MRP14 (S100A9) Protein Interacts with Alzheimer Beta- Amyloid Peptide and Induces Its Fibrillization Ce Zhang1*.¤ , Yonggang Liu3*., Jonathan Gilthorpe4, Johan R. C. van der Maarel2 1 Department of Medical Biochemistry and Biophysics, Umea˚ University, Umea˚, Sweden, 2 Department of Physics, National University of Singapore, Singapore, Singapore, 3 Laboratory of Stem Cell and Tissue Engineering, Chongqing Medical University, Chongqing, China, 4 Umea˚ Centre for Molecular Medicine (UCMM), Umea˚ University, Umea˚, Sweden Abstract Increasing evidence supports the contribution of local inflammation to the development of Alzheimer’s disease (AD) pathology, although the precise mechanisms are not clear. In this study, we demonstrate that the pro-inflammatory protein S100A9 interacts with the Ab1–40 peptide and promotes the formation of fibrillar b-amyloid structures. This interaction also results in reduced S100A9 cytotoxicity by the binding of S100A9 toxic species to Ab1–40 amyloid structures. These results suggest that secretion of S100A9 during inflammation promotes the formation of amyloid plaques. By acting as a sink for toxic species, plaque formation may be the result of a protective response within the brain of AD patients, in part mediated by S100A9. Citation: Zhang C, Liu Y, Gilthorpe J, van der Maarel JRC (2012) MRP14 (S100A9) Protein Interacts with Alzheimer Beta-Amyloid Peptide and Induces Its Fibrillization. PLoS ONE 7(3): e32953. doi:10.1371/journal.pone.0032953 Editor: Eugene A. Permyakov, Russian Academy of Sciences, Institute for Biological Instrumentation, Russian Federation Received November 28, 2011; Accepted February 2, 2012; Published March 22, 2012 Copyright: 2012 Zhang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: There are no current external funding sources for this study. Competing Interests: The authors have declared that no competing interests exist. * E-mail: phyzce@nus.edu.sg (CZ); liuyonggangcq01@yahoo.com (YL) . These authors contributed equally to this work. € Current address: Department of Physics, National University of Singapore, Singapore, Singapore Introduction Knockdown of S100A9 expression improves cognition function in Alzheimer’s disease model mice (Tg2576) and these animals also exhibit reduced amyloid plaque burden [1]. However, a mechanistic link between S100A9 and Alzheimers disease pathology has not been shown. S100A9 protein is a reliable marker of inflammation [2]. Infiltrating macrophages express S100A9 in the event of acute or chronic inflammation [3,4]. S100A9 and A8 are co-expressed mainly by phagocytes in a variety of inflammatory conditions, including rheumatoid arthritis, allograft rejection, inflammatory bowel, and lung diseases [5,6]. Inflammatory disorders such as chronic bronchitis, cystic fibrosis, and rheumatoid arthritis are also associated with elevated plasma levels of S100A9 [7,8]. It has been speculated that inflammation is directly related to Alzheimer’s disease. Studies performed in transgenic animals suggest that cerebral amyloid deposition is increased in inflam- matory conditions [9]. In these transgenic animals, amyloid plaques do not develop unless inflammation is induced. Heneka et al. reported that in certain conditions related to chronic stress, astrocytes secreting S100 proteins assemble at the site of amyloid plaques, which may serve to prolong neuroinflammation [10]. An additional report shows microglial activation in early stages of AD [11]. These results indicate that early and local inflammatory events contribute to neuronal dysfunction and the formation of amyloid plaques at this and later stages of AD, respectively. To investigate the relationship between S100A9 and amyloid plaques, we have studied the interaction between the main component of the amyloid plaque (Ab1–40) and S100A9 by exploring the conformational and structural transition of species generated between them by atomic force microscopy (AFM), circular dichorism (CD), polyacrylamide gel electrophoresis (PAGE) and the Thioflovin-T assay (ThT). We observed that, whilst retaining its native structure, S100A9 triggers and promotes the formation of b-amyloid aggregates. To investigate the effect of S100A9/Ab mixtures on cells of neural origin, we employed the WST-1 assay on the neuroblastoma SH-SY5Y cell line. Our results show that freshly dissolved Ab1–40 is not toxic to neural cells, whereas S100A9 shows strong cytotoxicity. The addition of Ab1–40 significantly reduces S100A9 cytotoxicity. Our study provides direct physical evidence that S100A9 triggers and accelerates Ab1–40 amyloid formation, which indicates that amyloid plaque formation is related to inflammation and may be the result of a protective response. Results Ab1–40 fibrillization induced by S100A9 Ab1–40/S100A9 mixtures containing various additions of Ab1– 40 were examined using atomic force microscopy (AFM). After 1 day (d) of incubation at 370C, massive aggregates were produced in the form of round-shaped oligomers, proto-filaments and elongated thick fibrils (Fig. 1). Randomly distributed on mica surface, the round-shaped oligomers had a diameter of 80 nm and a height of 10 nm. The proto-filaments carried a height of 2.7 nm, a width of 50 nm and an average length of 300 nm. They were dimensionally similar to those observed in the Ab control samples after 3 d of incubation under the same condition (Fig. S1). The thick fibrils were less abundant and had a height of 8 nm, a width PLoS ONE | www.plosone.org 1 March 2012 | Volume 7 | Issue 3 | e32953