Proteolytic release and nuclear translocation of Notch-1 are induced by presenilin-1 and impaired by pathogenic presenilin-1 mutations

Abstract

The Notch family of proteins consists of transmembrane receptors that play a critical role in the determination of cell fate. Genetic studies in Caenorhabditis elegans suggest that the presenilin proteins, which are associated with familial Alzheimer's disease, regulate Notch signaling. Here we show that proteolytic release of the Notch-1 intracellular domain (NICD), an essential step in the activation of Notch signaling, is markedly reduced in presenilin-1 (PS1)-deficient cells and is restored by PS1 expression. Nuclear translocation of the NICD is also markedly reduced in PS1-deficient cells, resulting in reduced transcriptional activation. Mutations in PS1 that are associated with familial Alzheimer's disease impair the ability of PS1 to induce proteolytic release of the NICD and nuclear translocation of the cleaved protein. These results suggest that PS1 plays a central role in the proteolytic activation of the Notch-1-signaling pathway and that this function is impaired by pathogenic PS1 mutations. Thus, dysregulation of proteolytic function may underlie the mechanism by which presenilin mutations cause Alzheimer's disease.

Figure 1

PS1 induces proteolytic release of the Notch-1 intracellular domain. (A) Notch-1 constructs utilized in assays of proteolytic cleavage. (B) Release of the Notch-1 intracellular domain is markedly reduced in PS1-KO cells. PS1-wt (WT) and PS1-KO (KO) cells were transfected with Myc epitope-tagged NδE, NδE(V1744K), or ICv1744, and immunoblot analysis was performed with monoclonal anti-Myc. Note that the cleaved form of NδE is generated in PS1-wt cells but not in PS1-KO cells. The NδE mutation V1744K prevents cleavage, and ICv1744 is truncated at the cleavage site (3). The uncleaved (uc) and cleaved (c) NδE proteins are indicated. Immunoreactivity was absent in immunoblots of untransfected cell lysates. (C) PS1 restores Notch-1 proteolytic release in PS1-KO cells. (Upper) NδE immunoblot. (Lower) PS1 immunoblot showing PS1 N-terminal fragments. (D) The NδEF construct containing the full-length Notch-1-intracellular domain is cleaved completely, and cleavage depends on PS1 and the presence of a PEST motif. Note that PS1-wt cells expressing NδEF show only the cleaved protein, whereas PS1-wt cells expressing the PEST deletion mutant show similar amounts of cleaved and uncleaved proteins. Shown are NδE immunoblots of PS1-wt and PS1-KO cells transfected with Myc-tagged NδEF or δPEST constructs. (E) Transcriptional activation of the HES-1 promoter is potentiated by PS1. NδEF-induced transcriptional activation of the HES-1 promoter was markedly reduced in PS1-KO cells. PS1-wt and PS1-KO cells were cotransfected with NδEF and HES-1-luciferase together with a β-galactosidase expression plasmid (5). Values represent the ratio of normalized luciferase activity to normalized activity determined with the luciferase plasmid alone and represent the mean ± SEM, n = 9. ∗, P < 0.01 relative to PS1-wt by ANOVA with post-hoc Student–Newman–Keuls test.

Figure 2

PS1 potentiates nuclear translocation of the Notch-1 intracellular domain. PS1-wt and PS1-KO cells were transfected with Myc-tagged NδEF. The cells were fixed 48 hr after transfection, and immunofluorescence microscopy was performed with monoclonal anti-Myc. (A) PS1-wt cells show predominantly nuclear immunoreactivity. (B) PS1-wt cell that shows prominent nuclear immunoreactivity and lower levels of cytoplasmic staining. (C) PS1-KO cells show cytoplasmic immunoreactivity in a vesicular distribution; most cells do not show nuclear immunoreactivity. (D) The cleaved Notch-1 fragment is detected in the nucleus of PS1-wt but not PS1-KO cells. Nuclear and membrane fractions were prepared from NδE-transfected PS1-wt and PS1-KO cells, and the distribution of cleaved and uncleaved Notch-1 proteins was determined by immunoblot analysis. Note that in PS1-wt cells, the cleaved Notch-1 fragment is detected in the nuclear extract but not in the membrane fraction, whereas uncleaved NδE is present in the membrane fraction. In PS1-KO cells, the cleaved fragment is not detected at significant levels in the nuclear extract, but appears after transfection of PS1. The cleaved fragment is not restored in the nuclear extract after transfection of the PS1 mutant C410Y. The difference in cytoplasmic membrane immunoreactivity for NδEF in A and B vs. NδE in D is due to the increased cleavage of the NδEF construct (Fig. 1D).

Figure 3

PS1 mutations associated with familial Alzheimer’s disease impair proteolytic release of the NICD. (A) PS1 mutations associated with familial Alzheimer’s disease impair PS1-induced cleavage. PS1-KO cells transfected with the indicated PS1 mutants exhibit reduced Notch-1 cleavage relative to PS1-KO cells transfected with wild-type PS1. (Upper) NδE immunoblot. (Lower) PS1 immunoblot showing transfected holo-PS1. (B) Quantitative analysis of the effects of six PS1 mutants on Notch-1 cleavage. The ratio of cleaved to uncleaved forms of NδE was quantitated after transfection of NδE in PS1-wt (Control) and PS1-KO (KO) cells and in PS1-KO cells cotransfected with wild-type PS1 (PS1wt) or the indicated PS1 mutants. Note that inhibition of Notch-1 cleavage ranges from 40–60% for PS1 N-terminal mutations to 70–100% for PS1 C-terminal mutations. Notch-1 cleavage was not significantly different between PS1-wt cells and PS1-KO cells transfected with wild-type PS1. Shown is the mean ± SEM, n = 4. ∗, P < 0.05 relative to KO+PS1wt by ANOVA with post-hoc Student–Newman–Keuls test.