Lori Isom - US grants

Voltage-sensitive sodium channels are responsible for the rising phase of the action potential in excitable cells, including central and peripheral neurons, heart, skeletal muscle and neuroendocrine cells. Sodium channels in brain are heterotrimeric complexes composed of alpha, beta-1 and beta-2 subunits, while sodium channels in heart and skeletal muscle are heterodimeric, composed of alpha and beta-1-like subunits. The structure of sodium c channels in neuroendocrine cells has not been identified. Previous work by this investigator has resulted in the molecular cloning and functional expression of the beta-1 subunit of the rat brain sodium channel. Co-expression of cloned beta-1 subunits with alpha subunits in Xenopus oocytes results in increased peak current, acceleration of inactivation, and a shift in the voltage dependence of inactivation to more negative membrane potentials. It is hypothesized that the level of expression and functional properties of sodium channels in excitable cells depends not only on the expression and properties of alpha subunits but also on the expression and functional interaction with beta subunits. The goal of these studies is to identify the sodium channel alpha and beta subunits that are expressed in adrenal medullary chromaffin cells by hybridization to known alpha and beta subunits, determine the structure of chromaffin cell beta subunits by molecular cloning and assess the role of beta subunits in chromaffin cell sodium channel functional expression, including stimulus-secretion coupling . Methods to be employed include: 1) molecular cloning of chromaffin cell sodium channel beta subunits, 2) identification of chromaffin cell sodium channel alpha subunits by hybridization studies, 3) blockade of chromaffin cell sodium channel beta subunit expression in vitro through the use of antisense RNA or antisense oligonucleotides and in vivo through the use of gene disruption experiments, and 4) overexpression of chromaffin cell sodium channel beta subunits by transfection in vitro and by transgenic expression in vivo. %%% Voltage-sensitive sodium channels are an important component of the mechanism by which excitable cells respond to and transmit signals. Examples of excitable cells include nerve cells, muscle cells and neuroendocrine cells. Sodium channels are composed of subunits. The subunit composition of sodium channels differs in different cell types. The subunit structure of sodium channels in neuroendocrine cells, such as chromaffin cells of the adrenal medulla, has not been determined. These studies will use techniques of molecular biology to identify the sodium channel subunits that are expressed in adrenal medullary chromaffin cells and assess their role in chromaffin cell function.

The goal of these proposed studies is to investigate the molecular basis of neurosecretion in adrenal chromaffin cells, with particular emphasis on the role played by voltage-sensitive Na+ channels. The specific aims of this proposal include determination of the molecular structure of adrenal chromaffin cell Na+ channels by molecular cloning techniques, expression of those Na+ channels in Xenopus oocytes and mammalian cells to assess their physiological and pharmacological properties, and finally, ablation of selected Na+ channel genes in adrenal chromaffin cells in primary culture using antisense mRNA technology to determine which aspects of catecholamine neurosecretion are contributed by specific Na+ channel subunits. I hypothesize that because Na+ channels in other tissues are functionally modulated by auxiliary beta subunits, Na+ channel beta subunits in adrenal chromaffin cells will modulate neuroendocrine Na+ channels and therefore make a significant contribution to the regulation of neurosecretion.

Lorie Isom
9975490

LAY SUMMARY

Voltage-gated sodium channels are the membrane proteins responsible for initiation of action potentials in excitable cells such as neurons, cardiac myocytes, or skeletal muscle myocytes. In neurons, sodium channels are localized at high density at axon initial segments and nodes of Ranvier. This arrangement allows for efficient, saltatory conduction of action potentials. The long-term goal of this proposal is to understand the molecular basis of sodium channel targeting and localization, with particular emphasis on the role of the auxiliary b subunits in this process. It is proposed that sodium channel a subunit isoforms contain an intrinsic molecular targeting domain in their amino acid sequence that directs newly synthesized channels toward specific plasma membrane locations. Furthermore, it is hypothesized that sodium channel localization in polarized cells is modulated by the combination of particular a and b subunits. Four specific aims are proposed: 1. To characterize the effects of b subunit expression on sodium channel a subunit membrane localization in polarized Madin-Darby canine kidney (MDCK) cells. MDCK cells have proven to be a powerful model system in which to study the membrane targeting of a variety of ion channels and receptors. This aim will reveal whether b subunits modulate a subunit localization or whether the information for sodium channel targeting is encoded in the a subunit isoform cDNA only. 2. To identify molecular targeting signals encoded in a and/or b subunit cDNAs. Truncation mutants and chimeras will be used in an attempt to localize the molecular targeting signals encoded in a and/or b subunit cDNAs. 3. To characterize the localization of sodium channel a and b subunits expressed endogenously by human NT-2 neurons. Recognizing the limitations of the use of epithelial cell models to study targeting of neuronal proteins to axonal membranes, the localization of a and b subunits that are endogenously expressed in a neuronal cell culture model system, NT-2 neurons, will also be studied. The results of these studies will be correlated with the results obtained from MDCK cells. 4. To analyze the contribution of b subunits to a subunit localization in targeted embryonic stem (ES) cells differentiated to neurons in culture. Targeted mouse ES cell lines that lack the gene for the sodium channel b2 subunit have recently been established in the lab. One of the cell lines was also doubly targeted in culture to produce the homozygous genotype using high concentrations of the antibiotic G418. These cells now lack both alleles of the b2 subunit gene. A similar experiment is underway that is designed to knockout the b1 subunit gene. The doubly targeted ES cells will be differentiated in culture to neurons using retinoic acid. Immunocytochemical localization of a subunits in the differentiated neurons will be performed to assess the contribution of b subunits to a subunit membrane targeting and localization. The results of these proposed studies will contribute to the fields of ion channel biology and brain development. A basic understanding of the mechanisms involved in sodium channel subunit assembly and membrane targeting is essential to understanding the establishment of neuronal polarity, formation of nodes of Ranvier, and efficient action potential conduction.

The long term goal of this research is to test the hypothesis that sodium channel beta subunits communicate between extracellular matrix or cell adhesion molecules and the neuronal cytoskeleton. Beta subunits are members of the immunoglobulin superfamily and contain cell adhesion molecule domains. An extracellular matrix protein, tenascin-R (TN-R) is a functional modulator of sodium channel beta and alpha subunits. Transfected cells expressing sodium channel beta1, beta2 or alpha subunits are initially attracted to and then are repelled from TN-R plated on a nitrocellulose substrate. Sodium channel beta subunits interact homophilically in drosophila S2 cells and recruit ankyrin to points of cell- cell contact. It is proposed that sodium channel beta subunits communicate extracellular signals to the neuronal cytoskeleton. This interaction may be important to sodium channel placement and function during normal formation in the CNS. Alternatively, this interaction may be important in axonal fasciculation or de-fasciculation or establishment of neuronal polarity. The goal of this proposal is to determine the molecular basis of the interactions of beta subunits with each other and with TN-R. The specific aims are proposed: 1. To determine the structural domains in beta1, beta1A, and beta2 that mediate the functional effects of TN-R on cell body migration. 2. To determine the structural domains present in beta1 and beta2 that mediate extracellular homophilic binding and intracellular recruitment of ankyrin. 3. To determine whether beta1 and beta2 exhibit heterophilic binding to each other or to neurofascin and, if so, whether the interaction is cis or trans. Truncation mutants which eliminate the intracellular COOH-terminal domains of beta1 and beta2, introduction of a COOH-terminal signal peptide which eliminates the transmembrane domain and adds a glycophosphatidyinositol lipid anchor to beta 1 or beta2, and point mutations of amino acids in beta1 and beta2 that are predicted to be located in the extracellular Ig fold will be constructed and expressed in mammalian cells and tested in cell migration assays. The effect of TN-R n beta1A, and beta1 isoform with a novel COOH terminal domain, will also be tested. These mutant constructs will be tested for homophilic binding and ankyrin recruitment in S2 cells. The experiments described in this proposal will lead to a greater understanding of the events involved in sodium channel placement during normal brain development, and may contribute to the understanding of nodal formation and axonal fasciculation. This basic knowledge may provide a framework for the development of therapeutic agents to treat demyelinating disease such as multiple sclerosis.

Chemistry (12)
The power and versatility of modern NMR spectroscopy make it an essential tool for chemists. The Department of Chemistry is adapting a variety of experiments from the research and educational literature that uses nuclear spin to obtain structural information and implementing them as discovery-based experiments. These experiments are providing the context for introducing NMR techniques in a sequence that gradually increases the difficulty of relating molecular structure to spectral information.

Organic chemistry students are being introduced to NMR early in the first semester through C-13, DEPT, and HETCOR experiments, followed by the characterization an organofluorine compound, using 2-D NMR to identify an unknown ester, probing the stereospecificity of a reduction reaction using NOE, and investigating the conformational dynamics of DEET. Students in Organic Spectroscopy are characterizing non-trivial unknowns using a variety of complementary spectroscopic techniques. Students in advanced courses are investigating phosphorus-phosphorus coupling and characterizing structures of enzyme inhibitors, among other laboratory projects. These discovery-based experiments require a nuclear magnetic resonance spectrometer with multinuclear capability and possessing the resolution and flexibility required by such experiments. Research in a variety of areas has become an integral part of the training of our undergraduate students and the high-field NMR is finding numerous applications.

It is well known that DNA contains the instructions for building live organisms. Less well known is that many of these instructions are not used directly. Which instructions are actually used is partially determined by how the DNA is bent and folded. Therefore, understanding the mechanisms by which DNA is deformed is important to understanding the function of DNA in the storage and expression of genetic information. This project investigates the tendency of cation binding to distort DNA. The experimental focus of the project involves using computational analysis to test the ability of cations to induce two types of DNA deformation: (1) phosphate crowding that occurs in bending and groove narrowing; and (2) base unstacking by ions interacting with base faces via cation-pi interactions. The ability of monovalent cationic protein side chains to induce DNA phosphate crowding in crystal structures of DNA/protein complexes will be analyzed. The project will also characterize cation-pi interactions in DNA by computational analysis of DNA crystal structures as well as determines the crystal structures of DNA complexes with various divalent cations.

Broader Impacts: Students engaged in this interdisciplinary research will learn skills important in experimentally determining crystal structures as well as computational skills to analyze structural information available in databases. The projects described herein will develop scientific thinking skills and programming skills that when combined with practical structure determination skills, will give the student well-rounded experience of these symbiotic aspects of structural biology. The students' education will be further expanded by close collaboration with and travel to two major research institutions to collect and analyze crystallographic data.

DESCRIPTION (provided by applicant): Rapid processing of information in the nervous system requires generation and saltatory conduction of action potentials along myelinated axons. In excitable cells, voltage-gated Na+ channels are responsible for action potential conduction and propagation. In myelinated axons, Na+ channels localize at high concentration in gaps between the myelin sheaths called nodes of Ranvier. Na+ channels are composed of a pore-forming a subunit and one or two betaa subunits. It is hypothesized that beta subunits serve as critical links between the extracellular environment and intracellular signal transduction, especially in the axo-glial apparatus where they may function to mark presumptive nodes and participate in axo-glial communication. Na+ channel beta1 and beta2 subunits are multifunctional. In addition to modulation of ion channel function and cell surface expression, beta1 and beta2 act as cell adhesion molecules of the immunoglobulin superfamily and participate in homophilic and heterophilic cell adhesion, interaction with extracellular matrix molecules, and interaction with the cytoskeleton. In the present exploratory grant application, it is proposed to develop the zebrafish (zf) system to identify zf beta subunits and to study Na+ channel a and a subunit targeting during nodal formation. The long-term goal will be to use reverse genetics to knockdown zf beta1 and beta2 expression to determine their roles in nodal formation. The proposed activities will lay the foundation for future success with this model system. Isoforms of zf beta1 and beta2 have been identified that are different from those previously cloned from mice. Thus, these studies may lead to significant advances in Na+ channel biology that were not thought to be possible using mouse models. The following specific aims are proposed: 1. To test whether novel a subunits identified in zf display unique subcellular localization, alpha subunit association, electrophysiological properties, or cell adhesive properties. 2. To investigate the temporal order of Na+ channel alpha and beta subunit targeting to nodes of Ranvier. Using transgenes encoding fluorescent-tagged zfNav1.6 and zf beta subunits under control of the zf-tubulin promoter, the temporal order of Na+ channel subunit localization to zf optic nerve nodes of Ranvier during development will be examined. Transgenes expressing labeled forms of neurofascin186, contactin, and ankyrinG will then be used to determine the temporal order of Na+ channel subunit appearance relative to these nodal proteins. Development of this powerful genetic approach will make a number of significant contributions to the understanding of the formation and maintenance of CNS nodes of Ranvier, as similar experiments in mouse models have not yet been possible. This information will be critical to the future development of therapies to treat diseases involving neuronal hyperexcitability such as epilepsy or diseases involving action potential conduction blockade such as Multiple Sclerosis.

DESCRIPTION (provided by applicant): The long term goal of this research is to test the hypothesis that voltage-gated Na+ channel (Nav1) beta subunits are cell adhesion molecules (CAMs) that communicate between extra- and intra-cellular signaling molecules and cytoskeletal proteins. beta1 subunits are multifunctional molecules that participate in modulation of Nav1 kinetics, in extracellular matrix interactions resulting in cellular migration, in homophilic cell adhesion resulting in cytoskeletal recruitment, in heterophilic cell adhesive interactions resulting in stabilization of Nav1 complexes at the cell surface, and in neurite outgrowth from cerebellar granule neurons. beta1 can be tyrosine phosphorylated, resulting in its differential subcellular localization in cardiac myocytes. Finally, beta1 subunits contribute to the regulation of neurpnal excitability as evidenced by the effects of beta1C121W in GEFS+1 epilepsy and the severe epileptic phenotype observed in beta1 (-/-) mice. This laboratory was the first to characterize the beta subunits as CAMs and, in doing so, initiated an entirely new field in Nav1 biology. It is proposed that Nav-beta1 subunits, as CAMs, not only play important roles in cell-cell and cell- matrix adhesion, but also modulate intracellular signaling in neurons as a result of these interactions. Furthermore, it is proposed that tyrosine phosphoryation of beta1 regulates its differential subcellular targeting to differential subcellular domains in neurons and thus determines the availability of beta1 to associate with cytoskeletal and signaling molecules in those domains. The following specific aims are designed to test this hypothesis: 1. To investigate the mechanism of beta1- mediated neurite outgrowth from cerebellar granule neurons. 2. To determine the domains of beta1 that are required to mediate neurite outgrowth and to investigate whether deletion of the beta1 gene results in differences in neurite outgrowth in vivo by investigating the migration and neurite extension of corticospinal axons and cerebellar granule cells in beta1-/- mice. 3. To test the hypothesis that beta1 contains a tyrosine-based targeting domain that includes beta1Y181 and that phosphorylation of this residue results in differential localization of pYbeta1 to ankyrin-independent subcellular domains in neurons. The overall goal of this research is to investigate the novel idea that Nav1 beta subunits are not only channel modulators but also act to initiate signal transduction cascades as a result of cell adhesive interactions. A beta1 mutation that causes epilepsy in humans, beta1C121W, not only disrupts beta1-mediated channel modulation but also disrupts beta1-mediated cell adhesion. Thus, as genetic mutations that affect L1-CAM-mediated cell adhesion result in severe neurological defects such as hydrocephaly and mental retardation, it is proposed that human mutations that result in disruption of Nav1 beta subunit-mediated cell adhesion may also result in neurological disease, including epilepsy.

DESCRIPTION (provided by applicant): This application is a request for partial funding of the 4th and 5th biennial FASEB Summer Conferences on Ion Channel Regulation. The 4th conference will be held June 7-12, 2009 at Snowmass Village in Snowmass, Colorado, and will be organized by co-chairs Drs. Lori Isom and John Adelman. The location and exact dates for the 2011 Conference will be determined in conjunction with FASEB. The Ion Channel Regulation Conference is a unique, comprehensive and integrative meeting that brings together investigators from multiple disciplines, including cell and molecular biology, physiology, neuroscience, biophysics, biochemistry and medicine. The conference has a strong emphasis on signaling aspects of ion channel regulation in normal physiology and disease (channelopathies) in cardiac, vascular, blood, neuronal and other main systems. Presentations will highlight significant and recent developments in these fields, and will focus on the wide range of mechanisms of channel regulation, assembly and distribution of signaling complexes. Another objective of the conference is to make it attractive to a wider audience by inviting speakers that represent the full spectrum of ion channel research and to actively recruit the attendance of young investigators. The format of the meeting creates an environment that permits free interdisciplinary exchange of information and ideas via oral presentations, workshops, posters and close informal interactions between investigators at all career stages. We have made a special effort to increase the participation of junior and underrepresented groups (women and minorities) by inviting them to present a talk, and by providing them with travel awards. In summary, the 2009 and 2011 FASEB Ion Channel Regulation Conferences will be premier forums for interactions between young and established investigators from different biomedical fields. The meetings should encourage new collaborations, significantly advance interactions between different branches of biomedical field and promote the professional growth of all participants. PUBLIC HEALTH RELEVANCE: This application is a request for partial funding of the 4th and 5th biennial FASEB Summer Conferences on Ion Channel Regulation. The 4th conference will be held June 7-12, 2009 at Snowmass Village in Snowmass, Colorado, and will be organized by co-chairs Drs. Lori Isom and John Adelman. The location and exact dates for the 2011 Conference will be determined in conjunction with FASEB. The Ion Channel Regulation Conference is a unique, comprehensive and integrative meeting that brings together investigators from multiple disciplines, including cell and molecular biology, physiology, neuroscience, biophysics, biochemistry and medicine. The meetings should encourage new collaborations, significantly advance interactions between different branches of biomedical field and promote the professional growth of all participants.

DESCRIPTION (provided by applicant): The long-term goal of this research is to understand the role of Scn1b, encoding voltage-gated Na+ channel ¿1, in CNS myelinating glia. The objective is first, to measure ¿1 expression in wildtype myelin and to characterize myelinating glia-specific Scn1b null mice. This powerful model provides a unique opportunity to study the role of Scn1b in myelination and axo-glial communication. Second, the objective is to determine the signal transduction mechanisms initiated by ¿1-mediated cell-cell adhesion in oligodendrocyte precursor cells (OPCs) or oligodendrocytes (OLs). The overall goal of this proposal is to test the central hypothesis that ¿1 is expressed in OPCs/OLs, in addition to axons, where it contributes to axo-glial communication through cell adhesion and modulation of electrical activity. Two specific aims are proposed: Aim 1. To determine the role of glial-expressed Scn1b in myelination. Preliminary Data demonstrate that mice that lack Scn1b in myelinating glia have movement disorders, dysmyelination, delayed myelination or lack of myelination, and axonal degeneration. These data support the working hypothesis of this aim that Scn1b plays a critical role in OPC development and/or axo-glial communication. Aim 2. To determine the role of Scn1b in OPC/OL signal transduction. The working hypothesis here is that ¿1-¿1 interactions play a key role in recognition of axons by glia and subsequent initiation of myelination via a signaling cascade in glia that involves the CAM contactin as well as fyn kinase activation. Further, it is proposed that ¿1 modulation of Na+ currents plays a role in OPC development, including proliferation, migration, and spacing along axons, and thus may contribute to the initiation of myelination. With respect to expected outcomes, the work proposed in Aims 1 and 2 is expected define a novel role for ¿1 in OPC signaling, including axo-glial communication. These results are expected to have an important positive impact on the fields of demyelinating and hypomyelinating disease. Rapid processing of information in the CNS requires efficient saltatory conduction of action potentials along myelinated axons. Disruptions in myelination result in a range of human neurological diseases, including multiple sclerosis in adults and leukodystrophies in children. While human mutations in SCN1B associated with demyelination, dysmyelination, or hypomyelination have not yet been identified, they have been determined to play a role in other neurological diseases, including severe epilepsy associated with mental retardation. A detailed understanding of the basic mechanisms underlying the role of SCN1B in axo-glial communication is essential to the future development of novel therapeutic regimens for demyelinating disease. PUBLIC HEALTH RELEVANCE: Disruptions in myelination and axo-glial communication result in a range of human neurological diseases, including Multiple Sclerosis. While mutations in Scn1b associated with central demyelination or dysmyelination have not yet been identified, a detailed understanding of the basic mechanisms underlying communication between the axon and the myelin sheath is essential to the future development of novel therapeutic regimens for Multiple Sclerosis. In the present application, it is proposed to investigate the role of Scn1b in CNS myelin formation, ultrastructure of the myelin sheath, and axonal degeneration, with the future goal of developing novel therapies to promote remyelination and axon protection in pathophysiology.

? DESCRIPTION (provided by applicant): Early onset pediatric epileptic encephalopathies (EEs) such as Dravet Syndrome (DS) are devastating to families because of the high degree of neurodevelopmental compromise, including developmental delay, cognitive decline, and intellectual disability. Most concerning are the severe seizures and high risk of sudden unexpected death in epilepsy (SUDEP). Mutations in voltage-gated Na+ channel (VGSC) ? and ? subunit genes are linked to DS. While the majority of DS cases are linked to SCN1A haploinsufficiency, SCN1B homozygous mutations are also linked to DS (or a DS-like EE). The objective of this work is to understand the mechanism of hyperexcitability in human SCN1B-linked DS/EE. It is hypothesized that the mechanism of Scn1b signaling in neurons involves cell type and subcellular domain specific changes in Na+ current (INa) and K+ current (IK) as well as cell adhesion-mediated effects on neuronal pathfinding and transcriptional regulation of ion channels and transporters. It is proposed that human SCN1B-DS mutations result in defects in neuronal cell-cell communication and ionic currents that are similar to those observed in Scn1b-/- mice. Three Specific Aims are designed to test this hypothesis: 1. To determine whether human SCN1B-linked DS mutations result in loss-of-function in vivo. SCN1B-DS mutations are assumed to result in a functional null phenotype, however, this has not been tested in vivo. A family with an inherited, recessive, SCN1B-DS mutation has donated skin biopsies for induced pluripotent stem cell (iPSC) generation. This mutation, as well as the previously identified SCN1B-R125C human mutation, will be introduced into the mouse Scn1b locus to test homozygous progeny for changes in excitability, neuronal pathfinding, INa, IK, and GABAergic signaling in comparison with Scn1b-/- mice. In parallel, SCN1B-linked human DS iPSC neurons and cerebral organoids will be generated and tested for similar deficits. Gene editing will be used to make isogenic controls, and to generate homozygous null patient-derived neurons to directly compare SCN1B-DS mutant and null cells in the same, isogenic, iPSC line of human neurons. 2. To determine localized changes in INa or IK in Scn1b-/- brain cortical slices. It is possible that the cause of seizures in SCN1B-DS is not disrupted neuronal pathfinding, as previously proposed, but instead neuronal subtype specific changes in INa or IK. Here, a combination of nucleated patch, pulled patches from the AIS, and immunofluorescence staining will be used to determine differential changes in INa and VGSC expression in -/- vs. +/+ cortex. Changes in IK and voltage-gated K+ channel (VGKC) expression will also be tested. 3. To determine whether disruption of Scn1b-mediated neuronal pathfinding plays a role in hyperexcitability in DS. Scn1b-/- mice have neuronal pathfinding defects that precede seizure onset. It was proposed that these defects might lead to the development of seizures. An inducible, pan-neuronal Cre line will be used to delete Scn1b past the critical period of mouse brain development to determine whether seizures and early mortality occur as in Scn1b-/- mice. Here, changes in INa, IK, neuronal patterning, and GABAergic signaling will be investigated following Scn1b deletion at progressive developmental time points. ?1-mediated neurite outgrowth requires trans homophilic ?1-?1 cell adhesion leading to intracellular association of ?1 with ankG in vitro. In a second set of experiments, mutations will be introduced to the mouse Scn1b locus that interrupt ?1-ankG association or ?1 tyrosine phosphorylation to ask whether disruption of the ?1-CAM signaling cascade leads to seizures in vivo. Even though SCN1B- linked DS/EE is a rare disease, this work is important because it will provide new information regarding how deficits in brain development and regulation of ionic currents can synergize to result in hyperexcitability.

? DESCRIPTION (provided by applicant): Application 6 of this SUDEP Research Alliance Centers Without Walls (CWOW) grant proposal, iPSC and Mouse Neurocardiac Models, explores cardiac arrhythmia and autonomic dysfunction as potential causes of SUDEP. Although SUDEP is the most devastating consequence of epilepsy and the leading cause of epilepsy mortality, astonishingly little is understood about its causes and no biomarkers exist to identify at risk epilepsy patients. To advance our understanding of these critical issues, we will focus on Dravet Syndrome (DS), a severe childhood epileptic encephalopathy associated with a high SUDEP incidence. DS is most frequently caused by mutations in the voltage-gated Na+ channel (VGSC) gene SCN1A, encoding NaV1.1. As NaV1.1 is expressed in brain, heart, and peripheral nerves, a compelling idea is that altered Na+ currents (INa) in DS cardiac myocytes (CMs) or autonomic neurons, in addition to central neurons, lead to arrhythmias and SUDEP. We used the induced pluripotent stem cell (iPSC) method to derive central and peripheral neurons and CMs from fibroblasts of DS subjects. Preliminary data from DS patient CMs suggest that a subset of DS subjects shows abnormal CM INa and excitability. In studies of a DS human mutant SCN1A knock-in mouse model, we observed spontaneous seizures and SUDEP, increased ventricular CM INa density, and ventricular arrhythmias at the time of SUDEP. Similarly, we found increased ventricular CM INa density, spontaneous seizures and SUDEP in a Scn1b null DS mouse model. Our work, studies of Scn1a heterozygous null DS mice, and clinical ECG studies in DS also show altered cardiac autonomic function. Thus, we hypothesize that SUDEP in DS is caused by VGSC mutations that produce cardiac electrical and/or autonomic dysfunction, in addition to brain dysfunction. Furthermore, that combined insights from studies of DS patient-derived cells, mouse models and patient peri-ictal ECG data will yield biomarkers of SUDEP risk in DS. Four specific aims will test these hypotheses: 1) To understand the effects of DS-linked SCN1A mutations on cardiac excitability using DS patient iPSC-derived CMs and DS mice; 2) To determine how DS-linked SCN1A mutations influence the excitability of autonomic neurons, cardiac autonomic innervation, and autonomic control of cardiac function using DS patient iPSC-derived autonomic neurons and DS mice; 3) To investigate changes in autonomic excitability in a second mouse model of DS, Scn1b null mice, and in SCN1B-DS patient iPSC CMs and neurons; and 4) To determine whether cardiac electrical and/or autonomic function is altered in DS patients at baseline or peri-ictally. Our wor will synergize with the entire CWOW proposal to not only uncover SUDEP mechanisms in DS, but also to provide advances in understanding SUDEP causes and biomarkers that will be applicable to other refractory epilepsies due to ion channelopathies and perhaps other etiologies. This work will also show proof-of-principle for the use of multiple platforms (cellular and clinical data from the same patients, and multiple mouse models) to individualize SUDEP risk and develop patient-specific preventative treatments.

? DESCRIPTION (provided by applicant): The University of Michigan, Pharmacological Sciences and Bio-related Chemistry Training Program (PSTP) provides pre-doctoral students with a strong foundation in basic pharmacological principles and a broad knowledge of other bio-related basic science disciplines (e.g., medicinal chemistry). All students will complete a core curriculum consisting of courses in pharmacology, medicinal chemistry, physiology, and biostatistics, with other elective courses designed to fit the programmatic needs of individual students. Students may follow one of two general tracks with emphasis on biological or chemical research. Areas of research concentration within the program include cardiovascular/renal pharmacology, neuropharmacology, xenobiotic metabolism, growth and metabolic regulation, receptor structure and function, synthesis and pharmacology of therapeutic and diagnostic agents, antibiotic discovery, enzymology, transport mechanisms, drug absorption, drug delivery, and pharmacokinetics. Students obtain laboratory experience in several types of pharmacological research and learn how to design experiments, evaluate experimental data, and use appropriate statistical methods. The training program consists of two Phases. In Phase I, trainees engage in: graduate-level coursework, a qualifying examination for candidacy (halfway through Phase I), training in the responsible conduct of research, seminar programs, required research presentations at an Annual Symposium, and supervised laboratory investigation leading to the student's doctoral dissertation. In Phase II, th trainees participate in advanced training activities that address: career development (Career Night with PSTP alumni, Yellow School Bus trip to Pharma, Individual Career Development Plans), refresher RCR training, and professional development (Grant Writing Workshop, Peer Mentoring, Pedagogical Training). The training program culminates with a final oral examination during which the trainee defends his/her dissertation before their dissertation committee. Highly-qualified students, with an interest in obtaining advanced training in the pharmacological sciences that would not normally be available to them in their Ph.D. programs, are nominated by their Ph.D. program and mentor (PSTP faculty member) to the PSTP. Nominations are screened and nominees are assigned to either the Biological or Chemical Track based on their research and specific training interests. Nominees are evaluated (including interviews) by the PSTP Executive Committee, which makes the final appointment decisions. Students enter the PSTP at the beginning of their second year of graduate school and generally are financially supported by the training grant for two years during Phase I of the program. Trainees' progress is monitored by review of course grades, qualifying exams, laboratory experiences, and thesis work by both the trainees' Ph.D. programs and the PSTP to maintain a high level of quality. In Phase II, the trainee's progress is monitored by their mentor and by an additional PSTP faculty member via an annual dissertation committee meeting, with requisite written reports to the PSTP Directors. Support is requested for 14 trainees per year.

? DESCRIPTION (provided by applicant): Neuronal channelopathies cause various brain disorders including epilepsy, migraine and ataxia. Despite the development of mouse models, pathophysiological mechanisms for these disorders are poorly understood. One particularly devastating channelopathy is Dravet Syndrome (DS), a severe childhood epileptic encephalopathy typically caused by de novo dominant mutations in SCN1A, encoding the voltage-gated Na+ channel (VGSC) Nav1.1. Heterologous expression of mutant channels suggests haploinsufficiency, raising the question of how loss of VGSCs underlying action potentials (APs) produces hyperexcitability. Data from DS mouse models indicate both decreased Na+ current in interneurons, implicating disinhibition, and increased Na+ current in pyramidal cells, implicating hyperexcitability, depending on genetic background, brain area, and animal age. To understand the effects of SCN1A DS mutations in human neurons we derived forebrain-like neurons from two DS subjects by induced pluripotent stem cell (iPSC) reprogramming of patient fibroblasts and compared them with iPSC-derived neurons from human controls. We found that DS patient-derived neurons have increased Na+ current density in both bipolar- and pyramidal-shaped neurons. Consistent with increased Na+ current, both putative excitatory and inhibitory patient-derived neurons showed spontaneous bursting and other evidence of hyperexcitability. Our data provided some of the first evidence that epilepsy patient-specific neurons obtained via the iPSC method are useful for modeling epileptic-like hyperactivity. Moreover, our findings revealed a previously unrecognized potential epilepsy mechanism underlying DS and offered a platform for future screening of novel anti-epileptic therapies using patient-derived neurons. The long-term goal of this research is to understand the molecular basis of genetic epilepsies. Our objective is to determine epilepsy mechanisms of SCN1A-linked DS in humans. We will test the central hypothesis that SCN1A haploinsufficiency leads to paradoxically increased Na+ current in excitatory and inhibitory neurons, as well as alterations in other ionic currents that underlie neuronal hyperexcitability in DS. The rationale fr this work is that identifying the role of SCN1A haploinsufficiency in the development of hyperexcitability may lead to novel treatments for DS as well as related pediatric epilepsies. We will test our hypothesis by pursuing three specific aims: 1: To determine whether SCN1A haploinsufficiency causes alterations in the expression of other VGSC ?-subunits that lead to increased Na+ current in DS patient-specific iPSC neurons. 2: To investigate changes in synaptic function in DS patient-specific iPSC neurons. 3: To determine the electrophysiological characteristics of DS patient-specific and control iPSC neurons differentiated in the rodent brain. This work is expected to reveal how SCN1A haploinsufficiency contributes to epilepsy in humans. Our results will have positive impact because this work will lead to a greater understanding of the mechanisms of DS and related diseases and may lead to novel therapeutic agents for epilepsy.

ABSTRACT: Early onset pediatric epileptic encephalopathies such as Dravet Syndrome (DS) are devastating to families because of the high degree of neurodevelopmental compromise, including developmental delay, cognitive decline, and intellectual disability. Most concerning are the severe seizures and high risk of sudden unexpected death in epilepsy (SUDEP). Mutations in voltage-gated Na+ channel (VGSC) ? and ? subunit genes are linked to DS. While the majority of DS cases are linked to SCN1A haploinsufficiency, SCN1B homozygous mutations are also linked to DS. Scn1b-/- mice have a DS phenotype with SUDEP. SCN1B encodes VGSC ?1 subunits, which are developmentally regulated cell adhesion molecules and ion channel modulators that play critical roles in the regulation of excitability. Scn1b-/- mice have cell type specific changes in Na+ (INa) and K+ (IK) currents. In addition, Scn1b-/- mice have neuronal proliferation, migration, and pathfinding defects at postnatal day (P)5 that precede seizure onset at ~P10. These data suggested that alterations in CAM function may contribute to hyperexcitability, however, new data challenge this idea and offer the alternative explanation that defective cell adhesion in SCN1B-linked DS may not contribute to seizures but instead impact other co-morbidities. Preliminary data show that Scn1b-/- mice also have delayed maturation of neuronal Cl- gradients such that GABAergic signaling remains depolarizing and excitatory until ~P17-18, which may contribute to hyperexcitability in SCN1B-linked DS. The objective of this work is to understand the mechanism of hyperexcitability in SCN1B-linked DS. The central hypothesis is that the mechanism of hyperexcitability in the Scn1b-/- model of DS is cell type specific changes in INa, IK, and GABAergic signaling. Further, it is proposed that human SCN1B-DS mutations result in loss-of-function, with similar defects in ionic currents and delayed maturation of GABAergic signaling as observed in Scn1b-/- neurons. The experimental plan will test three Aims: 1. To determine the mechanism of hyperexcitability resulting from Scn1b deletion; 2. To determine whether human SCN1B-linked DS mutations result in loss-of-function in mouse models; 3. To determine the phenotype of SCN1B-linked DS patient-derived induced pluripotent stem cell (iPSC) neurons. Model choice is key to understanding epilepsy mechanisms. Importantly, mice are not small humans. Thus, patient-derived iPSC neuronal models provide essential information regarding human disease. On the other hand, mature brain networks cannot yet be replicated using iPSCs and so brain slice preparations from transgenic mouse models remain important to understanding circuitry. Rather than relying on a single model, this project will compare and contrast human and mouse models to understand key mechanistic aspects of the development of hyperexcitability in DS. Even though SCN1B-linked DS is rare compared to SCN1A-linked disease, this work may lead to the discovery of novel targets for therapeutic intervention in DS caused by multiple types of gene mutations.

Sudden Unexpected Death in Epilepsy (SUDEP) is a leading cause of death in patients with epilepsy. SUDEP mechanisms are not understood, although there is evidence to implicate apnea, autonomic dysfunction, and cardiac arrhythmias. The majority of SUDEP patients die during sleep and, by definition, autopsy findings are largely unremarkable. Here we will generate a novel animal model of genetic epilepsy to investigate the role of cardiac arrhythmias in this devastating outcome. Loss-of-function variants in SCN1A are identified in patients with Dravet syndrome (DS). DS patients have the highest SUDEP risk, up to 20%. SCN1A is expressed in both the heart and brain of humans and mice. Because of this, we proposed that cardiac arrhythmias contribute to the mechanism of SUDEP in DS. We were the first group to show evidence for altered cardiac myocyte (CM) sodium current (INa) density and action potentials (APs), as well as cardiac arrhythmias in mouse models of SCN1A-linked DS. We were also the first to show that induced pluripotent stem cell (iPSC)-derived CMs from DS patients have substrates for arrhythmias. Importantly, no single animal or iPSC model can completely replicate the human DS phenotype. Because cardiac APs in mice are very different than in humans, we used human iPSC-CM models to investigate cell autonomous effects of SCN1A haploinsufficiency to predict cardiac arrhythmias. In spite of our success with iPSC-CMs, cells in 2-D culture cannot replicate complex cardiac tissues, cardiovascular changes, or cardiac autonomic innervation. Thus, we propose to add a transgenic rabbit model to our work because rabbits more closely replicate the human cardiac AP than mice and provide a complete organismal system with which to work. Adding a rabbit model is critical to our ability to fully understand SUDEP and to develop biomarkers for SUDEP risk in the future. The goal of this application is to develop a rabbit model of Scn1a-linked DS that can be used to more accurately replicate human cardiac physiology to ultimately understand the mechanisms of SUDEP in the genetic epilepsies. Using donor funds, we generated a New Zealand White (NZW) rabbit Scn1a deletion model using CRISPR-Cas9 gene editing techniques. We found that Scn1a-/- rabbits seize and die by postnatal day 11, similar to Scn1a-/- mice, physiologically confirming gene deletion. However, because DS patients are haploinsufficient for SCN1A, it is critical to develop a reliable Scn1a+/- rabbit DS model. We propose 3 Aims to characterize our new model: 1. To record EEGs in NZW Scn1a+/- rabbits to determine whether the animals have electrographic seizures. 2. To determine whether hyperthermia- induced seizures in NZW Scn1a+/- rabbits progress to DS-like spontaneous seizures. 3. To determine whether NZW Scn1a+/- rabbits have cardiac arrhythmia. Accomplishment of this work will establish an important, new model for use in SUDEP research that can be shared with other investigators and provide critical guidance for the future generation of other rabbit models of genetic epilepsy.

Sudden Unexpected Death in EPilepsy, or SUDEP, is a leading cause of death in patients with epilepsy. SUDEP mechanisms are not understood, although there is evidence to implicate apnea, autonomic dysfunction, and cardiac arrhythmias. We will take advantage of recent progress in the understanding of SUDEP risk in the genetic epilepsies to investigate the role of cardiac arrhythmias. SUDEP risk varies in a gene-specific manner. Loss-of- function variants in the voltage-gated sodium channel (VGSC) genes, SCN1A or SCN1B, are identified in patients with Dravet syndrome (DS) and gain-of-function variants in the VGSC SCN8A are found in patients with Early Infantile Epileptic Encephalopathy 13 (EIEE13). DS and EIEE13 patients have the highest SUDEP risk, up to 20%. In contrast, variants in chromodomain helicase DNA binding protein 2 (CHD2) are also associated with early onset EE, but SUDEP has not been reported in this population. SCN1A-, SCN1B-, SCN8A-, and CHD2- linked epilepsies are developmental and epileptic encephalopathies (DEEs), severe childhood epilepsies associated with cognitive and behavioral impairments. The familial focal epilepsies, are attributed to pathogenic variants in DEPDC5, encoding a member of the GATOR complex in the mTOR pathway. SUDEP is reported in 10% of these patients. Because VGSC genes are expressed in both heart and brain, we have proposed that cardiac arrhythmias contribute to the mechanism of SUDEP in channelopathy-linked genetic epilepsies. Our overall goal is to understand the mechanisms of SUDEP in the genetic epilepsies. Our objectives are to use patient-derived or transgenic mouse cardiac myocytes (CMs) to understand how epileptic VGSC gene mutations alter CM function and arrhythmogenic potential, and to determine whether similar changes are found in non-ion channel epilepsy genes that are expressed in the heart. Our central hypothesis is that both ion channel and non- ion channel genetic epilepsies with high, but not low, SUDEP risk exhibit pro-arrhythmogenic changes in patient- derived CMs and mouse models. To ask whether abnormal CM excitability also occurs in a non-ion channel genetic epilepsy with high SUDEP risk, we will investigate DEPDC5 variant iPSC-CMs and Depdc5-/- mice. Finally, we will examine Chd2-/- mice and human iPSC-CMs with variants in CHD2, a non-ion channel gene with a low SUDEP risk, to test whether altered CM excitability is specific to genetic epilepsies with high SUDEP rates. Like the VGSCs, DEPDC5 and CHD2 are expressed in brain and heart. Our Specific Aims are: 1. To determine the effects of SCN1A, SCN1B, and SCN8A epilepsy variants on CM excitability using patient-derived iPSC-CMs. 2. To ascertain whether CMs from DEPDC5 patients or Depdc5+/- mice display abnormal excitability and whether Depdc5+/- mice have arrhythmia. 3. To determine whether CMs from CHD2 patients or Chd2+/- mice display abnormal excitability and whether Chd2+/- mice have arrhythmia. There are no effective therapies for any of the genetic epilepsies and no reliable biomarkers for SUDEP risk. This work may lead to the discovery of diagnostic biomarkers for SUDEP risk in the future.

The advent of next generation DNA sequencing has revolutionized gene discovery in human diseases, including epilepsy. Hundreds of genes have been implicated in epilepsy in the last decade, revealing the diversity of biological mechanisms that can go awry in this disorder. However, the rate at which we are identifying new genes involved in epilepsy is now outpacing our ability to study disease mechanisms. Moreover, clinical gene panel or exome sequencing has become standard practice for patients with early-onset, familial, and refractory epilepsies. This rapid assimilation of genetic testing into clinical care has led to a surge in the number of genetic variants of uncertain significance (VUS), particularly the occurrence of missense VUS. These VUS are assigned to an indeterminate spectrum between pathogenic and benign, which complicate interpretation for genetic counselors, clinicians, patients and families, as well as assessment of the need for further testing. Here we propose a Center without Walls, entitled Epilepsy Multiplatform Variant Prediction (EpiMVP), spanning 5 institutions and incorporating expertise from geneticists, clinicians, computational biologists, neuroscientists, stem cell biologists, pharmacologists and electrophysiologists who have a proven track record of collaborative publications and grants, as well as stature as leaders of national and international epilepsy organizations. EpiMVP will develop a modular, highly integrated platform approach to accelerate determination of the functional, pharmacological, neuronal network and whole animal consequences of genetic variants implicated in a range of clinical epilepsy types. We will study non-ion-channel, non-receptor genes commonly implicated in epilepsy, and that are involved in diverse biological processes. Our ultimate goals are to devise an effective experimental platform for testing the pathogenicity of VUS in genes implicated in epilepsy and to generate a computational model (EpiPred) that predicts the likelihood that a variant is pathogenic or benign. This work is crucial in the pursuit of novel therapeutics and the promise of personalized medicine. The overall milestones of the Center are: 1. Evaluate genes associated with epilepsy and select candidates for analysis, model data for, and analyze all project data for development of EpiPred an iterative machine learning model to classify variants in genes implicated in epilepsy. 2. Test selected VUS using medium throughput, in vitro approaches. 3. Test selected VUS in human cortical neurons or human brain organoids using induced pluripotent stem cell approaches. 4. Test selected VUS in pre-clinical, in vivo models. The expected outcomes are: 1. Provide a freely available prediction tool for clinicians to differentiate between pathogenic and benign variants for genes implicated in epilepsy; 2. Provide experimental models to study the functional consequences of specific variants; 3. Provide a reclassification of VUS in ClinVar/ClinGen and to develop new guidelines for incorporating functional readouts into the ACMG criteria; 4. Inform the future development of novel therapeutics to treat epilepsy.

The advent of next generation DNA sequencing has revolutionized gene discovery in human diseases, including epilepsy. Hundreds of genes have been implicated in epilepsy in the last decade, revealing the diversity of biological mechanisms that can go awry in this disorder. However, the rate at which we are identifying new genes involved in epilepsy is now outpacing our ability to study disease mechanisms. Moreover, clinical gene panel or exome sequencing has become standard practice for patients with early-onset, familial, and refractory epilepsies. This rapid assimilation of genetic testing into clinical care has led to a surge in the number of genetic variants of uncertain significance (VUS), particularly the occurrence of missense VUS. These VUS are assigned to an indeterminate spectrum between pathogenic and benign, which complicate interpretation for genetic counselors, clinicians, patients and families, as well as assessment of the need for further testing. Here we propose a Center without Walls, entitled Epilepsy Multiplatform Variant Prediction (EpiMVP), spanning 5 institutions and incorporating expertise from geneticists, clinicians, computational biologists, neuroscientists, stem cell biologists, pharmacologists and electrophysiologists who have a proven track record of collaborative publications and grants, as well as stature as leaders of national and international epilepsy organizations. EpiMVP will develop a modular, highly integrated platform approach to accelerate determination of the functional, pharmacological, neuronal network and whole animal consequences of genetic variants implicated in a range of clinical epilepsy types. We will study non-ion-channel, non-receptor genes commonly implicated in epilepsy, and that are involved in diverse biological processes. Our ultimate goals are to devise an effective experimental platform for testing the pathogenicity of VUS in genes implicated in epilepsy and to generate a computational model (EpiPred) that predicts the likelihood that a variant is pathogenic or benign. This work is crucial in the pursuit of novel therapeutics and the promise of personalized medicine. The overall milestones of the Center are: 1. Evaluate genes associated with epilepsy and select candidates for analysis, model data for, and analyze all project data for development of EpiPred an iterative machine learning model to classify variants in genes implicated in epilepsy. 2. Test selected VUS using medium throughput, in vitro approaches. 3. Test selected VUS in human cortical neurons or human brain organoids using induced pluripotent stem cell approaches. 4. Test selected VUS in pre-clinical, in vivo models. The expected outcomes are: 1. Provide a freely available prediction tool for clinicians to differentiate between pathogenic and benign variants for genes implicated in epilepsy; 2. Provide experimental models to study the functional consequences of specific variants; 3. Provide a reclassification of VUS in ClinVar/ClinGen and to develop new guidelines for incorporating functional readouts into the ACMG criteria; 4. Inform the future development of novel therapeutics to treat epilepsy.

The overall mission of the Epilepsy Multiplatform Variant Prediction (EpiMVP) Administrative Core is to oversee and coordinate the activities of the EpiMVP Center Without Walls (CWOW), including the work of the Gene and Variant Curation Core (GVCC), the Human Epilepsy Tools Core (HETC), the three Scientific Projects, as well as center-wide matters related to the program charter, publications, data sharing, communications with the scientific and lay communities, and ethical/legal issues. The Administrative Core?s Principal Investigators (PIs)/Directors, Drs. Lori Isom and Jack Parent, who have a successful track record of working together, coordinating groups of investigators, conducting multiple projects simultaneously, and disseminating research findings, will provide overall leadership to EpiMVP throughout the five years of the proposed Center. Drs. Isom and Parent will also serve as Co-Chairs of the EpiMVP Steering Committee, which will be composed of all EpiMVP PIs and Core Directors (Dr. Ross, Dr. Wang, Dr. Carvill, and Dr. Uhler) and have responsibility for the strategic direction and operational activities of the entire Center. The EpiMVP Program, centered at the University of MI and reaching out to the University of CA-San Francisco, Northwestern University, the University of WA, and Weill Cornell Medicine, is composed of international experts in genetics, clinical care, computational biology, neuroscience, pharmacology, and electrophysiology who are at the forefront of epilepsy research with broad expertise in the study of gene variants linked to developmental and epileptic encephalopathies and other genetic epilepsies. Long-term, on-going collaborations are in place between many investigators. The addition of three investigators to the epilepsy field, Dr. Ross, Dr. Schnell, and Dr. Bai, strengthens the CWOW and brings a fresh perspective to known problems in epilepsy research. Strong institutional support, provided to the group by the University of MI Medical School and the University of MI Departments of Pharmacology and Neurology in the form of administrative infrastructure, planning grant funding, and trainee support, further insures the success of this proposal. The EpiMVP Administrative Core seeks to accomplish the following Milestones: 1. To provide leadership and expertise for the planning, development, coordination, and overall administration of EpiMVP. 2. To oversee reporting of CWOW activities within its membership and to NINDS and to disseminate information regarding EpiMVP findings to a broad constituency, from scientists to clinicians to patients and families. 3. To help train and mentor the next generation of scientists to advance the understanding and treatment of genetic epilepsies. The expected outcomes of the EpiMVP Administrative Core are: 1) Leadership of an efficient, productive, and collaborative multi-institutional CWOW; 2) Effective communication between EpiMVP members and NINDS, as well as rapid dissemination of results to the research community, healthcare professionals, and patients/families, 3) Training and mentoring of the next generation of epilepsy researchers.

The advent of next generation DNA sequencing has revolutionized gene discovery in human diseases, including epilepsy. Hundreds of genes have been implicated in epilepsy in the last decade, revealing the diversity of biological mechanisms that can go awry in this disorder. However, the rate at which we are identifying new genes involved in epilepsy is now outpacing our ability to study disease mechanisms. Moreover, clinical gene panel or exome sequencing has become standard practice for patients with early-onset, familial, and refractory epilepsies. This rapid assimilation of genetic testing into clinical care has led to a surge in the number of genetic variants of uncertain significance (VUS), particularly the occurrence of missense VUS. These VUS are assigned to an indeterminate spectrum between pathogenic and benign, which complicate interpretation for genetic counselors, clinicians, patients and families, as well as assessment of the need for further testing. Here we propose a Center without Walls, entitled Epilepsy Multiplatform Variant Prediction (EpiMVP), spanning 5 institutions and incorporating expertise from geneticists, clinicians, computational biologists, neuroscientists, stem cell biologists, pharmacologists and electrophysiologists who have a proven track record of collaborative publications and grants, as well as stature as leaders of national and international epilepsy organizations. EpiMVP will develop a modular, highly integrated platform approach to accelerate determination of the functional, pharmacological, neuronal network and whole animal consequences of genetic variants implicated in a range of clinical epilepsy types. We will study non-ion-channel, non-receptor genes commonly implicated in epilepsy, and that are involved in diverse biological processes. Our ultimate goals are to devise an effective experimental platform for testing the pathogenicity of VUS in genes implicated in epilepsy and to generate a computational model (EpiPred) that predicts the likelihood that a variant is pathogenic or benign. This work is crucial in the pursuit of novel therapeutics and the promise of personalized medicine. The overall milestones of the Center are: 1. Evaluate genes associated with epilepsy and select candidates for analysis, model data for, and analyze all project data for development of EpiPred an iterative machine learning model to classify variants in genes implicated in epilepsy. 2. Test selected VUS using medium throughput, in vitro approaches. 3. Test selected VUS in human cortical neurons or human brain organoids using induced pluripotent stem cell approaches. 4. Test selected VUS in pre-clinical, in vivo models. The expected outcomes are: 1. Provide a freely available prediction tool for clinicians to differentiate between pathogenic and benign variants for genes implicated in epilepsy; 2. Provide experimental models to study the functional consequences of specific variants; 3. Provide a reclassification of VUS in ClinVar/ClinGen and to develop new guidelines for incorporating functional readouts into the ACMG criteria; 4. Inform the future development of novel therapeutics to treat epilepsy.