Alan L. Goldin, M.D., Ph.D. - US grants

The voltage-gated sodium channel is an integral component of impulse conduction by neuronal cells, transmitting the initial inward current during an action potential. The channel consists of one large subunit termed alpha that is associated in some tissues with one or two small subunits termed beta. There are multiple different isoforms of the alpha subunit, including at least 4 distinct forms in the central nervous system (CNS). The physiological significance of the different forms is not known. The overall objective of this research is to determine the importance of the different channel isoforms in the CNS. There are two major goals. The first aspect is to define the mechanism and physiological significance in the CNS of sodium channel inactivation. We have previously shown that the cytoplasmic linker between domains III and IV is critical for sodium channel inactivation, possibly forming the nucleus of an inactivating particle. We will examine the mechanism and physiological significance of sodium channel inactivation by testing three hypotheses. First, we will determine if the III-IV linker binds to the III S4-S5 region as an inactivating particle. Second, we will determine if incomplete inactivation of sodium channels in the CNS lead to epilepsy. Third, we will determine if local anesthetics bind to the same region of the channel that serves as the docking site for the inactivating particle. The second major goal of these studies is to determine the significance of the different sodium channel isoforms in the CNS by testing two hypotheses. First, we will determine if two different isoforms are localized in different regions of the axon because of specific amino acid sequences in the cytoplasmic linkers. Second, we will determine if rBl and Na6 channels mediate the transient and maintained currents in cerebellar Purkinje cells, respectively. These studies should enhance our knowledge concerning the normal function of the voltage-gated sodium channel in the CNS, ultimately helping to provide an understanding of the pathological processes that affect channel function in disease and to design pharmaceuticals that interact with channels of specific tissues.

The voltage-sensitive sodium channel is an integral component of impulse conduction by neuronal cells, transmitting the initial inward current during an action potential. The overall goal of Dr. Goldin's study is to determine the roles of specific structural regions of the channel that are involved in gating. There are four putative transmembrane amphipathic helices termed S4 in the sodium channel that have been proposed to function as voltage sensor regions involved in voltage-dependent activation of the channel. Dr. Goldin will test this by constructing mutations at comparable positions in each of the S4 regions, and by determining if each of the charges is contributing to the effective valence of activation. His second objective is to determine if these same voltage sensors are involved in the process of slow sodium channel inactivation. While fast inactivation of sodium channels is thought to be essentially independent of voltage, slow inactivation very likely involves some voltage sensitivity independent of activation. The effects of each of the S4 region mutations in slow inactivation will be tested to determine if the S4 regions are functioning as inactivation voltage gating charge of the channel. Dr. Goldin will accomplish this by using a newly developed voltage clamp to measure gating currents of channels containing mutations in each of the four S4 regions. These studies should enhance our knowledge about the normal function of the voltage-sensitive sodium channel, ultimately helping to understand the pathological processes that affect channel function in disease and to design pharmaceuticals that interact with channels of specific tissues.

DESCRIPTION (provided by applicant) The goal of the UCI Medical Scientist Training Program (MSTP) is to train students as biomedical research scientists who will make advances to improve the diagnosis, understanding and treatment of human diseases. Training is offered in a diverse number of medically related scientific areas. This is accomplished by means of a flexible, independently designed curriculum for the MSTP students. MSTP students can complete their Ph.D. studies in any graduate program at UCI. These include 8 programs in the School of Medicine: Anatomy & Neurobiology, Biological Chemistry, Environmental Toxicology, Epidemiology, Experimental Pathology, Microbiology & Molecular Genetics, Pharmacology, and Physiology & Biophysics; 4 departments in the School of Biological Sciences: Developmental & Cell Biology, Ecology & Evolutionary Biology, Molecular Biology & Biochemistry, and Neurobiology & Behavior; and departments in the Schools of Engineering, Information & Computer Science, Physical Sciences, Social Ecology and the Program in Public Health. Established in 1987, the MSTP has enrolled 143 students and has graduated 67. Typically, 6 new students are enrolled each year. Potential MSTP candidates are admitted based on: 1) academic performance reflected in grades and MCAT scores; 2) substantial research experience and potential; 3) personal characteristics and experiences assessed during personal interviews; 4) evidence of a strong commitment to a career in biomedical research; and 5) factors contributing to educational diversity. Primary facilities for MSTP students include abundant teaching and research space located in 25 buildings located on the UCI campus. The UCI Medical Center and the Long Beach Veterans' Administration Hospital serve as the primary teaching hospitals, and 118 affiliated hospital sites and the Beckman Laser Institute and Medical Clinic provide additional training experiences. Shared core facilities, such as the Molecular Biology Core Facility, Mass Spectrometry Facility, IMAGE Facility for electron microscopy and analysis, the Research Imaging Center, computing facilities, 5 libraries, and animal research facilities including a transgenic mouse facility, provide broad infrastructure.

DESCRIPTION (provided by applicant): The epilepsies are a diverse group of disorders characterized by abnormal electrical activity in the CNS, affecting up to 3% of the population. Of this group, approximately 40% are idiopathic in which the underlying cause is most likely a genetic abnormality. The specific abnormality has been identified in only a small minority of cases, most of which are channelopathies involving defects in ion channel function. Even in those cases, the mechanisms by which the defects result in epilepsy are not understood. One class of channelopathies that cause epilepsy are mutations in voltage-gated sodium channels, which cause a number of different syndromes including Generalized Epilepsy with Febrile Seizures Plus (GEFS+). The goal of this research is to determine how abnormal sodium channel function resulting from mutations causing GEFS+ leads to epilepsy. There are three goals. The first aim is to determine which neuronal populations express the GEFS+ mutant sodium channels and to characterize the mutant channels in those cells. This will be accomplished by identifying the specific interneuron cell types that express the R1648H mutant sodium channels and using electrophysiological techniques to analyze dissociated neurons from mice in which specific populations are tagged with fluorescent proteins. The second aim is to determine the mechanism by which altered sodium channel activity resulting from the R1648H mutation leads to greater seizure susceptibility in GEFS+. This will be accomplished by recording from cortical and hippocampal slices from knock-in mice expressing the mutation. We will determine the effects of the mutation on the firing properties of specific classes of neurons, on seizure-like activity in field recordings, and on intrinsic excitability of the excitatory and inhibitory neurons and the quantitative effects of synaptic input using laser scanning photostimulation. The final aim is to determine if a second GEFS+ mutation (D1866Y) also causes decreased firing of inhibitory neurons in the CNS and to determine the role of the b1 subunit in the phenotype of this mutation. This will be accomplished by recording from dissociated cells and slices from knock-in mice expressing the mutation, and examining the effects of the D1866Y mutation in mice lacking the b1 subunit. These studies should enhance our knowledge concerning the physiological and pathological events leading to one specific form of epilepsy.

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): 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.