Development of new fusion proteins for visualizing amyloid-β oligomers in vivo

Abstract

The intracellular accumulation of amyloid-β (Aβ) oligomers critically contributes to disease progression in Alzheimer's disease (AD) and can be the potential target of AD therapy. Direct observation of molecular dynamics of Aβ oligomers in vivo is key for drug discovery research, however, it has been challenging because Aβ aggregation inhibits the fluorescence from fusion proteins. Here, we developed Aβ1-42-GFP fusion proteins that are oligomerized and visualize their dynamics inside cells even when aggregated. We examined the aggregation states of Aβ-GFP fusion proteins using several methods and confirmed that they did not assemble into fibrils, but instead formed oligomers in vitro and in live cells. By arranging the length of the liker between Aβ and GFP, we generated two fusion proteins with "a long-linker" and "a short-linker", and revealed that the aggregation property of fusion proteins can be evaluated by measuring fluorescence intensities using rat primary culture neurons transfected with Aβ-GFP plasmids and Aβ-GFP transgenic C. elegans. We found that Aβ-GFP fusion proteins induced cell death in COS7 cells. These results suggested that novel Aβ-GFP fusion proteins could be utilized for studying the physiological functions of Aβ oligomers in living cells and animals, and for drug screening by analyzing Aβ toxicity.

Figure 1. Representative images of COS7 cells transfected with various Aβ-GFP DNA constructs.

(A) Basic structure of genes encoding fusion protein containing Aβ_1-42 fused to GFP with a long-linker sequence (14 amino acids). (B) COS7 cells were transfected with plasmids encoding Aβ-GFP (a) Aβmut-GFP (b) Aβ (E22Δ)-GFP (c), or GFP (d). To confirm the expression of Aβ proteins, transfected cells were immunostained with the 6E10 antibody (e–h). Merged images with GFP are shown in (i–l). The regions within the dotted rectangles in (a–d) are enlarged in (m–p). Aggregated Aβ proteins (dotted localizations) were observed in Aβ-GFP and Aβ (E22Δ)-GFP transfected cells, however, the Aβmut-GFP proteins did not form detectable aggregates in cells. Scale bars: 20 μm (a–d) 5 μm (m–p). (C) Immunostaining of COS7 cells expressing the Aβ-GFP or Aβmut-GFP fusion proteins with the 11A1 antibody. Merged images showed that almost all the Aβ-GFP fusion protein was labeled with the11A1 antibody, indicating that the Aβ-GFP fusion protein formed oligomers. In contrast, the Aβmut-GFP was only partially labeled with the11A1 antibody. Scale bars: 20 μm (a–f) 5 μm (g,h).

Figure 2. Comparison of Aβ-GFP fluorescence intensities according to the linker length in primary culture neurons.

(A) Basic structure of genes encoding fusion proteins containing Aβ_1-42 fused to GFP with short-linker sequences (0, 2 or 3 amino acids). (B) COS7 cells transfected with a short-linker Aβ-GFP (2 amino acids). Faint GFP fluorescence was detected in the nucleus and surrounding areas (a) even though the fusion protein was stained by the 6E10 antibody (b). Merged image of (a,b) is shown in (c). Scale bar: 20 μm. (C) Primary culture of rat hippocampal neurons transfected with Aβ-GFP plasmids containing long-linker (a) or short-linkers (b). GFP fluorescence was nearly undetectable in cells carrying the short-linker plasmids, even though the fusion protein was stained by the 6E10 antibody (c,d). Merged images with GFP are shown in (e,f). Relative fluorescence intensities from cells expressing each fusion protein with various linker lengths were measured (g,h). Statistical analyses showed that the detection of the Aβ protein in neurons was nearly identical with each plasmid (h) but GFP fluorescence intensities increased significantly as the linker became longer (g) (***p < 0.001, Kruskal-Wallis test, n = 10–14 cells each). Scale bar: 10 μm.

Figure 3. NMR analyses of structural changes in Aβ-GFP fusion proteins.

Shown are parts of 500-MHz NMR spectra mainly reflecting methyl groups for the Aβ peptide (A,E), Aβ-GFP (B), Aβmut-GFP (C), and GFP (D). Spectra in (A–D) were recorded at 20 °C, where the red, green and blue lines indicate intact peptides, those after incubation at 37 °C for 15.5 h, and those after incubation at 37 °C for 50 h or 63.5 h, respectively. Spectra in (E) were recorded at 37 °C after 5 min −19 h of incubation, where changes in the intensity of the highest peak at 0.91 ppm are shown in (F).

Figure 4. EM analysis of molecular feature of Aβ-GFP fusion proteins.

EM images (A) and analyses (B) of Aβ-GFP fusion proteins. GFP (a), monomeric Aβ peptide (b), Aβ-GFP (c), Aβmut-GFP (d), and Aβ (E22Δ)-GFP (e) are indicated by arrows in each panel. 24 h after incubation at 4 °C (pH8.5), Aβ peptide formed long fibrils (f) but Aβ-GFP (g,h) and Aβ (E22Δ)-GFP (j) formed oligomers with various sizes (g,j) or filamentous-looking aggregates (h). Almost all the Aβmut-GFP remained as small particles in the size of a monomer or a very small oligomer (i) without a clear sign of polymerization. The inset shows a magnified view of the dotted rectangle in (h–j) revealing single units of Aβ-GFP fusion protein oligomers (arrows). Measurement of the area of each unit (B) shows that a single unit of polymerized Aβ-GFP and Aβ (E22Δ)-GFP contains two to four molecules but the particles observed with Aβmut-GFP contain single to two molecules (n = 100 units). Scale bars: 5 nm (a–e) and insets), 20 nm (f–j).

Figure 5. Expression of Aβ-GFP fusion proteins in C. elegans.

(A) Schematic representation of the Aβ-GFP fusion construct. (B) GFP fluorescence in the cholinergic motor neurons of Aβ-GFP transgenic C. elegans. The left illustration depicts the expressed proteins shown in the right pictures. The right pictures show the expression patterns of fusion proteins in C. elegans. (a) GFP, (b) Aβ-GFP with a long-linker, (c) Aβ-GFP with a short-linker, (d) Aβmut-GFP with a short-linker, and (e) crucumin treatment of animals bearing a short linker protein. Blankets indicate the cell bodies of neurons and arrowheads indicate the axon in the ventral nerve cord. Asterisks indicate the autofluorescence from the intestine. The long-linker has 14 amino acids and the short-linker has only 2 amino acids sequences. Cells expressing the short-linker Aβ-GFP protein did not show fluorescence (c) but the long-linker one and Aβmut-GFP showed bright fluorescence (b,d). Short-linker Aβ-GFP transgenic C. elegans were treated with curcumin, which induces Aβ disaggregation. Disappeared fluorescence was recovered after treatment with curcumin (e). Scale bar: 10 μm. (C) Localization of the Aβ-GFP fusion protein at the presynaptic regions. Aβ-GFP (a) and presynaptic protein SNB-1 fused with mCherry (b) were simultaneously expressed in cholinergic neurons. Several GFP puncta were co-localized with SNB-1 on the axon (c) suggesting that the fusion protein may be strongly accumulated at synaptic sites. Scale bar: 10 μm.

Figure 6. Effect of Aβ-GFP fusion proteins on the survival of COS7 cells.

The numbers of dead COS7 cells were counted 48 h (blue) and 72 h (red) after transfection with plasmids encoding GFP, Aβ-GFP, Aβmut-GFP, or Aβ (E22Δ)-GFP. The number of dead cells increased significantly in Aβ-GFP and Aβ (E22Δ)-GFP transfected cells compared with the Aβmut-GFP and GFP control transfected cells. The data represents the mean ± SEM (100 cells), *p < 0.05, **p < 0.001 by one-way ANOVA.