Scott C. Baraban - US grants

DESCRIPTION (provided by applicant): Malformations of cortical development (MCD) are often associated with developmental delay, cognitive deficit and intractable epilepsy. Despite considerable effort, our understanding of synaptic function - a key component mediating these neurological symptoms - within a malformation remains limited. To investigate this issue, we study injury- and genetically-based animal models of MCD, as well as human tissue resected from epilepsy patients with MCD. Based on prior investigations, we focus this proposal on our overall hypothesis that aberrant network activity underlies cognitive dysfunction and seizures associated with a brain malformation. Here, our studies focus on mice with a heterozygous inactivation of LIS1 (Lis1) e.g., a genetically-based MCD model exhibiting seizures, severe disorganization of hippocampal architecture and enlarged ventricles. Preliminary data suggest a significant enhancement of excitatory synaptic transmission onto CA1 pyramidal neurons in disorganized regions of Lis1 mice. Because enhanced excitation appears to be associated with a striking increase in synaptic facilitation and vesicle density, studies are proposed to examine presynaptic mechanisms in Lis1 mice. Techniques will involve use of acute hippocampal slices maintained in vitro, and application of visualized patch clamp methods to study the physiological function of pyramidal neurons and granule cells within a disorganized hippocampus. Pharmacological and genetic manipulations will be made to assess presynaptic release mechanisms and potential pre-clinical treatment strategies. Video- EEG monitoring and immunohistochemistry techniques will also be applied. Three specific aims are proposed: (i) to further examine enhanced excitatory neurotransmission in Lis1 mice, (ii) to examine the function of newborn granule cells in Lis1 mice, and (iii) to examine synaptic function and neurogenesis in a conditional Lis1 mouse. Our studies are designed to elucidate mechanisms contributing to epileptogenesis in a malformed brain. Results of the proposed experiments could have translational impact on the development of new therapies for patients with MCD.

DESCRIPTION (provided by applicant): Genetic factors play a major role in the development of epilepsy. In fact, with recent advances in human genetics it is now clear that mutations resulting in ion channel dysfunction, neurotransmitter receptor alterations, or abnormal brain development can predispose an individual to seizure activity. In contrast to what is known about the genetics of seizure susceptibility, virtually nothing is known about mutations that confer seizure resistance. To address this problem, we propose an innovative strategy to identify genes that render an organism resistant to the development of seizure activity. By combining the expertise of an epilepsy laboratory (Dr. S.C. Baraban) with that of a zebrafish genetics laboratory (Dr. H. Baier), we plan to identify seizure resistance mutants using large-scale mutagenesis screening in zebrafish (Danio rerio). Identification of genetic determinants of seizure resistance could, ultimately, lead to novel gene-based approaches that are designed to prevent the initiation of epileptic activity and result in a "seizure-free" life. Zebrafish mutagenesis screens have already identified mutations that define the function of hundreds of essential vertebrate genes. Here we propose to perform a highly selective functional screen in which behavioral and physiological measures are used to identify mutagenized zebrafish that are resistant to the development of seizure activity following exposure to a common convulsant agent (i.e., pentylenetetrazol). In Aims I & II, we will identify seizure resistant fish by monitoring behavior and electrographic activity following exposure to pentylenetetrazol. In Aim III, we will map, clone, and sequence the genes that confer seizure resistance to these fish. The results of this unique collaboration promise to provide new insights into the genetic factors that influence epilepsy, and will, perhaps, lead to a cure for this devastating neurological disorder.

DESCRIPTION (provided by applicant): Multi-potent progenitor cells raise great expectations for the treatment of neurological disorders and have generated considerable interest in the general field of medicine. A number of recent studies, including those from our laboratory (Wichertle et al. 1999), suggest that transplanted progenitors migrate considerable distances and differentiate into morphologically mature neurons in the host central nervous system. For example, progenitor cells derived from medial ganglionic eminence (MGE) have a unique ability to disperse, migrate, and differentiate into GABAergic interneurons in multiple adult brain regions. Although anatomical studies on transplanted progenitors suggest a mature neuronal morphology, the function of these neurons in a host brain remains unknown. In response to a recent NIH program announcement, in this proposal we will asses the ability of transplanted MGE progenitor cells to integrate in the host nervous system and modify a dysfunctional state e.g., epilepsy. Techniques will involve use of acute brain slices maintained in vitro, and application of visualized patch clamp methods to study the physiological function of fluorescently-tagged transplanted MGE cells. Immunohistochemical and electron microscopy experiments will be performed to assess the phenotype of transplanted cells. Mutant mice exhibiting loss of cortical interneurons (uPARv-/-; Dlx-/-) or alterations in synaptic inhibition (GAD657-/-) will be used to evaluate whether transplanted MGE progenitor cells influence the development of acute seizure activity. Three specific aims are proposed: (i) to develop procedures for transplantation of MGE progenitors into the adult CNS, (ii) to determine whether MGE progenitors increase inhibitory (GABAergic) function in the adult CNS, and (iii) to assess the therapeutic potential of MGE progenitor grafts against rodent seizure models. Our results promise to provide new information about the function of transplanted MGE progenitors in a host brain and may provide a direct demonstration of the potential for progenitor cells to treat intractable forms of epilepsy.

DESCRIPTION (provided by applicant): Genetic factors play a major role in the development of epilepsy. With recent advances in human genetics it s now clear that mutations resulting in ion channel dysfunction, neurotransmitter receptor alterations, or abnormal brain development predispose an individual to seizure activity. Although considerable progress has been made in the genetics of seizure susceptibility, virtually nothing is known about mutations that confer seizure resistance. To address this problem, we recently developed an acute seizure model in zebrafish (Danio rerio) e.g., exposure to a common convulsant agent, pentylenetetrazol (PTZ). Using this simple vertebrate model, and pilot funding from an R21 exploratory grant, we completed a large-scale forward-genetic mutagenesis screen to isolate seizure resistant zebrafish mutants. Six seizure resistant zebrafish mutants were identified and confirmed in out-cross testing. Here we propose to continue our study of seizure resistant zebrafish and to identify the underlying gene mutations. Our overall goal is to obtain a better understanding of seizure resistance. A multi-disciplinary strategy is proposed. Techniques include video monitoring and locomotion tracking software to study behavior. Electrophysiological experiments to assess brain function in zebrafish exposed to convulsant agents. Molecular and immunohistochemical studies to monitor immediate early gene expression. Genetic linkage analysis and positional cloning techniques to identify underlying gene mutations in two or three seizure resistant zebrafish mutants. Two specific aims are proposed: (i) To use behavioral, molecular and electrophysiological assays to characterize seizure-resistant phenotypes in zebrafish mutants and (ii) To use molecular analysis to map and clone seizure resistance genes in zebrafish. The results promise to provide new information about the genetics of seizure resistance and may lead to the design of novel anticonvulsant treatments designed to prevent epilepsy.

? DESCRIPTION (provided by applicant): Transplantation of GABA progenitors into the central nervous system has shown great promise for the treatment of neurological disease. Our laboratories demonstrated that GABA-expressing interneurons, derived from the rodent embryonic medial ganglionic eminence (MGE), migrate, integrate, and increase inhibition following transplantation. Potential therapeutic benefits of these cells were reported in animal models of: epilepsy, Parkinson's disease, Alzheimer's disease, neuropathic pain, schizophrenia, anxiety and psychosis. During the previous NIH-supported funding cycle and in response to NINDS epilepsy research benchmarks, we developed an adult transplantation strategy using MGE progenitors harvested from mouse embryos, and published the first studies demonstrating that MGE transplantation dramatically suppressed spontaneous seizures and improved cognitive or behavioral co-morbidities in an animal model of acquired epilepsy. These findings are consistent with our hypothesis that enhancement of GABA-mediated inhibition - through the generation of new interneurons - provides therapeutic benefit in conditions featuring excess excitation such as epilepsy. Translation of these findings to the clinic, ultimately, requires a more complete understanding of underlying mechanism(s). However, studies of MGE transplantation have not adequately addressed the question of how distinct interneuron sub-population(s) influence host circuitry and which sub-types are necessary for the therapeutic activity observed. To address these issues, we propose experiments to study progenitors harvested from the medial and caudal ganglionic eminences. These cells will be transplanted in neonatal and adult hippocampus, and in a common rodent model of acquired epilepsy. Donor mice will incorporate interneuron-specific Cre-recombinase lines, as well as floxed, channelrhodopsin (CHR2), vesicular GABA transporter deficient (VGAT), and diphtheria toxin (DT) animals. Techniques will involve use of acute brain slices maintained in vitro, visualized patch clamp recording in combination with optogenetic stimulation, and viral synaptic tracers. Video-EEG monitoring, immunofluorescence, behavior and confocal microscopy techniques will also be applied. Two specific aims are proposed: (i) to evaluate integration of transplanted progenitor cells in the host brain, and (ii) to identify interneuron sub-population(s) necessary fo the therapeutic benefits of transplanted progenitor cells. Our results promise to advance our long-term goal to develop a novel interneuron-based cell therapy for intractable epilepsies.

DESCRIPTION (provided by applicant): Inhibitory interneurons synthesize GABA and play a critical regulatory role in the central nervous system. Recent observations suggest that development of these interneurons requires a family of Dlx transcription factors. In studies from our laboratory (Cobos et al. 2005), it was demonstrated that mice lacking Dlx1 progressively lose a sub-class of cortical and hippocampal interneurons resulting in reduced synaptic inhibition and epilepsy. This novel model of interneuron-deficient, late-onset epilepsy offers a unique opportunity to precisely define the role of interneurons in epileptogenesis, and ultimately test therapeutic interventions to interrupt or abrogate this process. In response to recent NINDS "benchmarks", we here propose experiments that will examine how Dlx1 deficiency (and subsequent interneuron loss) alters network excitability, and test a potential therapeutic intervention using GABA progenitor cells. Techniques will involve use of acute brain slices maintained in vitro, and application of visualized patch clamp methods to study the physiological function of hippocampal interneurons and their associated circuits. Video-EEG monitoring and cell grafting techniques will also be applied. Three specific aims are proposed: (i) determine whether Dlx1 is required for interneuron function, (ii) determine whether synaptic input to surviving interneurons is disrupted in Dlx1 mutant mice, and (iii) determine whether GABA progenitor cell grafts reduce seizure activity in Dlx1 mutant mice. Our results promise to provide critical information about the role of interneurons in epilepsy and may provide a direct demonstration of the potential for GABA progenitor cells to treat seizures. PUBLIC HEALTH RELEVANCE: Epilepsy is a common neurological disorder afflicting nearly 3 million Americans. Loss or reduction of inhibitory synaptic transmission in hippocampal and cortical circuits is one potential mechanism resulting in the emergence of epilepsy. Using a mouse mutant lacking inhibitory nerve cells, and a cell grafting strategy to generate new inhibitory nerve cells, we will examine the mechanisms underlying epilepsy and a potential treatment.

DESCRIPTION (provided by applicant): Traditional drug discovery programs for epilepsy target anticonvulsant effects and rely, almost exclusively, on induced seizure models in adult rodents. However, numerous genetic models that mimic many features of human epilepsies have now been described. These models provide important information but are not easily adapted to drug discovery programs. As a simple vertebrate species amenable to rapid genetic manipulation and high-throughput drug screening, we propose an alternative approach using mutant zebrafish (Danio rerio) with spontaneous recurrent seizure phenotypes (i.e., epilepsy) as a platform to identify new treatments for medically refractory epilepsy. We recently began to explore the possibility that spontaneous single-gene mutations in zebrafish - especially those mimicking catastrophic forms of epilepsy often seen in children - result in epileptic phenotypes. Zebrafish mutants featuring a loss-of-function sodium channel (Nav1.1/SCN1A) mutation (e.g., a gene family identified in children with Severe Myoclonic Epilepsy of Infancy and Dravet syndrome) were recently identified by our laboratory as epileptic zebrafish with phenotypes similar to the human condition. Using large-scale transcriptome analysis, automated behavioral tracking, in vivo electrophysiology and pharmacological approaches we describe a novel approach to further our understanding and potential treatment of debilitating epilepsy disorders associated with Nav1.1 mutation. In this EUREKA proposal we will use these mutant zebrafish in our efforts to (i) identify molecular targets for therapeutic treatment of DS/SMEI and (ii) identify drug candidates for therapeutic treatment of DS/SMEI. Our results promise to establish an alternative, zebrafish-based, approach for high-throughput small-molecule drug discovery targeted to monogenic epilepsy disorders seen primarily in children.

DESCRIPTION (provided by applicant): We propose a theoretical-experimental program to quantitate seizure activity with neuronal resolution in the intact larval zebrafish central nervous system. Our study is made possible by recent advances in light-sheet microscopy, and theoretical and algorithmic advances in the analysis of large neural imaging datasets. Light-sheet microscopy has both excellent spatial and temporal resolution and is capable of virtually complete volumetric coverage of the larval zebrafish central nervous system. This, along with advanced statistical and computational techniques, allows us to quantify neural dynamics in the zebrafish brain with unprecedented accuracy. Because of the structure of neural circuits, inhibitory neuronal populations typically surround excited regions, protecting the brain from runaway excitatory (ictal) activity that is generated when a seizure forms. However, repeated waves of ictal activity can break down the surround inhibition, allowing a seizure to propagate. With high-resolution microscopy and state-of-the-art computational analysis and simulation methods, we will study how coherent ictal activity generated during seizures interacts with the surround inhibition (often called the 'inhibitory restraint') that is the brain's response to the seizure. A precise understanding of how coherent excitations interact with and depress inhibition in interneuron populations would provide a powerful control paradigm for spatial and temporal intervention in seizure formation and propagation. Furthermore, although computer simulations have been performed using detailed synaptic connectivity reconstructions from anatomical data, simulations derived, then validated in the same organism would be a transformative contribution to the study of seizures and more generally to neuroscience. This study combines the experimental, theoretical, and validation aspects of a neuroscience investigation into a unified whole in the study of a large, intact neuronal network for the first time. Although, for technical reasons, this approach is limited to the larval zebrafish, a small, transparent organism, it could radically improve our understanding of how protective mechanisms in meso-scale neuronal systems can fail in vertebrates. Our proposed study will provide information that could guide future seizure interventions such as neuron transplantation, electrical stimulation, surgical tissue removal or drug targeting of neuronal populations and synapses that most effectively prevent seizure formation and propagation. The education and training of the graduate students and postdocs involved in our program will be integrated with every aspect of the research. Undergraduate students will be involved in the research and mentored. The investigation is multi-institutional and builds on existing interdisciplinary collaborations in engineering, developmental neuroscience, epilepsy and mathematics.

? DESCRIPTION (provided by applicant): Dravet syndrome (DS), a catastrophic childhood epilepsy, is associated with severe intellectual disability, impaired social development, persistent drug-resistant seizures and a high risk of sudden unexpected death in epilepsy. We recently began to explore the possibility that single-gene mutations in zebrafish can be used to advance our understanding of the pathophysiology and treatment of DS. Zebrafish mutants featuring a loss-of- function sodium channel (SCN1A) mutation (e.g., a gene family commonly identified in children with DS) were identified and characterized by our laboratory as epileptic zebrafish with phenotypes similar to the human condition. Using automated behavioral tracking and in vivo electrophysiology assays we screened more than 1300 compounds using these fish. With the data generated, we now propose to extend this research program to include novel DS zebrafish mutants, additional high-throughput screening, and mechanistic analysis of a small molecule lead compound that emerged from the first screening effort. Three specific aims are proposed: (i) to generate and characterize zebrafish DS mutants, (ii) to perform high-throughput drug screening using zebrafish DS mutants, and (iii) to examine the mechanism of action for clemizole in DS mutants. Techniques will include automated locomotion tracking, in vivo zebrafish electrophysiology recording, pharmacology, CRISPR/Cas9 genome editing and calcium imaging using genetically encoded calcium indicators. Our results promise to advance our long-term goal to better understand the pathophysiology of genetic epilepsies, and identify promising new treatment options for these intractable conditions.

Project Summary/Abstract Children with catastrophic epilepsies develop intractable forms of epilepsy with severe intellectual and behavioral disabilities, including autism. Many of these children are diagnosed, early in life, with genetic forms of epilepsy. With recent advances in gene sequencing approximately 60 loss-of-function (LOF) single-gene mutations have been identified in this patient population. Zebrafish LOF mutants designed to represent these human genetic conditions would provide valuable tools for elucidating basic disease mechanisms and drug discovery. Methods such as the CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeat/Cas9) now allow for rapid and efficient modification of endogenous genes in a range of animal models, including zebrafish (Danio rerio). Using this technique, we recently developed and characterized a stxbp1 mutant zebrafish line representing children with early infantile epileptic encephalopathy with burst suppression. In this proposal, we will generate pre-clinical mutant zebrafish lines for all known human LOF mutations using CRISPR/Cas9 gene editing techniques (Aim I). To determine whether these mutants exhibit functional phenotypes (including epilepsy), we will carefully monitor behavior and neural activity using unique assays and methodologies developed in our laboratory (Aim II). Our strategy includes (i) detailed analysis of behavior using automated locomotion tracking and high-speed imaging, (ii) recording brain activity using in vivo electrophysiology techniques and (iii) brain-wide calcium imaging of neural networks. Zebrafish lines with epileptic phenotypes will also be screened against a small panel of existing antiepileptic drugs. All zebrafish mutant lines generated and carefully characterized here will be made available to the scientific community via the NINDS-supported Zebrafish International Resource Center in Oregon. Both as a resource and offering insight into functional consequences of these single gene mutations, our proposal establishes a comprehensive zebrafish-based precision medicine approach targeted to monogenic epilepsy disorders seen primarily in children.

Although altered metabolism is rapidly emerging as a key feature of epilepsies, it has not been systematically investigated in any genetic form of pediatric epilepsy. Dravet syndrome (DS), a catastrophic childhood epilepsy associated with de novo mutations in a voltage-activated sodium channel, Nav1.1 is one of the most common genetic epilepsies. DS patients suffer with intractable early-life seizures, and debilitating comorbidities. Energy metabolism in comorbidities associated with DS remain virtually unexplored. To address this unmet need, recent collaborative research in our two laboratories revealed decreased glycolytic and oxygen consumption rates in a validated zebrafish model of DS i.e., scn1Lab mutants. This was accompanied by downregulation of key enzymes, pck1 and pck2, in the gluconeogenesis pathway. Here, we hypothesize that energy disruption occurs in DS due to glucose dysregulation resulting in seizures and/or comorbidities. The following aims are proposed to test this hypothesis. Aim 1 will determine if pharmacological inhibition of pck1 and/or pck2 phenocopies metabolic and behavioral deficits in wildtype zebrafish. Aim 2 will determine if pharmacological manipulation of pck1 and/or pck2 is therapeutic in scn1Lab mutant zebrafish. These studies promise to provide a mechanistic explanation of the metabolic defects observed in DS and could suggest novel avenues for therapeutic intervention.