Altered synapse formation and function in a novel Dravet syndrome mouse model

描述(申请人提供)：GABAA受体亚单位基因突变经常与癫痫有关。大多数这样的癫痫综合征是相当良性的，并随着患者年龄的增长而消失。然而，GABAA受体Gamma2亚单位基因GABRGamma2的截断突变经常但并不总是与更严重的表型Drave氏综合征(DS)相关。到目前为止，大多数DS患者与SCN1A功能突变的丧失有关，这些突变导致GABA能中间神经元活性和动作电位放电受损。突触GABAA受体介导抑制性GABA能传递，携带GABRGamma2(Q351X)突变的DS小鼠模型的特征将为DS和癫痫的发病机制提供新的见解。结合对SCN1A和GABRGamma2小鼠模型的理解，将有助于确定两组不同的突变汇聚在一起的最终共同的病理生理途径。GABRGamma2(Q351X)突变与两个DS家系相关。杂合子GABRGamma2(Q351X)敲门(KI)小鼠是DS的主要特征。小鼠表现出多种神经发育异常和多种形式的癫痫，包括全身性强直阵挛癫痫，表明突触形成和功能受损。由于单纯GABRGamma2基因敲除(KO)杂合子小鼠没有癫痫发作，这表明突变的Gamma2(Q351X)亚单位蛋白的存在及其相关病理导致了严重的DS表型。我们先前已经证明突变的Gamma2(Q351X)亚基功能丧失。此外，突变蛋白在细胞内积累，形成聚集体，并对野生型亚基产生显性-负效应。通过质谱仪鉴定突变聚集体，聚集体的成分与神经退行性疾病中发现的包涵体中鉴定的成分相似，例如帕金森氏病特有的路易小体。我们的实验数据表明，突变蛋白也在杂合子Ki小鼠中形成了大量聚集体。突变的亚基损害了GABA能突触发生和GABA能传递。单量子点成像显示突变型Ki小鼠GABAA受体的突触和突触外分布发生了改变。我们推测，突变的Gamma2(Q351X)亚基的积累和聚集损害了突触的发育和GABA能传递，从而导致了严重的DS表型。在这个提案中，我们将详细描述突变亚基的积累、聚集及其对突触形成、稳定、连接和传递的影响。我们还将研究突变型GABRG2(Q351X)杂合子Ki小鼠野生型GABAA受体表达、分布、迁移率和周转以及神经行为的适应性变化。我们发现热休克蛋白(HSP)70和HSP40的过表达降低了突变的总蛋白和聚集的Gamma2亚单位蛋白。因此，我们建议在突变的Ki小鼠中通过上调Hsp70和Hsp40等伴侣蛋白来测试一种新的治疗策略。

DESCRIPTION (provided by applicant): 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.