Avtar Roopra - US grants

DESCRIPTION (provided by applicant): Epilepsy afflicts around one percent of the world's population. Unfortunately approximately a third of all affected individuals do not have their seizures controlled by currently available drugs. Therefore there is a pressing need for new classes of therapeutic options, requiring an improved understanding of the mechanisms that regulate plasticity in the nervous system. Recent work from my lab suggests that perturbation of energy metabolism may represent a novel route to controlling neuronal plasticity (Garriga-Canut et al, 2006). This application will explore the mechanism by which metabolic perturbation controls aspects of neuronal plasticity - specifically, i) epigenetic regulation of activity- dependent genes and ii) metabolic regulation of signaling cascades that control Long Term Potentiation (LTP) in the mouse hippocampus. Our previously published work and more recent preliminary data suggests that two key sensors of energy metabolism -AMP activated Protein kinase (AMPK) and C-terminal Binding Protein (CtBP) act to regulate neuronal plasticity in an acute cytoplasmic and post-translational manner and also a chronic epigenetic manner respectively. The function of both AMPK and CtBP can be modulated with small molecule pharmaceuticals that are well tolerated in vivo and we propose that either might represent attractive novel therapeutic targets for the treatment of epilepsy. This proposal aims to address the broad question of how energy metabolism can control i) epigenetic regulation of gene transcription and ii) kinase regulation of neuronal signaling cascades to modulate neuronal plasticity in the hippocampus. The findings will facilitate the generation of therapies that work through controlling sensors of energy metabolism to control plasticity in conditions such as epilepsy. PUBLIC HEALTH RELEVANCE: Epilepsy afflicts around one percent of the world's population but unfortunately approximately a third of all affected individuals do not have their seizures controlled by currently available drugs. Therefore there is a pressing need for new classes of therapeutic options, requiring an improved understanding of the molecules and mechanisms that function in the nervous system. Recent work from my lab suggests that metabolism may represent a novel route to controlling epilepsy and this application will explore the mechanism by which 2 sensors of metabolism (CtBP and AMPK) control neuronal function or 'plasticity'. The mechanisms and drugs we study here will hopefully lead to therapies that result in 'no seizures and no side effects'for the millions of people around the world with epilepsy.

PROJECT SUMMARY/ABSTRACT Tuberous Sclerosis Complex (TSC) affects approximately 1 in 6,000 people and presents with multiple neurological symptoms including, mental disability, autism, and epilepsy. TSC is caused by autosomal dominant inactivating mutations in either the TSC1 or TSC2 genes. Normally, TSC1 and TSC2 proteins heterodimerize and form a protein complex that ultimately inhibits mammalian Target of Rapamycin (mTOR), a kinase that regulates activity-dependent dendritic protein translation in the nervous system. Disease associated mutations in TSC1 or TSC2 results in persistent activation of mTOR, which results in learning and memory deficits, aberrant synaptic plasticity, epilepsy and a host of other symptoms. Rapamycin based drugs (rapalogues) that inhibit mTOR show promise in treating TSC. However, long-term inhibition of mTOR is known to activate other growth factor pathways, the chronic effects of which are either unknown or are linked to cancer progression and malignancy. Thus, new classes of drugs are required to mitigate the neurological symptoms associated with TSC. Using novel bioinformatics approaches, we have found that the transcription factor RE-1 Silencing Transcription Factor (REST) displays heightened function in human patient TSC cortical samples: in a screen of 189 transcription factors, REST target genes are the most differentially expressed between human TSC cortical samples and non-TSC samples. Moreover, our preliminary data demonstrate that i) REST protein levels are elevated in the TSC2 mutant mouse hippocampus and ii) pharmacological inhibition of HDAC1/2 (obligate cofactors for REST) restores appropriate plasticity in a mouse model of TSC. In this proposal, we will test the hypothesis that loss of TSC1 or TSC2 leads to enhanced REST function (epigenetic disregulation), which results in the altered synaptic plasticity seen in TSC. The Aims will be: Aim 1. Test the hypothesis that elevated REST is necessary and sufficient for aberrant plasticity. Aim 2. Test the hypothesis that antagonizing REST corepressors suppresses aberrant LTD and LTP in TSC. Should the hypothesis ?REST function is heightened to promote aberrant plasticity in TSC? be supported, an entirely new repetoire of drugs could potentially become available to treat the neurological symptoms associated with TSC.

? DESCRIPTION (provided by applicant): Epilepsy is the third most prevalent neurological disorder after stroke and Alzheimer's disease with an incidence of 1 in 26 individuals. Epileptogenesis refers to the progressive decrease in seizure threshold that ultimately results in unprovoked, spontaneous seizures that may increase in frequency, severity and duration. Though there are a number of anti-convulsant drugs available, there are no anti-epileptogenic drugs that mitigate the progression of the disease. Long-term changes in gene expression that are associated with epileptogenesis imply that one or more master regulators of transcription may be coordinating the brain alterations. In order to uncover these genetic mechanisms, we turned to the genome-wide expression datasets generated by the Epilepsy Microarray Consortium. The datasets consist of mRNA expression profiles of mouse dentate granule cells assayed at various time points after Status Epilepticus (S.E). Because these datasets are derived from brains induced by 3 different convulsant stimuli, each in 2 different labs, and at various time-points, this gives us the opportunity to discern model-independent and lab-independent alterations in gene networks. We used an innovative and novel bioinformatics tool developed by us to reveal the transcription factors and nuclear proteins that drive gene changes observed in the Epilepsy Consortium dataset. We found that a master epigenetic complex called Polycomb drives the majority of gene changes across labs, models and time. This is exciting because Polycomb is a well-known driver of life-long changes in gene expression that works by epigenetically silencing genes during body plan establishment across the phyla. Our preliminary data shows that the epigenetic mark uniquely catalyzed by Polycomb is induced in the hippocampus within 24hrs after seizures and remains at least 5 days after the seizure. In this proposal we will test the hypothesis that an alteration in Polycomb output is a principle modifier of epileptogenesis: we will determine whether the increase in Polycomb activity is pathological or protective. We will test whether pharmacological modulation of Polycomb can alter the process of epileptogenesis and thus potentially identify a novel class of drugs. Small molecule inhibitors of Polycomb such as those in clinical trials for lymphoma will be tested for their efficacy in stalling epileptogenesis. We anticipate that these studies will establish Polycomb as a major orchestrator of the long-term changes associated with epileptogenesis. If so, approaches that modulate Polycomb function may be of benefit to the 65 million people world- wide that live with epilepsy.

Project Summary/Abstract Epilepsy is the 4th most prevalent neurological disorder after stroke, Alzheimer?s and migraine with an incidence of 1 in 26 individuals. Though there are a number of anti-convulsant drugs available, there are no anti-epileptogenic drugs that mitigate the progression of the disease. Using novel bioinformatic approaches, we have identified an endogenous, protective program launched by the brain after a prolonged seizure that functions to mitigate pathological changes. Epileptogenesis is associated with a plethora of changes in the brain including alterations in plasticity, cell death, neurogenesis, inflammation and axonal sprouting. These changes occur over timescales ranging from many minutes to years, but the orchestrating mechanisms are virtually unknown. Long-term changes in gene expression that are associated with epileptogenesis imply that one or more master regulators of transcription may be coordinating the brain alterations. In order to uncover these transcriptional mechanisms, we turned to our recently published genome-wide expression datasets generated by the Epilepsy Microarray Consortium (EMC). The datasets consist of mRNA expression profiles of rat dentate granule cells assayed at various time points after Status Epilepticus (SE). Using a novel bioinformatic tool that integrates whole genome transcription factor binding data with gene expression profiles, we analyzed datasets derived from brains induced by 3 different convulsant stimuli, each in 2 independent labs, and at various time-points. This analysis projected that Polycomb target genes represent the majority of chronically altered genes during epileptogenesis. REST targets represent a second, overlapping, group of repressed genes. Polycomb is a well-known driver of life-long changes in gene expression that works by epigenetically silencing genes across the phyla. Our data shows an extremely robust induction of EZH2 protein (the catalytic methylase subunit of Polycomb) over a 20 day window post SE in neurons. Further, we find that antagonizing EZH2 shortly after SE robustly accelerates the onset of spontaneous recurrent seizures in mice, suggesting a protective rather than pathological role for EZH2. How antagonism of EZH2 later after SE remains to be determined. In this project, we will test the hypothesis that an alteration in Polycomb output is a principal modifier of epileptogenesis. We will ascertain whether EZH2 upregulation is always protective or whether its role evolves during the latent period. We will test the effect of an order-of-magnitude change in EZH2 levels on corepressor function to see whether such upregulation augments or hampers the repressive abilities of two major EZH2 containing complexes: Polycomb and REST. We anticipate that these studies will establish Polycomb as a major orchestrator of the long-term changes associated with epileptogenesis. If so, approaches that modulate Polycomb function may be of benefit to the 65 million people world-wide that live with epilepsy.