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.
Scientific report
Molecular Genetics of Seizure Disorders
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 neurological development. We accomplish this by studying a variety of different kinds of mouse genetic models for epilepsy.
Genetically complex epilepsy from a single gene mutation: Brunol4Frequent-flyer
Several years ago we began characterizing a new mouse mutant caused by transgenic insertion. Heterozygotes have non-lethal, limbic and tonic-clonic seizures that recur from 2-3 months of age; the tonic-clonic phase can be quite wild, hence the mutant allele was dubbed "frequent-flyer" (Ff). Homozygotes suffer from postnatal lethality, and depending upon genetic background they experience convulsions at an earlier age (4-5 weeks onset) and also have spike-wave discharges (SWD), the hallmark of absence epilepsy. The transgene resides in an intron of the Brunol4 gene, greatly decreasing the amount of steady-state mRNA. Subsequently we created a targeted null mutation in Brunol4 and observed similar phenotypes, confirming the phenotype-gene association. Brunol4 encodes one of a family of six RNA binding proteins that are associated with a variety of RNA post-transcriptional events, including splicing, polyadenylation, transport and translation control. In adults, Brunol4 expression is limited to the brain. Other studies on Brunol4 have demonstrated that it has the ability to regulate mRNA splicing in vitro, but in vivo its function is not known. To explain the complex seizure disorder of Brunol4Ff mutants, we hypothesized that it was critical for regulating the processing of one—or more likely several—RNAs encoding proteins that are directly involved in control of neuronal excitability. A microarray was used to identify candidates, resulting in approximately 70 genes whose transcripts were modestly but significantly downregulated in mutant brain. Four interesting genes were noted and subsequently validated, encoding proteins that are either known to cause epilepsy when knocked out in mice (Htr2c, Syn2) or encode synaptic proteins that are good candidates (Nsf, Snca). Interestingly, we found no evidence for regulation of splicing in mutant mice, suggesting that Brunol4 acts at a different level of RNA processing in vivo. Comparison of seizure phenotypes of the various single mutants and analysis of double mutants showed that quantitative downregulation of any one of these genes is not sufficient to cause the severity or variability of the frequent-flyer seizure phenotype, suggesting that it is indeed caused by some combination of these (and likely additional) molecules. This prediction is consistent with the complex seizure phenotype observed in Brunol4Ff mice. It also seems to mimic the majority of familial epilepsy in humans, which is genetically complex but for which only a small fraction of genes are known to date. These studies were published in 2007 (Yang, Mahaffey et al. 2007).
Our recent studies of Brunol4Ff mutants focused on determining whether the seizures that accompany Brunol4 deficiency have their origins in postnatal development in order to narrow-down the number of potential physiological mechanisms. Importantly, using temporal conditional gene targeting we have found that it is sufficient to delete the gene in adults, at least for convulsive seizure phenotypes. This has implications for which downstream molecules and mechanisms should be pursued. By using conditional strategies, we are now determining the spatial requirements (e.g. excitatory vs. inhibitory neurons). We are also presently studying the molecular mechanism by which BRUNOL4 regulates target RNA in vivo. Further characterization of the cellular origin and downstream targets will lead to a better understanding of the complex seizure disorder in this unique new model for epilepsy.
Fitful: a novel single-gene model of idiopathic limbic epilepsy
We are studying the genetic basis of spontaneous seizures in fitful mice, the mutation for which arose in C57BL/6J and causes dominant limbic and generalized seizures. Recurrent seizure episodes in heterozygotes are first seen at two months of age, but the mice have no other obvious abnormalities, neurological or otherwise. The recessive phenotypes, however, include severe cerebellar ataxia, juvenile seizures without obvious pathology, hearing and vision impairment and lethality usually by three to four weeks of age (depending on strain background). We identified a novel mutation in dynamin-1 (Dnm1). Dnm1 is one of three genes encoding dynamins, which belong to a family of large GTPases that function in endocytosis, vesicle scission, membrane recycling, organelle division, cytokinesis and antiviral activity. Although it has been shown by others that dynamin-1 is required for activity-dependent synaptic vesicle endocytosis in neurons, a mechanism that seems like a good candidate for an excitability disorder such as epilepsy, dynamin-1 knockout mice or heterozygotes do not have a seizure disorder of any kind. We are examining the biochemical and physiological basis for epilepsy in fitful mice
Absence epilepsy in C3H/He and related mouse strains
In 2005 we showed that C3H/He mice exhibit spontaneous 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 between the C3H/HeJ (HeJ) substrain and C57BL/6J (B6) or even C3HeB/FeJ show SWD much more frequently, over 100 episodes per hour. SWD were associated with an arrest of normal behavior and were suppressed by the anti-absence seizure drug ethosuximide, characteristic of absence epilepsy. Unlike other mouse models for absence epilepsy, the brains of C3H/He mice appear to function normally except for SWD, and the mice behave normally, reproduce well and live long lives. We previously determined that spkw1 (spike-wave 1, Chromosome 9) was the major genetic locus underlying SWD and that the underlying mutation is an IAP insertion in the last intron of Gria4, which encodes one of the four subunits of the AMPA receptor (AMPAR), responsible for fast excitatory synaptic transmission in the brain (Beyer, Deleuze et al. 2008). The SWD phenotype of Gria4 knockout mice, along with complementation testing, confirmed the phenotype-gene association. In collaboration with John Huguenard (Stanford University School of Medicine), we determined that that Gria4spkw1 allele confers prolonged synaptic activation of the reticular thalamus, a brain structure that is central to the regulation of SWD within thalamocortical circuitry by providing physiologically regulated inhibition of thalamic relay neurons. Genetic and phenotypic analysis of Gria4/Gria3 /double mutant mice suggest that the prolonged synaptic activation may be related to the different kinetic properties known for AMPAR containing these respective subunits, with Gria4 encoding faster desensitization.
Surprisingly, only the HeJ substrain of C3H has the Gria4 mutation, even though two others (HeSnJ and HeOuJ) have the same overall incidence of SWD as C3H/HeJ! However, our initial efforts to map SWD susceptibility gene(s) from the HeOuJ substrain showed that the inheritance is genetically complex; no trait loci could be mapped definitively (Tokuda et al. 2008). Moreover, the high degree of SWD seen in crosses with HeJ (e.g. backcross with over 100 SWD per hour) was not observed in crosses with HeOuJ. Altogether, these data suggest that all C3H strains have a genetic predisposition to SWD, and that over the years various SWD susceptibility and resistance mutations arose and became fixed in different substrains. HeJ acquired both Gria4spkw1 and also further resistance mutations that suppress the Gria4spkw1 phenotype, and the other He substrains have susceptibility mutations with effects more subtle than those of Gria4spkw1. Our current efforts are focused on using different genetic and genomic strategies for identifying the different susceptibility and resistance genes in C3H substrains.
Absence epilepsy in Nav1.6 (Scn8a) sodium channel mutants
Several years ago we adopted from the JAX Reproductive Mutagenesis program (http://reproductivegenomics.jax.org) a new mutation with a recessive severe locomotor impairment, and determined homozygotes have very frequent SWD with concomitant behavioral arrest, characteristic of absence epilepsy. Surprisingly we then found that heterozygotes, i.e. mice without obvious locomotor phenotype, also have frequent SWD. In collaboration with Miriam Meisler (University of Michigan), we determined that the phenotypes were due to a point mutation in the Scn8a gene, encoding an amino acid substitution in the pore loop of the alpha subunit of the voltage gated sodium channel, Nav1.6. Unlike the other major Scn genes, Scn8a was not previously implicated in seizure disorders, perhaps because it is associated with absence epilepsy, which requires EEG for clinical diagnosis. We then collaborated with Andrew Escayg (Emory University) and determined that two classical Scn8a alleles, one a null mutation and the other a loss of function, also showed SWD as heterozygotes. Interestingly, and consistent with the results described earlier, we also determined that the SWD were much more frequent on the C3HeB/FeJ strain background than on C57BL/6J. Together with Drs. Escayg and Meisler, we published the study recently (Papale, Beyer et al. 2009). Because of its dominant, idiopathic absence seizure phenotype, SCN8A is a very attractive candidate for human childhood absence epilepsy, which is a complex genetic disorder for which only one gene has been identified to date. Interestingly, in the preliminary release of the Baylor Ion Channel resequencing project (http://www.hgsc.bcm.tmc.edu/ionchannel-snpList.xsp), several potential nonconserved coding SNPs were identified in SCN8A in human DNAs, some of which were likely to be inherited from patients with idiopathic generalized epilepsy. It will be important to learn whether SCN8A pathogenic variants can be found in future resequencing efforts of epilepsy patients.
New seizure threshold mutants
Altered seizure threshold may contribute to genetically complex familial epilepsy. Several years ago we screened for new mouse mutations that conferred altered seizure threshold. 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. 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—were heritable. Two (nmf88 and Nmf134) represented novel alleles of Kcnq2, bolstering the importance of this homologue of the human epilepsy gene to seizure threshold and epilepsy. More recently, Nmf350 and Nmf389 were genetically mapped to Chromosomes 1 and 5. These mutations not only have a low seizure threshold but also have sporadic convulsive seizures. We subsequently found a missense mutation in Nmf350 in the gene encoding the proto-oncogene Akt3. Since the neurological phenotypes we observe are quite different than those previously shown for Akt3 knockout mice, we are currently pursuing mechanisms that underlay the seizure disorder in Akt3Nmf350 mice. Last, we identified the gene defective in Szt2 mutant mice as a large, highly conserved molecule with no similarity to other proteins (Frankel et al. 2009.
Lab staff
Principal Investigator: Wayne N. Frankel, Ph.D.
Research Scientist: Verity A. Letts, Ph.D.
Research Scientist: Rebecca M. Boumil, Ph.D.
Associate Research Scientist: Wenzhi Sun, Ph.D.
Postdoctoral Fellow: Jacy Wagnon, Ph.D.
Research Assistant III: Connie L. Mahaffey, M.S.
Research Assistant II: Barbara J. Beyer, B.A.
Research Assistant I: Tracy McGarr, B.S.
Research Assistant I: Alexandra Buckley, B.S.
Graduate Student: Christian Richard, M.S.
Research Administrative Assistant: Heidi Stanton-Drew, 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.
Beyer B, Deleuze C, Letts VA, Mahaffey CL, Boumil RM, Lew TA, Huguenard JR, Frankel WN. 2008. Absence seizures in C3H/HeJ and knockout mice caused by mutation of the AMPA receptor subunit Gria4. Hum Mol Genet. 17:1738-1749.
Howell VM, de Haan G, Bergren S, Jones JM, Culiat CT, Michaud EJ, Frankel WN, Meisler MH. 2008. A Targeted Deleterious Allele of the Splicing Factor SCNM1 in the Mouse. Genetics 180:1419-1427.
Miki, T. Zwingman, TA, Wakamori, M, Lutz, CM, Cook, SA, Hosford, DA, Herrup, K, Fletcher, CF, Mori, Y, Frankel, WN, Letts, VA. 2008. Two novel alleles of tottering with distinct Ca(v)2.1 calcium channel neuropathologies. Neuroscience 155:31-44.
Frankel WN, Yang Y, Mahaffey CL, Beyer BJ, O'Brien TP. 2009. Szt2, a novel gene for seizure threshold in mice. Genes Brain Behav 8:568-576.
Papale LA, Beyer B, Jones JM, Sharkey LM, Tufik S, Epstein M, Letts VA, Meisler MH, Frankel WN, Escayg A. 2009. Heterozyous mutations of the voltage-gated sodium channel SCN8A are associated with spike-wave discharges and absence epilepsy in mice. Hum Mol Genet 18:1633-1641.
Tokuda S, Beyer BJ, Frankel WN. 2009. Genetic complexity of absence seizures in C3H mice. Genes Brain Behav 8:283-289.
Boumil RM, Letts VA, Roberts MC, Lenz C, Mahaffey CL, Zhang ZW, Moser T, Frankel WN. 2010. A missense mutation in a highly conserved alternate exon of Dynamin-1 causes epilepsy in fitful mice. PLoS Genetics 6(8):e1001046.
Hawkins NA, Martin MS, Frankel WN, Kearney JA, Escayg A. 2011. Neuronal voltage-gated ion channels are genetic modifiers of generalized epilepsy with febrile seizures plus. Neurobiol Dis 41(3):655-660.
Tokuda S, Mahaffey CL, Monks B, Faulkner CR, Birnbaum MJ, Danzer SC, Frankel WN. 2011. A novel Akt3 mutation associated with enhanced kinase activity and seizure susceptibility in mice. Hum Mol Genet 20(5):988-999.
Wagnon JL, Mahaffey CL, Sun W, Yang Y, Chao HT, Frankel WN. 2011. Etiology of a genetically complex seizure disorder in Celf4 mutant mice. Genes Brain Behav 10(7):765-777.
Book Chapters and Reviews
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.
Frankel WN, Barsh GS. 2008. PLoS Genetics turns three: looking back, looking ahead. PLoS Genet 4(7):e1000135
Frankel WN. 2009. Epilepsy genes that do not encode ion channels in: Philip A. Schwartzkroin, editors Encyclopedia of Basic Epilepsy Research, Vol 1. Oxford: Academic Press; 2009. p. 330-337.
Frankel WN. 2009. Human epilepsy as a complex genetic trait: Lessons from animal models and prospects for the future, in: Philip A. Schwartzkroin, editors Encyclopedia of Basic Epilepsy Research, Vol [1]. Oxford: Academic Press; 2009. p. 356-363.
Frankel, WN. 2009. Genetics of complex neurological disease: challenges and opportunities for modeling epilepsy in mice and rats. Trends Genet 25:361-367.
