Functional characterization of SSRI resistant genes using C. elegans

描述(申请人提供)：这项建议的目标是通过基因的遗传解剖阐明5-羟色胺信号通路和受选择性5-羟色胺再摄取抑制物(SSRI)调节的功能通路。5-羟色胺能系统的失调与广泛的神经发育和精神障碍有关，SSRIs对这些疾病的许多症状产生了重大影响。SSRIs被认为通过抑制5-羟色胺再摄取转运体(SERT)发挥治疗作用，从而增加5-羟色胺信号转运体，从而导致我们的中心假设，即SSRIs耐药基因的遗传解剖不仅将识别与药物直接结合的基因，而且将识别受药物调控的基因和功能通路，因此，将有助于阐明5-羟色胺信号转导的基础生物学及其在疾病中的作用。利用5-羟色胺缺陷型线虫的可用性，以及在活的线虫中监测已识别细胞中基因表达和突触结构的独特能力，该建议使用线虫的直接正向遗传学方法来识别整个动物中的SSRIs抗性基因。这一建议是基于我们的初步数据，表明SSRI氟西汀(Prozac)靶向5-羟色胺突触后靶向调节乙酰胆碱、GABA和谷氨酸神经传递的活性。在四个特定的目标中，拟议的实验将在全基因组范围内搜索对氟西汀具有抗药性的突变蠕虫，以阐明SSRI、5-羟色胺和其他神经递质之间的遗传相互作用。第一个目标是识别氟西汀耐药基因。我们已经建立了对氟西汀耐药突变进行基因筛查的行为范式。利用这个范例，我们已经确定了5个已知的参与5-羟色胺信号传递的基因。此外，我们还通过无偏向诱变筛选分离到了35个新的氟西汀耐药突变株。我们将对新的突变体进行鉴定，并克隆突变基因。第二个目标是确定新的氟西汀耐药基因在调节乙酰胆碱、GABA和谷氨酸神经传递中的作用，并将耐药基因组织在连接5-羟色胺和单个神经递质信号通路的功能通路中。第三个目标是确定氟西汀耐药基因在5-羟色胺下游通路中的作用，这些通路调节发育和对环境压力的生理反应。第四个目标是研究新的氟西汀耐药基因和5-羟色胺受体之间的遗传相互作用。通过检测5-羟色胺缺失突变体、SERT缺失突变体、5-羟色胺受体突变体和氟西汀抗性突变体，我们将评估5-羟色胺、SSRIs和SERT在调节其他神经递质突触活性、发育和应激生理中作用的可比性。公共卫生相关性：对模式生物体中SSRIs下游靶标的系统遗传解剖将揭示在不同生物体中发挥保守功能的5-羟色胺能系统的核心组件和主要功能途径，并对SSRIs的治疗效果至关重要。这些功能通路还可以为细分特定疾病提供生化标记，并揭示可能统一不同疾病的共同遗传脆弱性。这些反过来将提供关于发病机制的遗传基础的线索，并为发现新的治疗方法提供新的窗口。

DESCRIPTION (provided by applicant): The goal of this proposal is to elucidate the serotonin signaling pathways through genetic dissection of genes and functional pathways regulated by Selective Serotonin Reuptake Inhibitors (SSRIs). Dysregulation of the serotonergic system has been implicated in a wide-spectrum of neurodevelopmental and psychiatric disorders, and SSRIs have made a major impact on many symptoms of these diseases. SSRIs are thought to exert therapeutic effects by inhibiting the serotonin reuptake transporter (SERT), thereby increasing serotonin signaling, leading to our central hypothesis that genetic dissection of SSRIs-resistant genes will identify not only the genes directly binding the drugs, but also the genes and functional pathways regulated by the drugs, and therefore, will shed lights on the fundamental biology of serotonin signaling and its role in the diseases. Taking the advantage of the availability of serotonin-deficient C. elegans, and the unique ability to monitor gene expression and synapse structure in identified cells in the living worm, this proposal uses direct forward genetic approaches in C. elegans to identify SSRIs-resistant genes in a whole animal. This proposal is based on our preliminary data indicating that the SSRI fluoxetine (Prozac ) targets serotonin postsynaptic targets to regulate the activity of acetylcholine, GABA, and glutamate neurotransmission. In four specific aims, proposed experiments will search genome-wide for mutant worms that are resistant to fluoxetine to elucidate the genetic interactions between SSRIs, serotonin, and the other neurotransmitters. The first aim is to identify fluoxetine- resistant genes. We have established a behavioral paradigm for genetic screening for fluoxetine-resistant mutants. Using this paradigm, we have already identified 5 genes known to be involved in serotonin signaling. In addition, we have isolated 35 new fluoxetine-resistant mutants through an unbiased mutagenesis screen. We will characterize the new mutants and clone the mutant genes. The second aim is to characterize the role of new fluoxetine-resistant genes in regulating acetylcholine, GABA and glutamate neurotransmission, and to organize the drug-resistant genes in functional pathways that couple serotonin and individual neurotransmitter signaling pathways. The third aim is to characterize the role of the fluoxetine-resistant genes in serotonin downstream pathways that regulate developmental and physiological responses to environmental stresses. The fourth aim is to examine the genetic interactions between new fluoxetine-resistant genes and serotonin receptors. By testing serotonin deficient mutants, SERT-null mutants, serotonin receptor mutants, and fluoxetine resistant mutants, we will evaluate the comparability of serotonin, SSRIs and SERT on the function of serotonin receptors in regulating the synaptic activity of the other neurotransmitters, development, and stress physiology. PUBLIC HEALTH RELEVANCE: The systematic genetic dissection of SSRIs downstream targets in a model organism will reveal core components and principal functional pathways of the serotonergic system that exert conserved functions in disparate organisms and are important for the therapeutic effects of SSRIs. These functional pathways may also provide biochemical markers for subdividing a particular disease and reveal shared genetic vulnerability that might unify distinct disorders. These in turn will provide clues about the genetic basis underlying the pathogenesis and offer new windows for the discovery of novel therapeutics.