Proteolytic processing of amyloid precursor protein generates ␤-amyloid (A␤) peptides that are deposited in senile plaques in brains of aged individuals and patients with Alzheimer's disease. Presenilins (PS1 and PS2) facilitate the final step in A␤ production, the intramembranous ␥-secretase cleavage of amyloid precursor protein. Biochemical and pharmacological evidence support a catalytic or accessory role for PS1 in ␥-secretase cleavage, as well as a regulatory role in select membrane protein trafficking. In this report, we demonstrate that PS1 is required for maturation and cell surface accumulation of nicastrin, an integral component of the multimeric ␥-secretase complex. Using kinetic labeling studies we show that in PS1 ؊/؊ /PS2؊/؊ cells nicastrin fails to reach the medial Golgi compartment, and as a consequence, is incompletely glycosylated. Stable expression of human PS1 restores these deficiencies in PS1 ؊/؊ fibroblasts. Moreover, membrane fractionation studies show co-localization of PS1 fragments with mature nicastrin. These results indicate a novel chaperone-type role for PS1 and PS2 in facilitating nicastrin maturation and transport in the early biosynthetic compartments. Our findings are consistent with PS1 influencing ␥-secretase processing at multiple steps, including maturation and intracellular trafficking of substrates and component(s) of the ␥-secretase complex.Alzheimer's disease is pathologically characterized by the cerebral deposition of 39 -42 amino acid peptides, termed ␤-amyloid (A␤), 1 which are generated by proteolytic processing of amyloid precursor protein (APP) (1). Mutations in the genes encoding presenilin 1 (PS1) and presenilin 2 (PS2) account for the majority of the cases of familial early-onset Alzheimer's disease (2). Biochemical and genetic evidence demonstrate that PS1 and PS2 facilitate the final step in A␤ production, the intramembranous ␥-secretase cleavage of APP (3-5). Moreover, it has been proposed that PS1 deficiency alters the trafficking of select membrane proteins (4). Familial Alzheimer's diseaselinked mutant PS1 and PS2 elevate the levels of highly amyloidogenic A␤42 peptides, thus promoting A␤ deposition in senile plaques (6, 7). However, the specific mechanism(s) involved in the selective elevation of A␤42 production by familial Alzheimer's disease-linked PS variants has not been clearly understood.The precise role played by PS1 and PS2 in ␥-secretase cleavage of APP and Notch has been under intense scrutiny. Biochemical fractionation and ␥-secretase inhibitor studies have suggested a catalytic role for PS1 in the intramembraneous cleavage of substrates (5, 8 -10). On the contrary, biochemical evidence also suggests an indirect role for PS1 in facilitating ␥-secretase cleavage of substrates (11). It is unlikely that this apparent controversy can be resolved by biochemical analysis, since PS-derived N-terminal (NTF) and C-terminal (CTF) fragments are components of high molecular weight multimeric protein complexes (12). One of the more recent members of the g...

Presenilins (PS1/PS2) play a critical role in proteolysis of ␤-amyloid precursor protein (␤APP) to generate ␤-amyloid, a peptide important in the pathogenesis of Alzheimer's disease. Nevertheless, several regulatory functions of PS1 have also been reported. Here we demonstrate, in neuroblastoma cells, that PS1 regulates the biogenesis of ␤APP-containing vesicles from the transGolgi network and the endoplasmic reticulum. PS1 deficiency or the expression of loss-of-function variants leads to robust vesicle formation, concomitant with increased maturation and/or cell surface accumulation of ␤APP. In contrast, release of vesicles containing ␤APP is impaired in familial Alzheimer's disease (FAD)-linked PS1 mutant cells, resulting in reduced ␤APP delivery to the cell surface. Moreover, diminution of surface ␤APP is profound at axonal terminals in neurons expressing a PS1 FAD variant. These results suggest that PS1 regulation of ␤APP trafficking may represent an alternative mechanism by which FAD-linked PS1 variants modulate ␤APP processing.Alzheimer's disease (AD) 1 is characterized by the excessive generation and accumulation of ␤-amyloid peptides (A␤). The amyloidogenic A␤ peptide is proteolytically derived from the ␤-amyloid precursor protein (␤APP) within the secretory pathway by distinct enzymatic activities known as ␤-and ␥-secretase (1, 2). Full-length ␤APP is synthesized in the endoplasmic reticulum (ER) and transported through the Golgi apparatus. The major population of secreted A␤ peptides is generated within the trans-Golgi network (TGN) (3-5), also the major site of ␤APP residence in neurons at steady state. ␤APP can be transported in TGN-derived secretory vesicles to the cell surface if not first proteolyzed to A␤ or an intermediate metabolite.At the plasma membrane ␤APP is either cleaved to produce a soluble molecule, s␤APP (6) or, alternatively, reinternalized within clathrin-coated vesicles to an endosomal/lysosomal degradation pathway (7,8). Thus, the distribution of ␤APP between the TGN and cell surface has a direct influence upon the relative generation of s␤APP versus A␤. This phenomenon makes delineation of the mechanisms responsible for regulating ␤APP trafficking from the TGN relevant to understanding the pathogenesis of AD.Expression of autosomal dominant variants of either ␤APP, presenilin 1 (PS1), or presenilin 2 (PS2) results in increased A␤42 production and predispose individuals to early onset familial Alzheimer's disease (FAD) (9 -11). Presenilins (PSs), multitransmembrane proteins, accumulate as endoproteolyzed heterodimers of N-and C-terminal fragments and associate with other membrane proteins (e.g. nicastrin, APH-1, and PEN-2) to form high molecular weight complexes (9,(12)(13)(14)(15)(16). Several lines of evidence suggest that presenilin complexes play a crucial role in intramembranous ␥-secretase cleavage of type I membrane proteins including ␤APP and the signaling receptor, Notch-1. For example, genetic ablation of PS1, PS2, or other components of the PS complex dramatically im...

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...

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Proteolytic processing of amyloid precursor protein (APP) by ␤-and ␥-secretases generates ␤-amyloid (A␤) peptides, which accumulate in the brains of individuals affected by Alzheimer disease. Detergent-resistant membrane microdomains (DRM) rich in cholesterol and sphingolipid, termed lipid rafts, have been implicated in A␤ production. Previously, we and others reported that the four integral subunits of the ␥-secretase associate with DRM. In this study we investigated the mechanisms underlying DRM association of ␥-secretase subunits. We report that in cultured cells and in brain the ␥-secretase subunits nicastrin and APH-1 undergo S-palmitoylation, the post-translational covalent attachment of the long chain fatty acid palmitate common in lipid raft-associated proteins. By mutagenesis we show that nicastrin is S-palmitoylated at Cys 689 , and APH-1 is S-palmitoylated at Cys 182 and Cys 245 . S-Palmitoylation-defective nicastrin and APH-1 form stable ␥-secretase complexes when expressed in knock-out fibroblasts lacking wild type subunits, suggesting that S-palmitoylation is not essential for ␥-secretase assembly. Nevertheless, fractionation studies show that S-palmitoylation contributes to DRM association of nicastrin and APH-1. Moreover, pulse-chase analyses reveal that S-palmitoylation is important for nascent polypeptide stability of both proteins. Co-expression of S-palmitoylation-deficient nicastrin and APH-1 in cultured cells neither affects A␤40, A␤42, and AICD production, nor intramembrane processing of Notch and N-cadherin. Our findings suggest that S-palmitoylation plays a role in stability and raft localization of nicastrin and APH-1, but does not directly modulate ␥-secretase processing of APP and other substrates.