Two molecular pathways (NMD and ERAD) contribute to a genetic epilepsy associated with the GABA(A) receptor GABRA1 PTC mutation, 975delC, S326fs328X.
Two molecular pathways (NMD and ERAD) contribute to a genetic epilepsy associated with the GABA(A) receptor GABRA1 PTC mutation, 975delC, S326fs328X.
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DOI：
10.1523/jneurosci.4512-08.2009
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发表时间：
2009-03-04
期刊：
影响因子：
--
通讯作者：
Macdonald RL
中科院分区：
文献类型：
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作者：
Kang JQ;Shen W;Macdonald RL
大约三分之一的人类遗传性疾病是由产生过早终止密码子（PTC）的突变引起的。钠通道和GABAA受体基因的这些突变与特发性全身性癫痫有关，但PTC对突变通道亚基生物发生和功能的细胞后果尚不清楚。PTC可以导致截短亚基的翻译，或者更可能地，通过无义介导的mRNA衰变（NMD）触发mRNA降解，从而在转录水平上防止或减少突变亚基的产生。GABAA受体α1亚基突变，975 delC，S326 fs 328 X，是一种与儿童失神癫痫相关的常染色体显性突变，在内含子8上游74 bp的9个外显子GABRA 1基因的外显子8中产生PTC。使用内含子8包含minigene，支持NMD，我们证明，突变mRNA大幅减少，但不是不存在。通过核糖体抑制或通过沉默NMD必需基因hUPF-1来阻断突变体转录物的丢失。在神经元和非神经元细胞中，PTC通过NMD引起突变体α1（S326 fs 328 X）亚基mRNA的大量丢失，小部分mRNA逃避NMD并产生突变蛋白。翻译的突变蛋白由于内质网相关降解（ERAD）而具有降低的稳定性，并且具有增强的与分子伴侣的缔合。这项研究表明，由于NMD的激活和ERAD的突变蛋白激活mRNA的丢失可能有助于癫痫的发生。这里概述的分子机制描绘了许多PTC产生突变的发病机制模型。
About one third of human genetic diseases are caused by premature translation-termination codon (PTC)-generating mutations. These mutations in sodium channel and GABAA receptor genes have been associated with idiopathic generalized epilepsies, but the cellular consequences of the PTCs on the mutant channel subunit biogenesis and function are unknown. The PTCs could result in translation of a truncated subunit, or more likely, trigger mRNA degradation through nonsense-mediated mRNA decay (NMD), thus preventing or reducing production of mutant subunit at the transcriptional level. The GABAA receptor α1 subunit mutation, 975delC, S326fs328X, is an autosomal dominant mutation associated with childhood absence epilepsy that generates a PTC in exon 8 of the 9 exon GABRA1 gene that is 74 bp upstream of intron 8. Using an intron 8-inclusion minigene that supports NMD, we demonstrated that mutant mRNA was substantially reduced, but not absent. Loss of mutant transcripts was blocked by ribosome inhibition or by silencing the NMD-essential gene hUPF-1. In both neurons and nonneuronal cells, the PTC caused substantial loss of mutant α1(S326fs328X) subunit mRNA through NMD with a minor portion of the mRNA escaping NMD and producing a mutant protein. The translated mutant protein had reduced stability due to endoplasmic reticulum associated degradation (ERAD) and had enhanced association with molecular chaperones. This study suggests that loss of mRNA due to activation of NMD and activation of ERAD by the mutant protein may contribute to epileptogenesis. The molecular mechanisms outlined here delineate a model for the pathogenesis of many PTC-generating mutations.