胰高血糖素样肽-1调控对帕金森病α-突触核蛋白异常聚集的作用及机制

Parkinson’s disease (PD) is a progressive neurodegenerative disorder. Currently neuroprotective therapy for PD is still limited due to the lack of effective intervention of abnormal misfolded α-synuclein, which is a key hallmarker in PD pathology. Previous researches showed that GLP-1 R stimulation led to improvements in motor and non-motor deficits in PD models and patients. In this project, we will use GLP-1 agonist and DPP-IV to treat the PD rat models with pathological α-synuclein accumulation by injecting AAV-A53T-α-synuclein into SN, and A53T transgenic mice. At the baseline and 3w、6w、12w、18w after the injection for rat, or 3m，6m，9m，12m for transgenic mice respectively, we will evaluate their effect by assessing behavioral test of cylinder test and rotarod, immunohistochemistry of tyrosine hydroxylase and α-synuclein and its quantification with sterology, ultrastructural morphology on Electron Microscope, Western blotting of tyrosine hydroxylase and α-synuclein, neuroimaging on dopamine transporter/VMAT2 by microPET/CT, and analysis of microdialysis on dopamine and other neurotransmitters. We will investigate the potential upstream mechanism by exploring RNA Seq, and then Gene Ontology/pathway enrichment analysis, and downstream processes by regulating autophagy/lysosomal pathways including macroautophagy, chaperone mediated autophagy，and ubiquitin-proteasome system. Additionally, we will identify different α-synuclein toxic species via AlphaLISA assay. Finally, we will perform the experiments on human iPS cell-derived dopamine neuron from PD patients with α-syn mutation, to validate the efficacy and mechanism of GLP-1 agonist and DPP-IV. This study will provide an important experimental basis for exploring the neuroprotective therapy in PD with abnormalα-synuclein aggregation.

帕金森病目前缺乏神经保护治疗,是由于对PD核心病理环节α-突触核蛋白(α-syn)异常聚集缺乏有效干预。前期证据显示调节胰高血糖素样肽-1（GLP-1）具有减低α-syn聚集改善PD功能的前景。本项目应用GLP-1激动剂和阻断其降解的DPP-IV抑制剂, 应用行为学评估、免疫组化及体视学、电镜、免疫印迹定量分析；microPET/CT、微透析神经递质分析，探讨它们对于具有α-syn异常聚集的大小鼠PD模型的作用；应用RNA Seq转录组测序和Gene Ontology/通路富集分析、蛋白酶体抑制剂及泛素连接酶抑制剂干预，以及AlphaLISA assay检测不同毒性形式α-syn，探讨GLP-1调控α-syn可能的上下游作用机制；最后在源于病人的α-syn突变iPS转化的多巴胺神经元细胞模型上验证上述作用及机制。本研究将为探索作用于α-syn异常聚集的PD神经保护新疗法提供重要的实验依据。

目前在帕金森病领域尚无有效的疾病修饰治疗，而在已有广泛应用的临床药物中寻找对帕金森病有疾病修饰作用的药物是一种高效而经济的方法。在现有文献的支持下，我们围绕帕金森病致病的核心蛋白--α突触核蛋白，利用在糖尿病领域中已有广泛应用的胰高血糖素样肽-1（GLP-1）激动剂艾塞那肽，探究其在帕金森病大鼠模型上的神经保护作用，并深入探讨其作用机制。我们的研究表明，Exendin-4可以有效清除模型大鼠脑内过度累积的致病性α突触核蛋白，并显著减少大鼠中脑黑质多巴胺能神经元的死亡以及纹状体多巴胺能神经元末梢的丢失和功能损伤，缓解大鼠的帕金森病样运动症状，并且通过体内和体外实验发现Exendin-4可能是通过抑制PI3K/Akt/mTOR通路进而激活自噬-溶酶体途径来发挥作用的。目前已有临床试验证明了艾塞那肽对于帕金森病具有治疗作用，而本研究结果从帕金森病的核心发病机制层面为艾塞那肽的疾病修饰作用提供了新的证据，鉴于GLP-1激动剂具有较高的安全性，提示其将来有望填补帕金森病疾病修饰药物的空白。