PROTEOGLYCANS/GLYCOSAMINOGLYCANS IN ALZHEIMER'S DISEASE

特异性硫酸肝素蛋白聚糖，串珠素，是一个常数 β-淀粉样蛋白（Abeta）的伴侣和组成部分- 阿尔茨海默病（AD）大脑中含有淀粉样蛋白沉积物。 的 本提案的总体目标是阐明和定义 串珠素参与β-淀粉样蛋白正常功能的机制 前体蛋白（betaPP），与Abeta相关， AD的发病机制 在目标#1中，抗体介导的细胞表面加帽 β PP，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是否与细胞上的串珠蛋白聚糖相关起作用 表面或细胞外基质中的神经突生长（通过 形态测定法）或细胞粘附（通过粘附测定法）。 在目标#2中，我们将 通过以下方式表征串珠素-Abeta相互作用的机制：1） 鉴定分离的淀粉样斑块核心中的串珠素（和其他PG）， 使用特定溶解技术和PG的神经纤维缠结 结构生物化学，和2）鉴定特异性串珠素核心蛋白 结构域和GAG链结构参与A β结合（通过 分析与Abeta偶联的SASD-125 I-串珠素的胰蛋白酶消化产物 肽，并通过亲和共电泳，亲和层析和 固相结合测定）。 此外，我们将使用后两者 评价串珠素与载脂蛋白E4相互作用的技术 对比E3。 在目标3中，我们将确定水平（通过北方分析 和mRNA狭缝印迹）和串珠素的位点（通过原位杂交） 在AD和正常老年脑中的合成，以及在原代培养物中， 鉴定串珠素产生细胞（通过结构PG生物化学，Western 和北方分析）结合Abeta或betaPP（通过亲和柱 色谱法）。 在目标4中，我们将确定1）形成的影响 串珠素-Abeta/betaPP或GAG-Abeta复合物（使用聚集测定 通过电子显微镜、刚果红染色、硫磺素T 荧光分光光度法、X射线衍射法和红外光谱法）和2） 利用HPLC，SDS-PAGE， 蛋白质印迹和扫描光密度法。 此外，我们将使用 PG合成的化学调节剂，并将外源性PG/GAG引入 原代培养，以评估改变PG合成对 β PP代谢和Abeta产生。 此外，我们将注入Abeta 或串珠素进入大鼠脑中以确定（通过免疫细胞化学，北方 分析和结构生物化学）Abeta是否引起上调 在串珠素的表达和合成，以及串珠素是否影响 体内β PP表达和合成。 破译机制 基底串珠素与Abeta/betaPP的相互作用对于 了解AD发病机制中的几个关键步骤， 未来治疗干预的相关目标。

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.