Proposed Role for Neuronal Serotonin N-acetyltransferase

描述：（由申请人提供）合成N-乙酰丙酮（NAS） 通过血清素N-乙酰转移酶（AANAT）的作用从血清素中分离。虽然 海马和小脑定位/合成NAS的证据是 几年前提供的，仍然认为在哺乳动物中，AANAT是 几乎只在前列腺和视网膜中表达， 神经元定位仅限于低等生物，如水果 飞翔紧紧我们最近报道AANAT mRNA在大鼠海马中表达， 并且它被抗抑郁药氟西汀（给药）上调 慢性）;其他人发现，NAS具有抗抑郁样活性， 啮齿动物行为绝望测试此外，我们a）在 表达AANAT mRNA大鼠CNS，包括小脑颗粒神经元 (CON)，B）建立了原代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3 H标记的5-羟色胺处理的培养物合成NAS，和d）发现， 这些神经元NAS可能作为一氧化氮（NO）的功能性抑制剂， 合成.我们假设NAS可以通过影响神经元的功能， 抑制NO合酶（NOS）活性和/或表达（可能通过 对NOS辅因子四氢生物蝶呤BH 4的合成的作用）， AANAT的神经元表达可能是抗抑郁药作用的靶点 治疗。将在以下6个AIM中检验这些假设：（1） 在原代大鼠CON培养物和BV-2小胶质细胞培养物中， 参与NOS的NAS触发抑制的途径，包括合成 四氢生物蝶呤，BH 4;（2）在体外表征AANAT的作用， 将5-羟色胺代谢成NAS和在BH 4和/或NOS的合成中使用 来自AANAT突变小鼠（表达酶失活的AANAT）的培养物和 （3）在AANAT突变和正常小鼠中表征基础和 刺激BH 4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） 在大鼠中研究除氟西汀以外的抗抑郁药是否增加 AANAT mRNA的脑含量，以及是否所有表达AANAT的脑区域和 松果体同样受到影响;（6）表征是否 AANAT突变小鼠对抗抑郁药的反应与正常小鼠不同， 强迫游泳的模式拟采用的技术包括： AANAT的RT-PCR和原位RT-PCR mRNA; NOS活性、亚硝酸盐和BH_4含量测定;以及强迫游泳 test.我们希望这些结果能够阐明AANAT/NAS在神经元凋亡中的作用。 从长远来看，在抑郁症的病理生物学中发挥作用。

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.