Amy R. Brooks-Kayal - US grants

Gamma-Aminobutyric acid (GABA) is the neurotransmitter at most inhibitory synapses in the vertebrate forebrain. The GABA(A) receptor is a ligand- gated anion channel composed of multiple receptor subunits. Alterations in GABAergic transmission have been implicated as a possible etiologic factor in some forms of epilepsy, and developmental changes in expression of GABA(A) receptor subunits may play an important role in both normal development and certain developmental neurological disorders such as age- related epilepsies. The GABA(A) receptor is the site of action of phenobarbital and benzodiazepines, two of the most commonly used anticonvulsants during infancy and early childhood. Little is known of processes regulating developmental expression of the GABA(A) receptor under normal conditions, and the effects of pathophysiological states such as seizures and anticonvulsant treatment on developmental expression of GABA(A) receptors have not been studied. A novel approach is proposed for addressing these questions by combining an in vitro system of forebrain neuronal development with the technique of single cell mRNA amplification which permits simultaneous molecular, electrophysiological, and morphological characterization of individual developing neurons. To evaluate potential mechanisms involved in developmental regulation of GABA(A) receptor subunit gene expression it is proposed: 1. To examine the developmental expression of the GABA(A) individual forebrain neurons differentiating in vitro and in vivo. 2. To examine the role of synapse formation and neuronal activity in the regulation of GABA(A) receptor subunit expression during development. 3. To determine the effects of seizure activity and treatment with anticonvulsant medications on the pattern of developmental expression of GABA(A) receptor subunit mRNAs utilizing an in vitro seizure model. It is anticipated that these studies will provide insight into mechanisms regulating developmental expression of the GABA(A) receptor both during normal neurodevelopment and in developmental neurological disorders such as the pediatric epilepsies.

DESCRIPTION (provided by applicant): Epilepsy is a common and disabling condition that frequently begins in infancy and childhood. The cellular and molecular changes that underlie the development of epilepsy (epileptogenesis) are not fully understood. GABAA receptors (GABARs) are the most abundant inhibitory neurotransmitter receptors in forebrain, and several lines of evidence indicate that alterations in these receptors may play a critical role in epileptogenesis. During the initial funding period of this grant, we demonstrated long-term, age-specific changes in expression of GABAR subunits in hippocampal dentate granule neurons (DGNs) following lithium-pilocarpine induced status epilepticus (SE) during early postnatal development. To fully understand the importance of these GABAR alterations in developmental epileptogenesis, however, we must determine 1) if these changes precede development of epilepsy or are a response to ongoing seizure activity, 2) if these changes occur in other developmental models of epilepsy and other regions of the hippocampus, and 3) if these changes are a critical determinant of later epilepsy development. In our first competitive renewal, studies are proposed to address these three fundamental questions. We will determine whether SE-induced changes in GABAR subunit expression precede or follow onset of epilepsy and if manipulation of GABAR subunit expression alters the frequency of epilepsy development. We will also expand our studies to examine GABAR subunit expression following early-life SE in two additional developmental epilepsy models (kainic acid-induced seizures and hyperthermia-induced seizures) and in three different regions of the hippocampus (CA1 and CA3 regions in addition to dentate gyrus). The proposed studies are expected to provide evidence that hippocampal GABAR subunit changes occur in a variety of developmental epilepsy models, precede onset of spontaneous seizures and play a critical role in the process of epileptogenesis after early-life SE. Results of these studies should advance our understanding of the role of GABAR changes in developmental epileptogenesis and facilitate development of new therapies for the prevention or cure of epilepsy after early-life insults by identifying potential new therapeutic targets.

DESCRIPTION (provided by applicant): Temporal lobe epilepsy (TLE) is the most common form of epilepsy &is often medically intractable. A large body of evidence indicates that abnormalities in inhibitory neurotransmission play an important role in TLE. GABAA receptors (GABARs) are pentamers composed of subunits from multiple subunit families that display developmental, regional and disease specific differences in expression, however, little is known regarding their regulation either in health or in disease. Our laboratories have identified long-term changes in GABAR a subunit gene expression, including decreases in expression of the a1 gene (Gabra1) in rat hippocampal dentate granule neurons following status epilepticus (SE) that are associated with later development of epilepsy. In the previous funding period, we established that decreased transcription of Gabra1 after SE is mediated by inducible cAMP early repressor (ICER) and phosphorylated CREB that bind to the Gabra1 CRE site. We further showed that ICER transcription is activated by the Janus Kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) signaling cascade, via actions of brain derived neurotrophic factor (BDNF). The JAK/STAT pathway has been little studied in epilepsy, and beyond its role in Gabra1 regulation, it is known to be an important regulator of neuronal proliferation, survival and gliogenesis, all of which may be important contributors to epileptogenesis. In the current proposal we will examine how BDNF signals through the JAK/STAT pathway to control GABAR subunit expression in the brain and its potential role in seizure- susceptibility. Specifically, we propose to: I. Determine how BDNF activates the JAK/STAT pathway. II. Determine the region and cell specificity of JAK/STAT activation in vivo after SE. III. Determine whether animals can be rescued from epilepsy development via manipulation of the BDNF-induced JAK/STAT pathway before or after SE. Results of these studies will provide new information about the dynamic interactions of BDNF and the JAK/STAT signaling cascade in its regulation of brain inhibition, especially as it pertains to the formation of a1 containing GABARs, and have the promise of facilitating the development of new therapies for the prevention, treatment or cure of epilepsy, as well as other nervous system disorders that share a change in the functional expression of a1 containing GABARs. PUBLIC HEALTH RELEVANCE: Epilepsy and seizures affect over 3 million Americans of all ages, and 50,000,000 people worldwide. Over thirty percent of patients with epilepsy have seizures that cannot be controlled with current treatments and up to 50,000 people die each year from seizures and related causes. The proposed studies seek to find new ways of treating and preventing epilepsy by identifying and then reversing the changes in brain cells that lead to this condition.

DESCRIPTION (provided by applicant): Advancing the care of children with nervous system disorders is critically dependent on training the next generation of academic pediatric neurologists to be both outstanding clinicians and successful independent basic and translational researchers who can work effectively in multidisciplinary research teams. The Colorado Neurosciences Academic Development Award (NSADA K12) program based at the University of Colorado Anschutz Medical Campus and Children's Hospital Colorado will leverage the combined resources of an outstanding neuroscience research community, our extensive university infrastructure for research training and one of the best children's hospitals in the country to provide exceptional basic-translational pediatric neuroscience research training for the next generation of academic pediatric neurologists. The overall goal is to provide our Scholars the scientific and professional skills needed for productive academic research careers as independent investigators and leaders who will make significant impacts in Child Neurology research. We will meet this goal with two aims: 1) provide the highest quality, personally designed training and career development opportunities focused in basic and translational science research; 2) provide training experiences that will educate the Scholars in multidisciplinary, integrated, team science required for translation of basic discoveries into opportunities to help solve and prevent major unmet problems in Child Neurology. To accomplish these aims, we have engaged outstanding research faculty in basic and translational science that have highly successful research and research training programs with proven track records of excellence to provide our scholars with optimal research training and career development. During this 5 year program we will recruit 3 Scholars from graduating pediatric neurology fellows and junior faculty (Instructors or Assistant Professors) who have strong academic records, are motivated and committed to establishing independent research careers, and have demonstrated evidence of research potential, and provide each of them with 3 years of uninterrupted research time. We will create a leadership and training program structure that will establish: a) criteria and processes for recruitment of scholars, research mentors, and individual mentorship teams; b) required research and career development training; c) evaluation of all aspects of the program. The Scholars will be exposed to a broad array of research and career development opportunities, and will learn the mechanisms involved in multidisciplinary, collaborative team science. We are committed to preparing the next generation of independent Pediatric Neurology/Neuroscience investigators to discover mechanisms of disease and effectively translate these discoveries into improvements in care for children with neurological disease.

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.