Thesis for the Degree of Master Microsomal epoxide hydrolase gene is a novel endogenous protectant against beta amyloid (1-42)-induced cognitive impairments in mice by Ngo Thi Ngoc Yen Department of Pharmacy Graduate school Kangwon National University August, 2011 Under the Guidance of Professor Hyoung-Chun Kim Microsomal epoxide hydrolase gene is a novel endogenous protectant against beta amyloid (1-42)-induced cognitive impairments in mice A THESIS Submitted to the Graduate School of Kangwon National University in Partial Fulfillment of the Requirements for the Degree of Master of Pharmacy by Ngo Thi Ngoc Yen Department of Pharmacy August, 2011 Approved by Committee of the Graduate School of Kangwon National University in Partial Fulfillment of the Requirements for the Degree of Master of Pharmacy Ngo Thi Ngoc Yen August, 2011 Thesis Committee; Kim, Hyoung-Chun (Chairman of Committee) Signature Shin, Eun-Joo (Committeeman) Signature Jhoo, Jin-Hyeong (Committeeman) Signature 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 II Material and methods III Results 13 IV Discussion 31 V References 36 List of figures Fig Experimental schedule for measuring Aβ (1-42)-induced cognitive impairment in the mEH (+/+)-, CHO-treated mEH (+/+)-, and mEH (-/-)-mice 18 Fig 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 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 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 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 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 AchE expression in the hippocampus of mEH (+/+)-, and mEH (-/-)-mice 25 Fig ChAT expression in the hippocampus of mEH (+/+)-, and mEH (-/-)-mice 26 Fig 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 wildtype and APPswe/PS1dE9 double transgenic mice 28 Fig 11 mEH expression in the hippocampus of APPswe/PS1dE9 wildtype 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 30 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 βpeptide (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 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, 12dimethylbenz[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 MPTPinduced 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 Cholinergic neurotransmitter has been accorded an important role in supporting learning and memory processes in the hippocampus, and its alteration has been suggested one of main causes of cognitive disorders such as Alzheimer’s disease (AD) (Collerton, 1986) The findings of the investigators using full-length Aβ -peptides showing that chronic infusion of Aβ (1-40) decreased ACh level and ChAT activity in the frontal cortex and hippocampus, and the injection of Aβ (1-42) in the nucleus basalis reduced ChAT in the cortex (Nabeshima and Nitta, 1994; Yamaguchi and Kawashima, 2001; Harkany et al., 1999), and enhanced AChE activity (Shin et al., 2005) In this connection, the findings by (Yamaguchi and Kawashima, 2001) that the number of choline acetyltransferase-immunoreactive cells in the medial septum was decreased by a single i.c.v injection of Aβ (25–35), and injection of amyloid- Aβ (25–35) or Aβ (1–40) to the septum of rats resulted in a marked decrease in basal and potassium-evoked acetylcholine release (Abe et al., 1994) These results altogether suggest that amyloid-β induces the dysfunction of cholinergic neuronal systems In line with previous findings, Aβ (1-42) injection impaired cholinergic transmission in this study And these changes were more pronounced in mEH (-/-)-, and CHO-treated mice than mEH (+/+)-mice It was reported that double transgenic mice show cognitive impairments with aging (Chapman et al., 1999; Holcomb et al., 1998; Perez et al., 2011) and that the Aβ accumulation can be detected in the cortex and hippocampus after at age months and older in the double transgenic mice (Frautschy et al., 1998; Hsiao et al., 1996) Interestingly, we found a significant of mEH overexpression in the hippocampus and cortex of double transgenic mice This finding was in line with our previous data, mEH expression was activated in AD brain and trimethyl-tintreated rat (Liu et al., 2006) 32 We reported that mEH and astrocytes were colocalized in the same cell (Shin et al., 2009; Liu et al., 2008; Liu et al., 2006), and the mEH-positive astrocytes were usually associated with earlier amyloid plaques, but not late-stage neurofibrillary tangles, supporting the notion that mEH activation is not merely a response of astrogliosis Astrocytes are crucial in plaque degradation as evidenced by the ultrastructural three-dimensional reconstruction of human classical plaques in different stages of development (Wegiel et al., 2000) Aβ peptides are preferentially internalized by astrocytes, and astrocytic hypertrophic processes degrade Aβ-containing plaques (Kurt et al., 1999), thus preventing the formation of the deposits of extracellular Aβ (Wright et al., 2010) The precise mechanism by which astrocytes recognize and degrade Aβ is not known, but apolipoprotein E (ApoE), which is almost exclusively expressed in astrocytes, has been proposed to be responsible for this cellular action ApoE is essential for astrocytes to attract chemically, internalize, and degrade Aβ deposits in brain sections in vitro (Koistinaho et al., 2004) Astrocytes also exert protective effects in AD by inhibiting activated microglia Aβ-induced transforming growth factor-β derived from astrocytes can suppress inducible nitric oxide synthase activity in microglia (Vincent et al., 1997) Combined, mEH may relate to protective functions of astrocytes in AD pathology More research is required to clarify the neuroprotective effect of mEH gene Our previous data showed that mEH expression was found around certain amyloid plaques, and this can only be explained by Aβ stimulation Other factors are also responsible for the high mEH level in AD as indicated by the lack of concordant association with different types of amyloid plaques One aetiologic hypothesis regarding AD is the involment of oxidative stress and the inflammatory cascade in the pathogenetic cascade of neurodegeneration (Markesbery and Carney, 33 1999) The neurotoxic action of Aβ was found to rely on H2O2 production (Behl et al., 1994) In addition, the cerebral cortical laminar distribution of interleukin-1 microglia may be the local pathogenic factor that acts in concert with corticocortical projection field patterns to determine the cortical laminar distribution of Aβ plaques in AD It is reasonable to speculate that the mEH activation around some plaques is at least partially derived from the combining stimulation of Aβ and proinflammatory cytokines (Sheng et al., 1998) Addionally, it was reported that patients with AD displayed significantly higher intrathecal levels of tumor necrosis factor-alpha , but not tumor necrosis factor-beta, compared with the control subjects (Tarkowski et al., 2000) The reason for Aβ-mediated mEH activation remains unknown mEH is involved in metabolism of compound-containing epoxide induced cognitive impairment Recent reports suggest that cognitive deficit may be associated with chronic low-level ethylene oxide exposure (Crystal et al., 1988; Estrin et al., 1990; Estrin et al., 1987b) One of the many reported teratogenic effects of prenatal phenytoin exposure involves the disruption of hippocampal development (Vorhees, 1983; Ogura et al., 2002), and the subsequent long-term behavioral deficits associated with hippocampal dependent learning tasks (Weisenburger et al., 1990; Tsutsumi et al., 1998) In addition, It was reported that phenytoin-induced cognitive decline and oxidative stress in rat (Reeta et al., 2009; Mowery et al., 2008) mEH plays a detoxifying role in metabolizing phenytoin in to less toxic intermediates Thus, mEH gene may have a protective effect in neurodegeneration Taken together, our results suggest that mEH gene is an essential cognitive enhancer in response to Aβ neurotoxicity in mice Neuroprotective effects of mEH 34 gene are due to enhancing cholinergic system and relating to protective 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Fluorometric Determination of Trace Hydrogen Peroxide: Applications in Detecting the Activity of Phagocyte NADPH Oxidase and Other Oxidases Analytical Biochemistry 1997;253(2):162168 44 생쥐에서-Amyloid (1-42)에 의해 유도된 인지기억 능 장애에 대한 새로운 내인성 보호제로서 microsomal epoxide hydroxylase 유전자의 역할 Ngo Thi Ngoc Yen 강원대학교 대학원 약학과 Microsomal epoxide hydrolase (mEH)는 외인성 물질을 대사· 해독시키는 주요한 생체내 변화효소 중 하나로서, 환경적 요인에 의한 뇌퇴행성 변화의 발병기전 을 조절하는 역할을 한다고 보고되어 왔다 이번 연구에서는 mEH 유전자 결 핍 생쥐와 야생형 생쥐를 응용하여, β-amyloid (βA)로 유도된 인지기억능 저하 모델에서 mEH의 역할을 규명하고자 하였다 βA의 뇌실내 투여는 Morris water maze, passive avoidance test, Y-maze test, novel object recogtion test, water finding test에서 유의하게 인지기억능의 저하를 유도하였는데, 이러한 인지기 억능의 저하는 야생형 생쥐에 비하여 mEH 유전자 결핍 생쥐에서 현저하였다 또한, 해마 (hippocampus) 조직에서 βA에 의해 유도된 acetylcholine (ACh) 수치 의 감소, choline acetyltransferase (ChAT)의 활성 및 발현의 저하, acetylcholinesterase (AChE)의 활성 및 발현의 증가 역시 야생형 생쥐에 비하여 45 mEH 유전자 결핍 생쥐에서 현저하였다 mEH 억제제인 cyclohexene oxide (CHO)를 투여한 야생형 생쥐 역시 mEH 유전자 결핍 생쥐와 같은 결과를 보였 다 Amyloid precursor protein Swedish mutation/presenilin-1 (APPswe/PS-1) 이중 발현 생쥐에서도 야생형 생쥐에 비하여 해마조직에서 ACh 수치의 감소, ChAT 의 활성 및 발현의 저하, AChE의 활성 및 발현의 증가가 관찰되었으나, 예상과 는 달리 해마와 내후각피질 (entorhinal cortex)에서 mEH의 발현이 유의하게 증 가되어 있었으며, 이는 mEH가 보상적으로 유도되어 APPswe/PS-1 유전자를 조절함을 의미한다 이상의 결과는 βA에 의한 인지기억능 저하에 대한 mEH 의 신경보호 역할을 제시한다 46 ...Under the Guidance of Professor Hyoung-Chun Kim Microsomal epoxide hydrolase gene is a novel endogenous protectant against beta amyloid (1-42)-induced cognitive impairments in mice A THESIS Submitted... laminar distribution of A? ? plaques in AD It is reasonable to speculate that the mEH activation around some plaques is at least partially derived from the combining stimulation of A? ? and proinflammatory... cerebral ventricle induces learning impairment and neuronal and morphological degeneration Japanese journal of pharmacology 1997;73(1):51-57 Nitta, A. ; Itoh, A. ; Hasegawa, T.; Nabeshima, T [beta] -Amyloid