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

总结 这项提案的重点是UBQLN 2，错义突变，其中导致肌萎缩性肌萎缩性侧索硬化症的显性遗传。 侧索硬化伴额颞叶痴呆（ALS-FTD）。UBQLN蛋白促进错误折叠的细胞的清除。 通过自噬和蛋白酶体降解途径从细胞中清除蛋白质，包括ER相关的 降解（ERAD）。ERAD中的干扰导致ER应激的诱导，其慢性诱导是 参与神经变性疾病，包括ALS的发病机制。人们越来越欣赏 ER应激可能参与ALS发病机制，因为调节这种信号传导的元件的靶向作用 反应可以减轻疾病。然而，我们不仅缺乏对潜在机制的良好理解， ALS基因突变触发ER应激，但也是突变的良好动物模型。我们最近 产生表达野生型（WT）或P497S或P506T UBQLN 2突变的转基因小鼠品系 导致ALS-FTD。表达UBQLN 2突变的细胞系发生运动神经元疾病， 认知缺陷，概括了人类疾病的关键特征。相比之下，WT系缺乏 疾病检查早期和终末期突变UBQLN 2系的脊髓中的蛋白质变化 揭示了ER应激和自噬标记物的强烈升高，表明蛋白质稳态已经 心烦意乱ER应激触发Ire1 α、PERK和ATF6信号通路的激活，统称为 未折叠蛋白反应（Unfolded protein response，UPR）普遍定期审议分两个阶段：适应阶段， 以恢复蛋白质稳态，如果不成功，就会触发细胞死亡的终末阶段。研究 表明遗传或药理学方法可以延长适应期或阻止细胞死亡 阶段，延迟ALS的SOD1小鼠模型中的疾病。在这个应用程序中，我们将利用类似的策略， 直接测试UPR信号传导或自噬的调节是否会减轻我们的UBQLN 2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测定来获得 UBQLN2突变如何干扰ERAD的机制见解。在目标2中，我们将评估是否延长 通过GADD 34的基因缺失，ER应激的适应性阶段，减轻了小鼠的疾病症状 携带P497S UBQLN 2突变。在目标2中，我们将评估CHOP的遗传缺失， 参与细胞死亡的执行，在突变型P497S小鼠中引起疾病。在目标4中，我们将评估 ASK 1是一种在Ire 1 α下游作用以驱动细胞死亡信号传导的激酶， P497S突变小鼠的疾病。在目标5中，我们将评估自噬的增强是否会减轻任何疾病 在我们的UBQLN 2系列中。在目标6中，我们将使用共培养实验来确定来自我们的UBQLN 2的星形胶质细胞是否 小鼠模型可以诱导MN的非细胞自主死亡。这项研究的结果将提供 UBQLN2突变导致疾病的潜在机制的重要见解， 其可用于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.