Neuroprotection Against Parkinsonian Cell Death

描述（由申请人提供）：帕金森病（PD）的特征是黑质致密部多巴胺能神经元的进行性和选择性丧失。尽管PD的发病机制尚不清楚，但利用帕金森神经毒素（如MPTP）或病毒靶向PD相关脑特异性蛋白1-Synuclein在SNc中过表达诱导的动物模型研究，在这一问题上取得了突破。来自不同模型的证据表明，PD中多巴胺能神经元的退化至少部分是通过凋亡-执行级联介导的。此外，小胶质细胞的神经毒性激活也可能导致PD患者多巴胺能神经变性。因此，本研究的长期目标是寻找能够阻断多巴胺能神经元凋亡信号通路和帕金森神经毒素诱导的小胶质细胞的神经毒性激活的新型神经保护剂。HSP27是小热休克蛋白家族的一员，热休克蛋白是一组普遍存在的应激蛋白，几乎在所有生物体中都有表达。HSP27在多种损伤后的脑组织中被显著诱导表达，其神经保护作用已被证实。除了已知的作为蛋白伴侣的功能外，HSP27还具有有效的抗凋亡作用。似乎HSP27在经过丝氨酸特异性磷酸化后获得了抗凋亡特性，这可以由氧化应激诱导。然而，HSP27抗凋亡作用的确切机制尚不完全清楚。我们已经创建了过表达野生型HSP27或非磷酸化HSP27突变体的转基因小鼠。通过转基因和基因转染两种方法，我们已经获得了令人兴奋的初步结果，这些结果表明：1)过表达HSP27可以防止帕金森神经毒素或靶向过表达人类神经毒性突变体（A53T） 1- Synuclein诱导的多巴胺能细胞死亡；2) HSP27的神经保护作用依赖于磷酸化介导的蛋白活化；3) HSP27可能通过抑制多巴胺能神经元中ASK1/ jnk依赖的线粒体死亡信号通路和小胶质细胞中ASK1/p38依赖的促炎反应来实现其神经保护作用。本研究试图进一步探索HSP27作为抗多巴胺能细胞死亡的神经保护分子，并具有未来的治疗意义。这一提议的总体假设是，HSP27的表达增强和磷酸化依赖激活通过新的抗凋亡和抗炎机制保护多巴胺能细胞死亡。我们建议使用体内和体外PD模型来验证这一假设。虽然体内动物模型模拟PD病理生理变化的一些重要方面，但体外细胞模型将通过允许精确的机制研究来补充体内研究。提议的具体目标如下：目标1。验证HSP27的转基因过表达和磷酸化依赖性激活保护帕金森损伤后大脑中多巴胺能细胞死亡的假设。目标2。验证HSP27对多巴胺能细胞死亡的直接神经保护作用是通过一种新的抗凋亡机制介导的，该机制涉及破坏ASK1/ jnk依赖的线粒体信号通路。目标3。验证HSP27通过第二种机制保护多巴胺能细胞死亡的假设，该机制涉及破坏脑小胶质细胞中依赖ASK1/p38的促炎信号。

DESCRIPTION (provided by applicant): Parkinson's disease (PD) is characterized by progressive and selective loss of dopaminergic neurons in substantia nigra pars compacta. Although the pathogenesis of PD remains unclear, a breakthrough on this question has emerged from studies using animal models induced by Parkinsonian neurotoxins, such as MPTP, or viral-targeted overexpression of the PD-related brain-specific protein 1-Synuclein in the SNc. Evidence from different models suggests that the degeneration of dopaminergic neurons in PD is mediated at least in part via apoptosis-execution cascades. In addition, the neurotoxic activation of microglia may also contribute to dopaminergic neurodegeneration in PD. Thus, the long-term goal of this proposal is to identify novel neuroprotective agents that are capable of blocking the apoptosis-signaling pathways in dopaminergic neurons and the neurotoxic activation of microglia induced by Parkinsonian neurotoxins. HSP27 is a member of the small heat shock protein family, a group of ubiquitous stress proteins that are expressed in virtually all organisms. The expression of HSP27 is markedly induced in the brain after several forms of injury, and its neuroprotective role has been demonstrated. In addition to its known function as a protein chaperone, HSP27 has potent anti-apoptotic effects. It appears that HSP27 gains its anti-apoptotic properties after undergoing serine-specific phosphorylation, which can be induced by oxidative stress. However, the precise mechanism underlying the anti-apoptotic effect of HSP27 is not fully understood. We have created transgenic mice overexpressing either the wild-type HSP27 or a non-phosphorylatable HSP27 mutant. Using both transgenic and gene-transfection approaches, we have obtained exciting preliminary results which suggest that: 1) overexpression of HSP27 protects against dopaminergic cell death induced by Parkinsonian neurotoxins or by targeted overexpression of the neurotoxic human mutant (A53T) 1- Synuclein; 2) the neuroprotective effect of HSP27 is dependent on phosphorylation-mediated activation of the protein; and 3) HSP27 may achieve its neuroprotective effects via inhibiting both the ASK1/JNK-dependent mitochondrial death-signaling pathway in dopaminergic neurons and ASK1/p38-dependent pro-inflammatory reactions of microglia. This proposal attempts to further explore HSP27 as a neuroprotective molecule against dopaminergic cell death, with future therapeutic implications. The overall hypothesis underlying this proposal is that enhanced expression and phosphorylation- dependent activation of HSP27 protects against dopaminergic cell death via novel anti-apoptotic and anti-inflammatory mechanisms. We propose to test the hypothesis using both in vivo and in vitro models of PD. While the in vivo animal models mimic some important aspects of pathophysiological changes in PD, the in vitro cellular models will complement the in vivo studies by allowing for precise mechanistic studies. The following specific objectives are proposed: Aim 1. Test the hypothesis that transgenic overexpression and phosphorylation-dependent activation of HSP27 protects against dopaminergic cell death in the brain following Parkinsonian insults. Aim 2. Test the hypothesis that the direct neuroprotective effect of HSP27 against dopaminergic cell death is mediated via a novel anti-apoptotic mechanism involving the disruption of ASK1/JNK-dependent mitochondrial signaling pathways. Aim 3. Test the hypothesis that HSP27 protects against dopaminergic cell death via a second mechanism which involves the disruption of ASK1/p38- dependent pro-inflammatory signaling in brain microglia.