Extra-nigral Neurodegeneration in Experimental Parkinson's Disease

描述(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)多巴胺能神经元的丢失有关，导致功能障碍。目前的治疗方法L-多巴并不能阻止疾病的进展，因此，必须开发新的治疗方法。因此，我们的目的是研究脑和脊髓(SC)中的炎症事件及其在帕金森病中的变性，并表征帕金森病患者的SC完整性和神经元是否丢失，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。虽然帕金森病的病因还不完全清楚，但神经毒素已经参与了帕金森病的发病机制。有毒的1-甲基-4-苯基-1，2，3，6-四氢吡啶(MPTP)已被广泛用作实验模型。由于MPTP的活性毒性代谢产物1-甲基-4-苯基吡啶离子(MPP+)可升高细胞内游离钙离子水平，促进线粒体功能障碍，故提出了一种钙离子介导的帕金森病发病机制。Ca~(2+)水平升高会促进钙蛋白酶的激活，增加炎症反应，损伤脑/SC神经元、轴突和髓鞘，最终导致功能障碍。我们的初步研究结果表明，PD小鼠SC中MPP+的直接检测到，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，表明SC也受到了影响。这些发现得到了初步数据的证实，即帕金森病患者SC的运动神经元也受到了损害。腹侧SC运动神经元细胞(VSC4.1)经MPP+处理后，细胞内[Ca~(2+)]浓度升高，钙蛋白酶活性升高，膜电位降低，细胞死亡，而钙蛋白酶抑制剂(CalPeptin，SJA6017)对细胞功能有保护和恢复作用。根据这些发现，我们推测，由于SC协调身体的运动和感觉，除了SN，SC神经元、轴突和髓鞘的损伤可能是PD的一个重要因素，而Calain通过促进炎症和细胞死亡在这种功能障碍中发挥关键作用，并可能成为治疗的靶点。三个具体目标将检验这些假说。具体目标1将研究MPTP在SC中是否直接转化为MPP+，从脑内通过退行性轴突进入，还是两者的组合；检测MPTP(MPP+)对急慢性帕金森病患者SN和SC神经元和白质的影响；评估Calain的表达和活性以及随后的炎症和细胞损伤；以及检测死后PD患者SC中神经元、轴突和髓鞘的状态。具体目的2探讨神经毒性MPP+对分化的VSC4.1细胞的影响，并利用电生理技术检测钙蛋白酶抑制剂对VSC4.1细胞的保护作用。具体目标3将检查Calain抑制剂治疗是否可以减轻MPTP诱导的PD小鼠的炎症，防止脑和SC神经元的凋亡，保护细胞，保护轴突和髓鞘，并改善功能。这些研究将阐明钙蛋白酶在MPTP诱导的帕金森病炎症和神经退行性变中的作用，以及作为治疗药物的钙蛋白酶抑制剂在帕金森病中可能的神经保护作用。 公共卫生相关性：由于脊髓协调身体的运动和感觉，除了黑质(脑)外，脊髓神经元的损伤、白质完整性的改变(即轴突和髓鞘变性)以及髓鞘形成细胞的丢失可能是帕金森病(PD)的重要因素，而Calain可能在这种功能障碍中发挥关键作用。这项研究将在动物模型中检测细胞和轴突损伤中钙蛋白酶在疾病进展中的作用，钙蛋白酶抑制剂在体内和体外作为治疗剂的有效性，以及死后帕金森病组织中神经元的状态。阐明钙蛋白酶在疾病进展中的作用可能会导致新的治疗靶点，因为最有效的疗法L-多巴并不能阻止帕金森病的进展。

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.