APP intracellular domain derived from amyloidogenic β- and γ-secretase cleavage regulates neprilysin expression

Abstract

Alzheimer's disease (AD) is characterized by an accumulation of Amyloid-β (Aβ), released by sequential proteolytic processing of the amyloid precursor protein (APP) by β - and γ-secretase. Aβ peptides can aggregate, leading to toxic Aβ oligomers and amyloid plaque formation. Aβ accumulation is not only dependent on de novo synthesis but also on Aβ degradation. Neprilysin (NEP) is one of the major enzymes involved in Aβ degradation. Here we investigate the molecular mechanism of NEP regulation, which is up to now controversially discussed to be affected by APP processing itself. We found that NEP expression is highly dependent on the APP intracellular domain (AICD), released by APP processing. Mouse embryonic fibroblasts devoid of APP processing, either by the lack of the catalytically active subunit of the γ-secretase complex [presenilin (PS) 1/2] or by the lack of APP and the APP-like protein 2 (APLP2), showed a decreased NEP expression, activity and protein level. Similar results were obtained by utilizing cells lacking a functional AICD domain (APPΔCT15) or expressing mutations in the genes encoding for PS1. AICD supplementation or retransfection with an AICD encoding plasmid could rescue the down-regulation of NEP further strengthening the link between AICD and transcriptional NEP regulation, in which Fe65 acts as an important adaptor protein. Especially AICD generated by the amyloidogenic pathway seems to be more involved in the regulation of NEP expression. In line, analysis of NEP gene expression in vivo in six transgenic AD mouse models (APP and APLP2 single knock-outs, APP/APLP2 double knock-out, APP-swedish, APP-swedish/PS1Δexon9, and APPΔCT15) confirmed the results obtained in cell culture. In summary, in the present study we clearly demonstrate an AICD-dependent regulation of the Aβ-degrading enzyme NEP in vitro and in vivo and elucidate the underlying mechanisms that might be beneficial to develop new therapeutic strategies for the treatment of AD.

Keywords: AICD; APP intracellular domain; Alzheimer's disease; Aβ degradation; gene regulation; neprilysin.

Figure 1

Reduction of NEP gene expression, protein level and activity in PS1/2 and APP/APLP2 deficient mouse embryonic fibroblasts (MEF). (A) Reduced NEP gene expression (71.6 ± 6.4%, p ≤ 0.001), protein level (26.2 ± 3.5%, p ≤ 0.001) and activity (66.6 ± 1.9%, p ≤ 0.001) in MEF cells devoid of PS1 and PS2 (MEF PS1/2 ^−/−) compared to MEF PS1/2 ^−/− retransfected with PS1 wt (MEF PS1rescue). Level of NEP gene expression in MEF wt: 111.2 ± 9.2%, p = 0.005 when compared to MEF PS1/2 ^−/−. (B) Decreased NEP gene expression (20.4 ± 13.3%, p ≤ 0.001), protein level (50.4 ± 10.9%, p = 0.012) and activity (74.9 ± 6.4%, p ≤ 0.001) in MEF lacking APP and the APP-like protein APLP2 (MEF APP/APLP2 −/−) in comparison to wild-type cells (MEF wt). (C) Decreased NEP gene expression in MEF wt treated with γ-secretase inhibitor (87.8 ± 3.6%, p = 0.015). Unaltered NEP gene expression in MEF APP/APLP2 −/− after incubation with γ-secretase inhibitor (20.8 ± 1.3%, p = 0.73 compared to 20.4% in cells treated with solvent control). (D) No effect of Aβ40/42 peptide long term incubation on NEP gene expression in MEF PS1/2^−/− (80.2 ± 9.9%, p = 0.43 compared to 71.6% in cells treated with solvent control) and MEF APP/APLP2^−/− (18.8 ± 2.5%, p = 0.43 compared to 20.4% in cells treated with solvent control). Asterisks show the statistical significance (^*p ≤ 0.05, ^**p ≤ 0.01 and ^***p ≤ 0.001, n.s., not significant). All quantified data represent an average of at least three independent experiments. Error bars represent standard deviation of the mean.

Figure 2

NEP gene expression and activity is regulated by AICD. (A) Reduced NEP gene expression (23.4 ± 8.1%, p ≤ 0.001), protein level (68.0 ± 4.1%, p ≤ 0.001), activity (55.3 ± 3.4%, p ≤ 0.001) and total Aβ degradation (−Thiorphan: 54.7 ± 2.7%, p ≤ 0.001 compared to wt; +Thiorphan: 68.5 ± 4.1%, p ≤ 0.001 compared to wt; p = 0.02 for effects −Thiorphan vs. +Thiorphan) in MEF expressing an APP construct lacking the last 15 C-terminal amino acids (aa) and therefore a functional AICD domain (MEF APPΔCT15) compared to control fibroblasts. (B) Measurement of FITC-AICD uptake in lysates of MEF APPΔCT15 after short term (2.8 ± 0.1%, p ≤ 0.001, shown in x fold change of fluorescence) and long term incubation with FITC-AICD (18.0 ± 5.1%, p = 0.03, shown in x fold change of fluorescence) compared to cells treated with solvent control. (C) Enhanced NEP gene expression in MEF APPΔCT15 after lipofection based short term (12 h) (143.6 ± 29.8%, p = 0.193 compared to solvent control) and after long term (9 days) (168.0 ± 18.7%, p = 0.002 compared to solvent control) incubation with AICD peptides. Level of NEP gene expression in MEF wt cells (427.0 ± 74.0%, p ≤ 0.001 when compared to MEF APPΔCT15) indicates a partial rescue of NEP gene expression after AICD incubation. (D) Increased NEP activity in MEF APPΔCT15 after lipofection based short term (12 h) (115.8 ± 5.5%, p = 0.014 compared to solvent control) and after long term (9 days) (120.4 ± 4.9%, p = 0.008 compared to solvent control) incubation with AICD peptides. Level of NEP activity in MEF wt cells (180.0 ± 6.12%, p ≤ 0.001 when compared to MEF APPΔCT15) indicates a partial rescue of NEP activity after AICD incubation. (E) Unaltered viabiliy (99.98 ± 0.11%, p = 0.86) and total protein level (101.5 ± 0.87%, p = 0.33) in MEF APPΔCT15 incubated with AICD peptides. (F) Increase in NEP gene expression (201.9 ± 21.4%, p = 0.009) in MEF APPΔCT15 retransfected with the last 50 C-terminal aa of APP (MEF APPΔCT15 + C50) compared to MEF APPΔCT15. Level of NEP gene expression in MEF wt cells (427.0 ± 74.0%, p ≤ 0.001 when compared to MEF APPΔCT15) indicates a partial rescue of NEP gene expression due to expression of C50. Statistical significance as described for Figure 1.

Figure 3

NEP gene expression and activity is influenced by amyloidogenic, but not by non-amyloidogenic APP processing due to degradation of α-/γ-APP-cleavage derived AICD by IDE. (A) Increased NEP gene expression (139.6 ± 52.3%, p = 0.49) and activity (127.5 ± 3.3%, p ≤ 0.001) in SH-SY5Y cells overexpressing APP carrying the swedish mutation (APPswe) compared to SH-SY5Y APP cells. Level of NEP gene expression in SH-SY5Y wt cells is indicated by a dotted line (p = 0.01 for NEP gene expression in SH-SY5Y APPswe compared to SH-SY5Ywt). (B) Reduction of NEP gene expression (64.0 ± 4.4%, p = 0.0012) and activity (50.0 ± 5.6%, p ≤ 0.001) in MEF PS1/2 deficient cells retransfected with PS1 carrying the T354I mutation (MEF PS1-T354I) compared to MEF PS1/2 deficient cells retransfected with PS1 wt (MEF PS1rescue). Level of NEP gene expression in MEF wt cells is indicated by a dotted line. (C) NEP gene expression and activity in SH-SY5Y overexpressing the APP α-cleaved C-terminal fragment (α-CFT) (RNA-level: 86.9 ± 9.5%, p = 0.241; activity: 100.3 ± 6.0%, p = 0.967) and the APP β-cleaved C-terminal fragment (β-CFT) (RNA-level: 223.5 ± 36.3%, p = 0.027; activity: 192.5 ± 3.9%, p ≤ 0.001). (D) Increased NEP gene expression (157.2 ± 12.2%, p ≤ 0.001) in SH-SY5Y α-CFT cells with reduced IDE expression (SH-SY5Y α-CTF + IDE-KD). (E) Enhanced NEP expression (195.2 ± 6.9%, p ≤ 0.001) in SH-SY5Y α-CTF cells after pharmacological IDE inhibition (SH-SY5Y α-CTF + IDE inhibitor). (F) NEP gene expression in SH-SY5Y wt cells treated with α- (98.3 ± 5.4%, p = 0.764), β - (80.6 ± 3.5%, p = 0.005) or γ- (80.7 ± 0.6%, p ≤ 0.001) secretase inhibitor. (G) Reduction in NEP gene expression (63.0 ± 5.1%, p = 0.002) and activity (71.5 ± 4.0%, p ≤ 0.001) in SH-SY5Y Fe65 knock-down (SH-SY5Y Fe65-KD) compared to mock-transfected control cells. Statistical significance as described for Figure 1.

Figure 4

NEP gene expression in brain tissue of transgenic mice. (A) NEP gene expression in brain tissue of APP knock-out (APP^−/−) (81.3 ± 8.5%, p = 0.049), APLP2 knock-out (APLP2^−/−) (106.2 ± 6.1%, p = 0.317) and in APP/APLP2-double knockout mice (APP/APLP2^−/−) (83.8 ± 6.1%, p = 0.015). (B) NEP gene expression in mice devoid of the last 15 APP C-terminal aa and hence AICD (APPΔCT15) (75.8 ± 5.7%, p ≤ 0.001). (C) NEP gene expression in APPswe (149.5 ± 19.4%, p = 0.016) and APPswe/PS1ΔE9 (78.6 ± 10.5%, p = 0.058) mouse brains. Statistical significance as described for Figure 1.