Extra-nigral Neurodegeneration in Experimental Parkinson's Disease

描述（由申请人提供）：帕金森病（PD）是一种进行性退行性运动障碍，与黑质（SN）多巴胺能神经元的丧失相关，导致功能障碍。目前的治疗方法左旋多巴不能阻止疾病进展；因此，必须开发新的治疗方法。因此，目的是研究PD患者脑和脊髓（SC）中的炎症事件及其变性，并表征PD患者是否丢失SC完整性和神经元，从而导致功能障碍。了解损伤的机制可能有助于开发新的治疗策略。虽然帕金森氏病的病因尚不完全清楚，但神经毒素已参与帕金森氏病的发病机制。毒性1-甲基-4-苯基-1,2,3,6-四氢吡啶（MPTP）被广泛用作实验模型。由于1-甲基-4-苯基吡啶离子（MPP+）， MPTP的活性毒性代谢物，增加细胞内游离Ca2+水平并促进线粒体功能障碍，Ca2+介导的PD病理已经被假设。Ca2+水平升高会促进钙蛋白酶激活，增加炎症反应，损伤脑/SC神经元、轴突和髓鞘，最终导致功能缺陷。我们在PD小鼠SC中直接检测到MPP+，星形胶质细胞和小胶质细胞的激活，以及神经元中钙蛋白酶活性和表达的增加，初步发现SC也受到影响。初步数据显示PD患者SC的运动神经元也受到损伤，证实了这些发现。MPP+处理腹侧SC运动神经元细胞（VSC4.1）显示细胞内[Ca2+]和calpain活性增加，膜电位丧失和死亡，而calpain抑制剂（calpeptin, SJA6017）保护和恢复细胞功能。根据这些发现，我们假设，由于SC协调身体的运动和感觉，除了SN外，SC神经元、轴突和髓磷脂的损伤可能是PD的一个重要因素，而钙蛋白酶通过促进炎症和细胞死亡在这种功能障碍中起着至关重要的作用，可能是治疗的靶点。三个具体目标将检验这些假设。特异性目的1将研究MPTP是直接在SC中转化为MPP+，还是通过大脑退化轴突进入，还是两者兼而有之；研究急性和慢性帕金森病患者中MPTP （MPP+）对SN、SC神经元和白质的影响；评估钙蛋白酶的表达和活性以及随后的炎症和细胞损伤；并检测死后PD患者SC中神经元、轴突和髓磷脂的状态。特异性目的2将探讨神经毒性MPP+对分化的VSC4.1细胞的影响，并利用电生理技术在体外测试calpain抑制剂的神经保护作用。特异性Aim 3将检测calpain抑制剂治疗是否会减轻mptp诱导的PD小鼠的炎症，防止脑和SC神经元凋亡，保护细胞，保存轴突和髓磷脂，并改善功能。这些研究将描述calpain在mptp诱导的PD炎症和神经退行性变中的作用，以及calpain抑制剂作为治疗药物在PD中可能的神经保护作用。

DESCRIPTION (provided by applicant): Parkinson's disease (PD), a progressive degenerative movement disorder associated with loss of dopaminergic neurons in substantia nigra (SN), leads to dysfunction. The current therapy, L-dopa, does not block disease progression; therefore, new therapies must be developed. Thus, the aim is to investigate inflammatory events in brain and spinal cord (SC) and their degeneration in PD and characterize whether SC integrity and neurons are lost in PD, contributing to dysfunction. Understanding the mechanisms of damage may help develop new therapeutic strategies. While the etiology of PD is not fully understood, neurotoxins have been im- plicated in PD pathogenesis. Toxic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been extensively used as an experimental model. Since 1-methyl-4-phenylpyridinium ion (MPP+), the active toxic metabolite of MPTP, increases intracellular-free Ca2+ level and promotes mitochondrial dysfunction, a Ca2+-mediated pathology in PD has been hypothesized. Increased Ca2+ levels will promote calpain activation, increase inflammatory responses, and damage brain/SC neurons, axons, and myelin, ultimately leading to functional deficit. Our preliminary findings of direct detection of MPP+ in PD mouse SC, activation of astrocytes and microglia, and increased calpain activity and expression in neurons indicate that SC is also affected. These findings were corroborated by preliminary data showing motoneurons from SC of PD patients are also damaged. MPP+ treatment of ventral SC motor neuron cells (VSC4.1) showed increased intracellular [Ca2+] and calpain activity with loss of membrane potential and death while calpain inhibitors (calpeptin, SJA6017) protected and restored cell function. From these findings, we hypothesize that, since SC coordinates movement and sensation of the body, damage to SC neurons, axons, and myelin, in addition to SN, may be an important factor in PD, and calpain plays a crucial role in this dysfunction by promoting inflammation and cell death and may be a target for therapy. Three specific aims will test the hypotheses. Specific Aim 1 will investigate whether MPTP is directly converted into MPP+ in SC, enters through the degenerating axons from brain, or a combination of both; examine the effects of MPTP (MPP+) on SN and SC neurons and white matter in acute and chronic parkinsonism; assess calpain expression and activity and subsequent inflammation and cell damage; and examine the status of neurons, axons, and myelin in SC of postmortem PD patients. Specific Aim 2 will explore the effects of neurotoxic MPP+ in differentiated VSC4.1 cells and test the neuroprotective efficacy of calpain inhibitors in vitro employing electrophysiological technique. Specific Aim 3 will examine whether calpain inhibitor treatment will attenuate inflammation, prevent apoptosis of brain and SC neurons, protect cells, preserve axons and myelin, and improve function in MPTP-induced PD mice. These studies will delineate the role of calpain in inflammation and neurodegeneration in MPTP-induced PD and the probable neuroprotective efficacy of calpain inhibitors in PD as therapeutic agents. PUBLIC HEALTH RELEVANCE: Since the spinal cord coordinates movement and sensation of the body, damage to spinal cord neurons, in addition to the substantia nigra (brain), and alteration in white matter integrity (i.e., axonal and myelin degeneration) as well as loss of myelin-forming cells may be important factors in Parkinson's disease (PD), and calpain could play a crucial role in this dysfunction. This study will examine the role of calpain in cell and axon damage in the progression of disease in an animal model, the efficacy of calpain inhibitor as a therapeutic agent in vivo and in vitro, and the status of neurons in post-mortem PD tissue. Delineating a role for calpain in the progression of disease could potentially lead to new therapeutic targets since the most potent therapy, L-dopa, does not block the progression of Parkinson's disease.