Recent studies have demonstrated an association between the gene dosage of apoE type 4 and late onset Alzheimer's disease (AD). Our long term goals are to elucidate the molecular mechanisms by which the apoE receptor system affects CNS development and function. ApoE transports lipoproteins via binding to low density lipoprotein (LDL) receptor and LDL receptor- related protein (LRP). ApoE is synthesized within the CNS. Additional ligands for LRP include tissue-type plasminogen activator and alpha2- macroglobulin, as well as a 39kDa protein which serves as a potent regulator of LRP function. We have recently demonstrated the expression, endocytotic function and regulation of LRP. the apoE clearance receptor, in rat brain using a variety of cellular and molecular approaches. Our reagents include isolated LRP, 39kDa protein and other ligands, as well as antibodies, cDNA's and both in vivo and isolated cellular systems. Thus the present aims are as follows: 1) Elucidate the pattern of expression of all components of the LRP system in brain specimens from the WUMS ADRC tissue bank. We shall examine LRP, 39kDa protein, apoE and other ligands using immunohistochemistry and quantitative colloidal gold immunoelectron microscopy. 2) Examine the function and regulation of the LRP system in murine primary neurons and astrocytes as well as differentiated cell lines. We shall define the biosynthesis, intracellular itinerary, cell surface function and endocytotic fate of LRP, the 39kDa protein and ligands using cellular biological approaches including biosynthetic labeling, receptor-ligand kinetics, chemical crosslinking, fluorescent microscopy, etc. 3) Define the expression and function of the LRP system in vivo and in primary CNS cello from young adult and aged a) normal mice, b) transgenic mice for apoE overexpression as well as c) apoE knockout mice. We shall use whole embryos as well as isolated tissues together with in situ hybridization and RNase protection assays, immunohistochemistry and Western analyses to define the components at the RNA and protein levels. 4) Define the molecular regions of apoE isoforms and of the 39kDa protein which recognize the ligand binding sites on the LRP molecule. We shall examine apoE isoforms and generate mutants of the 39kDa protein which will be examined both in binding competition studies as well as in direct binding studies to isolated LRP. Thus these studies taken together provide a basis for the initial approach to elucidating the molecular mechanisms by which apoE, the LRP clearance receptor and its regulator 39kDa protein contribute to CNS pathobiology in Alzheimer's disease.
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