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，以脑和脊髓运动神经元过早死亡为特征。肌萎缩侧索硬化症无法治愈，因此有必要更好地了解这种疾病的发病机制，以便为这种致命疾病开发有效的补救措施。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（SOD1）基因的突变占所有家族形式的约20%。散发性和家族性ALS在临床上是相似的，因此有必要研究家族性ALS以了解散发性病例。尽管ALS的确切病因尚不清楚，但有证据表明，细胞内突变SOD1 （mtSOD1）聚集体对运动神经元和邻近的非神经元细胞具有毒性，而运动神经元和邻近的非神经元细胞也与ALS密切相关。提出的mtSOD1毒性机制之一涉及触发内质网（ER）应激及其自适应信号转导途径，包括综合应激反应（ISR）。PERK通路是ISR的核心，ISR是一种细胞存活机制，由内质网中错误折叠或未折叠蛋白质的积累激活，从而威胁到细胞。本研究旨在探讨两种重要的ISR成分：PERK （ER激酶）和CHOP（参与应激介导的细胞死亡的转录因子）在ALS发病机制中的作用。在先前发现PERK通路对mtsod1诱导的ALS具有保护作用的基础上，提出的工作将验证运动神经元和/或少突胶质细胞（ALS中死亡的两种细胞类型）中过度的ISR是导致其死亡的主要因素的假设。PERK通路在少突胶质前体细胞（OPCs）和雪旺细胞中的作用也将被研究。为此，将从mtSOD1 ALS小鼠模型的运动神经元、少突胶质细胞、OPCs和雪旺细胞中基因删除PERK。此外，本研究将探讨CHOP在ALS发病机制中的作用，并验证其促进细胞凋亡的假设。CHOP的作用将在mtSOD1转基因小鼠中确定，这些小鼠在所有细胞（全局敲除）以及不同的细胞类型中都存在CHOP缺陷，如上所述。将使用临床和神经学评估、ISR成分的生化分析、免疫组织化学细胞存活的组织学分析以及电子显微镜下髓磷脂完整性的超微结构分析来评估具有PERK或CHOP缺陷的mtSOD1转基因小鼠的疾病进展。由于内质网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。这些研究也应该有助于确定与ALS相关的原代细胞类型，这对于开发新的治疗方法至关重要，包括干细胞移植，以防止这种毁灭性疾病的运动神经元退化。

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.