A2: PROJ 2: NEUROGENESIS IN THE ADULT BRAIN: ROLE OF BASAL LAMINA NETWORK

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。本研究的主要目的是探讨神经干细胞在成人脑神经原性微环境中增殖和分化的机制。我们最近通过共聚焦和透射电子显微镜表征了在神经原性龛中直接接触神经祖细胞和神经干细胞的特化基底层细胞（fractones）（Mercier等人，2002年; 2003年）。我们的假设是，fractones的细胞外基质/生长因子的相互作用，最终触发邻接的神经干细胞的增殖和分化的网站。去年，我们报道了fractones含有普遍存在的细胞外基质成分的基底层，如胶原蛋白IV，层粘连蛋白γ-1和β-1，巢蛋白，硫酸乙酰肝素蛋白聚糖（HSPG）串珠。串珠素是肝素结合生长因子成纤维细胞生长因子-2（FGF-2）的高亲和力结合剂和激活剂。我们今年的工作假设是，fractones捕获肝素结合生长因子，以触发神经发生。我们研究了FGF-2，双调蛋白（AR），肝素结合表皮生长因子（HB-EGF），三个强大的神经原性因子的结合活性。生物素化后，将每种生长因子在成年大鼠脑的冷冻切片上孵育（原位结合），或通过脑室内注射引入体内（体内结合）。使用这两种方法，我们证明了FGF-2和AR强烈和排他性地结合到碎片。相反，心室壁中HB-EGF的扩散结合位点表明HB-EGF通过不涉及碎片结合的方式调节干细胞命运。FGF-2和AR与碎片的结合对于神经干细胞领域是非常重要的，首次揭示了生长因子在神经原性龛中触发干细胞活化的机制。未来的研究将集中在鉴定与FGF-2结合的Fractone HSPG和其他分子，以及这些分子相互作用触发神经干细胞激活的精确机制。了解神经干细胞生态位中发生的机制将允许准确地设计神经创伤和神经退行性疾病的干细胞疗法。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The major goal of this project is to identify the mechanisms leading to neural stem cell proliferation and differentiation in the neurogenic niche of adult brain. We recently characterized by confocal and transmission electron microscopy specialized basal lamina labyrinths (fractones) that directly contact neural progenitor cells and neural stem cells in the neurogenic niche (Mercier et al., 2002; 2003). Our hypothesis is that fractones are sites of extracellular matrix/growth factor interactions that ultimately trigger proliferation and differentiation of abutting neural stem cells. Last year we reported that fractones contain ubiquitous extracellular matrix components of basal laminae such as collagen IV, laminin gamma-1 and beta-1, nidogen, and the heparan sulfate proteoglycan (HSPG) perlecan. Perlecan is a high affinity binder and activator of the heparin-binding growth factor fibroblast growth factor-2 (FGF-2). Our working hypothesis for this year was that fractones capture heparin-binding growth factors to trigger neurogenesis. We studied the binding activity of FGF-2, amphiregulin (AR), and heparin-binding epidermal growth factor (HB-EGF), three powerful neurogenic factors. After biotinylation, each growth factor was either incubated on frozen sections of the adult rat brain (in situ binding), or introduced in vivo by intracerebroventricular injection (in vivo binding). Using both methods, we demonstrated that FGF-2 and AR strongly and exclusively bind to fractones. In contrast, the diffuse binding sites of HB-EGF in the ventricle walls indicated that HB-EGF regulates stem cell fate by means that do not involve fractone binding. The binding of FGF-2 and AR to fractones is highly important for the neural stem cell field, revealing for the first time a mechanism by which growth factors trigger stem cell activation in the neurogenic niche. Future research will focus on identifying fractone HSPG and other molecules that bind to FGF-2, and the precise mechanisms by which these molecules interact to trigger neural stem cell activation. Understanding the mechanisms occurring in the neural stem cell niche will permit to accurately design stem cell therapies for neurotrauma and neurodegenerative diseases.