Proposed Role for Neuronal Serotonin N-acetyltransferase

描述：(申请人提供)合成N-乙酰5-羟色胺(NAS) 通过5-羟色胺N-乙酰转移酶(AANAT)的作用从5-羟色胺产生。虽然 NAS在海马和小脑的定位/合成的证据是 几年前提供的，人们仍然相信在哺乳动物中，AANAT是 几乎只在眼膜腺和视网膜中表达，而且它的 神经元的定位仅限于低等生物，如水果。 飞。我们最近报道了AANAT mRNA在大鼠海马区的表达 抗抑郁剂氟西汀(给药)上调了它的表达 慢性)；其他人发现NAS在 啮齿动物行为绝望测试。此外，我们a)确定了这些神经元在 表达AANAT mRNA的大鼠中枢神经系统，包括小脑颗粒神经元 (CON)，b)建立了AANAT mRNA正常表达的原代CON培养 异丙肾上腺素的表达和上调，c)证明了Con 用~3H标记的5-羟色胺处理的培养物合成NAS，d)发现在 这些神经元NAS可能是一氧化氮(NO)的功能性抑制物。 综合。我们假设NAS可以通过 抑制一氧化氮合酶(NOS)活性和/或表达(可能是通过 对一氧化氮合酶辅因子四氢生物蝶呤合成的作用)，以及 AANAT的神经元表达可能是抗抑郁药物作用的靶点 治疗。这些假设将在以下6个目标中得到检验：(1) 大鼠原代CON和BV-2小胶质细胞培养的特性 参与NAS触发的一氧化氮合酶抑制的通路，包括合成 四氢生物蝶呤，BH4；(2)在体外表征AANAT在 将5-羟色胺代谢成NAS并在BH4和/或NOS的合成中使用 AANAT突变小鼠的培养(表达酶失活的AANAT)和 正常小鼠；(3)AANAT突变小鼠和正常小鼠的基础和 刺激BH4和NO的合成；(4)大鼠CON培养的特性 调节AANAT表达的神经递质系统；(5) 在大鼠身上研究氟西汀以外的抗抑郁药物是否会增加 AANAT mRNA的脑含量，以及是否所有表达AANAT和AANAT的脑区 松果体也受到同样的影响；(6)表征 AANAT突变小鼠对抗抑郁剂的反应与正常小鼠不同 强迫游泳的典范。要使用的技术包括：定量逆转 转录/聚合酶链式反应和原位逆转录聚合酶链式反应检测AANAT 大鼠强迫游泳、一氧化氮合酶活性、亚硝酸盐和BH4含量的测定 测试。我们期望结果能够阐明AANAT/NAS在神经元中的作用 功能，从长远来看，在抑郁症的病理生物学中。

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.