Abstract
Amyloid-beta (Abeta) peptides, generated by the proteolysis of beta-amyloid precursor protein by beta- and gamma-secretases, play an important role in the pathogenesis of Alzheimer disease. Inflammation is also important. We recently reported that prostaglandin E(2) (PGE(2)), a strong inducer of inflammation, stimulates the production of Abeta through EP(2) and EP(4) receptors, and here we have examined the molecular mechanism. Activation of EP(2) and EP(4) receptors is coupled to an increase in cellular cAMP levels and activation of protein kinase A (PKA). We found that inhibitors of adenylate cyclase and PKA suppress EP(2), but not EP(4), receptor-mediated stimulation of the Abeta production. In contrast, inhibitors of endocytosis suppressed EP(4), but not EP(2), receptor-mediated stimulation. Activation of gamma-secretase was observed with the activation of EP(4) receptors but not EP(2) receptors. PGE(2)-dependent internalization of the EP(4) receptor was observed, and cells expressing a mutant EP(4) receptor lacking the internalization activity did not exhibit PGE(2)-stimulated production of Abeta. A physical interaction between the EP(4) receptor and PS-1, a catalytic subunit of gamma-secretases, was revealed by immunoprecipitation assays. PGE(2)-induced internalization of PS-1 and co-localization of EP(4), PS-1, and Rab7 (a marker of late endosomes and lysosomes) was observed. Co-localization of PS-1 and Rab7 was also observed in the brain of wild-type mice but not of EP(4) receptor null mice. These results suggest that PGE(2)-stimulated production of Abeta involves EP(4) receptor-mediated endocytosis of PS-1 followed by activation of the gamma-secretase, as well as EP(2) receptor-dependent activation of adenylate cyclase and PKA, both of which are important in the inflammation-mediated progression of Alzheimer disease.
Highlights
Alzheimer disease (AD)2 is the most common neurodegenerative disorder of the central nervous system and the leading
These results suggest that the EP2 receptor mediates PGE2stimulated production of A through activation of cAMP and protein kinase A (PKA) without an increase in - and ␥-secretase activity and that the EP4 receptor mediates prostaglandin E2 (PGE2)-stimulated A production through different mechanisms, which involve an activation rather than induction of expression of ␥-secretase
We showed that the inhibitors for adenylate cyclase and PKA, we showed that the expression and maturation of amyloid precursor protein (APP) (the ratio of the mature form of APP to the immature form of APP) and the ␣- and -secretase activities in extracts prepared from the brains of APPsw/EP4Ϫ/Ϫ mice were similar to those from APPsw/EP4ϩ/ϩ mice (Fig. 7B and C). (CTFs of APP that are generated by ␣- or -secretase (CTF␣ or C-terminal fragment (CTF), respectively) cAMP-PKA pathway is involved in EP2-mediated but not EP4mediated stimulation of A production by PGE2
Summary
Materials—Dulbecco’s modified Eagle’s medium (DMEM) and Ham-F12 medium were obtained from Nissui Pharmaceutical Co. Sandwich Enzyme-linked Immunosorbent Assay for A and ␥-Secretase- or -Secretase-mediated Peptide Cleavage Assay— Cells were cultured for 24 h, and the conditioned medium was subjected to a sandwich enzyme-linked immunosorbent assay using three types of specific monoclonal antibodies as described previously [35, 39]. Biotinylated proteins were precipitated using UltraLink immobilized Neutravidin beads and eluted by boiling in SDS sample buffer. Statistical Analysis—All values are expressed as the mean Ϯ incubated for 1 h at 37 °C in 200 l of 50 mM acetate buffer S.D. Two-way analysis of variance followed by the Tukey test or (pH 4.1) containing 100 mM sodium chloride, 0.025% bovine the Student’s t test for unpaired results was used to evaluate serum albumin, and 10 M fluorescent -secretase substrate. Differences were considered to be significant for values of p Ͻ 0.05
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