Extra-nigral Neurodegeneration in Experimental Parkinson's Disease

帕金森病（PD）是一种与多巴胺缺乏相关的进行性退行性运动障碍， 黑质（substantia nigra，SN）神经元的损伤导致功能障碍。目前的治疗，左旋多巴，不阻断dis-dopa- 因此，必须开发新的治疗方法。因此，目的是研究炎症 脑和脊髓（SC）中的事件及其在PD中的变性，并表征SC完整性和 神经元在PD中丢失，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PD的病因尚未完全了解，但神经毒素已被发现。 在PD发病机制中的作用有毒的1-甲基-4-苯基-1，2，3，6-四氢吡啶（MPTP）已被广泛应用于 用作实验模型。由于1-甲基-4-苯基吡啶鎓离子（MPP+）， MPTP增加细胞内游离Ca 2+水平并促进线粒体功能障碍，这是一种Ca 2+介导的病理学。 PD中的ogy已经被假设。Ca 2+水平的增加会促进钙蛋白酶的激活，增加炎症反应， tory反应，并损伤脑/SC神经元、轴突和髓鞘，最终导致功能缺陷。我们 PD小鼠SC中MPP+直接检测的初步结果，星形胶质细胞和小胶质细胞的活化， 神经元中钙蛋白酶活性和表达的增加表明SC也受到影响。这些发现 初步数据证实，PD患者SC的运动神经元也受损。MPP+ 腹侧SC运动神经元细胞（VSC4.1）的处理显示细胞内[Ca 2 +]和钙蛋白酶活性增加 钙蛋白酶抑制剂（calpeptin，SJA 6017）保护并恢复了细胞膜电位的丧失和死亡 细胞功能根据这些发现，我们假设，由于SC协调运动和感觉， 除SN外，SC神经元、轴突和髓鞘的损伤可能是机体损伤的重要因素 钙蛋白酶通过促进炎症和细胞死亡在这种功能障碍中起关键作用 并且可以是治疗的目标。三个具体目标将检验这些假设。具体目标1将调查 MPTP是否在SC中直接转化为MPP+，是否通过变性轴突从脑进入，或者是否通过神经元进入。 观察MPTP（MPP+）对急性脑缺血后SN和SC神经元以及白色物质的影响。 和慢性帕金森综合征;评估钙蛋白酶表达和活性以及随后的炎症和细胞 损伤;并检查死后PD患者SC中神经元，轴突和髓鞘的状态。具体 目的2探讨MPP+对分化的VSC4.1细胞的神经毒性作用，并检测MPP+对VSC4.1细胞的神经保护作用。 钙蛋白酶抑制剂在体外使用电生理技术的功效。具体目标3将审查 钙蛋白酶抑制剂治疗是否会减轻炎症，防止脑和SC神经元的凋亡， 在MPTP诱导的PD小鼠中保护细胞，保护轴突和髓鞘，并改善功能。这些研究将 描述钙蛋白酶在MPTP诱导的PD中炎症和神经变性中的作用，以及可能的 钙蛋白酶抑制剂作为治疗剂在PD中的神经保护功效。

Parkinson's disease (PD), a progressive degenerative movement disorder associated with loss of dopaminer- gic neurons in substantia nigra (SN), leads to dysfunction. The current therapy, L-dopa, does not block dis- ease 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 de- velop 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 pathol- ogy in PD has been hypothesized. Increased Ca2+ levels will promote calpain activation, increase inflamma- tory 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.