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

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.

. In addition, COS7 cells were transfected with a GFP construct alone as a control (Fig. 1Bd). To confirm the expression of the Aβ proteins, transfected cells were immunostained with an anti-β amyloid antibody (6E10; Fig. 1Be-h), which recognizes all species of Aβ , i.e., monomer, oligomer, and fibril forms of it 19 . As shown in Fig. 1Bi-k, almost all GFP fluorescence in the cytoplasm of transfectants were colocalized with fluorescence of 6E10 antibody, except for cells transfected with GFP alone (Fig. 1Bl), indicating that GFP signals coincide with the localization sites of each Aβ fusion protein.
Cells transfected with GFP alone showed almost uniform GFP expression in the cytoplasm and nucleus (Fig. 1Bd,p), as also observed with Aβ mut-GFP transfected cells (Fig. 1Bb,n). In contrast, cells expressing the Aβ -GFP fusion protein showed aggregates of various sizes and shapes of Aβ -GFP in the cytoplasm (Fig. 1Ba,m), although the nuclear distribution appeared uniform. The expression patterns of the Aβ (E22Δ)-GFP fusion protein were similar to that of the Aβ -GFP fusion protein, as aggregates of various sizes and shapes of Aβ (E22Δ)-GFP were also observed throughout the cells (Fig. 1Bc,o).
To assess the polymerization states of Aβ -GFP fusion proteins inside of cells, each transfectant was immunostained using the 11A1 antibody, which was developed against E22P-Aβ 10-35 as an antigen and recognizes oligomeric forms of Aβ specifically 20 . Almost all GFP signals were double-labeled by the 11A1 antibody in Aβ -GFP transfected cells (Fig. 1C), suggesting that the Aβ -GFP aggregates were oligomer (Fig. 1Cg). However, the Aβ mut-GFP fusion proteins were only partially double-labeled with the 11A1 antibody, especially in the peripheral regions of the cell (Fig. 1Ch), indicating that most of the Aβ mut-GFP proteins do not form oligomers inside of cells. Immunoblot analysis following native-PAGE also supports the results of immunostaining. Non-denaturing protein lysates from COS7 cells that expressed each Aβ -GFP fusion protein were separated on a gel. We used anti-GFP and anti-Aβ (6E10) antibodies to detect the fusion proteins. A clear single band close to the GFP signal was detected in Aβ mut-GFP lysate by both antibodies. In contrast, smear and ladder-like signals were observed in both Aβ -GFP and Aβ (E22Δ)-GFP lysates by these antibodies. These results indicated that most of the Aβ mut-GFP proteins exist as small-sizes molecules, probably monomers but Aβ -GFP and Aβ (E22Δ)-GFP proteins exist as oligomers of different sizes in the cell (see Supplementary Fig. S1 and methods online).
We investigated the specific subcellular localization site of the Aβ -GFP fusion protein by double labeling with antibodies against marker proteins specific for mitochondria, Golgi apparatus, or endoplasmic reticulum, however, no double labeling was detected in those intracellular organelles (data not shown). To observe when and how the fusion proteins are expressed and accumulated in cells, we performed the time-laps imaging of COS7 cells transiently expressing Aβ -GFP ( Supplementary Fig. S2). The Aβ -GFP fusion protein gradually aggregated in a time-dependent manner.
Comparison of GFP fluorescence intensity of Aβ-GFP fusion proteins according to the length of the linker sequence. We considered that proper folding of GFP in fusion proteins may depend on the linker length between Aβ and GFP, and that the folding efficacy may affect the fluorescent intensity. To determine the effect of the linker length on the fluorescence intensities of fusion proteins, Aβ -GFP plasmids with a short-linker (0, 2, or 3 amino acid) or a long-linker (14 amino acids) were transfected into COS7 cells and rat hippocampal primary culture neurons, and the fluorescence intensities of the GFP fusion proteins were compared. Figure 2 shows images of cells expressing fusion proteins with a 2-amino acid linker (short-linker) or a long-linker. Twenty-four hours after transfection, both COS7 cells and primary neurons were immunolabeled by the 6E10 antibody, and confocal images were taken under the completely same condition as described in the "Methods" section. GFP fluorescence showed uniform cytoplasmic distribution in both cell types transfected with the long-linker construct, (Figs 1Ba and 2Ca). However, in cells transfected with the short-linker construct, GFP fluorescence was undetectable in the cytoplasm and was very faint in the nucleus (Fig. 2Ba,Cb), even though the immunolabeling signals of the 6E10 antibody were detected strongly (Fig. 2Bb,Cd). Comparison of the staining intensities observed with the 6E10 antibody and that of GFP fluorescence was performed in neurons expressing Aβ -GFP proteins with differing linker length (Fig. 2Cg,h). The immunofluorescence intensities observed with 6E10 were nearly identical for each fusion protein, but the GFP fluorescence intensities decreased as the length 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 Aβ mut-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 Aβ mut-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 Aβ mut-GFP was only partially labeled with the11A1 antibody. Scale bars: 20 μm (a-f) 5 μm (g,h).    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 shortlinker 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. of the linker became shorter. These results indicated that GFP fused to Aβ via long-linker can fold normally and fluorescence robustly, whereas GFP cannot fluorescence robustly in the short-linker forms, probably because of misfolding.

Oligomerization of the Aβ-GFP fusion protein in vitro.
To analyze the molecular characteristics of the Aβ -GFP fusion proteins in detail, we performed nuclear magnetic resonance (NMR) measurements and electron microscopy (EM) observations. We focused on the long-linker fusion proteins because only these proteins appeared to be folded normally. NMR spectra of synthetic peptide (Fig. 3A,E,F), Aβ -GFP (B), Aβ mut-GFP (C) and GFP (D) were collected to examine aggregation. We used Hou's method 21 to generate the monomeric Aβ fusion proteins, as described in the "Methods" section. The NMR spectral intensities of the synthetic Aβ peptide decreased in a time-dependent manner and approached zero following approximately 8 h incubation period at 37 °C (Fig. 3A,E,F), indicating that nearly all peptides aggregated and formed fibrils. This is because the spin relaxation rate during 1 H NMR detection is inversely correlated to the overall rotational motion of the molecule 22 , resulting in impaired NMR signals to be observed for very large molecules, such as fibrils. For the Aβ mut-GFP, the spectra were unchanged even after a 63.5 h incubation at 37 °C (Fig. 3C), indicating that the monomeric state persists at 37 °C. The spectral intensity of the Aβ -GFP protein decreased by approximately 20% during the first 15.5 h incubation at 37 °C (green line), but not significantly during the subsequent 48 h (blue line, Fig. 3B). These data suggested that the aggregation stopped before fibril formation.
Next, we examined the molecular features of each Aβ -GFP fusion protein by negative-stain EM. The images of GFP showed round or rectangular particles ~3-4 nm in size (Fig. 4Aa), consistent with the atomic structure of GFP 23 . Under unpolymerizing conditions for Aβ , the Aβ peptide was observed as smaller, round or elongated particles (Fig. 4Ab), possibly corresponding to single Aβ peptides. In the images obtained for Aβ -GFP, Aβ mut-GFP, and Aβ (E22Δ)-GFP, some particles were only slightly larger than GFP, probably corresponding to single fusion proteins ( Fig. 4Ac-e). Under the conditions that promote polymerization, the synthetic Aβ peptides formed fibrils of 7.34 ± 0.36 nm (n = 30) in width (arrowhead), or thicker filaments (arrow), which appeared to form following entwining of the fibrils with each other (Fig. 4Af). In contrast, Aβ -GFP, Aβ (E22Δ)-GFP, and Aβ mut-GFP did not form regular fibrils. Instead, Aβ -GFP was observed as oligomers of various sizes ( Fig. 4Ag) or as filamentous-looking aggregates (Fig. 4Ah), and Aβ (E22Δ)-GFP was also observed as oligomers of various sizes (Fig. 4Aj). Magnified views of the dotted rectangles (inset of Fig. 4Ah,j) reveal that these aggregates are composed of small oligomeric clusters of ~10 nm. We call the single oligomeric cluster as 1 unit (arrows in inset). In the case of Aβ mut-GFP, however, these clusters were rarely observed and most of the molecules seemed to be in monomers or very small oligomers ( Fig. 4Ai and magnified view of dotted rectangle in i).
We examined how many molecules were present in single units of Aβ -GFP and Aβ (E22Δ)-GFP aggregates, or in a particle observed with Aβ mut-GFP . The estimated area of a single Aβ -GFP fusion protein was 13.7 nm 2 . Therefore, the actual measured values of areas for each single unit of Aβ -GFP aggregates were divided by 13.7 nm 2 . Figure 4B showed that the main species of single units of Aβ -GFP and Aβ (E22Δ)-GFP fusion protein oligomers contained 2-4 molecules. The averaged number of molecules in one unit of Aβ (E22Δ)-GFP seemed to be slightly larger than that of Aβ -GFP. The main species of Aβ mut-GFP was monomer to dimer, consistent with the idea that this mutation suppresses aggregation of Aβ . With all of the three fusion proteins, some small aggregates were observed, but long fibrils were not formed. Thus, both NMR and EM studies suggest that Aβ -GFP fusion proteins form small oligomers.

Fluorescence correlation spectroscopy (FCS) analysis of Aβ-GFP fusion protein in living cells.
To further confirm the oligomeric state of the Aβ -GFP fusion protein in living cells, we performed FCS analysis on cells expressing each fusion protein as well as on their lysates. First, we examined the properties of each fusion protein in aqueous solution, which were extracted from transfected COS7 cells. Compared to the diffusion constant for the GFP protein (111.0 μm 2 /s), that of the Aβ -GFP fusion protein was significantly lower (74.8 μm 2 /s, Cell lysate in Table 1), indicating that the protein mobility of the fusion protein was significantly decreased. The Aβ mut-GFP protein showed an intermediate diffusion constant (86.6 μm 2 /s) between that of GFP and Aβ -GFP. Since the estimated molecular weight of single Aβ -GFP fusion protein is not markedly different from that of GFP (33 kDa vs 27 kDa), the decreased diffusion mobility observed with the Aβ -GFP fusion protein suggested that some molecular complex, presumably oligomers composed of several Aβ -GFP fusion protein molecules, are formed within cells. Interestingly, the count per molecule (CPM) value of the Aβ -GFP fusion protein was decreased compared to that of GFP, whereas that of Aβ mut-GFP showed similar value to that of GFP, suggesting that an increase in fluorescent intensity of a particle does not simply occur, even if the fusion protein forms oligomers (see the "Discussion" section).
We applied this observation directly to living COS7 cells and the results were similar to those obtained in aqueous condition (Live cell in Table 1). Because of restricted diffusion in the cell, autocorrelation functions were fitted using a two-component diffusion model for this analysis. The diffusion constant of Aβ -GFP in both the cytoplasm and nucleus was significantly lower than that of GFP, again suggesting decreased diffusion mobility due to the formation of larger molecular complex compared to GFP (estimated Mw; 27 kDa in GFP vs 77 kDa in Aβ -GFP. See the "Methods" section for calculation). The two-component diffusion model analysis applied for living cells also showed that remarkable decrease of fast component fraction in Aβ -GFP expressing cells (84%), compared to both GFP and Aβ mut-GFP (around 95%). This suggests that interactions between the Aβ -GFP protein and other intracellular species may be increased and/or the amount of large soluble aggregates formed by the Aβ -GFP protein may be increased within cells. The CPM value from cells expressing Aβ -GFP was decreased compared to that of GFP and Aβ mut-GFP, suggesting that fluorescence in the large soluble aggregates/oligomers may be quenched (see the "Discussion" section for further explanation). We have also applied this observation to examine the molecular dynamics of Aβ (E22Δ)-GFP in cell lysate and in living cells. In both conditions, this mutant form showed significantly slower mobility than GFP, but the mobility was not significantly different from that of the wild-type Aβ -GFP in any conditions. These results suggest that the E22Δ mutation also causes the formation of large protein complex similar to the wild-type Aβ -GFP. These FCS data confirmed that our fusion  proteins generated by a long-linker sequence showed robust fluorescence and can be used to monitor the molecular dynamics of Aβ containing various types of mutations.

Expression of Aβ-GFP fusion proteins in transgenic C. elegans.
Both the in vitro analyses of the molecular state of Aβ -GFP fusion proteins and the in vivo analyses of living cultured cells suggested that the fusion proteins probably exist as oligomers. These results also indicated that the fluorescence of the fusion proteins can be altered dependent on their aggregation properties when a short-linker is used. To examine whether these phenomena can also be observed in neuronal cells of a living animal, we expressed our fusion proteins in C. elegans neurons and observed their dynamics in vivo. A schematic representation of the Aβ -GFP fusion construct used for transgenic C. elegans strains is shown in Fig. 5A. Aβ -GFP was specifically expressed in the cholinergic neurons by the acr-2 promoter. GFP fluorescence was detected steadily inside of the neurons in GFP transgenic animals (Fig. 5Ba). In transgenic animals expressing long-linker Aβ -GFP, GFP fluorescence was observed in both the cell bodies and their neurites, but showed accumulated or aggregated expression patterns of the fusion protein (Fig. 5Bb). However, GFP fluorescence was absent in the short-linker Aβ -GFP transgenic worms (Fig. 5Bc), which is similar to the expression patterns observed in COS7 cells and rat hippocampus primary neurons (Fig. 2).
We also wondered whether the fluorescence intensities in transgenic animals expressing short-linker Aβ -GFP reflect the aggregation properties of fusion proteins. To examine this, we expressed Aβ mut-GFP fusion protein with the short-linker, and GFP fluorescence was clearly and uniformly detected in the neuronal cells of Aβ mut-GFP transgenic worms (Fig. 5Bd). This finding indicates that non-fibril and soluble forms of Aβ do not affect the folding of GFP and that GFP fluorescence can be observed in living neurons if aggregation of the fusion protein is inhibited.
Therefore we examine whether these phenomena could be used to screen for substance that inhibit Aβ aggregation. It is known that curcumin can inhibit polymerization of Aβ . Thus we added it to the culture medium and the molecular state of short-linker forms of Aβ -GFP was observed in transgenic worms. In the animals reared on Table 1. FCS analysis of Aβ-GFP proteins in living cells. 1 CPM values from lysate samples are normalized by that of GFP, and live-cell CPMs are normalized by that of GFP in cytoplasm. 2 Kruskal-Wallis and post-hock tests are performed among each condition. Only the significant differences are shown: ***P < 0.001, **P < 0.01, *P < 0.05.
Scientific RepoRts | 6:22712 | DOI: 10.1038/srep22712 plates containing curcumin, bright and uniform GFP fluorescence was observed in both cell bodies and neurites, similar to animals expressing the Aβ mut-GFP protein (Fig. 5Be). These findings indicated that the inhibition of Aβ aggregation induced by curcumin results in the recovery of GFP fluorescence. This fusion protein can be also used to examine the subcellular localization of Aβ protein (Fig. 5C). The presynaptic VAMP2 protein (SNB-1 in C. elegans) was fused to mCherry and simultaneously expressed with the long-linker Aβ -GFP fusion protein, under the control of the same promoter. Several strong accumulations of the Aβ -GFP fusion protein correlated well with the position of RFP localization, meaning that the fusion protein tended to accumulate at the synaptic regions when the protein is expressed in presynaptic neurons.

Effect of Aβ-GFP oligomers on survival rate of COS7 cells. To examine whether the Aβ -GFP fusion
protein caused cellular toxicity in living cells, we measured cell death ratios in COS7 cells transfected with each Aβ -GFP fusion plasmid or the GFP plasmid (Fig. 6). Compared with cells expressing GFP, the ratios of dead cells significantly increased in both Aβ -GFP and Aβ (E22Δ)-GFP transfected COS7 cells until 72 h after transfection, but it was not changed in Aβ mut-GFP expressing cells. These results indicate that Aβ -GFP and Aβ (E22Δ)-GFP oligomer may cause cellular toxicities like wild-type Aβ oligomers.

Discussion
The intracellular accumulation of Aβ 1-42 has been proposed as an event responsible for early pathogenesis of AD. Especially, Aβ oligomer has been the subject of much attention as a target for studying the pathophysiological role of AD 24,25 , because it has been proposed to be a key mediator of cognitive decline in AD 11 . In this study, we developed new cellular and animal models of AD, which showed an accumulation of small sized Aβ oligomers inside of cells. This molecular state can be achieved by fusing Aβ and GFP, and this method can be used to visualize the molecular dynamics of Aβ in living cells by arranging the linker sequence between Aβ and GFP.
Previous report using yeast lysate that expresses Aβ -GFP fusion proteins suggested that Aβ 1-40 -GFP and the Aβ -GFP mutant that contains substitution Ile 41 to Glu and Ala 42 to Pro are less prone to aggregation and a portion of those fusion proteins exhibit soluble and non-aggregated forms, but Aβ 1-42 -GFP exhibit insoluble aggregate only 26 . We confirmed the molecular features of GFP-fused Aβ proteins through several strategies. In NMR experiments, we started the measurement for all samples under monomer conditions and the same concentration of proteins. Previous findings indicated that GFP is stable at pH 6-10 27,28 and that NaOH does not affect the conformational, tinctorial, morphological, and physiological functions of Aβ 29 . Therefore, our methods to form monomers should not affect the protein properties of Aβ -GFP fusion proteins. Our results indicated that the synthetic peptide formed fibrils within 8 h incubation period, however, the multimerization of Aβ -GFP proteins stopped before 15.5 h, consequently they could not form fibrils and remained as oligomers. In the EM experiments, the synthetic peptides formed long fibrils, but Aβ -GFP formed oligomers consisting of mainly 2-4 molecules, which did not assemble further into fibrils or large aggregates. These results were consistent with the NMR results and showed that Aβ -GFP form oligomers in vitro. The fusion protein is composed of a 27 kDa GFP component and a 4.5 kDa Aβ , thus a GFP molecule is much larger than the Aβ molecule. Therefore, aggregation of Aβ might be sterically hindered by GFP and, as a result, Aβ -GFP fusion protein could form only oligomers.
FCS analysis also suggested that the same molecular states of Aβ -GFP fusion proteins exist in living cells. In cultured living cells, the estimated molecular size of Aβ -GFP calculated from the diffusion constant was 77 kDa in the cytoplasm and 64 kDa in the nucleus. The larger molecular sizes probably result from either the formation of oligomers or molecular complexes with intracellular proteins. Contrary to the slow diffusion mobility of the Aβ -GFP fusion proteins, their CPM values, which refer to fluorescence intensities per single particle, were smaller than that of GFP as well as a non-aggregating mutant, Aβ mut-GFP (Table 1). These results suggest that homo-oligomeric species of Aβ -GFP may emit low fluorescence intensity because of quenching of GFP fluorescence. By fusing Aβ  to the N-terminus of GFP, the folding properties of GFP could be altered, and only a small fraction of the fusion proteins can express fluorescence 17,18 . Due to this fusion protein's nature, the CPM values of the Aβ -GFP fusion protein may not appear to correspond to the multimerization state of the fusion protein. Based on this argument, we focused on the values of diffusion constant and estimate that the major forms of oligomers of the Aβ -GFP protein may be trimers to tetramers in live cells. In contrast, Aβ mut-GFP showed a fraction of the fast component similar to that of GFP, but the estimates molecular weight (41 kDa) was between that of a monomer and a dimer, suggesting that the fusion protein probably exists as a mixture of monomers and dimers. The molecular state of the Aβ (E22Δ)-GFP mutant did not show a clear difference in molecular mobility from the wild-type Aβ -GFP, although it causes AD and shows an age-dependent intraneuronal accumulation of oligomers 16 . We believe that, however, our methods enable us to understand the intracellular dynamics of these kinds of mutant proteins of Aβ . Further investigations of appropriate linkers to generate Aβ -GFP with a less quenching property will clearly improve detection sensitivity of the Aβ oligomers in live cells using FCS. In COS7 cells transfected with the Aβ -GFP plasmid, almost all Aβ -GFP fusion proteins were labeled with the 11A1 antibody, which recognizes oligomeric Aβ specifically 20 . The decrease of the fast fraction in Aβ -GFP (84% and 83%) also means that larger complexes probably exist in living cells. Taken together, these data suggested that Aβ -GFP fusion proteins formed small oligomers inside cells and that this fusion protein is a new useful tool for assessing the intracellular function and toxicity of the Aβ oligomers.
Previously, it was reported that insoluble aggregates of N-terminal fusion partners with GFP attenuated the fluorescence of GFP 17 . A linker was constructed using 12 amino acids to make a reporter construct for evaluating protein folding, which was designed to avoid large bulky hydrophobic residues. Wurth et al. 18 used the same construct to make a fusion protein composed of a N-terminal GFP fused to C-terminal end of Aβ  . In this case, the wild type Aβ -GFP fusion protein did not fluoresce in E. coli whereas strong fluorescence was observed in the mutated Aβ -GFP fusions containing substitutions in the hydrophobic region responsible to aggregation of Aβ . Nair et al. 26 also reported using mutant Aβ -GFP fusion constructs with which aggregation is reduced that the fluorescence intensity in yeast reflects the aggregation state of Aβ . In the current study, we inserted 14 random amino acids as a linker sequence between Aβ and GFP (long-linker), and succeeded in developing new Aβ -GFP fusion proteins that fluoresce even when the wild type Aβ is aggregated. We do not fully understand the exact reasons why this linker sequence can generate stable fluorescence in an aggregated condition. Using this construct, however, it is possible to observe the molecular dynamics of Aβ -GFP oligomers in living cells. There are some reports suggesting that Aβ provokes ER stress and oxidative stress and induces cellular toxicities [30][31][32][33][34][35] . In particular, the E22Δ mutant caused ER stress, which induced apoptosis in HEK293 cells 36 . We also found that both Aβ -GFP and Aβ (E22Δ)-GFP caused increased cell death in transfected COS7 cells. These findings indicated that GFP-tagged Aβ fusion proteins may have similar physiological properties as wild type Aβ suggesting that they are quite applicable for examining "oligomer hypothesis" in AD. From our EM and FCS results, the molecular states of wild type Aβ -GFP and Aβ (E22Δ)-GFP are not greatly different from each other. Therefore there is a possibility that larger aggregates have a potential to induce strong toxicity compared to smaller aggregates, but the toxicity of these fusion proteins against cells may not depend solely on the aggregation sizes of Aβ . Although the difference in the structures of these fusion proteins are not known, there might be unknown interactive factors with Aβ oligomers that induce the toxicity inside cells.
To evaluate the property of our fusion proteins in living animals, we used C. elegans as an experimental model and observed in vivo Aβ dynamics. Although invertebrate is phylogenetically far removed from mammals, C. elegans possesses several genes homologous to the human AD-related genes such as nicastrin 37 , presenilin 38,39 , APH-1 40 and neprilysin 41 . In addition to these genetic relationships, over expression of Aβ exhibits an increased level of reactive oxygen species (ROS) in C. elegans 42 similar to those observed in AD patients. In C. elegans neurons, we confirm that our fusion proteins showed fluorescence properties quite similar to those in mammalian cells including rat primary cultured hippocampal neurons and COS7 cells, i.e., the protein with the short linker decreases its fluorescence when it aggregates, whereas the long linker retains fluorescence in spite of its aggregation. Therefore, C. elegans, as well as mammalian models, is an useful tool to investigate the basic pathogenesis of AD. Using this model system, we can perform both the genetic and chemical screening for endogenous or exogenous factors that are possible to regulate the Aβ aggregation in live animals. For example, the curcumin experiment in short-linker Aβ -GFP transgenic worms revealed that it is possible to monitor the effect of drugs against deposition and disaggregation of Aβ directly in living animals, suggesting that our new fusion proteins are useful tools for screening candidate synthetic chemical and naturally occurring products against AD in living neuron.
Mutant forms of Aβ -GFP were generated using GeneArt ® Site-Directed Mutagenesis System (Life Technologies) with the pAct-Aβ 1-42 -GFP plasmid serving as a template. The Aβ mut-GFP is a mutant generated by substituting Phe19 with Ser and Leu34 with Pro. The Aβ 1-42 (E22Δ)-GFP is a deletion mutant constructed by removing glutamate-22 of Aβ 1-42 sequence from the pAct-Aβ 1-42 -GFP plasmid.
To express Aβ -GFP fusion genes in C. elegans, the human Aβ 1-42 coding sequence was inserted into the Fire's C. elegans GFP expression vector (a kind gift from A. Fire). A 3.0 kb upstream region of the acr-2 gene was used to specifically express the fusion proteins in cholinergic motor neurons. The same promoter region was inserted into the mCherry vector to generate a Pacr-2: snb-1: mCherry fusion construct. The C. elegans snb-1 cDNA fragment was amplified by RT-PCR and was subcloned in frame into the Pacr2: mCherry vector. All plasmid DNAs were sequenced, and the sequences are available on request.
Scientific RepoRts | 6:22712 | DOI: 10.1038/srep22712 Generation of transgenic C. elegans. Transgenic worms were generated using standard microinjection methods for C. elegans. The pbLH98 [lin-15(+ )] or Pacr-2:: RFP plasmid was used as a co-injection marker at 50 ng/μl. Each fusion plasmid was injected at 10-25 ng/μl. At least 3 independent stable transgenic lines were used in the experiments performed in this study.
Cell culture and transfection. All experimental procedures performed on animals were carried out in accordance with the approved guidelines in ethical permit approved by the Institutional Animal Care and Use Committee of the National Institute of Advanced Industrial Science and Technology (Permission No. 2014-143) and in accordance with the Law No. 105 passed by and the Notification No. 6 released by the Japanese Government.
Primary cultures were prepared from the hippocampi of embryonic day 17 Wister rats (Nihon SLC, Shizuoka, Japan). Briefly, embryos were removed by cesarean sectioning, after which hippocampi were isolated and digested in 1 U/ml papain (Worthington Biochemical corporation, NJ, USA) in HEPES-buffered Hank's Balanced Salt Solution containing cysteine and BSA (1 mg/ml each) at 37 °C for 12 min. Dissociated cells were plated on poly-L-lysine coated glass cover slips at a density of 30,000 cells/ well in a 6-well plate in plating medium containing Dulbecco's Modified Eagle Medium (DMEM, Wako, Osaka, Japan) and 10% fetal bovine serum (FBS, Gibco, NY, USA). Two hours after plating, the medium was changed to Neurobasal ® Medium (Invitrogen, CA, USA) containing B27 supplement (Invitrogen) and 0.5 mM L-glutamine.
COS7 cells were cultured in DMEM containing 10% FBS at 37 °C in a humidified atmosphere containing 5% CO 2 /95% air. For transfection of plasmid DNAs, cells were plated at a density of 15,000 cells/cm 2 in a 6-well plate.
COS7 cells and 4DIV primary hippocampal neurons were transfected with plasmids using Lipofectamine 2000 (Invitrogen), as described previously 44 . Dead cells were counted 48 h and 72 h after transfection by staining the living COS7 cells with DAPI. The DAPI solution (Dojindo, Kumamoto, Japan) was added to the medium of each transfected cells and incubated for 20 min. After washed by PBS, five images were taken from each culture dish, and cells stained by DAPI were counted as dead. The total number of cells in images were also counted and used to calculate the number of dead cells per 100 cells. Statistical analysis was performed by one-way analysis of variance (ANOVA).

Immunohistochemistry and imaging of cells.
To confirm the expression of each Aβ -GFP fusion protein, transfected COS7 cells and primary neurons were immunostained with an anti-β amyloid (6E10) antibody (Covance, WI, USA) or 11A1 antibody (IBL, Gunma, Japan). Twenty-four hours after transfection, cells were fixed with 4% paraformaldehyde and 4% sucrose in PBS for 15 min at room temperature. Then, the cells were washed with PBS and permeabilized for 5 min in 0.25% Triton X-100. After washing the cells in PBS, they were blocked in 3% normal goat serum (Vector Laboratories, Inc., CA, USA) for 30 min and incubated with the 6E10 antibody (×800 dilution) or 11A1 antibody (×50 dilution) overnight at 4 °C. Each protein was visualized with an Alexa 568 conjugated secondary antibody (Invitrogen), and the nuclei were labeled with DAPI (Vector Laboratories, Inc.). Cell imaging was performed using Olympus FluoView 1000 (Olympus, Tokyo, Japan) and Nikon A1R (Nikon, Tokyo, Japan) confocal laser scanning microscopes. Each image was obtained at a resolution of 1020 × 1020 pixels. To quantify fluorescence intensities, the confocal scanning settings of pinhole, laser power, brightness, and contrast were held constant for all images. The fluorescence intensities of cell bodies were measured using Image J software (NIH, Bethesda, MD, USA). Statistical analysis of fluorescence intensities was performed by the Kruskal-Wallis test (n = 10-14 cells each).
Time laps imaging was performed using COS7 cells transfected with Aβ -GFP plasmid containing long-linker. Eighteen hours after transfection, fluorescent images of cells were taken every 10 min for 24 h in a humidified atmosphere containing 5% CO 2 / 95% air at 37 °C using a × 20 objective lens (KEYENCE corporation, Osaka, Japan).
Curcumin treatment of Aβ-GFP transgenic C. elegans. Curcumin were dissolved in ethanol, and 100 μl of 3 mM solution was spread onto NGM plates. Young-adult transgenic worms expressing short-linker Aβ -GFP protein were placed on the curcumin plates, and the fluorescence of their progeny was examined.

Purification of the Aβ-GFP fusion protein.
Purifications of recombinant proteins were performed according to the manufacturer's instructions (New England BioLabs, MA, USA) and Chong et al. 45 . Briefly, the coding sequences of Aβ -GFP, Aβ mut-GFP, and GFP were cloned into the pTXB1 vector (New England BioLabs) and the resulting plasmid DNAs were transformed into E. coli BL21 cells. The cells were grown in LB media at 37 °C until the culture reached an OD 600 nm of 0.5, and then the cells expressed the fusion protein by adding of 0.2 mM IPTG and incubated at 30 °C for 4 h. Cells were harvested and resuspended in Tris buffered solution (buffer A: 20 mM Tris-HCl, pH 8.5, 300 mM NaCl and 10% glycerol). After adding 0.002% CHAPS, 0.05 mM EDTA (pH 8.0) and 0.1 mM PMSF, the cell suspensions were incubated for 30 min at 4 °C and then ultrasonic disruption were performed on ice, using a BRANSON SONIFIRE 250. The lysed cell suspensions were centrifuged at 9600 × g for 20 min at 4 °C, after which the supernatants were loaded onto equilibrated Chitin beads (New England BioLabs) and incubated for 1 h on rotator at 4 °C. The beads were loaded in a column and washed with buffer A, and then the fusion proteins were eluted by Tris buffered solution (buffer B: buffer A containing 50 mM DTT) for 16 h at room temperature. The concentrations of proteins were measured using the BCA protein assay kit (Pierce). Purified fusion proteins were stored at − 80 °C until use.

NMR analysis.
To produce the monomeric Aβ -GFP and Aβ mut-GFP, 1 N NaOH were added to the purified protein solution until the pH reached 10-11, and then the protein solution was sonicated for 1 min 21 . Synthetic Aβ peptide (Peptide Institute, Inc. Osaka, Japan) was predissolved in 10 mM NaOH solution at a concentration of 500 μM. Then, the buffer of each protein was displaced to 20 mM deuterated Tris-HCl (d 11 , 99%; CDN isotope,