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

新型 Dravet 综合征小鼠模型中突触形成和功能的改变

• 批准号: 8596269
• 负责人: Jing-Qiong Kang
• 金额: $ 34.13万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8596269
• 关键词: Abbreviations; Action Potentials; Age; Alleles; Attenuated; Behavior; Benign; Biochemistry; Biogenesis; Brain; Characteristics; Comorbidity; Data; Development; Disease; Dominant-Negative Mutation; Epilepsy; Frequencies; Gene Mutation; Genes; Heat shock proteins; Heat-Shock Proteins 70; Human; Image; Immunohistochemistry; Impairment; In Vitro; Injury; Interneurons; Knock-out; Knockout Mice; Label; Lead; Lewy Bodies; Mass Spectrum Analysis; Mediating; Molecular Chaperones; Multiple Abnormalities; Mus; Mutation; Neurodegenerative Disorders; Neurons; Outcome; Parkinson Disease; Pathogenesis; Pathology; Pathway interactions; Patients; Phenotype; Protein Subunits; Proteins; Psyche structure; Quantum Dots; Relative (related person); Seizures; Slice; Sodium Channel; Symptoms; Synapses; Synaptic Transmission; Syndrome; Testing; Therapeutic; Tonic-Clonic Epilepsy; Vertebral column; disease phenotype; genetic pedigree; in vivo; insight; knockout gene; loss of function; loss of function mutation; mouse model; mutant; nervous system disorder; neurobehavior; novel; novel therapeutics; overexpression; postsynaptic; protein aggregate; protein aggregation; public health relevance; receptor; receptor expression; synaptic function; synaptogenesis; transmission process

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.

描述（由申请人提供）：GABAA受体亚单位基因突变通常与癫痫相关。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过时。然而，GABAA受体γ 2亚基基因GABR γ 2的截短突变通常但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SCN 1A功能缺失突变相关，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，具有GABR γ 2（Q351X）突变的DS小鼠模型的特征将为DS以及癫痫的发病机制提供新的见解。结合对SCN 1A和GABRgamma2小鼠模型的理解，将有助于确定两组不同突变会聚的最终共同病理生理途径。GABRgamma2（Q351X）突变与两个DS家系相关。杂合GABRgamma2（Q351X）基因敲入（KI）小鼠（患者状况）概括了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，包括全身强直阵挛性癫痫，表明突触形成和功能受损。由于单纯GABRgamma2基因敲除（KO）杂合子小鼠不发生癫痫发作，这表明突变体gamma2（Q351X）亚基蛋白的存在及其相关病理学导致重度DS表型。我们以前已经证明，突变体γ 2（Q351X）亚基的功能丧失。此外，突变蛋白质积累细胞内，形成聚集体，并施加显性负效应的野生型亚基。通过质谱鉴定突变体聚集体，并且聚集体的组分类似于在神经退行性疾病中发现的包涵体中鉴定的那些，例如帕金森病的特征性路易体。我们的试验数据表明，突变蛋白质也形成了大量的聚集在杂合KI小鼠。突变亚基损害GABA能突触发生和GABA能传递。单量子点成像表明突变KI小鼠中GABAA受体的突触与突触外分布发生了改变。我们推测突变体γ 2（Q351X）亚基的积累和聚集损害突触发育和GABA能传递，从而导致严重的DS表型。在这个提议中，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。我们还将表征野生型GABAA受体表达、分布、移动性和周转率以及突变GABRG2（Q351X）杂合KI小鼠中神经行为的适应性变化。我们发现热休克蛋白（Hsp）70和Hsp 40的过度表达减少了总的突变体和聚集的γ 2亚基蛋白。因此，我们建议通过上调突变KI小鼠中的Hsp70和Hsp40等分子伴侣来测试一种新型治疗策略。