Alzheimer's disease is a human cerebral degenerative disease manifested clinically by dementia. Pathologically, deposits of an unusual protein, Beta amyloid (BetaA4), are found in the cerebral vasculature and in senile plaques. BetaA4 is a 40 amino acid peptide which is derived by proteolytic cleavage from a larger precursor termed Beta-amyloid precursor protein (BetaAPP). Beta APP is a transmembrane protein with 1-2 N-linked oligosaccharides and 15-20 O-linked oligosaccharides. The role that glycosylation plays in the biosynthesis, intracellular trafficking, and surface expression of BetaAPP is not clear. In addition, it is not known whether alterations in BetaAPP glycosylation can influence proteolytic processing of BetaAPP leading to increased BetaA4 formation. This project will examine these issues by using two cell culture model systems and BetaAPP purified from human brain and cerebrospinal fluid. The NT2/D1 human teratocarcinoma cell line can be induced to undergo neuronal differentiation by incubation with retinoic acid. These cells synthesize abundant amounts of BetaAPP and the isoforms and electrophoretic mobility of BetaAPP are altered following differentiation. Therefore, NT2/D1 cells will allow us to biochemically analyze glycosylation of human BetaAPP and to examine the hypothesis that differentiation of these cells results in modification of the oligosaccharides on this protein. In addition, they will allow us to test the hypothesis that altering the glycosylation of BetaAPP in differentiated NT2/D1 cells changes the synthesis, secretion, and proteolytic processing of this protein. Wild-type Chinese hamster ovary (CHO) cells transfected with human BetaAPP CDNA also express abundant amounts of BetaAPP. Thus, by using glycosylation inhibitors and by transfecting BetaAPP CDNA into lectin- resistant CHO cell lines with defined defects in protein glycosylation, we will alter the glycosylation of BetaAPP. This approach will allow us to test the hypothesis that covalently bound N- and O-linked oligosaccharides are important in modulating the synthesis, secretion, and proteolytic processing of BetaAPP. Finally, it will be important to correlate the results found with the cell culture model systems to those found with human brain. BetaAPP will be purified from human brain and cerebrospinal fluid and the bound oligosaccharides analyzed. This will allow us to test the hypothesis that glycosylation of human brain BetaAPP changes during aging and is altered in patients with Alzheimer's disease. These studies are likely to clarify the role that the glycosylation of BetaAPP plays in the pathobiology of Alzheimer's disease.
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