PS1 deficiency and expression of PS1 with substitutions of two conserved transmembrane aspartate residues ("PS1 aspartate variants") leads to the reduction of A␤ peptide secretion and the accumulation of amyloid precursor protein (APP) C-terminal fragments. To define the nature of the "dominant negative" effect of the PS1 aspartate variants, we stably expressed PS1 harboring aspartate to alanine substitutions at codons 257 (D257A) or 385 (D385A), singly or in combination (D257A/ D385A), in mouse neuroblastoma, N2a cells. Expression of the PS1 aspartate variants resulted in marked accumulation of intracellular and cell surface APP C-terminal fragments. While expression of the D385A PS1 variant reduced the levels of secreted A␤ peptides, we now show that neither the PS1 D257A nor D257A/D385A variants impair A␤ production. Surprisingly, the stability of both immature and mature forms of APP is dramatically elevated in cells expressing PS1 aspartate variants, commensurate with an increase in the cell surface levels of APP. These findings lead us to conclude that the stability and trafficking of APP can be profoundly modulated by coexpression of PS1 with mutations at aspartate 257 and aspartate 385.Mutations in genes encoding presenilin 1 (PS1) 1 and presenilin 2 (PS2) cause the autosomal dominant form of familial Alzheimer's disease. PS1 and PS2 are highly homologous proteins with eight transmembrane domains (1, 2) that accumulate as ϳ30-kDa N-terminal fragments and ϳ20-kDa C-terminal fragments (CTFs) (3-5) that associate in high molecular weight complexes in vivo (6 -10). Expression of familial Alzheimer's disease-linked mutant PS elevates production of highly fibrillogenic A␤42 peptides (11-14) derived from amyloid precursor protein (APP), and these species are selectively deposited in brains of patients of Alzheimer's disease. A␤ peptides are generated by the concerted action of ␤-and ␥-secretases. Recent studies have confirmed that BACE I, a novel transmembrane aspartyl protease, is ␤- secretase (15-19).On the other hand, the identity of the ␥-secretase has been an enigma. Several lines of evidence, however, have lent strong support for the view that the PS1 and PS2 play an essential role in ␥-secretase processing of APP and the signaling receptor, Notch 1. First, cells with genetic ablations of PS1 and PS2 do not exhibit ␥-secretase activity (20, 21); A␤ secretion is completely abolished, and intramembraneous processing of Notch 1 is fully abrogated (22-24), although very recent reports have shown that A␤ derived from endogenous APP appears not to be altered by PS deficiency (25) and that the generation of N-terminally truncated forms of A␤ (A␤-(2-42)) are not dependent on PS1 expression (26). Interestingly, cells expressing PS1 or PS2 with amino acid substitutions of two conserved aspartate residues within predicted transmembrane domains 6 and 7 largely mimic the PS-null state with respect to ␥-secretase cleavage of APP and Notch 1 (24,(27)(28)(29)(30). These data have been interpreted to suggest that PS are u...

The presenilin-␥-secretase complex plays a critical role in mediating intramembranous proteolysis of several type I membrane proteins, including ␤-amyloid precursor protein (APP) and Notch. We now show that deleted in colorectal cancer (DCC) is subject to proteolysis within the ectodomain segment both in cultured cells and in vivo and that the residual membrane-tethered DCC "stub" is subsequently processed by ␥-secretase to generate a derivative termed DCC-intracellular domain (ICD). The production of DCC-ICD is inhibited by selective ␥-secretase inhibitors, and by the expression of the dominant negative PS1 D385A variant. Moreover, the membrane-tethered DCC "stubs" accumulate to high levels in PS1-deficient embryos. We also demonstrate that expression of a DCC-Gal4 chimera is capable of activating transcription in a luciferase-based reporter assay and this activity is dependent on ␥-secretase activity. Our findings offer the proposal that DCC performs dual roles both as a cell surface receptor that modulates intracellular signaling pathways and as a transcriptional coactivator that relies on ␥-secretase-dependent production and nuclear translocation of the cytoplasmic domain.Presenilin 1 and 2 (PS1 and PS2) 1 are polytopic membrane proteins that are mutated in the vast majority of pedigrees with early-onset familial Alzheimer's disease (1). PS plays an essential role in intramembranous, "␥-secretase" processing of several type I membrane proteins, including the ␤-amyloid precursor protein (APP) (2, 3), Notch1 (4, 5), ErbB-4 (6), N-and E-cadherins (7), low density lipoprotein receptor-related protein (8), CD44 (9), and nectin-1␣ (10). It is now well established that ␥-secretase processing of transmembrane proteins is preceded by proteolysis near the interface of the ectodomain and transmembrane segments that generates a soluble ectodomain that is "shed" and one, or more, membrane-tethered derivatives. In the case of APP, a set of membrane-tethered APP derivatives, termed APP-CTFs, are the substrates for ␥-secretase, and intramembranous processing leads to the production of A␤ peptides. Similarly, ␥-secretase is responsible for processing of a membrane-tethered Notch1 derivative, termed S2/ NEXT, resulting in the generation of a Notch derivative, termed S3/NICD that translocates to the nucleus and activates transcription of target genes (4, 11).Intrigued by the observation that deleted in colorectal cancer (DCC) is a substrate for cleavage by a metalloprotease that leads to "shedding" of the ectodomain segment (12), we asked whether the resulting membrane-tethered "stub" might be processed by ␥-secretase. In the present report, we show that in cultured mammalian cells, the truncated DCC stub is subject to a PS-dependent processing event that is inhibited by a highly potent ␥-secretase inhibitor. Moreover, the DCC stub accumulates to high levels in spinal cords of PS1-deficient mice, leading us to postulate that this species is also a substrate for ␥-secretase processing in vivo. Finally, we demonstrate that the ␥...

Intramembranous "␥-secretase" processing of ␤-amyloid precursor protein (APP) and other transmembrane proteins, including Notch, is mediated by a macromolecular complex consisting of presenilins (PSs), nicastrin (NCT), APH-1, and PEN-2. We now demonstrate that in cells coexpressing PS1, APH-1, and NCT, full-length PS1 accumulates to high levels and is fairly stable. Upon expression of PEN-2, the levels of PS1 holoprotein are significantly reduced, commensurate with an elevation in levels of PS1 fragments. These findings suggest that APH-1 and NCT are necessary for stabilization of fulllength PS1 and that PEN-2 is critical for the proteolysis of stabilized PS1. In N2a and 293 cell lines that stably overexpress PS1, APH-1, NCT, and PEN-2, PS1 fragment levels are elevated by up to 10-fold over endogenous levels. In these cells, we find a marked accumulation of the APP-CTF␥ (AICD) fragment and a concomitant reduction in levels of both APP-CTF␤ and CTF␣. Moreover, the production of the ␥-secretase-generated Notch S3/NICD derivative is modestly elevated. However, we failed to observe a corresponding increase in levels of secreted A␤ peptides in the medium of these cells. These results lead us to conclude that, although the PS1, APH-1, NCT, and PEN-2 are essential for ␥-secretase activity, the proteolysis of APP-CTF and Notch S2/NEXT are differentially regulated and require the activity of additional cofactors that promote production of AICD, NICD, and A␤.

The ␥-secretase complex, consisting of presenilins (PS), nicastrin (NCT), APH-1, and PEN-2, catalyzes the intramembranous proteolysis of truncated ␤-amyloid precursor protein (APP) and Notch derivatives to generate the APP intracellular domain (AICD) and Notch intracellular domain (NICD), respectively. To examine the intracellular sites in which active ␥-secretase resides, we expressed NCT variants harboring either an endoplasmic reticulum (ER) retention signal (NCT-ER) or a trans-Golgi network (TGN) targeting motif (NCT-TGN) along with PS1, APH-1, and PEN-2 and examined ␥-secretase activity in these settings. In cells expressing NCT-ER and the other components, PS1 fragments hyperaccumulated, but AICD levels were not elevated. On the other hand, upon coexpression of an ER-retained APP variant or a constitutionally active Notch mutant, N⌬E, we observed enhanced production of AICD or NICD, respectively, in cells expressing NCT-ER. Moreover, we show that membranes from cells expressing NCT-ER, NCT-TGN, or NCT-WT contain identical levels of PS1 derivatives that can be photoaffinity cross-linked to a biotinylated, benzophenone-derivatized ␥-secretase inhibitor. Finally, our cell-free ␥-secretase assays revealed nearly equivalent ␥-secretase activities in cells expressing PS1, APH-1, PEN-2, and either NCT-WT or NCT-ER. Taken together, we interpret these findings as suggesting that active ␥-secretase complex is generated in the early compartments of the secretory pathway but that these complexes are transported to late compartments in which substrates are encountered and subsequently processed within respective transmembrane segments.

There is mounting evidence indicating that overexpression or aberrant processing of amyloid precursor protein (betaAPP) is causally related to Alzheimer's disease. betaAPP is principally cleaved within the amyloid beta protein domain to release a large soluble ectodomain (betaAPPs) that has been known to have a wide range of trophic and protective functions. Activation of phospholipase C-coupled receptors has been shown to increase the release of betaAPPs through protein kinase C and calcium. Here we have examined whether nicotine can modulate the expression and processing of betaAPP in PC12 cells. Treatment of PC12 cells with nicotine increased the release of a carboxyl-terminally truncated, secreted form of betaAPP into the conditioned medium without affecting the expression level of betaAPP mRNA. The effect of nicotine on the secretion of betaAPPs is concentration (>50 microM)- and time (>2 hr)-dependent and attenuated by cotreatment with either mecamylamine, a specific nicotinic receptor antagonist, or EGTA, a calcium chelator, indicating calcium entry through the neuronal nicotinic acetylcholine receptor is essential in enhanced betaAPPs release by nicotine. However, nicotine did not significantly change the amyloid beta protein secretion from Swedish mutant betaAPP-transfected PC12 cells. These results imply that nicotinic receptor agonist might be beneficial in the treatment of Alzheimer's disease by not only supplementing the deficient cholinergic neurotransmission but also stimulating the release of betaAPPs.