Abstract
Presenilin-1 (PS1) facilitates gamma-secretase cleavage of the beta-amyloid precursor protein and the intramembraneous cleavage of Notch1. Although Alzheimer's disease-associated mutations in the homologous presenilin (PS2) gene elevate amyloid beta-peptide (Abeta42) production like PS1 mutations, here we demonstrate that a gene ablation of PS2 (unlike that of PS1) in mice does not result in a severe phenotype resembling that of Notch-ablated animals. To investigate the amyloidogenic function of PS2 more directly, we mutagenized a conserved aspartate at position 366 to alanine, because the corresponding residue of PS1 is known to be required for its amyloidogenic function. Cells expressing the PS2 D366A mutation exhibit significant deficits in proteolytic processing of beta-amyloid precursor protein indicating a defect in gamma-secretase activity. The reduced gamma-secretase activity results in the almost complete inhibition of Abeta and p3 production in cells stably expressing PS2 D366A, whereas cells overexpressing the wild-type PS2 cDNA produce robust levels of Abeta and p3. Using highly sensitive in vivo assays, we demonstrate that the PS2 D366A mutation not only blocks gamma-secretase activity but also inactivates PS2 activity in Notch signaling by inhibiting the proteolytic release of the cytoplasmic Notch1 domain. These data suggest that PS2 is functionally involved in Abeta production and Notch signaling by facilitating similar proteolytic cleavages.
Highlights
Presenilin-1 (PS1) facilitates ␥-secretase cleavage of the -amyloid precursor protein and the intramembraneous cleavage of Notch1
Using highly sensitive in vivo assays, we demonstrate that the PS2 D366A mutation blocks ␥-secretase activity and inactivates PS2 activity in Notch signaling by inhibiting the proteolytic release of the cytoplasmic Notch1 domain
From our experiments we conclude that loss of function mutations of PS2 interfere with A production as well as Notch signaling, suggesting that PS2 is functionally involved in the proteolytic release of A and NICD
Summary
Cell Culture and Cell Lines—Human embryonic kidney 293 cells [293] were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, 200 g/ml G418 (to select for APP expression), and 200 g/ml zeocin (to select for presenilin expression). 293 cells stably expressing PS2 D366A were generated by transfection of 293 cells stably expressing APP containing the Swedish mutation [13]. 293 cells stably co-expressing Swedish APP695 and wt PS2 were described previously [14]. Combined Immunoprecipitation/Western Blotting—Extracts from brains or stably transfected 293 cell lines were prepared and subjected to immunoprecipitation using the polyclonal antibody 3027 to PS1 [18], 3711 to PS2 [19], or C7 to APP [22]. For the analysis of A in conditioned media, cells were metabolically labeled with 450 Ci [35S]methionine (Promix, Amersham Pharmacia Biotech) for 2 h, and chased for 2 h in medium containing excess amounts of unlabeled methionine. To analyze cleavage of Notch, cells were starved for 1 h in methionine- and serum-free minimum Eagle’s medium, subsequently metabolically labeled with 300 Ci [35S]methionine (Promix, Amersham Pharmacia Biotech) for 20 min, and chased for 1 h in medium containing excess amounts of unlabeled methionine. The numbers of egg laid by individual transgenic animals were counted and placed into four categories.: Eglϩϩϩ, robust egg laying, more than 30 eggs laid; Eglϩϩ, 15–30 eggs laid; Eglϩ, 5–15 eggs laid; EglϪ, no eggs laid
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