Our goal is to unravel molecular mechanisms of neurological disease, with emphases on inherited epilepsy and other neurological disorders that have recurrent seizures as a major feature. We accomplish this by studying a variety of mouse genetic models for epilepsy with genetic, genomic, cell biology and physiology tools.
Genetics of Epilepsy in Mouse Models
Complex neurological disease from a single gene: Celf4 (W. Frankel)
RNA-binding proteins have emerged as causal agents of complex neurological diseases, including epilepsy, autism and intellectual disabilities. CELF4 (a.k.a. BRUNOL4, CUGBP or ELAVL4) is a brain-specific member of six mammalian CELF family proteins that function in mRNA metabolism. In 2007 we reported that hypomorphic and null mutations in the Celf4 locus causes a loss of function by reducing the amount of normal Celf4 transcript and protein, resulting in a complex neurological phenotype (Yang et al. 2007). Adult Celf4 null homozygotes and heterozygotes have a low seizure threshhold and recurrent routine handling-associated convulsive seizures with severity and penetrance dependent on mouse strain background. On strains other than C57BL/6J, homozygotes also experience non-convulsive (absence-like) seizures, showing that Celf4 is involved in different types of seizure circuits. In 2011 (Wagnon et al., 2011), we determined that CELF4 is expressed predominantly in excitatory neurons in the cytoplasm. By studying spatially conditional Celf4 mutants we also found that convulsive seizures require deletion from excitatory neurons. Interestingly, temporal conditional mutations revealed that deleting Celf4 from adults is sufficient for convulsive seizures, but that absence seizures require deletion in the first postnatal week, indicating etiological complexity even within a single gene mutation. Recently, a Danish research group described a human CELF4 mutation that confirmed clinical neurological and behavioral features closely resembling those of Celf4 deficient mice (Halgren et al. 2012).
A key step in understanding CELF4 functions was to identify its target mRNAs. In 2012, in collaboration with Jernej Ule (U. Cambridge), we used individual nucleotide resolution UV-crosslinking and immunoprecipitation (iCLIP) to identify a vast array of mRNAs (>2000) directly bound by CELF4 in adult mouse brain. CELF4 preferentially binds its targets in the 3' UTR at a (U)GU motif similar to that known for other CELFs. We characterized the fate of these "targets" in Celf4 mutants using microarray and RNAseq. Functional annotation clustering shows that CELF4 targets are enriched for many neuronal functions, including both postsynaptic and presynaptic neurotransmission. Whole-cell changes in Celf4 null mutants were modest, but significant shifts in target abundance between cell body and neuropil (CELF4 protein is present in both), supports a role for CELF4 in mRNA regulation within and beyond the cell body, into axons and dendrites. We also found that CELF4 protein most tightly associates with very large RNA granule subcellular fractions, and in the absence of CELF4, many target transcripts shift from monosomes to polysomes. Together these data suggest that CELF4 functions in translational silencing, analogous to its Drosophila ortholog, Bruno. Furthermore, functional annotation clustering also revealed that many CELF4 targets that shifted between cell body and neuropil in mutants associate with regulation of synaptic plasticity, suggesting the possibility that by binding to so many targets with functions in the same pathways, CELF4 coordinates homeostasis or other response mechanisms. These studies will be published in Novmeber 2012 (Wagnon, Briese et al., 2012).
In parallel we assessed the intrinsic properties of CELF4 deficient layer V cortical pyramidal neurons in acute brain slices. Neurons from mutant heterozygotes and homozygotes have a lower action potential (AP) initiation threshold and a larger AP gain compared to wildtype. Celf4 mutant neurons also demonstrate an increase in persistent sodium current (INaP) and a hyperpolarizing shift in the voltage dependence of activation. Interestingly, one of CELF4's target mRNAs encodes sodium channel Nav1.6, the primary instigator of AP at the axon initial segment (AIS) and the main carrier of INaP in excitatory neurons. CELF4 deficiency results in a dramatic elevation in the expression of Nav1.6 protein at the AIS in both null and heterozygous neurons. These results suggest that activation of Nav1.6 plays a direct role in disease by elevating intrinsic neuronal excitability. These results were recently published online (Sun et al., 2012).
Together these two studies imply that seizures and possibly other CELF4 phenotypes arise from a combination of a) hyperexcitation intrinsic to affected neurons, and b) a systemic inability to compensate to maintain a balance between inhibition and excitation. We plan to gain experimental support for this idea and also to examine the broader range of neurobehavioral phenotypes in Celf4 deficient mice, led by the symptoms of CELF4 patients. We have also begun proteomic studies to determine the composition of the high density RNA complexes with which CELF4 is associated.
Genetic modifiers of epilepsy in common mouse strains and substrains (W. Frankel)Previously we described two models for absence epilepsy in mice caused by mutations in known genes--Gria4 (encoding GluR4, an AMPA receptor subunit) and Scn8a (encoding Nav1.6, a voltage-gated sodium channel). In both, seizures are significantly more attenuated on the C57BL/6J (B6J) strain background than on C3H, where the seizures are more frequent and last longer. Attenuation of seizures on the B6J background was described by others for a knockin of a human epilepsy mutation, GABRG2R43Q, encoding a GABA-A receptor subunit (Tan et al. 2008), and we have confirmed that they are much worse in C3H mice (unpublished results). We have now crossed the Celf4 null allele to C3H and observe a similar phenomenon. We are currently exploiting our extensive prior knowledge about mouse strains and seizure susceptibility to determine whether the modifier effects for Gria4, Scn8a, Gabrg2 and Celf4 have a common molecular basis. This would suggest common mechanisms and potentially more attractive targets and would also contribute to understanding how the thalamocortical rhythms that go haywire during absence seizures are regulated. In traditional crosses we have mapped and validated six chromosomal regions that contribute to C3H vs. B6J modifying effects on Gria4, Scn8a and Gabrg2 (unpublished results). At least three are shared between at least two mutants. We have begun to examine genetic recombinants to reduce the interval sizes and to test candidate genes.
We are also looking for suppressors of absence seizures in C3H/HeJ (HeJ) mice, where the primary mutation is a substrain-specific IAP retrotransposon insertion in Gria4. Initial evidence for suppressor mutation(s) within the C3H strain family came from observing a much higher incidence of seizures in intersubstrain Gria4 mutant crosses than in HeJ itself. To identify candidate mutations that differ between these two otherwise very closely related substrains, we are exploiting the fact that IAPs are known to be very active as germline mutagens in C3H strains and the increasing ease of genome and exome sequencing. In collaboration with the Sanger Centre and Wellcome Trust, we characterized the endogenous retrotransposon composition, including IAPs, of 17 mouse strains, including C3H/HeJ (Nellaker et al. 2012). These results and others from our bench led to the discovery of a substrain-specific IAP insertion mutation in a novel, putative multitransmembrane protein encoding gene on distal Chr 8, where we suspected a Gria4 SWD suppressor to reside from earlier work. Congenic strains have confirmed the effect of this chromosomal region on SWD and we are developing a knockout to confirm this, while characterizing protein properties.
Synaptic vesicle recycling defects contribute to epilepsy in fitful mice (R. Boumil)In 2010 we described a novel mutation in the dynamin-1 gene (Dnm1) that causes a complex seizure phenotype (Boumil et al., 2010). In the past year we have begun to elucidate the underlying mechanisms. Dnm1 encodes a large multimeric GTPase necessary for activity-dependent membrane recycling in neurons, including synaptic vesicle endocytosis. Mice heterozygous for the Dnm1 mutation "fitful" (Dnm1Ftfl) experience recurrent seizures, and homozygotes have more debilitating, often lethal seizures in addition to severe ataxia and neurosensory deficits. Dnm1Ftfl is a missense mutation in an exon that defines the Dnm1a isoform, leaving intact an alternatively spliced exon that encodes Dnm1b. We hypothesize that endocytosis is stalled in Dnm1Ftfl mice at a checkpoint that requires proper function of the Dnm1 isoforms to proceed from early invagination to later stage fission events. A delay in synaptic vesicle endocytosis would result in a deficiency of the readily available pool of vesicles for neurotransmission. Indeed, ultrastructure analysis of wildtype and fitful mutant brain sections shows a decrease in the number of synaptic vesicles with an overall increase in vesicle size heterogeneity in mutants (unpublished results). We are also investigating the genetic interactions involved in the endocytic checkpoint, by crossing Dnm1Ftfl with other mutants deficient in proteins known to interact with Dnm1 at different stages of endocytosis, to ask how disruption of endocytosis/fission at different points affects seizure phenotype differentially. Preliminarily, we observe that perturbing SV recycling upstream of dynamin-1 ameliorates the seizure phenotype while disrupting other pathways exacerbates seizures (unpublished results).
Overall, these studies aim to define the involvement of Dnm1 in synaptic vesicle endocytosis. Additionally, they will give insight into the role that genes encoding endocytic proteins play in contributing to epilepsy. Previous studies have examined the function and mechanisms of action of dynamin-1 extensively, but Dnm1Ftfl remains the first identified Dnm1 mutation that leads to epilepsy in mammals and thereby allows us to study the complex genetic pathway of SV recycling while further supporting disruption of this pathway as a potential mechanism for human epilepsy.
Principal Investigator: Wayne N. Frankel, Ph.D.
Full-time laboratory staff:Research Scientist: Rebecca M. Boumil, Ph.D.
Postdoctoral Fellow: Jacy Wagnon, Ph.D.
Research Assistant III: Connie L. Mahaffey, M.S.
Research Assistant I: Sam Asinof, B.A., Tracy McGarr, B.A.
Ph.D. Student (U.Maine, GSBS): Christian D. Richard, M.S.
Part-time laboratory staff:Associate Research Scientist: Verity A. Letts, Ph.D.
Research Assistant II: Barbara J. Beyer, B.A
Laboratory Technician III: Joanne Smith
Research Administrative Assistant: Heidi Stanton-Drew, B.A.
Park HJ, Hong M, Bronson RT, Israel MA, Frankel WN, Yun K. 2013. Elevated Id2 expression results in precocious neural stem cell depletion and abnormal brain development. Stem Cells doi: 10.1002/stem.1351. (Epub ahead of print)
Wagnon JL, Briese M, Sun W, Mahaffey CL, Curk T, Rot G, Ule J, Frankel WN. 2012. CELF4 regulates translation and local abundance of a vast set of mRNAs, including genes associated with regulation of synaptic function. PLOS Genetics 8(11): e1003067.
Sun, W., Wagnon, JL, Mahaffey, CL, Briese, M, Ule, J., Frankel, WN. 201_. Aberrant sodium channel activity in the complex seizure disorder of CELF4 deficient mice. J. Physiol 591:241-255.
Nellaker C, Keane TM, Yalcin B, Wong K, Agam A, Belgard TG, Flint J, Adams DJ, Frankel WN, Ponting CP. 2012. The genomic landscape shaped by selection on transposable elements across 18 mouse strains. Genome Biol 13(6):R45.
Paz JT, Bryant AS, Peng K, Fenno L, Yizhar O, Frankel WN, Deisseroth K, Huguenard JR. 2011. A new mode of corticothalamic transmission revealed in the Gria4(-/-) model of absence epilepsy. Nat Neurosci 14(9): 1167-1173.
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.
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.
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.
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.
Tokuda S, Beyer BJ, Frankel WN. 2009. Genetic complexity of absence seizures in C3H mice. Genes Brain Behav 8:283-289.
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.
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.
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.
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.
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.
Yang Y, Mahaffey CL, Berube N, Maddatu TP, Cox GA, Frankel WN. 2007. Complex seizure disorder caused by Brunol4 deficiency in mice. PLoS Genet 3(7):e124.
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, 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.
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.
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.
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.
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.
Book Chapters and Reviews
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. 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 . Oxford: Academic Press; 2009. pp. 356-363.
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, Barsh GS. 2008. PLoS Genetics turns three: looking back, looking ahead. PLoS Genet 4(7):e1000135
Frankel WN. 2005. Introducing PLoS Genetics. PLoS Genet 1:e21.
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.
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.