Nicastrin is required for assembly of presenilin/gamma-secretase complexes to mediate Notch signaling and for processing and trafficking of beta-amyloid precursor protein in mammals

Abstract

Recent studies indicate that nicastrin (NCT) and presenilins form functional components of a multimeric gamma-secretase complex required for the regulated intramembraneous proteolysis of Notch and beta-amyloid (Abeta) precursor protein (APP). To determine whether nicastrin is required for proteolytic processing of Notch and APP in mammals and the role of nicastrin in presenilin/gamma-secretase complex assembly, we generated nicastrin-deficient (NCT-/-) mice and derived fibroblasts from NCT-/- embryos. Nicastrin-null embryos died by embryonic day 10.5 and exhibited several patterning defects, including abnormal somite segmentation, phenotypes that are reminiscent of embryos lacking Notch1 or both presenilins. Importantly, secretion of Abeta peptides is abolished in NCT-/- fibroblasts, whereas it is reduced by approximately 50% in NCT+/- cells; the failure to generate Abeta peptides in NCT-/- cells is accompanied by destabilization of the presenilin/gamma-secretase complex and accumulation of APP-C-terminal fragments. Moreover, APP trafficking analysis in NCT-/- fibroblasts revealed a significant delay in the rate of APP reinternalization compared with that of control cells. Together, these results establish that nicastrin is an essential component of the multimeric gamma-secretase complex in mammals required for both gamma-secretase activity and APP trafficking and suggest that nicastrin may be a valuable therapeutic target for Alzheimer's disease.

Fig. 1.

Targeted disruption of nicastringene by homologous recombination. A, Maps of the wild-type NCT locus, the targeting vector, and the disrupted NCT allele. Exon 9 (E9) and exon 10 are indicated by black boxes. The targeting vector shows the replacement of the exon 9, part of exon 10, and the intron 9 sequences by neomycin gene (Neo). Arrows indicate the sites within the targeted and the wild-type alleles from which PCR primers were chosen for genotyping. Lines below denote expected sizes forHindIII-digested fragments detected by a 3′-flanking probe (a 0.3 kb PCR fragment; black bar) from targeted and endogenousNCT alleles. B, Analysis of genomic DNA from mouse embryos by Southern blot. TheHindIII fragment detected for wild-type (6.8 kb) and targeted (5.8 kb) NCT alleles with the 3′ probe are indicated. C, PCR analysis of DNA extracted from embryos using primers indicated in Figure 1A. The 395 or 229 bp fragment is specific to the targeted or endogenousNCT allele, respectively. E, EcoRI; X,XbaI; BG, BglII; BA, BamHI; N,NotI; TK, thymidine kinase; +/+, wild type; +/−, Nicastrin heterozygous; −/−, Nicastrin knock-out.

Fig. 2.

Phenotype of nicastrin mutant embryos.NCT^−/− embryos at E9 (A) and E9.5 (B) display severe growth retardation and abnormal somite segmentation compared with NCT^+/− orNCT^+/+ embryos. E8NCT^−/− embryos show a twisted neural tube and no somite segmentation (F, G), whereas the E8NCT^+/− orNCT^+/+ embryos show clear somite segmentation (E). Although the yolk sac vasculature is well formed in the E10NCT^+/− embryo (D), vascular morphogenesis is abnormal in theNCT^−/− embryo (C). TheNCT^−/− embryos also show an underdeveloped heart (H). Transverse sections through E9.5 embryos show disorganization of the trunk and the ventral neural tube and small unlooped hearts in theNCT^−/− embryo (J), whereas in theNCT^+/− embryo (I), segmented somites and a well developed heart are observed (note that I andJ are shown in different scales). Solid arrow, Neural tube; arrow, heart; arrowhead, somite.

Fig. 3.

Nicastrin is required for assembly of PS1 into the γ-secretase complex. A, Cell lysates fromNCT^+/+,NCT^+/−, andNCT^−/− embryonic fibroblasts were immunoblotted using an antiserum specific for nicastrin and reprobed with antiserum against PS1-CTF or BACE1. Note that nicastrin and PS1 levels are markedly reduced inNCT^+/− cells, whereas they are absent in NCT^−/− fibroblasts.B, Lysates from membrane fractions of control,PS1^−/−, andNCT^−/− cells were solubilized with DDM, subjected to BN-PAGE, and analyzed by immunoblotting using antibodies specific to either PS1-NTF or NCT. Note that cells deficient in PS1 or NCT fail to form a high molecular weight complex, and the asterisk denotes an apparent intermediate NCT-containing complex independent of PS1. The middle panel is a darker exposure of the left panel. WT, Wild type. C, Lysates of wild-type (PS1^+/+),PS1^+/−, andPS1^−/− cells were immunoblotted using antisera specific for NCT. The same blot was reprobed with antisera specific for PS1 (PS1-loop) or actin. Note that the ratio of immature to mature NCT in PS1^+/− andPS1^−/− cells is markedly altered compared with that in control fibroblasts. PS1-CTF, PS1 C-terminal fragment.

Fig. 4.

APP processing and trafficking defects inNCT^−/− fibroblasts.A, Cell lysates fromNCT^+/+,NCT^+/−, andNCT^−/− embryonic fibroblasts infected with recombinant adenovirus expressing APPswe were immunoblotted using CT15, an antiserum recognizing the C terminus of APP, and reprobed with antisera against NCT and actin. The cells were harvested 48 hr after infection. B, Conditioned media collected from NCT^+/+,NCT^+/−, andNCT^−/− fibroblast cultures infected with adenovirus expressing APPswe for 48 hr were subjected to Aβ40 and Aβ42 ELISA assays. Bars represent average of eight determinations ± SEM. C, Conditioned media collected from NCT^+/+ andNCT^+/− fibroblast cultures infected with different doses of adenovirus expressing APPswe for 24 hr were subjected to Aβ40 ELISA assays. Bars represent average of four determinations ± SEM. D, IP-mass spectrometry analysis of secreted Aβ peptides fromNCT^+/+,NCT^+/−, andNCT^−/− fibroblasts expressing APPswe. Asterisks denote peaks corresponding to human Aβ peptides; the mass of each peptide is in parentheses. M/Z, Mass-to-charge ratio.

Fig. 5.

APP trafficking defects inNCT^−/− fibroblasts. Endocytosis of APP is delayed in NCT^−/− fibroblasts (D–F) compared withNCT^+/+ cells (A–C). NCT^+/+and NCT^−/− cells were transiently expressing APPswe, labeled with antiserum P2-1, chased for 5 and 15 min at 37°C, and fixed for immunofluorescence.