Deciphering the role of ER stress in ALS pathogenesis caused by UBQLN2 mutations

摘要 这项建议关注的是UBQLN2，错义突变导致肌营养不良的显性遗传 侧索硬化伴额颞叶痴呆(ALS-FTD)。UBQLN蛋白促进错误折叠的清除 来自细胞的蛋白质通过自噬和蛋白酶体降解途径，包括内质网相关的 退化(ERAD)。ERAD的紊乱导致内质网应激的诱导，这种应激的慢性诱导是 与包括肌萎缩侧索硬化症在内的神经退行性疾病的发病有关。越来越多的人意识到 内质网应激可能参与肌萎缩侧索硬化症的发病机制，因为调节这一信号转导的元件 回应可以减轻疾病。然而，我们不仅缺乏对潜在机制的良好理解 通过ALS基因的突变触发内质网应激，也是很好的突变动物模型。我们最近 产生了表达野生型(WT)或P497S或P506T UBQLN2突变的转基因小鼠品系 导致肌萎缩侧索硬化症-FTD。表达UBQLN2突变的品系会患上运动神经元病 认知缺陷，概括了人类疾病的主要特征。相比之下，WT线路没有 疾病。早期和末期突变UBQLN2系脊髓蛋白质变化的检测 揭示了内质网应激和自噬标志物的强劲上升，表明蛋白质的动态平衡 心烦意乱。内质网应激触发IRE1α、PERK和ATF6信号通路的激活，统称为 未折叠蛋白反应(UPR)。普遍定期审议有两个阶段：适应阶段，尝试是 以恢复蛋白质的动态平衡，如果不成功，就会触发细胞终末死亡阶段。研究表明， 表明延长适应阶段或阻止细胞死亡的遗传或药物方法 肌萎缩侧索硬化症小鼠模型的分期、延迟性疾病。在本应用程序中，我们将使用类似的策略来 直接测试UPR信号的调制或自噬是否会减轻我们的UBQLN2小鼠的疾病 ALS-FTD模型。有六个目标。在目标1中，我们将使用体外细胞和生化分析来获得 对UBQLN2突变如何干扰ERAD的机械性洞察。在目标2中，我们将评估是否延长 内质网应激适应阶段Gadd34基因缺失可减轻小鼠的疾病症状 携带P497S UBQLN2突变。在目标2中，我们将评估CHOP分子的基因缺失 参与细胞死亡的执行，减轻突变P497S小鼠的疾病。在目标4中，我们将评估 ASK1是一种作用于IRE1α下游以驱动细胞死亡信号的激酶，其基因缺失可缓解 P497S突变小鼠的疾病。在目标5中，我们将评估增强自噬是否可以缓解任何疾病。 在我们的UBQLN2线上。在目标6中，我们将使用共培养实验来确定UBQLN2中的星形胶质细胞 小鼠模型可诱导MN的非细胞自主性死亡。这项研究的结果将提供 对UBQLN2突变导致疾病的潜在机制的重要洞察， 可用于ALS-FTD的治疗干预。

Summary This proposal focuses on UBQLN2, missense mutations in which cause dominant inheritance of amyotrophic lateral sclerosis with frontotemporal dementia (ALS-FTD). UBQLN proteins facilitate clearance of misfolded proteins from cells through the autophagy and proteasome degradation pathways, including in ER-associated degradation (ERAD). Disturbances in ERAD lead to induction of ER stress, chronic induction of which is implicated in the pathogenesis of neurodegenerative diseases, including ALS. There is growing appreciation that ER stress may be involved in ALS pathogenesis because targeting of elements that regulate this signaling response can alleviate disease. However, we not only lack good understanding of the underlying mechanisms by which mutations in ALS genes trigger ER stress, but also good animal models of the mutations. We recently generated transgenic mice lines that express either wild type (WT) or the P497S or P506T UBQLN2 mutations that cause ALS-FTD. The lines expressing the UBQLN2 mutations develop motor neuron disease and cognitive deficits, recapitulating key features of the human disease. By contrast, the WT lines are devoid of disease. Examination of protein changes in the spinal cord of early and the end-stage mutant UBQLN2 lines revealed robust elevation of ER stress and autophagy markers, suggesting protein homeostasis has been perturbed. ER stress triggers the activation of Ire1α, PERK and ATF6 signaling pathways, collectively called the unfolded protein response (UPR). There are two phases of UPR: an adaptive phase, where attempts are made to restore protein homeostasis, which, if unsuccessful, triggers a terminal cell death phase. Studies have shown that genetic or pharmacological methods that prolong the adaptive phase, or which block the cell death phase, delay disease in SOD1 mouse models of ALS. In this application we will utilize similar strategies to directly test whether modulation of UPR signaling, or autophagy, will alleviate disease in our UBQLN2 mouse models of ALS-FTD. There are six aims. In Aim 1 we will use in vitro cell and biochemical assays to gain mechanistic insight into how UBQLN2 mutations interfere with ERAD. In Aim 2 we will evaluate if prolongation of the adaptive phase of ER stress, by genetic deletion of GADD34, alleviates disease symptoms in mice carrying the P497S UBQLN2 mutation. In Aim 2 we will evaluate if genetic deletion of CHOP, a molecule involved in execution of cell death, alleviates disease in mutant P497S mice. In Aim 4 we will evaluate whether genetic deletion of ASK1, a kinase that acts downstream of Ire1α to drive cell death signaling, alleviates disease in P497S mutant mice. In Aim 5 we will assess if enhancement of autophagy will alleviate any disease in our UBQLN2 lines. In Aim 6 we will use co-culture experiments to determine if astrocytes from our UBQLN2 mouse models can induce non-cell autonomous death of MN. The results of this research will provide important insight into the underlying mechanisms by which UBQLN2 mutations cause disease, the lessons of which could be exploited for therapeutic intervention in ALS-FTD.