Daniel Lowenstein - US grants

Status epilepticus is associated with significant long-term morbidity, including intellectual impairment, focal neurologic dysfunction, and recurrent seizures. Part of this morbidity is related to the neuronal injury resulting from prolonged, excessive electrical activity. Although various lines of evidence suggest that excitatory amino acids and their receptors are important mediators of seizure-induced injury, very little is known about the cellular defense against this excitotoxic injury. The central theme of the proposed research plan is to explore the relationship between stress protein (SP) expression and protection from seizure-induced neuronal injury. The overall hypothesis is that heat-shock proteins and other stress proteins are expressed by CNS cells in response to prolonged seizures, and that this expression is part of the cell's protective mechanism against excitotoxic injury. There are two major goals of the proposed research. The first is to characterize the scope, temporal sequence, and cellular location of stress proteins induced by prolonged seizures within the hippocampus. Two well-characterized in vivo models of seizures will be used, and SP expression will be analyzed via one and two-dimensional polyacrylamide gel electrophoresis, immunoblotting, and immunocytochemistry. The second goal is to use cell culture systems to determine whether stress proteins protect CNS cells against toxic factors, such as excitatory amino acids, that may be involved in the pathogenesis of seizure-induced neuronal damage. SP expression (induced by traditional methods and by exposure to excitatory amino acids) will first be characterized in primary cultures of rat neurons and glia. Various approaches will then be used to modulate the activity of SPs, in order to determine whether there is a causal relationship between SP expression and protection of CNS cells. The research training is designed to build upon the applicant's expertise in the clinical aspects of status epilepticus, and his fellowship training in molecular biology. Upon completion of the program, he will have obtained the experience and background necessary to develop an independent research program investigating the pathophysiology of status epilepticus at the molecular level.

Status epilepticus is associated with significant long-term morbidity, including intellectual impairment, focal neurologic dysfunction, and recurrent seizures. Part of this morbidity is related to the neuronal injury resulting from prolonged, excessive electrical activity. Although various lines of evidence suggest that excitatory amino acids and their receptors are important mediators of seizure-induced injury, very little is known about the cellular defense against this excitotoxic injury. The central theme of the proposed research plan is to explore the relationship between stress protein (SP) expression and protection from seizure-induced neuronal injury. The overall hypothesis is that heat-shock proteins and other stress proteins are expressed by CNS cells in response to prolonged seizures, and that this expression is part of the cell's protective mechanism against excitotoxic injury. There are two major goals of the proposed research. The first is to characterize the scope, temporal sequence, and cellular location of stress proteins induced by prolonged seizures within the hippocampus. Two well-characterized in vivo models of seizures will be used, and SP expression will be analyzed via one and two-dimensional polyacrylamide gel electrophoresis, immunoblotting, and immunocytochemistry. The second goal is to use cell culture systems to determine whether stress proteins protect CNS cells against toxic factors, such as excitatory amino acids, that may be involved in the pathogenesis of seizure-induced neuronal damage. SP expression (induced by traditional methods and by exposure to excitatory amino acids) will first be characterized in primary cultures of rat neurons and glia. Various approaches will then be used to modulate the activity of SPs, in order to determine whether there is a causal relationship between SP expression and protection of CNS cells. The research training is designed to build upon the applicant's expertise in the clinical aspects of status epilepticus, and his fellowship training in molecular biology. Upon completion of the program, he will have obtained the experience and background necessary to develop an independent research program investigating the pathophysiology of status epilepticus at the molecular level.

Calcium homeostasis has a major role in the normal physiology of neurons, and perturbations of intracellular calcium underlie a variety of neurologic diseases including epilepsy, stroke, and chronic degenerative diseases. For example, there is strong evidence that injury of hilar neurons, which is a critical feature of temporal lobe epilepsy, is related to calcium-mediated toxicity. Recent work has suggested that one component of the neuronal calcium homeostasis system involves cytoplasmic calcium binding proteins. Calbindin-D(28K) (CaBP) is one of several of these proteins expressed in the mammalian central nervous system (CNS). Although little is known about the actual functions of CaBP, it has been suggested that CaBP acts as a calcium buffer under normal conditions, and CaBP may also play a role in the protection of neurons from the untoward effects of abnormally high concentrations of intracellular calcium. This latter tenet is based primarily on reports describing a relationship between the presence or absence of CaBP in neuronal subpopulations and selective vulnerability to certain forms of acute and chronic CNS diseases. Nonetheless, the normal calcium buffering properties of CaBP in neurons are unknown, and direct proof of the protective function of CaBP remains lacking. Similarly, although there is some indirect evidence that CaBP has other functions beyond the buffering of calcium, these have not yet been identified. The goal of this research plan is to explore the function of CaBP using both in vitro and in vivo systems. For the in vitro studies, we are creating neural cell lines that overexpress CaBP. We plan to study the calcium-buffering properties of these cells directly with fura-2 analysis, and determine whether CaBP expression confers protection from the toxic effects of abnormal increases in intracellular calcium. Next, we will create similar neural cell lines that overexpress mutated forms of CaBP, and study the effects of these mutations on calcium-buffering and calcium- mediated toxicity. Finally, the properties of CaBP will be studied in vivo by generating transgenic mice that express CaBP in specific hippocampal neurons that both l) do not normally express the protein, and 2) are exquisitely vulnerable to various forms of CNS injury such as seizures, ischemia, and trauma. Electrophysiological and anatomical techniques will then be used to study the effects of the CaBP transgene expression on dentate granule cell physiology, and to determine whether hilar neurons expressing CaBP are relatively resistant to injury induced by prolonged stimulation of the perforant path.

Status epilepticus (SE) occurs in 45,000 to 65,000 persons annually in the United States and is a medical emergency that requires prompt, effective intervention. Despite the development of effective anticonvulsant therapies, and the recognition that prolonged SE is associated with poor neurological outcome, there is often a significant delay between the onset of SE and successful termination of seizures with conventional treatment. This is due both to the time required for transport of patients to an emergency care facility and, in a significant number of patients, a lack of response to initial anticonvulsant treatment. In 1990, the San Francisco Emergency Medical Services (EMS) system developed a protocol that allowed paramedics to administer intravenous diazepam for the treatment of SE in the field. A preliminary analysis of this protocol suggests that patients who received pre-hospital diazepam therapy had fewer seizures in the emergency department, SE of shorter duration, and were intubated less often than patients in whom conventional anticonvulsant therapy was initiated after hospital arrival. These data indicate that pre-hospital treatment of SE may simplify subsequent management and have a beneficial impact on the clinical course and outcome of patients. Nonetheless, the potential benefits and complications of this treatment strategy have not been assessed in a rigorous manner. This is of particular concern because the respiratory complications of intravenous diazepam may be severe, and reports from EMS systems elsewhere in the U.S. suggest that pre-hospital benzodiazepine therapy of SE is gaining popularity despite the lack of a well designed clinical trial to assess its value. This grant proposal describes a multicenter, prospective, randomized, double-blind study designed to compare the safety and efficacy of intravenous diazepam or lorazepam versus placebo when administered in the field by paramedics to adult patients in SE. The study takes advantage of the highly-centralized San Francisco EMS system which is based at San Francisco General Hospital (SFGH). Paramedics will verify the diagnosis of SEE, administer study drugs, and assess patients during transport while having continual radio contact with an attending physician and Medical Intensive Care Nurse at SFGH. The assessment of patients will include observations of clinical seizure activity, level of consciousness, vital signs, cardiac rhythms, and oxygenation via pulse oximetry. Paramedics will also hook-up an ambulatory EEG recording unit to each study patient at the time of initial contact, and continuous recordings will be maintained throughout the transit and emergency department phase of the study. These recordings will be subsequently interpreted and used to determine the validity of the SE diagnosis by the EMS system. Patients will receive standardized anticonvulsant treatment at the destination hospital, and will be monitored for seizure recurrence, cardiovascular and respiratory complications, need for subsequent intensive care, and neurological condition at discharge. Univariate and multivariate statistical comparisons will be used to determine whether pre-hospital therapy has a significant impact on the management and outcome of patients during transport and subsequent hospitalization.

Status epilepticus is associated with significant long-term morbidity, including intellectual impairment, focal neurologic dysfunction, and recurrent seizures. Part of this morbidity is related to the neuronal injury resulting from prolonged, excessive electrical activity. Although various lines of evidence suggest that excitatory amino acids and their receptors are important mediators of seizure-induced injury, very little is known about the cellular defense against this excitotoxic injury. The central theme of the proposed research plan is to explore the relationship between stress protein (SP) expression and protection from seizure-induced neuronal injury. The overall hypothesis is that heat-shock proteins and other stress proteins are expressed by CNS cells in response to prolonged seizures, and that this expression is part of the cell's protective mechanism against excitotoxic injury. There are two major goals of the proposed research. The first is to characterize the scope, temporal sequence, and cellular location of stress proteins induced by prolonged seizures within the hippocampus. Two well-characterized in vivo models of seizures will be used, and SP expression will be analyzed via one and two-dimensional polyacrylamide gel electrophoresis, immunoblotting, and immunocytochemistry. The second goal is to use cell culture systems to determine whether stress proteins protect CNS cells against toxic factors, such as excitatory amino acids, that may be involved in the pathogenesis of seizure-induced neuronal damage. SP expression (induced by traditional methods and by exposure to excitatory amino acids) will first be characterized in primary cultures of rat neurons and glia. Various approaches will then be used to modulate the activity of SPs, in order to determine whether there is a causal relationship between SP expression and protection of CNS cells. The research training is designed to build upon the applicant's expertise in the clinical aspects of status epilepticus, and his fellowship training in molecular biology. Upon completion of the program, he will have obtained the experience and background necessary to develop an independent research program investigating the pathophysiology of status epilepticus at the molecular level.

mossy fiber; neuronal guidance; neurotrophic factors; dentate gyrus; partial seizure; temporal lobe /cortex disorder; granule cell; nucleic acid sequence; nucleic acid probes; complementary DNA; developmental neurobiology; gene expression; pilocarpine; pyramidal cells; hippocampus; inhibitor /antagonist; nervous system regeneration; neuropharmacology; northern blottings; laboratory rat; western blottings; molecular cloning; genetic library; in situ hybridization; mixed tissue /cell culture;

Status epilepticus (SE) is a medical emergency that requires prompt, effective medical intervention. In the United States, SE occurs in over 100,000 persons annually. Morbidity and mortality after SE are affected by the condition precipitating SE, systemic stress from repeated generalized convulsions and central nervous system injury from continuous electrical seizure activity within the brain. Despite the development of effective anticonvulsant therapies, SE continues to be a major cause of death and neurologic disability. The goals of this study are to: (1) evaluate the safety and efficacy of intravenous benzodiazepine agents (diazepam and lorazepam) when given as pre-hospital therapy to patients in status epilepticus; (2) to establish whether pre-hospital therapy of status epilepticus alters the in-hospital management of patients and ultimately has an effect on patient outcome; (3) to determine the effectiveness of the EMS system in making the diagnosis of SE in the field; and (4) to more completely characterize the out-of-hospital seizure population. The primary reason for this competing continuation application is to request funding for an additional year of enrollment in order to achieve our original target goal of 210 unique enrollments.

DESCRIPTION (provided by applicant): Epilepsy is one of the most common neurological disorders and has enormous impact, both medical and social, for the individual as well as for the family. Treatments developed for epilepsy have largely been empirical rather than derived from knowledge of basic mechanisms, because the mechanisms underlying seizure occurrence and epileptogenesis are poorly understood. The Epilepsy Phenome/Genome Project (EPGP) is a large-scale, national, multi-institutional, collaborative research project aimed at advancing our understanding of the genetic basis of the most common forms of idiopathic and cryptogenic epilepsies and a subset of symptomatic epilepsy;i.e. epilepsies that are probably related to genetic predispositions or developmental anomalies rather than endogenous, acquired factors such as CNS infection, head trauma or stroke. The overall strategy of EPGP is to collect detailed, high quality phenotypic information on 3,750 epilepsy patients and 3,000 controls, and to use state-of-the-art genomic and computational methods to identify the contribution of genetic variation to: 1) the epilepsy phenotype, 2) developmental anomalies of the brain, and 3) the varied therapeutic response of patients treated with AEDs. The EPGP Consortium was formed 3 years ago and is comprised of 15 U.S. academic institutions and organized into administrative and scientific cores. Through funding from a planning grant, the Consortium has already planned enrollment protocols, data collection methods, analytical approaches, and much of the infrastructure necessary for carrying out the proposed studies. The application of powerful high-throughput methodologies will permit us to efficiently perform the large- scale genotyping and other analyses described in this application. These studies will allow us to address critical unresolved questions concerning the underlying genomic mechanisms behind the most common forms of epilepsy, which are poorly understood, and to advance our understanding of the genetics of variable drug response. Timely data-sharing and the establishment of patient cell lines through the NINDS Human Genetics Repository will greatly facilitate the work of other epilepsy investigators throughout the country. Importantly, EPGP directly matches one of the high-priority, strategic objectives of NINDS as specified in Benchmark B2 of the Benchmarks for Epilepsy Research: "Organize a national group of scientists to work together in search of genes that might contribute to epilepsy by doing a large screening project that links people with epilepsy to particular gene patterns."

DESCRIPTION (provided by applicant): The primary goal of the Epi4K Center Without Walls is to increase understanding of the genetic basis of human epilepsy in order to improve the well-being of patients and family members living with these disorders. This improvement will come in the form of better diagnostics, treatments and cures. To accomplish this goal, Epi4K aims to analyze the genomes of a large number of well-phenotyped epilepsy patients and families collected by investigators from several major research groups. The specific goals of this project (1 of 7 - Administrative Core) are to provide overall leadership to the Center, specifically regarding the Center's charter, universal protocols, plans for data sharing, internal and external communications, and tracking of scientific progress of all cores and projects within the Center.

DESCRIPTION (provided by applicant): The primary goal of the Epi4K Center Without Walls is to increase understanding of the genetic basis of human epilepsy in order to improve the well-being of patients and family members living with these disorders. This improvement will come in the form of better diagnostics, treatments and cures. To accomplish this goal, Epi4K aims to analyze the genomes of a large number of well-phenotyped epilepsy patients and families collected by investigators from several major research groups. A critical aspect of this enterprise, and the main goal of the Epi4K Phenotyping and Clinical Informatics (PCI) Core described here, is to assemble, organize and validate the phenotypic information on all patients proposed for genomic analysis, and to insure that patients'DNA samples are available to the Epi4K Sequencing, Biostatistics and Bioinformatics (SBB) Core when needed. The availability of well-documented, high quality phenotype data is obviously crucial to the success of all four proposed projects in Epi4K, since the detection of meaningful phenotype:genotype associations will depend highly on phenotype validity. However, achieving this goal in Epi4K will require substantial effort for two main reasons. First, the diagnosis and classification of the epilepsies, including clinical characteristics such as seizure type, seizure semiology, therapeutic response, and additional features such as intellectual and neurological deficits, rests primarily on clinica observations, which are prone to subjectivity and often poorly described or interpreted. Second, Epi4K will assemble at least seven different cohorts of patients collected using varied phenotyping methodologies. To address these challenges, we will capitalize on the substantial experience of the PCI Core investigators, all of whom have devoted considerable portions of their careers to developing methods for accurately phenotyping epilepsy. In addition, through the work of the Epilepsy Phenome/Genome Project (EPGP), we have already created data review systems and a highly efficient informatics infrastructure that can be adapted to the needs of Epi4K. With these resources in hand, the PCI Core seeks to accomplish the following specific aims: 1) to establish standards for documentation of epilepsy phenotypes that can be used reliably across different sites for all subjects undergoing genetic analyses in Epi4K;2) to design and implement an informatics infrastructure for an Epi4K Phenotype Data Repository and DNA Sample Tracking System;and 3) to validate the phenotype data associated with every DNA sample submitted for genome analyses. PUBLIC HEALTH RELEVANCE: Epilepsy is one of the most common neurological disorders in humans, affecting up to 3% of the population. Although there is a strong genetic component for epilepsy, there are still only a few genes known. The Epi4K project will identify new genes and genetic pathways in epilepsy and will directly benefit individuals with epilepsy and their families through improved diagnostic, prognostic and recurrence risk information. Epi4K will also advance our understanding of the biological basis of epilepsy, which is essential for the development of new and effective treatments, as well as cures. Disclaimer: Please note that the following critiques were prepared by the reviewers prior to the Study Section meeting and are provided in an essentially unedited form. While there is opportunity for the reviewers to update or revise their written evaluation, based upon the group's discussion, there is no guarantee that individual critiques have been updated subsequent to the discussion at the meeting. Therefore, the critiques may not fully reflect the final opinions of th individual reviewers at the close of group discussion or the final majority opinion of the group. Thus the Resume and Summary of Discussion is the final word on what the reviewers actually considered critical at the meeting.

PROJECT SUMMARY Clinical genetic data suggests that specific categories of epilepsy have genetic contributors, and there may be some overlap between categories. The Epi25 Collaborative was formed among more than 40 cohorts from around the world to sequence as many as 25,000 genomes or exomes. As of 2017, the collaborative has sequenced more than 13,000 exomes and clinical data has been collected for more than 8,000 cases. This project will complete the collection and review of the clinical data for each sample in the Epi25 collection to facilitate the translation of genomic and clinical discoveries into improved care for patients. The clinical and genomic data from Epi25 will be a global resource, shared with the research community for years to come. Epi25's governance structure, membership, and other information are available online at www.epi-25.org. In this project, clinical data is entered by contributors into Red Cap forms or uploaded directly into the Epi25 database. The clinical data is then checked by a computer algorithm that looks for key eligibility criteria for each participant. Errors and missing data are sent to the Phenotyping Coordinator to review and resolve, with the help of the contributing site.