Evidence that the beta-catenin nuclear translocation assay allows for measuring presenilin 1 dysfunction.
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-3beta(GSK-3beta), two members of the wingless Wnt pathway. The nuclear translocation and the stability of beta-catenin, and the interaction between GSK3beta and PSEN1 were influenced.


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 GSK3beta by co-immunoprecipitation experiments.


RESULTS
We report reduced nuclear translocation of beta-catenin in cells stably expressing I143T, 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(Abeta). 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 GSK3beta was observed when GSK3beta was immunoprecipitated. Immunoprecipitation of PSEN1 in cells overexpressing PSEN1 and in native cells, however, did not result in co-immunoprecipitation of endogenous GSK3beta.


CONCLUSIONS
Our results further establish the nuclear translocation assay of beta-catenin as an adequate alternative for traditional Abeta measurement to evaluate the effect of PSEN1 mutations on biochemical processes. We detected no significant effect of overexpressed wt or mt PSEN1 on the stability of beta-catenin. Finally, co-immunoprecipitation between PSEN1 and GSK3beta was not observed in our experimental setup.
Van Gassen
G
G
Molecular Genetics Laboratory, Neurogenetics Group, Flanders Interuniversity Institute for Biotechnology (VIB), Born-Bunge Foundation, University of Antwerp, Belgium.
De Jonghe
C
C
Nishimura
M
M
Yu
G
G
Kuhn
S
S
St George-Hyslop
P
P
Van Broeckhoven
C
C
D021581:Active Transport, Cell Nucleus; D000544:Alzheimer Disease / Q000235:genetics*; D015153:Blotting, Western; D001923:Brain Chemistry; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D002458:Cell Fractionation; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism*; D038362:Glycogen Synthase Kinase 3; D038341:Glycogen Synthase Kinases; D006801:Humans; D018021:Lithium Chloride / Q000494:pharmacology; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011233:Precipitin Tests; D053764:Presenilin-1; D015534:Trans-Activators; D051176:beta Catenin
C495270:CTNNB1 protein, human; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; C508853:PSEN1 protein, human; D010446:Peptide Fragments; D053764:Presenilin-1; D015534:Trans-Activators; D051176:beta Catenin; D038341:Glycogen Synthase Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D038362:Glycogen Synthase Kinase 3; D018021:Lithium Chloride