The novel matrix structures fractones promote FGF-2 in the neural stem cell niche

描述（由申请人提供）：神经干细胞和祖细胞（NSPC）为治疗神经系统的退行性疾病或创伤提供了巨大的潜力。在成年哺乳动物物种中，NSC主要存在于侧脑室壁中，在那里它们增殖和分化以产生新的神经元和神经胶质细胞。现在很明显，微环境因素调节NSPC在细胞所在的“小生境”中的增殖。然而，管理神经原性生态位的内在系统知之甚少。我们的总体目标是了解内源性或移植的NSPC如何在个体内发挥作用，最终取代丢失的细胞。这一基本认识对于设计新的治疗方法来治疗因创伤、变性和脱髓鞘而死亡的神经细胞至关重要。基于我们已发表的数据，我们假设，新的细胞外结构的“fractones”，我们的特点是直接接触NSPC在成年哺乳动物大脑的神经原生态位，介导和调节整个成年期的新的神经元和神经胶质细胞的生产。我们的初步结果表明，涉及的机制属于生长因子/细胞外基质的相互作用在NSPC/fractone接口。本提案的目的是确定是否以及如何fractones干预神经原性生长因子FGF-2诱导的NSPC增殖的过程。我们发现，fractones结合和浓缩FGF-2在体内，通过硫酸乙酰肝素蛋白聚糖（HSPG）依赖的机制。我们的具体目标是确定：1）FGF-2与碎片酮的结合是否负责小生境中FGF-2诱导的神经发生; 2）NSPC是否在与结合FGF-2的碎片酮接触时增殖; 3）碎片酮组分串珠素（一种主要的HSPG）对小生境中FGF-2诱导的NSPC增殖的作用。对于具体目标1，我们将使用生物素化的FGF-2作为NSPC增殖的示踪剂和诱导剂，以及肝素酶，两者都在成年小鼠的脑中体内注射，以确定从碎片携带的HSPG中切割生物素化的FGF-2的结合是否消除FGF-2诱导的神经发生。免疫荧光将用于观察掺入溴脱氧尿苷和生物素化FGF-2的有丝分裂后细胞。对于具体目标2，我们将使用生物素化的FGF-2来鉴定结合生物素化的FGF-2的片段与响应于生物素化的FGF-2而增殖的新兴细胞之间的空间关系。对于具体目标3，我们将使用转基因动物（串珠素缺失小鼠）来确定串珠素的缺乏如何对由生物素化的FGF-2在小生境中诱导的NSPC增殖有害。这些实验将使我们能够确定是否fractones促进神经干细胞龛中FGF-2诱导的NSPC增殖。如果得到证实，fractones将被认为是调节整个成年期神经干细胞生态位的关键成分。

DESCRIPTION (provided by applicant): Neural stem and progenitor cells (NSPC) offer enormous potential for treating degenerative diseases or traumas of the nervous system. In adult mammalian species, NSC primarily reside in the lateral ventricle walls, where they proliferate and differentiate to produce new neurons and glial cells. It is now evident that microenvironmental cues regulate NSPC proliferation in the "niche" where the cells reside. However, the intrinsic systems that govern the neurogenic niche are poorly understood. Our overall goal is to understand how NSPC, endogenous or transplanted, function within an individual, to eventually replace lost cells. This fundamental understanding is crucial for designing new therapies for neural cells dying due to trauma, degeneration and demyelination. Based on our published data, we hypothesize that the new extracellular structures "fractones" we characterized in direct contact with NSPC in the neurogenic niche of the adult mammalian brain, mediate and regulate the production of new neurons and glial cells throughout adulthood. Our preliminary results indicate that the mechanisms implicated pertain to growth factor/extracellular matrix interactions at the NSPC/fractone interface. The objective of this proposal is to determine whether and how fractones intervene in the process of NSPC proliferation induced by the neurogenic growth factor FGF-2. We found that fractones bind and concentrate FGF-2 in vivo, by a mechanism that is heparan sulfate proteoglycan (HSPG) dependent. Our specific aims are to determine: 1) whether FGF-2 binding to fractones is responsible for FGF-2 induced neurogenesis in the niche; 2) whether NSPC proliferate in contact with fractones that have bound FGF-2; 3) the role of the fractone component perlecan (a major HSPG) on FGF-2-induced NSPC proliferation in the niche. For specific aim 1, we will use biotinylated FGF-2 as a tracer and inducer of NSPC proliferation, and heparatinase, both injected in vivo in the brain of adult mice to determine whether cutting the binding of biotinylated FGF-2 from fractone-borne HSPG annihilates neurogenesis induced by FGF-2. Immunofluorescence will be used to visualize post-mitotic cells that have incorporated bromodeoxyuridine and biotinylated FGF-2. For specific aim 2, we will use biotinylated FGF-2 to identify the spatial relationships between fractones that have bound biotinylated FGF-2 and the emerging cells that have proliferated in response to biotinylated FGF-2. For specific aim 3, we will use transgenic animals (perlecan-null mice) to determine how the lack of perlecan is detrimental to NSPC proliferation induced by biotinylated FGF-2 in the niche. These experiments will allow us to determine whether fractones promote NSPC proliferation induced by FGF-2 in the neural stem cell niche. If demonstrated, fractones will be considered as crucial components that regulate the neural stem cell niche throughout adulthood.