Changes in zinc level in APP/PS1 mice fed a zinc diet.

<p>(<b>A</b>) At the age of 9 months, the Zn group mice showed an increased level of serum zinc. (<b>B</b>) Brain zinc levels were also significantly increased in the Zn group mice. ** <i>p</i><0.01 versus control group (Student's <i>t</i> test).</p

Expression level of APP cleavage enzymes and products in APP/PS1 mice.

<p>(<b>A</b>) The expression levels of ADAM10, BACE1 and PS1 in transgenic mouse brain were determined by Western blot analyses. GAPDH was used as an internal control. The level of ADAM10 was markedly reduced, whereas the level of BACE1 was significantly increased in Zn-treated mice, compared with controls. (<b>B</b>) Zinc treatment significantly reduced the level of sAPPα and C83, and increased the level of sAPPβ and C99 compared with the control group. ** <i>p</i><0.01 versus control group (Student's <i>t</i> test).</p

Morris water maze assessment of APP/PS1 transgenic mice.

<p>APP/PS1 mice at the age of 3 months were given either a standard diet and deionized water (Con), or a standard diet and deionized water containing 20 mg/ml ZnSO4 (Zn). Morris water maze tests were performed to evaluate whether high dietary zinc treatment affects learning and memory in APP/PS1 mice at the age of 9 months. (<b>A</b>, <b>B</b>) In the visible platform training from day 1 to 2, mice in different groups exhibited a similar escape latency and path length to find the visible platform. At day 3, 5, and 7 of the hidden platform tests, Zn-treated mice showed the longest latency and escape length. (<b>C</b>) In the probe trial on the last day, the Zn-treated mice exhibited the lowest passing times into the northwest quadrant, where the hidden platform was previously located. * <i>p</i><0.05, ** <i>p</i><0.01 versus control group (repeated measures ANOVA).</p

Expression level of APP and Aβ in APP/PS1 mice.

<p>(<b>A</b>) RT-PCR showed that no difference in APP mRNA levels between groups was detected in the transgenic mouse brain. GAPDH was used as an internal control. (<b>B</b>) Western blot analysis showed that high zinc treatment significantly increased the level of APP695protein. GAPDH was used as an internal control. (<b>C</b>, <b>D</b>) ELISA assay showed that the levels of Aβ1-40 and Aβ1-42 were significantly increased in Zn-treated mice compared with controls. * <i>p</i><0.05, ** <i>p</i><0.01 versus control group (Student's <i>t</i> test).</p

Cell viability and zinc accumulation in APPsw cells treated with zinc and TPEN.

<p>(<b>A</b>, <b>B</b>) MTT analyses were performed on the SHSY-5Y cells stably transfected with human APPsw, to select appropriate concentrations of zinc and TPEN for the <i>in vitro</i> studies. The cells were treated with indicated concentrations of zinc and TPEN for 8 h. Based on the cell viability, we chose a concentration of 1 µM ZnSO4 as “low zinc” and 70 µM as “high zinc” treatment, and 1 µM TPEN for zinc chelation treatment, respectively. (<b>C, D</b>) Zinquin fluorescence staining showing that zinc treatments enhanced the Zn-fluorescence accumulation, while TPEN reduced the density of fluorescence in APPsw cells. Fluorescence values were obtained during the period of basal conditions and the status at the end of each indicated administration. The y-axis data describe F/F0 fluorescence values. ** <i>p</i><0.01 versus control group; ## <i>p</i><0.01 versus 70 µM Zn treatment group (one-way ANOVA <i>Post hoc</i> Fisher's PLSD).</p

Zinc treatment enhances neuritic plaque formation in APP/PS1 mice.

<p>(<b>A</b>) Aβ immunohistochemical images showing the Aβ-positive plaques in the transgenic mouse brain. There were more neuritic plaques in Zn-treated mice compared with controls. (<b>B</b>) Quantification of neuritic plaques showed that both the number and size of neuritic plaques were increased in Zn-treated mice compared with controls. ** <i>p</i><0.01 versus control group (Student's <i>t</i> test).</p

Expression level of APP cleavage enzymes and products in APPsw cells.

<p>SHSY-5Y cells stably overexpressing APPsw were exposed to 1 µM zinc, 70 µM zinc, 1 µM TPEN, and 70 µM zinc plus 1 µM TPEN, respectively, for 8 h. (<b>A</b>) Western blot was performed to determine the expression levels of APP cleavage enzymes, including ADAM10, BACE1 and PS1. GAPDH was used as an internal control. Low (1 µM) and high zinc (70 µM) treatment showed different effects on the expression of ADAM10. ADAM10 was markedly increased after low zinc treatment, but significantly reduced after high zinc treatment. There were no significant changes in ADMA10 levels in the TPEN or Zn + TPEN group, compared with controls. High zinc (70 µM) treatment significantly increased the levels of BACE1 as well as PS1. TPEN treatment reduced the BACE1 and PS1 levels. In Zn + TPEN group, the levels of BACE1 and PS1 were significantly reduced compared with the high zinc (70 µM) treatment group. (<b>B</b>) The expression levels of APP cleavage products, including sAPPα, sAPPβ, C83 and C99, were determined by Western blot analysis. GAPDH was used as an internal control. Low zinc (1 µM) exposure significantly enhanced the expression level of sAPPα, however, high zinc (70 µM) treatment markedly reduced the sAPPα expression level. There were no significant changes in sAPPα levels in the TPEN or Zn + TPEN treatment group compared with controls. The expression level of sAPPβ was significantly decreased after low zinc (1 µM) treatment, but was significantly increased after high zinc (70 µM) treatment. (<b>C</b>) ELISA results showed the Aβ1-42 level in the medium of APPsw cells following the indicated treatments. High zinc (70 µM) treatment significantly increased the levels of Aβ1-42, whereas low zinc (1 µM) and TPEN treatment reduced the levels of Aβ1-42. * <i>p</i><0.05, ** <i>p</i><0.01 versus control group; <i># p</i><0.05, ## <i>p</i><0.01 versus 70 µM Zn treatment group (one-way ANOVA <i>Post hoc</i> Fisher's PLSD).</p

Zinc accumulation in neuritic plaques in APP/PS1 mice.

<p>(<b>A</b>) Zinc ions stained by AMG showing the distribution patterns of zinc ions in the cortex and hippocampus, especially mossy fibers. Importantly, the number and size of the zinc-containing plaques were increased in Zn-treated mice. (<b>B</b>) Statistical analysis showing that the number and size of zinc-containing neuritic plaques were significantly increased in Zn-treated mice compared with controls. ** <i>p</i><0.01 versus control group (Student's <i>t</i> test).</p

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

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