Retention of the Alzheimer's Amyloid Precursor Fragment C99 in the Endoplasmic Reticulum Prevents Formation of Amyloid β-Peptide

Abstract

␥-Secretase is a membrane-associated endoprotease that catalyzes the final step in the processing of Alzheimer's ␤-amyloid precursor protein (APP), resulting in the release of amyloid ␤-peptide (A␤). The molecular identity of ␥-secretase remains in question, although recent studies have implicated the presenilins, which are membrane-spanning proteins localized predominantly in the endoplasmic reticulum (ER). Based on these observations, we have tested the hypothesis that ␥-secretase cleavage of the membrane-anchored C-terminal stump of APP (i.e. C99) occurs in the ER compartment. When recombinant C99 was expressed in 293 cells, it was localized mainly in the Golgi apparatus and gave rise to abundant amounts of A␤. Co-expression of C99 with mutant forms of presenilin-1 (PS1) found in familial Alzheimer's disease resulted in a characteristic elevation of the A␤ 42 /A␤ 40 ratio, indicating that the N-terminal exodomain of APP is not required for mutant PS1 to influence the site of ␥-secretase cleavage. Biogenesis of both A␤ 40 and A␤ 42 was almost completely eliminated when C99 was prevented from leaving the ER by addition of a di-lysine retention motif (KKQN) or by co-expression with a dominant-negative mutant of the Rab1B GTPase. These findings indicate that the ER is not a major intracellular site for ␥-secretase cleavage of C99. Thus, by inference, PS1 localized in this compartment does not appear to be active as ␥-secretase. The results suggest that presenilins may acquire the characteristics of ␥-secretase after leaving the ER, possibly by assembling with other proteins in peripheral membranes.Amyloid ␤-peptide (A␤) 1 is the major molecular component of the cerebral amyloid plaques associated with Alzheimer's disease. The cellular pathways involved in the biogenesis of A␤ have been the subject of intense investigation since the discovery that A␤ originates from intracellular endoproteolytic processing of a type I membrane-spanning glycoprotein termed amyloid precursor protein (APP) (1-4). Extensive studies have established that APP can be processed via two alternative routes, one of which yields the 4-kDa A␤, whereas the other yields a truncated non-amyloidogenic peptide (p3) (5, 6). In most cells the non-amyloidogenic pathway predominates. The first step involves the cleavage of APP within the A␤ domain by a protease termed ␣-secretase (7-9). After release of the Nterminal exodomain, the residual 83-amino acid membranespanning C-terminal fragment is further processed by another protease termed ␥-secretase to remove the cytoplasmic tail and generate p3 (6, 10). Because the latter cleavage occurs within the predicted membrane spanning region of APP (11-13), ␥-secretase is generally thought to be an intramembrane protease. In the alternative amyloidogenic pathway, APP is initially cleaved proximal to the A␤ sequence by ␤-secretase, leaving a 99-amino acid C-terminal fragment (C99) that contains the intact A␤ sequence and the cytoplasmic tail (1,14,15). Thus, when ␥-secretase cuts the latter substrate, A␤ is rel...

Human CRB2 Inhibits γ-Secretase Cleavage of Amyloid Precursor Protein by Binding to the Presenilin Complex

“…Several lines of data suggest that APP-C99 is processed by ␥-secretase activity mainly at the plasma membrane or in endocytotic compartments, although a major pool of the ␥-secretase complex resides at early secretory compartments (37,38). On the other hand, previous reports (39,40) estimated that only a small portion of the total proteolyzed PS1 fragments in cellular membranes is engaged in active ␥-secretase complexes.…”

γ-Secretase Cleavage Site Specificity Differs for Intracellular and Secretory Amyloid β

“…Moreover, transgenic mice show accelerated neurodegeneration without extracellular amyloid deposition (24), indicating that intracellular amyloid-␤ peptides may play a crucial role in the development of AD. Intracellular sites with ␥-secretase activity were identified in primary neurons, neuronal cell lines, and peripheral cells (25)(26)(27)(28)(29)(30). Evidence has been obtained for the generation of A␤42 within the ER (27)(28)(29)31), where PS is predominantly localized (14 -16).…”

“…Evidence has been obtained for the generation of A␤42 within the ER (27)(28)(29)31), where PS is predominantly localized (14 -16). However, it has also been reported that A␤42 is produced in different organelles later in the secretory pathway and that the ER is not the major intracellular site of A␤42 generation (26,30,(32)(33)(34)74). Additionally, recent work has suggested that the A␤42 generation in the ER may be independent of PS (35).…”

Trafficking and Proteolytic Processing of APP

“…The g-secretase subunits found in the ER/early secretory pathway most likely represent unassembled or partially assembled subcomplexes, but not the active enzyme. The evidence supporting such a model is manifold and, because of space constraints, only key arguments will be highlighted: (1) APP/C99/C83 are not cleaved by g-secretase in the ER (Cupers et al 2001;Maltese et al 2001;Grimm et al 2003;Kaether et al 2006b); (2) by immuno-electron microscopy, PS1 is not detected in Golgi or TGN but in ER and PM (Rechards et al 2003); whereas the ER pool most likely reflects unassembled PS1, the PM pool has assembled, active complex; (3) only the mature, glycosylated NCT, not immature unglycosylated NCT, is present in the fully assembled, active g-secretase complex Kaether et al 2002), and therefore g-secretase-associated NCTmust have passed the Golgi; (4) g-secretase has been shown by various methods, including cell surface biotinylation, binding of biotinylated inhibitors specific for the active complex, and microscopic techniques, to be present at the PM (Kaether et al 2002;Tarassishin et al 2004;Chyung et al 2005). It has been shown that the 1-cleavage of APP differs in endosomes and PM (Fukumori et al 2006), suggesting that g-secretase has different properties depending on its subcellular localization, maybe because of differences in pH or lipid composition.…”