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The 5-hydroxymethylcytosine (5hmC) epigenetic modification is highly enriched in the CNS and a critical modulator of neuronal function and development. We found that cortical 5hmC was enhanced from 5 min to three days of reperfusion following focal ischemia in adult mice. Blockade of the 5hmC-producing enzyme ten-eleven translocase 3 (TET3) increased edema, infarct volume, and motor function impairments. To determine the mechanism by which TET3 provides ischemic neuroprotection, we assessed the genomic regions where TET3 modulates 5hmC. Genome-wide sequencing analysis of differentially hydroxymethylated regions (DhMRs) revealed that focal ischemia robustly increased 5hmC at the promoters of thousands of genes in a TET3-dependent manner. TET3 inhibition reduced 5hmC at the promoters of neuroprotective genes involved in cell survival, angiogenesis, neurogenesis, antioxidant defense, DNA repair, and metabolism demonstrating a role for TET3 in endogenous protection against stroke. The mRNA expression of several genes with known involvement in ischemic neuroprotection were also reduced with TET3 knockdown in both male and female mice, establishing a correlation between decreased promoter 5hmC levels and decreased gene expression. Collectively, our results indicate that TET3 globally increases 5hmC at regulatory regions and overwhelmingly modulates 5hmC in several neuroprotective pathways that may improve outcome after ischemic injury.

Intermittent fasting (IF) is a lifestyle intervention comprising a dietary regimen in which energy intake is restricted via alternating periods of fasting and ad libitum food consumption, without compromising nutritional composition. While epigenetic modifications can mediate effects of environmental factors on gene expression, no information is yet available on potential effects of IF on the epigenome. In this study, we found that IF causes modulation of histone H3 lysine 9 trimethylation (H3K9me3) epigenetic mark in the cerebellum of male C57/BL6 mice, which in turn orchestrates a plethora of transcriptomic changes involved in the robust metabolic switching processes commonly observed during IF. Interestingly, both epigenomic and transcriptomic modulation continued to be observed after refeeding, suggesting that memory of the IF-induced epigenetic change is maintained at the locus. Notably though, we found that termination of IF results in a loss of H3K9me3 regulation of the transcriptome. Collectively, our study characterizes a novel mechanism of IF in the epigenetic-transcriptomic axis, which controls myriad metabolic process changes. In addition to providing a valuable and innovative resource, our systemic analyses reveal molecular framework for understanding how IF impacts the metaboloepigenetics axis of the brain.Highlights○ Intermittent fasting (IF) and refeeding modifies epigenome in the cerebellum○ Integrative epigenomic and transcriptomic analyses revealed metabolic switching○ IF affects the metaboloepigenetics axis in regulating metabolic processes○ Integrative analyses revealed a loss of epigenetic reprogramme following refeeding

Introduction: We recently showed in a rodent model of transient focal cerebral ischemia that matrix metalloproteinase-12 (MMP-12) induction in the ischemic brain promotes post-stroke blood-brain barrier disruption, apoptosis, demyelination, and infarction. The purpose of the present study is to investigate the role of elevated MMP-12 on post-stroke neurological function and to identify the time window of therapeutic opportunity for MMP-12 suppression. Methods: Adult male Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion and reperfusion. Cohorts of rats (n =8-15/group) were administered with either MMP-12 shRNA or scrambled shRNA sequence (vehicle control) expressing plasmids (1 mg/Kg; intravenous) formulated as nanoparticles. The differences in sample size of various cohorts were attributed to the exclusion criteria followed, high mortality rate in vehicle control-treated group, and sample size required for statistical analysis. To assess the reflex, balance, sensory, and motor functions, rats from various cohorts were subjected to modified neurological severity scoring (mNSS), adhesive removal test, beam walk test and rotarod test at day 1, 3 and 5 of reperfusion. To assess the time window of therapeutic opportunity, various cohorts of rats were treated at 5 min, 3h, and 6h of reperfusion. Investigators blinded to study groups analyzed all outcome parameters. Results: The post-stroke percent survival rate in cohorts treated with MMP12shRNA expressing plasmids range from 82 to 89 as compared to 67 in vehicle control-treated group. The cohort of rats treated at 5 min of reperfusion with MMP-12snRNA expressing plasmids showed significantly better functional recovery as assessed by various neurological tests. However, delayed administration of MMP-12snRNA expressing plasmid (either at 3h or 6h of reperfusion) failed to promote any significant improvement in post-stroke neurological recovery. Conclusions: Post-stroke induction of MMP-12 in the ischemic brain contributes to neurological deficits and impedes recovery. MMP-12 targeting treatments immediately after reperfusion could offer substantial therapeutic benefits.

Intermittent fasting (IF) showed a substantial benefit in preventing vascular complications in both humans and animal models. We currently evaluated the effect of IF on post-stroke ischemic progression and motor dysfunction. Cohorts of adult male C57BL/6 mice were subjected to IF (16 h fasting and 8 h feeding) or ad libitum (AL) feeding for 45 days. Mice were subjected to one hour transient middle cerebral artery occlusion (MCAO) on day 45. Infarct size was measured on day 1 and 7 with T2-weighted 4.5T magnetic resonance imaging and Image J software with multi FDF opener plugin. Post-stroke motor deficits were evaluated with rotarod and beam walk tests between days 1 to 14 of reperfusion. Long-term post-stroke survival (till 90 days of reperfusion) was noted in both groups. IF group showed a significant decrease in body weight (by ~5.3g), soft weight (by ~5.6g), and lean weight (~8.1g) compared to AL group as identified by DEXA scanning on day 45 (n=6/group, *p

Degeneration of sympathetic innervation of the heart occurs in numerous diseases, including diabetes, idiopathic REM sleep disorder, and Parkinson’s disease (PD). In PD, cardiac sympathetic denervation occurs in 80-90% of patients and can begin before the onset of motor symptoms. Today, there are no disease-modifying therapies for cardiac sympathetic neurodegeneration, and biomarkers are limited to radioimaging techniques. Analysis of expression levels of coding mRNA and noncoding RNAs, such as microRNAs (miRNAs), can uncover pathways involved in disease, leading to the discovery of biomarkers, pathological mechanisms, and potential drug targets. Whole blood in particular is a clinically relevant source of biomarkers, as blood sampling is inexpensive and simple to perform. Our research group has previously developed a nonhuman primate model of cardiac sympathetic denervation by intravenous administration of the catecholaminergic neurotoxin 6-hydroxydopamine (6-OHDA). In this rhesus macaque (Macaca mulatta) model, imaging with positron emission tomography showed that oral administration of the peroxisome proliferator-activated receptor gamma (PPARγ) agonist pioglitazone (n=5; 5 mg/kg daily) significantly decreased cardiac inflammation and oxidative stress compared to placebo (n=5). Here, we report our analysis of miRNA and mRNA expression levels over time in the whole blood of these monkeys. Differential expression of three miRNAs was induced by 6-OHDA (mml-miR-16-2-3p, mml-miR-133d-3p, and mml-miR-1262-5p) and two miRNAs by pioglitazone (mml-miR-204-5p and mml-miR-146b-5p) at 12 weeks posttoxin, while expression of mRNAs involved in inflammatory cytokines and receptors was not significantly affected. Overall, this study contributes to the characterization of rhesus coding and noncoding RNA profiles in normal and disease-like conditions, which may facilitate the identification and clinical translation of biomarkers of cardiac neurodegeneration and neuroprotection.

Supplemental material, sj-pdf-1-jcb-10.1177_0271678X20943804 for Deletion of ubiquitin ligase Nedd4l exacerbates ischemic brain damage by TaeHee Kim, Anil K Chokkalla and Raghu Vemuganti in Journal of Cerebral Blood Flow & Metabolism

Supplemental material, sj-pdf-1-jcb-10.1177_0271678X20912965 for TET3 regulates DNA hydroxymethylation of neuroprotective genes following focal ischemia by Kahlilia C Morris-Blanco, Anil K Chokkalla, Mario J Bertogliat and Raghu Vemuganti in Journal of Cerebral Blood Flow & Metabolism

Oxidative stress plays a crucial role in traumatic brain injury (TBI) pathogenesis. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) formed in excess after TBI synergistically contribute to secondary brain damage together with lipid peroxidation products (reactive aldehydes) and inflammatory mediators. Furthermore, oxidative stress, endoplasmic reticulum stress and inflammation potentiate each other. Following TBI, excessive oxidative stress overloads the endogenous cellular antioxidant system leading to cell death. To combat oxidative stress, several antioxidant therapies were tested in preclinical animal models of TBI. These include free radical scavengers, activators of antioxidant systems, Inhibitors of free radical generating enzymes and antioxidant enzymes. Many of these therapies showed promising outcomes including reduced edema, blood-brain barrier (BBB) protection, smaller contusion volume, and less inflammation. In addition, many antioxidant therapies also promoted better sensory, motor, and cognitive functional recovery after TBI. Overall, preventing oxidative stress is a viable therapeutic option to minimize the secondary damage and to improve the quality of life after TBI.

Cerebral ischemia is known to activate the repressor element-1 (RE1)-silencing transcription factor (REST) which silences neural genes via epigenetic remodeling and promotes neurodegeneration. We presently determined if REST inhibition derepresses target genes involved in synaptic plasticity and promotes functional outcome after experimental stroke. Following transient focal ischemia induced by middle cerebral artery occlusion (MCAO) in adult rats, REST expression was upregulated significantly from 12 h to 1 day of reperfusion compared to sham control. At 1 day of reperfusion, REST protein levels were increased and observed in the nuclei of neurons in the peri-infarct cortex. REST knockdown by intracerebral REST siRNA injection significantly reduced the post-ischemic expression of REST and increased the expression of several REST target genes, compared to control siRNA group. REST inhibition also decreased post-ischemic markers of apoptosis, reduced cortical infarct volume, and improved post-ischemic functional recovery on days 5 and 7 of reperfusion compared to the control siRNA group. REST knockdown resulted in a global increase in synaptic plasticity gene expression at 1 day of reperfusion compared to the control siRNA group and significantly increased several synaptic plasticity genes containing RE-1 sequences in their regulatory regions. These results demonstrate that direct inhibition of the epigenetic remodeler REST prevents secondary brain damage in the cortex and improves functional outcome potentially via de-repression of plasticity-related genes after stroke.

Hepatic encephalopathy (HE) represents a serious complication of acute liver failure (ALF) in which cerebral edema leading to brainstem herniation as a result of increased intracranial hypertension is a major consequence. Long non-coding RNAs (lncRNAs) play a significant role in coordinating gene expression, with recent studies indicating an influence in the pathogenesis of several diseases. To investigate their involvement in the cerebral pathophysiology of ALF, we profiled the expression of lncRNAs in the frontal cortex of mice at coma stage following treatment with the hepatotoxin azoxymethane. Of the 35,923 lncRNAs profiled using microarrays, 868 transcripts were found to be differentially expressed in the ALF-treated group compared to the sham control group. Of these, 382 lncRNAs were upregulated and 486 lncRNAs downregulated. Pathway analysis revealed these lncRNAs target a number of biological and molecular pathways that include cytokine-cytokine receptor interaction, the mitogen activated protein kinase signaling pathway, the insulin signaling pathway, and the nuclear factor-κB signaling pathway. False discovery rate adjustment identified 9 upregulated lncRNAs, 2 of which are associated with neuroepithelial transforming gene 1 (NET1) and the monocarboxylate transporter 2 (Slc16a7), potential contributors to astrocyte cytoskeletal disruption/swelling and lactate production, respectively. Our findings suggest an important role for lncRNAs in the brain in ALF in relation to inflammation, neuropathology, and in terms of the functional basis of HE. Further work on these non-coding RNAs may lead to new therapeutic approaches for the treatment and management of cerebral dysfunction resulting from this potentially life-threatening disorder.

The role of matrix metalloproteinase-12 (MMP-12) in the pathogenesis of several inflammatory diseases such as chronic obstructive pulmonary disease, emphysema, and asthma is well established. Several new studies and recent reports from our laboratory and others highlighted the detrimental role of MMP-12 in the pathogenesis of several neurological diseases. In this review, we discuss in detail the pathological role of MMP-12 and the possible underlying molecular mechanisms that contribute to disease pathogenesis in the context of central nervous system diseases such as stroke, spinal cord injury, and multiple sclerosis. The available information on the specific MMP-12 inhibitors used in several preclinical and clinical studies is also reviewed. Based on the reported studies to date, MMP-12 suppression could emerge as a promising therapeutic target for several CNS diseases that were discussed in this review.

Epigenetic modifications are emerging as major players in the pathogenesis of neurodegenerative disorders and susceptibility to acute brain injury. DNA and histone modifications act together with non-coding RNAs to form a complex gene expression machinery that adapts the brain to environmental stressors and injury response. These modifications influence cell-level operations like neurogenesis and DNA repair to large, intricate processes such as brain patterning, memory formation, motor function and cognition. Thus, epigenetic imbalance has been shown to influence the progression of many neurological disorders independent of aberrations in the genetic code. This review aims to highlight ways in which epigenetics applies to several commonly researched neurodegenerative diseases and forms of acute brain injury as well as shed light on the benefits of epigenetics-based treatments.

BACKGROUND AND PURPOSE: Adenosine in many types of RNAs can be converted to N(6)-methyladenosine (m(6)A) 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 12h and 24h of reperfusion compared to sham. While 139 transcripts (122 mRNAs and 17 lncRNAs) were hypermethylated, 8 transcripts (5 mRNAs and 3 lncRNAs) were hypomethylated (>5-fold compared to sham) in the ischemic brain at 12h reperfusion. Inflammation, apoptosis and transcriptional regulation are the major biological processes modulated by the post-stroke 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 to sham. CONCLUSIONS: This is the first study to show that stroke alters the cerebral m(6)A epitranscriptome which might have functional implications in post-stroke pathophysiology.

Chronic kidney disease has a graded and independent inverse impact on cerebrovascular health. Both thrombotic and hemorrhagic complications are highly prevalent in chronic kidney disease patients. Growing evidence suggests that in chronic kidney disease patients, ischemic strokes are more common than hemorrhagic strokes. Chronic kidney disease is asymptomatic until an advanced stage, but mild to moderate chronic kidney disease incites various pathogenic mechanisms such as inflammation, oxidative stress, neurohormonal imbalance, formation of uremic toxins and vascular calcification which damage the endothelium and blood vessels. Cognitive dysfunction, dementia, transient infarcts, and white matter lesions are widespread in mild to moderate chronic kidney disease patients. Uremic toxins produced after chronic kidney disease can pass through the blood–brain barrier and mediate cognitive dysfunction and neurodegeneration. Furthermore, chronic kidney disease precipitates vascular risk factors that can lead to atherosclerosis, hypertension, atrial fibrillation, and diabetes. Chronic kidney disease also exacerbates stroke pathogenesis, worsens recovery outcomes, and limits the eligibility of stroke patients to receive available stroke therapeutics. This review highlights the mechanisms involved in the advancement of chronic kidney disease and its possible association with stroke.

Background and Purpose— Epigenetics play a significant role in brain pathologies. We currently evaluated the role of a recently discovered brain-enriched epigenetic modification known as 5-hydroxymethylcytosine (5hmC) in regulating transcriptomic and pathogenic mechanisms after focal ischemic injury. Methods— Young and aged male and female mice were subjected to transient middle cerebral artery occlusion, and the peri-infarct region was analyzed at various times of reperfusion. Two days before middle cerebral artery occlusion, short-interfering RNA against an isoform of the 5hmC producing enzyme TET (ten-eleven translocase) was injected intracerebrally. Ascorbate was injected intraperitoneally at 5 minutes, 30 minutes, or 2 hours of reperfusion. Motor function was tested with rotarod and beam-walk test. Results— Focal ischemia rapidly induced the activity of TET, the enzyme that catalyzes the formation of 5hmC and preferentially increased expression of the TET3 isoform in the peri-infarct region of the ischemic cortex. Levels of 5hmC were increased in a TET3-dependent manner, and inhibition of TET3 led to wide-scale reductions in the postischemic expression of neuroprotective genes involved in antioxidant defense and DNA repair. TET3 knockdown in adult male and female mice further increased brain degeneration after focal ischemia, demonstrating a role for TET3 and 5hmC in endogenous protection against stroke. Ascorbate treatment after focal ischemia enhanced TET3 activity and 5hmC enrichment in the peri-infarct region. TET3 activation by ascorbate provided robust protection against ischemic injury in young and aged mice of both sexes. Moreover, ascorbate treatment improved motor function recovery in both male and female mice. Conclusions— Collectively, these results indicate the potential of TET3 and 5hmC as novel stroke therapeutic targets. Visual Overview— An online visual overview is available for this article.

Despite the immeasurable burden on patients and families, no effective therapies to protect the CNS after an acute injury are available yet. Furthermore, the underlying mechanisms that promote neuronal death and functional deficits after injury remain to be poorly understood. The prevalence, age of onset, pathophysiology, and symptomatology of many CNS insults differ significantly between males and females. In the case of stroke, younger males tend to show a higher risk than younger females, while this trend reverses with age. Accumulating evidence from preclinical studies have shown that sex hormones play a crucial role in providing neuroprotection following ischemic stroke and other acute CNS injuries. Estrogen, in particular, exerts a neuroprotective effect by modulating the immune responses after injury. In addition, there exists a sexual dimorphism in cell death pathways between males and females that are independent of hormones. Meanwhile, recent studies suggest that microRNAs are critically involved in the sex-specific mechanisms of cell death. This review discusses the current knowledge on the contribution of sex and age to outcome after stroke. Implication of the interplay between these two factors on other CNS injuries (spinal cord injury and traumatic brain injury) from the experimental evidence were also discussed.

Mitochondria are dynamically active organelles, regulated through fission and fusion events to continuously redistribute them across axons, dendrites, and synapses of neurons to meet bioenergetics requirements and to control various functions, including cell proliferation, calcium buffering, neurotransmission, oxidative stress, and apoptosis. However, following acute or chronic injury to CNS, altered expression and function of proteins that mediate fission and fusion lead to mitochondrial dynamic imbalance. Particularly, if the fission is abnormally increased through pro-fission mediators such as Drp1, mitochondrial function will be impaired and mitochondria will become susceptible to insertion of proapototic proteins. This leads to the formation of mitochondrial transition pore, which eventually triggers apoptosis. Thus, mitochondrial dysfunction is a major promoter of neuronal death and secondary brain damage after an insult. This review discusses the implications of mitochondrial dynamic imbalance in neuronal death after acute and chronic CNS insults.

The health benefits of the plant-derived polyphenol resveratrol were established in multiple disease systems. Notably, pre-treatment with resveratrol was shown to be neuroprotective in several models of cerebral ischemia. Mechanisms of resveratrol-mediated neuroprotection have been explored in the context of canonical resveratrol targets, but epigenetic and non-coding RNA processes have not yet been evaluated. Resveratrol was shown to alter microRNAs in cancer and cardiac ischemia. Previous studies also showed that ischemic preconditioning that induces ischemic tolerance significantly alters cerebral microRNA levels, particularly those that target neuroprotective pathways. Therefore, we tested if resveratrol-mediated ischemic tolerance also alters microRNA expression with a goal to identify microRNAs that are amenable to manipulation to induce neuroprotection after cerebral ischemia. Hence, we tested the microRNA profiles in mouse brain following intraperitoneal administration of resveratrol that induced significant tolerance against transient focal ischemia. We analyzed microRNA profiles using microarrays from both Affymetrix and LC Sciences that contain probes for all known mouse microRNAs. The results show that there is no consistent change in any of the microRNAs tested between resveratrol and vehicle groups indicating that microRNAs play a minimal role in resveratrol-mediated cerebral ischemic tolerance.

α-Synuclein (α-Syn), one of the most abundant proteins in the CNS, is known to be a major player in the neurodegeneration observed in Parkinson9s disease. We currently report that transient focal ischemia upregulates α-Syn protein expression and nuclear translocation in neurons of the adult rodent brain. We further show that knockdown or knock-out of α-Syn significantly decreases the infarction and promotes better neurological recovery in rodents subjected to focal ischemia. Furthermore, α-Syn knockdown significantly reduced postischemic induction of phospho-Drp1, 3-nitrotyrosine, cleaved caspase-3, and LC-3 II/I, indicating its role in modulating mitochondrial fragmentation, oxidative stress, apoptosis, and autophagy, which are known to mediate poststroke neuronal death. Transient focal ischemia also significantly upregulated serine-129 (S129) phosphorylation (pα-Syn) of α-Syn and nuclear translocation of pα-Syn. Furthermore, knock-out mice that lack PLK2 (the predominant kinase that mediates S129 phosphorylation) showed better functional recovery and smaller infarcts when subjected to transient focal ischemia, indicating a detrimental role of S129 phosphorylation of α-Syn. In conclusion, our studies indicate that α-Syn is a potential therapeutic target to minimize poststroke brain damage. SIGNIFICANCE STATEMENT Abnormal aggregation of α-synuclein (α-Syn) has been known to cause Parkinson9s disease and other chronic synucleinopathies. However, even though α-Syn is linked to pathophysiological mechanisms similar to those that produce acute neurodenegerative disorders, such as stroke, the role of α-Syn in such disorder is not clear. We presently studied whether α-Syn mediates poststroke brain damage and more importantly whether preventing α-Syn expression is neuroprotective and leads to better physiological and functional outcome after stroke. Our study indicates that α-Syn is a potential therapeutic target for stroke therapy.

In mammals, many classes of noncoding RNAs (ncRNAs) are expressed at a much higher level in the brain than in other organs. Recent studies have identified a new class of ncRNAs called circular RNAs (circRNAs), which are produced by back-splicing and fusion of either exons, introns, or both exon-intron into covalently closed loops. The circRNAs are also highly enriched in the brain and increase continuously from the embryonic to the adult stage. Although the functional significance and mechanism of action of circRNAs are still being actively explored, they are thought to regulate the transcription of their host genes and sequestration of miRNAs and RNA binding proteins. Some circRNAs are also shown to have translation potential to form peptides. The expression and abundance of circRNAs seem to be spatiotemporally maintained in a normal brain. Altered expression of circRNAs is also thought to mediate several disorders, including brain-tumor growth, and acute and chronic neurodegenerative disorders by affecting mechanisms such as angiogenesis, neuronal plasticity, autophagy, apoptosis, and inflammation. This review discusses the involvement of various circRNAs in brain development and CNS diseases. A better understanding of the circRNA function will help to develop novel therapeutic strategies to treat CNS complications.

Glioblastoma multiforme (GBM) is the most aggressive and most frequently diagnosed malignant human glioma. Despite the best available standard of care (surgery, radiation, and chemotherapy), the median survival of GBM patients is less than 2 years. Many recent studies have indicated that microRNAs (miRNAs) are important for promoting or reducing/limiting GBM growth. In particular, we previously showed that GBMs express decreased levels of miR-100 relative to control tissue and that restoring miR-100 expression reduced GBM tumorigenicity by modulating SMRT/NCOR2 (Nuclear Receptor Corepressor 2). Here, we demonstrate that miR-100 overexpression decreases expression of the stem cell markers, nestin and L1CAM, and decreases proliferation of GBM tumor-initiating cells (GBM stem-like cells). We further show that miR-100-mediated anti-tumorigenic activity limits the activity of SMARCA5 and its downstream target STAT3 (known as mTOR-STAT3-Notch pathway). In addition, we report ErbB3 (Her3) as a putative miR-100 target, including inhibition of its downstream AKT and ERK signaling pathways.

Background and Purpose— Social isolation increases mortality and impairs recovery after stroke in clinical populations. These detrimental effects have been recapitulated in animal models, although the exact mechanism mediating these effects remains unclear. Dysregulation of microRNAs (miRNAs) occurs in both strokes as well as after social isolation, which trigger changes in many downstream genes. We hypothesized that miRNA regulation is involved in the detrimental effects of poststroke social isolation in aged animals. Methods— We pair-housed 18-month-old C57BL/6 male mice for 2 weeks before a 60-minute right middle cerebral artery occlusion or sham surgery and then randomly assigned mice to isolation or continued pair housing immediately after surgery. We euthanized mice either at 3, 7, or 15 days after surgery and isolated the perilesional frontal cortex for whole microRNAome analysis. In an additional cohort, we treated mice 1 day after stroke onset with an in vivo-ready antagomiR-141 for 3 days. Results— Using whole microRNAome analysis of 752 miRNAs, we identified miR-141-3p as a unique miRNA that was significantly upregulated in isolated mice in a time-dependent manner up to 2 weeks after stroke. Posttreatment with an antagomiR-141-3p reduced the postisolation-induced increase in miR-141-3p to levels almost equal to those of pair-housed stroke controls. This treatment significantly reduced mortality (by 21%) and normalized infarct volume and neurological scores in poststroke-isolated mice. Quantitative PCR analysis revealed a significant upregulation of Tgfβr1 (transforming growth factor beta receptor 1, a direct target of miR-141-3p) and Igf-1 (insulin-like growth factor 1) mRNA after treatment with antagomiR. Treatment also increased the expression of other pleiotropic cytokines such as Il-6 (interleukin 6) and Tnf-α (tumor necrosis factor-α), an indirect or secondary target) in brain tissue. Conclusions— miR-141-3p is increased with poststroke isolation. Inhibition of miR-141-3p improved mortality, neurological deficits, and decreased infarct volumes. Importantly, these therapeutic effects occurred in aged animals, the population most at risk for stroke and poststroke isolation.

Background and Purpose— Human and rodent genomes diverged ≈75 million years ago. However, 481 regions of their genomes (200–779 nucleotide each) remained absolutely conserved and form noncoding RNAs known as transcribed ultraconserved regions (T-UCRs). The functional significance of T-UCRs is not apparent, but their altered expression is associated with many diseases, and thus thought to be critical for life. We presently investigated the poststroke temporal changes in the expression of T-UCRs with potential functional significance. Methods— Male, spontaneously hypertensive rats were subjected to transient middle cerebral artery occlusion. Expression profile of T-UCRs was determined at 3, 6, and 12 hours of reperfusion using microarrays and real-time polymerase chain reaction in the peri-infarct cortex. The putative functional significance of stroke-responsive T-UCRs was identified by bioinformatics. Results— Ischemia altered expression of 69 T-UCRs at ≥1 time points of reperfusion compared with sham. Poststroke expression of the intragenic T-UCRs is independent of the expression of their parent gene mRNAs. Bioinformatics showed that the upstream/downstream and the parent genes of the T-UCRs modulate several biological and molecular functions, including metabolism, response to stimuli, cell communication, protein and nucleic acid binding. Conclusions— This first report shows that ischemic stroke temporally alters the noncoding ultraconserved RNAs in spontaneously hypertensive rats, but their functional significance is yet to be evaluated.

The middle cerebral artery occlusion (MCAO) model is widely used for inducing a focal cerebral ischemic insult (stroke) in rodents. Here, we describe a method for transient MCAO technique without craniotomy in both mice and rats. In our laboratory, this technique yields consistent secondary brain damage that evolves over a period of 3-7 days of reperfusion after transient MCAO. We also describe the methods for analyzing postischemic motor dysfunction and infarct volume in rodents subjected to transient MCAO.

The middle cerebral artery occlusion (MCAO) model is widely used for inducing a focal cerebral ischemic insult (stroke) in rodents. Here, we describe a method for transient MCAO technique without craniotomy in both mice and rats. In our laboratory, this technique yields consistent secondary brain damage that evolves over a period of 3-7 days of reperfusion after transient MCAO. We also describe the methods for analyzing postischemic motor dysfunction and infarct volume in rodents subjected to transient MCAO.

Ischemia induces extensive temporal changes in cerebral transcriptome that influences the neurologic outcome after stroke. In addition to protein-coding RNAs, many classes of noncoding RNAs, including long noncoding RNAs (LncRNAs), also undergo changes in the poststroke brain. We currently evaluated the functional significance of an LncRNA called Fos downstream transcript (FosDT) that is cogenic withFosgene. Following transient middle cerebral artery occlusion (MCAO) in adult rats, expression ofFosDTandFoswas induced. FosDT knockdown significantly ameliorated the postischemic motor deficits and reduced the infarct volume. Focal ischemia also increased FosDT binding to chromatin-modifying proteins (CMPs) Sin3a and coREST (corepressors of the transcription factor REST). Furthermore, FosDT knockdown derepressed REST-downstream genesGRIA2,NFκB2, andGRIN1in the postischemic brain. Thus, FosDT induction and its interactions with REST-associated CMPs, and the resulting regulation of REST-downstream genes might modulate ischemic brain damage. LncRNAs, such as FosDT, can be therapeutically targeted to minimize poststroke brain damage.SIGNIFICANCE STATEMENTMammalian brain is abundantly enriched with long noncoding RNAs (LncRNAs). Functional roles of LncRNAs in normal and pathological states are not yet understood. This study identified that LncRNA FosDT induced after transient focal ischemia modulates poststroke behavioral deficits and brain damage. These effects of FosDT in part are due to its interactions with chromatin-modifying proteins Sin3a and coREST (corepressors of the transcription factor REST) and subsequent derepression of REST-downstream genesGRIA2,NFκB2, andGRIN1. Therefore, LncRNA-mediated epigenetic remodeling could determine stroke outcome.

Background and Purpose— Matrix metalloproteinases (MMPs) have a central role in compromising the integrity of the blood–brain barrier (BBB). The role of MMP-12 in brain damage after ischemic stroke remains unknown. The main objective of the current study is to investigate the effect of MMP-12 suppression at an early time point before reperfusion on the BBB damage in rats. Methods— Sprague–Dawley rats were subjected to middle cerebral artery occlusion and reperfusion. MMP-12 shRNA–expressing plasmids formulated as nanoparticles were administered at a dose of 1 mg/kg body weight. The involvement of MMP-12 on BBB damage was assessed by performing various techniques, including Evans blue dye extravasation, 2,3,5-triphenyltetrazolium chloride staining, immunoblot, gelatin zymography, and immunofluorescence analysis. Results— MMP-12 is upregulated ≈31-, 47-, and 66-fold in rats subjected 1–, 2-, or 4-hour ischemia, respectively, followed by 1-day reperfusion. MMP-12 suppression protected the BBB integrity by inhibiting the degradation of tight-junction proteins. Either intravenous or intra-arterial delivery of MMP-12 shRNA-expressing plasmid significantly reduced the percent Evans blue dye extravasation and infarct size. Furthermore, MMP-12 suppression reduced the endogenous levels of other proteases, such as tissue-type plasminogen activator and MMP-9, which are also known to be the key players involved in BBB damage. Conclusions— These results demonstrate the adverse role of MMP-12 in acute brain damage that occurs after ischemic stroke and, thereby, suggesting that MMP-12 suppression could be a promising therapeutic target for cerebral ischemia.

Resveratrol, a stilbene formed in many plants in response to various stressors, elicits multiple beneficial effects in vertebrates. Particularly, resveratrol was shown to have therapeutic properties in cancer, atherosclerosis and neurodegeneration. Resveratrol-induced benefits are modulated by multiple synergistic pathways that control oxidative stress, inflammation and cell death. Despite the lack of a definitive mechanism, both in vivo and in vitro studies suggest that resveratrol can induce a neuroprotective state when administered acutely or prior to experimental injury to the CNS. In this review, we discuss the neuroprotective potential of resveratrol in stroke, traumatic brain injury and spinal cord injury, with a focus on the molecular pathways responsible for this protection.

Neuropathic pain, especially that resulting from spinal cord injury, is a tremendous clinical challenge. A myriad of biological changes have been implicated in producing these pain states including cellular interactions, extracellular proteins, ion channel expression, and epigenetic influences. Physiological consequences of these changes are varied and include functional deficits and pain responses. Developing therapies that effectively address the cause of these symptoms require a deeper knowledge of alterations in the molecular pathways. Matrix metalloproteinases and tissue inhibitors of metalloproteinases are two promising therapeutic targets. Matrix metalloproteinases interact with and influence many of the studied pain pathways. Gene expression of ion channels and inflammatory mediators clearly contributes to neuropathic pain. Localized and time dependent targeting of these proteins could alleviate and even prevent neuropathic pain from developing. Current therapeutic options for neuropathic pain are limited primarily to analgesics targeting the opioid pathway. Therapies directed at molecular targets are highly desirable and in early stages of development. These include transplantation of exogenously engineered cell populations and targeted gene manipulation. This review describes specific molecular targets amenable to therapeutic intervention using currently available delivery systems.

Toll-like receptor 2 (TLR2) and TLR4 belong to a family of highly conserved pattern recognition receptors and are well-known upstream sensors of signaling pathways of innate immunity. TLR2 and TLR4 upregulation is thought to be associated with poor outcome in stroke patients. We currently show that transient focal ischemia in adult rats induces TLR2 and TLR4 expression within hours and shRNA-mediated knockdown of TLR2 and TLR4 alone and in combination decreases the infarct size and swelling. We further show that TLR2 and TLR4 knockdown also prevented the induction of their downstream signaling molecules MyD88, IRAK1, and NFκB p65 as well as the pro-inflammatory cytokines IL-1β, IL-6, and TNFα. This study thus shows that attenuation of the severity of TLR2- and TLR4-mediated post-stroke inflammation ameliorates ischemic brain damage.

Background and Purpose— Circular RNAs (circRNAs) are a novel class of noncoding RNAs formed from many protein-coding genes by backsplicing. Although their physiological functions are not yet completely defined, they are thought to control transcription, translation, and microRNA levels. We investigated whether stroke changes the circRNAs expression profile in the mouse brain. Methods— Male C57BL/6J mice were subjected to transient middle cerebral artery occlusion, and circRNA expression profile was evaluated in the penumbral cortex at 6, 12, and 24 hours of reperfusion using circRNA microarrays and real-time PCR. Bioinformatics analysis was conducted to identify microRNA binding sites, transcription factor binding, and gene ontology of circRNAs altered after ischemia. Results— One thousand three-hundred twenty circRNAs were expressed at detectable levels mostly from exonic (1064) regions of the genes in the cerebral cortex of sham animals. Of those, 283 were altered (>2-fold) at least at one of the reperfusion time points, whereas 16 were altered at all 3 time points of reperfusion after transient middle cerebral artery occlusion compared with sham. Postischemic changes in circRNAs identified by microarray analysis were confirmed by real-time PCR. Bioinformatics showed that these 16 circRNAs contain binding sites for many microRNAs. Promoter analysis showed that the circRNAs altered after stroke might be controlled by a set of transcription factors. The major biological and molecular functions controlled by circRNAs altered after transient middle cerebral artery occlusion are biological regulation, metabolic process, cell communication, and binding to proteins, ions, and nucleic acids. Conclusions— This is a first study that shows that stroke alters the expression of circRNAs with possible functional implication to poststroke pathophysiology.

Both Parkinson’s disease (PD) and stroke are debilitating conditions that result in neuronal death and loss of neurological functions. These two conditions predominantly affect aging populations with the deterioration of the quality of life for the patients themselves and a tremendous burden to families. While the neurodegeneration and symptomology of PD develop chronically over the years, post-stroke neuronal death and dysfunction develop rapidly in days. Despite the discrepancy in the pathophysiological time frame and severity, both conditions share common molecular mechanisms that include oxidative stress, mitochondrial dysfunction, inflammation, endoplasmic reticulum stress, and activation of various cell death pathways (apoptosis/necrosis/autophagy) that synergistically modulate the neuronal death. Emerging evidence indicates that several proteins associated with early-onset familial PD play critical roles in mediating the neuronal death. Importantly, mutations in the genes encoding Parkin, PTEN-induced putative kinase 1 and DJ-1 mediate autosomal recessive forms of PD, whereas mutations in the genes encoding leucine-rich repeat kinase 2 and α-synuclein are responsible for autosomal dominant PD. This review discusses the significance of these proteins with the emphasis on the role of α-synuclein in mediating post-ischemic brain damage.

Stroke is the number one cause of neurological dysfunction in adults and has a heavy socioeconomic burden worldwide. The etiological origins of ischemic stroke and resulting pathological processes are mediated by a multifaceted cascade of molecular mechanisms that are in part modulated by posttranscriptional activity. Accumulating evidence has revealed a role for microRNAs (miRNAs) as essential mediators of posttranscriptional gene silencing in both the physiology of brain development and pathology of ischemic stroke. In this review, we compile miRNAs that have been reported to regulate various stroke risk factors and pre-disease mechanisms, including hypertension, atherosclerosis, and diabetes, followed by an in-depth analysis of miRNAs in ischemic stroke pathogenesis, such as excitotoxicity, oxidative stress, inflammation, apoptosis, angiogenesis and neurogenesis. Since promoting or suppressing expression of miRNAs by specific pharmaceutical and non-pharmaceutical therapies may be beneficial to post-stroke recovery, we also highlight the potential therapeutic value of miRNAs in clinical settings.

Accumulating evidence indicates that various classes of non-coding RNAs (ncRNAs) including microRNAs (miRNAs), PIWI-interacting RNAs (piRNAs) and long non-coding RNAs (lncRNAs) play important roles in normal state as well as the diseases of the CNS. Interestingly, ncRNAs have been shown to interact with messenger RNA, DNA and proteins, and these interactions could induce epigenetic modifications and control transcription and translation, thereby adding a new layer of genomic regulation. The ncRNA expression profiles are known to be altered after acute CNS injuries including stroke, traumatic brain injury and spinal cord injury that are major contributors of morbidity and mortality worldwide. Hence, a better understanding of the functional significance of ncRNAs following CNS injuries could help in developing potential therapeutic strategies to minimize the neuronal damage in those conditions. The potential of ncRNAs in blood and CSF as biomarkers for diagnosis and/or prognosis of acute CNS injuries has also gained importance in the recent years. This review highlighted the current progress in the understanding of the role of ncRNAs in initiation and progression of secondary neuronal damage and their application as biomarkers after acute CNS injuries.

Stroke is the leading cause of disability in the adult population throughout the world. The molecular mechanisms that promote poststroke brain damage are not yet understood completely. Studies showed that stroke rapidly alters the expression profiles of many classes of noncoding RNAs in brain. Studies also showed the functional significance of various classes of noncoding RNAs in promoting and/or protecting the brain after stroke.

Endoplasmic reticulum (ER) stress induces a variety of neuronal cell death pathways that play a critical role in the pathophysiology of stroke. ER stress occurs when unfolded/misfolded proteins accumulate and the folding capacity of ER chaperones exceeds the capacity of ER lumen to facilitate their disposal. As a consequence, a complex set of signaling pathways will be induced that transmit from ER to cytosol and nucleus to compensate damage and to restore the normal cellular homeostasis, collectively known as unfolded protein response (UPR). However, failure of UPR due to severe or prolonged stress leads to cell death. Following acute CNS injuries, chronic disturbances in protein folding and oxidative stress prolong ER stress leading to sustained ER dysfunction and neuronal cell death. While ER stress responses have been well studied after stroke, there is an emerging need to study the association of ER stress with other cell pathways that exacerbate neuronal death after an injury. In this review, we summarize the current understanding of the role for ER stress in acute brain injuries, highlighting the diverse molecular mechanisms associated with ER stress and its relation to oxidative stress and autophagy. We also discussed the existing and developing therapeutic options aimed to reduce ER stress to protect the CNS after acute injuries.

Stroke is one of the leading causes of death and disability worldwide. Experimental and clinical studies showed that sex and age play an important role in deciding the outcome after stroke. At younger ages, males were shown to have a higher risk for stroke than females. However, this trend reverses in older ages particularly when females reach menopause. Many preclinical studies indicate that steroid hormones modulate the age‐dependent differential stroke outcome. In addition, patterns of cell death pathways activated following cerebral ischemia are distinct between males and females, but independent of steroid hormones. Recent studies also indicate that microRNAs play important roles in mediating sex‐specific stroke outcome by regulating stroke‐related genes. This review discusses the contribution of sex and age to outcome after stroke with particular emphasis on the experimental studies that examined the effects of steroid hormones, differential cell death pathways, and involvement of sex‐specific microRNAs following cerebral ischemia. Current understanding of the role of thrombolytic agents in stroke therapy is also discussed.

Emerging evidence indicates that 481 regions of the genome (>200 bp) that actively transcribe noncoding RNAs shows 100% homology between humans, rats and mice. These transcribed ultraconserved regions (T-UCRs) are thought to control the essential regulatory functions basic for life in rodents and mammals. Using microarray analysis, we presently show that 107 T-UCRs are actively expressed in adult rat cerebral cortex. They are grouped into intragenic (61) and intergenic (46) based on their genic location. Interestingly, 10 T-UCRs are expressed at unusually high levels in cerebral cortex. Additionally, many T-UCRs also showed cogenic expression. We further analyzed the correlation of intragenic T-UCRs with their host protein coding genes. Surprisingly, most of the expressed intragenic T-UCRs (54 out of 61) displayed a negative correlation with their host gene expression. T-UCRs are thought to control the splicing and transcription of the protein-coding genes that host them and flank them. Bioinformatics analysis indicated that the protein products of majority of these genes are nuclear in localization, share protein domains and are involved in the regulation of diverse biological and molecular functions including metabolism, development, cell cycle, binding and transcription factor regulation. In conclusion, this is the first study to shows that many T-UCRs are expressed in rodent brain and they might play a role in physiological brain functions.

Stroke is one of the major contributors of mortality and morbidity worldwide. It results due to sudden blockade of blood flow into the brain thereby leading to breakdown of hemodynam - ic, biochemical and metabolic processes in the affected territory. These alterations are dependent on the severity and duration of the ischemia and modulated by pathophysiologic mechanisms that in- clude excitotoxicity, calcium overload, oxidative stress, endoplasmic reticulum stress, mitochondrial dysfunction, acidosis, inflammation and activation of cell death pathways. As there is no current therapy to minimize post-stroke brain damage and most of the clinical trials failed, it is impera- tive to design novel approaches including glutamate receptor antagonists, free radical scavengers/ anti-oxidants, anti-inflammatory and anti-apoptotic agents, and growth factors to prevent the pro - gression and increase the plasticity of neuronal damage after stroke. Additionally, rigorous in vitro and in vivo animal testing of potential drugs molecules is necessary to reduce the failure of clinical trials.

α-synuclein (α-syn) is one of the most abundant proteins in mammalian brain that is known to be a major player in the neurodegeneration observed in chronic conditions like Parkinson’s disease (PD). Several mechanisms were proposed for α-syn-induced neuronal death in PD including inflammation, oxidative stress, mitochondrial fission, and autophagy. Interestingly, all these pathophysiologic mechanisms also mediate neuronal death after acute CNS insults like stroke. Therefore, we examined whether α-syn contributes to post-stroke neuronal death and neurological dysfunction. Rodents were subjected to transient middle cerebral artery occlusion (tMCAO) and α-syn induction was silenced with a cocktail of α-syn-specific siRNAs. The levels of α-syn were estimated with qPCR and Western Blots. Post-ischemic motor deficit was evaluated with rotarod, beam walk and adhesive removal test 1 to 7 days after ischemia, and infarct volume was measured on cresyl violet stained brain sections. Cellular changes after ischemia were examined using immunofluorescence staining. Following tMCAO, α-syn levels were significantly up-regulated and phosphorylated at serine-129 in ischemic penumbra. Interestingly, in the ischemic brain non-phosphorylated and phosphorylated α-syn species translocated into the neuronal nuclei. We also observed that silencing α-syn by α-syn siRNA cocktail before tMCAO significantly decreased the infarction and improved the motor function. The neuroprotective effects were still observed when α-syn siRNAs were administered 30 min after the tMCAO. We found that the rate of survival at 7 days after a 90 min tMCAO was significantly higher in α-syn KO mice compared to wild-type control mice. Furthermore, α-syn suppression significantly mitigated the post-ischemic oxidative stress (8-OHdG and 3-NT), apoptosis (cleaved Caspase-3) and mitochondrial dysfunction (phospho-Drp1). Thus, we show that α-syn plays a critical role in neuronal death following stroke. Furthermore, phospho-S129 α-syn in the neuronal nuclei might be a potential mediator of the induction of ischemic brain damage. Preventing α-syn expression is a potential therapeutic target to minimize post-stroke brain damage.

The mammalian genome harbors 481 DNA elements (200-779 bp) that show 100% homology between humans, rats and mice despite millions of years of phylogenetic separation. These elements are actively transcribed into noncoding RNAs called transcribed ultraconserved regions (T-UCRs). Physiological function of T-UCRs is not yet evaluated in detail. Their role in various brain pathologies is also not known. Using microarray analysis, we currently evaluated the expression profiles of T-UCRs at 3h, 6h and 12h of reperfusion following transient middle cerebral artery occlusion (MCAO) in adult rats. Under normal physiological conditions, 61 intragenic and 46 intergenic T-UCRs were expressed in rat cerebral cortex. Surprisingly, 54 of the 61 intragenic T-UCRs displayed a negative correlation with their host gene expression. Following transient MCAO, expression of 67 T-UCRs (20 up- and 47 down-regulated) were altered at one or the other reperfusion time points. Of those altered, 39 are intragenic and 28 are intergenic. Temporally, 55 (11 up and 44 down) were altered at 3h, 53 (15 up and 38 down) at 6h and 62 (16 up and 46 down) at 12h of reperfusion. Interestingly, 36 T-UCRs were altered at all-time points of reperfusion and 4 of those were altered by >5 fold (uc.34 and uc.226 were up-regulated, and uc.286 and uc.417 were down-regulated). In addition, we observed a congenic expression of some T-UCRs (uc.208/uc.209, uc.460/uc.462/uc.465). Functional significance of T-UCRs altered after stroke was predicted by clustering multiple bioinformatics analysis tools (GeneMANIA, PANTHER, and WebGestalt) using the host genes and the nearest upstream and downstream genes as anchors. The products of T-UCR associated genes are involved in the regulation of diverse biological functions including metabolism, development and cellular process. This first time observation suggests that T-UCRs altered after ischemic stroke could have functional significance in post-stroke brain damage and plasticity.

Addition of a small peptide called ISG15 is known as ISGylation, which is an ubiquitin (ub)-like posttranslational modification. We currently show that focal ischemia induced by transient middle cerebral artery occlusion (MCAO) in adult mice significantly induces cortical protein ISGylation between 6 and 24 hours reperfusion. With two-dimensional western blotting, 45 proteins were observed to be significantly increased in ISGylation (by 1.8- to 9.7-fold) after focal ischemia compared with sham control. Immunochemistry showed that ISGylated proteins are localized in neurons within the ipsilateral striatum and in astroglia within the peri-infarct cortex of ischemic mice. When subjected to transient MCAO, ISG15−/− mice showed increased mortality, exacerbated infarction, and worsened neurologic recovery than did wild-type controls. In addition, mice lacking UBE1L (ub-activating enzyme E1-like protein, the first enzyme of the ISGylation cycle) also showed bigger infarcts when subjected to transient MCAO. Regional cerebral blood flow or other physiologic parameters were not significantly different in both knockouts compared with wild-type controls. These studies indicate that increased protein ISGylation might be an endogenous neuroprotective adaptation to minimize poststroke brain damage.