Andrew P. Escayg - US grants

Epilepsy is a common neurological disorder that has a significant impact on the quality of life and imposes a tremendous burden on patients and the healthcare system. Many antiepileptic drugs produce undesirable side effects and a strong need exists for the development of medications that specifically target the molecular defects that lead to seizure generation. Our long-term goal is to facilitate the development of more effective epilepsy treatments through a better understanding of the genetics of epilepsy and the processes that lead to seizure generation. We previously demonstrated that missense mutations in the voltage-gated sodium channel gene, SCN1A, are responsible for Generalized Epilepsy with Febrile Seizures Plus type 2 (GEFSP2). GEFSP2 is an inherited autosomal dominant form of epilepsy that is often characterized by febrile seizures in childhood followed by the development of adult epilepsy. Loss-of-function SCN1A mutations are responsible for Severe Myoclonic Epilepsy of Infancy (SMEI), a debilitating syndrome that includes seizures, ataxia and mental retardation. Affected individuals with the same SCN1A mutation display different types of epilepsy, disease severity and response to medication. We hypothesize that this clinical variability is due to additional genetic factors (modifier genes) that modulate seizure susceptibility. To test this hypothesis and to gain insight into the role of sodium channel dysfunction in epilepsy, we will generate an allelic series of BAC transgenic mice with three different human GEFSP2 mutations. The BAC transgene will also be engineered to enable us to study the biophysical properties of transgene-derived Scn1a channels in neurons without interference from endogenous sodium channels and to restrict the expression of the transgene to discrete brain regions. The mice will be evaluated for spontaneous seizures, seizure susceptibility and altered brain morphology. We will also use the GEFSP2 mice to map genetic modifiers of seizure susceptibility. This study will provide a better understanding of the genetic and molecular factors that underlie seizure generation and aid in the identification of novel targets for therapeutic intervention. The GEFSP2 mice will be a valuable resource and will be made available to the scientific community.

DESCRIPTION (provided by applicant): Epilepsy is a common disorder of aberrant neuronal excitability that has long-term consequences for health and intellectual and social development. Many antiepileptic medications have undesirable side effects and only target the symptoms of epilepsy leading to ineffective seizure control. Our long-term goal is to facilitate the development of more effective epilepsy treatments through a better understanding of the factors that affect neuronal excitability. Mutations in the voltage-gated sodium channel gene SCN1A, a critical regulator of neuronal excitability, have been identified in two forms of dominant idiopathic generalized epilepsy: Generalized Epilepsy with Febrile Seizures Plus (GEFSP2) and Severe Myoclonic Epilepsy of Infancy (SMEI). GEFSP2 is characterized by febrile (fever induced) seizures that persist beyond the age of six and the development of adult epilepsy. SMEI is a severe, debilitating childhood epilepsy characterized by febrile and afebrile seizures, mental retardation and ataxia. Many loss-of-function SCN1A mutations have been identified in SMEI patients, suggesting an important relationship between SCN1A expression levels and neuronal excitability. We hypothesize that sequence variation in critical SCN1A regulatory elements can also lead to altered expression and represent an important, but as yet unexplored, component of severe childhood epilepsies. We will test this hypothesis by functional analysis of SCN1A promoter variants identified in patients and in unaffected controls. By multi-species sequence analysis, we have identified 9 evolutionarily conserved non-coding sequences (> 143 bp in length) in the SCN1A gene. We hypothesize that these represent additional regulatory elements. The biological functions of these regions will be examined using a combination of in vitro assays and by targeted deletion in the mouse. To further investigate the relationship between SCN1A expression and seizure susceptibility, we will develop a series of mouse lines with 10-80% of endogenous Scnla expression levels. These expression levels will be generated by crossing well-characterized Scnla BAG transgenic lines to available heterozygous Scnla knock-out mice. This study will provide new, clinically relevant insights into the regulation of SCN1A and the mechanisms that determine neuronal excitability.

DESCRIPTION (provided by applicant): Several epilepsy syndromes, including severe myoclonic epilepsy in infancy (SMEI) and generalized epilepsy with febrile seizures plus (GEFS+), are caused by mutations in the voltage-gated sodium channels. Mutations in the sodium channel SCN1A are a major cause of GEFS+ and SMEI, even though three other sodium channels (SCN2A, SCN3A, and SCN8A) are expressed in the central nervous system (CNS). Mice with mutations in Scn1a and Scn2a exhibit lower seizure thresholds and spontaneous seizures. In marked contrast, we have shown that mutations in the mouse Scn8a gene lead to elevated seizure thresholds. Furthermore, altering Scn8a activity can restore normal seizure thresholds and life spans in mice with Scn1a mutations that serve as models of GEFS+ and SMEI. The goal of this application is to test the hypothesis that mutations in Scn8a can protect against seizures and to investigate the mechanism underlying such protection. This can be achieved in two specific aims. In the first aim we will determine the mechanism by which decreased Scn8a expression leads to higher seizure thresholds and protection against seizure induction. The first objective will establish whether the increase in seizure thresholds is due either to a direct effect of decreased Scn8a expression on neuronal excitability or an indirect effect of a compensatory increase in the expression of any of the other three CNS sodium channels, or possibly both. We will then determine whether reduced Scn8a expression leads to altered network excitability in hippocampal and cortical brain slices using electrophysiological recordings. The purpose of the second aim is to identify the neuronal cell types responsible for the elevation in seizure thresholds of Scn8a mutant mice. This will be accomplished by selectively deleting Scn8a from either pyramidal cells or interneurons in the cortex and hippocampus, and then evaluating the mice for elevated seizure thresholds and altered network excitability. Finally, we will use molecular genetic and electrophysiological approaches to investigate the mechanism by which altered Scn8a activity leads to the dramatic improvements seen in the seizure phenotype of an SMEI mouse model. These studies will provide important and clinically relevant insight into the mechanism by which altered Scn8a function leads to elevated seizure thresholds, enabling the pursuit of much-needed translational studies into the development of novel treatments for epilepsy. PUBLIC HEALTH RELEVANCE: We have observed that mice with reduced activity of the sodium channel gene Scn8a are more seizure resistant. We will investigate the mechanism that underlies this observation. This study will provide important, clinically relevant information on the contribution of Scn8a to seizure resistance and will lay the foundation for further research on the feasibility of reducing the activity of the human SCN8A gene as a treatment for human epilepsy.

DESCRIPTION (provided by applicant): The voltage-gated sodium channels SCN1A, SCN2A, SCN3A, and SCN8A are key regulators of neuronal excitability in the central nervous system (CNS). Mutations in SCN1A, SCN2A, and SCN3A are associated with several epilepsy subtypes, including generalized epilepsy with febrile seizures plus (GEFS+) and severe myoclonic epilepsy of infancy (SMEI), a debilitating childhood disorder characterized by refractory seizures, mental retardation, and ataxia. In striking contrast, we have observed elevated seizure thresholds in mice with mutations in Scn8a. Furthermore, we were able to restore normal seizure thresholds and lifespans in Scn1a mutants that model GEFS+ and SMEI by genetically altering the activity of Scn8a. Based on these observations, we hypothesize that neuronal excitability can be modulated by selectively reducing the expression level of Scn8a, thereby providing a potentially new approach to the treatment of epilepsy. Since current anti- epilepsy drugs (AEDs) cannot selectively target SCN8A, we will evaluate the possibility of altering neuronal excitability by selectively reducing the expression level of Scn8a using an in vivo shRNA interference strategy. This will be accomplished in two specific aims. In the first aim we will generate an adeno-associated virus (AAV) short-hairpin RNA (shRNA) expression vector against Scn8a (AAV- sh8a). The delivery of this reagent into the mouse hippocampus will be optimized to achieve maximum knockdown of Scn8a expression. In the second aim we will evaluate the ability of AAV-sh8a to ameliorate the seizure phenotype in a mouse model of SMEI. If successful, this proof-of-principle R21 proposal would open up an important new direction for the treatment of refractory epilepsy subtypes. PUBLIC HEALTH RELEVANCE: Despite advances in anti-epilepsy drug development, 20-40% of epilepsy patients still do not achieve adequate seizure control or do not respond to treatment at all. In this proof-of-principle study, we will develop and test a potential new strategy for the treatment of severe epilepsy. If successful, this study may offer new hope for the treatment of epilepsy subtypes that do not respond to current medications.

DESCRIPTION (provided by applicant): The idiopathic generalized epilepsies (IGEs) encompass several syndromes that are characterized by age- related, recurrent, and unprovoked generalized seizures in the absence of detectable brain lesions or metabolic abnormalities. The most common IGE subtypes are childhood absence epilepsy (CAE), juvenile absence epilepsy (JAE), juvenile myoclonic epilepsy (JME), and epilepsy with generalized tonic-clonic seizures (EGTCS). The etiology of IGE is genetically determined, and approximately 18 IGE genes have been identified to date. However, these genes are responsible for rare, Mendelian forms of dominant epilepsy and most of the genetic contributions to the IGEs remain unidentified, presenting a bottleneck to the development of more effective treatments. Copy number variants (CNVs) have recently emerged as an important source of both benign and pathogenic genetic variation. Within the last year, at least four CNVs have been identified in patients with common IGE subtypes. In fact, microdeletions on human chromosome 15q11.2 and 15q13.3 are estimated to account for 1% of IGE, making these the most common causes of IGE identified to date. Interestingly, these deletions have also been identified in autism spectrum disorders, schizophrenia and intellectual disability, suggesting a shared, but yet unidentified mechanism. The goal of this R03 proposal is to generate and characterize mouse models of the human 15q11.2 and 15q13.3 deletions. Homologous recombination in mouse embryonic stem cells (ES) will be used to introduce loxP sites flanking the syntenic intervals on mouse chromosome 7. Cre recombinase mediated deletion of the target intervals will recapitulate the human deletion event. In addition to providing the opportunity to study the mechanisms of these important epilepsy susceptibility loci, the mutant mice will also facilitate a better understanding of genetically complex, clinically challenging disorders such as autism and schizophrenia. We will characterize the seizure phenotype of the mice by evaluating seizure thresholds and spontaneous seizure frequency. These mice will provide a unique resource for detailed phenotypic analysis and mechanistic studies. PUBLIC HEALTH RELEVANCE: Genetic mutations are known to play an important role in the etiology of the idiopathic generalized epilepsies (IGEs);however, most currently identified epilepsy genes contribute to rare forms of epilepsy, and the genes responsible for common forms of IGE remain largely unknown. Within the last year, deletions on human chromosome 15q11.2 and 15q13.3 were estimated to be responsible for approximately 1% of cases of common IGE. In order to understand the mechanism by which these deletions lead to seizure generation, we will generate and characterize mouse lines that carry the same deletions that were observed in the patients.

DESCRIPTION (provided by applicant): PROJECT SUMMARY Mutations in the SCN1A voltage-gated sodium channel (VGSC) are responsible for a growing number of disorders, including genetic epilepsy with febrile seizures plus (GEFS+), Dravet syndrome (DS, or severe myoclonic epilepsy of infancy), and familial hemiplegic migraine. To better understand the mechanism by which SCN1A dysfunction leads to epilepsy, we generated transgenic and knock-in mice with the human SCN1A mutation R1648H. These mutants exhibit spontaneous seizures, reduced seizure thresholds, and shortened life spans. Electrophysiological analysis of cortical neurons revealed reduced function in both excitatory and inhibitory neurons, but the biophysical mechanisms were different - negatively shifted voltage dependence of fast inactivation in excitatory neurons versus slowed recovery from inactivation in inhibitory neurons. Our results and data from other groups led to the hypothesis that reduced GABAergic inhibition plays the major role in the pathogenesis of GEFS+ and DS. However, a direct causal link between SCN1A function in interneurons or pyramidal cells and seizure generation has not yet been established. In Aim 1, we will selectively delete Scn1a from either interneurons or pyramidal cells to directly establish the relative contribution of each cell type to seizure generation. In Aim 2, we will test the hypothesis that early-life febrile seizures (FSs), which are a prominent clinical feature of both GEFS+ and DS, have an impact on disease progression. We will also explore the mechanistic basis for the relationship between FSs and disease outcome and possible pharmacological interventions. Of further relevance to our ultimate goal of finding better treatments for epilepsy is our observation that altered function of the Scn8a VGSC can restore normal seizure thresholds and life spans to an Scn1a knockout model of DS. This observation led us to hypothesize that selective targeting of SCN8A may make an effective treatment for DS, which is often refractory to available medications. In Aim 3 we will investigate whether altering Scn8a function can also ameliorate other genetic forms of epilepsy and types of seizures. This study will provide important information on the role of VGSCs in the maintenance of normal neuronal excitability and in the development of epilepsy. These experiments are innovative, clinically relevant, and will stimulate much-needed translational research.

DESCRIPTION (provided by applicant): Mutations in the SCN1A voltage-gated sodium channel (VGSC) are responsible for a growing number of disorders, including Dravet syndrome (DS) and genetic epilepsy with febrile seizures plus (GEFS+). DS is a catastrophic early-life encephalopathy associated with prolonged and recurrent early-life febrile seizures (FSs), treatment-resistant afebrile epilepsy, ataxia, and intellectual disability. GEFS+ is an inherited disorder characterized by FSs that persist beyond the age of six and the development of a wide range of adult epilepsy subtypes. To date, research has focused mainly on the seizure component of these disorders; however, we now recognize that seizure onset is often followed by cognitive stagnation/decline, hyperactivity, and the development of a number of behavioral comorbidities, including autistic and psychotic traits. Although seizure frequency does tend to decline during adulthood, these comorbidities persist and are a major challenge in the clinical management of this patient population. Currently, the cognitive and behavioral outcomes associated with SCN1A dysfunction are poorly characterized, and the factors that influence the severity of these deficits unknown. Mitigating the impact of these comorbidities on quality-of-life outcomes will require broader research efforts to better characterize these cognitive and behavioral deficits, as well as identify factors that influence their severity. Towards this end, te proposed experiments in this R21 application will use Scn1a mouse models developed in our laboratory to address several fundamental gaps in our knowledge. Specifically, we will 1) better define the cognitive and behavioral deficits that result from altered SCN1A function, 2) determine whether prolonged early-life FSs, which are a common clinical feature of SCN1A-derived epilepsies, are likely to contribute to the worsening of cognitive and behavioral outcomes, and 3) determine whether the emergence of cognitive and behavioral abnormalities requires altered SCN1A function during the early period of postnatal brain development or is an invariant (age-independent) outcome of altered SCN1A function. These experiments are both innovative and clinically relevant, and will stimulate much-needed translational research.

? DESCRIPTION (provided by applicant): Epilepsy is a common neurological disorder that affects 50 million people worldwide. Approximately 30% of epileptic patients have treatment resistant (refractory) seizures, thereby presenting a major clinical challenge and burden. The most common form of refractory epilepsy is mesial temporal lobe epilepsy (MTLE), characterized by spontaneous seizures, neuropsychological deficits, and hippocampal sclerosis. At present, surgical resection of the epilepsy focus is the best treatment strategy for this disorder; however, this procedure is only used in a subset of cases. Consequently, there is an urgent need to develop alternative treatments. Mutations in the voltage-gated sodium channels (VGSCs) SCN1A, SCN2A, and SCN3A are associated with several epilepsy subtypes including Dravet syndrome (DS) and genetic epilepsy with febrile seizures plus (GEFS+). Gain of function mutations in the VGSC SCN8A have recently identified in individuals with epileptic encephalopathies. However, our laboratory has demonstrated that mice with Scn8a mutations that reduce channel activity or expression are more resistant to induced seizures when compared to their wild-type littermates. In addition, we were able to dramatically ameliorate seizure severity and restore normal lifespans to Scn1a mutants that model DS and GEFS+ by either co-segregation of an Scn8a mutation or hippocampal knockdown of Scn8a expression. Since the hippocampus is the major site of seizure generation and morphological changes in MTLE, we hypothesize that selective reduction of SCN8A expression in the hippocampus will provide an effective strategy for the treatment of MTLE. We will test this hypothesis with three specific aims. In Aim 1, we will establish the effect on spontaneous seizure frequency and severity of reducing hippocampal Scn8a expression in the widely used intra-hippocampal kainic acid mouse model of MTLE. Reduced Scn8a expression will be achieved by hippocampal injection of an adeno-associated viral vector expressing a short hairpin RNA against Scn8a (AAV-3). Seizure activity will be monitored in AAV-3 treated mice using continuous video/EEG analysis and will be compared to control mice injected with a scrambled construct (AAV-GFP). In Aim 2, we will determine if hippocampal reduction of Scn8a expression could also prevent or ameliorate the changes in behavior and hippocampal morphology and that are observed in this model of MTLE. Finally, in Aim 3, we compare the biophysical properties of hippocampal slices from the AAV-3 and AAV-GFP treated mice in order to directly examine neuronal excitability. We will also test if partial pharmacological block of Nav1.6, using novel compounds, can reduce seizure-like bursting activity in hippocampal slices from the MTLE mouse model, and we will explore the contribution of the different VGSCs to the development of MTLE. This clinically relevant proposal will provide important insight into the feasibility of targeting SCN8A as a treatment for MTLE, and more broadly, for other forms of refractory epilepsy.

ABSTRACT Epilepsy is a family of chronic neurologic disorders characterized by periodic, unpredictable seizures. Current pharmacotherapy for epilepsy relies on ion channel inhibitors, GABAergics, and compounds of unknown mechanism. Following first-line or dual treatment with these drugs, more than 30% of patients continue to experience seizures. A critical barrier in the epilepsy field is the poor brain penetrance of many promising therapeutics. Indeed, there are a number of large or lipophobic compounds that do not readily enter the brain when given systemically, but which are well known in animal studies to show great promise for controlling seizures when they are administered through direct cannulation of the brain. In the proposed studies, we intend to focus on the development of a new technology that will use endogenous biological mechanisms to actively transport these compounds into the brain. We will focus our efforts on neuropeptide Y (NPY) and oxytocin (OT) because 1) these compounds do not readily enter the brain through passive diffusion, 2) there is evidence that these compounds control seizure activity, 3) we have generated exciting preliminary data supporting the use of our technology to transport these compounds into the brain, and 4) these compounds are excellent prototypes with which to conduct proof-of-concept feasibility studies. The long-term goal of this research program is to develop a safe and effective approach to the delivery of neuropeptide treatments for epilepsy and other neurological disorders to the central nervous system. The completion of these studies will provide a solid foundation for further studies to develop even more advanced formulations. These formulations would further refine our approach to increase the brain penetrance, sustain the release, or compartmentalize to the brain novel neuropeptide therapeutics. As such, this proposal will form the foundation of a new research program which we hope will support a new wave of therapeutics for epilepsy.

PROJECT SUMMARY De novo loss-of-function mutations in the voltage-gated sodium channel SCN1A (encoding Nav1.1) are the main cause of Dravet syndrome (DS), a catastrophic early-life encephalopathy associated with prolonged and recurrent febrile seizures (FSs), treatment-resistant afebrile epilepsy, cognitive and behavioral deficits, and a 15-20% mortality rate. SCN1A mutations also lead to genetic epilepsy with febrile seizures plus (GEFS+), an inherited disorder characterized by early-life FSs and the development of a wide range of adult epilepsy subtypes. Current anti-epilepsy drugs often fail to provide adequate protection against the severe seizures and neuropsychiatric comorbidities that occur in patients with SCN1A mutations. Furthermore, almost a third of all epilepsy patients do not achieve adequate seizure control, highlighting the urgent need to develop multimodal treatments that can effectively mitigate the broad spectrum of clinical features associated with refractory epilepsies, while minimizing unwanted side effects. In this exploratory R21 proposal, we will test the hypothesis that Huperzine A (Hup A), a naturally occurring sesquiterpene Lycopodium alkaloid, will be efficacious in the treatment of DS. This hypothesis is based on the biological properties of Hup A, its demonstrated clinical safety, tolerability, ability to improve cognitive function, and our preliminary data. We will use heterozygous Scn1a knockout mice (a model of DS) to evaluate the potential of Hup A to increase seizure thresholds and prevent spontaneous seizure generation (Aim 1) and to ameliorate cognitive and behavioral deficits (Aim 2). This clinically relevant proposal could lay the foundation for the development of a novel therapy to treat SCN1A-derived epilepsies. Furthermore, since SCN1A mutations lead to reduced neuronal inhibition, which is a shared mechanism underlying many common forms of epilepsy, the outcome of this study may have important, broad implications for the treatment of refractory epilepsies.

PROJECT SUMMARY Epilepsy, characterized by recurrent spontaneous seizures, affects over 50 million people worldwide and is one of the most common neurological disorders. Heterozygous loss-of-function mutations in the voltage-gated sodium channel ? subunit gene, SCN1A (encoding the protein Nav1.1), are associated with multiple forms of treatment-resistant epilepsy, including Dravet syndrome (DS), a catastrophic, early-life encephalopathy. As 70- 80% of cases of DS can be attributed to a reduction in functional Nav1.1, strategies that can increase SCN1A transcription from the wild-type allele are predicted to improve patient outcomes. Despite the important role of SCN1A in epilepsy and, more broadly, in the normal regulation of neuronal excitability, little is currently known about the machinery underlying the regulation of SCN1A transcription. Therefore, we are limited in our ability to develop strategies to increase SCN1A transcription. We identified three predicted Functional Genomic Elements (FGEs) within the SCN1A locus that significantly increased reporter activity in a dual luciferase assay: FGE2, FGE17, and FGE23. In Aim 1, we will test the hypothesis that FGE2, FGE17, and FGE23 endogenously act as transcriptional enhancer elements for SCN1A. We will use the Synergistic Activation Mediator (dCas9SAM) system to localize transcriptional activators to these FGEs in neuronal cell culture. Previous studies have shown that localizing transcriptional activators to gene promoters or enhancers can elicit an increase in gene expression, making dCas9SAM a powerful tool for evaluating putative regulatory elements. Additionally, we will perform ATAC-seq on GABAergic interneurons, which preferentially express SCN1A, in an effort to identify additional FGEs that contribute to SCN1A transcriptional regulation. Our preliminary data also show that the minor allele of the epilepsy-associated SNP, rs6732655, located in SCN1A intron 16, significantly decreases reporter activity in a dual luciferase assay, compared to the major allele. In Aim 2, we will test the hypothesis that the rs6732655 minor allele introduces a silencer element, thereby reducing SCN1A expression. We will examine SNP allele-dependent protein-binding in order to identify candidate factors that bind to the minor allele. We will also knock-in the minor allele of this SNP to SH-SY5Y human neuroblastoma cells to evaluate the endogenous impact on SCN1A expression. Our long-term goal is to translate these findings into clinically-relevant strategies for increasing Nav1.1 levels, thereby providing new therapeutic options.

PROJECT SUMMARY Epilepsy is a common neurological disorder that affects 50 million people worldwide. Approximately 30% of epileptic patients have treatment resistant (refractory) seizures, thereby presenting a major clinical challenge and burden. Temporal lobe epilepsy (TLE) is the most common form of refractory human epilepsy, and mesial temporal lobe epilepsy (MTLE) is the most common form of TLE. Approximately 20% of patients with MTLE fail to achieve adequate seizure control. MTLE is characterized by spontaneous seizures, hippocampal sclerosis, and neuropsychological deficits. At present, surgical resection of the epilepsy focus is the best treatment strategy for this disorder; however, this procedure is only used in a subset of cases. Consequently, there is a need to develop alternative treatments that can effectively mitigate the broad spectrum of clinical features associated with MTLE, while minimizing unwanted side effects. Reversible acetylcholinesterase inhibitors (rAChEIs) (e.g., Huperzine A and donepezil) are most widely used in the treatment of dementia and Alzheimer's disease; however, there is increasing evidence that this class of compounds might be therapeutic in the treatment of epilepsy. Recently published data from our group demonstrated that the Huperzine A provides robust and sustained protection against induced seizures in a mouse model of the catastrophic, treatment-resistant encephalopathy Dravet syndrome (DS). In addition, we now provide preliminary data demonstrating 1) that donepezil can also increase resistance to induced seizures in the DS model, and 2) that Hup A dramatically reduces spontaneous seizure development in the intra- hippocampal kainic acid (IH-KA) mouse model of MTLE. Based on these observations, and the ability of rAChEIs to also protect against inflammation and cell death, and promote neurogenesis, we hypothesize that this class of compounds will be particularly efficacious in the treatment of MTLE. We will test this hypothesis by evaluating and comparing the ability of Hup A and donepezil to reduce spontaneous seizure frequency and severity (Aim 1) and ameliorate behavioral abnormalities and neuron loss (Aim 2) in the IH-KA mouse model of MTLE. This study will also have broader implications for the treatment of other forms of refractory epilepsy.

Project Summary Neuronal function of huntingtin-associated protein Huntingtin-associated protein-1 (Hap1) was first identified as a neuronal protein that interacts with the Huntington's disease (HD) protein, huntingtin (Htt). Hap1's binding to Htt is enhanced by expanded polyglutamine repeats in the N-terminal region of Htt. Moreover, unlike Htt, which is ubiquitously expressed, Hap1 is expressed primarily in neuronal cells, suggesting that it is a good candidate for involvement in the selective neurodegeneration in HD. Extensive studies have shown that Hap1 and Htt associate with each other in the intracellular trafficking of various vesicles and proteins. Also, like Htt, Hap1 is essential for early development and neurogenesis. Our recent studies showed that the function of both Hap1 and Htt is cell type and age dependent. We also know that neurogenesis is important for early brain development and the repair of neuronal damage in the adult brain. Although both Htt and Hap1 participate in neurogenesis, and impaired neurogenesis is seen in HD brains, whether Hap1 and Htt work together to regulate neurogenesis and whether mutant Htt affects neurogenesis via its interaction with Hap1 remain unknown. We hypothesize that Hap1 and Htt participate in age- and environmental stress-dependent neurogenesis and that mutant Htt affects this function by its interaction with Hap1. To test this hypothesis, we will use Hap1 KO and Htt KO mice as well as HD140Q knock-in mice to examine the role of the interaction of Hap1 and Htt in postnatal and adult neurogenesis. We will focus on neurogenesis in the hypothalamus and hippocampus, as loss of Hap1 in these two regions is found to cause body weight loss and depression, two well-known phenotypes that also occur in HD patients. We will use CRISPR/Cas9 to selectively deplete Htt expression or to increase the production of N-terminal mutant Htt in HD140Q KI mice to examine whether they have any effects on neurogenesis. Using these approaches, we propose two aims in this application. Aim 1 will investigate whether Hap1 and Htt work together to promote neurogenesis. Aim 2 will explore whether mutant Htt affects neurogenesis via its abnormal interaction with Hap1. The studies seek to provide new insight into the cell type- and age-dependent function of Hap1 and Htt, as well as the selective neuropathology of HD. Findings from these studies will also help us uncover therapeutic strategies for the specific neuropathology and phenotypes in HD. !

PROJECT SUMMARY Temporal lobe epilepsy (TLE) is the most common form of refractory human epilepsy, and mesial temporal lobe epilepsy (MTLE) is the most common form of TLE. MTLE is characterized by focal seizures, neuropsychological deficits, neuroinflammation, and hippocampal alterations that include neuronal loss. MTLE is often refractory to available antiepileptic drugs (AEDs), creating a need for the development of treatments that can more effectively mitigate the broad spectrum of clinical features associated with MTLE, while minimizing unwanted side effects. It is increasingly recognized that dysregulated neuroinflammatory responses can contribute to the pathogenesis of several forms of epilepsy, including MTLE, and is thus an important consideration in the development of new drug targets. Cannabinoid 2 receptors (CB2Rs) are receiving increasing attention for their ability to safely confer anti-inflammatory and neuroprotective properties in models of neurological disorders such as traumatic brain injury and stroke. While much less research has been conducted on the role of CB2Rs in epilepsy, pre-treatment with CB2R agonists has been shown to increase resistance to acutely induced seizures in mice. In addition, we provide Preliminary Data demonstrating that CB2R knockout mice are seizure susceptible, and resistance to induced seizures in wildtype mice is increased following the administration of EC21a, a novel CB2R positive allosteric modulator (PAM). Given that reactive gliosis and increased pro-inflammatory cytokine release in MTLE are important contributors to the development of spontaneous seizures, we hypothesize that that activation of CB2Rs will effectively ameliorate these inflammatory changes thereby reducing seizure generation and co- morbid behavioral abnormalities in MTLE. We will test this hypothesis by evaluating and comparing the ability of two CB2R selective compounds, JWH-133 (a CB2R agonist) and EC21a (a PAM), to reduce inflammation and neuron loss (Aim 1) and to mitigate the development of spontaneous seizures and behavioral abnormalities (Aim 2) in the intrahippocampal-kainic acid (IH-KA) mouse model of MTLE. We will also compare the effect of early and delayed treatment initiation. If successful, this proposal will establish the potential of CB2Rs as a novel therapeutic target for the treatment of MTLE and potentially other forms of epilepsy associated with significant neuroinflammation.

PROJECT SUMMARY Dysfunction of voltage-gated sodium channels (VGSCs) is responsible for several forms of catastrophic childhood encephalopathies. Over 1000 loss-of-function mutations in the VGSC SCN1A have been identified during the last two decades and are the main cause of Dravet syndrome (DS), characterized by recurrent early- life febrile seizures (FSs), severe afebrile epilepsy, cognitive and behavioral deficits, and a 15-20% mortality rate. Mutations in the VGSC SCN8A were more recently identified in 2012, and already over 200 gain-of-function SCN8A mutations have been reported in patients with a range of clinical features including catastrophic treatment-resistant childhood epilepsy, autism, intellectual disability and developmental delay. Unfortunately, most anti-epileptic drugs (AEDs) fail to adequately treat the broad range of severe seizures and behavioral phenotypes in patients with SCN1A- and SCN8A-derived epilepsy. Thus, despite recent progress in pharmacological treatments for DS, there remains a need to develop more effective, longer lasting treatments with fewer side effects. Neuropeptides are well known in animal studies to show great promise for controlling seizures and ameliorating behavioral abnormalities; however, they do not readily cross the blood brain barrier and are rapidly metabolized when given systemically. Thus, poor brain penetrance is a critical barrier to the clinical application of these promising therapeutics. To overcome this challenge, we developed and validated an approach based on the encapsulation of neuropeptides in nanoparticles conjugated to rabies virus glycoprotein (RVG). Using this approach, we have found that intranasal delivery of nanoparticle-encapsulated oxytocin (NP- OT) greatly increases brain penetrance and the capacity of OT to confer robust and sustained increases in resistance to seizures in mouse models of SCN1A and SCN8A dysfunction. We have also extended our strategy to encapsulate neuropeptide Y (NP-NPY), and similarly observed a robust improvement in its ability to confer seizure resistance. In the proposed study, we will establish the ability of NP-OT and NP-NPY to ameliorate spontaneous seizures and behavioral abnormalities in Scn1a and Scn8a mouse mutants (Aim 1). While the role of OT in social behavior is well-studied, less is known about the mechanisms by which it modulates seizure susceptibility. Thus, we will also identify the cellular and neural circuit mechanisms that contribute to the ability of OT to increase seizure resistance in the Scn1a and Scn8a mutants (Aim 2). Our long-term goal is to develop safe and effective approaches for the brain delivery of neuropeptides for the treatment of epilepsy and other neurological disorders.

Huntington's disease (HD) is a devastating neurodegenerative disease caused by expansion of a polyglutamine (polyQ) domain in distinct proteins with different functions. In HD, the polyQ domain is located in the N-terminal region of huntingtin (Htt). This N-terminal region is well conserved in a wide range of species, but polyQ expansion can lead to misfolding and subsequent toxicity of N-terminal fragments of Htt. Since a lack of Htt causes embryonic lethality in mice, Htt is also thought to be essential for animal development and survival. Reducing the expression of mutant Htt is widely accepted as an important strategy for treating HD, so considerable efforts have gone into developing siRNA and anti-sense oligonucleotides to suppress the expression of mutant Htt. These approaches have also raised concerns that markedly suppressing Htt expression could lead to side effects by diminishing the normal function of Htt; however, whether Htt can preserve critical functions without the N-terminal domain that contains the polyQ domain remains unknown. Addressing this issue is important if we are to develop a new strategy to treat HD: if the N-terminal polyQ domain is not required for essential Htt functions and can be removed, complete elimination of the N-terminal region of Htt is now possible since the recent development of the genomic editing tool, CRISPR/Cas9. In this competitive renewal application, we will use CRISPR/Cas9 to investigate the toxicity of N-terminal mutant Htt fragments and therapeutic effects by removing the polyQ-containing N-terminal region. In Aim 1, we will use CRISPR/Cas9 to introduce mutations in the mouse Htt gene in embryos from HD 140Q KI mice to generate truncated mutant Htt genes that express different N-terminal Htt fragments and can be transmitted to offspring via the germline. Using the newly established HD KI mice that express different N-terminal mHtt fragments containing the same polyQ repeat (140Q) at the endogenous level, we will examine the relationship between the length of N-terminal mutant Htt fragments and their nuclear accumulation and toxicity in striatal neurons. In Aim 2, we will use CRISPR/Cas9 to remove the N-terminal polyQ domain as a therapeutic strategy. We will explore whether removing the N-terminal polyQ domain in Htt can eliminate neuropathology without affecting neuronal survival and function in adult mice. These studies will use HD knock-in mice in which mutant Htt is expressed at the same endogenous level as in HD patients. We hope these studies will not only provide new insight into the pathogenesis of N-terminal mutant Htt fragments, but also allow us to develop a novel therapeutic strategy to treat Huntington's disease and other polyQ diseases.