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: 8851698
• 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 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8851698
• 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; Health; 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; 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受体亚基基因突变经常与癫痫有关。大多数这样的癫痫综合征相当良性，并且随着患者的年龄而长大。然而，GABAA受体γ2亚基基因Gabrgamma2中的截短突变通常是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功能突变的丧失有关，从而导致GABA能中间神经元活动和动作潜在的射击受损。突触GABAA受体介导了携带Gabrgamma2（Q351X）突变的DS小鼠模型的抑制性GABA能传递和表征，将为DS的发病机理以及一般癫痫发作提供新的见解。 SCN1A和Gabrgamma2小鼠模型的理解的结合将有助于查明最终的常见病理生理途径，两种不同的突变群会融合。 Gabrgamma2（Q351X）突变与两个DS血统有关。患者状况的杂合Gabrgamma2（Q351X）敲击蛋白（Ki）小鼠概括了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病，包括广泛的隆隆声癫痫，表明突触的形成和功能受损。由于简单的Gabrgamma2基因敲除（KO）杂合小鼠没有癫痫发作，因此这表明突变γ2（Q351X）亚基蛋白的存在及其相关病理有助于严重的DS表型。我们先前已经证明了突变γ2（Q351X）亚基是功能丧失。此外，突变蛋白会在细胞内积聚，形成聚集体，并对野生型亚基产生显性阴性作用。通过质谱法鉴定了突变骨料，并且聚集体的成分与神经退行性疾病中发现的夹杂物中发现的成分相似，例如帕金森氏病的特征的刘易身体。我们的试点数据表明，突变蛋白在杂合Ki小鼠中也形成了大量骨料。突变亚基损害了GABA能突触发生和GABA能传播。单个量子点成像表明突变Ki小鼠中GABAA受体的突触与GABAA受体的外肺外分布的分布发生了变化。我们假设突变γ2（Q351X）亚基的积累和聚集会损害突触的发展和GABA能传播，从而导致严重的表型为Ds。在此提案中，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，连通性和传播的影响。我们还将表征野生型GABAA受体表达，分布，迁移率和周转率以及突变体Gabrg2（Q351X）杂合子Ki小鼠中的神经行为的自适应变化。我们发现热休克蛋白（HSP）70和HSP40的过表达降低了总突变体和聚集的Gamma2亚基蛋白。因此，我们建议通过上调突变Ki小鼠中HSP70和HSP40（HSP40）的伴侣来测试一种新型的治疗策略。