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. 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 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. CONCLUSION: 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.

Molecular genetics of Alzheimer's disease: what have we learned?

Alzheimer's disease (AD), by far the most common form of dementia in the elderly, is clinically characterized by gradual, progressive loss in cognitive functioning and changes in personality, ultimately leading to death. It is now well established that genetic factors play an important role in AD. So far, three genes have been identified in which mutations cause autosomal-dominant AD: the amyloid precursor protein (APP) gene on chromosome 21, the presenilin 1 (PSEN1) gene on chromosome 14, and the homologous presenilin 2 (PSEN2) gene on chromosome 1. A major susceptibility gene, the apolipoprotein E (APOE) gene, was identified on chromosome 19.

Binding partners of Alzheimer's disease proteins: are they physiologically relevant?

Protein-protein interactions are a molecular basis for the structural and functional organization within cells. They are mediated by a growing number of protein modules that bind peptide targets. Alterations in binding affinities can have serious consequences for some essential cellular processes. The three proteins identified to have mutations in their corresponding genes leading to presenile Alzheimer dementia (AD)-the amyloid precursor protein (APP) and presenilin 1 and 2-all interact with other proteins. The nature and function of these interacting proteins may contribute to elucidating the proper physiological functions of the AD proteins. APP-interacting proteins are pointing toward a function of APP in cell adhesion and neurite outgrowth and signaling. Proteins interacting with the presenilins however are more diverse in nature linking presenilin function to regulation in different signaling pathways including Wnt and Notch but also in apoptosis and Ca(2+) homeostasis. Further research however is still needed to delineate the exact functional relevance of these interactions with respect to the physiological functions of the AD proteins in particular and the contribution of these proteins to AD pathogenesis in general.

Genetic association of the presenilin-1 regulatory region with early-onset Alzheimer's disease in a population-based sample.

Genetic association has been reported between a di-allelic polymorphism in intron 8 of presenilin-1 (PSEN1) and Alzheimer's disease (AD) in some studies but not in others. In a population-based series of 102 patients with early onset AD and 118 community controls we examined whether polymorphisms in linkage disequilibrium with intron8 of PSEN1 may explain the association. In addition to the intron 8 polymorphism (P = 0.05), a promoter polymorphism (P = 0.03) and the simple tandem repeat (STR) polymorphism D14S1028 located upstream of PSEN1 (P = 0.04) were found to be marginally significantly associated to AD. When excluding PSEN1 mutation cases (n = 6), the intron 8 association was explained by linkage disequilibrium to the dominant PSEN1 mutations. In the non-mutation cases, the weak associations between the polymorphisms in the regulatory region remained. Our study suggests that a polymorphism/mutation in the promoter or regulatory region of PSEN1 rather than the polymorphism in intron 8 of PSEN1 is associated with early onset AD.

Alzheimer's disease associated presenilin 1 interacts with HC5 and ZETA, subunits of the catalytic 20S proteasome.

Proteolytic processing and degradation tightly regulate the amount of stable, functional presenilin 1 (PSEN1) in the cell. The approximately 46-kDa PSEN1 holoprotein is cleaved into a approximately 30-kDa N-terminal fragment (NTF) and a approximately 20-kDa C-terminal fragment (CTF) by an unknown protease. The fragments are stabilized in a high molecular weight complex and nonincorporated fragments and excess holoprotein are degraded by the 26S proteasome. The tight balance between, on the one hand, processing and incorporation into the stable complex and, on the other hand, proteolytic degradation of excess PSEN1, indicates that minor changes in one of these two processes could be pathologically relevant. Here we demonstrate the direct physical interaction between PSEN1 and two subunits, HC5 and ZETA, of the 20S proteasome. These interactions were identified using an interaction trap screening and were further established in an in vitro binding assay. Furthermore, we were able to coimmunoprecipitate the transfected binding partners, as well as the endogenous PSEN1 and ZETA proteins from HEK 293T cells. Finally, degradation of ubiquitinated wild-type and mutant PSEN1 by the 26S proteasome was demonstrated. In conclusion, we report a direct interaction between PSEN1 and subunits of the 20S catalytic particle of the 26S proteasome, further establishing the involvement of proteasomal degradation in the regulation of PSEN1 turnover.

Identification of caspases that cleave presenilin-1 and presenilin-2. Five presenilin-1 (PS1) mutations do not alter the sensitivity of PS1 to caspases.

Mutations in the presenilin (PS) genes PSI and PS2 are involved in Alzheimer's disease (AD). Recently, apoptosis-associated cleavage of PS proteins was identified. Here we demonstrate that PS1 as well as PS2 are substrates for different members of the caspase protein family. Remarkably, the caspases acting on PS1 could be subdivided in two groups. One group, containing caspase-8, -6 and -11, cleaved PSI after residues ENDD329 and to a lesser extent after residues AQRD341. A second group consisting of caspase-3, -7 and -1 acted uniquely on AQRD341. Importantly, these two cleavage sites were also recognized by caspases in the C-terminal PS1 fragment produced by constitutive proteolysis. In decreasing order of activity, caspase-8, -3, -1, -6 and -7 proteolysed PS2 at the recognition site D326SYD329. Caspase-8 and -3 exhibited the highest proteolytic activity on both PS1 and PS2. PS1 and PS2 were not hydrolyzed by caspase-2 and PS2 also not by caspase-11. None of five missense mutations affected the sensitivity of PSI to caspase-mediated cleavage. This suggests that AD pathogenesis associated with PS1 missense mutations cannot be explained by a change in caspase-dependent processing.

Molecular genetic analysis of familial early-onset Alzheimer's disease linked to chromosome 14q24.3.

Genetic linkage studies have indicated that chromosome 14q24.3 harbours a major locus for early-onset (onset age <65 years) Alzheimer's disease (AD3). Positional cloning efforts have identified a novel gene S182 or presenilin 1 as the AD3 gene. We have mapped S182 in the AD3 candidate region between D14S277 and D14S284 defined by genetic linkage studies in the two chromosome 14 linked, early-onset AD families AD/A and AD/B. We have shown that S182 is expressed in lymphoblasts and have determined the complete cDNA in both brain and lymphoblasts by RT-PCR sequencing. S182 is alternatively spliced in both brain and lymphoblasts within a putative phosphorylation site located 5' in the coding region. We identified two novel mutations, Ile143Thr and Gly384la located in, respectively, the second transmembrane domain and in the sixth hydrophilic loop of the putative transmembrane structure of S182. As families AD/A and AD/B have very similar AD phenotype our observation of two mutations in functionally different domains suggest that onset age and severity of AD may not be very helpful predictors of the location of putative S182 mutations.

Mutation analysis of the chromosome 14q24.3 dihydrolipoyl succinyltransferase (DLST) gene in patients with early-onset Alzheimer disease.

Linkage analysis studies have indicated that the chromosome band 14q24.3 harbours a major gene for familial early-onset Alzheimer's disease (AD). Recently we localized the chromosome 14 AD gene (AD3) in the 6.4 cM interval between the markers D14S289 and D14S61. We mapped the gene encoding dihydrolipoyl succinyltransferase (DLST), the E2k component of human alpha-ketoglutarate dehydrogenase complex (KGDHC), in the AD3 candidate region using yeast artificial chromosomes (YACs). The DLST gene is a candidate for the AD3 gene since deficiencies in KGDHC activity have been observed in brain tissue and fibroblasts of AD patients. The 15 exons and the promoter region of the DLST gene were analysed for mutations in chromosome 14 linked AD cases and in two series of unrelated early-onset AD cases (onset age < 55 years). Sequence variations in intronic sequences (introns 3, 5 and 10) or silent mutations in exonic sequences (exons 8 and 14) were identified. However, no AD related mutations were observed, suggesting that the DLST gene is not the chromosome 14 AD3 gene.