ELISA analysis of beta-secretase cleavage of the Swedish amyloid precursor protein in the secretory and endocytic pathways.

Limiting beta amyloid (Abeta) production could become an important therapeutic target in Alzheimer's disease (AD). Abeta is derived by the sequential cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases. A double missense mutation in APP found in a Swedish pedigree (APPsw) elevates Abeta40 and Abeta42 production. Abeta production and, therefore, beta-secretase cleavage of APPsw reportedly occur in the endoplasmic reticulum (ER), Golgi and endocytic compartments. However, the relative contribution of beta-secretase cleavage occurring in each compartment has not been determined. Experiments described here use a novel ELISA to measure the beta-cleaved product, APPswbeta. Using this ELISA, we provide new information regarding the relative amount of beta-secretase cleavage of APPsw that occurs in secretory and endocytic pathways. Using a dilysine retrieval motif to retain APPsw in the ER, we discovered that less than 15% of the beta-secretase cleavage was still detected. Experiments utilizing endocytosis-impaired mutants of APPsw revealed that little or no beta-secretase cleavage of APPsw appears to take place through endocytosis. Surprisingly, deletion of the entire cytoplasmic tail increased both APPswbeta and Abeta secretion, suggesting that protein interactions with this region normally impede beta-secretase cleavage. These results suggest that APPsw is cleaved by beta-secretase late in the secretory pathway.