Proposed Role for Neuronal Serotonin N-acetyltransferase

神经元血清素 N-乙酰转移酶的拟议作用

• 批准号: 6697071
• 负责人: HARI MANEV
• 金额: $ 23.38万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-01-10 至 2006-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6697071
• 关键词: acyltransferase; antidepressants; behavior test; biological signal transduction; cerebellum; enzyme activity; enzyme mechanism; glutamate receptor; granule cell; high performance liquid chromatography; laboratory mouse; laboratory rat; lipopolysaccharides; mental disorder chemotherapy; messenger RNA; neuroendocrine system; neurophysiology; neurotransmitter metabolism; nitric oxide synthase; pineal body; polymerase chain reaction; serotonin; tetrahydrobiopterin; tissue /cell culture

DESCRIPTION: (provided by applicant) N-acetylserotonrn (NAS) is synthesized from serotonin by an action of serotonin N-acetyltransferase (AANAT). Although evidence of hippocampal and cerebellar localization/synthesis of NAS was provided several years ago, it is still believed that in mammals, AANAT is almost exclusively expressed in the pmeal gland and in the retina, and that its neuronal localization is restricted to the lower organisms, such as the fruit fly. We recently reported that AANAT mRNA is expressed in the rat hippocampus and that it is up-regulated by the antidepressant fluoxetine (administered chronically); others found that NAS possesses antidepressant-like activity in a rodent behavioral despair test. Furthermore, we a) identified those neurons in the rat CNS that express AANAT mRNA, including cerebellar granule neurons (CON), b) established primary CON cultures in which AANAT mRNA is normally expressed and is up-regulated by isoproterenol, c) demonstrated that CON cultures treated with 3H-labeled serotonin synthesize NAS, and d) found that in these neurons NAS may act as a functional inhibitor of nitric oxide (NO) synthesis. We hypothesize that NAS can affect neuronal functioning via the inhibition of NO synthase (NOS) activity and/or expression (possibly by an action on the synthesis of the NOS cofactor tetrahydrobiopterin BH4), and that neuronal expression of AANAT could be a target for the action of antidepressant treatments. These hypotheses will be tested in the following 6 AIMs: (1) Characterize in primary rat CON cultures andin BV-2 microglial cultures the pathways involved in NAS-triggered inhibition of NOS, including the synthesis of tetrahydrobiopterin, BH4; (2) Characterize in vitro the role of AANAT in metabolizing serotonin into NAS and in the synthesis of BH4 and/or NOS using cultures from AANAT mutated mice (expressing enzymatically inactive AANAT) and normal mice; (3) Characterize in AANAT-mutated and normal mice the basal and the stimulated BH4 and NO synthesis; (4) Characterize in rat CON cultures the neurotransmitter systems capable of regulating AANAT expression; (5) Investigate in rats whether antidepressants other than fluoxetine increase the brain content of AANAT mRNA, and whether all brain regions expressing AANAT and the pineal glands are equally affected; (6) Characterize whether the AANAT-mutated mice respond to antidepressant differently from normal mice in a model of forced swimming. Techniques to be used include: quantitative reverse transcription/polymerase chain reaction (RT-PCR) and in situ RT-PCR for AANAT mRNA; assays of NOS activity, nitrite and BH4 contents; and the forced swimming test. We expect the results to elucidate the role of AANAT/NAS in neuronal functioning, and in the long term, in the pathobiology of depression.

描述：（由申请人提供）N-乙酰基索龙（NAS）合成 通过5-羟色胺N-乙酰转移酶（AANAT）的作用从5-羟色胺出发。虽然 海马和小脑定位/NAS合成的证据为 提供几年前，仍然相信在哺乳动物中，Aanat是 几乎只在PMEAL腺和视网膜中表达 神经元定位仅限于较低的生物，例如水果 飞。我们最近报道说AANAT mRNA在大鼠海马中表达 并由抗抑郁剂氟西汀上调（给予 长期）;其他人发现NAS在A中具有抗抑郁药样活性 啮齿动物行为绝望测试。此外，我们a）在 表达AANAT mRNA的大鼠CNS，包括小脑颗粒神经元 （con），b）建立的原发性培养物，其中aanat mRNA通常为 表达并被异丙肾上腺素上调，c）证明了con 用3H标记的5-羟色胺合成NAS的培养物，d）发现 这些神经元NAS可以充当一氧化氮的功能抑制剂（NO） 合成。我们假设NAS可以通过 抑制非合酶（NOS）活性和/或表达的抑制（可能是通过 对NOS辅助因子四氢无菌蛋白BH4的合成的作用 AANAT的神经元表达可能是抗抑郁药作用的靶标 治疗。这些假设将在以下6个目标中进行检验：（1） 特征在原发性大鼠con培养物和In In In bv-2小胶质细胞培养物中 NOS抑制NOS的途径，包括合成 Bh4的四氢无菌蛋白酶； （2）在体外表征AANAT在 将5-羟色胺代谢为NAS以及使用BH4和/或NOS合成 来自AANAT突变的小鼠的培养物（表达酶促无活性的AANAT）和 正常小鼠； （3）以Aanat突变和正常小鼠为特征为基础和 刺激的BH4且无合成； （4）在大鼠培养物中表征 能够调节AANAT表达的神经递质系统； （5） 在大鼠中研究氟西汀以外的抗抑郁药是否会增加 AANAT mRNA的大脑含量，以及所有表达AANAT和 松果体同样受到影响； （6）表征是否 AANAT突变的小鼠对抗抑郁药的反应与正常小鼠不同 强迫游泳的模型。要使用的技术包括：定量反向 转录/聚合酶链反应（RT-PCR）和AANAT的原位RT-PCR mrna; NOS活性，亚硝酸盐和BH4含量的测定；和强迫游泳 测试。我们希望结果能阐明AANAT/NAS在神经元中的作用 从长远来看，在抑郁症的病理生物学中发挥作用。