多巴胺醌蛋白导致多巴胺细胞死亡的途径与分子机理研究

The theory that the accumulation of dopamine quinone (DAQ)-modified proteins (quinoprotein) in aged substantia nigra contributes to cell death is the most plausible theory explaining the selective loss of nigral dopaminergic neurons in Parkinson’s disease. Yet, the exact molecular mechanism remains elusive, which hampers the research progress in this area. We found five quinoproteins that were specifically increased in aged substantia nigra of rat midbrain. Interestingly, the majority of these quinoproteins are key enzymes involved in energy metabolism. Based on our preliminary results, we propose: the increase of DAQ-modified metabolic enzymes in aged nigral dopaminergic neurons disrupts cellular energy homeostasis, making these cells more vulnerable to the neurotoxic factors in Parkinson’s disease and resulting in the selective loss of nigral dopaminergic neurons...The following approaches are proposed in the application: (1) We will express and purify the four proteins identified in our preliminary studies, verify their modification by DAQ using in clean in vitro settings, and determine the relationship between DAQ modification and their enzyme activity. (2) We will detect if these four proteins will be modified by DAQ in cultured cells incubated with exogenous DA, and determine whether cellular energy metabolism is disrupted. (3) We will reduce the expression of these proteins in cultured cells, determine their influence on cellular energy metabolism, and more importantly, determine their influence on the susceptibility of these cells to the neurotoxic factors in Parkinson’s disease. (4) We will determine whether increase of phase II enzyme expression is able to reduce DAQ-modified proteins in aged substantia nigra and protect the dopaminergic neurons in the substantia nigra. (5) We will analyze the quinoproteins in the substantia nigra of differently aged primates and confirm our preliminary results in primates. ..The experiments proposed in this application will help us to understand the molecular mechanism for the selective loss of nigral dopaminergic neurons in Parkinson’s disease, and provide a new avenue to prevent the pathogenic changes in Parkinson’s disease.

多巴胺醌蛋白导致黑质多巴胺神经元特异性死亡的假说是重要的帕金森病（PD）发病学说，但其分子机理至今不明。我们在对大鼠中脑黑质的醌蛋白分析中，首次发现5种在老龄黑质脑区特异性增加的醌蛋白，其中4种为细胞能量代谢的关键酶。据此，我们提出：随年龄增加而累积的能量代谢相关酶的多巴胺醌化是导致PD病人黑质多巴胺能神经元特异性死亡的关键所在。项目从如下方面证明此假说：①重组表达这四种蛋白，观察其与多巴胺醌的结合特性及功能变化；②细胞学研究多巴胺醌对这些蛋白的醌化及能量代谢的影响；③下调这些蛋白的表达对细胞能量代谢的影响，以及是否使细胞更易于被PD的致病因子所损伤；④使动物高表达Ⅱ相酶抑制醌的形成，检测其对4种蛋白的醌化抑制和对多巴胺神经元的保护作用；⑤检测不同年龄灵长类中脑黑质醌蛋白的特性，并与鼠脑的结果进行比较。项目对揭示PD多巴胺神经元特异性死亡的具体机理具有重要意义，并探讨一种新的PD防治策略。