Glycines from the APP GXXXG/GXXXA Transmembrane Motifs Promote Formation of Pathogenic Aβ Oligomers in Cells

Abstract

Alzheimer's disease (AD) is the most common neurodegenerative disorder characterized by progressive cognitive decline leading to dementia. The amyloid precursor protein (APP) is a ubiquitous type I transmembrane (TM) protein sequentially processed to generate the β-amyloid peptide (Aβ), the major constituent of senile plaques that are typical AD lesions. There is a growing body of evidence that soluble Aβ oligomers correlate with clinical symptoms associated with the disease. The Aβ sequence begins in the extracellular juxtamembrane region of APP and includes roughly half of the TM domain. This region contains GXXXG and GXXXA motifs, which are critical for both TM protein interactions and fibrillogenic properties of peptides derived from TM α-helices. Glycine-to-leucine mutations of these motifs were previously shown to affect APP processing and Aβ production in cells. However, the detailed contribution of these motifs to APP dimerization, their relation to processing, and the conformational changes they can induce within Aβ species remains undefined. Here, we describe highly resistant Aβ42 oligomers that are produced in cellular membrane compartments. They are formed in cells by processing of the APP amyloidogenic C-terminal fragment (C99), or by direct expression of a peptide corresponding to Aβ42, but not to Aβ40. By a point-mutation approach, we demonstrate that glycine-to-leucine mutations in the G(29)XXXG(33) and G(38)XXXA(42) motifs dramatically affect the Aβ oligomerization process. G33 and G38 in these motifs are specifically involved in Aβ oligomerization; the G33L mutation strongly promotes oligomerization, while G38L blocks it with a dominant effect on G33 residue modification. Finally, we report that the secreted Aβ42 oligomers display pathological properties consistent with their suggested role in AD, but do not induce toxicity in survival assays with neuronal cells. Exposure of neurons to these Aβ42 oligomers dramatically affects neuronal differentiation and, consequently, neuronal network maturation.

Keywords: Alzheimer’s disease; GXXXG motifs; amyloid precursor protein; beta-amyloid peptide; neuronal differentiation; oligomers.

FIGURE 1

Schematic representation of the different amyloid precursor protein (APP) mutants and APP constructs used for the study. Schematic representation of human APP and APP C-terminal fragments including the different mutants generated. Numbering corresponds to the amino acid position in the Aβ sequence. C99 corresponds to the APP β C-terminal fragment, C42 to the Aβ42 peptide, and C40 to the Aβ40 peptide. All C-terminal constructs are fused to the human APP signal peptide (SP). TM, Transmembrane region; JM, Juxtamembrane region; AICD, APP IntraCellular Domain; ext, extracellular; int, intracellular. The amino acid substitutions generated by site-directed mutagenesis are in red. The cleavage sites of α (α)-, β (β)-, and γ (γ and ε)-secretases are indicated by arrows. C99-hGLuc1 and C99-hGLuc2 correspond to C99 constructs fused to hGLuc moieties used for the analysis of C99 dimerization in split luciferase assays. The epitopes recognized by the human-specific W0-2 antibody, the APP C-terminal and hGLuc antibodies are indicated.

FIGURE 2

Detection of oligomeric bands in cells expressing C99 and C42. (A) Expression of APP, C99 and C42 in CHO cells analyzed in total cell lysates by Western blotting with the W0-2 antibody (left panel) and the APP C-terminal antibody (right panel). The presence of APP, βCTF (C99) and αCTF are indicated by arrows. The star (^∗) indicates the presence of an unexpected band at ∼30 kDa. (B) Cell fractions from C42- (left panel) and C99- (right panel) transfected cells were analyzed by Western blotting with the W0-2 antibody. Total cell lysates (e) were fractionated into soluble (s) and membrane-enriched vesicular fractions (MLP). The presence of the higher molecular weight band (^∗) is indicated by arrows.

FIGURE 3

Detection of oligomeric bands and measurement of Aβ in the extracellular medium of cells expressing C99 and C42. (A) Culture media of CHO cells expressing APP, C99, and C42 were analyzed by Western blotting using the W0-2 antibody. The ∼30 kDa oligomer band (^∗) detected in culture media -similar to the one observed in cell lysates- is indicated by an arrow. (B) Soluble monomeric Aβ38, Aβ40, and Aβ42 were quantified by ECLIA in the culture media of transfected cells. Values (means ± SEM) given in pg/ml are representative of three independent experiments (n = 3 in each experiment). ^∗∗∗p < 0.001, ^∗p < 0.05, as compared to control (mock-transfected cells).

FIGURE 4

Oligomers detected in cells and not re-captured from the culture medium. CHO cells were either transfected by the C42 construct (transfected cells) or treated (treated cells) for 48 h with the conditioned culture medium of C42-transfected cells (mock, non-transfected), as depicted on the top of the figure. The cell lysates and culture media of transfected and treated cells were recovered and analyzed by Western blotting with the W0-2 antibody. The ∼30 kDa oligomer band (^∗) detected in cell lysates and culture media is indicated by an arrow.

FIGURE 5

Impact of GXXXG/GXXXA mutations on C99 dimerization and Aβ oligomerization. (A) Schematic representation of human the C99 and C42 constructs used. Numbering corresponds to amino acid position in the C99 sequence. The amino acid substitutions generated by site-directed mutagenesis are in red. Glycine and alanine residues of GXXXG/GXXA sequences are underlined. TM, Transmembrane region; ext, extracellular; int, intracellular. (B) Expression of C99 and Aβ oligomers analyzed in cell lysates by Western blotting with the APP C-terminal antibody and the W0-2 antibody, respectively. Oligomers (^∗) and C99 are indicated by arrows. (C) Expression of C99 and Aβ oligomers analyzed in lysates of cells expressing the different C99 mutants by Western blotting with the W0-2 antibody. Oligomers (^∗) and C99 are indicated by an arrow. (D,E) Dimerization of C99 and C99 mutants (m5, mA) was measured in living cells by the split-luciferase complementation assay. Cells were transfected with C99-coding sequences fused to two hGLuc moieties (hGluc1 and 2, see Figure 1). Bioluminescence (luciferase activity) was measured as RLU and given as percentage of bioluminescence detected in cells co-expressing C99-hGLuc1 and C99-hGLuc2. Values (means ± SEM) are representative of three independent experiments (n = 4 in each experiment). ^∗∗∗p < 0.001, n.s. (non-significant), as compared to control (mock-transfected cells). Expression of the fusion proteins was checked in cell lysates by Western blotting with the hGLuc antibody and the W0-2 antibody. (F) Analysis of Aβ oligomerization was monitored by Western blotting with the W0-2 antibody in cell lysates or culture media of cells expressing C42 or C42 mutants. Actin was used as loading probe (cell lysates). Oligomers (^∗) are indicated by an arrow.

FIGURE 6

Effects of Aβ oligomers on neuronal cell differentiation and survival. (A) Neuronal NG108-15 cells were treated with media of control CHO cells (mock) or CHO cells transfected with C42, C42 mutant 5 (m5) or C42 mutant A (mA) during the differentiation process. (B) The presence of Aβ oligomers in the media of CHO producing cells and NG108-15 cells (at day 5) was assessed by Western blotting with the W0-2 antibody. Oligomers (^∗) are indicated by arrows. (C) Neuronal differentiation was assessed by immunostaining of NG108-15 treated cells with the MAP2 antibody. Nuclei were stained with the DAPI. Scale: 200 μm. (D) Quantification of MAP2 immunostaining was performed and expressed as percentage of intensity measured in NG108-15 cells treated with the control medium (mock). Values (means ± SEM) are representative of three independent experiments (n = 2 in each experiment). ^∗∗∗p < 0.0001, ^∗∗p < 0.001, ns (non-significant) as compared to cells treated with the control medium. (E) Survival of the NG108-15 cells was assessed by MTS assay and given as percentage of survival measured in cells treated with the control medium. Values (means ± SEM) are representative of three independent experiments (n = 3 in each experiment). Statistical analysis showed non-significant differences between the conditions.