Methylene Blue Reduced Abnormal Tau Accumulation in P301L Tau Transgenic Mice Masato Hosokawa 1 , Tetsuaki Arai 1,2 , Masami Masuda-Suzukake 3 , Takashi Nonaka 3 , Makiko Yamashita 3 , Haruhiko Akiyama 1 *, Masato Hasegawa 3 1 Department of Dementia and Higher Brain Function, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan, 2 Department of Psychiatry, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Japan, 3 Department of Pathology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan Abstract In neurodegenerative disorders, abnormally hyperphosphorylated and aggregated tau accumulates intracellularly, a mechanism which is thought to induce neuronal cell death. Methylene blue, a type of phenothiazine, has been reported to inhibit tau aggregation in vitro. However, the effect of methylene blue in vivo has remained unknown. Therefore, we examined whether methylene blue suppresses abnormal tau accumulation using P301L tau transgenic mice. At 8 to 11 months of age, these mice were orally administered methylene blue for 5 months. Subsequent results of Western blotting analysis revealed that this agent reduced detergent-insoluble phospho-tau. Methylene blue may have potential as a drug candidate for the treatment of tauopathy. Citation: Hosokawa M, Arai T, Masuda-Suzukake M, Nonaka T, Yamashita M, et al. (2012) Methylene Blue Reduced Abnormal Tau Accumulation in P301L Tau Transgenic Mice. PLoS ONE 7(12): e52389. doi:10.1371/journal.pone.0052389 Editor: Andrea C. LeBlanc, McGill University, Canada Received June 12, 2012; Accepted November 14, 2012; Published December 20, 2012 Copyright: ß 2012 Hosokawa 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: This research was partially supported by the Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research (C), grant number 21591536 to HA and 24591738 to M. Hosokawa. The additional part of the funding of the authors9 study has come from institute budget. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: akiyama-hr@igakuken.or.jp Introduction In neurodegenerative disorders such as Alzheimer’s disease, corticobasal degeneration, and supranuclear palsy, the microtu- bule–associated protein tau is abnormally phosphorylated and redistributed into paired helical filaments (PHFs) forming neuro- fibrillary tangles, a process that correlates with pyramidal cell destruction and dementia. Abnormal tau accumulation is charac- terized by hyperphosphorylation, conformational change and aggregation with changes in solubility. Wischik et al. have identified a nonneuroleptic phenothiazine which reverses the proteolytic stability of protease-resistant PHFs by blocking tau-tau binding through the repeat domain [1]. Moreover, phenothiazines, including methylthioninium chloride (methylene blue (MB)), polyphenols and porphyrins, inhibited heparin-induced tau filament formation in vitro [2]. Based on these results, tau aggregation inhibitors are considered to be strong candidates for the treatment of tauopathy. TauRx Pharmaceuticals recently announced the completion of MB phase II clinical trials. They conducted MB dosing and efficacy studies involving 321 people with mild to moderate Alzheimer’s disease. Over a 50-week period, the cognitive decline of those on the drug appeared to be 81% slower than those taking a placebo. These results were presented at a conference [3] but have not been published. Currently, a large-scale phase III trial is in planning [4]. To date, there are two reports on the effect of MB on tau aggregation in vivo. In one study, MB did not alter abnormal tau phosphorylation and failed to inhibit tau-dependent neuronal cell toxicity in zebrafish [5]. The other study employed a distinct tau transgenic mouse line and mice received a 2–3-week treatment with oral MB. In this latter study, MB acted as a tau aggregation inhibitor. Although, these findings have been described in brief in a conference abstract, the details are unavailable [6]. More recently, Congdon et al. demonstrated that MB could induce autophagy in primary neurons, organotypic slice cultures and tau transgenic mice (JNPL3) [7]. They also showed a 2 week oral administration of MB attenuated the total tau levels in the absence of significant changes in sarkosyl-insoluble tau levels. In the present study, we investigated whether MB could reduce abnormal tau accumulation by carrying out long-term oral administration of MB using tau transgenic mice with the P301L mutation as a tauopathy model. Our results suggested that oral intake of MB could be a potential treatment for tauopathy. Materials and Methods Ethics Statement This study was carried out in strict accordance with the recommendations provided in the Guide for the Care and Use of Laboratory Animals of the Ministry of Health, Labour and Welfare of Japan and the Ministry of Education, Culture, Sports, Science and Technology of Japan. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Tokyo Metropolitan Institute of Medical Science (Permit Numbers: 22– 23 and 11-028). All experiments were performed under sodium pentobarbital anesthesia and every effort was made to minimize suffering. PLOS ONE | www.plosone.org 1 December 2012 | Volume 7 | Issue 12 | e52389