Abstract
Recently, we have shown that green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) exerts a beneficial role on reducing brain Abeta levels, resulting in mitigation of cerebral amyloidosis in a mouse model of Alzheimer disease. EGCG seems to accomplish this by modulating amyloid precursor protein (APP) processing, resulting in enhanced cleavage of the alpha-COOH-terminal fragment (alpha-CTF) of APP and corresponding elevation of the NH(2)-terminal APP product, soluble APP-alpha (sAPP-alpha). These beneficial effects were associated with increased alpha-secretase cleavage activity, but no significant alteration in beta-or gamma-secretase activities. To gain insight into the molecular mechanism whereby EGCG modulates APP processing, we evaluated the involvement of three candidate alpha-secretase enzymes, a-disintegrin and metalloprotease (ADAM) 9, 10, or 17, in EGCG-induced non-amyloidogenic APP metabolism. Results show that EGCG treatment of N2a cells stably transfected with "Swedish" mutant human APP (SweAPP N2a cells) leads to markedly elevated active ( approximately 60 kDa mature form) ADAM10 protein. Elevation of active ADAM10 correlates with increased alpha-CTF cleavage, and elevated sAPP-alpha. To specifically test the contribution of ADAM10 to non-amyloidogenic APP metabolism, small interfering RNA knockdown of ADAM9, -10, or -17 mRNA was employed. Results show that ADAM10 (but not ADAM9 or -17) is critical for EGCG-mediated alpha-secretase cleavage activity. In summary, ADAM10 activation is necessary for EGCG promotion of non-amyloidogenic (alpha-secretase cleavage) APP processing. Thus, ADAM10 represents an important pharmacotherapeutic target for the treatment of cerebral amyloidosis in Alzheimer disease.
Highlights
Gression of Alzheimer disease (AD) [1,2,3,4]
EGCG Treatment Enhances ADAM10 Activation in Cultured CNS Cells—To determine whether EGCG modulates expression of candidate ␣-secretases ADAM9, -10, or -17, we treated N2a cells stably transfected with Swedish mutant amyloid precursor protein (APP) (SweAPP N2a cells) with various doses of EGCG and measured the respective levels of protein expression
A corollary of this hypothesis is that therapeutics aimed at decreasing A generation/deposition through suppression of amyloidogenic APP processing or promotion of non-amyloidogenic APP processing are probable future treatments for AD [29, 37]. ␣-Secretases process APP into sAPP-␣, ␣/␥-secretase-processed soluble fragments, and corresponding carboxyl-terminal fragment (CTF), all of which are not known to contribute directly to amyloid plaques in the brains of AD patients [38]
Summary
Reagents—Green tea-derived EGCG (95% purity by high performance liquid chromatography) was purchased from Sigma. Following 3 washes, biotinylated antibody, 4G8 (0.5 g/ml in PBS with 1% bovine serum albumin), was added to the plates and incubated for 2 h at room temperature. Primary culture microglial cells were isolated from mouse cerebral cortices and were grown in RPMI 1640 medium supplemented with 5% fetal calf serum, 2 mM glutamine, 100 units/ml penicillin, 0.1 g/ml streptomycin, and 0.05 mM 2-mercaptoethanol according to previously described methods [32, 33]. One-way ANOVA revealed no significant differences between EGCG-treated cells and control cultures on the ratio of ADAM10 to ␥-actin (p Ͼ 0.05). The cells were transfected with 50 –200 nM anti-ADAM9, -10, or -17 siRNA or anti-green fluorescent protein (non-targeting control; Dharmacon) using Code-Breaker transfection reagent (Promega) and cultured for an additional 18 h in serum-free minimal essential medium. The statistical package for the social sciences release 10.0.5 (SPSS Inc., Chicago, IL) was used for all data analysis
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