Monocytes derived from AD patients over-expressed CXCL1.

<p>Monocytes of 23 AD patients and 21 age-matched elderly controls were isolated and total RNA was extracted. Real-time RT-PCR was described in Material and Methods. CXCL1/GAPDH means the relative expression level of CXCL1 after correction for the expression of GAPDH. Data was the mean ± S.D. *<i>p</i> < 0.05 as compared with age-matched elderly controls.</p

AD patient’s monocytes or CXCL1-overexpressing THP-1 cells had an enhanced ability to cross HBMEC monolayer in response to Aβ.

<p>(A) 125nM Aβ were added to the lower chamber of the Transwell insert cultured with HBMEC, AD patients’ monocytes were loaded on upper chamber of the Transwell insert and incubated for 8h or 24h. The cells transmigrated into the lower chamber were harvested and counted. Data were the mean±S.D. *<i>p</i> < 0.05, **<i>p</i> < 0.01 as compared with age-matched elderly controls (n=8); (B) Transendothelial migration assays were performed in the presence of Aβ in 0, 1.25, 12.5, and 125nM respectively in the lower chamber of the Transwell insert cultured with HBMEC. Monocytes derived from AD patients were added to the upper chamber of Transwell insert for 24h. Transmigrated cells were harvested and counted. The results represent three independent experiments. Data were the mean±S.D. *<i>p</i> < 0.05, **<i>p</i> < 0.01; (C) AD patients’ monocytes with anti-CXCL1 neutralizing antibody were loaded into the upper chamber. The cells transmigrated into the lower chamber were counted after 24h. Data were mean±S.D. **<i>p</i> < 0.01. (D) THP-1 cells was transduced by lentivirus based vectors and efficiently expressed GFP. (E) 1×10⁶ transduced THP-1 cells were analyzed by flow cytometry and GFP fluorescence intensity was measured on the FL1 channel. Approximately 97% of the THP-1 cells were GFP-positive. Real-time RT-PCR (F) or ELISA (G) was done to detect the expression of CXCL1 in THP-1 cells transduced with lentivirus based CXCL1. (H) Transendothelial migration assays were performed in the presence of Aβ in 125nM in the lower chamber of the Transwell insert cultured with HBMEC. CXCL1-overexpressing THP-1 cells were added to the upper chamber of Transwell insert for 8h or 24h. (I) In the presence of Aβ in 0, 1.25, 12.5, and 125nM respectively in the lower chamber of the Transwell insert cultured with HBMEC, CXCL1-overexpressing THP-1 cells was added to the upper chamber of Transwell insert for 24h. (J) CXCL1-overexpressing THP-1 cells with anti-CXCL1 neutralizing antibody were loaded into the upper chamber in the presence of Aβ in 125nM in the lower chamber of the Transwell insert cultured with HBMEC. The cells transmigrated into the lower chamber were counted after 24h. Transmigrated cells were harvested and counted. The results represented three independent experiments. Data was the mean±S.D. *<i>p</i> < 0.05, **<i>p</i> < 0.01.</p

ROCK inhibitor Y27632 could block CXCL1-overexpressing THP-1 cells transendothelial migration.

<p>HBMEC were pretreated with specific inhibitors as described in Material and Methods and then co-cultured with CXCL1-overexpressing THP-1 cells for 24h. Transmigrated CXCL1-overexpressing THP-1 cells (A), TEER (B) and HRP flux (C) were measured, respectively. The different detergent solubility of endothelial occludin were analyzed by Western blot (D). Data were means ± S.D. of three independent experiments. *<i>p</i> < 0.05, **<i>p</i> < 0.01. (E) Confluent HBMEC was co-cultured with CXCL1-overexpressing THP-1 cells for 24h. The Rho activity in HBMEC was analyzed.</p

CXCR2 in HBMEC was up-regulated by Aβ sitmulation and contributed to CXCL1-overexpressing THP-1 cells transendothelial migration.

<p>(A) HBMEC were exposed to Aβ for indicated time at a concentration of 125 nM or (B) indicated concentrations for 1h. CXCR2 expression on HBMEC was detected by Western blot using anti- CXCR2 antibody. Untreated HBMEC were used as control. (C) HBMEC were incubated with Aβ for indicated times at a concentration of 125 nM, or with indicated concentrations for 1h (D), and the total RNA of HBMEC was extracted for real-time RT-PCR. Expression was normalized to GAPDH and the group without Aβ was set to 1. Data were the mean ± SD (n=3). *<i>p</i> < 0.05. The HBMEC monolayer cultured on the Transwell insert was placed on a 24-well plate while Aβ was present in the lower chamber. CXCL1-overexpressing THP-1 cells（E) or AD patients’ monocytes（F) with anti-CXCR2 neutralizing antibody were loaded into the upper chamber. The cells transmigrated into the lower chamber were counted after 24h. Data were mean±S.D. **<i>p</i> < 0.01. (G) The HBMEC expressing full-length ORF of CXCR2 by transient transfection was identified by Western blot using anti-CXCR2 antibody. HBMEC transfected with vector were used as control. CXCL1-overexpressing THP-1 cells (H) or AD patients’ monocyts (I) were loaded into upper chamber of Transwell insert cultured with HBMEC transfected with CXCR2. The transmigrated monocytes were counted. Data were the mean ± S.D. *<i>p</i> < 0.05, **<i>p</i> < 0.01.</p

Disassembly of tight junction between brain endothelium over-expressed CXCR2 were induced by CXCL1-overexpressing THP-1 cells.

<p>(A) HBMEC transfected with CXCR2 or vector control was seeded into the upper chamber of Transwell insert until they reached confluence. CXCL1-overexpressing THP-1 cells was added to the upper chamber of Transwell insert for the indicated time. Then the TEER was measured by EVOM volt-ohmmeter. The TEER value was expressed as the percent of the 0h. *<i>p</i> < 0.05, **<i>p</i> < 0.01. (B) HBMEC transfected with CXCR2 or vector control was seeded into the upper chamber of Transwell insert until they reached confluence. CXCL1-overexpressing THP-1 cells was added to the upper chamber of Transwell insert for the indicated time. The medium in the upper chamber was replaced by RPMI-1640 containing 0.5 μM of HRP, and then the medium in the lower chamber was collected and the HRP concentration was assessed. Data were means ± S.D. of three independent experiments. *<i>p</i> < 0.05, **<i>p</i> < 0.01. (C) CXCL1-overexpressing THP-1 cells was co-cultrued with HBMEC transfected with CXCR2 or vector control on sterilized coverslips for 24h. The distribution of ZO-1 in the HBMEC was visualized by immunofluorescence. Nuclei were counterstained with DAPI. Scale bar: 40 μm. (D) CXCL1-overexpressing THP-1 cells was co-cultrued with HBMEC transfected with CXCR2 or vector control for the indicated time. The soluble and insoluble forms of occludin were analyzed by Western Blot. *<i>p</i> < 0.05, **<i>p</i> < 0.01.</p

Flow cytometry analysis of microglial accumulation in APP mouse.

<p>(A) Monocytes of APP/PS1 tansgenetic mouse and wild type controls were isolated and total RNA was extracted. Real-time RT-PCR was described in Material and Methods. CXCL1/GAPDH means the relative expression level of CXCL1 after correction for the expression of GAPDH. Data was the mean ± S.D. *<i>p</i> < 0.05. (B) and (C) representative dot plots for flow cytometry of CD11b and CD45 stained brain cells. (D) Quantitation of the percent of total brain cells positive for both CD11b and CD45 (CD11b⁺CD45⁺) by flow cytometry (n = 3 mice per group, *<i>p</i> < 0.05).</p

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

<div><p>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.</p></div

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

<p>(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.</p

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

<p>(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.</p

CysC enhances proteasomal degradation of BACE1 in HBMEC.

<p>(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.</p