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

描述（由申请人提供）：GABAA受体亚基基因突变通常与癫痫有关。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亚基基因GABRgamma2的截断突变通常但并不总是与更严重的表型Dravet综合征（DS）相关。迄今为止，大多数DS患者与SCN1A功能突变缺失相关，导致gaba能中间神经元活动受损和动作电位放电。突触GABAA受体介导抑制性gaba能传递，GABRgamma2 （Q351X）突变的DS小鼠模型的表征将为DS以及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突变聚集的最终共同病理生理途径。GABRgamma2 （Q351X）突变与两个DS家系相关。患者状态的杂合GABRgamma2 （Q351X）敲入（KI）小鼠再现了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，包括全身性强直性阵挛性癫痫，表明突触形成和功能受损。由于简单GABRgamma2基因敲除（KO）杂合小鼠没有癫痫发作，这表明突变的gamma2 （Q351X）亚基蛋白的存在及其相关病理导致了严重的DS表型。我们之前已经证明突变的gamma2 （Q351X）亚基丧失了功能。此外，突变蛋白在细胞内积累，形成聚集体，并对野生型亚基施加显性负作用。通过质谱法鉴定了突变聚集体，聚集体的成分与神经退行性疾病（如帕金森病的路易体特征）中发现的内含物相似。我们的初步数据表明，突变蛋白在杂合KI小鼠中也形成了大量的聚集体。突变亚基破坏gabaergy突触发生和gabaergy传递。单量子点成像显示突变KI小鼠GABAA受体突触与突触外分布发生改变。我们假设突变体gamma2 （Q351X）亚基的积累和聚集损害了突触发育和gaba能传递，从而导致了严重的DS表型。在本研究中，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、连通性和传递的影响。我们还将描述突变GABRG2 （Q351X）杂合KI小鼠中野生型GABAA受体表达、分布、迁移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的过表达减少了总突变体和聚集的gamma2亚基蛋白。因此，我们提出通过上调突变型KI小鼠的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.