Functional characterization of SSRI resistant genes using C. elegans

描述（由申请人提供）：本提案的目标是通过对选择性5 -羟色胺再摄取抑制剂（SSRIs）调控的基因和功能途径的基因解剖来阐明5 -羟色胺信号通路。5 -羟色胺能系统的失调与广泛的神经发育和精神疾病有关，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）来发挥治疗作用，从而增加5 -羟色胺信号传导，这导致了我们的中心假设，即对SSRIs耐药基因的遗传解剖不仅可以识别直接结合药物的基因，还可以识别药物调节的基因和功能途径，因此，将揭示5 -羟色胺信号传导的基础生物学及其在疾病中的作用。利用5 -羟色胺缺乏的秀丽隐杆线虫的可用性，以及在活的线虫中鉴定细胞中监测基因表达和突触结构的独特能力，本建议在秀丽隐杆线虫中使用直接正向遗传方法来鉴定整个动物的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（百忧解）靶向5 -羟色胺突触后靶点，以调节乙酰胆碱、GABA和谷氨酸神经传递的活性。在四个特定目标中，拟议的实验将在全基因组范围内搜索对氟西汀有抗性的突变蠕虫，以阐明SSRIs、血清素和其他神经递质之间的遗传相互作用。第一个目标是鉴定氟西汀耐药基因。我们已经建立了对氟西汀耐药突变体进行遗传筛查的行为范例。使用这种模式，我们已经确定了5个已知参与血清素信号传导的基因。此外，我们通过无偏诱变筛选分离出35种新的氟西汀耐药突变体。我们将描述新的突变体并克隆突变基因。二是研究新型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，梳理耐药基因在5 -羟色胺与个体神经递质信号通路耦合的功能通路。第三个目的是确定氟西汀抗性基因在调节发育和生理对环境应激反应的血清素下游通路中的作用。第四个目的是研究新的氟西汀抗性基因与血清素受体之间的遗传相互作用。通过测试5 -羟色胺缺陷突变体、SERT缺失突变体、5 -羟色胺受体突变体和氟西汀耐药突变体，我们将评估5 -羟色胺、SSRIs和SERT对5 -羟色胺受体在调节其他神经递质突触活性、发育和应激生理方面的功能的可比性。公共卫生相关性：对模式生物中SSRIs下游靶点的系统遗传解剖将揭示在不同生物中发挥保守功能的5 -羟色胺能系统的核心成分和主要功能途径，并对SSRIs的治疗效果至关重要。这些功能途径也可能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易感性。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.