Jack Parent - US grants

DESCRIPTION (provided by applicant): The overall goal of this research plan is to elucidate how focal ischemic brain injury alters the regulation of neural stem cells in the adult forebrain sub-ventricular zone (SVZ). Advances over the past 3 decades firmly establish the persistence of neural stem cells and neurogenesis in the adult mammalian rostral SVZolfactory bulb pathway, a system that offers a potential source for neuronal replacement after brain injury or degeneration. However, the normal regulation of neurogenesis and the endogenous SVZ neural precursor response to brain injury in the adult are poorly understood. Knowledge of neurogenic mechanisms in the normal and injured mature brain is essential for developing novel therapies using endogenous or transplanted neural stem cells. We have recently discovered that focal ischemia increases neurogenesis in the adult rat rostral SVZ. Moreover, a sub-population of the newly generated neurons migrates toward damaged areas, suggesting that factors produced by injury influence the proliferation and migration of endogenous precursors. Based on these findings, the primary hypothesis of the proposed research is that forebrain SVZ neural stem cells respond to focal ischemic injury of the mature brain by generating increased numbers of new neurons that migrate toward sites of damage, survive, and integrate into residual networks. The secondary hypothesis is that specific growth factors or chemotropic guidance cues, produced normally or induced by injury, influence the proliferation and migration of neuronal precursors generated in the postnatal forebrain SVZ. The Specific Aims are: 1) To characterize the cell proliferation, migration, differentiation and integration of rostral SVZ neural precursors in the normal adult rat brain and after focal ischemic injury, 2) to determine the molecular factors that regulate rostral SVZ neuronal precursor proliferation and migration in the intact rodent brain or after ischemic injury; and 3) to determine the effects of delayed basic fibroblast growth factor (bFGF) or insulin-like growth factor1 (IGF1) administration on adult forebrain SVZ neurogenesis, and on ischemiainduced neurogenesis and infarct size after focal ischemic injury. The experimental design includes mitotic labeling and cell fate analysis of in vivo SVZ neurogenesis in the normal adult rat and in an adult rat transient-focal ischemia/stroke model, as well as studies of in vitro neurogenesis in slice cultures of the neonatal SVZolfactory bulb pathway. Progress in these Aims will advance our understanding of adult SVZ neurogenesis in the normal and ischemic brain, and may provide insight into the potential use of neural stem cells for stroke therapy.

[unreadable] DESCRIPTION (provided by applicant): The goals of this proposal are to examine the integration and regenerative capacity of adult-born telencephalic neurons using zebrafish models of conditional gene expression and brain injury. Neural stem/progenitor cells (NPCs) and neurogenesis persist throughout life in the adult vertebrate subventricular zone (SVZ)-olfactory bulb pathway, but the regulation and biological function of adult-born neurons are poorly understood. This neurogenic pathway in mammals is stimulated by stroke and other brain injuries. SVZ neuroblasts are diverted from their normal olfactory bulb target to the injured striatum and differentiate into neurons with an apparent striatal phenotype. NPCs in the adult therefore appear to contribute to a regenerative response that, if augmented, may improve recovery from forebrain injuries. The consequences of injury-induced SVZ neurogenesis remain obscure, however, as little progress has been made in establishing the long-term survival and functional integration of adult-born neurons after brain insults. Zebrafish is an attractive, albeit underutilized, model system for the study of adult neurogenesis in the intact or injured forebrain. The central nervous system (CNS) regenerative response of adult teleost fish, including zebrafish, is much more robust than in mammals. Understanding how regeneration is achieved after brain injury in the zebrafish is likely to provide insight into why mammalian CNS regeneration is limited, and how this limitation might be reversed to achieve restorative NPC therapies. These and other advantages of the zebrafish system have led our group and others to begin characterizing SVZ-olfactory bulb neurogenesis in adult zebrafish. Our data suggest that adult-born neurons integrate in the bulb and that SVZ neurogenesis is stimulated by excitotoxin-induced telencephalic injury. We also have begun developing inducible transgenic zebrafish lines for fate mapping to examine the long-term integration of adult-born telencephalic neurons. Using these lines and the excitotoxic forebrain injury model, we propose to test the following hypotheses: 1) Adult-born SVZ neuroblasts migrate to the olfactory bulb and generate olfactory neurons that integrate into preexisting networks; and 2) Excitotoxic telencephalic injury stimulates adult SVZ neurogenesis to replace damaged neurons. Two specific aims are proposed to test these hypotheses. In Aim 1, transgenic zebrafish lines with inducible Cre recombinase under the control of NPC-specific promoters will be crossed with a reporter line to conditionally label adult-born neurons in the telencephalic SVZ and examine their structural and functional integration. In Aim 2, fish will undergo excitotoxic telencephalic lesioning to determine whether injury stimulates the proliferation, migration, and long-term integration of adult-born neurons identified by mitotic labeling or transgenic approaches. These studies will shed light on the long-term fate and regenerative potential of adult-born forebrain NPCs and will provide valuable tools to study adult neurogenesis in the intact and injured vertebrate forebrain. [unreadable] PUBLIC HEALTH RELEVANCE: Neural stem cells and the birth of new nerve cells persist in the adult brain. These cells have therapeutic potential and may be stimulated to repair the brain after injury. The reasons why repair is often incomplete after acute brain insults are unknown, but zebrafish have a greater nervous system regenerative capacity than mammals and may shed light on the factors that limit repair. Because the same pathways of nerve cell birth are present in the forebrains of zebrafish and mammals, progress in understanding neural stem cell behavior in the intact or injured fish brain may lead to therapies for neural repair after stroke or other brain insults. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The overall goals of this proposal are to determine the extent to which human embryonic stem cell (hESC)- or induced pluripotent stem cell (iPSC)-derived neural progenitor cell (NPC) grafts improve recovery after experimental stroke, and to begin addressing the critical safety and efficacy questions necessary for eventual human stroke therapy. hESCs offer many advantages as a source of NPCs for regenerative therapy, such as a readily available supply, vast differentiation potential and ease of genetic manipulation. iPSCs offer the added advantage of autologous grafting that obviates the need for immunosuppression. For either source, however, the ideal donor cell types and developmental states required to achieve CNS regeneration are poorly understood. Whether NPC grafting restores function after stroke and the mechanisms by which it might do so also are unknown. We have developed methods to enrich for specific NPC populations derived from hESCs, and have generated NPCs and multiple neuronal subtypes using iPSCs derived from human fibroblasts. Using these techniques, we propose to test the following hypotheses: 1) purified populations of multipotent NPCs (mpNPCs) and neuronal restricted precursors (NRPs) can be derived from hESCs or from human somatic cells via iPSCs. These populations will differ in their migration, differentiation and integration after transplantation into the intact or injured adult rat brain;and 2) grafting of mpNPCs or NRPs after experimental stroke will enhance functional recovery directly by neuronal replacement or by stimulating repair via endogenous NPCs. Specific Aims 1 and 2 are to purify and characterize specific NPC populations using promoter-based reporter or cell surface antigen-based selection. Aim 3 is to examine the behavior of these NPC populations after grafting into neurogenic and non-neurogenic regions of the adult rat brain, and Aim 4 is to examine the influence of hESC- and iPSC (human and rat)-derived NPC grafts on recovery after experimental stroke. Progress in these aims will advance our knowledge of how graft factors influence the capacity of hESC- or iPSC-derived NPCs to repair the injured brain and promote recovery after experimental stroke, and will provide insight into the untapped reparative potential and possible risks of these therapies. PUBLIC HEALTH RELEVANCE: Stroke is a common and potentially devastating neurological disorder with no proven regenerative therapies. This proposal aims to derive neural stem cells (NSCs) from human embryonic stem cells or reprogramming of human adult skin cells, and to use a stroke model to identify the optimal NSC grafts for brain reparative stroke therapy. Progress in this area offers advances toward novel cell-based restorative therapies for stroke and other brain insults.

? 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.

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.

PROJECT SUMMARY ? PROJECT 2 Next generation DNA sequencing (NGS) has led to the rapid discovery of large numbers of epilepsy genes, and the list of epilepsy genes has grown well beyond ion channels to those that affect a wide array of cellular functions. Our understanding of how any specific gene mutation leads to epilepsy, however, increasingly lags behind gene discovery. Moreover, NGS has led to increased numbers of genetic variants of uncertain significance (VUS) that are difficult to interpret diagnostically. We lack the tools to assay VUS effects or effectively study pathogenic mechanisms for these epilepsy genes. To address these shortfalls, the EpiMVP will optimize cutting-edge multiplatform assays for epilepsy genes that include cell lines (Project 1), human pluripotent stem cells (hPSCs; Projects 1 and 2), human cortical organoids (hCOs; Project 2), and in vivo rodent and zebrafish models (Project 3). The Gene and Variant Curation Core (GVCC) will interact with the projects to select and refine specific genes and variants for testing as the VUS list is streamlined from Projects 1 to 2 to 3. Key to this effort is the Human Epilepsy Tools Core (HETC) which will provide cell lines (for Projects 1 and 2) and variant expression vectors (for all 3 projects). The long-term goal of our work is to deliver an in vitro testing pipeline in human neuronal models to assay clinically relevant VUS for all non-ion channel epilepsy genes. We have identified relevant morphological/functional 2-D or hCO phenotypes for 6 genes in the top 10 most commonly diagnosed non-ion channel genetic epilepsies, as well as reagents for several others, using: 1) 2-D hPSC cultures, including small molecule differentiation into excitatory or inhibitory cortical neurons, excitatory induced neurons and induced GABA neurons (iNeurons/iGNs) generated by transcription factor expression, and mixed cultures (iNeurons, iGNs and glia); and 2) 3-D hCO cultures, including multi-rosette, single rosette, excitatory, inhibitory and fusion hCOs. We will use these assays to test the hypothesis that our platforms will predict VUS pathogenicity and effectively prioritize variants for in vivo testing in Project 3. Our immediate goals are to optimize assays for 1-2 genes per year, determine VUS pathogenicity in vitro for these genes and, in concert with the VGCC, refine the VUS list for further in vivo testing in Project 3. The goals will be accomplished using 2-D hPSCs for Milestone 1 and 3-D hCOs in Milestone 2, and will include structural and functional assays for each model system. These studies will provide the following deliverables: 1) multiple optimized, cross-validated (between Parent and Ross labs) hPSC platforms to interrogate epilepsy genes; 2) determination of in vitro human neuronal VUS pathogenicity for at least 5 non-ion channel epilepsy genes; 3) human neuronal models for each epilepsy gene; and 4) optimized platforms for future mechanistic and precision therapeutic studies.