Suppression of the yeast elongation factor Spt4 ortholog reduces expanded SCA36 GGCCUG repeat aggregation and cytotoxicity
Suppression of the yeast elongation factor Spt4 ortholog reduces expanded SCA36 GGCCUG repeat aggregation and cytotoxicity
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DOI：
10.1016/j.brainres.2018.12.045
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发表时间：
2019-05-15
期刊：
影响因子：
2.9
通讯作者：
Ikeda, Yoshio
中科院分区：
文献类型：
--
作者：
Furuta, Natsumi;Tsukagoshi, Setsuki;Ikeda, Yoshio
核仁蛋白56基因内含子1中的六核苷酸GGCCTG重复扩增导致脊髓小脑共济失调36型（SCA 36），这是一种相对纯粹的小脑共济失调，伴有进行性运动神经元受累。在这项研究中，通过将扩增的GGCCTG/CAGGCC重复序列引入培养的Neuro 2A细胞中来产生SCA 36细胞模型。在细胞中检测到有义（GGCCUG）（exp）而非反义（CAGGCC）（exp）RNA灶，这与SCA 36患者尸检脑中的观察结果一致。甘氨酸-脯氨酸二肽重复序列（DPR）的形成，由于重复相关的非ATG翻译很少发生在细胞表达扩大GGCCTG重复序列;相反，细胞窝藏扩大c9 orf 72 GGGGCC/GGCCCC重复序列稳健表达DPR蛋白。目前还没有有效的治疗微卫星重复扩增疾病，包括SCA 36。为了鉴定潜在有用的疗法，我们筛选了五种候选化合物，以确定其降低扩展的SCA 36重复序列的毒性的能力，并基于RNA焦点定量和细胞毒性测定来评估小干扰RNA介导的Supt 4a/Supt 5（酵母转录延伸因子Spt 4/Spt 5的鼠直系同源物）沉默是否具有治疗潜力。Supt 4a敲除和红霉素处理通过调节（GGCCUG）（exp）mRNA抑制（GGCCUG）（exp）RNA灶的形成和DPR蛋白的形成，从而改善SCA 36细胞模型中的细胞毒性。这些数据为开发治疗SCA 36和其他重复扩增疾病的有效治疗策略提供了基础。
A hexanucleotide GGCCTG repeat expansion in intron 1 of the nucleolar protein 56 gene causes spinocerebellar ataxia type 36 (SCA36), which is a relatively pure cerebellar ataxia with progressive motor neuron involvement. In this study SCA36 cell models were generated by introducing expanded GGCCTG/CAGGCC repeats into cultured Neuro2A cells. Sense (GGCCUG)(exp) but not antisense (CAGGCC)(exp) RNA foci were detected in the cells, consistent with observations in autopsied brains of patients with SCA36. Glycine-proline dipeptide repeat (DPR) formation due to repeat-associated non-ATG translation rarely occurred in cells expressing expanded GGCCTG repeats; in contrast, cells harboring expanded c9orf72 GGGGCC/GGCCCC repeats robustly expressed DPR proteins. There are currently no effective treatments for microsatellite repeat expansion diseases including SCA36. In order to identify potentially useful therapies, we screened five candidate chemical compounds for their ability to diminish the toxicity of expanded SCA36 repeats and evaluated whether small interfering RNA-mediated silencing of Supt4a/Supt5, the murine ortholog of the yeast transcriptional elongation factor Spt4/Spt5, has therapeutic potential based on RNA foci quantification and cytotoxicity assays. Supt4a knockdown and erythromycin treatment suppressed the formation of (GGCCUG)(exp) RNA foci and DPR protein formation via regulation of (GGCCUG)(exp) mRNA, thereby ameliorating the cytotoxicity in SCA36 cell models. These data provide a basis for developing effective therapeutic strategies for the treatment of SCA36 and other repeat expansion disorders.