Spinocerebellar Ataxia type 3 (SCA3, also known as Machado-Joseph disease) is a neurodegenerative disorder caused by a CAG repeat expansion encoding an abnormally long polyglutamine (polyQ) tract in the disease protein, ataxin-3 (ATXN3). No preventive treatment is yet available for SCA3. Because SCA3 is likely caused by a toxic gain of ATXN3 function, a rational therapeutic strategy is to reduce mutant ATXN3 levels by targeting pathways that control its production or stability. Here, we sought to identify genes that modulate ATXN3 levels as potential therapeutic targets in this fatal disorder. We screened a collection of siRNAs targeting 2742 druggable human genes using a cell-based assay based on luminescence readout of polyQ-expanded ATXN3. From 317 candidate genes identified in the primary screen, 100 genes were selected for validation. Among the 33 genes confirmed in secondary assays, 15 were validated in an independent cell model as modulators of pathogenic ATXN3 protein levels. Ten of these genes were then assessed in a Drosophila model of SCA3, and one was confirmed as a key modulator of physiological ATXN3 abundance in SCA3 neuronal progenitor cells. Among the 15 genes shown to modulate ATXN3 in mammalian cells, orthologs of CHD4, FBXL3, HR and MC3R regulate mutant ATXN3-mediated toxicity in fly eyes. Further mechanistic studies of one of these genes, FBXL3, encoding a F-box protein that is a component of the SKP1-Cullin-F-box (SCF) ubiquitin ligase complex, showed that it reduces levels of normal and pathogenic ATXN3 in SCA3 neuronal progenitor cells, primarily via a SCF complex-dependent manner. Bioinformatic analysis of the 15 genes revealed a potential molecular network with connections to tumor necrosis factor-α/nuclear factor-kappa B (TNF/NF-kB) and extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathways. Overall, we identified 15 druggable genes with diverse functions to be suppressors or enhancers of pathogenic ATXN3 abundance. Among identified pathways highlighted by this screen, the FBXL3/SCF axis represents a novel molecular pathway that regulates physiological levels of ATXN3 protein.

中文翻译：

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可药用的基因组筛选确定了ATXN3（脊髓小脑共济失调3型疾病蛋白）的丰度和毒性的新调节剂。

脊髓小脑共济失调3型（SCA3，也称为Machado-Joseph疾病）是一种神经退行性疾病，由CAG重复扩增引起，该疾病在疾病蛋白ataxin-3（ATXN3）中编码异常长的聚谷氨酰胺（polyQ）道。尚无针对SCA3的预防性治疗。因为SCA3可能是由ATXN3功能的毒性获得引起的，所以一种合理的治疗策略是通过靶向控制其产生或稳定性的途径来降低突变型ATXN3的水平。在这里，我们寻求鉴定调节ATXN3水平的基因作为这种致命疾病的潜在治疗靶标。我们使用基于细胞的检测方法，基于基于polyQ扩增的ATXN3的发光读数，筛选了针对2742个可药物治疗的人类基因的siRNA集合。从初步筛选中鉴定的317个候选基因中，选择了100个基因进行验证。在二次测定中确认的33个基因中，有15个在独立细胞模型中被确认为病原性ATXN3蛋白水平的调节剂。然后在果蝇SCA3模型中评估了其中的10个基因，其中1个被确认为SCA3神经元祖细胞中生理ATXN3丰度的关键调节剂。在15种显示可在哺乳动物细胞中调节ATXN3的基因中，CHD4，FBXL3，HR和MC3R的直系同源基因调节蝇眼中突变ATXN3介导的毒性。对其中一个基因FBXL3进行进一步的机理研究，该基因编码F-box蛋白，该蛋白是SKP1-Cullin-F-box（SCF）泛素连接酶复合物的组成部分，表明它降低了SCA3神经元中正常和致病性ATXN3的水平。祖细胞，主要是通过SCF依赖复合物的方式。对15个基因的生物信息学分析显示，潜在的分子网络与肿瘤坏死因子-α/核因子-κB（TNF / NF-kB）和细胞外信号调节激酶1和2（ERK1 / 2）途径有关。总体而言，我们确定了具有多种功能的15种可药物治疗的基因，可作为致病性ATXN3丰度的抑制剂或增强剂。在此屏幕突出显示的已鉴定途径中，FBXL3 / SCF轴代表了一种调节ATXN3蛋白质生理水平的新型分子途径。