Ethan M. Goldberg, M.D., Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): [unreadable] Epilepsy affects 1-2% of the world's population. Given the role of voltage-gated ion channels in the regulation of neuronal excitability, there is general agreement that ion channels are involved in the pathogenesis of at least some forms of this disease. In fact, various types of epilepsy are due to mutation of genes that encode for components of voltage-gated channels selective for potassium (K+). Mice defective in the voltage-gated K+ channels Kv3.1 and Kv3.2 are epileptic, likely due to impaired cortical inhibition. This project seeks to study the role of Kv3.1/Kv3.2 in the properties of fast-spiking GABAergic interneurons (FS cells) - where these channels are specifically expressed - using dual whole-cell patch clamp recordings in the neocortex of mouse. Of particular interest are the roles of Kv3.1 and Kv3.2 in (1) neurotransmission at the FS cell terminal, and (2) the network behavior of interconnected FS cells. This project will explore the dynamics of GABA release at the FS cell terminal and its derangement in Kv3.1/3.2 knockout mice, and the disruption of synchronous FS cell behavior in these mice. This work may have implications for normal cognitive functions as well as neuropathology involving the GABAergic system, including epilepsy. [unreadable] [unreadable]

PROJECT SUMMARY This mentored career development award proposal describes an integrated training program designed to advance my career towards the goal of running an independent R01-funded biomedical research laboratory focused on the study of epilepsy. Currently, there is no way to prevent epilepsy in at-risk individuals prior to the appearance of seizures, and there are limited treatment options for patients with medically intractable epilepsy. With the guidance of my mentor, Dr. Coulter, I have designed a training plan to successfully learn and apply a coordinated, powerful set of state-of-the-art techniques ? including electrophysiology, optogenetics, and two- photon calcium imaging ? in vitro and then in awake, behaving experimental animals in vivo. The proposed research tests the hypothesis that brain circuit dysfunction in a well-established model of epilepsy is due to abnormal activity of a defined subtype of inhibitory interneuron, the fast-spiking cells (?FS cells?). This multimodal analysis of circuit-level mechanisms of epilepsy will yield novel results that will contribute to the development and application of novel therapeutic strategies to prevent and treat epilepsy. Candidate: I am currently Assistant Professor in the Division of Neurology at The Children's Hospital of Philadelphia (CHOP) and Departments of Neurology and Neuroscience at The Perelman School of Medicine at the University of Pennsylvania (UPenn). I am an M.D./Ph.D. physician-scientist with a strong background in neuroscience, having received a Ph.D. in Physiology & Neuroscience from NYU in the laboratory of Dr. Bernardo Rudy. I completed a five-year clinical training program in pediatric neurology at CHOP/UPenn and now take care of children with epilepsy in General Neurology and Neurogenetics Clinic at CHOP. This proposal builds on my long-standing interest in the neurobiology of disease and established interests in synaptic inhibition and GABAergic inhibitory interneurons in the cerebral cortex. This K08 award will provide me with critical training and support to insure a successful transition to independence and long-term achievement and productivity as a neuroscientist and academic pediatric neurologist in the field of epilepsy. My goal is to become an R01-funded independent investigator studying epilepsy in mouse models to inform the development of mechanistically oriented therapies that could be translated to, and transform, patient care. Environment: My mentor is Dr. Douglas Coulter, an established investigator in the field of epilepsy and a pioneer in the application of dynamic imaging methods to the study of epilepsy mechanisms. Dr. Coulter is Director of the Center for Dynamic Imaging of Nervous System Function at CHOP/UPenn and the Translational Research Epilepsy Program at CHOP; he has multiple RO1 grants studying epilepsy. Dr. Coulter also has a robust track record of mentoring trainees who have gone on themselves to be leaders in the field of epilepsy. His laboratory is located in the Abramson Research Building, where the 4th and 5th floors are dedicated to neuroscience research and include a collaborative group of highly successful scientists who are interested in and committed to my career development and success. Dr. Coulter and I have constructed an outstanding mentorship team to guide the execution of the proposed studies and my overall career development. I will attain mastery in the clinical field of epilepsy neurogenetics under the guidance of Eric Marsh, M.D., Ph.D., Head of the Section on Neurogenetics, Division of Neurology, at CHOP, who also runs an R01-funded basic neuroscience laboratory. Training will occur at CHOP/UPenn, an academically enriching neuroscience community with extensive resources and opportunities for scientific interaction, including a wide range of available coursework and multiple ongoing neuroscience-, neurology-, and epilepsy-related seminar series. My career development plan involves rigorous training in dynamic imaging, optogenetics, and the study of epilepsy in animal models, coursework in crucial subject areas, as well as formal and informal training in how to properly conduct science and run a research laboratory. This application is supported enthusiastically by the Division of Neurology at CHOP and Department of Neurology at UPenn. Research: My preliminary results show that there is abnormal GABAergic synaptic inhibition in the hippocampus in a well-established animal model of temporal lobe epilepsy; namely, with failure of the so-called ?dentate gate.? Rather than being a general failure of inhibition, I have determined that a defined subset of GABAergic inhibition interneuron in dentate gyrus exhibits abnormal activity in epilepsy. This proposal will build on my preliminary data to test the hypotheses that: (1) the mechanistic basis of the dentate gate is feed-forward inhibition specifically provided by fast-spiking interneurons, and (2) manipulation of FS cell activity in the epileptic brain using optogenetics can reconstitute normal circuit activity. I predict that targeted silencing of fast-spiking cells in control conditions will reproduce epileptic circuit pathology and augmenting the activity of these cells in epileptic brain will recover normal inhibition. These outcomes will provide novel information regarding the normal function of fast-spiking interneurons and role of synaptic inhibition in dentate gyrus, as well as establishing important mechanistic contributions to the pathogenesis of temporal lobe epilepsy. This mentored career development award will ultimately position me to translate the insights gleaned from basic neuroscience research to inform and motivate future attempts at the targeted treatment of epilepsy based on manipulation of GABAergic interneurons.

How activity of individual brain neurons results in the emergence, spread, and eventual termination of seizures is a basic and unsolved issue. Extensive research has examined brain activity during seizures using invasive and non-invasive tools, but there is a gap in our understanding of the cellular-level basis of large-scale phenomena such as seizures. Here, Dr. Sarah F. Muldoon (University at Buffalo, SUNY) and Dr. Ethan M. Goldberg (The Children's Hospital of Philadelphia and The University of Pennsylvania) use methods for recording neural activity from multiple single neurons in mice, as well as methods for recording large-scale neuroelectric activity (EEG), to identify groups of neurons whose activity is associated with time-evolving features of seizures. This project combines newly developed theoretical and computational neuroscience approaches with state-of-the art experimental techniques for imaging and manipulating brain activity. It evaluates a novel hypothesis that transitions to seizure onset and between seizure sub-states represent changes in the activity of small, identifiable clusters of neurons, and that such clusters can be identified and manipulated to modulate seizures. This multidisciplinary collaborative approach is expected to produce novel insights into the mechanisms of seizure generation and propagation, inform novel treatments for epilepsy, and provide a framework generalizable to larger efforts to link data related to changes in brain state across scales.

Recent work suggests a fine-grained and evolving heterogeneity in individual neuronal dynamics during seizures. However, relatively little is known about the relationship between single neuron activity and large-scale seizure dynamics. Here, we combine multilayer network theory with two-photon calcium imaging in an experimental in vivo epilepsy model to identify functional cell assemblies (small groups of neurons with similar, functionally-relevant activity patterns) associated with transition to and between seizure states. We characterize the ability of defined subsets GABAergic inhibitory interneurons to impact cell assembly dynamics, using optogenetics to manipulate interneurons to interrupt transition to seizure. The overall goal of this proposal is to develop a means for rapid detection of spatially- and functionally-defined neuronal assemblies associated with these sub-state transitions so as to predict and manipulate such transitions. The results will provide the first large-scale imaging data on the cellular architecture of epileptic seizures in vivo as well as a set of novel tools for the analysis of such data. This work will lead to the development of neural control strategies designed to specifically target subpopulations of neurons that have been functionally identified as key elements in driving epileptic dynamics.

This project is funded by Integrative Strategies for Understanding Neural and Cognitive Systems (NSF-NCS), a multidisciplinary program jointly supported by the Directorates for Computer and Information Science and Engineering (CISE), Education and Human Resources (EHR), Engineering (ENG), and Social, Behavioral, and Economic Sciences (SBE).

PROJECT SUMMARY Dravet syndrome is a severe neurodevelopmental disorder that affects 1 in 16,000 children and is defined by treatment-resistant epilepsy, developmental delay, intellectual disability, autism spectrum disorder, and a high rate of sudden death. Dravet syndrome is caused by mutation in the gene SCN1A, which encodes the sodium (Na+) channel Nav1.1 How SCN1A mutation leads to the clinical entity known as Dravet syndrome remains unclear; this gap in knowledge has profoundly limited the practical impact that such a diagnosis has on treatment, quality of life, and long-term outcome for patients with this disorder. Prior work in experimental animal models of Dravet syndrome including Scn1a+/- mice suggests that loss of Nav1.1 leads to epilepsy via dysfunction of GABAergic inhibitory interneurons in the cerebral cortex, with the most prominent identified abnormalities being impaired action potential generation in a critical subtype of interneuron known as the parvalbumin-positive fast-spiking interneuron (PV-IN). However, data presented here indicates that, surprisingly, PV-IN dysfunction is transient, being restricted to a brief time window in early development, with subsequent recovery of high frequency firing. Preliminary data suggests that the specific locus of pathology in Dravet syndrome is actually PV-IN axons, with abnormal action potential propagation leading to conduction delay and synaptic failure, even though PV-INs have recovered the ability to generate action potentials at high frequency. This finding has important implications for the development of novel treatment approaches for Dravet syndrome, such as cell transplantation, gene therapy, or precision medicine. This new 5-year application from the lab of an early stage investigator uses innovative neuroscience approaches to test this new hypothesis as to the mechanism of pathology in Dravet syndrome. Proposed experiments will establish the molecular identity and physiological properties of Na+ channels in PV-IN axons in Scn1a+/- mice as compared to wild-type controls using targeted recordings from interneuron axons and detailed immunohistochemistry of axonal Na+ channels (Aim 1); determine the impact of PV-IN axonal dysfunction on the timing of feedforward inhibition in cerebral cortical circuits (Aim 2); and assess the activity of defined subsets of neurons in awake, behaving Scn1a+/- mice using in vivo imaging and electrophysiology to corroborate in vitro findings (Aim 3). The overall outcome of the proposed experiments will set forth a unifying hypothesis as to the pathophysiology of Dravet syndrome. Such knowledge is critical to the development of novel, targeted therapies for this currently incurable and untreatable disease. The long-term objective of this line of research is to apply preclinical data from experimental model systems to the development of new, mechanistically oriented therapies in human patients.

PROJECT SUMMARY Recently-described SCN3A-related neurodevelopmental disorder (SCN3A-NDD) is caused by pathogenic variants in the gene SCN3A, which encodes the sodium (Na+) channel subunit Nav1.3. SCN3A-NDD is a devastating condition defined by treatment-resistant epilepsy and severe/profound intellectual disability (ID); surprisingly, many patients also exhibit malformation of cortical development (MCD), a developmental disturbance in the structural formation of the cerebral cortex of the brain, suggesting functional roles for Nav1.3 during embryological development. How genetic variants in SCN3A leads to epilepsy and neurodevelopmental disability, and how SCN3A variants lead to MCD, is unknown. Research is required to clarify the functional role of Nav1.3 during early brain development and to progress towards novel therapies or preventative measures for SCN3A-NDD, which is currently and untreatable disorder. This 5-year collaborative application employs novel tools and innovative neuroscience approaches to test the hypothesis that pathogenic variants in SCN3A lead to a disorder that includes epilepsy and MCD via dysregulated Na+ currents in migrating neurons of the developing cerebral cortex. Electrophysiological recordings in heterologous cell systems indicate that pathogenic SCN3A variants found in patients with SCN3A-NDD largely produce Na+ channels that exhibit gain of function due to increased persistent current and alterations in the voltage dependence of channel activation, which increase channel activity. However, the mechanistic basis of observed variability in epilepsy severity and presence or absence of MCD, is unclear. And how altered channel activity impacts the function of neurons has not been investigated. Proposed experiments will determine the relationship between specific SCN3A variants and correlated clinical phenotype (epilepsy, MCD, severity of ID) in a large cohort of human patients with SCN3A-NDD. To link SCN3A variants to dysfunction of ion channels and neurons, we will compare the biophysical properties of normal Na+ channels to channels containing variant Nav1.3; test cell-intrinsic effects of SCN3A variants in neurons generated from induced pluripotent stem cells from human SCN3A-NDD patients; and test effects of variant overexpression via in utero electroporation of mouse embryo followed by electrical recording in brain slices (Aim 1). The impact of variant SCN3A on the morphology of immature neurons and cytoarchitecture of the developing cerebral cortex will inform the role of SCN3A in development (Aim 2). To translate these findings towards clinical applications, we will attempt to ameliorate features of SCN3A-NND in advanced model systems, including a newly generated conditional point mutant mouse, via targeted manipulation of pathogenic Nav1.3-mediated Na+ current (Aim 3). Results will provide novel information on the role of Nav1.3 during brain development, and will define the pathogenic mechanisms of SCN3A-NDD towards development of novel, targeted therapies in human patients.