Faculty Bibliography

In murine L cells, treatment with calpeptin or calpain inhibitor III increased Abeta42, but not Abeta40, secretion in a dose-dependent fashion. This correlated with an increase in the levels of amyloid precursor protein (APP) carboxyl-terminal fragments (CTFs). Immunoprecipitation with novel mAbs directed against the carboxyl-terminus of APP or specific for the beta-cleaved CTF showed that generation of both alpha- and beta-cleaved CTFs increase proportionately following inhibition of calpains. Pulse-chase metabolic labeling confirmed that inhibiting calpains increases the production of alpha- and beta-cleaved APP metabolites. Immunolabeling showed greater betaCTF signal in calpeptin-treated cells, primarily in small vesicular compartments that were shown to be predominantly endosomal by colocalization with early endosomal antigen 1. A second mAb, which recognizes an extracellular/luminal epitope found on both APP and betaCTFs, gave more cell surface labeling of calpeptin-treated cells than control cells. Quantitative binding of this antibody confirmed that inhibiting calpains caused a partial redistribution of APP to the cell surface. These results demonstrate that 1) calpain inhibition results in a partial redistribution of APP to the cell surface, 2) this redistribution leads to an increase in both alpha- and beta-cleavage without changing the ratio of alphaCTFs/betaCTFs, and 3) the bulk of the betaCTFs in the cell are within early endosomes, confirming the importance of this compartment in APP processing

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