Article Figures & Data
Figures
- Figure 1.
EGCG treatment inhibits Aβ generation in cultured neuronal cells. Aβ1–40,42 peptides were analyzed in conditioned media from SweAPP N2a cells (a, c, d) or TgAPPsw mouse-derived primary neurons (b) by ELISA (n = 3 for each condition). Data are represented as a percentage of Aβ1–40,42 peptides secreted 12 h after EGCG treatment relative to control (untreated). a, b, One-way ANOVA followed by post hoc comparison revealed significant differences between EGCG and the other compounds at 40, 20, 10, and 5 μm treatment concentrations (p < 0.001). c, When comparing EGCG (20 μm) treatment with cotreatment of SweAPP N2a cells with EGCG (20 μm) plus GC (80 μm), C (80 μm), or GC/C, a significant difference was noted for each comparison (p < 0.001). d, SweAPP N2a cells were treated with EGCG at a comparable concentration with that found in GT (GT contains 30% EGCG), and a significant difference was noted between GT and EGCG treatments (40 μg/ml vs 20 μm; 20 μg/ml vs 10 μm; 10 μg/ml vs 5 μm) on inhibition of Aβ generation (p < 0.001 for each comparison). Reduction for each treatment condition is indicated for c and d.
- Figure 2.
EGCG treatment alters APP cleavage in vitro. a, b, SweAPP N2 a cells were treated with EGCG at 20 μm or PBS (control) for 12 h. Cell lysates were prepared and subjected to Western blot (WB) analysis of APP CTFs (a), and conditioned media were collected for immunoprecipitation (IP)/WB (b). c, d, Cell lysates were prepared from SweAPP N2a cells treated with EGCG at 20μm for a range of time points (c) or EGCG at various doses for 12 h (d) and subjected to WB for APP CTFs. e, g, Cell lysates were prepared from SweAPP N2a cells treated with EGCG (-) EC, (+) EC, GC, C, or GT at the doses indicated for 12 h. For f, SweAPP N2a cells were cotreated with EGCG (20 μm) and GC, C, or GC/C at 80 μm for 12 h. For a and c–g, WB analysis using antibody 369 against the cytoplasmic tail of APP shows full-length holo APP and two bands corresponding to β-CTF (C99) and α-CTF (C83). For b, WB analysis using antibody 22C11 against the N terminus of APP shows sAPP-α (IP with antibody 6E10 directed against Aβ1–17) and sAPP-β [following immunodepletion (ID) with 6E10 and subsequent IP with 22C11]. a, c, d, g, Western blot analysis with anti-actin antibody shows actin protein (as an internal reference control). Densitometry analysis shows the ratio of α-CTF to β-CTF as indicated below the figures. a, A t test revealed a significant difference between EGCG treatment and control (n = 3 for each condition; p < 0.001). f, One-way ANOVA showed significant between-groups differences (p < 0.01) with n = 4 for each condition, and post hoc comparison revealed a significant difference between EGCG and EGCG/GC/C treatments (p < 0.001). Similar results were observed in N2a cells transfected with human wild-type APP695 using 369 antibody and in SweAPP N2a cells using Calbiochem polyclonal APP C-terminal APP antibody (data not shown).
- Figure 3.
EGCG treatment promotes α-secretase cleavage of APP in vitro. a, b, Cell lysates were prepared from SweAPP N2a cells treated with EGCG (20μm) for different time points as indicated. a, Western blot analysis by anti-TACE antibody shows TACE and cleaved fragments. b, α-, β-, and γ-secretase cleavage activities were analyzed in cell lysates using secretase cleavage activity assays. Data are presented as a percentage of fluorescence units/milligrams protein activated 1, 2, or 3 h after EGCG treatment relative to control (PBS). A t test revealed a significant difference betweenα-secretase and either β- or γ-secretase cleavage activity at 1, 2, and 3 h after EGCG treatment (p < 0.001). c–e, SweAPP N2a cells were treated with EGCG (20 μm) or PBS (control) in the presence or absence of TAPI-1 at various doses (c) or at 25 μm (d, e) for 4 h. Cell-cultured supernatants were collected, and cell lysates were prepared from cultured cells. c, Western blot analysis by antibody 369 shows holo APP and two bands corresponding to β-CTF and α-CTF. d, Data are represented as percentage of α-secretase cleavage activity calculated in terms of fluorescence units/milligrams protein after EGCG treatment relative to control (PBS) 4 h after EGCG treatment in the presence or absence of TAPI-1. A t test revealed a significant difference between EGCG treatment and cotreatment with EGCG and TAPI-1 (p < 0.001); increased levels of activity are indicated. e, Data are presented as percentage of Aβ secretion relative to PBS control 4 h after EGCG treatment in the presence or absence of TAPI-1. A t test revealed a significant difference between EGCG and EGCG plus TAPI-1 treatment (p < 0.001); reduction for each treatment condition is indicated.
- Figure 4.
EGCG promotes nonamyloidogenic APP processing and reduces cerebral amyloidosis in TgAPPsw mice. Brain homogenates were prepared from female TgAPPsw mice treated with EGCG (n = 11) or PBS (n = 9). a, Top, Western blot analysis by antibody 369 shows holo APP and two bands corresponding to β-CTF and α-CTF. a, Middle and bottom, Western blot analysis by antibody 22C11 shows holo APP (middle; following ID/C-terminal APP antibody) and sAPP-α (bottom; following ID/C-terminal APP antibody and IP/6E10). Detergent-soluble Aβ1–40,42 (b) and insoluble Aβ1–40,42 prepared with 5 m guanidine (c) were analyzed by ELISA. Data are presented as mean ± 1 SEM of Aβ1–40 or Aβ1–42 (pg/mg protein) separately. For b and c, a t test revealed a significant between-groups difference for either soluble or insoluble Aβ1–40,42 (p < 0.001 for each comparison). d, α-, β-, and γ-secretase cleavage activities were analyzed by secretase cleavage activity assays. Data are presented as mean ± 1 SEM of fluorescence units/mg protein. A t test revealed a significant difference between EGCG- and PBS-treated Tg APPsw mice for α-secretase activity (p < 0.001). e, Mouse brain coronal paraffin sections were stained with anti-human Aβ antibody (4G8). Left, Control PBS-treated mice. Right, EGCG-treated mice. The top panels are from the cingulate cortex (CC), the middle panels are from the hippocampus (H), and the bottom panels are from the entorhinal cortex (EC). f, Percentages of 4G8-immunoreactive Aβ plaques (mean ± 1 SEM) were calculated by quantitative image analysis, and reduction for each brain region is indicated. g, Mouse brain sections from the indicated brain regions were stained with thioflavin S. Left, Control PBS-treated mice. Right, EGCG-treated mice. h, Percentage of thioflavin S plaques (mean ± 1 SEM) were quantified by image analysis, and reduction for each brain region is indicated. A t test for independent samples revealed significant differences (p < 0.005) between groups for each brain region examined in f and h. Scale bar, 50 μm.
