Figure 1.
Characterization of gallate moiety in increasing amyloid precursor protein (APP) α-proteolysis and reducing amyloid-β (Aβ) generation.
Given (−)-epigallocatechin-3 gallate (EGCG) significantly reduces Aβ generation from N2a/APPsw cells, we screened other gallate containing phenolic compounds, namely (−)-gallocatechin (GC), methyl gallate (MG), propyl gallate (PG), butyl gallate (BG), octyl gallate (OG), and atranorin (AN) (an ester of gallic acid) and two non-gallate containing compounds, (−)-epicatechin (EC) and (−)-catechin (C), for their ability to reduce Aβ generation (Table 1). (A-D) N2a cells overexpressing wild-type APP (N2a/APPwt) were plated in 24 well-plates (200,000 cells/well) and treated with each of these compounds at 10 µM (our previous studies indicated that this is the minimum concentration of EGCG required to noticeably inhibit Aβ production in N2a/APPsw cells) in addition to PBS control. Data are represented as pg of Aβ40, Aβ42 (A, B) or ng of sAPPα (C) in the conditioned media secreted throughout the 4 h of administration for each compound, normalized to intracellular protein (mg). sAPPα ELISA results were further supported by immunoblotting analysis (IB) using an anti-Aβ1–17 antibody (6E10) (D). One-way ANOVA followed by post hoc comparison revealed significant differences when comparing EGCG, AN and OG to each of other compounds. These results are representative of three independent experiments with three replicates for each condition. We have summarized these data in Table 1. Together, they suggest that the gallate moiety may be an important functional component in EGCG, AN and OG, consequently playing a role in reducing Aβ generation and elevating APP α-processing. We have replicated these results in cells overexpressing the Swedish mutant form of APP (N2a/APPsw cells, data not shown).
Table 1.
Characterization of gallate moiety in increasing amyloid precursor protein (APP) α-proteolysis and reducing amyloid-β (Aβ) generation.
Figure 2.
OG significantly promoted α-secretase cleavage of APP and reduced Aβ production in N2a cells overexpressing wild-type APP (N2a/APPwt cells).
N2a/APPwt cells were plated in 24 well-plates (100,000 cells/well) and treated with OG or EGCG at 10 µM, a most effective dose as determined by our previous and preliminary studies, for 12 hours. The conditioned media were then collected from these cells and subjected to: (A) ELISA for sAPPα, (B) IB analyses for sAPPα, (C) ELISA for Aβ40 and Aβ42and (D) IB analysis for total Aβ species using 6E10. In addition, cell lysates (Cell lysa.) were prepared and subjected to IB analysis for carboxyl-terminal fragments of APP (CTFs) using an anti-carboxyl-terminal APP antibody (pAb751/770) (E, top) and for ADAM10 using an anti-carboxyl-terminal ADAM10 antibody (ADAM10) (E, bottom). As shown below each IB panel, densitometry analysis shows the band density ratios (mean ± SD) of α-CTF to β-actin and mature ADAM10 (mADAM10) to β-actin. For A and C, data are presented as mean ± SD and one-way ANOVA followed by post hoc comparison revealed significant differences in promotion of sAPPα and inhibition of Aβ productions between OG versus EGCG (***P<0.001 with n = 3 for each condition).
Figure 3.
Estrogen receptor (ER) may mediate OG-promoted α-secretase cleavage of APP.
Cultured N2a/APPwt cells were treated with OG at the most effective dose (10 µM) in presence of an ERα selective antagonist (MPP dihydrochloride) or an ERβ selective inhibitor (PHTPP) at various doses as indicated for 12 hours. Cell lysates were then prepared from these cells and subjected to IB analysis for APP processing into α-CTF (A and B, left) using an APP-carboxyl-terminal antibody (pAb396) and ADAM10 activation (A, B, right) using an anti-carboxyl-terminal ADAM10 antibody (ADAM10). Most notably, co-treatment of cultured N2a/APPwt cells with MPP (A), but not PHTPP (B), significantly suppresses OG’s promotion of ADAM10 activation and α-CTF cleavage. In addition, cultured N2a/APPwt cells were also treated with OG at 10 µM in presence of PI3K (wortmannin, WM) or Akt (TCN) inhibitor at various doses as indicated for 12 hours. Cell lysates were prepared from these treated cells and subjected to IB analysis for APP processing into α-CTF (C and D, left) and ADAM10 maturation (C and D, right). Both PI3K (WM) (C) and Akt (TCN) (D) inhibitors markedly reduced OG’s promotion of ADAM10 maturation and α-CTF cleavage. As shown below each IB panel, densitometry analysis shows the band density ratios (mean ± SD) of α-CTF to β-actin and mature ADAM10 (mADAM10) to pre-mature ADAM10 (pADAM10).
Figure 4.
OG fails to promote α-secretase cleavage of APP in estrogen receptor α (ERα) deficient cells.
We cultured primary cortical neuronal cells from brain tissues of one-day-old ERα deficient and intact mice. One week after the primary culture, these neuronal cells were transiently transfected with human wild-type APP695 plasmid DNA (hAPPwt) with Lipofectiamine™ LTX Reagent (as detailed in the method section). Twenty four hours later, these neuronal cells were treated with OG at 10 µM for 12 hours. Cell lysates were then prepared and subjected to IB analyses for α- and β-CTF (A) using pAb396 and ADAM10 maturation (B) using an anti-carboxyl-terminal ADAM10 antibody (ADAM10). Most notably, OG’s promotion of anti-amyloidogenic APP processing was significantly attenuated in the ERα null mouse-derived primary neuronal cells overexpressing hAPPwt. Human APP expression was examined after transient transfection by IB analysis using an anti-Aβ1–17 antibody (6E10) (data not shown). As shown below each IB, densitometry analysis shows the band density ratio (mean ± SD) of α-CTF to β-CTF and mature ADAM10 (mADAM10) to pre-mature ADAM10 (pADAM10).
Figure 5.
Tg2576 mice treated with OG showed markedly increased α-secretase cleavage of APP and significantly decreased levels of Aβ generation.
A total of 13 Tg2576 female mice were used in this study over a period of one week. Five mice received OG or EGCG, and the remaining three received PBS. Beginning at 6 months of age (adult), Tg2576 mice were intracerebroventricular (i.c.v.) injected with OG or EGCG (10 µg in 5 µl PBS; this dose was suggested by our preliminary rangefinder study) or PBS (5 µl) daily through the implanted cannulae. Mice were sacrificed 24 h after the last injection for analysis of sAPPα, CTFs of APP, Aβs, and ADAM10 activation in brain homogenates using IB analyses and ELISA. IB analysis results show (A, top) holo APP and two bands corresponding to β-CTF and α-CTF by an APP-carboxyl-terminal antibody (pAb396), (A, bottom) mature ADAM10 (mADAM10) by an anti-carboxyl-terminal ADAM10 antibody (ADAM10) and (B) sAPPα production by a specific sAPPα antibody (2B3). As shown below each IB, densitometry analysis shows the band density ratios (mean ± SD) of α-CTF to β-CTF, mature ADAM10 (mADAM10) to β-actin and sAPPα to β-actin. (C) Detergent-soluble Aβs were analyzed by ELISA. Data are represented as mean ± SD of Aβ (pg/mg protein). For (C), a t test revealed a significant difference between OG- and EGCG-treated or PBS-injected Tg2576 mice for soluble total Aβ (Mean ± SD; **P<0.005; ***P<0.001). Most notably, OG-treated Tg2576 mice show much higher levels of APP processing into sAPPα and α-CTF as well as mADAM10 compared with either PBS- or EGCG-treated mice.
Figure 6.
Female and male Tg2576 mice do not show a significant difference in terms ofα-secretase processing of APP and soluble cerebral Aβ levels upon OG treatment.
A total of 13 Tg2576 mice were used in this study over a period of one week. Five female and five male Tg2576 mice received OG, and the remaining three mice (2 male/1 female) received PBS. Beginning at 6 months of age, these Tg2576 mice were i.c.v. injected with OG (10 µg in 5 µl PBS) or PBS (5 µl) daily through the implanted cannulae for one week. Mice were sacrificed 24 hours after the last injection for analysis of CTFs of APP, ADAM10 activation and Aβs in brain homogenates using IB analyses and ELISA. IB analysis show (A, top) holo APP and two bands corresponding to β-CTF and α-CTF by an APP-carboxyl-terminal antibody (pAb396) and (A, bottom) mADAM10 by an anti-carboxyl-terminal ADAM10 antibody (ADAM10). As shown below each IB, densitometry analysis shows the band density ratios (mean ± SD) of α-CTF to β-CTF and mADAM10 to β-actin. (B) Detergent-soluble Aβs were analyzed by ELISA. Data are represented as mean ± SD of Aβ (pg/mg protein). For (B), a t-test revealed no significant difference between female and male OG-treated Tg2576 mice for total soluble Aβ (P>0.05). Most notably, both OG-treated female and male Tg2576 mice show much higher levels of APP processing into α-CTF as well as mADAM10 compared with PBS-injected mice.