WDR91在内吞体运输及神经发育中的功能和机制研究

Normal neuronal development is essential for the establishment of neural networks and the maintenance of brain function. WDR91 is a novel protein located on endosomes, and brain-specific WDR91 inactivation impairs neuronal development, however the underlying mechanisms are still limited. Endosomal trafficking plays a crucial role in neuronal development, protein sorting and transport are regulated by the Retromer complex and small GTPase Rab proteins. Therefore, investigating the modulation of WDR91 on Retromer complex and Rab protein is helpful to reveal the function of WDR91 for endosomal trafficking and neuronal development. Previous work in this project show that loss of WDR91 causes defects of Retromer-dependent receptors trafficking. In addition, the above trafficking pathway can be rescued by re-expressing WT WDR91 but not the Rab7 binding-deficient WDR91. In this project, we propose to explore the regulatory relationship among WDR91, Retromer complex and Rab7 in detail, and further elucidate the mechanism of WDR91 in neuronal development. Through mechanistic insights into the role of endosomal trafficking in neuronal development, our work may provide effective ways to maintain cellular protein homeostasis and provide potential ways to prevent and treat neurological diseases caused by homeostatic disorders.

神经发育对神经网络的建立和大脑功能的维持至关重要。WDR91是在内吞体定位的新蛋白，特异性敲除WDR91的小鼠出现神经发育障碍，但其潜在原因仍知之甚少。由内吞体介导的运输通路是神经发育的必要环节，Retromer复合体和小GTP酶Rab蛋白参与内吞体蛋白分选及转运过程，因此研究WDR91对Retromer复合体和Rab蛋白的影响有利于从内吞体运输的角度揭示WDR91在神经发育中的功能。前期工作发现，缺失WDR91使内吞体依赖于Retromer复合体的逆向运输过程受阻，并且与Rab7结合存在缺陷的WDR91无法恢复这一运输过程。本项目后续工作将深入探究WDR91、Retromer复合体和Rab7三者的调控关系，并进一步阐明WDR91在神经发育中的作用机理。相关结果有望深化内吞体运输对神经发育的作用的认识，为维护胞内蛋白稳态提供有效的方法，为预防和治疗稳态紊乱引发的神经疾病提供潜在的新思路。

逆向蛋白运输复合体Retromer介导内吞货物蛋白逆向回收到细胞膜或高尔基体，其缺陷与神经发育障碍密切相关。Retromer由VPS26-VPS29-VPS35三聚体和分选连接蛋白Sorting Nexins（SNXs）组成，并且其受到Rab7和WASH复合体等因子的调控。伴随着早-晚期内吞体转化，Retromer依赖的运输过程在内吞体特定的局部区域发生。然而，Retromer及其已知的调控因子如何在内吞体上相互协调形成用于循环蛋白的局部区域仍知之甚少。WDR91作为小GTP酶Rab7的效应因子被招募到内吞体，负向调控内吞体上磷脂酰肌醇-3-磷酸（PI3P）的水平，促进早晚期内吞体转化。在此基础上，本项目发现敲除WDR91（KO-91）的细胞中Retromer介导的膜表面受体及胞内受体的运输过程均受阻，并且Retromer在内吞体整个表面发生累积。当在KO-91细胞中表达野生型WDR91而非与Rab7结合有缺陷的WDR91突变体时，Retromer重新定位在内吞体的局部区域，Retromer依赖的运输过程也被成功挽救。这些结果表明WDR91的缺失通过破坏内吞体上用于循环蛋白的局部区域的形成以损害Retromer依赖的逆向运输过程，并且与Rab7结合对于WDR91功能的正常发挥是必不可少的。进一步研究发现WDR91使Rab7与Retromer的连接加强，WDR91还与内吞体上控制微丝聚合的WASH复合体的核心亚基FAM21相互作用，进而将Rab7,Retromer和WASH复合体限制在WDR91定位的特定区域，促进内吞体上用于循环蛋白的局部区域的形成，有利于Retromer在该区域发挥作用最终形成管状结构离开。项目研究成果拓展了对Retromer调控机制的认识，为控制内吞体分选转运和维持胞内蛋白稳态提供宝贵线索，为预防和治疗内吞体运输障碍导致的神经疾病提供科学的理论依据。