Evidence that the beta-catenin nuclear translocation assay allows for measuring presenilin 1 dysfunction

Background: Mutations in the presenilin (PSEN) genes are responsible for th e majority of early-onset Alzheimer disease (AD) cases. PSEN1 is a componen t of a high molecular weight, endoplasmic reticulum, membrane-bound protein complex, including beta-catenin. Pathogenic PSEN1 mutations were demonstra ted to have an effect on beta-catenin and glycogen synthase kinase-3 beta(G SK-3 beta), two members of the wingless Wnt pathway. The nuclear translocat ion and the stability of beta-catenin, and the interaction between GSK3 bet a and PSEN1 were influenced. Materials and Methods: Stably transfected human embryonic kidney (HEK) 293 cells overexpressing wild-type (wt) and mutant (mt) PSEN1, treated with and without LiCl, were used to isolate cytoplasmic and nuclear fractions. By W estern blot analysis, endogenous beta-catenin levels were examined. By anal yzing cytosolic fractions of PSEN1, transfected and nontransfected HEK 293 cells, and total brain extracts of AD patients and controls, we evaluated t he effect of PSEN1 overexpression on beta-catenin stability. Finally, we an alyzed the effect of pathogenic PSEN1 mutations on the interaction between PSEN1 and GSK3 beta by co-immunoprecipitation experiments. Results: We report reduced nuclear translocation of beta-catenin in cells s tably expressing 1143T, G384A, and T113-114ins PSEN1. The G384A PSEN1 mutat ion showed a similar pronounced effect on nuclear translocation of beta-cat enin, as reported for processing of amyloid precursor protein (APP) into am yloid beta(A beta) Overexpression of PSEN1 and the presence of pathogenic m utations in PSEN1 had no significant effect on the stability of beta-cateni n. Nonspecific binding of overexpressed PSEN1 to endogenous GSK3 beta was o bserved when GSK3 beta was immunoprecipitated. Immunoprecipitation of PSEN1 in cells overexpressing PSEN1 and in native cells, however, did not result in co-immunoprecipitation of endogenous GSK3 beta. Conclusion: Our results further establish the nuclear translocation assay o f beta-catenin as an adequate alternative for traditional A beta measuremen t to evaluate the effect of PSEN1 mutations on biochemical processes. We de tected no significant effect of overexpressed wt or mt PSEN1 on the stabilt y of beta-catenin. Finally, co-immunoprecipitation between PSEN1 and GSK3 b eta was not observed in our experimental setup.