FINAL REPORT For Japan-Korea Joint Research Project

Transcription

1 (Form4-2) FINAL REPORT For Japan-Korea Joint Research Project AREA 1. Mathematics & Physics 2. Chemistry & Material Science 3. Biology 4. Informatics & Mechatronics 5. Geo-Science & Space Science 6. Medical Science 7. Humanities & Social 1. Research Title: The role of specific genes in neurotoxicity of abuse drugs and development of therapeutic Strategies. 2. Term of Research: From July.1st.2010 to June.30th.2012 ( 24 months ) 3. Total Budget a. Financial Support by JSPS: Total amount: 2,400 thousand yen 1 st Year 800 thousand yen 2 nd Year 1,200 thousand yen 3 rd Year 400 thousand yen b. Other Financial Support : Total amount: 0 thousand yen 4. Project Organization a. Japanese Principal Researcher Name Toshitaka Nabeshima Institution / Department Position Meijo University / Graduate School of Pharmaceutical b. Korean Principal Researcher Name Hyoung-Chun Kim Institution / Department Position / College of Pharmacy, 1

2 c. List of Japanese-side Participants (Except for Principal Researcher) Name Institution/Department Position Kiyofumi Yamada Department of Neuropsychopharmacology and Yukihiro Noda Hospital Pharmacy, Meijo University, Division of Clinical and Neuropsychopharmacology, Faculty of Pharmacy Masayuki Hiramatsu Meijo University, Graduate School of Associate Taku Nagai Environmental and Human Associate Department of Neuropsychopharmacology and Takayoshi Mamiya Akihiro Mouri Kazuya Toriumi Daisuke Ibi Takenao Koseki Shinnosuke Yamada Yun Juesuk Tsuyoshi Nakai Yuki Aoyama Yurie Matsumoto Hiroyuki Watanabe Hospital Pharmacy, Assistant Assistant Researcher Researcher Graduate Student Yu Ando Lingling Lu Ping Lu Rui Wang d. List of Korean-side Participants (Except for Principal Researcher) Name Institution/Department Position Keewon Kim Eun-Joo Shin Hwa-Jung Kim Choon-Gon Jang Chae-Ha Yang Ki-Wan Oh Myung-Bok Wie Bae-Dong Jung Ji Hoon Jeong Yoon Hee Chung Sung-Kwon Ko Jae-Hyung Bach Zhengyi Li Chonbuk National University Ewha Woman s University Sungkyunkwan University Daegu Haany University Chungbuk National University Chung-Ang University Chung-Ang University Semyung University Assistant Associate Associate Assistant Associate 2

3 5. Number of Exchanges during the Final Fiscal Year* a. from Japan to Korea *Japanese fiscal year begins April 1. Name Home Institution Duration Host Institution Takayoshi Mamiya Meijo University April 23- April 28 Toshitaka Nabeshima Meijo University June 25 June 30 Taku Nagai Nagoya University June 25 June 30 Total: 3 persons Total: 18 man-days Numbers of Exchanges during the Past Fiscal Years FY2010: Total 4 persons FY2011: Total 9 persons None b. from Korea to Japan Name Home Institution Duration Host Institution Total: 0 persons Total: 0 man-days Numbers of Exchanges during the Past Fiscal Years FY2010: Total 5 persons FY2011: Total 0 persons 3

4 6. Objective of Research Abuse drugs such as phencyclidine (PCP) and methamphetamine (METH) are well-known to induce psychological dependence and neurotoxicity. It is one of the most serious social problems that young drug abusers have been increasing in not only Japan and Korea, but also all over the world. Because it is unclear that the mechanism how those drugs lead them to drug dependence in detail, there are no complete therapies and drugs to the patients. Therefore, in this project, we are eager to clarify the common neuronal mechanism of abuse drugs based on specific genes, and to develop new therapy and medicines. Previously, the Japanese group has developed the animal models of PCP and METH dependence, and identified shati as a novel molecule which is increased dramatically by METH treatment in the nucleus accumbens (NAc) in those models and inhibits METH-induced behavioral alterations. Recently, it has been reported that shati has aspartate N-acetyl transferase activity, an enzyme catalyzing aspartate and acetyl-coa to N-acetyl aspartate (NAA). Therefore, first of all, the Japan group clarified the relationship shati and NAA and whether NAA attenuates the behavioral abnormality in the animal model. Furthermore, the Japan group investigated other novel functions and localization of shati, analyzes by molecular biological and neurochemical methods. Glutathione pathways have reported to suppress the development of toxicity by abuse drugs such as METH and PCP. The Korean group determined serial changes in the parameters of glutathione (GSH) pathway after repeated administration of abuse drugs using GPx1, an antioxidant enzyme with GSH, knockout (KO) mice. Additionally, to examine the potency of compounds modulating the GSH pathway as a preventive and therapeutic agent of drug abuse, the Korea group evaluated whether the administration of a precursor of glutathione, N-acetyl-L-cycteine (NAC), could suppress the neurochemical changes and the behavioral impairment induced by PCP. Furthermore, the Korean group focused on protein kinase C (PKC) as a novel key molecule related to drug dependence, and uncovered its role in METH-induced neurotoxicity. 4

5 7. Methodology [Behavioral Pharmacology] Shati or GPx1 KO and their wild-type (WT; C57BL/6J strain background) mice were housed in plastic cages and were kept in a regulated environment (25 ± 1 C, 50 ± 5 % humidity), with a 12 hours light/dark cycle (lights on at 8:00 AM, off at 8:00 PM). Food and tap water were available ad libitum. To evaluate dependence and sensitization induced by abuse drugs, we used locomotor activity test and conditioned place preference test. - Locomotor activity test Locomotor activity was measured using an infrared detector (Scanet SV40, Melquest, Japan) in a plastic box (W45x L25x H40 cm). After habituation, locomotor activity immediately after drug administration was measured for a several hours. - Conditioned place preference test The conditioned place preference procedure is classically used to test the addictive liability of abuse drugs in research. The apparatus used for the place conditioning task consisted of two compartments: one transparent and one black Plexiglas boxes (both W15x L15 x H15 cm). To enable mice to distinguish easily the two compartments, the floors of the transparent and black boxes were covered with white plastic mesh and black frosting Plexiglas, respectively. Each box could be divided by a sliding door. In a preconditioning session, animals are allowed to freely explore the apparatus. During conditioning, a drug is administered to the subjects, which are then confined in one assigned compartment. Finally, in the postconditioning test, the subjects access freely to the whole apparatus. The amount of time spent in the conditioned compartment is recorded by Scanet SV40, Melquest). When the drug has marked rewarding properties, the animal will spend more time in the compartment of which was paired to the drug. [Molecular Biology and Biochemistry] - GST-pull down assay and MASS spectrometry To clarify a novel function of shati, we identified shati-binding proteins by GST-pull down. GST-His and GST-shati-His proteins were expressed and purified from E. Coli. They were independently incubated with the lysate of mouse brain, and 5

6 the proteins that bound to GST-His or GST-shati-His were separated by SDS-PAGE. Then, protein bands were excised from the gel, and the proteins were identified by MASS spectrometry after digestion by trypsin. - HPLC The quantification of N-acetyl aspartate (NAA) in brain was carried out by HPLC after pre-column derivatization with 4-N,N-dimethylaminosulfonyl-7-N- (2-aminoethyl)amino-2,1,3-benzoxadiazole (DBD-ED). [Neurological and Cell Biology] - Cell culture COS7 cells were cultured in a humidified atmosphere of 95% air and 5% CO 2 and collected 48 hours after transfection with Fugene 6 regent (Roche). - Primary culture Primary cortical neuronal cultures were prepared from mouse fetal brain on E15.5. Briefly, cortices were dissected out from fetal brain and then mechanically dissociated by gently pipetting. The cells were plated onto Ø10 cm poly-l-lysine-coated dishes (Asahi Techno Glass, Japan) in Neurobasal medium supplemented with 2% B-27 supplement (Life Technologies) and 0.5 mm Glutamax (Life Technologies) at a density of 0.5 x 10 4 cells/cm 2. The cultures were then maintained at 37 ºC in a humidified atmosphere of 95% air and 5% CO 2 and were used after 2 and 14 days in vitro (DIV). The immunostaining was performed with anti-map2 antibody (1:1000; Millipore). - Golgi staining Mice on 8 weeks were stained using FD Rapid GolgiStain kit (FD Neuro Technologies) along accompanying protocol. Images were acquired with BIOREVO BZ-9000 (Keyence, Japan), and then the morphology of staining pyramidal neuron in layer III in the PFC was analyzed by Neuroleucida (MBF Bioscience, Japan). A length of dendrite and axon and a soma area were traced, and the exact length of the dendritic segment was calculated. A Scholl analysis of ring intersections was used to estimate dendritic length, and for cortical cells, dendritic length was also estimated using the branch order method. 6