Jennifer A. Kearney - US grants

The voltage-sensitive sodium channel alpha subunit, Scn8a, which is expressed in brain and spinal cord, was discovered in our laboratory as the gene responsible for motor endplate disease (med) in mice. Mice lacking Scn8a show ataxia and progressive hind limb paralysis, leading to death by 3-4 weeks. Although neurons contain several voltage-gated sodium channels, the loss of this single sodium channel alpha subunit results in a lethal phenotype, suggesting that Scn8a has unique properties which are essential for survival. These unique properties could be the result of a distinctive subcellular distribution and/or functional characteristics. The major goal of this proposal is to identify the unique functional domain(s) of Scn8a by construction of chimeric sodium channels and assay of their ability to correct the lethal phenotype of med mice in vivo.

The voltage-sensitive sodium channel alpha subunit, Scn8a, which is expressed in brain and spinal cord, was discovered in our laboratory as the gene responsible for motor endplate disease (med) in mice. Mice lacking Scn8a show ataxia and progressive hind limb paralysis, leading to death by 3-4 weeks. Although neurons contain several voltage-gated sodium channels, the loss of this single sodium channel alpha subunit results in a lethal phenotype, suggesting that Scn8a has unique properties which are essential for survival. These unique properties could be the result of a distinctive subcellular distribution and/or functional characteristics. The major goal of this proposal is to identify the unique functional domain(s) of Scn8a by construction of chimeric sodium channels and assay of their ability to correct the lethal phenotype of med mice in vivo.

DESCRIPTION (provided by applicant): Mutations in voltage-gated sodium channels are responsible for some forms of human epilepsy. Q54 transgenic mice have a mutation in the voltage-gated sodium channel Scn2a and are a model for this disorder. The disorder in these mice begins with short-duration partial seizures and progresses to status epilepticus and premature death. There is extensive cell loss and gliosis in the hippocampus, which resembles the hippocampal sclerosis in temporal lobe epilepsy patients. Clinical severity of the epilepsy phenotype is influenced by genetic background. Q54 mice that are congenic on the resistant C57BL/6J background have delayed onset of seizures and increased survival, compared to susceptible [SJL/J x C57BL/6J]F1s. This suggests that dominant genetic modifiers underlie the severity of the phenotype and may influence the susceptibility of the hippocampus to pathological changes. We will identify the modifier gene(s) that influence seizure susceptibility in Q54 transgenic mice by positional cloning. Although hippocampal neuronal loss and reorganization have been reported in patients for many years, it has been difficult to determine if the pathological changes precede or result from seizures. We will compare the time course of cell loss, synaptic reorganization and dentate granule cell neurogenesis in the hippocampus of resistant and susceptible Q54 mice to determine the relationship between cellular pathology and clinical severity. The interaction of the mutated sodium channel gene and the modifier gene(s) in Q54 transgenic mice provides a model of gene interaction affecting susceptibility to inherited epilepsy. Understanding of this modifier gene(s) will provide insights into the pathogenesis of epilepsy and may suggest novel therapeutic targets for the treatment of human epilepsy patients. This proposal addresses two important areas in epilepsy research pertinent to the program announcement (PAR-01-111 ): the genetic complexity of inherited epilepsy, and the role of neuronal injury in seizure susceptibility. These studies will provide a foundation for an independent epilepsy research program for Dr. Jennifer Kearney and will establish a new collaboration between Dr. Kearney and Dr. Jack Parent. This collaboration will combine Dr. Kearney's expertise in mouse genetics with Dr. Parent's expertise in hippocampal pathology.

DESCRIPTION (provided by applicant): Mutations in voltage-gated sodium channels have been associated with several types of human epilepsy, including Genetic (Generalized) Epilepsy with Febrile Seizure Plus (GEFS+) and Dravet Syndrome. Within these genetic epilepsies, there is variable penetrance and expressivity of the clinical phenotype, suggesting a role for genetic modifiers. We have developed mouse models with mutations in voltage-gated sodium channels and seizure-related phenotypes with different underlying mechanisms. Scn2aQ54 transgenic mice have a gain-of-function mutation that results in spontaneous, adult-onset partial motor seizures and models features of GEFS+. Heterozygous Scn1a+/- null mice are a model of Dravet Syndrome, a severe, infant-onset epilepsy with progressive worsening accompanied by psychomotor regression. A common feature of these mouse models is that epilepsy severity varies depending on the genetic strain background, suggesting that genetic modifiers influence the phenotype. Scn2aQ54 mice on the resistant C57BL/6J background have delayed onset, decreased severity and improved survival compared to the susceptible (C57BL/6J x SJL/J)F1 background. Conversely, the epilepsy phenotype of Scn1a+/- mice is more severe on the C57BL/6J background, while they have delayed onset and improved survival on the 129S6/SvEvTac strain background. Based on these observations, we hypothesize that multiple genetic modifiers act to influence penetrance and expressivity of the primary epilepsy mutation. Previously we mapped two modifier loci that are responsible for the strain difference in Scn2aQ54 mice: Moe1 (modifier of epilepsy 1) on Chromosome 11 and Moe2 on Chromosome 19. During the previous funding period we performed fine mapping and candidate gene analysis of the Moe1 region, identified the Moe2 modifier gene, and identified additional modifier loci that influence the Scn2aQ54 phenotype. We propose to continue our analysis of epilepsy modifiers using the Scn2aQ54 and Scn1a+/- mouse models. First, we will identify the responsible genes at Scn2aQ54 modifier loci. Next, we will determine the molecular basis of the Moe2 modifier effect. Finally, we will map genetic modifier loci in the Scn1a+/- mouse model and perform transcriptome analysis via RNA-seq for accelerated identification of candidate genes. The major goal of our studies is to identify and characterize modifier genes that influence epilepsy susceptibility and severity. These genes are likely to contribute to common epilepsy syndromes with more complex genetics. Identification of epilepsy modifier genes will provide insight into the basic biology of epileptogenesis and may identify novel therapeutic targets for the treatment of human patients.

DESCRIPTION (provided by applicant): Mutations in voltage-gated sodium channels have been implicated in several types of human epilepsy with varying degrees of clinical severity. Mutations in SCN1A were first identified in Generalized Epilepsy with Febrile Seizures Plus (GEFS+), a benign, childhood-onset syndrome in which family members have febrile seizures in childhood and may go on to develop other seizure types as adults. SCN1A mutations have also been identified in Severe Myoclonic Epilepsy of Infancy (SMEI), an infant-onset syndrome characterized by generalized tonic-clonic or hemiclonic seizures. As the syndrome progresses, SMEI patients develop other seizure types including myoclonic, absence and partial seizures, and a decline of psychomotor and mental development. Overall more than 300 mutations of SCN1A have been reported in patients with epilepsy, making it the most common genetic cause of epilepsy. In vitro expression studies of SCN1A mutations have revealed a variety of functional defects. However, there is not an obvious correlation between Nav1.1 dysfunction in heterologous expression systems and severity of the clinical phenotype. The lack of a clear genotype-phenotype correlation may reflect a limitation of in vitro expression systems to evaluate neuronal sodium channel mutations. The most reliable data on functional consequences of mutations can be obtained from mice engineered to carry the mutations. However, the resources and time required for generating knock-in mice by homologous recombination is prohibitive. Recombination-mediated cassette exchange (RMCE) allows for rapid and efficient production of an allele series of mice carrying mutant DNAs at the target locus. In this method, a cassette acceptor containing a selectable marker flanked by lox sites is targeted to the endogenous mouse locus by homologous recombination. Subsequent exchange of the cassette acceptor for the sequence of interest occurs by cre-mediated recombination in the ES cells, which is much more efficient than homologous recombination. The gain in efficiency decreases the time and resources required to generate multiple variants, allowing for parallel generation of an allelic series of mice. Specific aim 1 will generate a mouse ES cell line in which Scn1a exon 1 containing the translation start site is replaced by a loxed cassette acceptor via homologous recombination. Subsequent exchange with SCN1A cDNAs will allow expression of the cDNA under the endogenous regulatory control while ablating expression of the mouse gene. Specific aim 2 will generate mice by cre-mediated cassette exchange in which mouse Scn1a is replaced with the human SCN1A cDNA as a critical proof-of-principle experiment for this approach. Development and validation of this approach will enable in vivo characterization of human epilepsy mutations and provides a valuable resource for understanding the mechanisms underlying epilepsy. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to develop a system for rapid and efficient production of mouse models carrying human epilepsy mutations. Mouse models will provide insight into the molecular and genetic events that underlie epilepsy and will be valuable tools for developing novel therapeutic strategies.

DESCRIPTION (provided by applicant): Mutations in voltage-gated sodium channels are responsible for several human epilepsies with varying degrees of clinical severity. Over 800 mutations in SCN1A, encoding the neuronal voltage-gated sodium channel Nav1.1, have been reported patients. SCN1A mutations are associated with epilepsy phenotypes on the genetic epilepsy with febrile seizures plus (GEFS+) spectrum. The GEFS+ spectrum ranges from simple febrile seizures on the mild end of the spectrum to Dravet syndrome on the severe end. Heterozygous loss-of-function mutations in SCN1A result in Dravet syndrome, an infant-onset epileptic encephalopathy characterized by a variety of seizure types, developmental delay and elevated mortality risk. A common feature of monogenic epilepsies is variable expressivity in individuals carrying the same mutation, suggesting that clinical severity is influenced by genetic modifiers. Mice with heterozygous deletion of Scn1a (Scn1a+/-) model a number of features of Dravet syndrome, including spontaneous seizures and increased mortality risk. Loss of Scn1a results in reduced sodium current in hippocampal GABAergic interneurons, which is predicted to increase excitability due to failure of inhibition. Phenotype severity in Scn1a+/- mice is strongly dependent on strain background. Scn1a+/- mice on the resistant 129 strain background (129.Scn1a+/-) have no overt phenotype and live a normal lifespan. In contrast, Scn1a+/- mice on a (129xB6)F1 strain background (F1.Scn1a+/-) exhibit spontaneous seizures and premature lethality, with 50% dying by 1 month of age. Based on the strain- dependent difference in phenotype, we hypothesize that genetic modifiers influence Scn1a+/- phenotype severity. We recently mapped several modifier loci that influence premature lethality of Scn1a+/- mice. In the current proposal, we will perform fine mapping and candidate gene analysis with the goal of identifying the responsible modifier genes. In addition to the strain-dependent differences in clinical severity, we also observed strain-dependent differences in hippocampal neuron sodium currents (INa). GABAergic interneurons isolated from the F1.Scn1a+/- mice exhibit decreased INa density compared to wildtype littermate controls. In contrast, INa density is preserved in GABAergic interneurons isolated from 129.Scn1a+/- and is no different from wildtype littermates. This suggests that interneurons from strain 129 compensate for the loss of Nav1.1, while F1 interneurons do not. Based on this observation, we hypothesize that there are strain differences in compensatory capacity in the context of Scn1a heterozygous deletion. We propose to perform RNA-seq analysis to characterize hippocampal transcriptome differences during the critical phase of phenotype onset in susceptible F1.Scn1a+/- and resistant 129.Scn1a+/- mice. The results of this analysis will suggest candidate modifier genes and pathways that influence phenotype severity in Scn1a+/- mice. Identification of Dravet syndrome modifier genes will provide insight into the pathophysiology of epilepsy and will suggest novel therapeutic strategies for the improved treatment of human patients.

? DESCRIPTION (provided by applicant) Epilepsy is a common neurological disorder that affects approximately three million people in the United States. Approximately 30-40% of patients are unable to achieve adequate seizure control with currently available treatments. Understanding the causes of intractable epilepsy may suggest alternative therapeutic strategies that will address this critical unmet need. Epilepsy is presumed to have a genetic basis in approximately 70% of cases. Numerous genes associated with epilepsy have been identified, and most are components of neuronal signaling, including ion channels, ion channel-associated proteins, and synaptic proteins. Epileptic encephalopathies are particularly severe childhood epilepsies that often include intractable seizures, and are frequently attributable to de novo single gene mutations. Studying mouse models of epilepsy have increased our understanding of the etiology and pathogenesis of epilepsy. In the previous funding period, we used mouse models with voltage-gated sodium channel mutations to examine the effect of genome variation on epilepsy severity and identified several modifier loci and genes. A number of genes influenced phenotype severity of the Scn2aQ54 mouse model, including Kcnv2, Hlf, and Cacna1g. Kcnv2 encodes the Kv8.2 voltage-gated potassium channel subunit, which modulates delayed rectifier potassium currents by co- assembling in heteromeric channels with Kv2.1, encoded by Kcnb1. Delayed rectifier potassium current is an important mediator of neuronal excitability particularly under conditions of repetitive firing. We hypothesized that genetic variation in KCNV2 or KCNB1 may contribute to epilepsy risk in humans. Recently, we identified de novo mutations of KCNB1 as a novel cause early infantile epileptic encephalopathy type 26 (EIEE26), which is characterized by pharmacoresistant seizures with associated cognitive and motor deficits. We propose to continue our research program on the genetic basis of epilepsy with a focus on this newly described syndrome, EIEE26. First, we will perform functional studies on a large series of KCNB1 patient mutations in order to determine the range of functional defects and define genotype-phenotype relationships. In addition, we will develop and characterize murine models of EIEE26 in order to better understand how KCNB1 mutations lead to epilepsy. Finally, we will determine how genome background influences disease severity in Kcnb1 mouse models and identify modifier loci. The proposed studies will provide insight into the genetic architecture of epilepsy, and improve treatment of patients by enhancing precision of molecular diagnosis and suggesting novel pathways for therapeutic intervention.

Approximately 1 in 6 children in the United States are affected by neurodevelopmental disorders (NDD), which pose a significant burden to patients, families and society. Among NDD, autism spectrum disorder (ASD), intellectual disability (ID), developmental delay (DD) and early-onset epilepsy account for approximately 40% of cases. There is considerable evidence for a significant contribution of de novo genetic mutations to these disorders, and variants in SCN2A are a major genetic driver contributing to several NDD, including ASD, ID, DD, epileptic encephalopathies, and schizophrenia. This suggests proper function of Nav1.2 voltage-gated sodium channels, encoded by SCN2A, are important for normal brain development and function. Twenty-years ago, we developed the Scn2aQ54 mouse model that is hemizygous for a mutant Nav1.2 channel. Scn2aQ54 mice exhibit early-onset epilepsy and repetitive behaviors suggestive of autistic-like traits. Although this model exhibits some features of patients with SCN2A variants, it lacks construct validity. It was generated by random-insertion transgenesis of a mutated rat Scn2a cDNA under control of the neuron specific enolase (Eno2) promoter. Due to the nature of the transgene construct, the timing/location of expression may not faithfully recapitulate endogenous Scn2a and neonatal splicing events do not occur. In light of these limitations and transformative advances in genome engineering, we propose to make improved mouse models carrying human SCN2A variants, including an infantile spasms variant and a recurrent ASD/ID variant. We will generate two mouse lines using crispr/CAS9 and homology directed repair, and perform initial characterization of cellular, neurodevelopmental and epilepsy phenotypes on a C57BL/6J background (Aim 1). SCN2A variants are associated a wide-degree phenotype heterogeneity, even among individuals carrying the same mutation. This suggests that phenotype expressivity can be influenced by other factors, which may include genetic modifiers. Consistent with this, the epilepsy phenotype of Scn2aQ54 mice was strongly influenced by genetic background. To address the effect of genomic background variation, we will cross Scn2a mutant mice with several strains and survey neurodevelopmental and epilepsy phenotypes in F1 offspring (Aim 2). We will focus on a subset of parental strains of the collaborative cross and diversity outbred genetic mapping resources to support future leveraging of these resources. The proposed Scn2a strains and phenotype information will be deposited in the NIH-sponsored Mutant Mouse Regional Resource Center (MMRRC) and Mouse Genome Database (MGD/MGI). This proposal will deliver two novel mouse models of NDD with Scn2a driver mutations and make them readily available to the wider research community to enable independent studies of Scn2a channel variants in their native context at the cellular, circuit, systems and whole animal levels, as well as enable studies of gene-gene and gene-environment interactions. This will advance our understanding of the pathophysiology of brain development disorders and may ultimately lead to the development of novel therapies.

PROJECT SUMMARY ? PROJECT 3 Epilepsy is a common neurological disorder that affects over 3 million Americans and has a substantial genetic contribution to its etiology. Mutation of voltage-gated ion channel genes (?Channelopathies?), particularly voltage-gated sodium (NaV) and potassium (KV) channel genes, have emerged as a major cause of early onset epileptic encephalopathies. These severe epilepsy syndromes are often difficult to treat with existing therapies and are associated with adverse neurodevelopmental sequelae, making them a high priority for better treatment approaches like precision medicine. Functional characterization of a small number of epilepsy- associated voltage-gated ion channel mutations in heterologous expression systems have demonstrated a range of dysfunction, but it is presently difficult to extrapolate these results to in vivo effects. A major goal of our Center is to determine how well in vitro cellular models predict neuronal dysfunction and pharmacological responses in an intact brain. To accomplish this goal, Project 3 will focus on a series of representative mouse models with NaV and KV channel variants that cause prototypical patterns of dysfunction. We hypothesize that differences in the relative contribution of specific channels to excitability in various cell types within neuronal networks determine the net effect on excitation-inhibition balance and influence pharmacological response. Mouse models provide the opportunity to evaluate the effect of channel variants at the whole animal, cellular and network levels, as well as to investigate pharmacological responses. In Aim 1, we will develop mouse models to investigate NaV and KV channel variants associated with early onset epileptic encephalopathy. Mouse lines will be evaluated for neurological phenotypes and pharmacological response in vivo. In Aim 2, we will determine the impact of NaV and KV channel variants on channel properties and intrinsic cell excitability in acutely dissociated neurons isolated from mouse models, and then determine the effectiveness of pharmacological agents at normalizing channel activity and/or cell excitability in these neurons. These results will be compared with similar recordings from heterologous expression systems (Project 1) and patient-specific iPSC-derived neurons (Project 2) to establish important correlations between in vitro and in vivo models. In Aim 3, we will determine the impact of NaV and KV channel variants on intrinsic properties of neurons and consequent effects on network activity in brain slices, and then determine the effectiveness of pharmacological agents at normalizing aberrant cellular and network excitability. Results from Project 3 will provide mechanistic insight into the effects of channel dysfunction in intact brains, and determine therapeutic strategies that normalize excitation-inhibition balance and prevent/reduce seizures. Synergy between this project and Projects 1 and 2 include cross-platform comparisons of the same channelopathy-associated epilepsy variants, which will facilitate translation of results into valuable information for implementation of precision medicine in this common neurological disorder.

ABSTRACT Alzheimer?s disease and epilepsy are common age-related CNS disorders. Both Alzheimer?s disease and epilepsy are more frequent in the elderly compared to any other age groups, and a history of epilepsy is a risk factor for development of Alzheimer?s and related dementias. Further, patients with Alzheimer?s disease have unprovoked seizures and epilepsy at a significantly higher rate than non-demented elderly. These public health correlations are seen at the level of pathophysiology and manifested symptoms. For example, cognitive impairment is a definitive aspect of Alzheimer?s disease, and recurrent epileptic seizures are associated with cognitive impairment. Clearly, the increase in aging of the world?s population makes this comorbidity a major concern. This proposal is focused on addressing a common pathophysiological mechanism in Alzheimer?s and epilepsy ? dysregulated proinflammatory cytokine production. Proinflammatory cytokine overproduction from abnormally activated glia is a contributor to subsequent neurological damage and cognitive deficits in both epilepsy/seizure disorders and in Alzheimer?s and related dementias. Despite advances in our understanding of these molecular neuroinflammatory mechanisms underlying adverse neuronal sequelae in CNS disorders, approved therapeutics that target this pathological process are lacking. ImmunoChem Therapeutics (ICT) proposes to advance MW189, a novel small molecule candidate already in early phase clinical development, having successfully completed phase 1a and phase 1b clinical trials. MW189 is a selective suppressor of injury- and disease-induced proinflammatory cytokine overproduction associated with destructive glia inflammation/synaptic dysfunction cycles and their long-term neurotoxic effects. This proposed Fast-Track SBIR will deliver a phase 2a trial-ready portfolio for future first-in-patient (FIP) epilepsy treatment trials. Specifically, we will: 1. Develop a commercial-scale version of a validated GMP clinical grade drug production approach, produce a multi-Kg drug substance lot, and obtain its release for future patient clinical trials, 2. Obtain preclinical efficacy data for dosing information and the biological rationale required to support future phase 2a proof-of-concept studies in patients with drug-resistant epilepsy, 3. Prepare required documents and submit a phase 2 IND for a future clinical trial in patients with drug-resistant epilepsy. Our milestones and their associated key tasks are organized as SBIR Phase I activities (year 01) and SBIR Phase II activities (years 02-03). The Fast-Track structure will allow us to immediately move to SBIR Phase II activities that flow seamlessly from preparation and technology transfer to essential milestones for a future FIP safety trial including pharmacokinetics and a pharmacodynamic arm. Success will also further de-risk MW189 for future phase 2 trials in Alzheimer?s disease or other age-related disorders that involve dysregulated neuroinflammation as a driver of disease progression.