Figure 5. Electrophoresis of S100A9 incubated with NHSY5Y cells. (a) and (c) SDS PAGE; (b) Native PAGE. Panel (a) has the following designations: column 1 corresponds to the ladder; 2 to S100A9 (0.2 mM); 3 to cell culture buffer DMEM; 4 to S100A9 (0.04 mM) with cells in DMEM for 3 d; 5 to S100A9 (0.01 mM) with cells in DMEM. In panel (b), 1 to S100A9 (0.2 mM); 2 to DMEM; 3 to S100A9 (0.04 mM) with cells in DMEM for 3 d. (c) 0.2 mM S100A9 at pH 3.0 and 7.4. The data was obtained by several experiments, and integrated together according to the ladders. The quantities of S100A9 monomer and homodimer were estimated by integrating the intensities of all bands using Image J software. Based on the SDS PAGE data, the freshly dissolved S100A9 contained 74% monomers and 26% homodimers. After incubation with cells for 3 d, the fraction of homodimer dropped to 8.4%, which suggests that the reduction in the quantity of S100A9 dimers is caused by its interaction with cells. A similar decrease in the S100A9 dimer content was observed when S100A9 was mixed with various concentrations of Ab1–40 (Fig. 6). Accompanied by a dramatic increase in the quantity of large aggregates at the top of the gel, a declining intensity in the dimer bands was observed for increasing Ab1–40 doses, which indicates the consumption of S100A9 dimers by Ab1–40. The fraction of S100A9 dimers was 30% at 0.002 mM Ab1–40, 18% at 0.01 mM Ab1–40, 14% at 0.02 mM Ab1–40 and 10% with 0.1 mM Ab1– 40. Native PAGE revealed a band for Ab1–40 monomers when the concentration of Ab1–40 exceeded 0.1 mM. Given that S100A9 alone contained 74% monomers and 26% homodimers, 0.2 mM S100A9 solution contains 0.15 mM monomers and 0.025 mM dimers. If Ab1–40 interacts with the S100A9 monomer, the reaction could consume at least 0.15 mM of Ab1–40, which is inconsistent with the existence of an Ab1–40 band in the presence of 0.1 mM Ab1–40. Conversely, 0.025 mM S100A9 dimers could consume 0.02 mM Ab1–40. Considering the decrease in the amount of S100A9 dimers and the absence of an Ab1–40 band for Ab1–40 concentrations below 0.02 mM, we believe that there is a specific interaction between the S100A9 dimer and Ab1–40 that does not involve S100A9 monomer. Moreover, there was no additional band for S100A9/Ab1–40 hetero-dimer or any other visible complexes. Thus, it can be assumed that these proteins are not stabilized in low-molecular- weight structures. It is possible that S100A9 can serve at the Ab nucleation or fibrillation stages. The rapid formation of Ab1–40 amyloid structures contributes to the absence of specific stable complexes between S100A9 and Ab1–40 in low-molecular-weight forms. Discussion The induction of the S100A9 gene was found in the brains of AD patients and AD animal models, Tg2576 and CT-Tg mice [1]. This observation, together with our results, suggests that S100A9 is responsible for the formation of AD amyloid plaques. The detailed molecular mechanism remains however, unknown. Therefore, we focused our research on the role of S100A9 in Ab amyloid formation and interaction with neuron origin cells. Inflammation and Alzheimer’s Disease (AD) The consequences of S100A9/Ab1–40 interaction were inves- tigated using AFM and CD. When S100A9 was mixed with Ab1– 40, large quantities of amyloid aggregates (fibrils and oligomers) were formed (Fig. 1). The height of the fibrils was 2 nm, which is similar to the values pertaining to mature Ab1–40 amyloid fibrils (Fig. S1). Conformational variations with different Ab concentra- tions identified Ab1–40 as the origin of amyloid formation (Fig. 1). In contrast to the lack of transition in the S100A9 secondary structure, Ab1–40 is responsible for the small changes seen in CD spectra (Fig. 3). The possibility of S100A9 amyloid formation was excluded by comparing S100A9 CD spectra in Fig. 3 with those of S100A9 exposed to harsh conditions (pH 3.0, shaking) (Fig. S3). The formation of S100A9 amyloid structure not only induced the transition to b-sheet structure accompanied by the formation of small filaments (1 nm in height), but also increased cell viability (Fig. S4). The reduction in S100A9 cytotoxicity induced by the addition of Ab1–40 was further verified by a luminescent cytotoxicity assay of neuron cells (Fig. S5). Sharing the same protective effect against S100A9 cytotoxicity, Ab1–40 monomer was more efficient than Ab1–40 plaques. The higher cell survival rate with Ab1–40 monomers compared to the ones of the plaques is consistent with our AFM observation that S100A9 interacted with freshly dissolved Ab peptides. Possibly, at the same peptide concentration, the pre-formed amyloid plaques decrease Ab1–40 monomer quantity and S100A9 consumption, which was observed as an increased cytotoxicity in the luminescent cytotoxicity assay. The Ab/S100A9 interaction was further studied using the peptides Ab1–16, Ab12–14 and Ab1–42 (Fig. S5). Similar to the case with Ab1–40, S100A9 cytotoxicity was greatly reduced upon addition of Ab12–14 and Ab1–42. This was accompanied by the formation of large quantities of amyloid aggregates (Fig. S6). It is worth noting that the Ab/S100A9 interaction did not only inhibit S100A9 cytotoxicity, but also suppressed the toxicity of Ab1–42. In contrast, the addition of Ab1–16 brought no effect on S100A9 cytotoxicity and no emergence of amyloid fibrillar structures. As the major difference between Ab1–16 and other Ab peptides is the absence of a hydrophobic core, we conclude that the hydrophobic core (L17V18F19F20A21) is essential for the S100A9 and Ab peptide interaction. In summary, S100A9 interacts with Ab1–40 and induces Ab amyloid formation whilst retaining its native structures. This specific interaction reduces the quantity of toxic S100A9 species and thus decreases S100A9-mediated cytotoxicity. With a constant amount of S100A9 monomers, we observed a great decrease in S100A9 dimer content from 26% to 8% (Fig. 5). Such variation reflects the consumption of S100A9 dimers via interaction with cells, which does not involve S100A9 monomers. Since S100A9, apparently via the activity of the dimmer, induces cell death, the reduction in cytotoxicity induced by the addition of Ab1–40 could be related to its specific interaction with S100A9 dimers. The hypothesis is supported by titrating S100A9 with different dose of Ab1–40. With increasing concentrations of Ab1– 40, amount of S100A9 dimer deceased dramatically (Fig. 6). Additionally, more aggregates are formed as indicated by the increasing band intensity corresponding to the wells. Therefore, it is possible that the interaction between S100A9 dimers and Ab1– 40 reduces the number of toxic S100A9 dimers, leading to an increase in cell viability. S100A9 homodimer is shown to be expressed independently during inflammation [15,16]. It has the ability to induce neutrophil immobilization and bind both Ca and Zn ions [17,18]. Structurally, the S100A9 monomer consists of 4 a-helices (H1,H2,H3 and H4), a long carboxy-terminal tail and a hinge region between H2 and H3. Through hydrophobic interactions, the C- and N-teminus of helices (H1 and H1’;H4 and H4’) from PLoS ONE | www.plosone.org 4 March 2012 | Volume 7 | Issue 3 | e32953