Evidence that the 42- and 40-amino acid forms of amyloid beta protein are generated from the beta-amyloid precursor protein by different protease activities

Abstract

Cerebral deposition of the amyloid beta protein (A beta) is an early and invariant feature of Alzheimer disease (AD). Whereas the 40-amino acid form of A beta (A beta 40) accounts for approximately 90% of all A beta normally released from cells, it appears to contribute only to later phases of the pathology. In contrast, the longer more amyloidogenic 42-residue form (A beta 42), accounting for only approximately 10% of secreted A beta, is deposited in the earliest phase of AD and remains the major constituent of most amyloid plaques throughout the disease. Moreover, its levels have been shown to be increased in all known forms of early-onset familial AD. Thus, inhibition of A beta 42 production is a prime therapeutic goal. The same protease, gamma-secretase, is assumed to generate the C termini of both A beta 40 and A beta 42. Herein, we analyze the effect of the compound MDL 28170, previously suggested to inhibit gamma-secretase, on beta-amyloid precursor protein processing. By immunoprecipitating conditioned medium of different cell lines with various A beta 40- and A beta 42-specific antibodies, we demonstrate a much stronger inhibition of the gamma-secretase cleavage at residue 40 than of that at residue 42. These data suggest that different proteases generate the A beta 40 and A beta 42 C termini. Further, they raise the possibility of identifying compounds that do not interfere with general beta-amyloid precursor protein metabolism, including A beta 40 production, but specifically block the generation of the pathogenic A beta 42 peptide.

Figure 1

MDL 28170 influences βAPP metabolism. 0, Untreated K695sw cells. 200, K695sw cells treated with 200 μM MDL 28170. Lanes: 1 and 2, aliquot of total chase medium electrophoresed directly on the gel; 3 and 4, 1736 immunoprecipitation of chase medium; 5 and 6, 192sw immunoprecipitation of chase medium [note that β-cut APPs (lanes 5 and 6) runs slightly below α-cut APPs (lanes 3 and 4), as expected]; 7 and 8, C7 immunoprecipitation of cell lysates (N′ and N′/O′-glycosylated forms of full-length βAPP as well as the 10-kDa C-terminal fragment are indicated); 9 and 10, 1282 immunoprecipitation of cell lysates.

Figure 2

Differential inhibition of Aβ₄₂ and Aβ₄₀ formation. (A) Labeled K695sw cells were chased with the indicated concentrations of MDL 28170 and precipitated with 21F12 (Upper) followed by 1282 (Lower). (B) Quantitation of the effect of 200 μM MDL 28170 on Aβ and p3 by phosphorimaging. The bars show the pixel number relative to an untreated control (200); SDs are indicated. The decreases in Aβ_total and p3_total relative to an untreated control were significant (∗, two-tailed t test, n = 4, P < 0.001) and the decreases in Aβ₄₂ and p3₄₂ upon treatment with MDL 28170 did not reach significance. Moreover, the difference in inhibition of Aβ₄₂ versus total Aβ is significant (two-tailed t test, n = 4, P < 0.01) and the difference in inhibition of p3₄₂ versus total p3 is also significant (two-tailed t test, n = 4, P < 0.05).

Figure 3

Differential inhibition of Aβ₄₂ and Aβ₄₀ formation in K695sw cells under a variety of conditions. (A) Treatment with 1 μM phorbol 12,13-dibutyrate (lanes P) decreases both Aβ₄₂ and Aβ_total. (Upper) 21F12 precipitation. (Lower) Subsequent 1282 precipitation. (B–E) Cells were chased with (lanes M) or without (lanes 0) 200 μM MDL 28170 and the resultant media were immunoprecipitated as follows. (B) First with BC05 (Upper) and then with 1282 (Lower). (C) First with C42 (Upper) and then with 1282 (Lower). (D) First with 2G3 (Upper) and then with 21F12 (Lower). (E) In two separate aliquots, either with 21F12 (Upper) or with 1282 (Lower). (F) Cells were labeled for 3 h in the presence of 100 μM MDL 28170 and the medium was immunoprecipitated first with 21F12 (Upper) and then with 1282 (Lower). (G) Cells were chased with (lane M) or without (lane 0) 200 μM MDL 28170 and the resultant media were immunoprecipitated first with 21F12 (Upper) and then with 1963 (Lower).

Figure 4

Differential inhibition of Aβ₄₂ and Aβ₄₀ formation in different cell types. Labeled cells were chased with the indicated concentrations of MDL 28170, and the media were precipitated with 21F12 (Upper) followed by 1282 (Lower). (A) K695_717I cells. The relatively low βAPP expression in this line leads to a faint Aβ₄₂ band. However, the bands are not due to endogenous βAPP, because nontransfected 293 cells do not show any Aβ or p3 bands under the conditions of the experiment (not shown). (B) CHO695 cells. Note that in CHO cells, the p3 bands migrate as doublets, as described (30). (C) SKN695 cells.