Abstract
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 β-catenin. Pathogenic PSEN1 mutations were demonstrated to have an effect on β-catenin and glycogen synthase kinase-3β(GSK-3β), two members of the wingless Wnt pathway. The nuclear translocation and the stability of β-catenin, and the interaction between GSK3β 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 β-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 β-catenin stability. Finally, we analyzed the effect of pathogenic PSEN1 mutations on the interaction between PSEN1 and GSK3β by co-immunoprecipitation experiments.
Results
We report reduced nuclear translocation of β-catenin in cells stably expressing I143T, G384A, and T113-114ins PSEN1. The G384A PSEN1 mutation showed a similar pronounced effect on nuclear translocation of β-catenin, as reported for processing of amyloid precursor protein (APP) into amyloid β(Aβ). Overexpression of PSEN1 and the presence of pathogenic mutations in PSEN1 had no significant effect on the stability of β-catenin. Nonspecific binding of overexpressed PSEN1 to endogenous GSK3β was observed when GSK3β was immuno-precipitated. Immunoprecipitation of PSEN1 in cells overexpressing PSEN1 and in native cells, however, did not result in co-immunoprecipitation of endogenous GSK3β.
Conclusion
Our results further establish the nuclear translocation assay of β-catenin as an adequate alternative for traditional Aβ 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 β-catenin. Finally, co-immunoprecipitation between PSEN1 and GSK3β was not observed in our experimental setup.
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Acknowledgement
The authors want to thank Sofie Van Gestel for statistical assistance. This work was supported by the Fund for Scientific Research-Flanders (FWO-F), the Interuniversity Attraction Poles (IUAP p4/17), the International Alzheimer’s Research Foundation (IARF), and the Focused Giving Program of Johnson & Johnson. This work was further supported by grants of the Canadian Genetic Disease Network, the Medical Research Council of Canada, the Howard Hughes Medical Research Foundation (PH St GH), and the Department of Medicine Post-Doctoral fellowship award (MN). GVG is a grant holder of the Institute for Science and Technology (IWT). CDJ is a postdoctoral fellow at the FWO-F.
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Van Gassen, G., De Jonghe, C., Nishimura, M. et al. Evidence that the β-catenin Nuclear Translocation Assay Allows for Measuring Presenilin 1 Dysfunction. Mol Med 6, 570–580 (2000). https://doi.org/10.1007/BF03401795
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DOI: https://doi.org/10.1007/BF03401795