A Single Amino Acid Mutation in SNAP-25 Induces Anxiety-Related Behavior in Mouse Masakazu Kataoka 1. , Saori Yamamori 2. , Eiji Suzuki 3 , Shigeru Watanabe 2 , Taku Sato 2 , Hitoshi Miyaoka 2 , Sadahiro Azuma 2 , Shiro Ikegami 4¤a , Reiko Kuwahara 4 , Rika Suzuki-Migishima 4 , Yohko Nakahara 4 , Itsuko Nihonmatsu 4 , Kaoru Inokuchi 4¤b , Yuko Katoh-Fukui 4¤c , Minesuke Yokoyama 4¤d , Masami Takahashi 2 * 1 Department of Environmental Science and Technology, Faculty of Engineering, Shinshu University, Nagano-shi, Nagano, Japan, 2 Kitasato University School of Medicine, Sagamihara-shi, Kanagawa, Japan, 3 Department of Psychiatry, International University of Health and Welfare Atami Hospital, Atami-shi, Shizuoka, Japan, 4 Mitsubishi Kagaku Institute of Life Sciences, Machida-shi, Tokyo, Japan Abstract Synaptosomal-associated protein of 25 kDa (SNAP-25) is a presynaptic protein essential for neurotransmitter release. Previously, we demonstrate that protein kinase C (PKC) phosphorylates Ser 187 of SNAP-25, and enhances neurotransmitter release by recruiting secretory vesicles near to the plasma membrane. As PKC is abundant in the brain and SNAP-25 is essential for synaptic transmission, SNAP-25 phosphorylation is likely to play a crucial role in the central nervous system. We therefore generated a mutant mouse, substituting Ser 187 of SNAP-25 with Ala using ‘‘knock-in’’ technology. The most striking effect of the mutation was observed in their behavior. The homozygous mutant mice froze readily in response to environmental change, and showed strong anxiety-related behavior in general activity and light and dark preference tests. In addition, the mutant mice sometimes exhibited spontaneously occurring convulsive seizures. Microdialysis measurements revealed that serotonin and dopamine release were markedly reduced in amygdala. These results clearly indicate that PKC-dependent SNAP-25 phosphorylation plays a critical role in the regulation of emotional behavior as well as the suppression of epileptic seizures, and the lack of enhancement of monoamine release is one of the possible mechanisms underlying these defects. Citation: Kataoka M, Yamamori S, Suzuki E, Watanabe S, Sato T, et al. (2011) A Single Amino Acid Mutation in SNAP-25 Induces Anxiety-Related Behavior in Mouse. PLoS ONE 6(9): e25158. doi:10.1371/journal.pone.0025158 Editor: Thomas Claudepierre, Faculty of Medicine University of Leipzig, Germany Received April 13, 2011; Accepted August 27, 2011; Published September 20, 2011 Copyright: ß 2011 Kataoka 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 work was supported by Grants-in-Aid for Scientific Research on Priority Areas (B) (#18300127, 21300141) from the Japan Society for the Promotion of Science (JSPS), Grant-in-Aid 15082206 for Scientific Research on Priority Areas on ‘‘Elucidation of Glia–Neuron Network-Mediated Information Processing Systems’’ from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and a Grant-in-Aid for Core Research for Evolutional Science and Technology (CREST) from the Japan Science and Technology Agency (JST). 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: masami@med.kitasato-u.ac.jp . These authors contributed equally to this work. ¤a Current address: Department of Psychology, Saitama Institute of Technology, Fukaya-shi, Saitama, Japan ¤b Current address: Department of Biochemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama-shi, Toyama, Japan ¤c Current address: National Center for Geriatrics and Gerontology, National Institute for Longevity Science, Morioka-machi, Obu, Aichi, Japan ¤d Current address: Animal Resources Branch, Center for Bioresource-based Researches, Brain Research Institute, Niigata University, Niigata-shi, Niigata, Japan Introduction Synaptic transmission requires neurotransmitter release from presynaptic nerve terminals. Three SNARE proteins, VAMP-2/ synaptobrevin 2 in the synaptic vesicle membrane, and SNAP-25 and syntaxin 1 in synaptic plasma membrane, play crucial roles in the exocytotic release of neurotransmitters [1–4]. Neurotransmit- ter release is regulated both positively and negatively by various kinds of protein kinases [5–7], and these regulations are some of the important mechanisms of synaptic plasticity underlying learning and memory. In many neuronal preparations, neurotransmitter release is enhanced by the activation of protein kinase C (PKC) [8,9]. Previously, we showed that PKC activation induced enhancement of Ca 2+ -dependent release of dopamine (DA) and acetylcholine (ACh) from PC12 cells, and Ser 187 was specifically phosphorylated in these conditions [10,11]. We also showed that the recruitment of secretory vesicles containing DA and ACh was enhanced by the activation of PKC [12]. In adrenal chromaffin cells and insulin secreting cells, PKC activation enhanced exocytotic release of these hormones by increasing the size of the readily releasable vesicle pool and the highly Ca 2+ -sensitive vesicle pool (HCSP), and phosphorylation of SNAP-25 at Ser 187 was essential for these effects [13–16]. Immunoblotting analysis using phosphorylation- specific antibodies revealed that the phosphorylation of SNAP-25 at Ser 187 also occurred in brain [17–21] and interestingly the phosphorylation of SNAP-25 was dramatically changed in epilepsy [20,21]. However, the physiological roles of phosphorylation at this site are still obscure [18,22]. To address the issue, we generated a knock-in mouse with a single amino acid substitution of Ala at Ser 187 . We found that the mutant mouse displayed a variety of interesting behavioral phenotypes consistent with the conclusion that the phosphorylation of SNAP-25 plays an important role in synaptic function after birth. PLoS ONE | www.plosone.org 1 September 2011 | Volume 6 | Issue 9 | e25158