ApoE促进LRP1介导小胶质细胞内化降解Aβ的作用研究

Alzheimer’s disease (AD) is characterized by the accumulation and deposition of β-amyloid (Aβ) peptides within the brain, leading to the perturbation of synaptic function and neuronal loss that typifies the disease. Microglial internalization and degradation of Aβ are believed to be key mechanisms of the initial defense of the brain against the toxic accumulation of Aβ. Therefore, promoting microglia activation may be a powerful strategy to clear Aβ and prevent Aβ-associated pathological events and neuronal loss. In this study, we use immunoelectron microscopy, confocal microscopy, ELISA, immunoprecipitation, Western blotting, gene cloning, RNAi, and Morris water maze to investigate astroglial apolipoprotein E promotes low density lipoprotein receptor-related protein 1 (LRP1) mediated beta-amyloid protein internalization and degradation in microglia, and to assess phosphatidylinositol 3 kinase/Akt(PI3K/Akt) signaling pathways involving the regulation of Aβ internalization and degradation. First, astrocytes and microglia were obtained from postnatal apoe knock-out mouse (apoe-/-) by a standard enzyme treatment protocol. The astrocyte and microglia purity were determined by glial fibrillary acidic protein (GFAP) and ionizing calcium adapter binding molecule 1(Iba1) positivity respectively through immunofluorescence. Recombinant plasmid that carrying each apoe genotype (ε2, ε3, ε4) were transfected into apoe knocked out astrocytes. Then microglia was exposed to conditioned media from astrocytes. Choose an ApoE subtype that significant promotes Aβ internalization and degradation in microglia. Second, to confirm the effect of LRP1 on Aβ internalization and degradation in microglia, we observe Aβ level after overexpressing LRP1 and knockdown LRP1, and to evaluate this effect on learning and memory in AD transgenic mice. Third, inhibitors of neprilysin (NEP) , insulin degradation enzymes (IDE), lysosomal proteases and proteasome were used to study the intracellular Aβ degradation machinery. Fourth, we observe Aβ level after inhibitions of PI3K/Akt signaling pathway. Our study will provide valuable data and theory foundation for the pathogenesis and treatment of AD.

促进小胶质细胞内化降解Aβ是减少AD患者脑内Aβ沉积的有效途径。本研究拟首先培养apoe-/-鼠小胶质细胞和星形胶质细胞，利用定点突变及基因克隆技术构建携带人源性apoeε2、ε3、ε4基因片段的慢病毒颗粒并转染星形胶质细胞，收集条件培养基培养小胶质细胞，免疫荧光染色及ELISA方法筛选出与细胞内化降解Aβ最为相关的ApoE亚型；其次，构建LRP1过表达和RNA干扰慢病毒颗粒，在细胞和整体水平上调和下调LRP1后观察细胞内化Aβ的变化，Morris水迷宫检测AD转基因鼠学习记忆力的改变；再次，在细胞水平应用各种蛋白酶抑制剂研究内化入溶酶体内Aβ的降解途径；最后，应用PI3K抑制剂阻断PI3K/Akt通路，在细胞和整体水平观察Aβ的变化。通过该研究阐明ApoE对LRP1介导小胶质细胞内化降解Aβ的促进作用及PI3K/Akt通路对该过程的调控作用，并为临床治疗AD提供新的实验依据和理论基础。

本研究通过体内外实验，应用分子生物学、形态学等方法观察进入细胞内的Aβ的亚细胞结构定位；分析Aβ的细胞内化与LRP1间的关系；观察ApoE对LRP1介导小胶质细胞内化降解Aβ的促进作用及PI3K/Akt和MAPK信号通路对该过程的调控作用。首先内化的Aβ主要定位于细胞的溶酶体和线粒体中，内质网中有少量Aβ沉积；其次通过构建携带人源性apoeε2、ε3、ε4基因片段的慢病毒颗粒转染细胞并初步发现apoeε2促进小胶质细胞内化Aβ；再次发现Aβ与LRP1相互结合形成Aβ-LRP1复合物，LRP1基因敲低时Aβ内化减少；最后，Aβ-LRP1复合物能够激活PI3K/Akt和MAPK信号转导通路；p38特异性抑制剂SB202190和ERK1/2特异性抑制剂U0126预处理细胞，细胞内化Aβ被阻断，JNK特异性抑制剂SP600125预处理细胞，细胞内化Aβ不受影响。综合上述结果，本研究为探讨星形胶质细胞ApoE促进LRP1介导小胶质细胞内化降解Aβ及信号转导通路对该过程的调控作用的研究奠定了坚实的工作基础,对于认识小胶质细胞内化降解Aβ提供了重要的启示性线索,并为临床治疗AD提供新的实验依据和理论基础。该项目资助发表SCI收录论文5篇（影响因子分别为3.905、3.603、3.440、3.247、2.561），会议论文摘要3篇，国外学术交流1次，国内学术交流2次。培养硕士生3名，在读。项目投入经费17.5万元，支出13.6万元，各项支出与预算相符。剩余经费3.9万元，剩余经费将继续用于本项目后续研究的支出。