hnRNP A3 binds to GGGGCC repeats and is a constituent of p62-positive/TDP43-negative inclusions in the hippocampus of patients with C9orf72 mutations
hnRNP A3 binds to GGGGCC repeats and is a constituent of p62-positive/TDP43-negative inclusions in the hippocampus of patients with C9orf72 mutations
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DOI：
10.1007/s00401-013-1088-7
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发表时间：
2013-03-01
影响因子：
12.7
通讯作者：
Haass, Christian
中科院分区：
文献类型：
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作者：
Mori, Kohji;Lammich, Sven;Haass, Christian
遗传分析显示，C9orf72基因调控区域内的六核苷酸重复序列扩张GGGGCC是家族性肌萎缩侧索硬化症最常见的原因，也是额颞叶退行性变的第二常见原因。由于重复序列的扩展可能通过隔离RNA结合蛋白而导致RNA毒性，因此我们寻找能够与GGGGCC重复序列结合的蛋白质。在含有六核苷酸GGGGCC的体外转录的生物素化RNA中，或作为对照，将AAAACC重复序列与核蛋白提取物孵育。采用严格的筛选方法，鉴定了20种在RNA代谢、翻译和运输中具有不同功能的RNA结合蛋白。这些蛋白的一个子集被进一步用免疫组织化学方法在人类尸检脑中进行了研究。这表明hnRNP A3在C9orf72重复延长患者的海马区形成了神经元胞浆和核内包涵体。共聚焦显微镜显示，这些包涵体属于迄今在C9orf72重复扩增携带者尸检中所见的神秘的p62阳性/TDP-43阴性包涵体组。因此，我们已经确定了这些病原性包涵体的一种蛋白质成分。
Genetic analysis revealed the hexanucleotide repeat expansion GGGGCC within the regulatory region of the gene C9orf72 as the most common cause of familial amyotrophic lateral sclerosis and the second most common cause of frontotemporal lobar degeneration. Since repeat expansions might cause RNA toxicity via sequestration of RNA-binding proteins, we searched for proteins capable of binding to GGGGCC repeats. In vitro-transcribed biotinylated RNA containing hexanucleotide GGGGCC or, as control, AAAACC repeats were incubated with nuclear protein extracts. Using stringent filtering protocols 20 RNA-binding proteins with a variety of different functions in RNA metabolism, translation and transport were identified. A subset of these proteins was further investigated by immunohistochemistry in human autopsy brains. This revealed that hnRNP A3 formed neuronal cytoplasmic and intranuclear inclusions in the hippocampus of patients with C9orf72 repeat extensions. Confocal microcopy showed that these inclusions belong to the group of the so far enigmatic p62-positive/TDP-43 negative inclusions characteristically seen in autopsy cases of diseased C9orf72 repeat expansion carriers. Thus, we have identified one protein component of these pathognomonic inclusions.