Spiro[imidazo[1,2-a]pyridine-3,2-indan]-2(3H)-one (ZSET1446/ ST101) Treatment Rescues Olfactory Bulbectomy-Induced Memory Impairment by Activating Ca /Calmodulin Kinase II and Protein Kinase C in Mouse Hippocampus

Olfactory bulbectomy (OBX) in mice elicits impaired memory and cognitive functions. Here, we found that chronic oral administration of spiro[imidazo[1,2-a]pyridine-3,2-indan]-2(3H)one (ZSET1446/ST101) (0.1–1 mg/kg/day), a novel cognitive enhancer, significantly improved memory deficits as assessed by Y-maze and novel object recognition tasks in OBX mice. Immunostaining of cholinergic neurons in the medial septum by using an anti-choline acetyltransferase antibody indicated that chronic ZSET1446 treatment did not rescue cholinergic neurons. However, chronic treatment significantly restored OBXinduced decreases both in calcium/calmodulin-dependent protein kinase II (CaMKII) and protein kinase C (PKC) phosphorylation without improving decreased extracellular signalregulated kinase phosphorylation in the hippocampal CA1 region. Consistent with enhanced CaMKII and PKC phosphorylation, ZSET1446 treatment improved glutamate receptor 1 (Ser-831) phosphorylation in the hippocampal CA1 region. ZSET1446 treatment also significantly rescued impaired long-term potentiation (LTP) in the hippocampal CA1 region of OBX mice. Taken together, the cognition-enhancing effect of ZSET1446 is probably mediated in part by stimulation of CaMKII and PKC activities, which in turn rescue impaired hippocampal LTP in OBX mice. Several recent rodent studies suggest that impairment of the olfactory system leads to impaired memory and cognitive function (Sieck, 1972; Serby et al., 1985; Koss, 1986). For example, impaired performance on learning and memory tasks, such as passive avoidance and spatial reference memory, was observed in olfactory bulbectomized (OBX) mice and rats (Kovács et al., 2001; Hozumi et al., 2003; Attems et al., 2005; Han et al., 2008). In addition, elevated levels of -amyloid peptide are seen in extracts from neocortex and hippocampus in OBX mice (Aleksandrova et al., 2004). It is interesting that significant impairment of the olfactory system is observed in humans in the early stages of Alzheimer’s disease (AD) (Esiri and Wilcock, 1984; Doty, 1991). OBX injury causes retrograde degeneration of cholinergic neurons from the medial septum, leading to decreased cholinergic innervation of the cortex and hippocampus (Moriguchi et al., 2006; Nakajima et al., 2007). Indeed, levels of acetylcholinesterase (AChE) (Nakajima et al., 2007) and choline acetyltransferase (ChAT) (Han et al., 2008), both presynaptic markers of cholinergic neurons, significantly decrease in the cortex and hippocampus of OBX mice. Consistent with these observations, OBX-induced memory deficits are improved by stimulation of the cholinergic system (Yamamoto et al., 1997; Hozumi et al., 2003). We recently reported that a novel neuroprotective vanadium compound rescues cholinergic neuThis work was supported in part by Grants-in-Aid for Scientific Research 19390150 (to K.F.) and 1907187 (to F.H.) from the Ministry of Education, Science, Sports and Culture of Japan and the Smoking Research Foundation (to K.F.). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.108.137471. ABBREVIATIONS: OBX, olfactory bulbectomy; AD, Alzheimer’s disease; AChE, acetylcholinesterase; ChAT, choline acetyl transferase; LTP, long-term potentiation; CaMKII, calcium/calmodulin-dependent protein kinase II; PKC, protein kinase C; ERK, extracellular signal-regulated kinase; NMDA, N-methyl-D-aspartate; ZSET1446, spiro[imidazo[1,2-a]pyridine-3,2-indan]-2(3H)-one; ACh, acetylcholine; PBS, phosphatebuffered saline; fESPS, field excitatory postsynaptic potential; HFS, high-frequency stimulation; GluR1, glutamate receptor 1; MK-801, 5Hdibenzo[a,d]cyclohepten-5,10-imine (dizocilpine maleate); Veh, vehicle. 0022-3565/08/3261-127–134$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 326, No. 1 Copyright © 2008 by The American Society for Pharmacology and Experimental Therapeutics 137471/3348732 JPET 326:127–134, 2008 Printed in U.S.A. 127 at A PE T Jornals on July 8, 2017 jpet.asjournals.org D ow nladed from rons in the medial septum from OBX-induced cholinergic neurodegeneration and restores hippocampal cholinergic activity, thereby improving hippocampus-dependent memory deficits as in seen OBX mice (Han et al., 2008). Glutamatergic neurons in the central nervous system play important roles in memory processes (Staubli et al., 1994; Weisskopf and Nicoll, 1995; Gray et al., 1996). Hippocampal long-term potentiation (LTP) is thought to be a model reflecting synaptic plasticity changes seen in learning and memory (Frey and Morris, 1998; Fukunaga and Miyamoto, 2000). In addition, Ca /calmodulin-dependent protein kinase II (CaMKII) activity is essential for LTP induction as well as maintenance (Fukunaga and Miyamoto, 2000; Lisman et al., 2002). Likewise, protein kinase C (PKC), protein kinase A, and extracellular signal-regulated kinase (ERK) play pivotal roles in hippocampal LTP induction (Sweatt et al., 1998; Collingridge et al., 2004; Xia and Storm, 2005). We recently found that hippocampal LTP was impaired in OBX mice through N-methyl-D-aspartate (NMDA) receptor hypofunction, with a concomitant decrease in CaMKII autophosphorylation and PKC phosphorylation but without changes in PKA activity (Moriguchi et al., 2006). To develop a novel cognitive enhancer that does not inhibit AChE, we synthesized a new azaindolizinone derivative, ZSET1446 (ST101), which improves learning and memory by potentiating nicotine-induced acetylcholine (ACh) release in the hippocampus of amyloid(1-40)-infused rats (Yamaguchi et al., 2006). ZSET1446 treatment also rescues decreases in ChAT activity seen in the medial septum and hippocampus of the same model (Yamaguchi et al., 2006). In addition, MK801 treatment significantly blocked the improving effects of ZSET1446 on methamphetamine-induced memory impairment, suggesting that NMDA receptor activation is required for ZSET1446 effects (Ito et al., 2007). Thus, for conditions such as AD, ZSET1446 is a candidate therapeutic agent to improve cognitive impairment without affecting AChE. Little is known about precise molecular mechanisms in vivo underlying ZSET1446-mediated improvement of learning and memory in the central nervous system. Thus, we asked whether oral ZSET1446 administration protects septohippocampal cholinergic neurons from OBX-induced neurodegeneration in mice. We also investigated whether the drug improves impaired hippocampal LTP as well as decreased CaMKII and PKC activity in OBX mice. Our data suggest that stimulation of CaMKII and PKC activities in the hippocampal CA1 region by ZSET1446 treatment underlies its cognition-enhancing effects in OBX mice. Materials and Methods Animals. Adult male DDY mice weighing 23 to 26 g were obtained from Nippon SLC (Hamamatsu, Japan), and they were housed in polypropylene cages in a temperature(23 1°C) and humiditycontrolled environment maintained on 12-h light/dark schedules (lights on from 8:00 AM to 8:00 PM). Mice were provided food and water ad libitum. Experiments were performed according to the Guide for Care and Use of Laboratory Animals at Tohoku University. Bilateral Olfactory Bulbectomy and Drug Treatment. After an acclimatization period of 1 week, bilateral olfactory bulbectomy was performed. OBX mice were prepared as described by Hozumi et al. (2003). In brief, mice anesthetized with sodium pentobarbital (50 mg/kg i.p.; Dainippon, Osaka, Japan) were placed in a stereotaxic instrument. After exposure of the skull, 1-mm-diameter holes were drilled on either side of the olfactory bulbs. Olfactory bulbs were removed through the hole by gentle aspiration with a suction pump, and care was taken not to damage the frontal cortex. Holes were filled with a hemostatic sponge to avoid further bleeding, and the skin was closed. Sham-operated mice were treated similarly but bulbs were left intact. Drug administration began after a 3-day recovery period. ZSET1446/ST101 was kindly provided by Zenyaku Kogyo Co. Ltd. (Tokyo, Japan). ZSET1446 (0.001–1 mg/kg, dissolved in distilled water or vehicle alone) was administered p.o. daily for 10 consecutive days until mice were analyzed. After sacrifice by decapitation, brains were checked to confirm complete removal of olfactory bulbs and lack of cortical damage to exclude incomplete OBX mice from analysis. Hippocampal CA1 region and the medial septum were dissected out for further analyses. Behavioral Analyses. Spontaneous alternation behavior in a Y-maze task was recorded and evaluated as a spatial memory task. The apparatus consisted of three identical black Plexiglas arms (length width height, 50 16 32 cm). Each mouse was placed at the end of one fixed arm and allowed to move freely through the maze during an 8-min session. The sequence of arm entries was recorded visually, and three consecutive choices were defined as an alternation. The percentage of alternation was calculated as (actual alternations/maximal alternations) 100. In addition, the total number of arms entered during the session was determined. An object recognition task used to evaluate recognition memory (Ennaceur and Aggleton, 1997). This task is based on the tendency of rodents to discriminate a familiar from a new object. Mice were individually habituated to an open-field box (35 25 35 cm) for 2 consecutive days. The experimenter scoring behavior was blinded to the treatment. During the acquisition phases, two objects of the same material were placed in a symmetric position in the center of the chamber for 10 min. One hour after the acquisition phase training, one of the objects was replaced by a novel object, and exploratory behavior was again analyzed for 5 min. After each session, objects were thoroughly cleaned with 75% ethanol to prevent odor recognition. Exploration of an object was defined as rearing on the object or sniffing it at a distance of less than 1 cm, touching it with the nose, or both. Successful recognition of a previously explored object was reflected by preferential exploration of the novel object. Discrimination of spatial novelty was assessed by comparing the difference between time of exploration of the novel (right) and familiar object (left) and the total time spent exploring both objects, which made it possible to adjust for differences in total exploration time. Immunohistochemistry. Mice were anesthetized and transcardially perfusion-fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS). Whole brains were immediately removed and postfixed overnight at 4°C in the same fixative. Then, coronal brain sections at the coordinates of anterior or posterior to bregma (35 m in thickness) were prepared using a microslicer system (Vibratome, St. Louis, MO). Sections were incubated at room temperature with 0.01% Triton X-100 in PBS for 30 min and for another hour in 3% bovine serum albumin in PBS. For immunolabeling, slices were probed with anti-ChAT (goat polyclonal antibody, 1:500; Millipore Bioscience Research Reagents, Temecula, CA) overnight at 4°C. After washing, sections were incubated with biotinylated anti-goat IgG (1:5000) in TNB buffer (0.1 M Tris-HCl, 0.15 M NaCl, 0.5% blocking reagent; pH 7.4; PerkinElmer Life and Analytical Sciences, Boston, MA) for 1 h, followed by streptavidin-horseradish peroxidase (1:5000) labeled for 2 h. Sections were then stained with tetramethylrhodamine tyramide for 10 min using the TSA-Direct kit (PerkinElmer Life and Analytical Sciences). Immunofluorescent images were taken with a confocal laser-scanning microscope (TCS SP; Leica, Wetzlar, Germany). To normalize immunoreactivity with the anti-ChAT antibody, we analyzed cortical slices without changing confocal laser intensity and aperture, and we confirmed that the intensity of immunoreactivity with anti-ChAT antibody was the same between control and bulbectomized cortical slices. Slices were prepared from the medial septum region according to the nomencla128 Han et al. at A PE T Jornals on July 8, 2017 jpet.asjournals.org D ow nladed from ture of stereotaxic mouse brain atlas (Franklin and Paxinos, 1997). From each animal, the number of ChAT-positive neurons was assessed in two 35m sections obtained from anterior 1.0 mm relative to bregma. The amounts of ChAT-positive neurons in identical fields were analyzed with acquisition software QCapture 6.0 (QImaging, Burnaby, BC, Canada) and Adobe Photoshop 6.0 (Adobe Systems, San Jose, CA). Changes in amounts of ChAT-positive neurons were expressed as percentages of amounts seen in sham-operated animals. Electrophysiology. Hippocampal slices were prepared as described previously (Liu et al., 1999). Transverse slices (400 m in thickness) prepared using a Vibratome microslicer DTK-1000 were incubated 2 h in continuously oxygenized (95% O2, 5% CO2) artificial cerebrospinal fluid at room temperature. After a 2-h recovery period, a slice was transferred to an interface recording chamber and perfused at a flow rate of 2 ml/min with artificial cerebrospinal fluid warmed to 34°C. Field excitatory postsynaptic potentials (fEPSPs) were evoked by a 0.05-Hz test stimulus through a bipolar stimulating electrode placed on the Schaffer collateral/commissural pathway and recorded from the stratum radiatum of CA1 using a glass electrode filled with 3 M NaCl. Recording was performed using a singleelectrode amplifier (CEZ-3100; Nihon Kohden, Tokyo, Japan), and the maximal value of the initial fEPSP slope was recorded and averaged every 1 min (three traces) using an A/D converter (PowerLab 200; ADInstruments Pty Ltd., Castle Hill, Australia) and a personal computer. Stimulus intensity was adjusted to evoke a fEPSPs of 1.0-mV amplitude. After a stable baseline was obtained, high-frequency stimulation (HFS) of 100-Hz frequency of 1-s duration was applied twice with a 10-s interval, and test stimuli were continued for the indicated periods. Western Blotting Analysis. Hippocampal CA1 samples were homogenized in 70 l of buffer containing 50 mM Tris-HCl, pH 7.4, 0.5% Triton X-100, 4 mM EGTA, 10 mM EDTA, 1 mM Na3VO4, 40 mM sodium pyrophosphate, 50 mM NaF, 100 nM calyculin A, 50 g/ml leupeptin, 25 g/ml pepstatin A, 50 g/ml trypsin inhibitor, and 1 mM dithiothreitol. Insoluble material was removed by a 10min centrifugation (15,000 rpm). After determining supernatant protein concentration using Bradford’s solution, samples were boiled 3 min in Laemmli’s sample buffer. Samples containing equivalent amounts of protein were subjected to SDS-polyacrylamide gel electrophoresis. Proteins were transferred to an Immobilon polyvinylidene diflouride membrane for 2 h at 70 V. After blocking with TTBS solution (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween 20) containing 2.5% bovine serum albumin for 1 h at room temperature, membranes were incubated overnight at 4°C with anti-phospho-CaMKII (1:5000) (Fukunaga et al., 1988), anti-CaMKII (1:5000) (Fukunaga et al., 1988), anti-phospho-synapsin I (site 3) (1:2000; Millipore Bioscience Research Reagents), anti-synapsin I (1:2000) (Fukunaga et al., 1992), anti-phospho-GluR1 (Ser-831) (1:1000; Millipore, Billerica, MA), anti-GluR1 (1:1000; Millipore Bioscience Research Reagents), anti-phospho-PKC (1:2000; Millipore), antiPKC (1:2000; Millipore), anti-phospho-mitogen-activated protein kinase (ERK 1/2) (1:2000; Sigma-Aldrich, St. Louis, MO), anti-ERK (1:2000; Sigma-Aldrich), and anti-tubulin (1:10,000; Sigma-Aldrich). Bound antibodies were visualized using the enhanced chemiluminescence detection system (GE Healthcare, Chalfont St. Giles, UK) and analyzed semiquantitatively using the ImageJ program (National Institutes of Health, Bethesda, MD). Statistical Analysis. Data were represented as means S.E.M. Statistical significance was performed by one-way analysis of variance, followed by a Dunnett’s test for multigroup comparisons. P 0.05 indicated statistically significant differences.