Investigation of repeat-associated non-AUG translation and dipeptide repeat proteins in C9orf72-associated ALS/FTD

基因C9 orf 72中的六核苷酸（G4 C2）重复扩增是家族性和散发性肌萎缩侧索硬化（ALS）和额颞叶痴呆（FTD）的最常见的已知原因（DeJesus-Hernandez et al.，2011; Renton等人，2011年）。C9 orf 72在神经元中表达，其中重复扩增被双向转录成含有异常重复的RNA（DeJesus-Hernandez等人，2011年）。有义和反义转录物都聚集到细胞质和核内RNA病灶中（Mizielinska et al.，2014）。尽管缺乏常规起始密码子，但这些转录物通过重复相关的非AUG（RAN）翻译进行翻译（Ash等人，2013;绿色等人，2017).这产生五种二肽重复蛋白（DPR）：甘氨酸-丙氨酸（GA）、甘氨酸-精氨酸（GR）、脯氨酸-精氨酸（PR）、脯氨酸-丙氨酸（PA）和甘氨酸-脯氨酸（GP）（Mori等人，2013年）。DPR在广泛的脑区域中形成神经元内含物（Ash等人，2013年）。此外，大多数患者的脑显示RNA结合蛋白TDP-43从细胞核到细胞质聚集体的截短和易位，这一现象在非C9 orf 72 ALS和FTD中也很常见（Neumann等人，2006). RNA灶和DPR，特别是富含精氨酸的GR和PR，对多种模型系统都是有毒的（Donnelly et al.，2013; Mizielinska等人，2014; Lopez-Gonzalez等人，2016）。我们实验室最近的工作表明，通过将周期性终止密码子插入重复区域来抑制果蝇模型中的RAN翻译保留了mRNA病灶，但改善了神经变性（Mizielinska et al.，2014; Moens等人，2018年）。这表明减少DPRs的翻译可能对重复介导的疾病具有保护作用。在这项工作之后，我们的实验室与行业合作伙伴合作筛选了20，000种小分子（SM），以确定那些在过度表达G4 C2重复序列的HeLa细胞中减少RAN翻译的小分子。SM有机会通过血脑屏障，并且与目前正在开发的另一种治疗选择反义寡核苷酸相比，生产成本更低（Schludi和Edbauer，2017; Donnelly等人，2013).组织培养、ELISA、SIMOA、免疫细胞化学、共聚焦显微镜、Southern印迹、蛋白质合成测定（Click-iT AHA）、RNA FISH、转染/转导

A hexanucleotide (G4C2) repeat expansion in the gene C9orf72 is the commonest known cause of both familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (DeJesus-Hernandez et al., 2011; Renton et al., 2011). C9orf72 is expressed in neurons where the repeat expansion is transcribed bi-directionally into aberrant repeat-containing RNA (DeJesus-Hernandez et al., 2011). Both sense and antisense transcripts aggregate into cytoplasmic and intranuclear RNA foci (Mizielinska et al., 2014). Despite the lack of a conventional start codon, these transcripts are translated through repeat-associated non-AUG (RAN) translation (Ash et al., 2013; Green et al., 2017).This produces five dipeptide repeat proteins (DPRs): glycine-alanine (GA), glycine-arginine (GR), proline-arginine (PR), proline-alanine (PA) and glycine-proline (GP) (Mori et al., 2013). The DPRs form neuronal inclusions in widespread brain regions (Ash et al., 2013). Additionally, most patient brains show truncation and translocation of the RNA-binding protein TDP-43 from the nucleus to cytoplasmic aggregates, a phenomenon also common in non-C9orf72 ALS and FTD (Neumann et al., 2006).Both RNA foci and DPRs, particularly the arginine-rich GR and PR, were toxic to multiple model systems (Donnelly et al., 2013; Mizielinska et al., 2014; Lopez-Gonzalez et al., 2016). Recent work in our lab showed that inhibiting RAN-translation in a Drosophila model through inserting periodic stop codons into the repeat region retained mRNA foci, but ameliorated neurodegeneration (Mizielinska et al., 2014; Moens et al., 2018). This suggests that reducing the translation of DPRs may be protective against repeat-mediated disease.Following this work, our lab has collaborated with an industry partner to screen 20,000 small molecules (SMs) to identify those that reduce RAN-translation in HeLa cells overexpressing G4C2 repeats. SMs have a chance of passing the blood-brain barrier and are cheaper to produce compared to antisense oligonucleotides, another therapeutic option currently under development (Schludi and Edbauer, 2017; Donnelly et al., 2013).Techniques usedTissue culture, ELISA, SIMOA, immunocytochemistry, confocal microscopy, Southern blotting, protein synthesis assay (Click-iT AHA), RNA FISH, transfection/transduction