Familial Alzheimer's disease presenilin 1 mutations cause alterations in the conformation of presenilin and interactions with amyloid precursor protein

Abstract

Presenilin 1 (PS1) is a critical component of the gamma-secretase complex, an enzymatic activity that cleaves amyloid beta (Abeta) from the amyloid precursor protein (APP). More than 100 mutations spread throughout the PS1 molecule are linked to autosomal dominant familial Alzheimer's disease (FAD). All of these mutations lead to a similar phenotype: an increased ratio of Abeta42 to Abeta40, increased plaque deposition, and early age of onset. We use a recently developed microscopy approach, fluorescence lifetime imaging microscopy, to monitor the relative molecular distance between PS1 N and C termini in intact cells. We show that FAD-linked missense mutations located near the N and C termini, in the mid-region of PS1, and the exon 9 deletion mutation all change the spatial relationship between PS1 N and C termini in a similar way, increasing proximity of the two epitopes. This effect is opposite of that observed by treatment with Abeta42-lowering nonsteroidal anti-inflammatory drugs (NSAIDs) (Lleo et al., 2004b). Accordingly, treatment of M146L PS1-overexpressing neurons with high-dose NSAIDs somewhat offsets the conformational change associated with the mutation. Moreover, by monitoring the relative distance between a PS1 loop epitope and the APP C terminus, we demonstrate that the FAD PS1 mutations are also associated with a consistent change in the configuration of the PS1-APP complex. The nonpathogenic E318G PS1 polymorphism had no effect on PS1 N terminus-C terminus proximity or PS1-APP interactions. We propose that the conformational change we observed may therefore provide a shared molecular mechanism for FAD pathogenesis caused by a wide range of PS1 mutations.

Figure 1.

FLIM analysis of the subcellular distribution of the PS1 holoprotein and heteroprotein populations. A, B, Negative control. CHO cells double-immunostained with antibodies to the PS1 NT (A, intensity image) and to ER resident BiP are shown. B, The FLIM image shows pseudocolored PS1-FITC lifetime, which is ∼ 2300 ps. C, E, Intensity image of the FITC-PS1 immunostaining in the wild-type PS1 (C)- and D257A PS1 (E)-expressing CHO cell. D, F, Color-coded FLIM images of the FITC lifetimes in the cell shown in C and E, respectively. The continuous colorimetric scale shows color-coded fluorescence lifetime in picoseconds. A shorter lifetime represents closer proximity between the fluorophore-labeled PS1 NT and CT and is shown in red. Absence of FRET between the fluorophores on PS1 N and C termini results in a longer FITC lifetime and is indicated in blue. The cell profiles are shown by tracings.

Figure 2.

Detection of PS1 and Nct in cells treated with small interfering RNA (siRNA) or in cells expressing aspartate mutant PS1. A, CHO cells were transfected with control or Nct siRNA for 72 h, lysed in a buffer containing 1% Triton X-100, and resolved by electrophoresis on a 4-20% Tris-glycine gel (Invitrogen, Carlsbad, CA). To test the effect of aspartate mutations, we used CHO cells stably expressing D257A mutant PS1. The samples were probed with mouse α-PS1 antibody (Chemicon). PS1 endoproteolysis was significantly reduced in both Nct siRNA- and D257A PS1-transfected cells. B, Nct RNAi-treated cells probed with rabbit α-Nct antibody (Chemicon). The expression of Nct was nearly abolished in Nct siRNA-treated cells. The asterisk indicates nonspecific bands and serves as a loading control. CTF, C-terminal fragment.

Figure 3.

FLIM analysis of the FITC lifetime distribution in CHO cells expressing wild-type (wt) and FAD mutant PS1. The cells were double-immunostained with antibodies against the PS1 NT and CT. The intensity images show the FITC-immunostained N-terminal antibody. Pseudocolored FLIM images show the spatial distribution of the t2 FITC-PS1-NT lifetimes in cells expressing wt PS1 and selected representative PS1 mutations: M146L, G384A, and Δex.9. The pseudocolored FLIM images are presented for clarity using discrete colors for fluorescence lifetimes: red, lifetimes between 600 and 1600 picoseconds (psec); green, 1601-2000 psec; blue, 2001-2600 psec. Both red and green denote FRETing populations (shorter FITC lifetime reflects closer PS1 NT-CT proximity), representing functionally active PS1. A population with a majority of non-FRETing PS1 molecules (longer FITC lifetime, PS1 NT-CT far apart) is shown in blue.

Figure 4.

FLIM analysis of the PS1 NT-CT proximity in primary neurons expressing wild-type (wt) PS1 (A, B) and M146L PS1 (C, D). A, C, Intensity images of the FITC-PS1 immunoreactivity (x81 antibody). B, D, Pseudocolored FLIM images of the FITC lifetime distribution within neuronal cells. The FITC lifetimes are shown using discrete color-coding: red, 600-1600 picoseconds (psec); green, 1601-2000 psec; blue, 2001-2600 psec. Lifetimes significantly shorter than 2000 psec represent FRETing molecules with the PS1 NT and CT in close proximity.

Figure 5.

Schematic representation of the predicted PS1 conformation for immature PS1 holoprotein (A) and functionally active PS1 heterodimer complexes (B, C). We postulate two closely related conformations, which may exist in a dynamic equilibrium. In this model, FAD mutations in PS1 shift the equilibrium to favor the conformation shown in C and stabilize the alignment of the active site of PS1/γ-secretase with the APP substrate to favor cleavage at Aβ₄₂ position on APP. The black circles in TM6 and TM7 indicate the position of aspartate 257 and aspartate 385, respectively (a putative catalytic site). The stars in C indicate the position of analyzed FAD PS1 mutations (small star, M139V, M146L, L166P, L286V, G384A, and C410Y; large star, exon 9 deletion). APP C99 substrate is aligned with PS1 to be predominantly cleaved at the Aβ₄₀ (B) or Aβ₄₂ (C) position. For simplicity, we show only one component of the multimeric γ-secretase complex and PS1 as a monomer, but analogous models with PS1 as a dimer or multimer are also possible.