Mechanisms of Exercise-Induced Protection and Rescue in Models of Dopamine Loss

描述(申请人提供)：帕金森氏症(PD)仅在美国就至少有100万人患病。尽管人们对帕金森病的病理生理学了解很多，关于其病因的信息也在不断涌现，但没有任何药物治疗表明对帕金森病的预防或延缓其进展具有显著的持续影响。然而，临床证据表明，体育锻炼就是这样一种治疗方法，这一点得到了与帕金森病运动症状相关的多巴胺(DA)缺乏动物模型的研究的支持。此外，运动是一种实用和可持续的治疗干预措施，可能同时作用于大多数(如果不是全部)能够保护DA神经元和恢复DA功能的细胞事件。这项建议旨在检验运动增加DA功能和保护DA神经元免受毒性损伤的假设，部分原因是神经营养因子(NTF)信号增加，保护线粒体呼吸和刺激血管生成。一个MPTP小鼠模型将被用来检验这一假设。目的1.为了确定运动对MPTP对多巴胺能功能影响的影响，首先建立运动与毒素暴露之间的最佳时间关系，从而探索保护(先运动后毒素)和抢救(毒素后运动)。(1B)然后将评估最佳运动范式对DA细胞丢失以及突触前和突触后DA受体变化的影响。(1C)根据目标1a的规定，对MPTP动物进行运动治疗，将测量纹状体组织和细胞外液中DA和代谢物的水平。(1D)MPTP运动后对线粒体呼吸进行评估。目的2.为了评估NTF在运动诱导的保护中的作用，我们将研究四个不同的NTF家族成员对DA神经元的保护作用：GDNF、BDNF、MANF和VEGF。(2)在运动、MPTP及MPTP与运动的最佳组合后，将确定NTF变化的时间和解剖分布。(B)随后将评估外源性神经营养因子模仿目标1中确定的运动效果的能力。(2C)目标2a和2b建议的神经营养因子和/或其受体将被有条件地剔除，以确定这是否增加了MPTP的毒素影响和减少了运动的保护作用，从而强化了神经营养因子和运动诱导的保护之间存在因果关系的假设。目的3.为了探讨血管生成在运动和神经营养因子诱导的神经保护中的作用，(A)用BrdU测量新细胞和用PECAM-1(CD31)标记血管，评估黑质、纹状体和皮质的血管构筑。(3B)FITC标记的番茄凝集素将用于检测血管未闭。(3)进行血管密度三维分析。(3D)最后，将测量血管生成促进蛋白的表达，包括血管生成素1和2。这些结果将为下一轮专注于运动诱导保护的额外机制的研究计划提供基础，为运动诱导的帕金森病保护提供更有力的证据，并为该病的药物治疗建立靶标。

DESCRIPTION (provided by applicant): Parkinson's disease (PD) affects at least 1 million individuals in the US alone. Although much is known about its pathophysiology and information is emerging about its cause, there are no pharmacological treatments shown to have a significant, sustained impact on the prevention of PD or on attenuation of its progress. However, clinical evidence suggests that physical exercise is such a treatment, and this is supported by studies of animal models of the dopamine (DA) deficiency associated with the motor symptoms of PD. Moreover, exercise is a practical and sustainable therapeutic intervention likely to act simultaneously on most if not all of the cellular events capable of protecting DA neurons and restoring DA function. This proposal is designed test the hypothesis that exercise increases DA function and protects DA neurons against toxic insult due in part to increased neurotrophic factor (NTF) signaling, protection of mitochondrial respiration, and stimulation of angiogenesis. An MPTP mouse model will be used to test this hypothesis. Aim 1. To determine the effects of exercise on the impact of MPTP on dopaminergic function (1a) the optimal temporal relationship between exercise and toxin exposure will first be established, thus exploring both protection (exercise before toxin) and rescue (exercise after toxin). (1b) The impact of the optimal exercise paradigm on DA cell loss and on changes in pre- and post-synaptic DA receptors will then be assessed. (1c) Levels of DA and metabolites in striatal tissue and extracellular fluid will be measured in MPTP animals treated with exercise as dictated by Aim 1a. (1d) Mitochondrial respiration will be assessed after MPTP exercise. Aim 2. To assess the role of NTFs in exercise-induced protection members of four distinct NTF families known to protect DA neurons will be examined: GDNF, BDNF, MANF, and VEGF. (2a) The temporal and anatomical profile of NTF changes will be determined after exercise, MPTP, and the optimal combination of MPTP and exercise. (b) The ability of exogenous NTF to mimic the effects of exercise identified in Aim 1 will then be assessed. (2c) The NTFs and/or their receptors suggested by Aim 2a and 2b will be conditionally knocked out to determine if this increases the toxin impact of MPTP and reduces the protective impact of exercise, thereby strengthening the hypothesis of a causal relationship between NTF and exercise-induced protection. Aim 3. To examine the role of angiogenesis in exercise- and NTF-induced neuroprotection, (a) vasculature be assessed in substantia nigra, striatum, and cortex using BrdU to measure new cells and PECAM-1 (CD31), to mark blood vessels. (3b) FITC-conjugated tomato lectin will be used to detect patent blood vessels. (3c) A 3-dimensional analysis of vascular density will be performed. (3d) Finally, expression of angiogenesis-promoting proteins, including angiopoetins 1 and 2, will be measured. These results will provide the basis for the next iteration of a research program focusing on additional mechanisms underlying exercise-induced protection, provide stronger evidence for exercise-induced protection in PD, and establish targets for pharmacological treatment of the condition.