Journal of Neuroscience Research 90:1743–1755 (2012) Hepatocyte Growth Factor Overexpression in the Nervous System Enhances Learning and Memory Performance in Mice Takashi Kato,1 Hiroshi Funakoshi,2* Keiichi Kadoyama,3 Satsuki Noma,2,4 Masaaki Kanai,2 Wakana Ohya-Shimada,1,2 Shinya Mizuno,4 Nobutaka Doe,5 Taizo Taniguchi,3 and Toshikazu Nakamura1* 1Kringle Pharma Joint Research Division for Regenerative Drug Discovery, Center for Advanced Science and Innovation, Osaka University, Osaka, Japan 2Center for Advanced Research and Education, Asahikawa Medical University, Asahikawa, Japan 3Department of Pharmaceutical Health Care, Faculty of Pharmaceutical Sciences, Himeji Dokkyo University, Himeji, Japan 4Division of Virology, Department of Microbiology and Immunology, Osaka University Graduate School of Medicine, Osaka, Japan 5Laboratory of Neurogenesis and CNS Repair, Institute for Advanced Medical Sciences, Hyogo College of Medicine, Hyogo, Japan Hepatocyte growth factor (HGF) and its receptor, c- Learning and memory are among the most essential Met, play pivotal roles in the nervous system during activities for intelligent life. A large amount of evidence development and in disease states. However, the suggests that forebrain regions such as the hippocampus physiological roles of HGF in the adult brain are not and cerebral cortex are important for cognition (Sara, well understood. In the present study, to assess its 2000; Frankland and Bontempi, 2005). Compared with role in learning and memory function, we used trans- the accumulating knowledge of the mechanisms underly- genic mice that overexpress HGF in a neuron-specific ing learning and memory impairments under pathophys- manner (HGF-Tg) to deliver HGF into the brain without iological conditions (i.e., neuronal and vascular degener- injury. HGF-Tg mice displayed increased alternation ation/dysfunction in diseases including Alzheimer’s dis- rates in the Y-maze test compared with age-matched ease), the mechanisms regulating physiological learning wild-type (WT) controls. In the Morris water maze and memory performance remain a mystery at the mo- (MWM) test, HGF-Tg mice took less time to find the lecular level. Only limited numbers of molecules have platform on the first day, whereas the latency to been characterized, including CaM kinase II, NMDA re- escape to the hidden platform was decreased over ceptor subunit 2B (NR2B), CREB, and brain-derived training days compared with WT mice. A transfer test revealed that the incidence of arrival at the exact location of the platform was higher for HGF-Tg mice Additional Supporting Information may be found in the online version compared with WT mice. These results demonstrate of this article. that overexpression of HGF leads to an enhancement Contract grant sponsor: Japanese Ministry of Health Labor and Welfare of both short- and long-term memory. Western blot (to H.F.); Contract grant sponsor: Ministry of Education, Science, Sports, analyses revealed that the levels of N-methyl-D- and Culture of Japan (to H.F., T.N.); Contract grant sponsor: Global aspartate (NMDA) receptor subunits NR2A and NR2B, Centers of Excellence (COE) program of the Ministry of Education, Sci- but not NR1, were increased in the hippocampus of ence, and Culture of Japan (to T.N.). HGF-Tg mice compared with WT controls, suggesting W. Ohya-Shimada’s current address is Center for Advanced Research that an upregulation of NR2A and NR2B could repre- and Education, Asahikawa Medical University, Asahikawa, Japan sent one mechanism by which HGF enhances learn- *Correspondence to: Dr. Hiroshi Funakoshi, Center for Advanced ing and memory performance. These results demon- Research and Education, Asahikawa Medical University, Asahikawa 078- strate that modulation of learning and memory per- 8510, Japan. E-mail: [email protected] and Dr. Toshikazu formance is an important physiological function of Nakamura, Kringle Pharma Joint Research Division for Regenerative HGF that contributes to normal CNS plasticity, and Drug Discovery, Center for Advanced Science and Innovation, Osaka we propose HGF as a novel regulator of higher brain University, Osaka Japan, E-mail: [email protected] functions. VC 2012 Wiley Periodicals, Inc. Received 19 September 2011; Revised 1 March 2012; Accepted 13 March 2012 Key words: hepatocyte growth factor (HGF); c-Met; Published online 26 April 2012 in Wiley Online Library learning and memory; NMDA receptor; neurotrophin (wileyonlinelibrary.com). DOI: 10.1002/jnr.23065 ' 2012 Wiley Periodicals, Inc. 1744 Kato et al. neurotrophic factor (BDNF; Silva et al., 1992, 1998; tests, the Y-maze and the Morris water maze (MWM). Tang et al., 1999; Nakajo et al., 2008). Identification of Our results yield new insights into the regulatory mech- additional molecules affecting these molecular neuroa- anisms of adult learning and memory. daptations in the forebrain is crucial for a better under- standing of the physiological mechanisms that regulate MATERIALS AND METHODS learning and memory performance. Animals Hepatocyte growth factor (HGF) was initially iden- tified as a potent mitogen for primary hepatocytes Transgenic mice expressing rat HGF in the nervous sys- (Nakamura et al., 1984, 1989). HGF exhibits multiple tem (HGF-Tg mice) were prepared, bred, and maintained as biological effects, including mitogenic, motogenic, mor- previously described (Sun et al., 2002b). After back-crossing phogenic, and antiapoptotic activities in a wide variety with WT C57BL/6J mice (SLC, Shizuoka, Japan) for more of cells by binding to the c-Met/HGF receptor tyrosine than 10 generations, homozygous HGF-Tg mice were created kinase (c-Met) (Funakoshi and Nakamura, 2003; Naka- by the mating of heterozygotes and were confirmed either by mura and Mizuno, 2010). HGF plays pivotal roles as an dot blot hybridization or a PCR method, as previously organotrophic factor during development and in the described (Sun et al., 2002b; Kadoyama et al., 2007, 2009; protection and regeneration of a variety of organs under Benkhoucha et al., 2010). Further confirmation was accom- pathophysiological conditions (Funakoshi and Nakamura, plished by progeny tests. Age-matched WT C57BL/6J mice 2003; Nakamura and Mizuno, 2010). In the nervous sys- served as controls. HGF-Tg and WT mice were transferred tem, HGF and c-Met are expressed not only in develop- into experimental rooms (for Y-maze and MWM tasks) and ing brains but also in diseased brains and play functional maintained for more than 10 days before behavioral assessment. roles during development and in disease processes The acquisition, care, housing, use, and disposition of the ani- (Maina and Klein, 1999; Funakoshi and Nakamura, mals were in compliance with institutional laws and regulations 2011). However, the in vivo role of HGF in the adult of the Osaka University Graduate School of Medicine and Asa- brain is not well understood. hikawa Medical University. The experimental procedures for Notably, brain regions that express HGF and c-Met the MWM tests were conducted according to the National include forebrain regions such as the hippocampus and cer- Institute of Health guidelines. All efforts were made to mini- ebral cortex. Application of HGF in vitro enhances neurite mize animal discomfort and the number of animals used. extension and branching and maturation of the dendritic spine, induces the phosphorylation of Akt and CREB and Measurement of HGF Protein Levels in the translocation of NMDA receptors, and increases the phos- Hippocampus and Cerebral Cortex phorylation of NR2B and neuronal survival in primary After being deeply anesthetized with an overdose of so- hippocampal and/or cerebral cortical neurons (Honda dium pentobarbital, the hippocampus and cerebral cortex of et al., 1995; Sun et al., 2002a; Ishihara et al., 2005; Akita HGF-Tg heterozygous (1/2), HGF-Tg homozygous (1/1), et al., 2008; Lim and Walikonis, 2008). HGF enhances and age-matched WT mice were excised, quickly frozen, and NMDA currents and synaptic plasticity in the hippocam- stored at 2808C until use. Frozen tissue samples were ho- pus and also enhances synaptic long-term potentiation mogenized and centrifuged, and the supernatants were then (LTP) in the CA1 region of the hippocampus in vitro used to quantify HGF protein levels via enzyme-linked im- (Akimoto et al., 2004). Similarly to other neurotrophic fac- munosorbent assay (ELISA; Tokushu Meneki, Tokyo, Japan), tors (Zafra et al., 1990; Funakoshi et al., 1995), HGF par- as previously described (Sun et al., 2002b). ticipates in activity-dependent signaling, including signal- ing in primary hippocampal neurons (Tyndall and Waliko- Histological and Immunohistochemical Analyses nis, 2007). These results led us to hypothesize that Deeply anesthetized animals were transcardially perfused physiological activation of the HGF-c-Met system might with ice-cold phosphate-buffered saline (PBS; pH 7.4) followed underlie the activity-dependent regulation of physiological by ice-cold 4% paraformaldehyde (PFA) in PBS. The extracted learning and memory performance in the adult brain. brains were fixed in 4% PFA in PBS at 48Covernight.For In the present study, to test the possibility that the analyses of brain morphology and MAP2 immunostaining, the HGF-c-Met system plays a role in learning and memory brains were embedded in paraffin. Serial coronal sections (7 performance, we used transgenic mice overexpressing lm) were prepared, deparaffinized, and stained with Nissl or HGF under the control of the neuron-specific enolase