Weggen S, Eriksen JL, Das P, Sagi SA, Wang R, Pietrzik CU, Findlay KA, Smith TE, Murphy MP, Bulter T, Kang DE, Marquez-Sterling N, Golde TE, Koo EH. A subset of NSAIDs lower amyloidogenic Abeta42 independently of cyclooxygenase activity. Nature. 2001 Nov 8;414(6860):212-6. PubMed Abstract, View on AlzSWAN

REAGENTS/MATERIAL:
Cell cultures used: CHO cells stably transfected with human APP751, CHO cells transfected with both human APP751 and human mutant PS1 (M146L), CHO cells transfected with human mutant APP751 (V717F), human neuroglioma cells HS683 transfected with APP695, HEK 293 cells transfected with APP695, and spontaneously immortalized embryonic fibroblasts from COX1- and COX-2-deficient animals. Complete protease inhibitor cocktail (Roche) was added and Ab40 and Ab42 were quantified by BAN50/BA27 and BAN50/BC05 ELISAs or 3160/BA27 and 3160/BC05 ELISAs. All measurements were performed in duplicate. Primary embryonic fibroblasts derived from mice deficient in COX-1 and COX-2 were infected with 100 plaque-forming units per cell in serum-free medium for 2 h. The Myc-tagged, amino-terminal-truncated Notch-1 construct (NDEMV, in which methionine 1,726 has been mutated to valine to eliminate translation initiation at that site) and the construct containing only the NICD domain have been described Kopan, R. et al. Secreted Ab peptides were analysed by IPMS as described in Wang, R. et al. One millilitre of conditioned medium was immunoprecipitated with monoclonal antibody 4G8 (Senetek) and molecular masses and concentrations of Ab peptides were measured with an ultravioletlaser desorption/ionization time-of-night mass spectrometer. To compare the concentrations of individual Ab species in conditioned medium, synthetic Ab(12-28) peptide (Sigma) was added to the samples as an internal standard, and relative peak heights were calculated. Bicine/urea Ab western blot analysis was performed as described in Wiltfang, J. et al. One millilitre of conditioned medium was immunoprecipitated with APP monoclonal antibody 26D6 recognizing amino acids 1-12 of the Ab sequence. Samples were separated on bicine/urea gels, transferred to nitrocellulose membranes and immunoblotted with 26D6 antibody. Standard Ab40, Ab42 and Ab38 peptides (Sigma) were separated on the same gel for identification of the corresponding Ab species. Female Tg2576 mice overexpressing APP695 containing the `Swedish' mutation were treated at 3 months old. NSAIDs were dissolved in DMSO, mixed with a sucrose drink (Kool-Aid) and fed orally to the animals. Controls were administered Kool-Aid with DMSO. Fibroblasts deficient in COX-1 and COX-2 were provided by X. Zhang and D. A. Young Notch plasmids from R. Kopan; 26D6 antibody from M. Kounnas; BAN50, BA27 and BC05 antibodies from Takeda industries.

FUTURE DIRECTION:
Because the Ab42 effect described is independent of COX activity, compounds with optimized Ab42 reduction and little to no inhibition of COX-1 activity are likely to be identified. Such agents would represent a new generation of `anti-amyloid' drugs that selectively target production of the highly amyloidogenic Ab42 species without inhibiting either COX activity or the vital physiological functions of g-secretase.