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Post-stroke neuroinflammation is pivotal in brain repair, yet persistent inflammation can aggravate ischemic brain damage and hamper recovery. Following stroke, specific molecules released from brain cells attract and activate central and peripheral immune cells. These immune cells subsequently release diverse inflammatory molecules within the ischemic brain, initiating a sequence of events, including activation of transcription factors in different brain cell types that modulate gene expression and influence outcomes; the interactive action of various noncoding RNAs (ncRNAs) to regulate multiple biological processes including inflammation, epitranscriptomic RNA modification that controls RNA processing, stability, and translation; and epigenetic changes including DNA methylation, hydroxymethylation, and histone modifications crucial in managing the genic response to stroke. Interactions among these events further affect post-stroke inflammation and shape the depth of ischemic brain damage and functional outcomes. We highlighted these aspects of neuroinflammation in this review and postulate that deciphering these mechanisms is pivotal for identifying therapeutic targets to alleviate post-stroke dysfunction and enhance recovery., (© 2024 International Society for Neurochemistry.)

We previously showed that stroke alters circular RNA (circRNA) expression profiles. Many circRNAs undergo epitranscriptomic modifications, particularly methylation of adenosine to form N 6 -methyladenosine (m 6 A). This modification significantly influences the circRNA metabolism and functionality. Hence, we currently evaluated if transient focal ischemia in adult C57BL/6J mice alters the m 6 A methylation of circRNAs. Changes in m 6 A were profiled in the peri-infarct cortex following immunoprecipitation coupled with microarrays. Correlation and gene ontology analyses were performed to understand the association of m 6 A changes with circRNA regulation and functional implications after stroke. Many circRNAs showed differential regulation (up or down) after stroke, and this change was highest at 24h of reperfusion. Notably, most circRNAs differentially regulated after stroke also exhibited temporal changes in m 6 A modification patterns. The majority of circRNAs that showed post-stroke differential m 6 A modifications were derived from protein-coding genes. Hyper-than hypomethylation of circRNAs was most prevalent after stroke. Gene ontology analysis of the host genes suggested that m 6 A-modified circRNAs might regulate functions such as synapse-related processes, indicating that m 6 A epitranscriptomic modification in circRNAs could potentially influence post-stroke synaptic pathophysiology., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Preconditioning-induced cerebral ischemic tolerance is known to be a beneficial adaptation to protect the brain in an unavoidable event of stroke. We currently demonstrate that a short bout (6 weeks) of intermittent fasting (IF; 15 h fast/day) induces similar ischemic tolerance to that of a longer bout (12 weeks) in adult C57BL/6 male mice subjected to transient middle cerebral artery occlusion (MCAO). In addition, the 6 weeks IF regimen induced ischemic tolerance irrespective of age (3 months or 24 months) and sex. Mice subjected to transient MCAO following IF showed improved motor function recovery (rotarod and beam walk tests) between days 1 and 14 of reperfusion and smaller infarcts (T2-MRI) on day 1 of reperfusion compared with age/sex matched ad libitum (AL) controls. Diet influences the gut microbiome composition and stroke is known to promote gut bacterial dysbiosis. We presently show that IF promotes a beneficial phenotype of gut microbiome following transient MCAO compared with AL cohort. Furthermore, post-stroke levels of short-chain fatty acids (SCFAs), which are known to be neuroprotective, are higher in the fecal samples of the IF cohort compared with the AL cohort. Thus, our studies indicate the efficacy of IF in protecting the brain after stroke, irrespective of age and sex, probably by altering gut microbiome and SCFA production., Competing Interests: Declaration of competing interest The authors declare no conflicts., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

N 6 -Methyladenosine (m 6 A) is a neuronal-enriched, reversible post-transcriptional modification that regulates RNA metabolism. The m 6 A-modified RNAs recruit various m 6 A-binding proteins that act as readers. Differential m 6 A methylation patterns are implicated in ischemic brain damage, yet the precise role of m 6 A readers in propagating post-stroke m 6 A signaling remains unclear. We presently evaluated the functional significance of the brain-enriched m 6 A reader YTHDF1, in post-stroke pathophysiology. Focal cerebral ischemia significantly increased YTHDF1 mRNA and protein expression in adult mice of both sexes. YTHDF1 -/- male, but not female, mice subjected to transient middle cerebral artery occlusion (MCAO) showed worsened motor function recovery and increased infarction compared to sex-matched YTHDF1 +/+ mice. YTHDF1 -/- male, but not female, mice subjected to transient MCAO also showed significantly perturbed expression of genes related to inflammation, and increased infiltration of peripheral immune cells into the peri-infarct cortex, compared with sex-matched YTHDF1 +/+ mice. Thus, this study demonstrates a sexual dimorphism of YTHDF1 in regulating post-ischemic inflammation and pathophysiology. Hence, post-stroke epitranscriptomic regulation might be sex-dependent., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

We previously showed that knockdown or deletion of Fos downstream transcript (FosDT; a stroke-induced brain-specific long noncoding RNA) is neuroprotective. We presently tested the therapeutic potential of FosDT siRNA in rodents subjected to transient middle cerebral artery occlusion (MCAO) using the Stroke Treatment Academic Industry Roundtable criteria, including sex, age, species, and comorbidity. FosDT siRNA (IV) given at 30 min of reperfusion significantly improved motor function recovery (rotarod test, beam walk test, and adhesive removal test) and reduced infarct size in adult and aged spontaneously hypertensive rats of both sexes. FosDT siRNA administered in a delayed fashion (3.5 h of reperfusion following 1 h transient MCAO) also significantly improved motor function recovery and decreased infarct volume. Furthermore, FosDT siRNA enhanced post-stroke functional recovery in normal and diabetic mice. Mechanistically, FosDT triggered post-ischemic neuronal damage via the transcription factor REST as REST siRNA mitigated the enhanced functional outcome in FosDT -/- rats. Additionally, NF-κB regulated FosDT expression as NF-κB inhibitor BAY 11-7082 significantly decreased post-ischemic FosDT induction. Thus, FosDT is a promising target with a favorable therapeutic window to mitigate secondary brain damage and facilitate recovery after stroke regardless of sex, age, species, and comorbidity., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

The microRNA-21 (miR-21) levels in the brain are crucial in determining post-stroke brain damage and recovery. The miR-21 exerts neuroprotection by targeting mRNAs that translate proteins that mediate brain damage. We currently determined the efficacy and efficiency of intravenously administered miR-21 mimic after focal cerebral ischemia in mice. Adult male mice were intravenously administered with either control mimic or miR-21 mimic at 5 min/2 h after reperfusion following 1 h transient middle cerebral artery occlusion to determine the therapeutic window of miR-21 mimic. Adult female, type-2 diabetic male, aged male, and aged female mice were administered with control/miR-21 mimic at 5 min after reperfusion following 35 min/1 h transient middle cerebral artery occlusion. Early administration of miR-21 mimic significantly reduced brain damage and promoted long-term recovery after stroke. Further, miR-21 mimic is more effective in males than in females subjected to stroke. However, delayed treatment with miR-21 mimic is not efficacious, and type-2 diabetic subjects show no improvement with miR-21 mimic treatment., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Methylation of adenosine at N1 position yields N 1 -methyladenosine (m 1 A), which is an epitranscriptomic modification that regulates mRNA metabolism. Recent studies showed that altered m 1 A methylation promotes acute and chronic neurological diseases. We currently evaluated the effect of focal ischemia on cerebral m 1 A methylome and its machinery. Adult male C57BL/6J mice were subjected to transient middle cerebral artery occlusion, and the peri-infarct cortex was analyzed at 12 h and 24 h of reperfusion. The bulk abundance of m 1 A was measured by mass spectrometry and dot blot, and transcriptome-wide m 1 A alterations were profiled using antibody-independent m 1 A-quant-seq. Expression of the m 1 A writers and erasers was estimated by real-time PCR. Ischemia significantly decreased m 1 A levels and concomitantly upregulated m 1 A demethylase alkB homolog 3 at 24 h of reperfusion compared to sham. Transcriptome-wide profiling showed differential m 1 A methylation at 14 sites (8 were hypo- and 6 were hypermethylated). Many of those are located in the 3'-UTRs of unannotated transcripts proximal to the genes involved in regulating protein complex assembly, circadian rhythms, chromatin remodeling, and chromosome organization. Using several different approaches, we show for the first time that m 1 A epitranscriptomic modification in RNA is highly sensitive to cerebral ischemia., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Post-stroke secondary brain damage is significantly influenced by the induction and accumulation of α-Synuclein (α-Syn). α-Syn-positive inclusions are often present in tauopathies and elevated tau levels and phosphorylation promotes neurodegeneration. Glycogen synthase kinase 3β (GSK-3β) is a known promoter of tau phosphorylation. We currently evaluated the interaction of α-Syn with GSK-3β and tau in post-ischemic mouse brain. Transient focal ischemia led to increased cerebral protein-protein interaction of α-Syn with both GSK-3β and tau and elevated tau phosphorylation. Treatment with a GSK-3β inhibitor prevented post-ischemic tau phosphorylation. Furthermore, α-Syn interaction was observed to be crucial for post-ischemic GSK-3β-dependent tau hyperphosphorylation as it was not seen in α-Syn knockout mice. Moreover, tau knockout mice show significantly smaller brain damage after transient focal ischemia. Overall, the present study indicates that GSK-3β catalyzes the α-Syn-dependent tau phosphorylation and preventing this interaction is crucial to limit post-ischemic secondary brain damage., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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.

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., Competing Interests: The authors declare that they have no competing interests., (© 2022 The Authors.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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.

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., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Glutamate-induced excitotoxicity is a well-recognized cause of neuronal cell death. Nutritional supplementation with Coenzyme Q10 (CoQ10) has been previously demonstrated to serve neuro-protective effects against glutamate-induced excitotoxicity. The aim of the present study was to determine whether the protective effect of CoQ10 against glutamate toxicity could be attributed to stimulating mitochondrial biogenesis. Mouse hippocampal neuronal HT22 cells were incubated with glutamate with or without ubisol Q10. The results revealed that glutamate significantly decreased levels of mitochondrial biogenesis related proteins, including peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α and nuclear respiratory factor (NRF)2. Additionally, glutamate reduced mitochondrial biogenesis, as determined using a mitochondrial biogenesis kit. Pretreatment with CoQ10 prevented decreases in phosphorylated (p)-Akt, p-cAMP response element-binding protein, PGC-1α, NRF2 and mitochondrial transcription factor A, increasing mitochondrial biogenesis. Taken together, the results described a novel mechanism of CoQ10-induced neuroprotection and indicated a central role for mitochondrial biogenesis in protecting against glutamate-induced excitotoxicity., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Zimmerman et al.)

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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature.)

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.

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.

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., Competing Interests: Declaration of Competing Interest Authors declare no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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.

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.

Ischemic stroke, which is caused by a clot that blocks blood flow to the brain, can be severely disabling and sometimes fatal. We previously showed that transient focal ischemia in a rat model induces extensive temporal changes in the expression of cerebral microRNAs, with a sustained decrease in the abundance of miR-7a-5p (miR-7). Here, we evaluated the therapeutic efficacy of a miR-7 mimic oligonucleotide after cerebral ischemia in rodents according to the Stroke Treatment Academic Industry Roundtable (STAIR) criteria. Rodents were injected locally or systemically with miR-7 mimic before or after transient middle cerebral artery occlusion. Decreased miR-7 expression was observed in both young and aged rats of both sexes after cerebral ischemia. Pre- or postischemic treatment with miR-7 mimic decreased the lesion volume in both sexes and ages studied. Furthermore, systemic injection of miR-7 mimic into mice at 30 min (but not 2 hours) after cerebral ischemia substantially decreased the lesion volume and improved motor and cognitive functional recovery with minimal peripheral toxicity. The miR-7 mimic treatment substantially reduced the postischemic induction of α-synuclein (α-Syn), a protein that induces mitochondrial fragmentation, oxidative stress, and autophagy that promote neuronal cell death. Deletion of the gene encoding α-Syn abolished miR-7 mimic-dependent neuroprotection and functional recovery in young male mice. Further analysis confirmed that the transcript encoding α-Syn was bound and repressed by miR-7. Our findings suggest that miR-7 mimics may therapeutically minimize stroke-induced brain damage and disability., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Uncontrolled oxidative stress contributes to the secondary neuronal death that promotes long-term neurological dysfunction following traumatic brain injury (TBI). Surprisingly, both NADPH oxidase 2 (NOX2) that increases and transcription factor Nrf2 that decreases reactive oxygen species (ROS) are induced after TBI. As the post-injury functional outcome depends on the balance of these opposing molecular pathways, we evaluated the effect of TBI on the motor and cognitive deficits and cortical contusion volume in NOX2 and Nrf2 knockout mice. Genetic deletion of NOX2 improved, while Nrf2 worsened the post-TBI motor function recovery and lesion volume indicating that decreasing ROS levels might be beneficial after TBI. Treatment with either apocynin (NOX2 inhibitor) or TBHQ (Nrf2 activator) alone significantly improved the motor function after TBI, but had no effect on the lesion volume, compared to vehicle control. Whereas, the combo therapy (apocynin + TBHQ) given at either 5 min/24 h or 2 h/24 h improved motor and cognitive function and decreased cortical contusion volume compared to vehicle group. Thus, both the generation and disposal of ROS are important modulators of oxidative stress, and a combo therapy that prevents ROS formation and potentiates ROS disposal concurrently is efficacious after TBI.

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., (© 2018 American Heart Association, Inc.)

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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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., (© 2017 American Heart Association, Inc.)

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 Author(s) 2016.)

Unlabelled: 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 Parkinson's 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 Parkinson's 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., (Copyright © 2016 the authors 0270-6474/16/367055-11$15.00/0.)

Mitochondrial dysfunction and oxidative stress are the major events that lead to the formation of mitochondrial permeability transition pore (mPTP) during glutamate-induced cytotoxicity and cell death. Coenzyme Q10 (CoQ10) has widely been used for the treatment of mitochondrial disorders and neurodegenerative diseases. Comparing to traditional lipid-soluble CoQ10, water soluble CoQ10 (Ubisol-Q10) has high intracellular and intra-mitochondrial distribution. The aims of the present study are to determine the neuroprotective effects of Ubisol-Q10 on glutamate-induced cell death and to explore its functional mechanisms. HT22 neuronal cells were exposed to glutamate. Cell viability was measured and mitochondrial fragmentation was assessed by mitochondrial imaging. The mPTP opening was determined by mitochondrial membrane potential and calcium retention capacity. The results revealed that the anti-glutamate toxicity effects of Ubisol-Q10 was associated with its ability to block mitochondrial fragmentation, to maintain calcium retention capacity and mitochondrial membrane potential, and to prevent mPTP formation, AIF release, and DNA fragmentation. We concluded that Ubisol-Q10 protects cells from glutamate toxicity by preserving the integrity of mitochondrial structure and function. Therefore, adequate CoQ10 supplementation may be beneficial in preventing cerebral stroke and other disorders that involve mitochondrial dysfunction.

Chronic exposure to ultraviolet radiation (UVR) is linked to the development of cutaneous squamous cell carcinoma (SCC), a non-melanoma form of skin cancer that can metastasize. Tumor necrosis factor-alpha (TNFα), a pro-inflammatory cytokine, is linked to UVR-induced development of SCC. To find clues about the mechanisms by which TNFα may promote UVR-induced development of SCC, we investigated changes in the expression profiling of microRNAs (miRNA), a novel class of short noncoding RNAs, which affects translation and stability of mRNAs. In this experiment, TNFα knockout (TNFα KO) mice and their wild type (WT) littermates were exposed to acute UVR (2.0 kJ/m2) and the expression profiling of epidermal miRNA was determined 4hr post UVR exposure. TNFα deletion in untreated WT mice resulted in differential expression (log fold change>1) of seventeen miRNA. UVR exposure in WT mice induced differential expression of 22 miRNA. However, UVR exposure in TNFα KO mice altered only two miRNAs. Four miRNA, were differentially expressed between WT+UVR and TNFα KO+UVR groups. Differentially expressed selected miRNAs were further validated using real time PCR. Few of the differentially expressed miRNAs (miR-31-5p, miR-196a-5p, miR-127-3p, miR-206-3p, miR-411-5p, miR-709, and miR-322-5p) were also observed in UVR-induced SCC. Finally, bio-informatics analysis using DIANA, MIRANDA, Target Scan, and miRDB algorithms revealed a link with major UVR-induced pathways (MAPK, PI3K-Akt, transcriptional mis-regulation, Wnt, and TGF-beta).

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 with Fos gene. Following transient middle cerebral artery occlusion (MCAO) in adult rats, expression of FosDT and Fos was 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 genes GRIA2, NFκB2, and GRIN1 in 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 Statement: Mammalian 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 genes GRIA2, NFκB2, and GRIN1. Therefore, LncRNA-mediated epigenetic remodeling could determine stroke outcome., (Copyright © 2015 the authors 0270-6474/15/3516443-07$15.00/0.)

Silver nanoparticles (AgNP), one of the most commonly used engineered nanomaterial for biomedical and industrial applications, has shown a toxic potential to our ecosystems and humans. In this study, murine hippocampal neuronal HT22 cells were used to delineate subcellular responses and mechanisms to AgNP by assessing the response levels of caspase-3, mitochondrial oxygen consumption, reactive oxygen species (ROS), and mitochondrial membrane potential in addition to cell viability testing. Selenium, an essential trace element that has been known to carry protecting property from heavy metals, was tested for its ameliorating potential in the cells exposed to AgNP. Results showed that AgNP reduced cell viability. The toxicity was associated with mitochondrial membrane depolarization, increased accumulation of ROS, elevated mitochondrial oxygen consumption, and caspase-3 activation. Treatment with sodium selenite reduced cell death, stabilized mitochondrial membrane potential and oxygen consumption rate, and prevented accumulation of ROS and activation of caspase-3. It is concluded that AgNP induces mitochondrial stress and treatment with selenite is capable of preventing the adverse effects of AgNP on the mitochondria.

Coenzyme Q10 (CoQ10) acts by scavenging reactive oxygen species to protect neuronal cells against oxidative stress in neurodegenerative diseases. The present study was designed to examine whether CoQ10 was capable of protecting astrocytes from reactive oxygen species (ROS) mediated damage. For this purpose, ultraviolet B (UVB) irradiation was used as a tool to induce ROS stress to cultured astrocytes. The cells were treated with 10 and 25 μg/ml of CoQ10 for 3 or 24 h prior to the cells being exposed to UVB irradiation and maintained for 24 h post UVB exposure. Cell viability was assessed by MTT conversion assay. Mitochondrial respiration was assessed by respirometer. While superoxide production and mitochondrial membrane potential were measured using fluorescent probes, levels of cytochrome C (cyto-c), cleaved caspase-9, and caspase-8 were detected using Western blotting and/or immunocytochemistry. The results showed that UVB irradiation decreased cell viability and this damaging effect was associated with superoxide accumulation, mitochondrial membrane potential hyperpolarization, mitochondrial respiration suppression, cyto-c release, and the activation of both caspase-9 and -8. Treatment with CoQ10 at two different concentrations started 24 h before UVB exposure significantly increased the cell viability. The protective effect of CoQ10 was associated with reduction in superoxide, normalization of mitochondrial membrane potential, improvement of mitochondrial respiration, inhibition of cyto-c release, suppression of caspase-9. Furthermore, CoQ10 enhanced mitochondrial biogenesis. It is concluded that CoQ10 may protect astrocytes through suppression of oxidative stress, prevention of mitochondrial dysfunction, blockade of mitochondria-mediated cell death pathway, and enhancement of mitochondrial biogenesis.

The mechanisms that promote brain dysfunction after acute liver failure (ALF) are not clearly understood. The small noncoding RNAs known as microRNAs (miRNAs) significantly control mRNA translation and thus normal and pathological functions in the mammalian body. To understand their significance in ALF, we currently profiled the expression of miRNAs in the cerebral cortex of mice sacrificed at coma stage following treatment with azoxymethane. Of the 470 miRNAs profiled using microarrays, 37 were significantly altered (20 up-and 17 down-regulated) in their expression in the ALF group compared to sham group. In silico analysis showed that the ALF-responsive miRNAs target on average 231 mRNAs/miRNA (range: 3 to 840 targets). Pathways analysis showed that many miRNAs altered after ALF target multiple mRNAs that are part of various biological and molecular pathways. Glutamatergic synapse, Wnt signaling, MAP-kinase signaling, axon guidance, PI3-kinase-AKT signaling, T-cell receptor signaling and ubiquitin-mediated proteolysis are the top pathways targeted by the ALF-sensitive miRNAs. At least 28 ALF-responsive miRNAs target each of the above pathways. We hypothesize that alterations in miRNAs and their down-stream mRNAs of signaling pathways might play a role in the induction and progression of neurological dysfunction observed during ALF.

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., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Mitochondrial biogenesis is activated by nuclear encoded transcription co-activator peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), which is regulated by several upstream factors including protein kinase A and Akt/protein kinase B. We have previously shown that selenoprotein H enhances the levels of nuclear regulators for mitochondrial biogenesis, increases mitochondrial mass and improves mitochondrial respiratory rate, under physiological condition. Furthermore, overexpression of selenoprotein H protects neuronal HT22 cells from ultraviolet B irradiation-induced cell damage by lowering reactive oxygen species production, and inhibiting activation of caspase-3 and -9, as well as p53. The objective of this study is to identify the cell signaling pathways by which selenoprotein H initiates mitochondrial biogenesis. We first confirmed our previous observation that selenoprotein H transfected HT22 cells increased the protein levels of nuclear-encoded mitochondrial biogenesis factors, peroxisome proliferator-activated receptor γ coactivator-1α, nuclear respiratory factor 1 and mitochondrial transcription factor A. We then observed that total and phosphorylation of protein kinase A, Akt/protein kinase B and cyclic adenosine monophosphate response element-binding protein (CREB) were significantly increased in selenoprotein H transfected cells compared to vector transfected HT22 cells. To verify whether the observed stimulating effects on mitochondrial biogenesis pathways are caused by selenoprotein H and mediated through CREB, we knocked down selenoprotein H mRNA level using siRNA and inhibited CREB with napthol AS-E phosphate in selenoprotein H transfected cells and repeated the measurements of the aforementioned biomarkers. Our results revealed that silencing of selenoprotein H not only decreased the protein levels of PGC-1α, nuclear respiratory factor 1 and mitochondrial transcription factor A, but also decreased the total and phosphorylation levels of protein kinase A, protein kinase B, and CREB. Similarly, CREB inhibition reduced CREB activation and PGC-1α protein levels in selenoprotein H transfected cells. Moreover, selenoprotein H transfection increased the activity of mitochondrial complexes and prevented the ultraviolet B induced fall of mitochondrial membrane potential. We conclude that the effects of selenoprotein H on mitochondrial biogenesis and mitochondrial function are probably mediated through protein kinase A-CREB-PGC-1α and Akt/protein kinase B-CREB-PGC-1α pathways., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: Mitochondrial dysfunction is one of the major events responsible for activation of neuronal cell death pathways during cerebral ischemia. Trace element selenium has been shown to protect neurons in various diseases conditions. Present study is conducted to demonstrate that selenium preserves mitochondrial functional performance, activates mitochondrial biogenesis and prevents hypoxic/ischemic cell damage., Results: The study conducted on HT22 cells exposed to glutamate or hypoxia and mice subjected to 60-min focal cerebral ischemia revealed that selenium (100 nM) pretreatment (24 h) significantly attenuated cell death induced by either glutamate toxicity or hypoxia. The protective effects were associated with reduction of glutamate and hypoxia-induced ROS production and alleviation of hypoxia-induced suppression of mitochondrial respiratory complex activities. The animal studies demonstrated that selenite pretreatment (0.2 mg/kg i.p. once a day for 7 days) ameliorated cerebral infarct volume and reduced DNA oxidation. Furthermore, selenite increased protein levels of peroxisome proliferator-activated receptor-γ coactivator 1alpha (PGC-1α) and nuclear respiratory factor 1 (NRF1), two key nuclear factors that regulate mitochondrial biogenesis. Finally, selenite normalized the ischemia-induced activation of Beclin 1 and microtubule-associated protein 1 light chain 3-II (LC3-II), markers for autophagy., Conclusions: These results suggest that selenium protects neurons against hypoxic/ischemic damage by reducing oxidative stress, restoring mitochondrial functional activities and stimulating mitochondrial biogenesis.

Preischemic hyperglycemia exacerbates brain damage caused by cerebral ischemia. In the present experiment, we studied the effects of preischemic hyperglycemia on protein markers that are related to mitochondrial fission and fusion, mitochondrial biogenesis, and autophagy in mice subjected to 30-min transient focal ischemia. The fission proteins dynamin-related protein 1 (Drp1) and fission 1 (Fis1), fusion proteins optic atrophy 1 (Opa1) and mitofusin 2 (Mfn2), mitochondrial biogenesis regulators nuclear respiratory factor 1 (NRF1) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), and autophagy marker beclin 1 and microtubule-associated protein light chain 3 (LC3) were analyzed in control, 30 min middle cerebral artery occlusion (MCAO) plus 6-, 24-, and 72 h of reperfusion in normo- and hyperglycemic conditions. Cerebral ischemia increased the levels of Drp1 and decreased Fis1 after reperfusion. Preischemic hyperglycemia further augmented the increase of Drp1 and induced elevation in Fis1. Ischemia inhibited the levels of Opa1 and Mfn2 and hyperglycemia further decreased the level of Opa1. Further, NRF1 increased after reperfusion in both normo- and hyperglycemic animals. However, such increase was caused by reperfusion rather than glucose level. Finally, ischemia increased beclin 1 level at 6 and 24 h of reperfusion and hyperglycemia further increased the beclin 1 level and caused LC3-II increase as well. Hyperglycemia enhances the ischemia-induced mitochondrial dynamic imbalance towards fission that may favor mitochondrial fragmentation and subsequent damage. Hyperglycemia elevated autophagy markers may represent an adapting reaction to the severe damage incurred in hyperglycemic animals or a third pathway of cell death.

Supplementation of selenium has been shown to protect cells against free radical mediated cell damage. The objectives of this study are to examine whether supplementation of selenium stimulates mitochondrial biogenesis signaling pathways and whether selenium enhances mitochondrial functional performance. Murine hippocampal neuronal HT22 cells were treated with sodium selenite for 24 hours. Mitochondrial biogenesis markers, mitochondrial respiratory rate and activities of mitochondrial electron transport chain complexes were measured and compared to non-treated cells. The results revealed that treatment of selenium to the HT22 cells elevated the levels of nuclear mitochondrial biogenesis regulators PGC-1α and NRF1, as well as mitochondrial proteins cytochrome c and cytochrome c oxidase IV (COX IV). These effects are associated with phosphorylation of Akt and cAMP response element-binding (CREB). Supplementation of selenium significantly increased mitochondrial respiration and improved the activities of mitochondrial respiratory complexes. We conclude that selenium activates mitochondrial biogenesis signaling pathway and improves mitochondrial function. These effects may be associated with modulation of AKT-CREB pathway.

Glutamate-induced cytotoxicity is partially mediated by enhanced oxidative stress. The objectives of the present study are to determine the effects of glutamate on mitochondrial membrane potential, oxygen consumption, mitochondrial dynamics and autophagy regulating factors and to explore the protective effects of selenium against glutamate cytotoxicity in murine neuronal HT22 cells. Our results demonstrated that glutamate resulted in cell death in a dose-dependent manner and supplementation of 100 nM sodium selenite prevented the detrimental effects of glutamate on cell survival. The glutamate induced cytotoxicity was associated with mitochondrial hyperpolarization, increased ROS production and enhanced oxygen consumption. Selenium reversed these alterations. Furthermore, glutamate increased the levels of mitochondrial fission protein markers pDrp1 and Fis1 and caused increase in mitochondrial fragmentation. Selenium corrected the glutamate-caused mitochondrial dynamic imbalance and reduced the number of cells with fragmented mitochondria. Finally, glutamate activated autophagy markers Beclin 1 and LC3-II, while selenium prevented the activation. These results suggest that glutamate targets the mitochondria and selenium supplementation within physiological concentration is capable of preventing the detrimental effects of glutamate on the mitochondria. Therefore, adequate selenium supplementation may be an efficient strategy to prevent the detrimental glutamate toxicity and further studies are warranted to define the therapeutic potentials of selenium in animal disease models and in human.

Mitochondrial dysfunction plays an important role in mediating ischemic brain damage. Immp2l is an inner mitochondrial membrane peptidase that processes mitochondrial protein cytochrome c1 (Cyc1). Homozygous mutation of Immp2l (Immp2l(Tg(Tyr)979Ove) or Immp2l(-/-)) elevates mitochondrial membrane potential, increases superoxide (O(2)(-)) production in the brain and impairs fertility. The objectives of this study are to explore the effects of heterozygous mutation of Immp2l (Immp2l(+/-)) on ischemic outcome and to determine the influence of Immp2l deficiency on brain mitochondria after stroke. Male Immp2l(+/-) and wild-type (WT) mice were subjected to 1-h focal cerebral ischemia. Their brains were harvested after 5 and 24-h of reperfusion. The results showed that infarct volume and DNA oxidative damage significantly increased in the Immp2l(+/-) mice. There were no obvious cerebral vasculature abnormalities between the two types of mice viewed by Indian ink perfusion. The increased damage in Immp2l(+/-) mice was associated with early increase in O(2)(-) production. Mitochondrial respiratory rate, total mitochondrial respiratory capacity and mitochondrial respiratory complex activities were decreased at 5-h of recirculation in Immp2l(+/-) mice compared to WT mice. Our results suggest that Immp2l deficiency increases ischemic brain damage by enhancing O(2)(-) production and damaging mitochondrial functional performance., (Published by Elsevier Inc.)

Both preischemic hyperglycemia and suppression of SOD2 activity aggravate ischemic brain damage. This study was undertaken to assess the effect of SOD2 mutation on ischemic brain damage and its relation to the factors involved in autophagy regulation in hyperglycemic wild-type (WT) and heterozygous SOD2 knockout (SOD2(-/+)) mice subjected to 30-min transient focal ischemia. The brain samples were analyzed at 5 and 24 h after recirculation for ischemic lesion volume, superoxide production, and oxidative DNA damage and protein levels of Beclin 1, damage-regulated autophagy modulator (DRAM), and microtubule-associated protein 1 light chain 3 (LC3). The results revealed a significant increase in infarct volume in hyperglycemic SOD2(-/+) mice, and this was accompanied with an early (5 h) significant rise in superoxide production and reduced SOD2 activity in SOD2(-/+) mice as compared to WT mice. The superoxide production is associated with oxidative DNA damage as indicated by colocalization of the dihydroethidium (DHE) signal with 8-OHdG fluorescence in SOD2(-/+) mice. In addition, while ischemia in WT hyperglycemics increased the levels of autophagy markers Beclin 1, DRAM, and LC3, ischemia in hyperglycemic, SOD2-deficient mice suppressed the levels of autophagy stimulators. These results suggest that SOD2 knockdown exacerbates ischemic brain damage under hyperglycemic conditions via increased oxidative stress and DNA oxidation. Such effect is associated with suppression of autophagy regulators.

Overexpression of selenoprotein H (SelH) gene provides neuroprotection in neurons against UVB-induced cell death by blocking the mitochondrial-initiated apoptotic cell death pathway. This study examined the effects of SelH on mitochondrial biogenesis and mitochondrial function. The results demonstrated that overexpression of SelH gene in neuronal HT22 cells significantly increased the levels of mitochondrial biogenesis regulators, nuclear respiratory factor-1 (NRF-1), peroxisome proliferator-activated receptor-γ coactivator-1 alpha (PGC-1α) and mitochondrial transcription factor A (Tfam). Mitochondrial cytochrome c content was elevated, mass was increased and respiration was enhanced. SelH transfection ameliorated ultra violet B (UVB)-induced suppression of mitochondrial biogenesis markers and depolarization of mitochondrial membrane potential. Overexpression of SelH promotes mitochondrial biogenesis and improves mitochondrial functional performance., (Copyright © 2010 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

The efficient functioning of the ER is indispensable for most of the cellular activities and survival. Disturbances in the physiological functions of the ER result in the activation of a complex set of signaling pathways from the ER to the cytosol and nucleus, and these are collectively known as unfolded protein response (UPR), which is aimed to compensate damage and can eventually trigger cell death if ER stress is severe or persists for a longer period. The precise molecular mechanisms that facilitate this switch in brain damage have yet to be understood completely with multiple potential participants involved. The ER stress-associated cell death pathways have been recognized in the numerous pathophysiological conditions, such as diabetes, hypoxia, ischemia/reperfusion injury, and neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, and bipolar disorder. Hence, there is an emerging need to study the basic molecular mechanisms of ER stress-mediating multiple cell survival/death signaling pathways. These molecules that regulate the ER stress response would be potential drug targets in brain diseases., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Mitochondrial hyperpolarization inhibits the electron transport chain and increases incomplete reduction of oxygen, enabling production of reactive oxygen species (ROS). The consequence is mitochondrial damage that eventually causes cell death. Uncoupling proteins (UCPs) are inner mitochondrial membrane proteins that dissipate the mitochondrial proton gradient by transporting H(+) across the inner membrane, thereby stabilizing the inner mitochondrial membrane potential and reducing the formation of ROS. The role of UCP2 in neuroprotection is still in debate. This study seeks to clarify the role of UCP2 in transient focal ischemia (tFI) and to further understand the mechanisms of ischemic brain damage. Both wild-type and UCP2-knockout mice were subjected to tFI. Knocking out UCP2 significantly increased the infarct volume to 61% per hemisphere as compared with 18% in wild-type animals. Knocking out UCP2 suppressed antioxidant, cell-cycle, and DNA repair genes, including Sod1 and Sod2, Gstm1, and cyclins. Furthermore, knocking out UCP2 significantly upregulated the protein levels of the inflammatory cytokines, including CTACK, CXCL16, Eotaxin-2, fractalkine, and BLC. It is concluded that knocking out the UCP2 gene exacerbates neuronal death after cerebral ischemia with reperfusion and this detrimental effect is mediated by alteration of antioxidant genes and upregulation of inflammatory mediators.

Both preischemic hyperglycemia and reduction of manganese superoxide dismutase activity are known to enhance neuronal death induced by transient cerebral ischemia. Transcriptional factor hypoxia-inducible factor 1 (HIF-1) regulates multiple downstream genes that modulate cell metabolism, survival, death, angiogenesis, hematopoiesis, and other functions. The objectives of this study were to determine (i) whether hyperglycemia is able to increase ischemic brain damage in mutant manganese superoxide dismutase (SOD2) mice and (ii) whether the reduction of SOD2 activity has a profound effect on HIF-1 protein expression under hyperglycemic ischemic condition. Both wild type and mutant SOD deficient (SOD2(-/+)) mice were induced to hyperglycemia 30min before induction of a 30-min transient middle cerebral artery occlusion (tMCAO). Brains were extracted after 5 and 24h of reperfusion for immunohistochemistry and Western blot analyses. The results showed that preischemic hyperglycemia significantly increased infarct volume in SOD2(-/+)mice and that HIF-1alpha protein levels were significantly reduced in ischemic core area at 5- and 24-h of reperfusion in hyperglycemic SOD2(-/+) mice. However, the HIF-1alpha protein levels were not significantly decreased in hyperglycemic wild type animals subjected to stroke. The results suggest that the increased brain damage observed in hyperglycemic SOD2(-/+) mice is associated with HIF-1alpha suppression, while hyperglycemia per se does not seem to exert its detrimental effects on ischemic brain via modulating HIF-1 pathway.

The uncoupling proteins (UCPs) are mitochondrial transporter proteins involved in proton conductance across inner mitochondrial membrane (IMM). UCP2, which is one of the members of this class of proteins, has a wide but restricted tissue distribution including brain. Its physiologic role according to emerging evidences, although still not clear, indicate that distribution of UCP2 may be related to regulation of mitochondria membrane potential (DeltaPsim), production of reactive oxygen species (ROS), preservation of calcium homeostasis, modulation of neuronal activity, and eventually inhibition of cellular damage. These factors are very important in determining the fate of neurons and damage progression in the brain during various neurodegenerative diseases including cerebral stroke. Recent evidence indicates that an increased expression and activity of UCP2 are well correlated with neuronal survival after stroke and trauma. This review briefly covers the present understanding of UCP2, which eventually may be beneficial to understand the precise role of UCP2 to develop strategy to identify its potential therapeutic application.