Neurodevelopmental deficits and cell-type-specific transcriptomic perturbations in a mouse model of HNRNPU haploinsufficiency.

Heterozygous de novo loss-of-function mutations in the gene expression regulator HNRNPU cause an early-onset developmental and epileptic encephalopathy. To gain insight into pathological mechanisms and lay the potential groundwork for developing targeted therapies, we characterized the neurophysiologic and cell-type-specific transcriptomic consequences of a mouse model of HNRNPU haploinsufficiency. Heterozygous mutants demonstrated global developmental delay, impaired ultrasonic vocalizations, cognitive dysfunction and increased seizure susceptibility, thus modeling aspects of the human disease. Single-cell RNA-sequencing of hippocampal and neocortical cells revealed widespread, yet modest, dysregulation of gene expression across mutant neuronal subtypes. We observed an increased burden of differentially-expressed genes in mutant excitatory neurons of the subiculum-a region of the hippocampus implicated in temporal lobe epilepsy. Evaluation of transcriptomic signature reversal as a therapeutic strategy highlights the potential importance of generating cell-type-specific signatures. Overall, this work provides insight into HNRNPU-mediated disease mechanisms and provides a framework for using single-cell RNA-sequencing to study transcriptional regulators implicated in disease.

Independent Neuronal Origin of Seizures and Behavioral Comorbidities in an Animal Model of a Severe Childhood Genetic Epileptic Encephalopathy.

The childhood epileptic encephalopathies (EE's) are seizure disorders that broadly impact development including cognitive, sensory and motor progress with severe consequences and comorbidities. Recently, mutations in DNM1 (dynamin 1) have been implicated in two EE syndromes, Lennox-Gastaut Syndrome and Infantile Spasms. Dnm1 encodes dynamin 1, a large multimeric GTPase necessary for activity-dependent membrane recycling in neurons, including synaptic vesicle endocytosis. Dnm1Ftfl or "fitful" mice carry a spontaneous mutation in the mouse ortholog of DNM1 and recapitulate many of the disease features associated with human DNM1 patients, providing a relevant disease model of human EE's. In order to examine the cellular etiology of seizures and behavioral and neurological comorbidities, we engineered a conditional Dnm1Ftfl mouse model of DNM1 EE. Observations of Dnm1Ftfl/flox mice in combination with various neuronal subpopulation specific cre strains demonstrate unique seizure phenotypes and clear separation of major neurobehavioral comorbidities from severe seizures associated with the germline model. This demonstration of pleiotropy suggests that treating seizures per se may not prevent severe comorbidity observed in EE associated with dynamin-1 mutations, and is likely to have implications for other genetic forms of EE.

Unraveling genetic modifiers in the gria4 mouse model of absence epilepsy.

Absence epilepsy (AE) is a common type of genetic generalized epilepsy (GGE), particularly in children. AE and GGE are complex genetic diseases with few causal variants identified to date. Gria4 deficient mice provide a model of AE, one for which the common laboratory inbred strain C3H/HeJ (HeJ) harbors a natural IAP retrotransposon insertion in Gria4 that reduces its expression 8-fold. Between C3H and non-seizing strains such as C57BL/6, genetic modifiers alter disease severity. Even C3H substrains have surprising variation in the duration and incidence of spike-wave discharges (SWD), the characteristic electroencephalographic feature of absence seizures. Here we discovered extensive IAP retrotransposition in the C3H substrain, and identified a HeJ-private IAP in the Pcnxl2 gene, which encodes a putative multi-transmembrane protein of unknown function, resulting in decreased expression. By creating new Pcnxl2 frameshift alleles using TALEN mutagenesis, we show that Pcnxl2 deficiency is responsible for mitigating the seizure phenotype - making Pcnxl2 the first known modifier gene for absence seizures in any species. This finding gave us a handle on genetic complexity between strains, directing us to use another C3H substrain to map additional modifiers including validation of a Chr 15 locus that profoundly affects the severity of SWD episodes. Together these new findings expand our knowledge of how natural variation modulates seizures, and highlights the feasibility of characterizing and validating modifiers in mouse strains and substrains in the post-genome sequence era.

A missense mutation in a highly conserved alternate exon of dynamin-1 causes epilepsy in fitful mice.

Dynamin-1 (Dnm1) encodes a large multimeric GTPase necessary for activity-dependent membrane recycling in neurons, including synaptic vesicle endocytosis. Mice heterozygous for a novel spontaneous Dnm1 mutation--fitful--experience recurrent seizures, and homozygotes have more debilitating, often lethal seizures in addition to severe ataxia and neurosensory deficits. Fitful is a missense mutation in an exon that defines the DNM1a isoform, leaving intact the alternatively spliced exon that encodes DNM1b. The expression of the corresponding alternate transcripts is developmentally regulated, with DNM1b expression highest during early neuronal development and DNM1a expression increasing postnatally with synaptic maturation. Mutant DNM1a does not efficiently self-assemble into higher order complexes known to be necessary for proper dynamin function, and it also interferes with endocytic recycling in cell culture. In mice, the mutation results in defective synaptic transmission characterized by a slower recovery from depression after trains of stimulation. The DNM1a and DNM1b isoform pair is highly conserved in vertebrate evolution, whereas invertebrates have only one isoform. We speculate that the emergence of more specialized forms of DNM1 may be important in organisms with complex neuronal function.

Heterozygous mutations of the voltage-gated sodium channel SCN8A are associated with spike-wave discharges and absence epilepsy in mice.

In a chemical mutagenesis screen, we identified the novel Scn8a(8J) allele of the gene encoding the neuronal voltage-gated sodium channel Na(v)1.6. The missense mutation V929F in this allele alters an evolutionarily conserved residue in the pore loop of domain 2 of Na(v)1.6. Electroencephalography (EEG) revealed well-defined spike-wave discharges (SWD), the hallmark of absence epilepsy, in Scn8a(8J) heterozygotes and in heterozygotes for two classical Scn8a alleles, Scn8a(med) (null) and Scn8a(med-jo) (missense). Mouse strain background had a significant effect on SWD, with mutants on the C3HeB/FeJ strain showing a higher incidence than on C57BL/6J. The abnormal EEG patterns in heterozygous mutant mice and the influence of genetic background on SWD make SCN8A an attractive candidate gene for common human absence epilepsy, a genetically complex disorder.

Absence seizures in C3H/HeJ and knockout mice caused by mutation of the AMPA receptor subunit Gria4.

Absence epilepsy, characterized by spike-wave discharges (SWD) in the electroencephalogram, arises from aberrations within the circuitry of the cerebral cortex and thalamus that regulates awareness. The inbred mouse strain C3H/HeJ is prone to absence seizures, with a major susceptibility locus, spkw1, accounting for most of the phenotype. Here we find that spkw1 is associated with a hypomorphic retroviral-like insertion mutation in the Gria4 gene, encoding one of the four amino-3-hydroxy-5-methyl-4isoxazolepropionic acid (AMPA) receptor subunits in the brain. Consistent with this, Gria4 knockout mice also have frequent SWD and do not complement spkw1. In contrast, null mutants for the related gene Gria3 do not have SWD, and Gria3 loss actually lowers SWD of spkw1 homozygotes. Gria3 and Gria4 encode the predominant AMPA receptor subunits in the reticular thalamus, which is thought to play a central role in seizure genesis by inhibiting thalamic relay cells and promoting rebound burst firing responses. In Gria4 mutants, synaptic excitation of inhibitory reticular thalamic neurons is enhanced, with increased duration of synaptic responses-consistent with what might be expected from reduction of the kinetically faster subunit of AMPA receptors encoded by Gria4. These results demonstrate for the first time an essential role for Gria4 in the brain, and suggest that abnormal AMPA receptor-dependent synaptic activity can be involved in the network hypersynchrony that underlies absence seizures.

Stargazer--a mouse to seize!

The goals of this short review are to familiarize readers with the stargazer mouse and to outline the major functional defects associated with this mutant. The roles of the stargazin protein in calcium channel function and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-receptor trafficking are discussed; focus is placed on studies regarding the thalamus, whence absence seizures potentially originate, and the cerebellum, which is associated with the ataxic phenotype. Finally, two additional alleles of stargazer, waggler and stargazer 3Jackson (3J), illustrate the value of an allelic series for understanding stargazin function.

Development of a new genetic model for absence epilepsy: spike-wave seizures in C3H/He and backcross mice.

To characterize the genetic basis of spike-wave discharges (SWDs) detected by electroencephalography (EEG) in C3H/He mice, substrains of C3H mice were evaluated by EEG and sensitivity to ethosuximide. Crosses with the SWD-negative strain C57BL/6J were performed to map the underlying gene(s). C3H/He substrains exhibited a modest incidence (average of 19 SWDs per hour) of 7-8 Hz SWDs when at rest, compared with the C3HeB/Fe subline (four SWDs per hour). In the mapping backcross, however, many mice showed a very high incidence (50-220 SWDs per hour) throughout the recording period. SWDs were first detected at 3.5 weeks of age, were associated with behavioral arrest, were suppressed by ethosuximide, and were strongest in the cerebral cortex and thalamus. The major C3H determinant of SWDs, spkw1 (spike-wave 1), mapped to chromosome (Chr 9), and together with a C57BL/6J determinant on Chr 8, spkw2, accounted for more than one-half of the phenotypic variation in the backcross mice. The modest SWD incidence in C3H/He mice and the high incidence in backcrosses implies that SWD could be a confounding variable for other behaviors. Because C3H/He mice have no other brain abnormalities, they are an attractive alternative for studying idiopathic absence epilepsy.

A targeted mutation in Cacng4 exacerbates spike-wave seizures in stargazer (Cacng2) mice.

The voltage-dependent calcium channel gamma4 subunit protein, CACNG4, is closely related to the gamma2 subunit, CACNG2. Both are expressed primarily in the brain and share 53% amino acid identity. The Cacng2 gene is disrupted in the stargazer mouse, with its distinctive phenotype including ataxia, frequent absence seizure episodes, and head elevation. A disruption within Cacng4 was engineered to assess its particular function. The homozygous Cacng4-targeted mutant mouse appeared normal with no ataxic gait or absence seizures, suggesting that other members of the gamma subunit family might functionally compensate for the absence of CACNG4. To test this hypothesis, the targeted Cacng4 mutation was combined with alleles of Cacng2. Absence seizures were observed in combination with the stargazer 3J mutation, which itself does not have seizures, and increased seizure activity was observed in combination with the waggler allele. Furthermore, within the corticothalamic loop, where absence seizures arise, CACNG4 expression is restricted to the thalamus. Our studies show that the CACNG4 protein has seizure suppressing activity, but this effect is revealed only when CACNG2 expression is also compromised, suggesting that CACNG subunits have in vivo overlapping functions.

Phenotypic heterogeneity in the stargazin allelic series.

The stargazer mutant mouse is characterized by its ataxic gait, head tossing, and absence seizures. The mutation was identified in the gamma 2 subunit gene of the high voltage-dependent calcium channel, Cacng2. Subsequently, two allelic variants of stargazer have arisen, waggler and stargazer 3J. In this study, we have compared these new alleles to the original stargazer allele. All three mutations affect the Cacng2 mRNA levels as they all arise from disruptions within the introns of this gene. Our results show that the mutations cause reduced Cacng2 mRNA and protein levels. Stargazer and waggler mice have the least amount of mRNA and undetectable protein, whereas stargazer 3J appears to be the mildest allele, both in terms of the phenotype and protein expression. Electroencephalographic (EEG) analysis confirmed that stargazer has frequent spike-wave discharges (SWDs); the average duration of each discharge burst is 5 seconds and recurs every minute. The waggler allele causes a greater variation in SWD activity depending on the individual mouse, and the stargazer 3J mouse has no SWDs. The preliminary characterization of this heterogeneous allelic series provides a basis to explore more biochemical and physiological parameters relating to the role of the Cacng2 product in calcium channel activity, AMPA receptor localization, and cerebellar disturbances.

Spontaneous deletion of epilepsy gene orthologs in a mutant mouse with a low electroconvulsive threshold.

The electroconvulsive threshold (ECT) test has been used extensively to determine the protection conferred by antiepileptic drug candidates against induced seizures in rodents. Despite its clinical relevance, the potential of ECT to identify mouse epilepsy models in genetic studies has not been thoroughly assessed. We adopted the ECT test to screen the progeny of ethylnitrosourea treated male C57BL/6J mice. In a small-scale screen, several mutant lines conferring a low threshold to ECT minimal clonic seizures were mapped to the telomeric region of mouse chromosome 2 in independent founder families. This high incidence was suggestive of a single spontaneous event that pre-existed in the founders of mutagenized stock. Genetic and physical mapping led to the discovery that several lines shared a single mutation, Szt1 (seizure threshold-1), consisting of a 300 kb deletion of genomic DNA involving three known genes. Two of these genes, Kcnq2 and Chrna4, are known to be mutated in human epilepsy families. Szt1 homozygotes and heterozygotes display similar phenotypes to those found in the respective Kcnq2 knockout mutant mice, suggesting that Kcnq2 haploinsufficiency is at the root of the Szt1 seizure sensitivity. Our results provide a novel genetic model for epilepsy research and demonstrate that the approach of using ECT to study seizures in mice has the potential to lead to the identification of human epilepsy susceptibility genes.