Bidirectional transcripts of the expanded C9orf72 hexanucleotide repeat are translated into aggregating dipeptide repeat proteins
Bidirectional transcripts of the expanded C9orf72 hexanucleotide repeat are translated into aggregating dipeptide repeat proteins
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DOI：
10.1007/s00401-013-1189-3
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发表时间：
2013-12-01
影响因子：
12.7
通讯作者：
Edbauer, Dieter
中科院分区：
文献类型：
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作者：
Mori, Kohji;Arzberger, Thomas;Edbauer, Dieter
基因C9 orf 72的第一内含子中的大量GGGGCC重复扩增是家族性额颞叶变性（FTLD）和肌萎缩侧索硬化（ALS）的最常见已知原因。尽管其内含子定位且缺乏ATG起始密码子，但重复区在所有三个阅读框中被翻译成聚集的二肽重复（DPR）蛋白，聚-（Gly-Ala）、聚-（Gly-Pro）和聚-（Gly-Arg）。我们采用基于抗体的方法来进一步验证DPR蛋白的翻译。为了测试反义重复RNA转录物是否也被翻译，我们提出了针对预测产物聚-（Ala-Pro）和聚-（Pro-Arg）的抗体。这两种抗体染色p62阳性神经元胞质包涵体整个小脑和海马，表明不仅有义，而且反义链重复翻译成DPR蛋白在没有ATG起始密码子。两条链的蛋白质产物共聚集，表明两条链同时翻译。此外，靶向DPR蛋白质的推定羧基末端的抗体可以检测C9 orf 72重复扩增载体中的包涵体病理学，表明非ATG翻译继续通过整个重复序列和超越。一种高灵敏度的单克隆抗体对聚-（甘氨酸-精氨酸），可视化丰富的包涵体病理在所有皮质区域和一些包涵体也在运动神经元。总之，我们的数据表明，GGGGCC重复是双向翻译成五个不同的DPR蛋白，共聚集在特征p62阳性TDP-43阴性夹杂物中发现FTLD/ALS的情况下与C9 orf 72重复扩增。新型抗poly-（Gly-Arg）单克隆抗体将有助于C9 orf 72 FTLD/ALS的病理诊断。
Massive GGGGCC repeat expansion in the first intron of the gene C9orf72 is the most common known cause of familial frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Despite its intronic localization and lack of an ATG start codon, the repeat region is translated in all three reading frames into aggregating dipeptide-repeat (DPR) proteins, poly-(Gly-Ala), poly-(Gly-Pro) and poly-(Gly-Arg). We took an antibody-based approach to further validate the translation of DPR proteins. To test whether the antisense repeat RNA transcript is also translated, we raised antibodies against the predicted products, poly-(Ala-Pro) and poly-(Pro-Arg). Both antibodies stained p62-positive neuronal cytoplasmic inclusions throughout the cerebellum and hippocampus indicating that not only sense but also antisense strand repeats are translated into DPR proteins in the absence of ATG start codons. Protein products of both strands co-aggregate suggesting concurrent translation of both strands. Moreover, an antibody targeting the putative carboxyl terminus of DPR proteins can detect inclusion pathology in C9orf72 repeat expansion carriers suggesting that the non-ATG translation continues through the entire repeat and beyond. A highly sensitive monoclonal antibody against poly-(Gly-Arg), visualized abundant inclusion pathology in all cortical regions and some inclusions also in motoneurons. Together, our data show that the GGGGCC repeat is bidirectionally translated into five distinct DPR proteins that co-aggregate in the characteristic p62-positive TDP-43 negative inclusions found in FTLD/ALS cases with C9orf72 repeat expansion. Novel monoclonal antibodies against poly-(Gly-Arg) will facilitate pathological diagnosis of C9orf72 FTLD/ALS.