Recent data from our laboratory and others has established that normal processing of the amyloid beta protein precursor (BetaAPP) produces and releases 4 kD amyloid Beta protein (ABeta) that is essentially identical to the ABeta deposited as amyloid iA Alzheimer's disease (AD). Strong evidence that amyloid deposition plays a critical role in the development of AD has come from the identification of familial AD (FAD) kindreds in which the AD phenotype cosegregates with mutations in the betaAPP gene. Three of these mutations alter the valine located three residues carboxyl to Abeta43 (val717 in betaAPP) to isoleucine (delta I), phenylalanine (delta F), or glycine (delta G). A fourth double mutation (delta NL) alters the lysine-methionine located immediately amino to Abeta1 to asparagine-leucine. The location of these mutations in close proximity to A-beta immediately suggests that they may cause AD by altering betaAPP processing in a way that is amyloidogenic. To evaluate this possibility, we compared human neuroblastoma (M17) cells expressing normal or FAD- linked mutant betaAPP695. Cells expressing the betaAPPdeltaNL mutant showed a 5-fold increase in the relative amount of an approximate 11.4 kD A-beta-bearing carboxyl-terminal betaAPP derivative, and they released 6-fold more 4 kD Abeta into the medium. These observations provide strong evidence that betaAPPdeltaNL causes AD because it undergoes altered processing that releases increased amounts of ABeta. Significantly, transfected cells expressing betaAPPdeltaI showed no increase in the 11.4 kD Abeta-bearing COOH-terminal betaAPP derivative and no increase in secretion of 4 kD Abeta. To further examine the FAD-linked (betaAPP717 mutants (delta-I, delta-F), we analyzed transfected M17 cells by (i) isolating metabolically labeled 4 kD Abeta from conditioned medium, digesting with CNBr, and analyzing the COOH-terminal peptides released or (ii) assessing the A-beta in conditioned medium using sandwich ELISAs that discriminate Abeta1-40 from the longer Abeta1-42. Both methods demonstrated that the betaAPP717 mutations cause a 1.5 to 1.9-fold increase in the percentage of long Abeta1-42 generated. It is well established that long Abeta (e.g. Abeta1-42) forms insoluble amyloid fibrils more rapidly than Abeta1-40. Thus the betaAPP717 mutants, like the deltaNL mutant, undergo altered processing that enhances the likelihood of amyloid deposition. Taken together these observations provide strong evidence (i) that amyloid deposition is critical in AD, and (ii) that the pathway producing A- in cultured cells is highly relevant to amyloid deposition in AD. It is now evident that the rate of amyloid deposition will depend on (i) the rate at which BetaAPP is processed into secreted Abeta, (ii) the rate at which secreted Abeta is removed, and (iii) the rate at which insoluble amyloid fibrils are formed at any prevailing concentration of soluble, extracellular Abeta. In this proposal we focus on the factors that govern A- concentration because our studies of the genetic forms of AD indicate that Abeta concentration is critically important in determining whether enough amyloid is deposited to cause disease. Thus the first specific aim of this proposal is to identify the set of proteases that determine the rate at which the various Abeta peptides are released. Our second specific aim is to identify the mechanism(s) responsible for clearing secreted Abeta, mechanism(s) that currently are completely unknown. Our last specific aim is to analyze Abeta in plasma to determine whether the concentrations of total Abeta or Abeta ending at Abeta42 are correlated with AD.
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