PS1 Affects the Pathology of Alzheimer's Disease by Regulating BACE1 Distribution in the ER and BACE1 Maturation in the Golgi Apparatus

Abstract

Neuritic plaques are one of the major pathological hallmarks of Alzheimer's disease. They are formed by the aggregation of extracellular amyloid-β protein (Aβ), which is derived from the sequential cleavage of amyloid-β precursor protein (APP) by β- and γ-secretase. BACE1 is the main β-secretase in the pathogenic process of Alzheimer's disease, which is believed to be a rate-limiting step of Aβ production. Presenilin 1 (PS1) is the active center of the γ-secretase that participates in the APP hydrolysis process. Mutations in the PS1 gene (PSEN1) are the most common cause of early onset familial Alzheimer's disease (FAD). The PSEN1 mutations can alter the activity of γ-secretase on the cleavage of APP. Previous studies have shown that PSEN1 mutations increase the expression and activity of BACE1 and that BACE1 expression and activity are elevated in the brains of PSEN1 mutant knock-in mice, compared with wild-type mice, as well as in the cerebral cortex of FAD patients carrying PSEN1 mutations, compared with sporadic AD patients and controls. Here, we used a Psen1 knockout cell line and a PS1 inhibitor to show that PS1 affects the expression of BACE1 in vitro. Furthermore, we used sucrose gradient fractionation combined with western blotting to analyze the distribution of BACE1, combined with a time-lapse technique to show that PS1 upregulates the distribution and trafficking of BACE1 in the endoplasmic reticulum, Golgi, and endosomes. More importantly, we found that the PSEN1 mutant S170F increases the distribution of BACE1 in the endoplasmic reticulum and changes the ratio of mature BACE1 in the trans-Golgi network. The effect of PSEN1 mutations on BACE1 may contribute to determining the phenotype of early onset FAD.

Keywords: BACE1; Golgi; endoplasmic reticulum; mutants; presenilin 1.

Figure 1

PS1 overexpression upregulated endogenous BACE1 in MEF PS1^−/− cells. (A) Western blot of the lysates 48 h after transfection with pPS1-WT or without transfection. The protein expression levels of APP and BACE1 were detected. (B) Quantification of APP and BACE1 in MEF PS1^−/− cells. (C) PS1 overexpression increased the mRNA level of BACE1 in MEF PS1^−/− cell. (D) Western blot of lysates from cells treated with γ-secretase inhibitor L-685,458 for 0, 60, 90, and 180 min after 48 h of transfection with pPS1-WT. The protein expression levels of APP, BACE1, PS1, and CTFs (C99) were detected. Quantification of APP (E), BACE1 (F), PS1 (G), and C99 (H); Actin was used as a reference; Dunnett’s post-hoc test: ** p < 0.01, * p < 0.05, (n = 3).

Figure 2

The effect of PS1 on BACE1 subcellular fractionation in MEF PS1^−/− cell. Western blotting of samples after sucrose density gradient centrifugation with transient transfection of BACE1 (A) or cotransfection of pBACE1 and pPS1 (B). Their quantitative analyses are shown in (C) and (D), respectively. The Y-axis represents the percentage of target protein in one fraction against total fractions (n = 3).

Figure 3

Intracellular distribution of BACE1 with or without PS1 (scale bar = 25 μm). (A) BACE1. (B) Organelle (calnexin: endoplasmic reticulum; Syn-6: Golgi; EEA1: early endosomes). (C) Nucleus (Hoechst33258). (D) Merged picture. (E) Quantification of the colocalization ratio. Dunnett’s post-hoc test: ** p < 0.01, * p < 0.05, (n = 3).

Figure 4

Time-lapse imaging of BACE1 and the Golgi tracker with or without PS1. (A) The distribution of BACE1-EGFP without PS1. (B) The distribution of BACE1-EGFP with PS1. (C) Quantitative analysis of the colocalization of BACE1-EGFP and the Golgi tracker with and without PS1.

Figure 5

Time-lapse imaging of BACE1 and the ER tracker with or without PS1. (A) The distribution of BACE1-EGFP without PS1. (B) The distribution of BACE1-EGFP with PS1. (C) Quantitative analysis of the colocalization of BACE1-EGFP and the ER tracker with and without PS1.

Figure 6

The effect of PS1 mutations on BACE1 subcellular fractionation in MEF PS1^−/− cell. Western blotting of samples after sucrose density gradient centrifugation with transient cotransfection of pBACE1 and pPS1-D257A (A) or pPS1-S170F (B). Their quantitative analyses are shown in (C) and (D), respectively. The Y-axis represents the percentage of target protein in one fraction against total fractions (n = 3).

Figure 7

Effect of PSEN1 mutations on colocation of BACE1 and the ER (scale bar = 25 μm). (A) BACE1. (B) Calnexin. (C) Nucleus (Hoechst33258). (D) Merged picture. Quantification of the colocalization ratio of BACE1 and the ER under PSEN1 mutations D257A (E) and S170F (F). One-way ANOVA: ** p < 0.01, * p < 0.05 (n = 3).

Figure 8

Effect of PSEN1 mutations on colocation of BACE1 and the Golgi (scale bar = 25 μm). (A) BACE1. (B) Syn-6. (C) Nucleus (Hoechst33258). (D) Merged picture. Quantification of the colocalization ratio of BACE1 and the Golgi under the PSEN1 mutations D257A (E) and S170F (F). One-way ANOVA: ** p < 0.01, * p < 0.05 (n = 3).