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Title
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Evidence that the β-catenin nuclear translocation assay allows for measuring presenilin 1 dysfunction
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Author
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Abstract
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| Background: Mutations in the presenilin (PSEN) genes are responsible for the majority of early-onset Alzheimer disease (AD) cases. PSEN1 is a component of a high molecular weight, endoplasmic reticulum, membrane-bound protein complex, including beta-catenin. Pathogenic PSEN1 mutations were demonstrated to have an effect on beta-catenin and glycogen synthase kinase-3 beta(GSK-3 beta), two members of the wingless Wnt pathway. The nuclear translocation and the stability of beta-catenin, and the interaction between GSK3 beta 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 Western blot analysis, endogenous beta-catenin levels were examined. By analyzing cytosolic fractions of PSEN1, transfected and nontransfected HEK 293 cells, and total brain extracts of AD patients and controls, we evaluated the effect of PSEN1 overexpression on beta-catenin stability. Finally, we analyzed 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 stably expressing 1143T, G384A, and T113-114ins PSEN1. The G384A PSEN1 mutation showed a similar pronounced effect on nuclear translocation of beta-catenin, as reported for processing of amyloid precursor protein (APP) into amyloid beta(A beta) Overexpression of PSEN1 and the presence of pathogenic mutations in PSEN1 had no significant effect on the stability of beta-catenin. Nonspecific binding of overexpressed PSEN1 to endogenous GSK3 beta was observed 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 of beta-catenin as an adequate alternative for traditional A beta measurement to evaluate the effect of PSEN1 mutations on biochemical processes. We detected no significant effect of overexpressed wt or mt PSEN1 on the stabilty of beta-catenin. Finally, co-immunoprecipitation between PSEN1 and GSK3 beta was not observed in our experimental setup. |
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Language
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English
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Source (journal)
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Molecular medicine. - Oxford
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Publication
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Oxford
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2000
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ISSN
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1076-1551
1528-3658
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DOI
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10.1007/BF03401795
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Volume/pages
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6
:7
(2000)
, p. 570-580
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ISI
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000089104200002
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Full text (Publisher's DOI)
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Full text (open access)
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