Presenilin 1 Is Required for Maturation and Cell Surface Accumulation of Nicastrin

Abstract

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...

Cellular distribution of γ-secretase subunit nicastrin in the developing and adult rat brains

“…Earlier studies have shown that nicastrin can be co-immunoprecipitated and co-localized with PS1 in a variety of cells/neurons [1,30,37,44,49,55]. Consistent with previous reports [3,12,27,28,29,53], we find that PS1 is distributed widely in the adult rat brain.…”

“…The nicastrin and PS1 antisera used in the present study have been previously characterized [29,30,51]. The specificity of antisera is further confirmed by our immunoblotting experiment, which showed that nicastrin antiserum essentially reacted with one major band of approximately 120 kDa corresponding to the mature complex glycosylated forms of the protein in all brain regions examined.…”

“…Adult male rats (225-275 g) and postnatal rats from different developmental stages i.e., postnatal day 1 (P1), P7 and P21 were housed and maintained in accordance with the University of Alberta Policy on the handling and treatment of laboratory animals. The characterization of a polyclonal nicastrin antiserum raised against synthetic peptide corresponding to residues 689-709 (SP718) and a polyclonal PS1 antisera raised against N-terminal (PS1 NT ) fragment has been described previously [29,30,48]. Commercially available goat polyclonal nicastrin antiserum (N-19) was purchased from SantaCruz Biotechnology Inc. (California, USA).…”

“…In parallel, four rats from each of the following postnatal developmental stages i.e., P1, P7 and P21 were decapitated and brain regions of interest (i.e., cortex, hippocampus, cerebellum and brainstem) were dissected out and homogenized in Tris-lysis buffer. Tissues were then processed for immunoprecipitation by incubating with SP718 nicastrin antiserum as described earlier [30,51]. The immune complexes were precipitated by protein A/G PLUS-agarose, separated by 4-20% polyacrylamide gel electrophoresis for 90 min before being transferred to PVDF membranes.…”

“…Nevertheless, biogenesis, maturation, stability and the steady-state levels of γ-secretase components are co-dependent. For example, the heavily glycosylated type I membrane protein nicastrin does not mature or exit the ER in cells lacking PS1 expression [30]. The function of nicastrin within the γ-secretase complex is beginning to emerge.…”