GABA chaperoning of epilepsy-associated GABAA receptor mutants.

癫痫相关 GABAA 受体突变体的 GABA 陪伴。

• 批准号: 8260504
• 负责人: NANCY J LEIDENHEIMER
• 金额: $ 7.2万
• 依托单位: LOUISIANA STATE UNIV HSC SHREVEPORT
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-01 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8260504
• 关键词: 4-Aminobutyrate aminotransferase; Aminobutyric Acids; Anesthetics; Anxiety; Barbiturates; Benzodiazepines; Brain; Cell Surface Receptors; Cell surface; Cells; Cerebrum; Clinical Trials; Confocal Microscopy; Cystic Fibrosis; Disease; Endoplasmic Reticulum; Epilepsy; Epitopes; Fabry Disease; Flow Cytometry; GABA Receptor; Gene Mutation; Glutamate Decarboxylase; Health; Immunofluorescence Immunologic; Ligands; Long QT Syndrome; Measures; Mediating; Molecular Chaperones; Mutation; Neuraxis; Neurologic; Neurons; Neurotransmitter Receptor; Neurotransmitters; Pathway interactions; Process; Recombinants; Research; Retinitis Pigmentosa; Schizophrenia; Sleeplessness; Surface; Synapses; Testing; Therapeutic Agents; Vigabatrin; barbituric acid salt; base; gamma-Aminobutyric Acid; inhibitor/antagonist; mutant; neurotransmission; receptor; research study; synthetic enzyme

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.

描述（由申请人提供）：神经递质-氨基丁酸（GABA）是中枢神经系统中的主要抑制性神经递质。大脑中约30%的突触含有GABAA受体（Nutt，2006），这是一种介导快速抑制性神经传递的GABA受体亚型。GABAa受体与多种神经学（癫痫、失眠）和精神病学（焦虑、精神分裂症）障碍相关（Mohler，2006; Charych等人，2009），并且是包括苯二氮卓类、巴比妥类和麻醉剂的几类治疗剂的靶标（Whiting，2003）。 GABAa受体亚单位的突变与几种基于遗传的癫痫相关（Macdonald et al.，2010年）。这些突变体中的一些由于低效的折叠和加工而保留在内质网（ER）中，导致细胞表面表达的缺陷。这种“构象”疾病并不是唯一的，一些疾病相关的ER保留突变体的细胞表面表达可以通过配体或药理学伴侣来拯救。多种疾病突变体显示出通过药理学伴侣进行拯救的希望，包括与囊性纤维化、色素性视网膜炎、长QT综合征和法布里病相关的那些（Bernier等人，2004年）。用于治疗法布里病的药理学伴侣的临床试验目前正在进行中（Fan和石井，2010）。在这个建议中，我们假设，配体伴侣可用于拯救ER保留的癫痫相关GABAA受体突变体的表面表达。 为了支持这一观点，我们最近已经表明，GABA可以在早期分泌途径中充当配体伴侣，以增加重组野生型GABAA受体的细胞表面表达（Eshaq等人，2010年）。我们的上述假设将通过两个具体目标进行检验。在具体目标1中，我们将确定在HEK 293细胞中表达的癫痫相关GABAA受体突变体的细胞表面表达是否可以通过GABA处理来拯救。在具体目标2中，我们将确定原代大脑皮质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转氨酶抑制剂氨己烯酸提高细胞内GABA水平来拯救。对于这些实验，将使用免疫荧光共聚焦显微镜和流式细胞术测量受体细胞表面表达。 公共卫生关系：GABAA受体是一种神经递质受体，可以携带引起癫痫或与癫痫相关的各种基因突变。这些突变损害了细胞正确合成受体的能力。拟议的研究调查了帮助这些突变受体正确合成的可能机制。因此，这些研究与癫痫的治疗相关。