血管内皮细胞来源的淀粉样前体蛋白（APP）对成体神经干细胞增殖分化以及凋亡调控的分子机制研究

Endothelial cells have close interactions with neural stem cells in adult neurogenic regions and have been implicated to provide niche signals for adult neurogenesis. However, the identity of endothelial derived niche factors remains elusive. We took two different approaches to address this question. First we performed cDNA microarray analysis and obtained a list of around 80 genes by comparison of the expression profile of niche and non-niche endothelial cells using adult Tie-2 GFP transgenic mice. Next we used Retroviral Mediated Signal Trap technology to identify all cDNAs encoding secreted or membrane-bound protein in endothelial cells from subventricular zone (SVZ) of Tie-2 GFP transgenic mice. A total of 220 positive clones were screened. 94 secreted or membrane-bound proteins were identified. Interestingly, the APP (Amyloid Precursor Protein) was identified as highly overrepresented in both approaches. Our preliminary ex vivo cell culture experiments showed N-APP overexpression conditioned medium completely abolished de novo neurosphere formation, inhibited secondary neurosphere formation and induced neurosphere apoptosis. Although APP is well established for its neurotoxicity in neurodegenerative diseases, its function in adult neurogenic niche is never demonstrated. Given current findings, we speculate APP is a negative reglator in adult neurogenic niche microenvironment which helps to maintain a fine balance of neural progenitor cells by contributing to its normal apoptotic process. In the proposed study we will first perform Real-time PCR and immunohistochemistry experiments to reveal spatial and temporal APP expression in adult neurogenic niche and then we will establish brain endothelial cell-neural stem cell coculture system and perform both overexpression and siRNA mediated knockdown experiement to elucidate APP function ex vivo. We will next use RCAS-TVA system combined with stereotatic injection of RCAS plamids into adult mice SVZ regions to enable targeted APP overexpression or knockdown in niche endothelial cells to estimate APP function in vivo. Our research will provide new insights into endothelial cell interaction with neural stem cells in adult neurogenic niche and have important implications for neural stem cell function in aging and neurodegenerative diseases.

成体神经干细胞存在于血管内皮细胞构成的微环境中，微血管内皮细胞对成体神经干细胞特性的维持起重要作用，但具体调控机制不明。项目申请人的前期研究通过两种不同的筛查手段同时发现淀粉样前体蛋白(Amyloid Precursor Protein，APP）是微环境中可能的重要调控因子。初步的体外实验发现，过度表达APP可以诱导成体神经干细胞球凋亡，以及完全抑制原发性以及次级神经球的发生。因此APP可能是神经干细胞微环境中重要的反向调控因子。本研究拟在前期研究基础上进一步揭示APP的时空表达特点，通过体外EC-NSC共培养模型结合APP过表达以及靶向沉默技术观察APP对成体神经干细胞增殖，分化以及凋亡的影响，并创新性的运用RCAS-TVA系统以及脑内立体定向注射达到在体观察APP功能的目的。该研究将首次揭示血管内皮源性APP在神经干细胞微环境的调控作用，为老龄化以及神经退行性疾病的研究提供新的思路。

成体神经干细胞在体内主要分布在侧脑室下区SVZ和海马齿状回颗粒细胞下区SGZ,这两个区域被称为成年神经发生区（adult neurogenic zone）。 值得注意的是，在这两个脑区，神经干细胞均与微血管临近 ，但是目前没有任何一项研究对神经干细胞和微环境中血管内皮细胞分泌的调控因子进行系统全面的研究。..项目申请人美国的前期研究中，通过cDNA微阵列技术以及逆转录病毒介导的信号蛋白俘获技术这两种不同的筛查手段同时发现淀粉样前体蛋白(Amyloid Precursor Protein，APP）是成年神经发生区神经微血管niche中可能的重要反向调控因子。体外实验发现，bEnd 3脑血管内皮细胞系高表达APP； bEnd 3条件培养基中明显抑制原代以及次代成体神经干细胞球的产生次代成体神经干细胞球的产生并诱导其发生凋亡，初步证实了我们的判断。..本项目旨在进一步研究APP作用的具体机制。通过本项目的研究我们发现，脑血管内皮细胞来源的APP产物N末端128个氨基酸，简称N-APP可能是成年神经发生区的重要凋亡诱导者。过表达N-APP可以明显抑制原代以及次代成体神经干细胞球产生以及诱导其凋亡，机制可能是通过N-APP以配体-受体结合的方式与成体神经干细胞的DR6受体结合直接激活下游的Caspase3 介导的细胞内凋亡途径，这种负性调节方式目前未见报道，当然这种推测还需要进一步的在体实验来证实。脑血管内皮细胞有可能是通过这种负性调节方式保持着成年神经发生区的神经干细胞数目平衡。但是，我们的研究也表明，N-APP介导的APP-DR6途径并不是成年神经发生区唯一的负性调控机制，我们还在寻找其他的靶标。..本研究首次提出neurovascular niche中血管内皮细胞对成体神经干细胞存在负向调控，并首次提出neurovascular niche中血管内皮细胞源性的APP是成体神经干细胞凋亡的重要诱导者，其作用环节有可能是通过N-APP-DR6死亡通路，APP可能通过对成体神经干细胞凋亡的调控而在一定程度上促进阿尔茨海默病的发生，这些概念的提出对今后利用神经干细胞治疗神经损伤，神经系统退行性疾病（帕金森病，亨廷顿病，阿尔茨海默病），脑卒中等诸多疾病提供了新的思路。