Cystatin C Shifts APP Processing from Amyloid-β Production towards Non-Amyloidgenic Pathway in Brain Endothelial Cells

Abstract

Amyloid-β (Aβ), the major component of neuritic plaques in Alzheimer's disease (AD), is derived from sequential proteolytic cleavage of amyloid protein precursor (APP) by secretases. In this study, we found that cystatin C (CysC), a natural cysteine protease inhibitor, is able to reduce Aβ40 secretion in human brain microvascular endothelial cells (HBMEC). The CysC-induced Aβ40 reduction was caused by degradation of β-secretase BACE1 through the ubiquitin/proteasome pathway. In contrast, we found that CysC promoted secretion of soluble APPα indicating the activated non-amyloidogenic processing of APP in HBMEC. Further results revealed that α-secretase ADAM10, which was transcriptionally upregulated in response to CysC, was required for the CysC-induced sAPPα secretion. Knockdown of SIRT1 abolished CysC-triggered ADAM10 upregulation and sAPPα production. Taken together, our results demonstrated that exogenously applied CysC can direct amyloidogenic APP processing to non-amyloidgenic pathway in brain endothelial cells, mediated by proteasomal degradation of BACE1 and SIRT1-mediated ADAM10 upregulation. Our study unveils previously unrecognized protective role of CysC in APP processing.

Fig 1. CysC reduces Aβ secretion and promotes sAPPα secretion in HBMEC.

(A, B) HBMEC were treated with 0.4 μM CysC for indicated times (0, 2, 4, 8, 12 hr), and the concentrations of Aβ40 (A) and sAPPα (B) levels in the culture medium (supernatant) were determined by ELISA analysis. (C, D) HBMEC were treated with indicated concentrations of CysC for 8 hr. Then the concentrations of Aβ (C) and sAPPα (D) were measured by ELISA. (E, F) HBMEC were pretreated with CysC (0.4 μM) for 4 hr, followed by incubation with 50 μM H2O2 for indicated times (0, 2, 4, 8, 12 hr). Then the concentrations of Aβ (E) and sAPPα (F) were determined by ELISA. All values are presented as mean ± SEM for three independent experiments. Statistical significance was calculated by one-way ANOVA. *, p<0.05; **, p<0.01; ***, p<0.001.

Fig 2. CysC specifically attenuates the increased BACE1 expression induced by H₂O₂ in HBMEC.

(A) HBMEC were treated with 50 μM H₂O₂ for indicated times (0, 2, 4, 8, 12 hr) and then the expression of BACE1, BACE2, NICASTRIN, PS1, APH-1, PS2 and PEN-2 were detected by western blot, with GAPDH served as loading control (left panel). The protein levels were obtained by calculating the band densitometry and normalized to the band intensity of GAPDH, and the values were normalized to control defined as 1 (right panel). Statistical significance was analyzed using one-way ANOVA. *, p<0.05; **, p<0.01; ***, p<0.001. (B) HBMEC were pretreated with β-secretase inhibitor II (1 μM) and γ-secretase inhibitor IX (1 μM) for 1 hr, respectively, with DMSO served as vehicle control. Then the cells were treated with or without H₂O₂ (50 μM) for 8 hr. The concentrations of Aβ40 were determined by ELISA assays. Statistical significance was analyzed using one-way ANOVA. **, p<0.01; ***, p<0.001. (C) HBMEC were pretreated with CysC (0.4 μM) for 4 hr followed by incubation with 50 μM H2O2 for 8 hr, and then the expression of BACE1, NICASTRIN, PS1, PS2, APH-1 and PEN-2 were detected by western blot, with GAPDH as the loading control (left panel). The protein levels were obtained by calculating the band densitometry and normalized to the band intensity of GAPDH, and the values were normalized to control (right panel).*, p<0.05; **, p<0.01.

Fig 3. CysC enhances proteasomal degradation of BACE1 in HBMEC.

(A) HBMEC were treated with 50 μM H₂O₂ for indicated times in the absence or presence of CysC (0.4 μM) and the mRNA levels of BACE1 were analyzed by real-time RT-PCR, with GADPH as internal control. Data were normalized to control. (B) HBMEC were pretreated with CysC (0.4 μM) for 4 hr and then the cells were incubated with MG132 (5 μM), chloroquine (100 μM) or NH4Cl (20 mM) for 1 hr, followed by incubation with H2O2 (50 μM) for 8 hr. Then the protein levels of BACE1 were detected by western blot. GAPDH was used as the loading control. Statistical significance was calculated using two-way ANOVA. *, p<0.05. (C) HBMEC were pretreated with or without CysC (0.4 μM) for 4 hr followed by incubation with H₂O₂ (50 μM) for 8 hr. Total cells lysates were immunoprecipitated with BACE1 antibody and then the ubiquitinated BACE1 was detected by western blot with anti-ubiquitin antibody. Representative results from 3 independent experiments were presented.

Fig 4. CysC-induced sAPPα secretion is mediated by upregulation of ADAM10 in HBMEC.

(A) HBMEC were treated with CysC (0.4 μM) for 0, 2, 4, 8, 12 hr, respectively, and then the protein levels of ADAM10 were detected by western blot, with GAPDH as loading control. The band densitometry were measured and normalized to GAPDH, and the values were normalized to control. Statistical significance was analyzed with one-way ANOVA. *, p<0.05; **, p<0.01. (B, C) HBMEC were transiently transfected with ADAM10 siRNA#1(B) and ADAM10 siRNA#2 (C), with non-silencing siRNA as control. 48 hr later, the cells were treated with CysC (0.4 μM) for 8 hr. Then ADAM10 expression was analyzed by western blot, with GAPDH as loading control. The band densitometry were measured and normalized to GAPDH, and the values were normalized to control. Statistical significance was analyzed with one-way ANOVA. *, p<0.05; **, p<0.01. (D) The HBMEC transfected with ADAM10 siRNA were incubated with CysC (0.4 μM) for 8 hr, with non-silencing siRNA as a control. Then the secreted sAPPα were determined by ELISA assay. The values are means ± SEM of three independent experiments. **, P<0.01.

Fig 5. CysC enhances SIRT1 expression to upregulate ADAM10 mRNA levels, leading to increased sAPPα secretion.

(A) HBMEC were treated with CysC for indicated times (0, 2, 4, 8, 12 hr), and the mRNA expressions of ADAM10 were analyzed by real-time RT-PCR, with GADPH as internal control. Data were normalized to control. Statistical significance was analyzed using one-way ANOVA. *, p<0.05; **, p<0.01; ***, p<0.001. (B) HBMEC were incubated with CysC for indicated times (0, 2, 4, 8, 12 hr), then the protein levels of SIRT1 were detected by western blot with GAPDH as the loading control. The band densitometry were measured and normalized to GAPDH, and the values were normalized to control. Statistical significance was analyzed with one-way ANOVA. *, p<0.05. (C-E) HBMEC were transiently transfected with SIRT1 siRNA, with non-silencing siRNA as a control. 48 hr later, the cells were treated with CysC for 8 hr, and the mRNA (C) and protein (D) levels of ADAM10, as well as sAPPα secretion (E) were determined. Statistical significance was calculated with one-way ANOVA.*, p<0.05; **, p<0.01.