从CREB信号通路探讨δ-阿片受体对帕金森病转基因小鼠α-synuclein寡聚体形成调控的作用机制

In Parkinson disease(PD) research, there are three major difficulties:unknown etiology, pathogenesis uncertainty and lack of treatment.α-Synuclein protein is the main component of Lewy bodies. Strong evidence suggests that in PD,cytotoxic α-synuclein oligomers are key to pathogenicity.The aggregation ofα-synuclein in the brain has been implicated as a critical step in the development of PD.Studies have shown that, δ- opioid receptor (DOR) densely distributed in the striatum plays a role in the pathogenesis of PD. Our previous studies on the cellular model of PD found that δ- opioid receptors by inducing cAMP response element binding protein (CREB) phosphorylation, inhibited MPP+ induced overexpression of α-synuclein and oligomer formation in HEK293T cells.On the basis of our previous work，We will observe in vivo the effects of δ- opioid receptors on the behavioral changes in α-synuclein transgenic model of Parkinson disease. We will investigate the effects of the distribution of DOR on the expression of α-synuclein in mouse brain tissue and the formation of α-synuclein oligomers in striatum by using immunofluorescence, ELISA, etc. And also we will detect CREB Ser-133, TORC1 / SIK1 and target gene expression,etc.in order to explore the correlation between DOR and α-synuclein protein, the signaling pathways and molecular mechanisms may be involved in PD and the possible mechanism of anti-Parkinson disease and neuroprotection of δ- opioid receptor. These study may be key molecules for the development of therapeutics for PD.

帕金森病（PD）研究存在三大难点：发病原因不明，发病机制不确定，治疗措施匮乏。α-synuclein病理性聚集是PD临床症状发生、发展及受累脑区变性的主要原因。研究表明，δ-阿片受体（DOR）在纹状体分布密集，在PD的发病机制中起着一定作用。我们前期研究发现，DOR通过诱导转录因子CREB磷酸化，抑制了MPP+ 所致的细胞内α-synuclein过度表达及寡聚体形成，可能具有抗帕金森病的作用。课题拟在在此基础上，通过观察DOR对PD转基因小鼠行为学的改变，应用分子生物学方法检测DOR对小鼠脑组织中α-synuclein的表达分布、寡聚体形成的影响，检测CREB Ser-133 、TORC1/SIK1及靶基因等表达，探讨DOR与α-synuclein蛋白的相关性及两者参与PD的可能信号通路及分子机制，为进一步探索DOR抗帕金森病作用及神经保护的可能机制，为寻求PD临床治疗学的新靶点打下基础。

本研究通过观察δ-阿片受体在帕金森病细胞模型上对α-synuclein表达、α-synuclein寡聚体形成及其细胞内Lewy小体形成的影响，探讨δ-阿片受体与α-synuclein的相关性，进一步明确δ-阿片受体抗帕金森病作用及神经保护作用的可能机制；并通过观察α-synuclein寡聚体、δ-阿片受体对CREB Ser-133 、TORC1/SIK1及靶基因BDNF等表达的影响，探索α-synuclein、δ-阿片受体两者参与帕金森病的可能信号通路及分子机制。结果显示， DOR激活可能通过CREB途径减弱MPP+或严重缺氧诱导的突触核蛋白表达及病理性聚集；在α-synuclein过度表达和突变条件下通过TORC1/SIK1/CREB途径，抑制了毒性α-synuclein寡聚体的形成。DJ-1基因参与了帕金森病损伤的DOR保护。.在α-synuclein-pEGFP转基因帕金森病小鼠模型上，通过行为学检测、免疫荧光、HE染色观察δ-阿片受体对帕金森病小鼠不同时间段（4月龄、6月龄、8月龄）行为学的改变、对α-synuclein在小鼠脑组织中表达分布的影响、对脑纹状体α-synucl ein寡聚体形成的调控及其纹状体多巴胺能神经元细胞内Lewy小体的形成的影响；同时应用针刺干预，探讨针刺对帕金森病小鼠纹状体miRNA表达水平的影响及其可能机制 。结果显示：δ-阿片受体及针刺可能通过上调miRNA-124表达改善帕金森病转基因小鼠行为，通过miRNA-124/p-CREB/BDNF信号通路上调p-CREB、BDNF表达水平。为深入探索帕金森病的发病机制，寻求帕金森病临床治疗学的新靶点打下基础。