Overview
Our overall goal is to unravel mechanisms of brain disease, with emphases on inherited epilepsy and selected aspects of animal development. We combine a variety of conventional biological approaches, including whole-animal behavior, physiology and cell culture, with more modern genetic approaches such as gene discovery, gene expression, and analysis of transgenic animals. We have discovered several new mouse models for epilepsy, including two called fitful and frequent flyer that represent single-gene models associated with a complex form of common epilepsy on an inbred strain background. We are also continuing studies of a new mouse model for absence epilepsy involving C3H substrains of mice, and have begun to dissect the genetics and the underlying pathophysiology.
Research details
Molecular Genetics of Seizure Disorders
Overview: Genetics of epilepsy in mouse models
Our goal is to unravel molecular mechanisms of neurological disease, with emphases on inherited epilepsy and selected aspects of neurodevelopment.
Seizure threshold tests
Electroconvulsive threshold (ECT) is the application of electrical current to assess seizure threshold in vivo. It is a fast, robust, and relatively uninvasive procedure. ECT is also flexible and is designed for experimental intervention; it is the primary means of preclinical evaluation of antiepileptic drugs (AED). Given the large fraction of human epileptics who are refractory to AED therapy, there is a need to develop mouse strains that model AED response as well as altered seizure threshold. By exploiting the simple principles of a response curve, which is the basis for determination of a strain response, we have found that it is possible to screen for both low- and high-threshold seizure phenotypes (i.e., susceptibility or resistance) by setting the current (or the amount of AED) to detect mutants with outlying responses.
New seizure threshold mutants
In 2001, we surveyed ECT responses for various mouse strains, some of which are used in mutation screens and others that make important mating partners for genetic mapping. Pilot screens yielded new mutations, Szt1, Szt2 and Szt3(seizure threshold 1, 2 and 3), each associated with a low threshold (susceptibility) to forebrain clonic seizures. We had found that Szt1 was not ENU-induced but rather was a founder spontaneous mutation segregating in the parental C57BL/6J stocks. In addition, while Szt1 confers a dominant seizure phenotype, it confers a recessive perinatal lethal phenotype. After examining candidate genes in the vicinity of Szt1, we found that the mutation was a large genomic deletion involving at least five genes, two of which (Kcnq2, Chrna4) have human counterparts that are known to be mutated in epilepsy. This previously published study (Yang et al., 2003) validated the ECT screen as yielding tools that are directly relevant for human epilepsy. More recently, we have collaborated with the laboratories of Drs. Steve White and Karen Wilcox at the University of Utah to examine in finer detail the pharmacological and physiological properties of the KCNQ2 ion channel in Szt1 mutant mice (Otto et al, 2006).
In the Neuroscience Mutagenesis Facility (NMF) at The Jackson Laboratory, we identified seizure-threshold variants from a large-scale screen. At least seven-Nmf31, Nmf88, Nmf134, Nmf350, Nmf360, Nmf389, Nmf393-are heritable. Two (Nmf88 and Nmf134) represent novel alleles of Kcnq2, bolstering the importance of this homologue of a human epilepsy gene to seizure threshold and epilepsy. In a collaboration with investigators at the University of Michigan, we found that when Nmf134 is combined with a sodium channel variant, the double mutant mice have a very severe seizure syndrome (Kearney et al., 2006). Nmf350, Nmf360 and Nmf389 were genetically mapped to Chromosomes 1, 14 and 5, respectively. Nmf350, with the strongest phenotype and also occasional spontaneous seizures, has now been fine-mapped to a subcentimorgan region. It maps near a quantitative trait locus for induced seizures, Szs1, previously mapped between C57BL/6J and DBA/2J strains and under study by Dr. T. Ferraro and colleagues (U. Penn). We are presently trying to determine whether Nmf350 is an independent locus or corresponds to Szs1, by genetic fine-structure mapping.
In addition, in 2004 we identified the gene for Szt2, one of the three seizure threshold mutants from the pilot screen. The gene, which is expressed widely but is highest in the central nervous system (CNS) and in lymphoid tissue, is predicted to encode a completely novel, large (>3,400 amino acids) protein of unknown function. In the past year we have embarked on some exploratory studies, still underway, to determine if the encoded gene is involved in DNA transcription and whether any epilepsy-relevant downstream targets, such as ion channels, are dysregulated as a result.
Fitful and frequent flyer: New single-gene models of limbic epilepsy
We are also studying the genetic basis of spontaneous seizures in two new models. One mutation, called fitful, arose in C57BL/6J and causes dominant limbic seizures. Recurrent seizure episodes in heterozygotes are first seen at 2 months of age. The recessive phenotype is a severe cerebellar ataxia and juvenile seizures without obvious pathology. We have narrowed the gene search by high resolution mapping to a few candidate genes. The second mutation is a transgenic insertion that causes limbic and tonic-clonic seizures at 3-4 months. Because the tonic-clonic component was a bit wild, we called the allele frequent flyer. The insertion occurred in a gene encoding an RNA binding protein, novelly implicated for epilepsy. Although the seizure phenotype is dominant, homozygotes die just after birth. We think that this protein is involved in metabolizing target RNAs of proteins that are more direct effectors of neuroexcitability, and we are doing expression profiling to identify targets. In addition, heterozygotes have a low threshold to electroconvulsive seizure, a property that makes it feasible to test the efficacy of antiepileptic drugs. We are excited about fitful and frequent flyer because they represent tractable, single-gene models on an inbred strain background, each associated with a complex form of common epilepsy similar to seizure disorders in humans.
Absence epilepsy in a common inbred strain
In 2006, we continued studies of a new mouse model for absence epilepsy, the C3H/He inbred strain, and began to dissect the genetics (Frankel et al., 2005). C3H/He mice exhibit spontaneous spike-wave discharges (SWD) as determined by electroencephalography (EEG), approximately 20-30 episodes per hour. We noticed during the course of genetic analysis that almost half of backcross mice to other inbred strains show SWD much more frequently, over 100 episodes per hour. These SWD were associated with an arrest of normal behavior and were suppressed by the anti-absence seizure drug ethosuximide, suggesting that the mice provide a model of absence epilepsy. Unlike all other known mouse models for absence epilepsy, the brains of C3H/He mice appear to function normally except for SWD, and the mice behave normally, reproduce and live long lives. We previously mapped one genetic locus that is associated with the high SWD incidence of backcross mice, spkw1 (spike wave 1, Chromosome 9), and have also mapped another locus, spkw2 (spike wave 2, Chromosome 8), that interacts with it. Together these two loci account for more than half of the genetic variance. In the past year we have undertaken additional mapping crosses, microarray studies and transgenic/gene targeted mice and have identified the gene for the spkw1 mutation, which is very likely central to the regulation of SWD by the reticular thalamus. This mutation is only found in the HeJ substrain of C3H, suggesting that the other He substrains have SWD due to other mutation(s).
Lab staff
Principal Investigator: Wayne N. Frankel, Ph.D.
Research Scientist: Verity A. Letts, Ph.D.
Associate Research Scientist: Rebecca M. Boumil, Ph.D.
Postdoctoral Associate: Satoko Tokuda, Ph.D.
Research Assistant III: Connie L. Mahaffey, M.S.
Research Assistant II: Barbara J. Beyer, B.A.
Laboratory Technician III: Carolyne Dunbar
Research Administrative Assistant: Mary Robertson, B.A.
Publication listings
Frankel WN, Beyer B, Maxwell CR, Pretel S, Letts VA, Siegel SJ. 2005. Development of a new genetic model for absence epilepsy: Spike-wave seizures in C3H/He and backcross mice. J Neurosci 25:3452-3458.Letts VA, Mahaffey CL, Beyer B, Frankel WN. 2005. A Targeted mutation in Cacng4 exacerbates spike-wave seizures in stargazer (Cacng2) mice. Proc Natl Acad Sci USA 102:2123-2128.
Shin HW, Hayashi M, Christoforidis S, Lacas-Gervais S, Hoepfner S, Wenk MR, Modregger J, Uttenweiler-Joseph S, Wilm M, Nystuen A, Frankel WN, Solimena M, De Camilli P, Zerial M. 2005. An enzymatic cascade of Rab5 effectors regulates phosphoinositide turnover in the endocytic pathway. J Cell Biol 170:607-618.
Wooley CM, Sher RB, Kale A, Frankel WN, Cox GA, Seburn KL. 2005. Gait analysis detects early changes in transgenic SODI(G93A) mice. Muscle Nerve 32:43-50.
Yang Y, Mahaffey CL, Berube N, Nystuen A, Frankel WN. 2005. Functional characterization of fidgetin, an AAA-family protein mutated in fidget mice. Exp Cell Res 304:50-58.
Kearney JA, Yang Y, Beyer B, Bergren SK, Claes L, DeJonghe P, Frankel WN. 2006. Severe epilepsy resulting from genetic interaction between Scn2a and Kcnq2. Hum Mol Genet 15:1043-1048.
Otto JF, Yang Y, Frankel WN, White HS, Wilcox KS. 2006. A spontaneous mutation involving Kcnq2 (Kv7.2) reduces M-current density and spike frequency adaptation in mouse CA1 neurons. J Neurosci 26:2053-2059.
Sher RB, Aoyoma C, Huebsch KA, Ji S, Kerner J, Yang Y, Frankel WN, Hoppel CL, Wood PA, Vance DE, Cox GA. 2006. A rostrocaudal muscular dystrophy caused by a defect in choline kinase beta, the first enzyme in phosphatidylcholine biosynthesis. J Biol Chem 281:4938-4948.
Yang Y, Mahaffey CL, Berube N, Frankel WN. 2006. Interaction between fidgetin and protein kinase A-anchoring protein AKAP95 is critical for palatogenesis in the mouse. J Biol Chem 281:22352-22359.
Zhou X, Jen PH, Seburn KL, Frankel WN, Zheng QY. 2006. Auditory brainstem responses in 10 inbred strains of mice. Brain Res 1091:16-26.
Yang Y, Mahaffey CL, Bérube N, Maddatu TP, Cox GA, Frankel WN. 2007. Complex seizure disorder caused by Brunol4 deficiency in mice. PLoS Genet 3(7):e124.
Bult, CJ, Kibbe WA, Snoddy J, Vitaterna M, Swanson D, Pretel S, Li Y, Hohman MM, Rinchik E, Takahashi JS, Frankel Wn, Goldowitz D. 2004. A genome end-game: understanding gene function in the nervous system. Nat Neurosci 7:484-485.
Frankel WN. 2004. Mouse Genetics (27 part chapter published at Ergito, http://www.ergito.com).
O'Brien TP, Frankel WN. 2004. Moving forward with chemical mutagenesis in the mouse. J Physiol 554:13-21.
Yang Y, Frankel WN. 2004. Genetic approaches to studying mouse models of human seizure disorders. In: Recent Advances in Epilepsy Research, Binder DK, Scharfman HE (eds). Kluwer Academic/Plenum Publishers, New York.
Frankel WN. 2005. Introducing PLoS Genetics. PLoS Genet 1:e21.
