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: 6621076
• 负责人: HARI MANEV
• 金额: $ 27.28万
• 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/6621076
• 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：（申请人提供）N-乙酰血清素（NAS）的合成 通过血清素 N-乙酰转移酶 (AANAT) 的作用从血清素中提取。虽然 NAS 在海马和小脑定位/合成的证据是 几年前，人们仍然相信，在哺乳动物中，AANAT 几乎只在前膜腺和视网膜中表达，并且其 神经元定位仅限于低等生物，例如水果 飞。我们最近报道了 AANAT mRNA 在大鼠海马中表达 并且它被抗抑郁药氟西汀（服用 长期）；其他人发现 NAS 在以下方面具有抗抑郁样活性： 啮齿动物行为绝望测试。此外，我们 a) 识别出这些神经元 表达 AANAT mRNA 的大鼠 CNS，包括小脑颗粒神经元 (CON), b) 建立原代 CON 培养物，其中 AANAT mRNA 通常是 表达并被异丙肾上腺素上调，c) 证明 CON 用 3H 标记的血清素处理的培养物合成 NAS，并且 d) 发现 这些神经元 NAS 可能充当一氧化氮 (NO) 的功能性抑制剂 合成。我们假设 NAS 可以通过以下方式影响神经元功能： 抑制一氧化氮合酶（NOS）活性和/或表达（可能通过 对 NOS 辅因子四氢生物蝶呤 BH4 合成的作用，以及 AANAT 的神经元表达可能是抗抑郁药作用的目标 治疗。这些假设将在以下 6 个 AIM 中进行检验：(1) 在原代大鼠 CON 培养物和 BV-2 小胶质细胞培养物中表征 参与 NAS 触发的 NOS 抑制的途径，包括合成 四氢生物蝶呤，BH4； (2) 体外表征 AANAT 在 使用将血清素代谢为 NAS 并合成 BH4 和/或 NOS AANAT 突变小鼠的培养物（表达酶失活的 AANAT）和 正常小鼠； (3) 表征 AANAT 突变小鼠和正常小鼠的基础和 刺激 BH4 和 NO 合成； (4) 在大鼠 CON 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，以及是否所有大脑区域都表达 AANAT 和 松果体同样受到影响； (6) 表征是否 AANAT 突变小鼠对抗抑郁药物的反应与正常小鼠不同 强迫游泳模型。要使用的技术包括： 定量逆向 用于 AANAT 的转录/聚合酶链式反应 (RT-PCR) 和原位 RT-PCR 信使RNA； NOS活性、亚硝酸盐和BH4含量测定；以及强迫游泳 测试。我们期望结果能够阐明 AANAT/NAS 在神经元中的作用 从长远来看，在抑郁症的病理学中发挥作用。