Raghu Vemuganti - US grants

DESCRIPTION (provided by applicant): Focal cerebral ischemia is associated with a robust inflammation that contributes to the progression of ischemic neuronal damage. The mechanisms, which modulate the inflammation after focal ischemia, are not well-understood. Suppressor of cytokine signaling (SOCS) family of proteins controls the inflammation in the peripheral organs. Binding of pro-inflammatory cytokine IL-6 (formed in excess after ischemia) to its receptors induces transphosphorylation of the receptor-associated Janus kinases (JAKs). Phosphorylated JAKs in turn phosphorylate the down-stream STAT family of transcription factors, which dimerize and translocate into nucleus. Binding of phosphorylated STAT to DNA stimulates cytokine gene expression to generate more interleukins. This cycle leads to sustained inflammation unless controlled. Phosphorylated STAT also stimulates SOCS gene expression. SOCS proteins act as intracellular negative feedback regulators to inhibit JAK-STAT phosphorylation and thereby dampen the cytokine signal transduction. In the adult brain, SOCS proteins are expressed at a very low level, but can be induced rapidly. Our preliminary data showed an upregulation of SOCS-3 and STAT-3 expression after focal ischemia. We hypothesize that SOCS-3 induction is an endogenous neuroprotective event to control post-ischemic inflammation and neuronal damage. We also hypothesize that in the ischemic brain, SOCS-3 actions are mediated by STAT-3. Using antisense knockdown and adenovirus-induced overexpression of individual proteins, we will analyze the mechanism of action of the 3 control points of SOCS-3 pathway (IL-6, STAT-3 and SOCS-3) in modulating post-ischemic inflammation and neuronal damage. We will study (a) if SOCS-3 knockdown increases and overexpression decreases interleukin levels and STAT-3 phosphorylation after ischemia; (b) if IL-6 knockdown (starting point of the cascade) prevents STAT-3 phosphorylation and SOCS-3 expression after ischemia, and (c) if STAT-3 knockdown/overexpression modulates post-ischemic SOCS-3 induction. Using GeneChip in combination with antisense and adenovirus, we will analyze the ischemia-induced gene expression changes modulated by SOCS-3. The ultimate goal is to define the role of SOCS-3/STAT-3 in post-ischemic cerebral inflammation.

[unreadable] DESCRIPTION (provided by applicant): The role of transcription factors (TFs) in modulating post-ischemic cerebral inflammation is not evaluated in detail. While Egr1 (NGFI-A/Krox24) is a TF that promotes inflammatory gene expression, at least 2 other TFs can control Egr1. Of these, c-EBP-beta can stimulate and PPAR-gamma can inhibit Egr1 induction. We hypothesize that after focal ischemia (1) EgM induction contributes to inflammation and brain damage; (2) c-EBP-beta is an upstream TF that induces Egr1 expression and inflammation; and (3) PPAR-gamma activation can curtail Egr1 induction and inflammation. Preliminary studies showed (a) sustained upregulation of Egr1, c-EBP-beta and PPAR-gamma expression after focal ischemia; (b) smaller infarcts in Egr1 null mice and bigger infarcts in EgM adenoviral transfected rats after focal ischemia, (c) curtailed post-ischemic inflammatory gene expression in EgM null mice, (d) less brain damage, decreased inflammation and less Egr1 induction in c-EBP-beta knockout mice after focal ischemia, and (e) prevention of post-ischemic EgM induction, inflammation and infarction by treatment with PPAR-gamma agonists. Using antisense knockdown, adenovirus-induced overexpression, and null mice, we will evaluate the functional significance and the interactive mechanism of action of these transcription factors in modulating inflammation and neuronal damage after transient focal ischemia in rodent brain. We will study if (a) Egr-1 knockdown prevents and Egr-1 overexpression exacerbates post-ischemic inflammation and brain damage; (b) Ischemia in Egr-1 knockout mice results in less inflammation and smaller infarcts; (c) C-EBP beta knockout mice show curtailed EgM induction, decreased inflammation and less neuronal damage after ischemia; and (d) PPAR-gamma agonists decrease the post-ischemic inflammation and brain damage by preventing EgM induction. The ultimate goal is to define the role of EgM and its regulators to develop therapies to control cerebral inflammation at the level of transcription. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The molecular mechanisms that promote neuronal death and thus neurological dysfunction after stroke (focal cerebral ischemia) are not understood completely. We and others showed that focal ischemia leads to extensive temporally orchestrated changes in the mRNA expression in rodents. Due to their position between the genomic DNA and functional proteins, mRNAs are considered as the key controllers of the cellular functions. However, recent studies showed that several small, non-coding, evolutionarily conserved RNAs known as microRNAs (miRNAs) control mRNA levels and function in various organisms including vertebrates. No studies to date examined whether the cerebral miRNA profiles will be altered in the post-ischemic brain and if yes, those changes have any functional significance in promoting the brain damage. This R21 proposal wishes to analyze the following hypotheses. (1) Focal ischemia alters cerebral miRNA profiles that might contribute in part to the post-ischemic pathological events and thus brain injury, (2) Endogenous and exogenous neuroprotective strategies act in part by modulating the miRNA profiles in the post-ischemic brain and (3) preventing the function of specific miRNAs that are upregulated in the post-ischemic brain using miRNA inhibitors can affect the down-stream targets and the stroke outcome. The long-term goal is to understand the role of miRNA in ischemic brain damage and to develop novel molecular therapies to modulate miRNAs. PUBLIC HEALTH RELEVANCE: Stroke is the 3rd leading cause of death in adult population all over the world. Currently very few options are available for designing effective treatment paradigms to prevent stroke-induced neurological dysfunction. Understanding the molecular mechanisms that mediate the pathological events like inflammation, edema and ionic imbalance which ultimately promote neuronal death after stroke is important to develop novel therapies. While tens of thousands of messenger RNAs (mRNAs) control the formation of various proteins and thus cellular functions, few hundreds of small microRNAs control mRNA levels in cells. To date no studies evaluated the significance of miRNAs in stroke-induced brain damage. The goals of this proposal is to analyze if stroke alters miRNA profiles in rodent brain and if yes, whether those changes respond to pharmacological and endogenous neuroprotective paradigms. We will also analyze if miRNA levels and function can be modulated in brain to change the functional outcome of stroke in future. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Every year, thousands of humans suffer traumatic brain injury (TBI) and most of the survivors manifest moderate to severe neurological dysfunction. Currently no known therapies that can prevent secondary neuronal death and/or promote neurological recovery after TBI in humans are available. Inflammation that starts within minutes and sustains for days after brain injury is thought to promote the secondary neuronal death and thus motor dysfunction. As controlling inflammation at the level of transcription is an effective strategy to prevent the neuronal damage, the goal of this proposal is to evaluate the efficacy of the transcription factor PPAR3 agonist rosiglitazone in preventing neuronal death and neurological dysfunction following TBI in adult mice. Previous studies showed that acute treatment with statins also induce neuroprotection following TBI, which is independent of their capability to lower cholesterol. Statins increase PPAR expression which is thought to mediate some of the pleiotropic beneficial effects of statins after an injury. Hence, we wish to test if the therapeutic potential and the window of benefit of rosiglitazone treatment following TBI can be enhanced by co-treating the animals with simvastatin. As both these compounds are FDA-approved, if proven beneficial, the combination can be quickly translated into clinical use to minimize the neurological deficits following TBI in humans. PUBLIC HEALTH RELEVANCE: Traumatic brain injury is a leading cause of disability in the world with few current therapeutic options. This proposal wishes to evaluate if a combination therapy with rosiglitazone and simvastatin (two FDA-approved drugs) can prevent the secondary neuronal death and thus neurological dysfunction after traumatic brain injury in mice. The ultimate goal is to identify drugs that benefit the recovery of brain trauma patients.

DESCRIPTION (provided by applicant): The protein-coding genes represent <2% of the eukaryotic genome as ~98% of the transcriptional output is non-coding (nc) RNAs. The ncRNAs are currently considered as the master controllers of the transcription and translation and hence any disruption in their function could lead to severe compromises in cellular homeostasis. Despite their abundance and paramount functional importance, very few studies to date evaluated the significance of ncRNAs in acute brain damage. We and others recently showed that miRNA expression profiles alter extensively following stroke;and modulating specific miRNAs induce neuroprotection. These studies indicate the role of ncRNAs in ischemic pathophysiology, but the significance of other ncRNAs like piwi-interacting RNA (piRNA) to ischemic brain damage is not evaluated yet. The piRNAs (26 to 31 nt long) are the most abundant of all ncRNAs with >40,000 piRNAs identified so far in eukaryotes. The major function attributed to them is to selectively target and silence the RNAs formed by the retrotransposons (RTs). As RTs are the predominant class of transposons (jumping genes) which mutate and disrupt the protein-coding genes, the piRNAs balance the fitness of the genome to maintain the cellular equilibrium. We hypothesize that "Focal ischemia alters cerebral piRNAome with functional significance to ischemic brain damage". In preliminary studies, we observed that >10% of the 40,000 piRNAs evaluated are expressed in rat cerebral cortex. Furthermore, when rats were subjected to focal ischemia, 106 cortical piRNAs were either up- or down-regulated (>2.5 fold). Bioinformatics showed that stroke- responsive piRNAs have several RT targets distributed throughout the genome, and the promoters of those piRNAs contain binding sites for multiple transcription factors. Aim 1a is to study the temporal pattern of piRNA expression profiles following transient focal ischemia. Aim 1b is to conduct bioinformatics analysis to find RTs targeted by piRNAs altered after ischemia. Aim 1c is to conduct bioinformatics analysis to identify transcription factor binding sites in the promoters of the stroke-responsive piRNAs. Aim 2 is to test the effect of knocking-down specific piRNAs on the histological and behavioral outcomes after focal ischemia. Aim 1 results will generate a catalog of the piRNA profiles after experimental stroke, and will identify the putative down-stream targets and upstream controllers of stroke-responsive piRNAs. Aim 2 results will show if piRNAs have a functional significance to stroke outcome. PUBLIC HEALTH RELEVANCE: Stroke is a leading cause of death and disability in adult population. The mechanisms that lead to stroke-induced brain damage are not fully understood. This proposal wishes to evaluate if stroke-induced brain damage is mediated in part by a subtype of non- coding RNAs known as piRNAs.

DESCRIPTION (provided by applicant): The miRNAs are transcribed as primary miRNAs (primiRs) by RNA polymerase II from either independent miRNA genes or from the introns of protein-coding genes. The miRNA gene promoters are known to contain many transcription factor binding sites, but the role of transcription factors in miRNA biogenesis is not yet understood. We made an in silico observation that the promoters of certain miRNAs contain binding sites for the transcription factor PPAR? known as PPREs. Preliminary studies showed that several miRNAs that contain PPREs in their promoters were induced by PPAR? agonist rosiglitazone indicating that PPAR¿ might control the expression of miRNAs. In addition to targeting 3'-UTRs of mRNAs to repress translation, the miRNAs can also bind to the promoters of protein-coding genes in a sequence-specific manner. With bioinformatics, we observed binding sites for 4 miRNAs in PPAR? promoter indicating that those miRNAs might control PPAR? gene expression. Interestingly, PPAR? promoter contains binding sites for miRNAs that have PPREs in their promoters. For example, promoters of mir-329 and miR-145 showed 4 PPREs each while PPAR? promoter showed binding site for both miR-329 and miR-145. We hypothesize that PPAR? and specific miRNAs modulate each other with significant consequences in maintaining cellular equilibrium. Furthermore, some of the pleiotropic neuroprotective effects of PPAR? agonists might be due to their effect on miRNAs. Aim 1 is to test if PPAR? activation alters the expression of PPRE-containing miRNAs and to study if PPAR? down-stream miRNAs play a role in PPAR-mediated neuroprotection. Aim 2 is to test if specific miRNAs can induce PPAR? expression by promoter interaction and if that can potentiate the neuroprotection afforded by PPAR? agonists. The overall goal is to study if PPAR? is in a cyclical loop with certain miRNA and their mutual inducibility has functional significance. PUBLIC HEALTH RELEVANCE: Transcription factors and microRNAs are master controllers of gene and protein expression. This proposal wishes to evaluate the mutual interaction and the subsequent consequences of a transcription factor known as PPAR with certain miRNAs in mediating neuroprotection after ischemia.

DESCRIPTION (provided by applicant): Traumatic brain injury (TBI) leads to long-term neurological dysfunction. The extent of secondary neuronal death (mediated synergistically by pathophysiologic events that include but not limited to inflammation, oxidative stress, ER stress and ionic imbalance) dictates the functional outcome after TBI. The present proposal wishes to evaluate if controlling oxidative stress and the interconnected inflammation can minimize the secondary brain damage leading to improved neurological recovery in rodents subjected to TBI. We will test apocynin that inhibits NADPH oxidase subunit NOX2 and thus curtails reactive oxygen species (ROS) formation, and TBHQ that potentiates the transcription factor Nrf2 which is upstream to many antioxidant genes and thus efficiently neutralizes ROS. Our preliminary data provided the proof-of-principle for the efficacy of these 2 drugs in a rodent TBI model. In this proposal we will identify the minimal efficacious dose and the window of opportunity for the 2 drugs. As secondary brain damage after TBI is multifactorial, a combination therapy to achieve neuroprotection by targeting multiple interactive pathways might be more efficacious than mono-therapies that target single pathways. To efficiently control oxidative stress, it is essential to curtail the formation of ROS and at the same time increase the disposal of ROS. Hence, we will test if a combination of apocynin and TBHQ curtails neuronal death and neurological dysfunction after TBI more effectively.

DESCRIPTION (provided by applicant): Efficient functioning of the Endoplasmic reticulum (ER) is indispensable for normal cellular functions as ER plays an important role in the maintenance of intracellular Ca2+ homeostasis, proper folding of proteins, post-translation modifications and transport of nascent proteins to different destinies. Any disruption of ER results in the activation of a complex set of signaling pathways that propagate from the ER to the cytosol to the nucleus. These are collectively known as unfolded protein response (UPR), which is aimed to compensate damage and to restore the normal cellular homeostasis. While limited and transient UPR is beneficial, prolonged or severe UPR, and the ensuing ER stress leads to cell death. Furthermore, CNS insults leads to oxidative stress which is also neurotoxic. We hypothesize that following traumatic brain injury (TBI), ER stress and oxidative stress are coincidental, potentiate each other bi-directionally and synergistically exacerbate the secondary brain damage. Using a rodent model of controlled cortical impact injury, we wish to answer the following questions. (1) What is the role of PERK-mediated ER stress pathway after TBI? (2) In the post-injury brain, are ER stress and oxidative stress connected? In particular, if ER stress mediated by PERK and oxidative stress modulated by NADPH oxidase NOX2 influence each other? (3) What is the effect of knocking-out/inhibiting individual rate-limiting proteins of PERK pathway eif2¿, ATF4 and CHOP on oxidative stress and neuronal damage after TBI? Conversely, what is the effect of knocking-out/inhibiting NOX2 on ER stress and neuronal damage after TBI? The long-term goal is to understand the mutual interplay of ER stress and oxidative stress in post-TBI brain damage.

? DESCRIPTION (provided by applicant): The mechanisms that contribute to the secondary neuronal death and thereby the neurological dysfunction following stroke are not completely understood. Recent studies showed that cerebral ischemia rapidly alters the expression profiles of various classes of noncoding RNAs (ncRNAs). This observation has significant functional implications to post-stroke outcome as ncRNAs are currently considered as controllers of transcription and translation in mammals. In particular, our recent studies showed that expression of several long noncoding RNAs (lncRNAs; lincRNAs) that serve as scaffolding between chromatin-modifying proteins (CMPs), transcription factors, histones and DNA. In the present proposal, as a test case we wish to analyze the role of one such lncRNA named Fos Downstream Transcript (FosDT; MRAK159688), which is highly up-regulated in the ischemic brain. Based on the preliminary data, we hypothesize that (1) Increased FosDT expression contributes to post-stroke secondary brain damage and neurological dysfunction. (2) Mechanism of FosDT action is by its interaction with CMPs Sin3A and coREST and thereby modulating the REST-mediated suppression of GRIA2 and NFKB2 in the ischemic brain. FosDT knockdown protects brain after ischemia by de-repressing these REST-suppressed genes that prevent ischemic neuronal death. Aim 1 is to evaluate the functional significance of FosDT in promoting secondary brain damage and neurological dysfunction following experimental stroke using FosDT siRNA-mediated knockdown. Aim 2 is to evaluate if the mechanism of FosDT-mediated ischemic brain damage is by interaction with the REST-mediated repression of GRIA2 and NFKB2. Overall, this project will evaluate the significance of an lncRNA induced after stroke in post-ischemic brain damage and the downstream mechanisms that propagate the actions of the lncRNA after ischemia. These are the first proposed studies to our knowledge to evaluate the role of an lncRNA in post-ischemic brain damage. The long- term goal is to prioritize the experiments to decide if it is worth exploring this new class of RNAs as stroke therapeutics.

Alpha-synuclein (?-Syn) is one of the most abundant proteins in the CNS that is known to be a major player in the neurodegeneration observed in Parkinson?s disease. We show that stroke (transient focal ischemia) upregulates ?-Syn protein expression and nuclear translocation in neurons of adult rodents and humans. We further show that knockdown or knockout of ?-Syn significantly decreases the infarction and promotes better neurological recovery in rodents subjected to focal ischemia. Based on these exciting new leads, in this proposal we wish to test the therapeutic potential of targeting ?-Syn in post-stroke brain by following the criteria set by the Stroke Treatment Academic Industry Roundtable (STAIR) consortium. Aim 1 is to evaluate the window of therapeutic opportunity, effect of sex, age, route of administration and toxicity of ?-Syn siRNA therapy following focal ischemia in rodents. We further observed that a microRNA called miR-7a potently targets ?-Syn. Importantly miR-7a showed an inverse relation to ?-Syn (miR-7a levels were down-regulated while ?-Syn levels were upregulated after stroke). Hence, we will test the efficacy of replenishing miR-7a in the post-stroke brain to repress ?-Syn and thus decrease brain damage. Testing alternate approaches to target a protein gives better opportunities for future clinical translation. Hence, miR-7a mimic therapy will serve as an alternate approach to ?-Syn siRNA therapy. Aim 2 is to evaluate the window of therapeutic opportunity, effect of sex, age, route of administration, toxicity and long-term effects of miR-7a mimic after focal ischemia. The mechanisms that contribute to ?-Syn-mediated secondary brain damage after stroke are not well understood. We demonstrate that ?-Syn protein formed in excess in brain during the acute phase after stroke oligomerizes and forms aggregates with time. We further show that ?-Syn promotes brain damage by multiple pathologic mechanisms including mitochondrial fission. In chronic neurodegeneration, ?-Syn is known to act as an essential scaffolding molecule for the activation of GSK-3? and the subsequent Tau hyperphosphorylation that leads to activation of Drp1 which promotes mitochondrial fission. In preliminary studies we observed increased phosphorylation of GSK-3?, Tau and Drp1. In Aim 3, we will test if ?-Syn promotes post-ischemic mitochondrial fission and brain damage by involving GSK-3? and Tau. The long-term goal of these studies is to evaluate if targeting ?-Syn is a viable option for stroke therapy in both males and females and at different ages.

Mammalian genome encodes tens of thousands of long noncoding RNAs (lncRNAs) which are emerging as important regulators of transcription and translation. Any perturbation in the levels and function of lncRNAs can be expected to promote pathological changes. We recently showed that transient focal cerebral ischemia (stroke) in adult rodents rapidly changes the cerebral expression profiles of lncRNAs. We further show that post-ischemic brain damage and neurological dysfunction can be mitigated significantly by inhibiting an lncRNA called FosDT that was observed to be induced after stroke. Due to their functional importance as epigenetic modulators of transcription, lncRNAs has a tremendous potential to be developed as stroke therapies. Hence, we currently propose testing the efficacy of inhibiting FosDT in rodent stroke models. We hypothesize that ?FosDT is a novel therapeutic target to protect brain after stroke.? This is the first study to our knowledge to comprehensively test the significance of an lncRNA in protecting the brain after stroke. Recent guidelines suggest that stroke therapeutic development should follow the criteria suggested by the STAIR (Stroke Treatment Academic Industry Roundtable) consortium. Hence, we will incorporate many of those recommendations in the present FosDT therapeutic testing. Aim 1 is to test the dose and window of opportunity after stroke for FosDT therapy as a function of sex and age. Aim 2 is to test post-stroke FosDT therapy as a function of type of ischemia, route of administration, and systemic toxicity. Aim 3 will evaluate the efficacy of post-ischemic FosDT therapy as a function of species and diabetes (a comorbid condition) and to identify if this therapy influences the post-ischemic pathophysiologic mechanisms. Aim 4 will evaluate the mechanisms that are upstream and downstream of FosDT in ischemic brain. Overall, this is the first systematic study to test the therapeutic potential of targeting an lncRNA to protect brain after stroke.

Epigenetic changes in DNA and histones are known to significantly influence the gene expression and outcome after many diseases. The role of epigenetics in ischemic brain damage is not yet fully understood. Of particular interest, the cytosine in DNA undergoes methylation to form 5-methylcytosine (5mC) which is known to be a transcriptional silencer. Recent studies showed that 5mC will be oxidized by ten-eleven translocation (TET) hydroxylases to form 5-hydroxymethylcytosine (5hmC). This epigenetic change is considered as a transcriptional derepression mark that increases cell survival under adverse conditions. In particular, brain contains ~10 fold higher 5hmC levels than other organs of the body. Preliminary studies showed that transient focal ischemia in adult rodents significantly increase the genomic 5hmC levels in the peri-infarct cortex. TET3 knockdown decreased 5hmC levels, and exacerbated post-ischemic mortality and infarction in both male and female mice. On the other hand, increasing 5hmC levels by treatment with ascorbate (a TET inducer) significantly protected the brain after focal ischemia in a TET3-dependent manner. Hence, we hypothesize that ?Tet3 mediated induction of 5hmC is a neuroprotective adaptation that can be potentiated to protect brain after stroke.? The major neuronal isoform of TET3 lacks DNA binding domains. Our preliminary data show that TET3 binds to lncRNAs with high affinity. The lncRNAs are known to act as scaffolds to bring DNA/RNA/protein together enabling their action. LncRNAs are also known to modulate post-stroke outcome. Hence, we further hypothesize that ?lncRNAs play a vital role in scaffolding and guiding TET3 to specific genomic sites, and thus modulate 5hmC levels and functional outcome after stroke.? Aim 1: To evaluate if DNA hydroxymethylation is neuroprotective after stroke. We will test the functional significance of 5hmC in post-stroke pathophysiology by loss of function and gain of function of TET3. Genomic sites where 5hmC is increased after stroke will be mapped by chromatin immunoprecipitation combined with massively parallel DNA sequencing (ChIP-seq) following TET3 knockdown and induction. Aim 2: To study if lncRNAs regulate TET3-mediated DNA hydroxymethylation and the ensuing neuroprotection after stroke. We will determine the genomic locations modulated by the TET3-interacting lncRNAs by high throughput sequencing method chromatin isolation by RNA purification (ChiRP-seq). We will further study if lncRNA function is essential for TET3/5hmC mediated neuroprotection after stroke. The long-term goal is to define the role of 5hmC in post-ischemic pathology and to test if increasing 5hmC levels is beneficial after stroke.