Neuroprotection Against Parkinsonian Cell Death

描述(申请人提供)：帕金森病(PD)的特征是进行性和选择性的黑质致密部多巴胺能神经元的丢失。尽管帕金森病的发病机制尚不清楚，但通过使用帕金森病神经毒素诱导的动物模型(如MPTP)或在黑质中病毒靶向过度表达帕金森病相关脑特异性蛋白1-突触核蛋白的研究，已经在这个问题上取得了突破。来自不同模型的证据表明，帕金森病中多巴胺能神经元的退化至少部分是通过凋亡执行级联反应来调节的。此外，小胶质细胞的神经毒性激活也可能参与了帕金森病的多巴胺能神经退行性变。因此，这项提议的长期目标是寻找能够阻断多巴胺能神经元中的凋亡信号通路和帕金森神经毒素诱导的小胶质细胞神经毒性激活的新型神经保护剂。HSP27是小分子热休克蛋白家族的一员，该家族是一组普遍存在的应激蛋白，几乎在所有生物中都有表达。热休克蛋白27在几种形式的损伤后在脑内显著诱导表达，其神经保护作用已被证明。除了已知的蛋白质伴侣功能外，HSP27还具有很强的抗细胞凋亡作用。似乎HSP27在经历了丝氨酸特异的磷酸化后获得了抗凋亡的特性，这可以由氧化应激诱导。然而，HSP27抗细胞凋亡作用的确切机制尚不完全清楚。我们已经创造了过度表达野生型HSP27或非磷酸化HSP27突变体的转基因小鼠。我们利用转基因和基因转染两种方法获得了令人振奋的初步结果，表明：1)过表达HSP27可以保护帕金森病神经毒素诱导的多巴胺能细胞死亡，或通过靶向过表达神经毒性人类突变体(A53T)1-突变体(A53T)1-突变体(A53T)1-突触核蛋白来保护多巴胺能细胞；2)HSP27的神经保护作用依赖于磷酸化介导的蛋白激活；以及3)HSP27可能通过抑制依赖于ASK1/JNK的多巴胺能神经元线粒体死亡信号通路和依赖于ASK1/p38的小胶质细胞的促炎反应来实现其神经保护作用。这项建议试图进一步探讨HSP27作为神经保护分子对抗多巴胺能细胞死亡的作用，并具有未来的治疗意义。支持这一建议的总体假设是，HSP27的增强表达和磷酸化依赖的激活通过新的抗凋亡和抗炎机制来保护多巴胺能细胞的死亡。我们建议使用帕金森病的体内和体外模型来检验这一假说。虽然体内动物模型模拟了帕金森病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进行精确的机制研究来补充体内研究。提出了以下具体目标：目的1.验证转基因过表达和磷酸化依赖的HSP27激活对帕金森病后脑多巴胺能细胞死亡具有保护作用的假说。目的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破坏依赖于ASK1/JNK的线粒体信号通路的一种新的抗凋亡机制来实现的。目的3.验证热休克蛋白27通过第二种机制对抗多巴胺能细胞死亡的假说，该机制涉及破坏脑小胶质细胞中依赖于ASK1/p38的促炎信号。 公共卫生相关性：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丢失，目前还没有有效的神经保护治疗方法。这项建议的目的是研究一种小休克蛋白的增强表达是否可以改善帕金森神经毒素诱导的帕金森病细胞和动物模型中多巴胺能细胞的死亡。这些信息将对未来开发新的治疗策略来治疗帕金森氏病，并可能治疗其他神经疾病有价值。

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. PUBLIC HEALTH RELEVANCE: Parkinson's disease (PD) is characterized by progressive and selective loss of dopaminergic neurons in substantia nigra pars compacta, for which an effective neuroprotective treatment is currently unavailable. The objective of this proposal is to investigate whether enhanced expression of a small shock protein can ameliorate Parkinsonian neurotoxin-induced dopaminergic cell death in cellular and animal models of PD. This information will be valuable for future development of new therapeutic strategies for the treatment of Parkinson's disease and, possibly, other neurological disorders.