke. Expression of m 6 A writers, readers, and erasers was also estimated in the ischemic brain. Results— Global m 6 A levels increased significantly at 12 hours and 24 hours of reperfusion compared with sham. While 139 transcripts (122 mRNAs and 17 lncRNAs) were hypermethylated, 8 transcripts (5 mRNAs and 3 lncRNAs) were hypomethylated (>5-fold compared with sham) in the ischemic brain at 12 hours reperfusion. Inflammation, apoptosis, and transcriptional regulation are the major biological processes modulated by the poststroke differentially m 6 A methylated mRNAs. The m 6 A writers were unaltered, but the m 6 A eraser (fat mass and obesity-associated protein) decreased significantly after stroke compared with sham. Conclusions— This is the first study to show that stroke alters the cerebral m 6 A epitranscriptome, which might have functional implications in poststroke pathophysiology. Visual Overview— An online visual overview is available for this article.

RNA is more than just a combination of four genetically encoded nucleobases as it carries extra information in the form of epitranscriptomic modifications. Diverse chemical groups attach covalently to RNA to enhance the plasticity of cellular transcriptome. The reversible and dynamic nature of epitranscriptomic modifications allows RNAs to achieve rapid and context-specific gene regulation. Dedicated cellular machinery comprising of writers, erasers, and readers drives the epitranscriptomic signaling. Epitranscriptomic modifications control crucial steps of mRNA metabolism such as splicing, export, localization, stability, degradation, and translation. The majority of the epitranscriptomic modifications are highly abundant in the brain and contribute to activity-dependent gene expression. Thus, they regulate the vital physiological processes of the brain, such as synaptic plasticity, neurogenesis, and stress response. Furthermore, epitranscriptomic alterations influence the progression of several neurologic disorders. This review discussed the molecular mechanisms of epitranscriptomic regulation in neurodevelopmental and neuropathological conditions with the goal to identify novel therapeutic targets.

The mammalian brain expresses several classes of noncoding RNAs (ncRNAs), including long ncRNAs (lncRNAs), circular RNAs (circRNAs), and microRNAs (miRNAs). These ncRNAs play vital roles in regulating cellular processes by RNA/protein scaffolding, sponging and epigenetic modifications during the pathophysiological conditions, thereby controlling transcription and translation. Some of these functions are the result of crosstalk between ncRNAs to form a competitive endogenous RNA network. These intricately organized networks comprise lncRNA/miRNA, circRNA/miRNA, or lncRNA/miRNA/circRNA, leading to crosstalk between coding and ncRNAs through miRNAs. The miRNA response elements predominantly mediate the ncRNA crosstalk to buffer the miRNAs and thereby fine-tune and counterbalance the genomic changes and regulate neuronal plasticity, synaptogenesis and neuronal differentiation. The perturbed levels and interactions of the ncRNAs could lead to pathologic events like apoptosis and inflammation. Although the regulatory landscape of the ncRNA crosstalk is still evolving, some well-known examples such as lncRNA Malat1 sponging miR-145, circRNA CDR1as sponging miR-7, and lncRNA Cyrano and the circRNA CDR1as regulating miR-7, has been shown to affect brain function. The ability to manipulate these networks is crucial in determining the functional outcome of central nervous system (CNS) pathologies. The focus of this review is to highlights the interactions and crosstalk of these networks in regulating pathophysiologic CNS function.

Stroke induces the expression of several long noncoding RNAs (lncRNAs) in the brain. However, their functional significance in post-stroke outcome is poorly understood. We recently observed that a brain-specific lncRNA called Fos downstream transcript (FosDT) is induced rapidly in rodent brain following focal ischemia. We here show that FosDT expression is developmentally regulated with adults specifically display higher expression in the cerebral cortex than neonates. To understand its significance in ischemic brain damage, we developed FosDT knockout rats using CRISPR-Cas9 genome editing. We found that FosDT knockout rats did not show any anomalies in growth and development, fertility, brain cytoarchitecture and cerebral vasculature. However, when subjected to transient focal ischemia, FosDT knockout rats of both sexes showed enhanced sensorimotor recovery and reduced brain damage. To further understand the mechanistic implications of FosDT in the ischemic brain, we conducted RNA-seq analysis. The result showed that improved post-stroke functional outcome in FosDT knockout rats is partially associated with curtailed post-ischemic induction of inflammatory genes. When rats subjected to transient focal ischemia were treated with FosDT siRNA, there was significant neuroprotection and better functional outcome irrespective of sex and age. FosDT siRNA was efficacious when administered peripherally and also in a delayed manner. Thus, preventing FosDT activation is beneficial for the post-stroke outcome.

The antioxidant ascorbate is an enzyme cofactor with established roles in maintaining brain function and providing neuroprotection in response to oxidative stress. Recently ascorbate has been identified as an epigenetic regulator through its ability to induce the activity of the ten-eleven translocase (TET) enzymes that produce 5-hydroxymethylcytosine (5-hmC), a CNS-enriched epigenetic modification that is associated with transcriptional activation and neuroprotection. In the current study, we evaluated the role of ascorbate on 5-hmC and its therapeutic potential against ischemic brain injury in comorbid conditions associated with stroke in mice. Adult mice subjected to transient middle cerebral artery occlusion (MCAO) were injected intraperitoneally (i.p.) with ascorbate at 30 min of reperfusion. Young adult male and female mice showed robust induction of 5hmC in the peri-infarct region of the cortex, reduced infarct size and improved motor function. Knockdown of TET3 by intracerebral injection using siRNA blocked the ascorbate-induced increases in 5hmC and led to increased brain degeneration and motor function deficits. Genome-wide sequencing analysis of differentially hydroxymethylated regions (DhMRs) revealed that ascorbate increased 5hmC at the promoters of genes associated with protection against ischemia pathophysiology. Furthermore, ascorbate treatment reduced infarct and improved functional recovery in aged, obese diabetic and hypertensive male and female mice. Delayed ascorbate treatment at 6h of reperfusion was also protective against secondary brain damage and motor deficits following experimental stroke. Collectively, these results indicate that ascorbate regulates the 5hmC epigenetic modification in a neuroprotective manner and may represent a promising therapeutic target for stroke treatment.

The mechanistic role of Tenascin-C (TNC) in the pathogenesis of acute ischemic stroke is not known despite its prognostic association with cerebrovascular diseases. We currently observed that transient middle cerebral artery occlusion (MCAO) upregulated cerebral TNC mRNA and protein expression between 3h and 24h reperfusion in adult mice of both sexes. We then evaluated the effect of TNC knockdown on ischemic outcome in adult mice of both sexes by treating with either TNC siRNA or control siRNA (intravenous) at 5 min of reperfusion following transient MCAO. TNC siRNA treatment significantly reduced the post-ischemic TNC protein induction tested at 72h reperfusion compared with the sex-matched control siRNA treated cohorts (n=6/group/sex). TNC siRNA cohorts showed significantly improved post-stroke motor function identified by beam walk test and rotarod test between days 1 and 14 of reperfusion compared with the sex-matched control siRNA cohorts (n=7/group/sex). TNC siRNA cohorts of both sexes also showed decreased post-ischemic BBB disruption (evaluated with T1-weighted MRI with gadobenate dimeglumine as contrast agent) and reduced infarction (assessed with T2-weighted MRI) at 3 days of reperfusion compared with the sex-matched control siRNA treated cohorts (n =4/group for BBB and n =12/group/sex for infarct). At day 21 of reperfusion, the survival rate was observed to be higher in the TNC siRNA treated mice compared with the control siRNA treated mice (n =7/group/sex). These studies thus show that induction of TNC during the acute period after stroke might be a mediator of ischemic brain damage and its knockdown is neuroprotective. Importantly this effect is independent of sex. The study was funded by the Department of Neurological Surgery, Univ. of Wisconsin-Madison.

Following traumatic brain injury (TBI), increased production of reactive oxygen species (ROS) and the ensuing oxidative stress promotes the secondary brain damage that encompasses both grey matter and white matter. As this contributes to the long-term neurological deficits, decreasing oxidative stress during the acute period of TBI is beneficial. While NADPH oxidase (NOX2) is the major producer of ROS, transcription factor Nrf2 that induces antioxidant enzymes promotes efficient ROS disposal. We recently showed that treatment with an antioxidant drug combo of apocynin (NOX2 inhibitor) and TBHQ (Nrf2 activator) protects the grey matter in adult mice subjected to TBI. We currently show that this antioxidant combo therapy given at 2 h and 24 h after TBI also protects white matter in mouse brain. Thus, the better functional outcomes after TBI in the combo therapy treated mice might be due to a combination of sparing both grey matter and white matter. Hence, the antioxidant combo we tested is a potent therapeutic option for translation in future.

Cellular RNAs are pervasively tagged with diverse chemical moieties, collectively called epitranscriptomic modifications. The methylation of adenosine at N(6) position generates N(6)-methyladenosine (m(6)A), which is the most abundant and reversible epitranscriptomic modification in mammals. The m(6)A signaling is mediated by a dedicated set of proteins comprised of writers, erasers, and readers. Contrary to the activation–repression binary view of gene regulation, emerging evidence suggests that the m(6)A methylation controls multiple aspects of mRNA metabolism, such as splicing, export, stability, translation, and degradation, culminating in the fine-tuning of gene expression. Brain shows the highest abundance of m(6)A methylation in the body, which is developmentally altered. Within the brain, m(6)A methylation is biased toward neuronal transcripts and sensitive to neuronal activity. In a healthy brain, m(6)A maintains several developmental and physiological processes such as neurogenesis, axonal growth, synaptic plasticity, circadian rhythm, cognitive function, and stress response. The m(6)A imbalance contributes to the pathogenesis of acute and chronic CNS insults, brain cancer, and neuropsychiatric disorders. This review discussed the molecular mechanisms of m(6)A regulation and its implication in the developmental, physiological, and pathological processes of the brain.

Background and Purpose: Increased expression of α-Syn (α-Synuclein) is known to mediate secondary brain damage after stroke. We presently studied if α-Syn knockdown can protect ischemic brain irrespective of sex and age. Methods: Adult and aged male and female mice were subjected to transient middle cerebral artery occlusion. α-Syn small interfering RNA (siRNA) was administered intravenous at 30 minutes or 3 hour reperfusion. Poststroke motor deficits were evaluated between day 1 and 7 and infarct volume was measured at day 7 of reperfusion. Results: α-Syn knockdown significantly decreased poststroke brain damage and improved poststroke motor function recovery in adult and aged mice of both sexes. However, the window of therapeutic opportunity for α-Syn siRNA is very limited. Conclusions: α-Syn plays a critical role in ischemic brain damage and preventing α-Syn protein expression early after stroke minimizes poststroke brain damage leading to better functional outcomes irrespective of age and sex.

Ubiquitination by Nedd4 (neuronally expressed developmentally downregulated 4) family of HECT type E3 ligases plays a key role in degrading misfolded and damaged proteins, and its disruption leads to neurodegeneration. Parkinson's disease-causing protein α-Synuclein (α-Syn) is ubiquitinated by the Nedd4 family and degraded by endosomes. Nedd4l is the only Nedd4 homolog that showed upregulation in post-stroke surviving cortical neurons where it correlated with neuroprotection. We tested the role of Nedd4l after stroke by subjecting the Nedd4l−/− mice to transient middle cerebral artery occlusion. Focal ischemia significantly increased Nedd4l expression and poly-ubiquitinated α-Syn levels, and knockout of Nedd4l reduced post-ischemic poly-ubiquitinated α-Syn that is majorly located in the peri-infarct neurons. Co-immunoprecipitation further shows that focal ischemia enhances the α-Syn-Nedd4l interaction resulting in increased ubiquitination of α-Syn. Nedd4l knockout mice ( n = 7 mice/group) showed exacerbated post-ischemic motor dysfunction manifested by decreased time on the rotarod and increased number of foot faults, and significantly increased ischemic brain damage. This suggests that Nedd4l might be a potential therapeutic target to minimize α-Syn-mediated toxicity after cerebral ischemia.

Stroke leads to significant neuronal death and long-term neurological disability due to synergistic pathogenic mechanisms. Stroke induces a change in eating habits and in many cases, leads to undernutrition that aggravates the post-stroke pathology. Proper nutritional regimen remains a major strategy to control the modifiable risk factors for cardiovascular and cerebrovascular diseases including stroke. Studies indicate that nutraceuticals (isolated and concentrated form of high-potency natural bioactive substances present in dietary nutritional components) can act as prophylactic as well as adjuvant therapeutic agents to prevent stroke risk, to promote ischemic tolerance and to reduce post-stroke consequences. Nutraceuticals are also thought to regulate blood pressure, delay neurodegeneration and improve overall vascular health. Nutraceuticals potentially mediate these effects by their powerful antioxidant and anti-inflammatory properties. This review discusses the studies that have highlighted the translational potential of nutraceuticals as stroke therapies.

Traumatic brain injury (TBI) is known to promote significant DNA damage irrespective of age, sex, and species. Chemical as well as structural DNA modification start within minutes and persist for days after TBI. Although several DNA repair pathways are induced following TBI, the simultaneous downregulation of some of the genes and proteins of these pathways leads to an aberrant overall DNA repair process. In many instances, DNA damages escape even the most robust repair mechanisms, especially when the repair process becomes overwhelmed or becomes inefficient by severe or repeated injuries. The persisting DNA damage and/or lack of DNA repair contributes to long-term functional deficits. In this review, we discuss the mechanisms of TBI-induced DNA damage and repair. We further discussed the putative experimental therapies that target the members of the DNA repair process for improved outcome following TBI.