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. 2002 Oct 29;99(22):14452-7.
doi: 10.1073/pnas.222413999. Epub 2002 Oct 18.

Alleles at the Nicastrin Locus Modify Presenilin 1- Deficiency Phenotype

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Free PMC article

Alleles at the Nicastrin Locus Modify Presenilin 1- Deficiency Phenotype

Richard Rozmahel et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Presenilin 1 (PS1), presenilin 2, and nicastrin form high molecular weight complexes that are necessary for the endoproteolysis of several type 1 transmembrane proteins, including amyloid precursor protein (APP) and the Notch receptor, by apparently similar mechanisms. The cleavage of the Notch receptor at the "S3-site" releases a C-terminal cytoplasmic fragment (Notch intracellular domain) that acts as the intracellular transduction molecule for Notch activation. Missense mutations in the presenilins cause familial Alzheimer's disease by augmenting the "gamma-secretase" cleavage of APP and overproducing one of the proteolytic derivatives, the Abeta peptide. Null mutations in PS1 inhibit both gamma-secretase cleavage of APP and S3-site cleavage of the Notch receptor. Mice lacking PS1 function have defective Notch signaling and die perinatally with severe skeletal and brain deformities. We report here that a genetic modifier on mouse distal chromosome 1, coinciding with the locus containing Nicastrin, influences presenilin-mediated Notch S3-site cleavage and the resultant Notch phenotype without affecting presenilin-mediated APP gamma-site cleavage. Two missense substitutions of residues conserved among vertebrates have been identified in nicastrin. These results indicate that Notch S3-site cleavage and APP gamma-site cleavage are distinct presenilin-dependent processes and support a functional interaction between nicastrin and presenilins in vertebrates. The dissociation of Notch S3-site and APP gamma-site cleavage activities will facilitate development of gamma-secretase inhibitors for treatment of Alzheimer's disease.

Figures

Fig 1.
Fig 1.
Gross phenotype of Notch-severe and Notch-mild PS1−/− mice. (a) Notch-severe (Left) and Notch-mild (Right) B6 × 129 F2 PS1−/− mice at 5 weeks of age. Identical to previously reported 129 PS1−/− animals, the Notch-severe mice were significantly smaller than their Notch-mild or wild-type sibs and presented a grossly deformed vertebral column. The represented Notch-severe animal also manifested back-end paralysis commonly observed in this group. (b and d) Ventral and lateral x-rays, respectively, of a 6-week-old Notch-severe mouse alongside its Notch-mild sib (c and e).
Fig 2.
Fig 2.
Production of NICD by wild-type, PS1−/− Notch-mild and Notch-severe mice. (a) Six-day-old PS1−/− Notch-mild (Upper) and Notch-severe (Lower) mice showing reduced size and grossly deformed axial skeletons of the latter. (b) Cells from 6-day-old Notch-mild mice produced more NICD than cells from Notch-severe mice. (c) Comparison of relative NICD production (ratio of NICD/NICD+NotchΔE) from cells Notch-mild (n = 12) and Notch-severe (n = 18) mice shows a significant difference (P < 0.02) between the groups.
Fig 3.
Fig 3.
APP processing by wild-type, PS1−/− Notch-mild and Notch-severe mice. (a) Western blot of wild-type, Notch-severe, and Notch-mild B6 × 129 F2 PS1−/− brain protein lysates incubated with an APP-specific antibody. The result shows equal amounts of full-length APP (FL-APP) among all three brains. Although no APP C-terminal fragment (CTF-APP) was observed in the wild-type brains, it was equally evident among all Notch-severe and Notch-mild brains. (b) Results of ELISAs for Aβ40 and Aβ42 from PS1+/+, PS1+/−, and Notch-severe and Notch-mild PS1−/− brains.
Fig 4.
Fig 4.
Linkage of the modifier to chromosome 1. (a) A genome scan of the Notch-severe and Notch-mild mice mapped a modifier of the Notch phenotype to distal chromosome 1. Genetic analysis of the locus showed the maximum LOD score was 6.1, identified by D1Mit150 at position 100-cM. (b) Physical definition of the modifier confidence interval showing placement of Nicastrin and PS2.
Fig 5.
Fig 5.
Quantitation of PS2 protein. The same Western blot of wild-type, Notch-severe, and Notch-mild B6 × 129 F2 PS1−/− brain lysates shown in Fig. 3a was incubated with a PS2 C-terminal antibody. A signal corresponding to the PS2-CTF is seen in the wild-type lanes, whereas a more intense signal is evident in the lanes corresponding to the mutant animals. No consistent difference is observed between the Notch-severe and Notch-mild animals. Hybridization with the Rab8 antibody is shown for control of protein loading (identical to that in Fig. 3a).

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