Unusual phenotypic alteration of beta amyloid precursor protein (betaAPP) maturation by a new Val-715 --> Met betaAPP-770 mutation responsible for probable early-onset Alzheimer's disease

Abstract

We have identified a novel beta amyloid precursor protein (betaAPP) mutation (V715M-betaAPP770) that cosegregates with early-onset Alzheimer's disease (AD) in a pedigree. Unlike other familial AD-linked betaAPP mutations reported to date, overexpression of V715M-betaAPP in human HEK293 cells and murine neurons reduces total Abeta production and increases the recovery of the physiologically secreted product, APPalpha. V715M-betaAPP significantly reduces Abeta40 secretion without affecting Abeta42 production in HEK293 cells. However, a marked increase in N-terminally truncated Abeta ending at position 42 (x-42Abeta) is observed, whereas its counterpart x-40Abeta is not affected. These results suggest that, in some cases, familial AD may be associated with a reduction in the overall production of Abeta but may be caused by increased production of truncated forms of Abeta ending at the 42 position.

Figure 1

Organization of βAPP and mapping of epitopes recognized by antibodies. α, α-secretase; β, β-secretase; γ40, γ-secretase acting at the 40th aa of Aβ; γ42, γ-secretase acting at the 42nd aa of Aβ. Antibodies are shown in boxes.

Figure 2

Partial pedigree of the 074 family with the V715M mutation. Solid symbols indicate affected individuals. Arrow denotes the propositus. WT, wild type; 715, V715M mutation.

Figure 3

Effect of the V715M-βAPP mutation on the secretion of total Aβ by HEK293 cells. Stably transfected HEK293 cells overexpressing wild-type βAPP (wt-10 clone), V715M-βAPP (two clones, V715M-6 and V715M-9), or Swedish mutated (Sw)-βAPP were obtained and cultured as described in Experimental Procedures. βAPP expression in cell lysates was revealed with WO2 (A). Total secreted Aβ (B) was immunoprecipitated (with FCA18 antibody), submitted to SDS/PAGE and Western blot analysis (with mAbWO2), and then analyzed as described in Experimental Procedures. Bars in C correspond to densitometric analyses of secreted Aβ (normalized to the amount of endogenous βAPP content) and are the means ± SEM of three to four independent determinations. Mock indicates HEK293 cells stably transfected with the empty pcDNA3 vector. ∗, P < 0.001 (versus wt-10 cells).

Figure 4

Effect of the V715M-βAPP mutation on the secretion of Aβ40 and Aβ42 and their x-40/42-related products by HEK293 cells. Stably transfected HEK293 cells are as in Fig. 3 and metabolically labeled as described in Experimental Procedures. Expression of βAPP revealed as in Fig. 3 is shown A. Secreted Aβ42/x-42 (B) and Aβ40/x-40 (C) were obtained after sequential immunoprecipitation by means of FCA3542 and FCA3340, respectively, and quantified after radioautography and densitometric analysis. Bars in D and E represent the densitometric analysis, normalized to the amount of endogenous βAPP content, of Aβ42 or Aβ40 (solid bars) and x-42 or x-40 (open bars) and are expressed as the percentage of those obtained with wt-βAPP751-expressing cells taken as 100 (note the distinct scales of ordinates). Values are the means ± SEM of three to eight independent experiments. F illustrates the ratios of Aβ42/Aβ40 (solid bars) and x-42/x-40 (open bars). Mock indicates HEK293 cells stably transfected with the empty vector pcDNA3. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001; NS, nonstatistically significant (versus corresponding wt).

Figure 5

Effect of the V715M-βAPP mutation on the intracellular formation of total Aβ, p10, and p12. The indicated transfectants were obtained and metabolically labeled as in Figs. 3 and 4. Cells were lysed and centrifuged, and then supernatants were treated with FCA18 (A and C) or B11.4 (C) antibodies and analyzed as in Fig. 4. Quantification of total intracellular Aβ (B), p12 (D), and p10 (E) was performed as in Fig. 4. Values are expressed as the percentage of those obtained with wt-βAPP-expressing cells (taken as 100) and are the means ± SEM of three to four independent experiments. ∗, P < 0.1; ∗∗, P < 0.05; ∗∗∗, P < 0.01; ∗∗∗∗, P < 0.001; NS, nonstatistically significant (versus corresponding wt-10).

Figure 6

Effect of the V715M-βAPP mutation on the secretion of total sAPP and APPα by HEK293 cells. The indicated transfectants were obtained and metabolically labeled as in Figs. 3 and 4. Cells media were immunoprecipitated with the 207 antibody. Quantification of total sAPP [i.e., APPα + APPβ (A and C)] was performed after SDS/PAGE, Western blot analysis, and direct radioautography as in Fig. 4. APPα (B and D) was analyzed from the same nitrocellulose after hybridization with 10D5C as described in Experimental Procedures. Values are expressed as the percentage of those obtained with wt-βAPP-expressing cells (taken as 100) and are the means ± SEM of three to five independent experiments. ∗, P < 0.001 (versus corresponding wt).

Figure 7

Effect of the V715M-βAPP mutation on the secretion of total Aβ by cultured neurons. TSM1 neuronal cells were obtained (10), cultured, and transiently transfected with wt-, Sw-, or V715M-βAPP as described in Experimental Procedures. βAPP content was analyzed after Western blotting by means of mAbWO2 (A). Total secreted Aβ was analyzed after immunoprecipitation with FCA18 and Western blot analysis of immunoprecipitated proteins with mAbWO2 as described in Experimental Procedures (B).