CDK5磷酸化依赖的PMCA1泛素化降解介导慢性钙超载在帕金森病神经元死亡中的机制研究

The classic pathology of Parkinson’s disease was characterized the loss of dopamine-generating cells in the substantia nigra, a region of the midbrain. Many environmental factors and genetic factors maybe lead to the large numbers of dopaminergic neuronal death. Recent studies have demonstrated the chronic calcium overload emerged at early phase of Parkinson’s disease and accelerated the dopaminergic neuronal loss, but the molecular mechanism was still unknown. Here, this study would be elucidated phosphorylation-dependent ubiquitination and degradation of PMCA1 by CDK5 mediates chronic calcium overload in the progressive neuronal death of Parkinson's disease. Firstly, the integrated proteomics quantitative analysis of post-translational modifications were employed to study the relationship between phosphorylation and ubiquitination. Then we discovered the calcium signaling pathway was significant dysregulation by bioinformatics analysis and identified the key protein of PMCA1 as the targeted molecule. Furthermore, we explored the molecular mechanism that CDK5 mediates S1178 site phosphorylation of PMCA1 could give rise to the linkage of K452 site ubiquitination, and promotes degradation of PMCA1, which mediates the cellular chronic calcium overload and neuronal death in various Parkinson’s disease models, including cell models, animal models and clinic brain slices. Finally, inhibiting the pathway of phosphorylation-dependent ubiquitination and degradation of PMCA1 by CDK5 whether play a protective role for dopaminergic neurons in Parkinson’s disease models. In summary, this project would be put forward a novel pathophysiological mechanism and therapeutic target under the dopaminergic neuronal death in Parkinson’s disease.

中脑黑质多巴胺能神经元特异性丢失是帕金森病（PD）患者脑内最典型的病理学特征，多种环境因素与遗传因素均可导致其大量死亡。近年研究发现慢性钙超载在PD早期黑质神经元死亡过程中具有重要意义，但其分子机制尚不明确。本项目首先运用综合定量蛋白质组学检测PD转基因鼠脑黑质组织中磷酸化及泛素化异常情况并分析其关联性，通过生物信息学分析揭示PD中钙信号通路发生异常，进而筛选钙信号调节中关键分子PMCA1作为靶点；其次应用PD细胞、动物模型及临床脑片验证PD黑质神经元中PMCA1磷酸化、泛素化、蛋白降解程度升高及慢性钙超载；然后证明PMCA1蛋白K452位点泛素化介导的自身降解依赖于CDK5磷酸化S1178位点，其降解可引起慢性钙超载并导致神经元死亡；最后验证通过干扰PMCA1磷酸化依赖的泛素化降解对PD中黑质神经元的保护作用，旨在为阐明PD黑质神经元死亡的分子机制和干预治疗提供新的研究思路和药物靶点。

中脑黑质多巴胺能神经元特异性丢失是帕金森病（PD）患者脑内最典型的病理学特征，多种环境因素与遗传因素均可导致其大量死亡。近年研究发现慢性钙超载在PD早期黑质神经元死亡过程中具有重要意义，但其分子机制尚不明确。主要研究内容包括：（1）对早期帕金森病α-synuclein-A30P*A53T转基因小鼠（6月龄）黑质区组织蛋白磷酸化质谱结果进行生物信息学分析与数据挖掘；（2）对早期帕金森病α-synuclein-A30P*A53T转基因小鼠（6月龄）黑质区组织蛋白泛素化质谱结果进行生物信息学分析与数据挖掘；（3）PMCA1蛋白在MPP+处理不同时间点与MPTP动物模型中的mRNA表达水平检测；（4）PMCA1蛋白在MPP+处理不同时间点与MPTP动物模型中的蛋白表达水平检测；（5）鉴定Cdk5磷酸化PMCA1蛋白的氨基酸位点。数据结果显示：磷酸化与泛素化定量修饰蛋白质组学生物信息学分析发现PD中钙信号通路发生异常，并筛选钙信号调节中关键分子PMCA1作为靶点；同时p25/Cdk5信号通路在MPP+处理神经元细胞模型和MPTP药物诱导小鼠动物模型黑质区组织中被激活；PMCA1蛋白在MPP+处理神经元细胞模型和MPTP药物诱导小鼠动物模型黑质区组织中mRNA水平无变化，蛋白水平下调；实验鉴定PMCA1蛋白S1178位点被Cdk5磷酸化。本项目旨在为阐明PD黑质神经元死亡的分子机制和干预治疗提供新的研究思路和药物靶点。