The transcriptionally active amyloid precursor protein (APP) intracellular domain is preferentially produced from the 695 isoform of APP in a {beta}-secretase-dependent pathway

Abstract

Amyloidogenic processing of the amyloid precursor protein (APP) by β- and γ-secretases generates several biologically active products, including amyloid-β (Aβ) and the APP intracellular domain (AICD). AICD regulates transcription of several neuronal genes, especially the Aβ-degrading enzyme, neprilysin (NEP). APP exists in several alternatively spliced isoforms, APP(695), APP(751), and APP(770). We have examined whether each isoform can contribute to AICD generation and hence up-regulation of NEP expression. Using SH-SY5Y neuronal cells stably expressing each of the APP isoforms, we observed that only APP(695) up-regulated nuclear AICD levels (9-fold) and NEP expression (6-fold). Increased NEP expression was abolished by a β- or γ-secretase inhibitor but not an α-secretase inhibitor. This correlated with a marked increase in both Aβ(1-40) and Aβ(1-42) in APP(695) cells as compared with APP(751) or APP(770) cells. Similar phenomena were observed in Neuro2a but not HEK293 cells. SH-SY5Y cells expressing the Swedish mutant of APP(695) also showed an increase in Aβ levels and NEP expression as compared with wild-type APP(695) cells. Chromatin immunoprecipitation revealed that AICD was associated with the NEP promoter in APP(695), Neuro2a, and APP(Swe) cells but not APP(751) nor APP(770) cells where AICD was replaced by histone deacetylase 1 (HDAC1). AICD occupancy of the NEP promoter was replaced by HDAC1 after treatment of the APP(695) cells with a β- but not an α-secretase inhibitor. The increased AICD and NEP levels were significantly reduced in cholesterol-depleted APP(695) cells. In conclusion, Aβ and functional AICD appear to be preferentially synthesized through β-secretase action on APP(695).

FIGURE 1.

APP processing in SH-SY5Y cells expressing the different APP isoforms. A, Western blot of APP and actin in cell lysates and of sAPPα and sAPPβ in conditioned media from SH-SY5Y mock-transfected and APP₆₉₅-, APP₇₅₁-, or APP₇₇₀-expressing SH-SY5Y cells. B–D, densitometric analysis of APP (B), sAPPα (C), and sAPPβ (D). E, ELISA analysis of Aβ_1–40 and Aβ_1–42 levels in conditioned media from the cell lines. F, Western blot of AICD and actin in nuclear and non-nuclear fractions isolated from SH-SY5Y mock-transfected and APP₆₉₅-, APP₇₅₁-, or APP₇₇₀-expressing SH-SY5Y cells. G, densitometric analysis of AICD levels (normalized to actin) from the nuclear fraction of SH-SY5Y cells. Data represent mean ± S.E. (n = 3); *, p < 0.05 as compared with mock-transfected control, †, p < 0.05 as compared with other APP isoform cell lines.

FIGURE 2.

Analysis of NEP expression and NEP promoter occupancy in SH-SY5Y cells expressing the various APP isoforms in the presence or absence of the γ-secretase inhibitor, L685,458. A, comparative analysis by conventional PCR of NEP mRNA expression in the mock-transfected and the APP₆₉₅-, APP₇₅₁-, and APP₇₇₀-expressing SH-SY5Y cell lines with actin mRNA as loading control. B, effect of the γ-secretase inhibitor L685,458 on NEP expression in control and APP-overexpressing SH-SY5Y cell lines. Data are expressed as the ratio of NEP expression to actin expression and represent mean ± S.E. (n = 5); *, p < 0.05 as compared with mock-transfected control. C, chromatin immunoprecipitation analysis of SH-SY5Y cells expressing the various APP isoforms. Control and APP-expressing SH-SY5Y cells were fixed, and chromatin was immunoprecipitated with anti-AICD, -HDAC1, or -Aβ antibodies. DNA pulled down by the antibody was analyzed by real-time PCR with primers specific to the NEP promoter 2. The results represent enrichment of DNA pulled down with the anti-AICD, anti-HDAC1, or anti-Aβ antibody versus non-immune IgG. Data represent mean ± S.E. (n = 5); *, p < 0.05 as compared with mock-transfected control.

FIGURE 3.

Chromatin immunoprecipitation analysis of mock-transfected and APP-expressing SH-SY5Y cells with anti-AICD or anti-HDAC1 antibodies. Mock-transfected SH-SY5Y cells or cells expressing the various APP isoforms were incubated with or without the γ-secretase inhibitor, L685,458, as described under “Experimental Procedures.” Cells were fixed, and chromatin was immunoprecipitated with anti-AICD (A) or anti-HDAC1 (B) antibodies. DNA pulled down by the antibody was analyzed by real-time PCR with primers specific to NEP promoters 1 and 2 or for part of the coding region (exon 19). The results represent enrichment of DNA pulled down with the anti-AICD or anti-HDAC1 antibodies versus non-immune IgG. Data represent mean ± S.E. (n = 5); *, p < 0.05 as compared with mock-transfected control.

FIGURE 4.

Analysis of NEP expression in Neuro2a and HEK293 cells and of NEP promoter occupancy in Neuro2a cells expressing the different APP isoforms. A, NEP expression in control and APP-overexpressing N2a cell lines. B, NEP expression in control and APP-overexpressing HEK cell lines. C, chromatin immunoprecipitation analysis of control and APP-overexpressing N2a cells with anti-AICD or anti-HDAC1 antibodies. Data represent mean ± S.E. (n = 3); *, p < 0.05 as compared with mock-transfected control, †, p < 0.05 as compared with other APP isoform cell lines.

FIGURE 5.

The effects of α- or β-secretase inhibition on the soluble forms of APP and on NEP expression and ChIP analysis in SH-SY5Y cells expressing APP₆₉₅. A and C, Western blot (A) and densitometric analysis (C) of sAPPα and sAPPβ levels in the conditioned medium of cells treated with the α-secretase inhibitor, TAPI-2 (100 μm), for 5 h. B and C, Western blot (B) and densitometric analysis (C) of sAPPα and sAPPβ levels in the conditioned medium of cells treated with the β-secretase inhibitor, βIV (1 μm), for 24 h. D, effects of α-secretase and/or β-secretase inhibition on NEP expression in SH-SY5Y APP₆₉₅ cells. Cells treated as above with TAPI-2 or βIV were analyzed for NEP expression levels, and data are expressed as the ratio of NEP expression to GAPDH expression. Data are shown as mean ± S.E. (n = 3); *, p < 0.05 as compared with control levels. E, APP₆₉₅ cells were treated with the α-secretase inhibitor, TAPI-2 (100 μm), or with the β-secretase inhibitor, βIV (1 μm), as above and fixed, and chromatin was immunoprecipitated with either the anti-AICD or the anti-HDAC1 antibodies, as described under “Experimental Procedures.” DNA pulled down by the antibody was analyzed by real-time PCR with primers specific to the NEP promoter 2. The data are presented as the -fold of enrichment of DNA pulled down with anti-AICD or anti-HDAC1 antibodies versus non-immune IgG. Data represent mean ± S.E. (n = 5); *, p < 0.05 for APP₆₉₅ cells as compared with uninhibited control after treatment with β-secretase inhibitor.

FIGURE 6.

Changes in Aβ levels and NEP expression with the APP_Swe mutation. A, Western blot of APP and actin in cell lysates from SH-SY5Y mock-transfected and APP₆₉₅- and APP_Swe-expressing SH-SY5Y cells. B, densitometric analysis of APP levels. C, relative amounts of Aβ_1–40 and Aβ_1–42 in conditioned medium from the APP_Swe cells as compared with the APP₆₉₅ cells. D, NEP expression in SH-SY5Y mock-transfected (control) and APP₆₉₅- and APP_Swe-expressing SH-SY5Y cells. Data are expressed as the ratio of NEP expression to GAPDH expression. E, chromatin immunoprecipitation with the anti-AICD antibody, as described under “Experimental Procedures.” DNA pulled down by the antibody was analyzed by real-time PCR with primers specific to the NEP promoter 2. The data are presented as the -fold of enrichment of DNA pulled down with anti-AICD versus non-immune IgG. Data represent mean ± S.E. (n = 3); *, p < 0.05 as compared with the mock-transfected control; †, p < 0.05 as compared with the wild-type APP₆₉₅ cell line.

FIGURE 7.

Effects of cellular cholesterol depletion on AICD and NEP expression levels in SH-SY5Y cells expressing APP₆₉₅. SH-SY5Y cells overexpressing APP₆₉₅ were treated with 5 mm methyl-β-cyclodextrin as described under “Experimental Procedures,” and subsequently, lysates were prepared and analyzed by immunoblotting for levels of AICD and NEP protein. A, Western blot of AICD and actin in control cells and cells treated with mβCD. B, densitometric analysis of AICD levels (normalized to actin) from the SH-SY5Y cells. C, Western blot of NEP and actin in control cells and cells treated with mβCD. D, densitometric analysis of NEP levels (normalized to actin) from the SH-SY5Y cells. Data represent mean ± S.E. (n = 3); *, p < 0.05 as compared with untreated cells.

FIGURE 8.

A model for the endocytosis and nuclear delivery of transcriptionally active AICD. APP₆₉₅ is sequestered along with BACE1 and γ-secretase complexes into lipid raft domains, and processing of the APP occurs following endocytosis where the acidic interior environment favors the catalytic action of the secretases (46), which are aspartic proteinases. The AICD, in combination with Fe65, is delivered to the nucleus by retrograde transport (20), where it can facilitate specific gene transcription, e.g. of the NEP gene (21, 24). In contrast, the predominant action of the metalloenzyme α-secretase on APP isoforms occurs at the cell surface. The subsequent action of γ-secretase releases AICD into the cytosol, where it can be degraded by insulin-degrading enzyme (IDE) (41). Ac, acetyl.