衔接蛋白σ1A亚基参与阿尔兹海默氏症中APP内涵体分选转运的分子机制研究

APP trafficking along axons and then incorporated onto membranes. The inappropriate processing of APP leads to Abeta1-42 secretion and accumulation, which could cause synaptic defects, Abeta plaque formation and neuronal death. Therefore, it is important to understand the defects in APP transport in early AD pathogenesis. By using high thoughput proteomic screening iTRAQ approach in AD model mice, we have screened for the factors modulating APP expression and trafficking. We have identified consistent high expression level of σ1A adaptin in hippocampus region of two AD model mice, in both mRNA and protein level. Further study reveals that σ1A adaptin affects APP membrane expression level and cleavage in N2A cells. As σ1A adaptin and other adaptin subunits forms the adaptin complex-1 which plays important role in transgolgi sorting and recyclying endosome transport, we hypothesize that σ1A adaptin may regulate APP containing vesicle sorting from transgolgi network to recyclying endosome. In this study, we are planning to explore the underlying mechanism of σ1A adaptin-modulated APP trafficking by using live imaging, super-resolution microscopy and CRISPR/CAS9 knockout technologies. In addition, we are going make conditional knockout of σ1A adaptin in mice hippocampus region, and examine the role of σ1A adaptin in neural circuitry formation and learning and memory, in order to further explore the importance of σ1A adaptin in AD pathogenesis.

β-淀粉样蛋白（Aβ）积累发生在阿尔茨海默氏症（AD）病理早期并在突触障碍、Aβ斑块形成和神经元死亡中起重要作用。其前体蛋白APP在胞内存在多种转运途径，其转运及剪切异常可导致Aβ过多释放及神经元病变。APP转运机制研究是AD病理研究的重要内容，而目前APP内涵体在反面高尔基体到循环内涵体的转运途径尚不清晰。本项目通过蛋白组学手段发现衔接蛋白σ1A 亚基（σ1A adaptin）在早期AD模型小鼠海马区表达增高，且影响APP膜表达及剪切。我们推测σ1A adaptin参与APP在高尔基体及内涵体间的分选转运过程。本项目拟结合活细胞成像，高分辨显微成像，CRISPR/CAS9基因敲除等手段深入研究σ1A adaptin调控APP转运及剪切的分子机制。通过海马区基因敲除观察其对小鼠神经环路功能及行为的影响。本项目旨在发现APP内涵体的分选转运途径，并为AD早期诊断及干预提供可能的分子靶标线索。

β-淀粉样蛋白（Aβ）积累发生在阿尔茨海默氏症（AD）病理早期并在突触障碍、Aβ斑块形成和神经元死亡中起重要作用。其前体蛋白APP在胞内存在多种转运途径，其转运及剪切异常可导致Aβ过多释放及神经元病变。APP转运机制研究是AD病理研究的重要内容，而目前APP内涵体在反面高尔基体到循环内涵体的转运途径尚不清晰。本项目通过蛋白组学手段发现衔接蛋白σ1A 亚基（σ1A adaptin）在早期AD模型小鼠海马区表达增高，且影响APP膜表达及剪切。我们推测σ1A adaptin参与APP在高尔基体及内涵体间的分选转运过程。利用His标签蛋白琼脂糖纯化树脂纯化His-σ1A adaptin融合蛋白（以包涵体的形式存在），再用提纯得到的σ1A adaptin蛋白对免疫血清进行纯化分离；在HEK293T细胞内或原代培养神经元中共表达野生型APP和σ1A adaptin蛋白以及瑞士突变型APPswe和σ1A adaptin蛋白，利用免疫印迹法检验APP 剪切状态，酶联免疫吸附测定（ELISA）检验细胞外Aβ42释放及聚集状态，同时利用免疫荧光手段对APP和各类囊泡标记物进行共定位分析，观察APP在细胞内的分布改变。我们发现在与σ1A adaptin共表达后，APPwt、APPswe的总体表达量均有明显的下调，且σ1A adaptin对APPswe的下调作用更加明显，APPswe的C端水解产物总CTF量也有增加。ELISA检测到APPswe实验组产生的Aβ42也有明显的增加。在神经元胞体内的共定位实验中，我们发现σ1A adaptin会促进APPswe与AP-1复合体大亚基adaptin γ 、早胞内体共定位。过表达或敲减σ1A adaptin均会影响APP转运及剪切状态。因此，我们发现σ1A adaptin可在AD早期介导APP在高尔基体及内涵体之间的分选转运过程，并且对APPwt及APPswe存在差异性的转运调节作用。本课题发现了σ1A adaptin蛋白在AD早期对APP分选、转运及剪切的调控机制，解释了早期AD病变的分子机制，对早期AD检测及干预手段的研发具有积极意义。