The aspartate-257 of presenilin 1 is indispensable for mouse development and production of beta-amyloid peptides through beta-catenin-independent mechanisms

Abstract

To differentiate multiple activities of presenilin 1 (PS1), we generated transgenic mice expressing two human PS1 alleles: one with the aspartate to alanine mutation at residue 257 (hPS1D257A) that impairs the proteolytic activity of PS1, and the other deleting amino acids 340-371 of the hydrophilic loop sequence (hPS1Deltacat) essential for beta-catenin interaction. We show here that although hPS1Deltacat is fully competent in rescuing the PS1-null lethal phenotype, hPS1D257A does not exhibit developmental activity. hPS1D257A also leads to the concurrent loss of the proteolytic processing of Notch and beta-amyloid precursor protein (APP) and the generation of beta-amyloid peptides (Abeta). Further, by measuring the levels of endogenous Abeta(X-40) and Abeta(X-42) in primary neuronal cultures, we confirmed the concept that PS1 is indispensable for the production of secreted Abeta.

Figure 1

Biochemical characterization of hPS1Δcat and hPS1D257A transgenic and rescue mice. (A) Schematic representation of the transgenic constructs. hPS1Δcat is human PS1 cDNA deleting amino acids 340–371 of the hydrophilic loop sequence. hPS1D257A contains an aspartate to alanine mutation at residue 257 of PS1. Each construct was inserted into the pHZ024 vector, which includes the human Thy-1 (Thy) promoter and SV40 small t and poly(A) sequences (24). (B) Brain expression profiles of hPS1Δcat transgenic mice assayed by Western blot analysis using the human PS1-specific NTF antibody PSN2. Δcat-3 and Δcat-6 represents two hPS1Δcat transgenic lines: wt, wild-type nontransgenic mouse brain; and 17-3, a previously identified human wild-type PS1 transgenic line (24). Hybridization with an anti-β-actin was used as loading control. (C) Brain expression profiles of hPS1D257A (DA) transgenic mice (lines 3, 4, 6, and 7) by using the PSN2 antibody, which detected only full-length (FL) hPS1 in transgenic lines (Upper). The same brain lysates were also hybridized with the PS1 loop antibody 490D to detect PS1-CTF (Lower). wt, wild-type nontransgenic mouse brain. (D) Western blot analysis of hPS1Δcat (lines 3 and 6) and hPS1D257A (lines 3, 4, 6, and 7) transgenes expressed on mouse PS1-null (rescue or R) background. E14.5 embryonic brains were hybridized with either the PSN2 antibody (Upper) or the PS1-C antibody, which was raised against the C-terminus end of hPS1 (Lower). Both the wild-type hPS1 rescue (17-3R) and hPS1Δcat rescue (Δcat-3R and Δcat-6R) brains formed indistinguishable PS1-NTF, and normal or truncated CTF, respectively. However, the hPS1D257A rescue lines (DA-3R, DA-4R, DA-6R, and DA-7R) formed only FL protein. (E) Interaction of β-catenin by hPS1Δcat and hPS1D257A. Brain samples of DA-7R, Δcat-3R, or 17-3R were immunoprecipitated with an anti-β-catenin antibody and Western blotted with the PS1 antibody AB14 and the anti-β-catenin antibody. PS1Δcat failed to associate with β-catenin.

Figure 2

Histology and skeleton staining of hPS1Δcat and hPS1D257A rescue mice. (A) Hematoxylin and eosin staining of mid-sagittal sections of E14.5 embryos, showing CNS hemorrhage and axial skeleton defects in both PS1-knockout (PS1^−/−) and hPS1D257A rescue (PS1^−/−; hPS1D257A) mice, whereas the hPS1Δcat rescue (PS1^−/−; hPS1Δcat) mice looked normal. (B) Alcian blue and alizarin red staining of newborn skeletons revealing similar findings. WT, wild-type control.

Figure 3

Notch processing and downstream signaling by hPS1D257A mutant. (A) PS1^−/− fibroblast cells were cotransfected with the constitutively active Notch construct NΔE plus pcDNA3 empty vector (vector), pcDNA3-hPS1D257A (D257A), or pcDNA3-wild-type hPS1 (hPS1), respectively. PS1 proteolytic activity was measured by the appearance of Notch intracellular domain (NICD) by Western blotting. Transfection with NICD was used as size control. (B) Notch-mediated downstream signaling was determined by cotransfecting the PS1^−/− cells with the CBF-luciferase reporter plus empty vector (bar 1), hPS1D257A (bar 2), or wild-type hPS1 (bar 3), respectively, followed by quantifying the luciferase activity. AU, arbitrary unit.

Figure 4

APP processing and Aβ production by hPS1D257A and hPS1Δcat transgenes. (A) Western blot analysis of APP-FL and APP-CTF levels in E14.5 (lines 1–4) and adult (lanes 5–7) brains. DA-6R and DA-7R: PS1^−/−; hPS1D257A (lines 6 and 7), Δcat-3R: PS1^−/−; hPS1Δcat, (line 3), and 17-3: wild-type hPS1. (B and C) Endogenous Aβ_X-40 (B) and Aβ_X-42 (C) levels in conditioned medium of primary neuronal cultures of PS1^−/− (n = 8), PS1^−/−; hPS1D257A (line 7) (PS1^−/−; DA-7, n = 10), and PS1^+/+ (n = 10) brains. (D and E) Endogenous Aβ_X-40 (D) and Aβ_X-42 (E) levels in the brains of control (PS1^+/+, n = 8), hPS1Δcat (line 3) rescue (PS1^−/−; Δcat-3, n = 8), and wild-type hPS1 rescue (PS1^−/−; 17-3, n = 8) mice. Levels represent mean ± standard deviation.

Figure 5

Soluble β-catenin levels and stability in hPS1Δcat rescue mutant. (A) Higher steady-state levels of soluble β-catenin in PS1^−/−; hPS1Δcat (line 3) (Δcat-3R) neurons as compared with that of wild-type hPS1 rescue (17-3R) samples. In contrast, no appreciable differences in soluble β-catenin levels were detected between PS1^−/−; hPS1D257A (line 7) (DA-7R) and wild-type hPS1 rescue (17-3R). (B) Delayed β-catenin ubiquitination by PS1Δcat. Neuronal cultures of PS1^−/−; 17-3 or PS1^−/−; Δcat-3 mice were treated with the proteasome inhibitor MG132 (20 μM) for 0, 30, or 60 min followed by immunoblotting with the anti-β-catenin antibody to visualize nonubiquinated (β-catenin) and multiubiquitinated β-catenin (Ub-β-catenin). (C) Impaired β-catenin turnover by PS1Δcat. PS1^−/−; 17-3 and PS1^−/−; Δcat-3 cultures were treated with cycloheximide (25 mg/ml) for 0, 15, 30, or 60 min. Soluble β-catenin was extracted and subjected to immunoblotting with the anti-β-catenin antibody.