GABA-A Receptor Alpha 1 Subunit Degradation and Its Association with Epilepsy

描述（由申请人提供）：大约3%的人在一生中的某个时候会患上癫痫，其中三分之一的患者不会用抗癫痫药物控制癫痫发作。我们的长期目标是阐明遗传性癫痫的机制，以帮助开发更有效的抗癫痫治疗方法。青少年肌阵挛性癫痫是最常见的癫痫综合征之一，约占所有癫痫病例的5%。GABA-A受体α 1亚基基因（GABRA 1）中的非保守错义突变A322 D与常染色体显性形式的青少年肌阵挛性癫痫（ADJME）相关。最近，我们证明了在哺乳动物非神经元表达系统中，ADJME突变在蛋白质翻译后但在亚基寡聚化之前降低α 1亚基表达，数据表明该突变导致α 1亚基的增强的内质网相关降解（ERAD）。ERAD是一个复杂的过程，利用许多伴侣蛋白，因此它是可能的ERAD在神经元中将不同于在成纤维细胞系。虽然已经研究了神经元ERAD缺陷与一些神经退行性疾病的关系，但其作为错误折叠突变蛋白的正常反应的作用尚不清楚。我们建议阐明的作用，正常ERAD的天然α 1，重组野生型，和突变体α 1（A322 D）亚基在神经元细胞系（HT-22）和培养的皮层神经元。我们提出三个具体目标。1)我们将确定ADJME α 1亚基突变对α 1亚基生物合成和降解的影响及其与成纤维细胞和神经元中泛素蛋白酶体系统的相关性。2)我们将描述这种突变对神经元中GABA-A受体生理学的影响。3)我们将描述神经元对ADJME突变导致的α 1亚基降解的代偿反应;具体而言，我们将研究神经元对其他α亚基表达的改变以及由此产生的突触后GABA生理学变化。

DESCRIPTION (provided by applicant): Approximately 3% of people develop epilepsy at some point during their life and one third of these patients will not have their seizures controlled with antiepileptic medication. Our long term goal is to elucidate mechanisms of genetic epilepsy in an effort to help develop more effective antiepileptic therapies. Juvenile myoclonic epilepsy is one of the most common epilepsy syndromes and it accounts for approximately 5% of all cases of epilepsy. The non-conserved, missense mutation A322D in the GABA-A receptor alpha 1 subunit gene (GABRA1) is associated with an autosomal dominant form of juvenile myoclonic epilepsy (ADJME). Recently, we demonstrated that in a mammalian, non-neuronal expression system, the ADJME mutation reduces alpha 1 subunit expression after protein translation, but before subunit oligomerization, data which suggested that the mutation causes enhanced endoplasmic reticulum associated degradation (ERAD) of the alpha 1 subunit. ERAD is a complex process that utilizes numerous chaperone proteins and thus it is likely that ERAD in neurons would differ from that in fibroblast cell lines. Although neuronal ERAD deficiencies have been studied in relation to some neurodegenerative diseases, its role as a normal response to misfolded mutated proteins is unknown. We propose to elucidate the role of normal ERAD of native alpha 1, recombinant wild type, and mutant alpha 1(A322D) subunits in both a neuronal cell line (HT-22) and in cultured cortical neurons. We propose three Specific Aims. 1) We will determine the effect of the ADJME alpha 1 subunit mutation on alpha 1 subunit biosynthesis and degradation and its association with the ubiquitin proteasome system in both fibroblasts and neurons. 2) We will characterize the effect of this mutation on the GABA-A receptor physiology in neurons. 3) We will characterize neurons' compensatory response to degradation of the alpha 1 subunit resulting from the ADJME mutation; specifically, we will study neurons' alteration of expression of other alpha subunits and the resulting changes in postsynaptic GABA physiology.