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

基本信息

批准号：
9304342
负责人：
Jing-Qiong Kang
金额：
$ 34.56万
依托单位：
VANDERBILT UNIVERSITY MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-07-01 至 2019-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9304342
关键词：
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 70 Human Image Immunohistochemistry Impairment In Vitro Injury Interneurons Knock-in Knock-in Mouse Knock-out Knockout Mice Label Lead Lewy Bodies Mass Spectrum Analysis Mediating Molecular Chaperones Mus Mutation Neurodegenerative Disorders Neurons Outcome Parkinson Disease Pathogenesis Pathology Pathway interactions Patients Phase Phenotype Protein Subunits Proteins Psyche structure Quantum Dots 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

项目摘要

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相关。大多数此类癫痫综合征是相当良性的，并且随着患者年龄的增长而消失。然而，GABAA 受体 gamma2 亚基基因 GABRgamma2 的截短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 患者都与 SCN1A 功能缺失突变有关，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，并且具有 GABRgamma2 (Q351X) 突变的 DS 小鼠模型的特征将为 DS 以及一般癫痫的发病机制提供新的见解。结合 SCN1A 和 GABRgamma2 小鼠模型的理解将有助于查明两组不同突变汇聚的最终共同病理生理学途径。 GABRgamma2 (Q351X) 突变与两个 DS 谱系相关。作为患者状况的杂合 GABRgamma2 (Q351X) 敲入 (KI) 小鼠概括了 DS 的主要特征。这些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。由于简单的 GABRgamma2 基因敲除 (KO) 杂合小鼠没有癫痫发作，这表明突变型 gamma2 (Q351X) 亚基蛋白的存在及其相关病理学导致了严重的 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，并对野生型亚基产生显性负效应。通过质谱法鉴定了突变体聚集体，聚集体的成分与神经退行性疾病中发现的内含物相似，例如帕金森病的路易体特征。我们的试验数据表明，突变蛋白也在杂合 KI 小鼠中形成大量聚集体。突变亚基损害了 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。我们假设突变型 gamma2 (Q351X) 亚基的积累和聚集会损害突触发育和 GABA 能传递，从而导致严重的 DS 表型。在本提案中，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。我们还将表征突变型 GABRG2 (Q351X) 杂合 KI 小鼠中野生型 GABAA 受体表达、分布、移动性和更新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 的过度表达减少了总突变体和聚集的 γ2 亚基蛋白。因此，我们建议通过上调突变 KI 小鼠中的 Hsp70 和 Hsp40 等伴侣来测试一种新的治疗策略。