The Jackson Laboratory

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

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. We accomplish this by studying a variety of different kinds of mouse genetic models for epilepsy.

Genetically complex epilepsy from a single gene mutation: Frequent-flyer

Several years ago we began characterizing a new mouse mutant caused by transgenic insertion. Heterozygous mutants have non-lethal, tonic-clonic and generalized seizures that recur from 2-3 months of age; these seizures can be quite wild, hence the mutant was dubbed "frequent-flyer" (allele symbol Ff). Homozygotes suffer from postnatal lethality, depending upon genetic background, experience convulsions at an earlier age (4-5 wks onset), and also have spike-wave discharges (SWD), the hallmark of absence epilepsy. The insertion site was cloned and found to reside 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 is one of six genes that encode a family of RNA binding proteins, previously 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 Brunol4^Ff mutants, we hypothesized that it was critical for regulating the processing of one-or more likely several-RNAs encoding proteins that are more immediately 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 candidates 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 could find 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 heterogeneity of the frequent-flyer seizure phenotype, suggesting that it is indeed some combination of these (and likely additional) molecules that is responsible. Such a prediction is consistent with the complex seizure phenotype observed in Brunol4^Ff 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 recently (Yang, Mahaffey et al, 2007).

Our current studies of Brunol4^Ff mutants are focused on determining the developmental stage critical for the seizure phenotypes (by use of conditional knockout), on the mechanism by which BRUNOL4 regulates target RNA in vivo. We are also characterizing additional downregulated genes that may lead to a better understanding of the seizure disorder in this unique new model for epilepsy.

Fitful: a novel single-gene model of idiopathic limbic epilepsy

We are also 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 2 months of age, but the mice have no other obvious abnormalities, neurological or otherwise. The recessive phenotype, however, is severe cerebellar ataxia, juvenile seizures without obvious pathology, hearing and vision impairment and lethality usually by 3-4 wks of age (depending on strain background). We have identified a novel mutation in the gene encoding dynamin-1. Dynamin-1 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. We are currently examining the molecular and physiological basis for epilepsy in fitful mice.

Absence epilepsy in a common inbred strain, C3H/He

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) show SWD much more frequently, over 100 episodes per hour (we later determined that crosses between HeJ and another C3H substrain, FeJ, which by itself has only 4-5 SWD per hour, showed the same synergy). 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 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 and live long lives. We previously determined that spkw1 (spike-wave 1, Chromosome 9) was the major genetic locus underlying SWD. Very recently, we determined that spkw1 is an IAP insertion mutation 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. 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 Gria4^spkw1 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 feedforward and feedback inhibition of thalamic relay neurons. Preliminary 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. This work was published very recently (Beyer, Deleuze et al., 2008), and we are continuing our analysis of Gria3 and Gria4 knockout mice and compound mutants.

Much to our surprise, only the HeJ substrain of C3H has the Gria4 mutation, even though two other substrains (HeSnJ and HeOuJ) have a similar incidence of SWD. Since few other inbred mouse strains have SWD at all, this suggests that the "core" C3H genome is sensitized in favor of SWD (e.g. even the FeJ substrain has a few SWD per hour). Therefore, it may not be a coincidence that HeOuJ, HeSnJ and HeJ each have appreciable SWD, but for different reasons, if during the 60 years of breeding since these C3H substrains diverged, SWD-causing mutations would manifest more readily on C3H than on other backgrounds. However, preliminary efforts to map SWD susceptibility gene(s) from the HeOuJ substrain suggest that the inheritance is genetically complex; no trait loci could be mapped definitively. Moreover, the synergism seen in crosses with HeJ (producing backcross mice that have over 100 SWD per hour) was not observed in crosses with HeOuJ. Altogether, these data suggest that over the years both SWD susceptibility and resistance mutations have arisen and become fixed in different substrains of C3H; HeJ must have resistance mutations that suppress the Gria4^spkw1 phenotype, and the other He substrains must have susceptibility mutations more subtle than Gria4^spkw1. Our future efforts will be focused on using different genetic and genomic strategies for identifying modifiers of the Gria4^spkw1 phenotype, as well as SWD modifier genes in the other substrains.

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. In the past year we 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 Akt3^Nmf350 mice. Last, we previously identified the gene defective in Szt2 mutant mice as a large, highly conserved molecule with no similarity to other proteins. We will be resuming molecular characterization of Szt2 in the coming year.

Lab staff

Principal Investigator: Wayne N. Frankel, Ph.D.
Research Scientist: Verity A. Letts, Ph.D.
Research Scientist: Rebecca M. Boumil, Ph.D.
Postdoctoral Fellow: Satoko Tokuda, Ph.D., Jacy Wagnon, 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: Heidi Stanton-Drew

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. 2009. Genetics of complex neurological disease: challenges and opportunities for modeling epilepsy in mice and rats. Trends Genet 25:361-367.

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.
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

Search Staff Bibliography Database