GABAA receptor subunit gene mutations are frequently associated with epilepsy. Most such epilepsy syndromes are rather benign and are outgrown as patient age. However, truncation mutations in the GABAA receptor gamma2 subunit gene GABRgamma2 are often but not always associated with a more severe phenotype, Dravet syndrome (DS). To date, most DS patients are associated with SCN1A loss of function mutations that result in impaired GABAergic interneuron activity and action potential firing. Synaptic GABAA receptors mediate inhibitory GABAergic transmission and characterizations of DS mouse model harboring GABRgamma2 (Q351X) mutation would provide new insights into the pathogenesis of DS as well as epilepsy in general. Combination of understandings from both SCN1A and GABRgamma2 mouse models will help pinpoint the final common pathophysiologic pathway on which two distinct groups of mutations converge. GABRgamma2 (Q351X) mutation is associated with two DS pedigrees. The heterozygous GABRgamma2 (Q351X) knockin (KI) mice which are patient condition recapitulate the major features of DS. The mice displayed multiple neurodevelopmental abnormalities and multiple forms of epilepsy including generalized tonic clonic epilepsy, suggesting impaired synapse formation and function. Since simple GABRgamma2 gene knockout (KO) heterozygous mice do not have seizures, this suggests that presence of the mutant gamma2 (Q351X) subunit protein and its related pathology contribute to the severe DS phenotype. We have previously demonstrated that the mutant gamma2 (Q351X) subunits were loss of function. Additionally, the mutant protein accumulated intracellularly, formed aggregates, and imposed a dominant-negative effect on the wildtype subunits. The mutant aggregates were identified by mass spectrometry, and the components of the aggregates were similar to those identified in inclusions found in neurodegenerative diseases, such as the Lewy bodies characteristic of Parkinson's disease. Our pilot data demonstrated that the mutant protein also formed substantial aggregates in the heterozygous KI mice. The mutant subunits impaired GABAergic synaptogenesis and GABAergic transmission. The single quantum dot imaging indicated the synapse vs. extrasynapse distribution of GABAA receptors were altered in the mutant KI mice. We hypothesize that the accumulation and aggregation of the mutant gamma2 (Q351X) subunits impair synapse development and GABAergic transmission, thus resulting in the severe phenotype as DS. In this proposal, we will characterize in detail the mutant subunit accumulation, aggregation and its impact on synapse formation, stabilization, connectivity and transmission. We will also characterize adaptive changes of wildtype GABAA receptor expression, distribution, mobility and turnover as well as neurobehaviors in the mutant GABRG2 (Q351X) heterozygous KI mice. We found overexpression of heat shock protein (Hsp)70 and Hsp40 reduced the total mutant and aggregated gamma2 subunit protein. We thus propose to test a novel therapeutic strategy by upregulating chaperones like Hsp70 and Hsp40 in the mutant KI mice.
Seizure disorders (epilepsies) are very common and Dravet syndrome (DS) is the most severe kind among seizure disorders. The patients suffering from DS have intractable seizures and mental impairment and with poor outcome. In this proposal, we will use a mouse model which harbors a mutation causing DS in humans and manifests the symptoms seen in humans with DS to study the detailed underlying mechanisms of DS and to develop a novel therapeutic strategy against this disorder.
|Johnston, Ann J; Kang, Jing-Qiong; Shen, Wangzhen et al. (2014) A novel GABRG2 mutation, p.R136*, in a family with GEFS+ and extended phenotypes. Neurobiol Dis 64:131-41|