PROTEOGLYCANS/GLYCOSAMINOGLYCANS IN ALZHEIMER'S DISEASE

特异性的硫酸肝素蛋白多糖是一种恒定的 β-淀粉样蛋白(Abeta)的伴生物和组成部分- 在阿尔茨海默病(AD)的大脑中含有淀粉样沉积。这个 这项建议的总体目标是阐明和定义 Perlecan参与β-淀粉样蛋白正常功能的机制 前体蛋白(βPP)，及其与Abeta的关联，以及在 阿尔茨海默病发病机制。在目标1中，抗体介导的细胞表面封盖 双标记免疫荧光和共聚焦显微镜，将 用于大鼠小胶质细胞、星形胶质细胞和神经元的原代培养 评估BetaPP功能是否与细胞上的Perlecan相关 在轴突生长过程中表面或在细胞外基质中(通过 形态计量学)或细胞黏附(通过黏附分析)。在目标2中，我们将 通过1)描述Perlecan-Abeta相互作用的机制 鉴定分离的淀粉样斑块核心中的Perlecan(和其他PG) 应用特定增溶技术和PG的神经原纤维缠结 结构生物化学，以及2)鉴定特定的Perlecan核心蛋白 参与Aβ结合的结构域(S)和Gag链结构(S)(by SASD-125I-Perlecan偶联Abeta的胰酶分析 通过亲和电泳法、亲和层析法和亲和层析法 固相结合分析)。此外，我们还将使用后两者 评估Perlecan与载脂蛋白E4相互作用的技术 对阵E3。在目标3中，我们将确定水平(通过Northern分析 和mRNA插槽印迹)和位置(通过原位杂交) AD和正常衰老大脑的合成，以及在原代培养中将 鉴定Perlecan产生细胞(通过结构PG生物化学，Western 和Northern分析)，其结合Abeta或BetaPP(通过亲和柱 层析)。在目标4中，我们将确定1)队形的影响 Perlecan-Abeta/BetaPP或Gag-Abeta络合物(使用聚集分析 用电子显微镜、刚果红染色、硫代黄素T分析 荧光光谱、X射线衍射和红外光谱)和2) 用高效液相色谱、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法 Western blotting和扫描密度法。此外，我们将使用 PG合成的化学调节剂，并将外源PGs/GAG导入 评估PG合成改变对原代培养的影响 β-PP代谢和A-β的产生。此外，我们还会给阿贝塔注射 或Perlecan注入大鼠脑内以确定(通过免疫细胞化学，Northern 分析和结构生物化学)Abeta是否导致上调 在Perlecan的表达和合成中，以及Perlecan是否影响 β-PP在体内的表达和合成。破译机制 潜在的Perlecan与Abeta/BetaPP的相互作用将是必不可少的 了解AD发病机制中的几个关键步骤，并可能提供 为今后的治疗干预提供相关靶点。

The specific heparin sulfate proteoglycan, perlecan, is a constant companion and integral component of beta-amyloid protein (Abeta)- containing amyloid deposits in Alzheimer's disease (AD) brain. The overall objective of this proposal is to elucidate and define the mechanisms involving perlecan in the normal function of the beta-amyloid precursor protein (betaPP), in its association with Abeta, and in the pathogenesis of AD. In Aim #1, antibody-mediated cell surface capping of betaPP, double labelled immunofluorescence and confocal microscopy, will be used in rat primary cultures of microglia, astrocytes and neurons to assess whether betaPP functions in association with perlecan on the cell surface or in the extracellular matrix during neurite outgrowth (by morphometry) or cell adhesion (by adhesion assays). In Aim #2, we will characterize the mechanisms of perlecan-Abeta interactions by 1) identifying perlecan (and other PGs) in isolated amyloid plaque cores and neurofibrillary tangles using specific solubilization techniques and PG structural biochemistry, and 2) identifying specific perlecan core protein domain(s) and GAG chain structure(s) involved in Abeta-binding (by analyzing tryptic digestions of SASD-125 I-perlecan coupled to Abeta peptides, and by affinity coelectrophoresis, affinity chromatography and solid phase binding assays). In addition, we will use the latter two techniques to assess the interaction of perlecan with Apolipoprotein E4 versus E3. In Aim #3, we will determine the levels (by Northern analysis and mRNA slot blots) and sites (by in situ hybridization) of perlecan synthesis in AD and normal aged brain, and in primary cultures will identify perlecan producing cells (by structural PG biochemistry, Western and Northern analysis) which bind Abeta or betaPP (by affinity column chromatography). In Aim #4, we will determine 1) the effects of formation of perlecan-Abeta/betaPP or GAG-Abeta complexes (using aggregation assays analyzed by electron microscopy, congo red staining, Thioflavin T spectrofluorometry, X-ray diffraction and infrared spectroscopy) and 2) whether perlecan protects Abeta/betaPP degradation using HPLC, SDS-PAGE, Western blotting and scanning densitometry. Additionally, we will use chemical modulators of PG synthesis, and introduce exogenous PGs/GAGs into primary cultures to assess the consequences of altered PG synthesis on betaPP metabolism and Abeta production. Furthermore, we will infuse Abeta or perlecan into rat brain to determine (by immunocytochemistry, Northern analysis and structural biochemistry) whether Abeta causes an upregulation in perlecan expression and synthesis, and whether perlecan influences betaPP expression and synthesis in vivo. Deciphering the mechanisms underlying perlecan's interaction with Abeta/betaPP will be essential to understand several key steps in the pathogenesis of AD and may provide relevant targets for therapeutic intervention in the future.