The 4 kD (39-43 residue) amyloid beta protein (AB), which is deposited as amyloid in Alzheimer's disease (AD), is encoded as an internal peptide that begins 99 residues from the carboxyl terminus of a set of 695-770 residue glycoproteins referred to as the amyloid Beta protein precursor (BAPP). Recent data from our laboratory and others has established that normal processing of the BAPP involves (i) a constitutive secretory pathway in which the BAPP is cleaved within AB to produce a large, secreted, NH2-terminal derivative and an 8.7 kD COOH-terminal fragment, neither of which can produce amyloid because they do not contain the entire AB, (ii) endosomal/lysosomal processing which produces a complex set of COOH-terminal derivatives that includes potentially amyloidogenic forms with the entire AB a or near their NH2 terminus, and (iii) the production of 4 kD AB(essentially identical to the AB deposited as amyloid in AD) that is released from cultured cells and readily detected in CSF. 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 BAPP gene. Three of these mutations alter the valine mutation (NL) alters the lysine-methionine located immediately amino to AB1 (lys670=met671 in BAPP) to isoleucine, phenylalanine, or glycine. A fourth double mutation (NL) alters the lysine-methionine located immediately amino to A1 (lys670-met671 in BAPP770) to asparagine-leucine. The location of these mutations in close proximity to AB immediately suggests that they may cause AD by altering BAPP processing in a way that is amyloidogenic. To evaluate this possibility, we have recently compare human neuroblastoma (M17) cells expressing normal BAPP695 or FAD-linked mutant BAPP 695. Cells expressing the BAPP NL mutant showed a 5-fold increase in the relative amount of an 11.4 kD AB-bearing carboxyl- terminal BAPP derivative, and they released 6-fold more 5 kD AB into the medium. These observations provide strong evidence (i) that this mutant BPP causes AD because it undergoes altered processing that releases increased amounts of AB, and (ii) that the pathway producing AB in cultured cells is highly relevant to AD. In this application, we propose to expand our analysis of AB production by undertaking experimentation designed t o (1) test the hypothesis that the 4kD AB produced and released by normal cellular processing consists of several peptides with variable COOH-termini (AB39-43) similar to the AB peptides that have been identified in AD amyloid, (2) test the hypothesis that the BAPP717 mutants favor production of the longer AB1-42 or 43 forms, which selectivity deposit as plaque core amyloid, without increasing the production of AB as expected from the observation by our group and others that phorbol esters substantially increase the production of the large secreted BAPP derivative. In addition, we propose (4) to compare BAPP processing and the AB released by transfected mouse or human cells expressing wild type BAPP or the FAD-linked mutant BAPPs in order to (i) determine if the disappointing results obtained to date in transgenic mouse model of AD are related to low rates of AB production in mouse cells expressing human BAPPs, and (ii) identify constructs likely to significantly increase CNS AB production when they are introduced into transgenic mice. Finally, we plan (5) to carry out a systematic comparison of the amount of soluble AB in various regions of AD and control brains in an effort to determine if the amount of soluble AB is correlated with AD and/or amyloid deposition.
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