Numb/Notch信号通路调控一种新的蛋白修饰模式-"寡泛素化"参与Lewy小体形成及其在脑内播散的机制研究

Lewy body (LB) is known as the pathological hallmark of Parkinson' s disease (PD) . Recent studies have suggested that PD attributed to the formation and transmission of LB in the brain. The aggragation of α-synuclein oligomer is the initial step of LB formation; further, Monoubiquitylation of α-synuclein regulate the formation of α-synuclein oligomer and its endocytosis, which is the way of LB dissemination. Therefore, suppression monoubiquitylation of α-synuclein might be an effective way to inhibit the formation and transmission of LB. Numb is the decision factor of neural precursor cell differentiation, antagonism with Notch, takes park in apoptosis, endocytosis, ubiquitin-proteasome degradation physiological process. Our previous studies have confirmed that the Numb/Notch signaling upregulated the level of Tubulin protein, a interactor of a-synuclein and could activate the aggregation of α-synuclein; further, we found colocalization and interaction between Numb and α-synuclein in 293A cells. This study will yield valuable insights into the mechanism of Numb/Notch signaling involved in the formation and transmission of LB through regulates monoubiquitylation of α-synuclein in the brain; and find the new targets for inhibiting the formation and transmission of LB. Try to find a new direction for the prevention and treatment of PD.

路易小体(LB)是帕金森病(PD)的病理标志，最新研究显示PD致病与LB的形成及在脑内神经元间播散密切相关。LB形成和播散的关键步骤即"α-syn寡聚体"成核聚集；而"α-syn寡泛素化修饰"是α-syn寡聚体形成的启动因素，并调控其细胞间传递的重要环节"胞吞作用"。因此，抑制α-syn寡泛素化修饰，可能有效阻止LB形成及播散。Numb是神经前体细胞分化决定因子，最新研究发现其与Notch相互拮抗，参与凋亡、胞吞作用、泛素蛋白酶体降解等诸多过程。本课题组前期研究证实Numb/Notch信号通路上调α-syn相互作用蛋白Tubulin；而Tubulin可以启动α-syn成核聚集；进一步发现Numb与α-syn存在细胞内共定位及相互结合。本项目将深入探讨Numb/Notch通路调控α-syn寡泛素化参与其包涵体形成及通过胞吞传播的机制，寻找阻断LB形成及播散的干预靶点，为防治PD提供新的方向。

帕金森病（Parkinson's disease，PD）患者脑内的嗜伊红的路易小体（Lewy body,LB）被认为是PD的病理学标志。本项目深入探讨了Numb调控α-synuclein单泛素化修饰参与α-synuclein寡聚体形成的机制。在该项目研究中，为探求Numb蛋白对α-syn蛋白的泛素化调控，首先利用细胞免疫荧光技术检测Numb与 α-syn蛋白的共定位，结果显示二者存在亚细胞共定位。由于蛋白间存在复杂的相互作用，为进一步验证Numb与 α-syn的相互作用，我们分别将EGFP-N1或EGFP与HA-α-syn共同转染后利用免疫共沉淀技术及Western-blot检测，结果发现在过表达Numb的沉淀复合物中检测到α-syn蛋白的存在，因此，可证实二者在体内存在相互结合。之后，进一步研究Numb对α-syn寡泛素水平的调控，结果显示，EGFP-Numb与HA-α-syn共同转染后，通过Western blot 检测证实Numb上调了α-syn寡泛素化水平。最后，构建MPP+细胞模型，分别将EGFP-N1、EGFP-Numb转染SH-SY5Y细胞，利用细胞免疫荧光技术检测内源性α-syn包涵体。结果显示，与EGFP-N1组对比，EGFP-Numb组中过表达的Numb促使MPP+诱导的α-syn包涵体的形成。本研究完成了标书中的主要内容，且取得了有意义的成果，并证实了Numb蛋白上调α-syn蛋白的寡泛素化水平，并参与诱导α-syn包涵体的形成。本项目为寻找阻断LB播散的干预靶点及延缓帕金森病病程提供新的手段。