GABA chaperoning of epilepsy-associated GABAA receptor mutants.

描述（申请人提供）：神经递质-氨基丁酸（GABA）是中枢神经系统中主要的抑制性神经递质。大脑中大约30%的突触含有GABAA受体（Nutt, 2006），这是GABA受体的一种亚型，介导快速抑制性神经传递。GABAA受体与多种神经系统疾病（癫痫、失眠）和精神疾病（焦虑、精神分裂症）有关（Mohler, 2006; Charych et al., 2009），是包括苯二氮平类药物、巴比妥类药物和麻醉剂在内的几种治疗药物的靶点（Whiting, 2003）。GABAA受体亚基突变与几种遗传性癫痫有关（Macdonald et al., 2010）。其中一些突变体由于折叠和加工效率低下而保留在内质网（ER）中，导致细胞表面表达缺陷。这种“构象”疾病并不是唯一的，一些与疾病相关的、er保留的突变体的细胞表面表达可以通过配体或药理学伴侣来挽救。包括囊性纤维化、色素性视网膜炎、长QT综合征和法布里氏病（Bernier et al., 2004）相关的疾病突变体在内的多种疾病突变体都有望被药物伴侣拯救。目前正在进行药物伴侣治疗法布里氏病的临床试验（Fan and Ishii, 2010）。在这项提议中，我们假设配体伴侣可能用于挽救er保留的表面表达，癫痫相关的GABAA受体突变体。为了支持这一观点，我们最近发现GABA可以在早期分泌途径中作为配体伴侣，增加重组野生型GABAA受体的细胞表面表达（Eshaq et al., 2010）。我们的上述假设将通过两个具体目标来检验。在特异性目的1中，我们将确定GABA治疗是否可以挽救HEK 293细胞中癫痫相关GABAA受体突变体的细胞表面表达。在特异性目的2中，我们将确定在原代大脑皮层培养物中表达的癫痫相关GABAA受体突变体的细胞表面表达是否可以通过使用GABA转氨酶抑制剂vigabatrin提高细胞内GABA水平来挽救。对于这些实验，受体细胞表面表达将使用免疫荧光共聚焦显微镜和流式细胞术进行测量。

DESCRIPTION (provided by applicant): The neurotransmitter -aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the central nervous system. Approximately 30% of synapses in the brain contain GABAA receptors (Nutt, 2006), a subtype of GABA receptor that mediates fast inhibitory neurotransmission. The GABAA receptor is associated with a variety of neurological (epilepsy, insomnia) and psychiatric (anxiety, schizophrenia) disorders (Mohler, 2006; Charych et al., 2009) and is the target of several classes of therapeutic agents including benzodiazepines, barbiturates and anesthetics (Whiting, 2003). Mutations in GABAA receptor subunits are associated with several genetically- based epilepsies (Macdonald et al., 2010). Some of these mutants are retained in the endoplasmic reticulum (ER) due to inefficient folding and processing, resulting in deficits in cell surface expression. Such "conformational" diseases are not unique and the cell surface expression of some disease-associated, ER-retained mutants can be rescued by ligand or pharmacological chaperones. A variety of disease mutants show promise for rescue by pharmacological chaperones, including those associated with cystic fibrosis, retinitis pigmentosa, long QT syndrome and Fabry's disease (Bernier et al., 2004). Clinical trials with pharmacological chaperones for the treatment of Fabry's disease are now underway (Fan and Ishii, 2010). In this proposal, we hypothesize that ligand chaperoning may be used to rescue the surface expression of ER-retained, epilepsy-associated GABAA receptor mutants. In support of this notion, we have recently shown that GABA can act as a ligand chaperone in the early secretory pathway to increase the cell surface expression of recombinant, wild type GABAA receptors (Eshaq et al., 2010). Our above hypothesis will be tested by two specific aims. In Specific Aim 1 we will determine if the cell surface expression of epilepsy-associated GABAA receptor mutants expressed in HEK 293 cells can be rescued by GABA treatment. In Specific aim 2 we will determine whether the cell surface expression of epilepsy-associated GABAA receptor mutants expressed in primary cerebral cortical cultures can be rescued by elevating intracellular GABA levels with the GABA transaminase inhibitor vigabatrin. For these experiments, receptor cell surface expression will be measured using immunofluorescence confocal microscopy and flow cytometry. PUBLIC HEALTH RELEVANCE: The GABAA receptor is a neurotransmitter receptor that can carry various genetic mutations that either cause or are associated with epilepsy. These mutations impair the ability of the cell to properly synthesize the receptor. The proposed research investigates a possible mechanism for aiding the correct synthesis of these mutant receptors. Thus, these studies have relevance to the treatment of epilepsy.