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.
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