Shelley Russek - US grants

Destabilization of the delicate balance between inhibition and excitation in the nervous system may underlie many neurological disorders, including temporal lobe epilepsy (TLE). Gamma-aminobutyric acid (GABA) is the major transmitter at inhibitory chemical synapses in the central nervous system. Alteration in type A GABA receptor (GABA/AR) function due to change in subunit composition has been hypothesized to be a critical component of epileptogenesis. Little is known, however, about the genetic mechanisms that regulate granule cells of adult rats following pilocarpine-induced status epilepticus (SE), it has yet to be demonstrated that these changes are either necessary or sufficient for the development of epilepsy. The presence of an alpha4 subunit (GABRA4) and the lack of an alpha1 subunit (GABRA) in the GABAalphaR complex has been associated with a decrease in benzodiazepine sensitivity and a heightened sensitivity to blockade by zinc. Both of these features are also seen in adult rats with TLE following pilocarpine-induced SE. The broad objective of this project is to test the hypothesis that alterations in GABAA4 subunit gene expression play a critical role in the process of epileptogenesis by re- establishing normal levels of GABRA4 and GABRA1 following pilocarpine-induced SE and determining whether development of spontaneous seizures is subsequently prevented. To accomplish this objective we will further characterize the 5'flanking region of the GABRA4 gene to identify the boundaries of the promoter and its regulatory sequences that are critical for transcriptional activity in primary cultures of dentate granule cells. Adeno-associated parvovirus (AAV) vectors will then be designed to contain the GABRA4 promoter driving the transcription of GABRA1 transgene to up-regulate alpha1 subunit levels, or a GABRA4 antisense RNA, to down-regulate alpha4 levels. GABA/AR subunit levels will be examined following viral delivery of these vectors to dentate granule cells in culture and in vivo. An alternative strategy of decoy oligonucleotides containing regulatory sequences found in the GABRA4 promoter will also be tested in vitro and in vivo for its ability to down-regulate endogenous GABRA4 promoter will also be tested in vitro and in vivo for its ability to down- regulate sequences found in the GABRA4 promoter will also be tested in vitro and in vivo for its ability to down-regulate endogenous GABRA4 gene expression. These vectors will then be introduced into dentate granule cells of pilocarpine-treated rats to determine whether GABA/AR alpha-subunit expression can be normalized, and if so whether subsequent development of epilepsy can be prevented. Results of these studies should enhance our understanding of GABA/AR subunit gene regulation, establish if subunit changes are a necessary component of epileptogenesis and provide a basis for novel therapeutic strategies for the prevention or cure of epilepsy.

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GABA is the major transmitter at inhibitory chemical synapses in the CNS and alterations in type A GABA receptor (GABAR) function have been hypothesized to be a critical component of epileptogenesis. Alterations in GABAR subunit-specific mRNA levels in vivo underlie changes in functional properties of dentate granule cells following pilocarpine-induced status epilepticus (SE). Increased expression of the alpha-4 subunit and decreased expression of alpha-1 after pilocarpine treatment produces benzodiazepine insensitivity and increased zinc blockade of GABA currents. It has long been suspected that a change in the number or composition of GABARs can produce profound changes in inhibitory synaptic transmission, however, it is not known whether the changes in GABAR properties seen in animal models of temporal lobe epilepsy (TIE) are either necessary or sufficient for epileptogenesis. We have shown that the minimal promoter of the alpha-4 subunit gene (GABRA4) when contained within AAV vectors controls region-specific transcription and condition-specific SE up-regulation. In addition, we have shown that viral-mediated delivery of alpha-1 subunits using the GABRA4 promoter inhibits the development of spontaneous seizures, establishing for the first time a connection between the expression of GABAR subunits and epileptogenesis in animals. Results of functional promoter assays and in vivo chromatin immunoprecipitation (ChIP) show that the condition-specific response of the GABRA4 gene most likely reflects the binding of the early growth response factor 3 (EGR3). The experiments of this proposal are aimed at determining the role of prolonged synaptic activity and release of growth factors to Egr mediated changes in GABRA4 transcription that occur in cultured primary embronic and postnatal dentate granule cells. Using viral-mediated delivery of RNAi to target both endogenous Egr3 and GABRA4 gene expression, we will test whether increased alpha-4 subunits are required for development of spontaneous seizures in TLE rats.

? DESCRIPTION (provided by applicant): Brain derived neurotrophic factor (BDNF), a critical signaling molecule in the brain, is functionally linked to essential cellular processes, such as those associated with learning and memory. Altered BDNF signaling is thought to play a crucial role in dysregulated neuroplasticity underlying multiple neurological diseases, including epilepsy, yet the complex molecular determinants of these important brain processes are not fully understood. Our laboratories discovered that BDNF modulates inhibition, in part, through activation of the Janus Kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) signaling pathway. Employing primary cultured neurons and rodent models, we have reported that BDNF-induced JAK/STAT signaling represses the expression of synaptic ?1 containing GABAA receptors (GABARs) following epileptogenic brain injuries, including status epilepticus (SE) and brain trauma (TBI), and that JAK/STAT inhibition at the time of injury can reduce the severity of subsequent epilepsy. We also have preliminary evidence that BDNF-induced JAK/STAT signaling is mediated by TrKB activation at the cell surface promoting JAK2 autophosphorylation within an intracellular signalsome containing p75 neurotrophin receptors (a complex referred to as (i)p75NTRJ), leading to STAT3 recruitment and activation. Subsequent transport of the (i)p75NTRJ signalsome into the nucleus may alter transcription of multiple target genes, including inducible early cAMP repressor (ICER) that binds to and represses the ?1 GABAR gene after SE. To test our hypothesis that BDNF-induced JAK/STAT activation is dysregulated following brain injury causing a systematic change in the transcription of multiple genes that promote epileptogenesis, we will use an unbiased transcriptomic approach, including RNA-seq and ChIP-seq with antibodies to pSTAT3, p75NTR, JAK2, and ICER, in a preclinical epilepsy model utilizing wild-type and transgenic mice with inducible deletion of p75NTR or STAT3 genes. Specifically, we will: 1) Identify the target genes of (i)p75NTRJ that change their expression after epileptogenic brain injury and whether they are specific to neurons or part of a general cellular response using neuron selective or global p75NTR or STAT3 deletion/inhibition; 2) Mechanistically test the relationship of target genes identified in Aim 1 to the genomic response of individual neurons treated with BDNF or kainate; and 3) Determine the functional consequences of neuronal selective or global p75NTR or STAT3 removal on epileptogenesis and cognitive co-morbidities following brain injury. Combining transcriptomic analysis with mouse genetics in an animal model of epilepsy, we will expand our current knowledge of GABAR gene regulation after brain injury to appreciate the complex patterns of genomic changes that underlie epileptogenesis. Such understanding is essential for development of epilepsy modifying therapies targeting intracellular pathways that broadly regulate gene expression & may be more efficacious than those that target the products of single gene candidates in isolation.

DESCRIPTION (provided by applicant): Approximately 65 million people worldwide have epilepsy. Over one-third of these individuals do not respond to current medical therapy; consequently, novel therapeutic agents are needed. Although certain brain injuries such as traumatic brain injury, stroke and prolonged status epileptics (SE) are known to predispose to epilepsy, there are currently no effective interventions to reduce the risk of epilepsy after such injuries. We and others have established that activation of the Janus Kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) signaling pathway occurs in the hippocampus following brain injuries that lead to epilepsy. Using a rat model of temporal lobe epilepsy (TLE), we have preliminary evidence that this activation may be a critical mediator of acquired epileptogenesis. We have observed that peripheral administration of WP1066- a JAK/STAT pathway inhibitor -at the time of SE reduces both STAT activation & spontaneous seizure frequency for 4 weeks. While it was a useful molecule for displaying proof-of-concept, WP1066 is limited by highly unfavorable chemical & pharmacokinetic (PK) properties. Our initial structure- activity relationship studies have identified one novel analog of WP1066 that has increased stability, and we have preliminary evidence that this analog, as well as two known small molecular weight JAK/STAT inhibitors that are in clinical trial or FDA approved, result in higher brain concentrations and inhibit STAT3 activation after SE more effectively than WP1066. We propose to examine these novel JAK/STAT inhibitors to determine 1) their potency to inhibit JAK/STAT pathway activation and cellular toxicity in primary hippocampal neurons, 2) brain concentrations as a function of dose and time, ability to block acute seizure- induced JAK/STAT pathway activation in brain and specificity/off-target effects on other kinases, and 3) the effects of these novel JAK/STAT inhibitors on epilepsy development and cognitive co-morbidities in a rat TLE model. The expected outcome is identification of lead JAK/STAT inhibitors that can be advanced towards clinical testing for epilepsy disease modification.

ABSTRACT Growing evidence suggests that aberrant transcriptional regulation is a key component of the pathogenesis of multiple neuropsychiatric disorders, which are thought to stem from an individual's inability to cope with stressful and traumatic events. Indeed, while traumatic events are very common, only susceptible individuals go on to develop psychopathologies, while others have the ability to cope and therefore remain resilient. Research into the genetic and epigenetic mechanisms underlying the pathological response to stress is crucial for the development of effective treatments to either mitigate susceptibility or enhance resilience. The mechanisms underlying resilience to stress remain not fully understood. This project concerns the transcriptional repressor RE1-Silencing Transcription factor (REST), also known as neuron-restrictive silencer factor (NRSF). REST is expressed in the central nucleus of the amygdala (CeA), a key brain site for the behavioral and endocrine responses of stress. Recent studies have suggested that REST may be a protective factor which would make neurons more resistant to stressors via the repression of downstream genes. Interestingly, we have found that REST levels are significantly elevated in the CeA of rats which are resilient to the effects of chronic social defeat stress (SDS). Several gaps still exist: i) Although REST is elevated in the amygdala of SDS resilient rats, whether the increased levels reflect increased binding of REST to target genes is unknown, as well as whether such binding alters gene expression in a region-specific manner. ii) Furthermore, it is unknown whether REST has a role in the development of behavioral vulnerability to chronic stress. The provocative, high risk/high reward hypothesis of this application is that increased function of the REST transcriptional repressor in the CeA underlies the resilience to the adverse effects of chronic stress. This hypothesis will be tested by combining an established animal model with state-of-the-art molecular and epigenetic techniques to pursue the following specific aims: Aim 1 will determine whether elevated levels of REST in the CeA of SDS resilient rats reflect increased binding of REST to endogenous NRSE sites and the transcriptional repression of REST target genes in CeA neurons, by using chromatin immunoprecipitation assays followed by high density sequencing (ChIP-Seq), and RNA-seq. Aim 2 will determine whether CeA REST mediates resilience to chronic social defeat stress, by overexpressing REST in the CeA in vivo using an adeno-associated viral (AAV) vector in SDS animals, and qPCR to validate REST-mediated repression of specific gene targets in the CeA that are stress responsive. The proposed experiments lay the foundation for highly relevant studies to test the hypothesis that CeA REST is a substrate that confers resilience to stress-induced psychopathologies. This research may lead to a breakthrough in our understanding of the role of REST in the adult brain and of stress-related disorders.

Major barriers impede the translation of basic research findings from preclinical animal models of Alzheimer's disease (AD) into the discovery of methods for early detection of AD onset and the treatment of memory dysfunction. We hypothesize that early dysfunction of memory will be observable as dysfunction of hippocampal trisynaptic circuit dynamics (TCDs) that are specifically associated with spatial memory. The individual firing patterns of CA3 & CA1 pyramidal cells (or ?place cells?) within the trisynaptic circuit can be measured while acquisition, encoding, recall, and reconsolidation of spatial representations occurs. Our recent in vivo studies of aged animals, a model for amnestic mild cognitive impairment, have revealed age and novelty specific effects on CA3 & CA1 place cell dynamics that are rapidly reversed by acute administration of levetiracetam + valproic acid (Robitsek 2015). We also hypothesize that changes in properties of single neurons within the trisynaptic circuit will be an early signature for future behavioral impairment and for identifying genome responses that may be most relevant to AD during the prodromal phase. In this multidisciplinary application, we propose to use the novel TgF344-AD rat model (expressing mutant human amyloid precursor protein (APPsw) and presenilin 1 (PS1?E9)) to identify hippocampal TCDs that change during development of AD-associated memory dysfunction and to interrogate their underlying transcriptome and methylome. Prodromal changes in neural activity appear to play a role in the progression of neuropathological changes observed in AD by promoting the release of tau and the formation of neurofibrillary tangles. Accordingly, plasma tau levels are associated with cognitive decline and conversion of mild cognitive impairment into dementia. Less is known, however, about the cell specific response of the genome during the onset and course of AD progression that may factor significantly into disease etiology and resilience, nor the composition of the transcriptome of individual cells that respond dynamically to the onset and growing burden of inflammation. As a first step to fill this gap in knowledge, we propose the following two Aims: 1) To determine the temporal window for altered neural network activity in TgF344-AD rats that anticipates the age related progressive deterioration of spatial memory (in vivo electrophysiology); and 2) To determine the transcriptome of unique cell types in the TgF344-AD hippocampus (single cell RNA-sequencing (scRNA-seq) using a within-subjects design (Aim 1)) and the state of the TgF344-AD hippocampal genome as determined in parallel molecular studies (bulk RNA-seq and Methyl-seq). The proposed research has the potential to detect prodromal signatures of genomic events that underlie the onset of memory decline, uncovering relevant molecular determinants that may enable interventions to enhance memory and, conceivably, slow disease progression and human suffering.

ABSTRACT Temporal lobe epilepsy (TLE) develops after a period of ongoing molecular cascades and neural circuit remodeling in the hippocampus resulting in increased susceptibility to spontaneous seizures. Targeting these cascades in TLE patients could reverse their symptoms and have the potential to provide a viable disease- modifying treatment, especially for the large portion of over 30% of TLE patients who do not respond to any available treatments. In recent years, the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathway has been implicated in temporal lobe epilepsy (TLE). The JAK/STAT pathway is known to be involved in inflammation and immunity, and only more recently has been shown to be associated with neuronal functions such as synaptic plasticity. Our laboratories previously showed that a JAK inhibitor, WP1066, could greatly reduce the number of spontaneous seizures that animals went on to develop over time in the pilocarpine model of status epilepticus (SE). We have continued to investigate the mechanism of JAK/STAT- induced epileptogenic responses through the use of a new transgenic line we developed where STAT3 knockdown (KD) can be controlled by tamoxifen-induced CRE expression specifically in forebrain excitatory neurons via the Calcium/Calmodulin Dependent Protein Kinase II alpha (CamK2a) promoter. We now report that this knockdown of STAT3 (nSTAT3KD) markedly reduces spontaneous seizure frequency in the intrahippocampal kainate model (IHKA) and ?rescues? mice from KA-induced memory deficits as measured by Contextual Fear Conditioning. Recently, using deep RNA-sequencing we also discovered transcriptomic signatures 24 hours after SE that occur in response to IHKA injections (ipsilateral and contralateral to the injection site) and are reversed by nSTAT3KD, especially for those genes important in sphingolipid metabolism: a regulator of neuronal structure, and the trafficking, stability, and function of multiple membrane bound receptors, including ligand- and voltage-gated ion channels. These findings, taken together with our preliminary IHKA metabolome, brings us to propose the following unique hypothesis that there is a JAKx/STAT3 pathway in excitatory forebrain neurons that becomes activated in response to prolonged seizures and that identifying the cells most susceptible to STAT3 signaling during the epileptogenic process will provide a window on basic circuitry that underlies memory formation, and most importantly, the brain's susceptibility to epilepsy development. To test this hypothesis, we have three Aims using state of the art molecular technologies (metabolomic profiling, single nuclei RNA sequencing, and chromatin immunoprecipitation sequencing) to interrogate the molecular signature of the hippocampus (24 h, 2 wk, and 4 wk after IHKA SE) . The emerging transcriptome for STAT3 in the context of epilepsy suggests that it may be useful for identifying potential epileptogenic gene networks that were previously unknown, selecting early-detection biomarkers that inform seizure susceptibility, as well as choosing new targets for the future treatment of intractable epilepsies.