β-淀粉样蛋白对exosome分泌及其介导受体细胞效应的影响和机制研究

Alzheimer's disease (AD) is a common neurodegenerative disorder which is characterized by cognitive dysfunction and memory impairment. Most scholars believed that β-amyloid (Aβ) peptides deposition is the initial factors and the central link with AD pathology. However, up to date, complete conclusion has not been reached about AD pathogenesis. Exosomes are endosome-derived vesicles formed during the formation of multi-vesicular bodies (MVBs). Exosome can not only secrete contents but also present proteins or RNAs to the recipient cell. Previous research found that several β-amyloid precursor protein (APP) derivatives accumulate in exosomes. More recently, exosomes were demonstrated to contain Aβ peptides. Exosome-associated protein was also specifically enriched in senile plaques of AD brain sections. Our previous studies have shown that Aβ could increase the expression of exosome-associated proteins. However, the mechanism of Aβ on exosome secretion and the exosome-mediated effect on recipient cell remain to be elucidated. Based on the above research foundation, we hypothesized that "Aβ may involved in AD pathogenesis through influencing the secretion of exosome protein, RNAs and the exosome-mediated recipient cellular effects". This application project intends to investigate the effect of Aβ on exosome secretion and the mechanisms in regulation of exosome secretion. Further attention will be focused on the effect of exosome on recipient cell. This research will provide a novel viewpoint in Aβ neurotoxicity mechanisms. The results of this study are helpful to reveal the role of the exosome in AD pathological mechanism, and provide a theoretical and experimental basis for the use of exosome as a carrier for AD drug therapy.

阿尔茨海默病(AD)是常见的神经系统退行性疾病，Aβ介导的神经毒性是AD病理的中心环节，但其确切机制尚未阐明。内体起源的外体(exosome)不仅分泌内容物，还可递呈蛋白或RNA到受体细胞。研究发现，exosome内分布有APP和Aβ，exosome蛋白在AD老年斑有沉积。我们前期的研究发现，Aβ可诱导exosome蛋白表达的增加，但Aβ对exosome分泌的影响及exosome介导的受体细胞效应尚未得到解析。我们推测"Aβ可能通过影响exosome蛋白和RNA的分泌，以及exosome介导的细胞效应参与AD的病理进展"。本项目拟观察Aβ对exosome分泌的影响，以及调控exosome分泌的分子机制；并进一步探讨分泌的exosome对受体细胞的效应及其机制。本课题研究将从exosome的视角进一步揭示Aβ的神经毒性作用机制，并为从exosome的角度进行AD治疗提供理论参考和实验依据。

阿尔茨海默病(AD)是常见的神经系统退行性疾病，Aβ介导的神经毒性是AD病理的中心环节。本研究旨在探讨Aβ是否影响外体exosome的分泌，及调控exosome分泌的Munc-18机制，并探讨受体细胞表面DR6在Aβ介导的神经毒性中的作用及其机制。.细胞培养基提取exosome组分，western blot结果显示有Alix和Flotillin等exosome蛋白的存在，且有APP和DR6的分布。Aβ可增加exosome中Alix水平。Alix在AD转基因鼠Aβ免疫阳性的老年斑中及其周围有分布。硫磺素荧光结果显示exosome可促进Aβ纤维化聚集。mAβ和exosome共孵育可增加Annexin和PI阳性细胞数量，显著增加LDH的释放，且可诱导caspase-3和caspase-6的激活，以及BAX表达的上调，此外，exosome标志蛋白Alix亦有显著增加。.对于Aβ影响exosome分泌的Munc-18机制，western blot结果显示，Munc-18在细胞体和细胞核皆有分布，KCL诱导的兴奋刺激可诱导Munc-18的出核。免疫沉淀结果显示转录因子UXT可与Munc-18结合，KA介导的兴奋可诱导UXT的出核。Aβ可诱导Munc-18表达和分布的增加，在AD转基因鼠有Munc-18和UXT表达的增加。Munc-18干扰可增加Alix及APP和DR6等在exosome中的表达，Munc-18干扰可诱导转录因子Mecp2磷酸化改变，且NRSF和REST水平有显著降低。.我们接下来进一步探讨了exosome的受体细胞效应。结果显示， APP-N片段可激活凋亡通路增强Aβ毒性，Aβ可诱导APP剪切片段的产生。Aβ可诱导DR6表达的增加，DR6在AD鼠有表达和分布的增加。Pull-down结果显示APP和DR6有免疫共沉淀，在AD转基因鼠脑中，APP与DR6有共沉淀。DR641-341、DR6抗体及DR6 RNAi等干预可抑制Aβ/APP18-286共孵育诱导的细胞损伤，其机制可能是通过抑制caspase-3,6的激活及BAX表达。.综上所述，我们研究的显示Aβ可诱导exosome的分泌，exosome可促进Aβ纤维聚集，Munc-18可参与调节exosome的分泌，DR6可作为受体细胞上潜在的exosome作用靶点。该研究对于认识AD的病理机制提供了一个新的视角。