β‐Amyloid peptide in regulated secretory vesicles of chromaffin cells: evidence for multiple cysteine proteolytic activities in distinct pathways for β‐secretase activity in chromaffin vesicles
Authors: H. Hook Vivian Y. , Toneff Thomas , Aaron Wade , Yasothornsrikul Sukkid , Bundey Richard , Reisine Terry 
Published: May 2003

A key factor in Alzheimer's disease (AD) is the β‐secretase activity that is required for the production of beta‐amyloid (Aβ) peptide from its amyloid precursor protein (APP) precursor. In this study, the majority of Aβ secretion from neuronal chromaffin cells was found to occur via the regulated secretory pathway, compared with the constitutive secretory pathway; therefore, β‐secretase activity in the regulated secretory pathway was examined for the production and secretion of Aβ in chromaffin cells obtained from in vivo adrenal medullary tissue. The presence of Aβ(1–40) in APP‐containing chromaffin vesicles, which represent regulated secretory vesicles, was demonstrated by radioimmunoassay (RIA) and reverse‐phase high‐performance liquid chromatography. These vesicles also contain Aβ(1–42), measured by RIA. Significantly, regulated secretion of Aβ(1–40) from chromaffin cells represented the majority of secreted Aβ (> 95% of total secreted Aβ), compared with low levels of constitutively secreted Aβ(1–40). These results indicate the importance of Aβ production and secretion in the regulated secretory pathway as a major source of extracellular Aβ. β‐secretase activity in isolated chromaffin vesicles was detected with the substrate Z‐Val‐Lys‐Met‐↓MCA (methylcoumarinamide) that contains the β‐secretase cleavage site. Optimum β‐secretase activity in these vesicles required reducing conditions and acidic pH (pH 5–6), consistent with the in vivo intravesicular environment. Evidence for cysteine protease activity was shown by E64c inhibition of Z‐Val‐Lys‐Met‐MCA‐cleaving activity, and E64c inhibition of Aβ(1–40) production in isolated chromaffin vesicles. Chromatography resolved the β‐secretase activity into two distinct proteolytic pathways consisting of: (i) direct cleavage of the β‐secretase site at Met‐↓Asp by two cysteine proteolytic activities represented by peaks II‐A and II‐B, and (ii) an aminopeptidase‐dependent pathway represented by peak I cysteine protease activity that cleaves between Lys‐↓Met, followed by Met‐aminopeptidase that would generate the β‐secretase cleavage site. Treatment of chromaffin cells in primary culture with the cysteine protease inhibitor E64d reduced the production of the β‐secretase product, a 12–14 kDa C‐terminal APP fragment. In addition, BACE 1 and BACE 2 were detected in chromaffin vesicles; BACE 1 represented a small fraction of total β‐secretase activity in these vesicles. These results illustrate that multiple cysteine proteases, in combination with BACE 1, contribute to β‐secretase activity in the regulated secretory pathway. These results complement earlier findings for BACE 1 as β‐secretase for Aβ production in the constitutive secretory pathway that provides basal secretion of Aβ into conditioned media. These findings suggest that drug inhibition of several proteases may be required for reducing Aβ levels as a potential therapeutic approach for AD.