Microsomal epoxide hydrolase gene is a novel endogenous protectant against beta amyloid (1-42)-induced cognitive impairments in mice Ngo Thi Ngoc Yen Department of Pharmacy Graduate School, Kangwon National University Abstract Microsomal epoxide hydrolase (mEH) is one of critical biotransformation enzymes in xenobiotic metabolism and detoxification. In the early study, it was suggested that mEH may play a modulatory role in pathogenesis of neurodegeneration in response to environmental stress. To extend this understanding, the role of mEH in the β amyloid (βA)-induced memory impairment was examined by using mEH (-/-) mice and wild-type mice (WT). The cognitive performance was assessed using Morris water maze, passive avoidance, Y maze, novel object recognition test and water finding test after intracerebroventricular (i.c.v) injection with βA (1-42). The result showed that functional deficits of learning and memory in mEH (-/-) groups were significant impaired compared to the WT. Decreases in the acetylcholine (ACh) and choline acetyltransferase (ChAT) activity and its expresstion, while increases in the acetylcholinesterase activity and its expression were observed in the hippocampus of mEH (-/-) mice. Consistently, effects of cyclohexene-oxide (CHO), a mEH inhibitor were comparable to mEH (-/-) case. In addition, ChAT expression was observed lower and AChE expression was higher in the APPswe/PS1 double transgenic mice. Unexpectedly, a significant increase in mEH expression in the APPswe/PS1 double transgenic mice was observed in the hippocampus and entorhinal cortex reflecting that compensative induction of mEH to modulate of APPswe/PS1 gene. Combined, these data suggests that mEH plays a neuroprotective role against cognitive dysfunction induced by βA. CONTENTS I. Introduction 1 II. Material and methods 4 III. Results . 13 IV. Discussion 31 V. References . 36 List of figures Fig. 1. Experimental schedule for measuring Aβ (1-42)-induced cognitive impairment in the mEH (+/+)-, CHO-treated mEH (+/+)-, and mEH (-/-)-mice. . 18 Fig. 2. Effects of mEH gene deficiency and CHO (100mg/kg, i.p.) on the water maze performance [hidden platform performance (A), probe performance (B), and working memory performance (C)] after Aβ (1-42) infusion. . 20 Fig. 3. Effects of mEH gene deficiency and CHO (100mg/kg, i.p.) on the Y-maze performance (A) and novel object recognition performance (B after Aβ (1-42) infusion. . 21 Fig. 4. Effects of mEH gene deficiency and CHO (100mg/kg, i.p.) on the water finding performance (A) and passive avoidance performance (B) after Aβ (1-42) infusion. 22 Fig. 5. Effects of mEH gene deficiency and CHO (100mg/kg, i.p.) on the ACh level (A), activities of AChE (B) and ChAT (C) in the hippocampus of mice after Aβ (1-42) infusion 23 Fig. 6. Effects of mEH gene deficiency on the gene expressions of AchE (A) and ChAT (B) in the hippocampus of mice after Aβ (1-42) infusion 24 Fig. 7. AchE expression in the hippocampus of mEH (+/+)-, and mEH (-/-)-mice 25 Fig. 8. ChAT expression in the hippocampus of mEH (+/+)-, and mEH (-/-)-mice 26 Fig. 9. AchE expression in the hippocampus of APPswe/PS1dE9 wild- type and APPswe/PS1dE9 double transgenic mice . 27 Fig. 10. ChAT expression in the hippocampus of APPswe/PS1dE9 wild- type and APPswe/PS1dE9 double transgenic mice . 28 Fig. 11. mEH expression in the hippocampus of APPswe/PS1dE9 wild- type and APPswe/PS1dE9 double transgenic mice 29 Fig. 12. Flow chart describing current hypothesis on the roles of mEH gene in the Aβ (1-42)-induced memory dysfunction . 301 I. Introduction Alzheimer's disease (AD) results from neurodegeneration characterized by the deposition of senile plaques, development of neurofibrillary tangles, inflammation, and neuronal loss. The senile places are composed of amyloid β- peptide (Aβ), a 40-42 amino acid peptide fragment of the β-amyloid precursor protein that plays an important role in the development of AD. It has been demonstrated that a continuous intracerebroventricular (i.c.v) infusion of Aβ (1-42) into the cerebral ventricle in rats results in learning and memory deficit (Estrin et al., 1987a; Nitta et al., 1994). We have demonstrated that a single i.c.v. injection of Aβ (1-42) causes the memory deficits in mice (Crystal et al., 1988; Jhoo et al., 2004; Estrin et al., 1990). It has been demonstrated that cholinergic system may play a crucial role in modulating cognitive performance and learning memory processes (Winkler et al., 1995) and deterioration of acetylcholine (ACh) function contribute to cognitive decline, which might be associated with AD (Mesulam MM., 1996). Moreover, in addition to significant neuronal cell loss within this brain region, evidence implicating the basal forebrain cholinergic system in AD neuropathology comes from numerous studies demonstrating decreases in choline acetyltransferase (ChAT) activity (Bowen et al., 1976; Davies and Maloney, 1976; Perry et al., 1977; Whitehouse et al., 1982), high affinity choline uptake (Rylett et al., 1983), acetylcholine (ACh) release (Nilsson et al., 1986), and both nicotinic and muscarinic ACh receptor binding (Araujo et al., 1988) in post-mortem brain tissue of AD patients compared to non-pathological control brains. These evidences 2 implicate basal cholinergic system involvement in AD pathogenesis and its accompanying cognitive deficits. Microsomal epoxide hydrolase (mEH), a member of epoxide hydrolase (EH; EC3.3.2.3) family, is one of several xenobiotic biotransformation enzymes. mEH catalyzes the trans-addition of water to a broad range of epoxide subtracts (Fretland and Omiecinski, 2000). mEH appears to have universal expression in all tissues studies to date include the brain. mEH is thought to play a pivotal role in protection against the toxicity of reactive epoxide intermediates, because metabolism of epoxides by this enzyme results in the production of less reactive and less toxic dihyrodiol intermediates of drug such as phenytoin and carbamazepine (Gaedigk et al., 1994 ). Futhermore, mEH is involved in metabolism of compound-containing epoxide induced cognitive impairment (Brashear et al., 1996; Estrin et al., 1987a; Fennell and Brown, 2001). In contrast to this protective effect, it was suggested that mEH is required for the metabolic activation of the potent carcinogen 7, 12- dimethylbenz[a]anthracene (DMBA), a widely studied experimental prototype for the polycylic aromatic hydrocarbon class of chemical carcinogens (Miyata et al., 1999). We hypothesized that the induction of mEH in the striatal complex after drug dependence is a compensative/protective response to low dose of methamphetamine (Shin et al., 2009). However, mEH is involved in the MPTP- induced doparminergic toxicology (Liu et al., 2008). Importantly, we reported an increase in mEH expression in the hippocampus and the entorhinal and transentorhinal cortex of AD cases, where severe pathology is usually found. mEH colocalized with astrocytes but did not colocalize with amyloid plaques (Liu et al., 2006). As a result, mEH remains to be charaterised whether mEH has an important role in neuroprotection or neurodegeneration in response to environmental stress. 3 To achieve better understandings, we examined the role of mEH using mEH (-/-) mice with mEH inhibitor-cyclohexene oxide (CHO) in the presence of Aβ (1-42) exposure. In addition, we examined mEH expression in the APPswe/PS1dE9 double transgenic mice. It was assessed Aβ (1-42)-induced behavioral changes, ACh level, AChE and ChAT activity and their expressions in the mEH (+/+)-, and mEH (-/-)-mice. This study suggested that mEH gene plays an essential role to maintain cognitive function in the presence of Aβ (1-42)-induced cognitive impairment. 4 II. Materials and methods 2.1. Animals and Drug treatments All mice were treated in strict accordance with the NIH Guide for the Humane Care and Use of Laboratory Animals. Male six month-old mEH (-/-)-mice and male ten month-old APP/PS2dE9 double transgenic mice [B6C3-Tg (APPswe, PSEN1De9) 85Dbo/J] were maintained on a 12:12 h light: dark cycle and fed ad libitum. Also, they were adapted to these conditions for 2 weeks before the experiment. Aβ (42-1) and Aβ (1-42) were dissolved in 35% acetonitrile containing 0.1% trifluoroacetic acid. The Aβ (42-1) or Aβ (1-42) administration [400 pmol, intracerebroventricular injection (i.c.v.)] was performed according to the procedure established by Laursen and Belkap (Laursen and Belknap, 1986). Briefly, each mouse was injected consciously at bregma with a 10 àl Hamilton microsyringe fitted with a 26-gauge needle that was inserted to a depth of 2.4 mm. The injection volume was 1.8 àl. The injection placement or needle track was visible and was verified at the time of dissection. CHO (100mg.kg, i.p) was dissolve in saline and administered to Aβ treated mice for 11 consecutive days. The experimental schedule is shown in Fig. 1. CHO or saline administration began 1 h before Aβ i.c.v injection and behavioral test. The behavioral study began on day 3 after Aβ i.c.v. infusion, and carried out sequentially. All mice were sacrificed immediately after behavioral tests. For histological analysis, animals were anesthetized with 60% urethane and perfuse 5 transcardially with 200 ml of 50 mM phosphate buffered saline (PBS), followed by 50 ml of paraformaldehyde in PBS. The brains were fixed at 4 0 C for 24 h in the same fixative and then cryoprotected in 30% sucrose. The brains were sectioned on a horizontal sliding microstome into 35 àm transverse free-floading sections. For western blot analysis and biochemical assay, the brains were quickly removed, the hippocampus tissues were dissected out and samples were instantly frozen in liquid nitrogen and kept at -79 0 C until needed for analysis. 2.2. Morris water test The apparatus was circular water, 97 cm in diameter and 60 cm height. During testing, the tank was filled with water (23 ± 2 °C) that was clouded with powered milk. A transparent platform was set inside the tank and its top was submerged 2 cm below the water surface in the center of one among the four quadrants of the maze. The tank was located in a large room with many extramaze cues that were constant throughout the study (Morris, 1984). The movements of the animal in the tank were monitored with a video tracking system (EthoVision, Noldus, The Netherlands). 2.2.1. Reference memory test For each training trial, the mouse was put into the pool at one of the five positions, the sequence of the positions being selected randomly. The platform was located a constant position throughout the test period in the middle of one quadrant, equidistant from the center and edge of the pool. In each training session, the latency to escape on to the hidden platform was recorded. If the mouse found the platform, it was allowed to remain there for 10 s and was then returned to its home cage. If the mouse was unable to find the platform within 60 s, the training was 6 terminated and a maximum score of 60 s was assigned. Training was conducted for four consecutive days, four times a day, from day 3 to 6 after the Aβ i.c.v. injection. 2.2.2. Probe test On day 7 after Aβ i.c.v. injection, a double probe trial was conducted. The platform was removed from the pool and each mouse was allowed to swim for 60 s in the maze. The time spent by the animal searching for the missing platform on the target quadrant was recorded. 2.2.3. Working memory (repeated acquisition) test Working memory test was conducted three consecutive days from day 8 to 10 and consisted of five trials per day. The working memory test was procedurally similar to reference memory test except that the platform location was changed daily. The first trial of the day was an informative sample trial in which the mouse was allowed to swim to the platform in its new location. Spatial working memory was regarded as the mean escape latency of the second to fifth trials. 2.3. Y-maze test The Y-maze was carried out on day 3 after Aβ (1-42) injection as described previously (Maurice et al., 1994). Briefly, the maze was made of black painted wood; each arm was 40 cm long, 12 cm high, 3 cm wide at the bottom and 10 cm wide at the top. The arms converged at an equilateral triangular central area that was 4 cm at its longest axis. Each mouse was placed at the end of one arm and allowed to move freely through the maze during an 8 min session. The series of arm entries were recorded visually. Alternation was defined as successive entry into the three arms, on overlapping triplet sets. The alternation percentage was 7 calculated as the ratio of actual alternations to possible alternations (defined as the total number of arm entries minus 2), multiplied by 100. 2.4. Novel object recognition test This task, based on the spontaneous tendency of rodents to explore a novel object more often than a familiar one (Ennaceur and Delacour, 1988) was performed with a slight modification as described previously (Dodart et al., 2002). A plastic chamber (35 cm×35 cm×35 cm) was used in low light condition during the light phase of the light/dark cycle. The general procedure consisted of three different phases: a habituation phase, an acquisition phase, and a retention phase. On the 1st day (habituation phase), mice were individually subjected to a single familiarization session of 10 min, during which they were introduced in the empty arena, in order to become familiar with the apparatus. On the 2nd day (acquisition phase) animals were subjected to a single 10 min session, during which floor-fixed two objects (A and B) were placed in a symmetric position from the center of the arena, 15 cm from each and 8 cm from the nearest wall. The two objects, made of the same wooden material with the similar color and smell, were different in shape but identical in size. Mice were allowed to explore the objects in the open field. A preference index for each mouse was expressed as a ratio of the amount of time spent exploring object A (TA ×100)/(TA + TB), where TA and TB are the time spent on exploring object A and object B, respectively. On the 3rd day (retention phase), mice were allowed to explore the open field in the presence of two objects: the familiar object A and a novel object C in different shape but in similar color and size (A and C). A recognition index, calculated for each mouse, was expressed as the ratio (TC ×100)/(TA + TC), where TA and TC are the time spent during retention phase on object A and object C, respectively. The time spent exploring