Calpain cleavage of α-synuclein and T-cell reactivity in Parkinson’s disease

摘要 帕金森病(PD)是一种衰弱的进行性退行性运动障碍，与缺乏 黑质(SN)多巴胺能神经元与α-突触核蛋白(α-SYN)的积聚 大脑，小胶质细胞的激活，炎性细胞因子/趋化因子的产生，CD4T细胞的渗透， 和神经退化。最有效的疗法L-多巴并不能阻止疾病的发展，而 其递进性机制尚不清楚。钙蛋白酶是一种受钙调节的半胱氨酸蛋白酶，它发挥着 在帕金森病脊髓变性中的关键作用及其在α-SYN聚集、活化中的作用 小胶质细胞、T细胞及其迁移表明钙蛋白酶在促进炎症过程和 疾病的发展。虽然α-SYN的CalPain-1裂解促进了PD样疾病中的突触核蛋白聚集， 两个主要的Calain亚型，CalPain-1和CalPain-2，在α-SYN表达中的精确参与 CD4T细胞仍不清楚。初步研究发现MPTP小鼠体内存在一种亚型的CD4T细胞， 这一点被Calain抑制剂取消，这表明Calain和CD4T细胞的激活可能在 在帕金森病的炎症过程和疾病进展中的关键作用。初步数据还表明， SiRNA介导的Calain-2基因敲除减少人B细胞的抗原提呈并抑制 CD4T细胞的活化。因此，我们假设不同的钙蛋白亚型的激活可能有利于 帕金森病患者α同源反应性CD4T细胞亚型的扩增。我们还假设CalPain 抑制α-SYN可能减弱炎性T细胞的聚集和扩张，减轻炎症和 支持帕金森病患者的神经元存活和改善预后。提出了两个具体的目标来测试 假设：(目的1)研究calain-1或calain-2的激活是否与小胶质细胞有关 α-SYN递呈给CD4T细胞导致帕金森病样疾病进展和神经变性 小鼠的疾病。(目的2)确定抑制Calain-2是否会减少α-SYN的小胶质细胞提呈 并产生致病的CD4T细胞，延缓帕金森病小鼠模型的疾病进展。目标是 本研究的目的是探讨CalPain-1和CalPain-2在α-SYN反应性致病CD_4形成中的作用。 T细胞，以及来自MPTP小鼠的CD4T细胞亚群是否能在 免疫缺陷小鼠。计划进行研究以确定CalPain-1的缺失是否会减弱α-SYN 利用Calain-1基因敲除(KO)小鼠聚集和扩增CD4T细胞。此外，还包括 Calain-2抑制剂将在炎性CD4T细胞反应和炎症反应的改变研究中进行评估 Calain-1 KO小鼠细胞因子/趋化因子的产生测试不同的钙蛋白亚型在体内的作用 α-SYN反应性致病T细胞的产生和诱导神经元的死亡和变性可能导致 开发一种治疗帕金森病和其他神经退行性疾病的新方法。

Summary Parkinson's disease (PD) is a debilitating progressive degenerative movement disorder associated with loss of dopaminergic (DA) neurons in the substantia nigra (SN) along with the accumulation of α-synuclein (α-syn) in the brain, activation of microglia, production of inflammatory cytokines/chemokines, infiltration of CD4+ T-cells, and neurodegeneration. The most potent therapy, L-dopa, does not block disease progression, and the mechanism of the progressive nature is unclear. Calpain, a cysteine protease regulated by calcium, plays a pivotal role in SN and SC (spinal cord) degeneration in PD, and its role in α-syn aggregation, activation of microglia, T cells and their migration indicate calpain to be crucial in promoting the inflammatory process and disease progression. While calpain-1 cleavage of α-syn promotes synuclein aggregation in PD-like diseases, the precise involvement of the two major calpain isoforms, calpain-1 and calpain-2, in α-syn presentation to CD4+ T-cells remains unknown. Preliminary studies here identified a subtype of CD4+ T cells in MPTP mice, which was abolished by calpain inhibitor, suggesting that activation of calpain and CD4+ T cells may play critical roles in the inflammatory process and disease progression in PD. Preliminary data also suggest that siRNA-mediated knockdown of calpain-2 diminishes antigen presentation by human B-cells and inhibits activation of CD4+ T cells. Thus, we hypothesize that activation of distinct calpain isoforms may favor expansion of a subtype of α-syn-reactive CD4+ T cells in PD-like disease. We also hypothesize that calpain inhibition may attenuate α-syn aggregation and expansion of inflammatory T cells, reduce inflammation and support neuronal survival and improved outcome in PD patients. Two specific aims are proposed to test the hypothesis: (Aim 1) To investigate whether activation of calpain-1 or calpain-2 is linked with microglial presentation of α-syn to CD4+ T cells resulting in disease progression and neurodegeneration in PD-like disease in mice. (Aim 2) To determine whether inhibition of calpain-2 reduces microglial presentation of α-syn and generation of pathogenic CD4+ T cells, attenuating disease progression in mouse models of PD. The goal of this study is to investigate the role of calpain-1 and calpain-2 in generating α-syn-reactive pathogenic CD4+ T cells, and whether a subpopulation of CD4+ T cells from MPTP mice can induce PD-like disease in immunodeficient mice. Studies are planned to determine whether deletion of calpain-1 attenuates α-syn aggregation and expansion of CD4+ T cells using calpain-1 knockout (KO) mice. In addition, the role of calpain-2 inhibitor will be assessed in the study of alteration of inflammatory CD4+ T cell response and production of cytokines/chemokines in calpain-1 KO mice. Testing the effects of distinct calpain isoforms in the generation of α-syn-reactive pathogenic T cells and induction neuronal death and degeneration may lead to development of a novel approach for treating PD as well as other neurodegenerative disorders.