The PERKplexity of the integrated stress response in motor neurons and myelinatin

描述（由申请人提供）：肌萎缩性侧索硬化症（ALS）是一种进行性神经退行性疾病，其特征在于大脑和脊髓中运动神经元的过早死亡。ALS无法治愈，需要更好地了解疾病的发病机制，以便为这种致命的疾病开发有效的治疗方法。大多数ALS病例（90%）没有明显的遗传成分（称为散发性），而约10%是遗传性的（家族性）。Cu/Zn超氧化物歧化酶（SOD 1）基因突变占所有家族型的约20%。散发性和家族性ALS在临床上是相似的，因此有理由对家族性ALS进行研究以了解散发性病例。虽然ALS的确切原因尚不清楚，但有证据表明细胞内突变的SOD 1（mtSOD 1）聚集体对运动神经元和邻近的非神经元细胞具有毒性，这些细胞也与疾病密切相关。mtSOD 1毒性的机制之一涉及触发内质网（ER）应激及其适应性信号转导途径，包括整合应激反应（ISR）。PERK通路是ISR的核心，ISR是一种细胞存活机制，由ER中错误折叠或未折叠蛋白质的积累激活，这些蛋白质可能会压倒细胞。拟议的研究旨在调查ALS发病机制中两个突出的ISR组分的作用：PERK（ER激酶）和CHOP（参与应激介导的细胞死亡的转录因子）。基于先前的研究结果，即PERK通路对mtSOD 1诱导的ALS具有保护作用，拟议的工作将测试运动神经元和/或少突胶质细胞（ALS中死亡的两种细胞类型）中不堪重负的ISR是导致其死亡的主要因素的假设。PERK通路的作用也将在少突胶质细胞前体细胞（OPC）和雪旺细胞中进行研究。为此，PERK将从mtSOD 1 ALS小鼠模型中的运动神经元、少突胶质细胞、OPC和许旺细胞中遗传缺失。此外，该提案将解决CHOP在ALS发病机制中的作用，并测试其作用是促凋亡的假设。如上所述，CHOP的作用将在所有细胞（整体敲除）以及不同细胞类型中具有CHOP缺陷的mtSOD 1转基因小鼠中确定。将使用临床和神经学评估、ISR组分的生化分析、通过免疫组织化学进行的细胞存活的组织学分析和通过电子显微镜进行的髓鞘完整性的超微结构分析来评价具有PERK或CHOP缺陷的mtSOD 1转基因小鼠的疾病进展。由于ER应激存在于散发性和家族性ALS中，因此拟议的研究将为这两种形式的疾病提供重要的见解。这些研究还应该有助于确定参与ALS的主要细胞类型，这对于开发新的治疗方法（包括干细胞移植）以预防这种毁灭性疾病中的运动神经元变性至关重要。

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease, characterized by premature death of motor neurons in the brain and spinal cord. There is no cure for ALS, necessitating a better understanding of the disease pathogenesis in order to develop effective remedies for this fatal disorder. Most cases of ALS (90%) have no apparent genetic component (referred to as sporadic), whereas ~10% are inherited (familial). Mutations in the Cu/Zn superoxide dismutase (SOD1) gene comprise ~20% of all the familial forms. Sporadic and familial forms of ALS are clinically similar, thus justifyig research on familial ALS to understand sporadic cases. Although the precise cause of ALS remains unclear, evidence suggests that intracellular mutant SOD1 (mtSOD1) aggregates are toxic to motor neurons and neighboring non-neuronal cells, which are also critically involved in the disease. One of the proposed mechanisms for mtSOD1 toxicity involves triggering endoplasmic reticulum (ER) stress and its adaptive signaling transduction pathways, including integrated stress response (ISR). The PERK pathway is central to the ISR, which is a cellular survival mechanism activated by accumulation of misfolded or unfolded proteins in the ER that threaten to overwhelm a cell. The proposed research studies aim to investigate the roles of two prominent ISR components in ALS pathogenesis: PERK (ER kinase) and CHOP (transcription factor involved in stress-mediated cell death). Building upon previous findings that the PERK pathway is protective against mtSOD1-induced ALS, the proposed work will test the hypothesis that an overwhelmed ISR in motor neurons and/or oligodendrocytes, the two cell types that die in ALS, is a major factor that contributes to their demise. The role of the PERK pathway will also be investigated in oligodendrocyte precursor cells (OPCs) and in Schwann cells. To this end, PERK will be genetically deleted from motor neurons, oligodendrocytes, OPCs, and Schwann cells in the mtSOD1 ALS mouse model. Additionally, this proposal will address the role of CHOP in the pathogenesis of ALS and test the hypothesis that its effects are pro-apoptotic. The role of CHOP will be determined in mtSOD1 transgenic mice generated with CHOP deficiency in all cells (global knockout), as well as in distinct cell types, as described above. The mtSOD1 transgenic mice with PERK or CHOP deficiencies will be evaluated for disease progression using clinical and neurological assessment, biochemical analysis of ISR components, histological analysis of cell survival by immunohistochemistry, and ultrastructural analysis of myelin integrity by electron microscopy. Because ER stress is present in sporadic and familial ALS, the proposed research will provide important insights into both forms of the disease. These studies should also help identify the primary cell type involved in ALS, which is critical for development of novel therapeutic approaches, including stem cell transplantation, to prevent motor neuron degeneration in this devastating disease.