Revealing Novel Mechanisms of Amphetamine Action in a Drosophila Model

揭示果蝇模型中苯丙胺作用的新机制

基本信息

批准号：
8902527
负责人：
ZACHARY FREYBERG
金额：
$ 18.62万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-08-01 至 2016-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8902527
关键词：
Address Affect Amphetamines Animal Model Arrestins Award Behavior Behavioral Behavioral Genetics Biochemical Biochemical Pathway Biological Assay Biological Models Biosensor Brain Clinical Dependence Development Dopamine Dopamine D2 Receptor Drosophila genus Drosophila melanogaster Drug Addiction Drug abuse Event Functional disorder Genetic Genetic Techniques Glycogen Synthase Kinase 3 Goals Health Image Imaging Device Investigation K-Series Research Career Programs Knowledge Larva Length Life Life Cycle Stages Light Locomotion Measures Mediating Mentors Microscopy Modeling Molecular Molecular Mechanisms of Action Molecular Target Nerve Neurobiology Neurons Neurosciences Research Neurotransmitters Orthologous Gene Pathway interactions Pertussis Toxin Pharmaceutical Preparations Phosphotransferases Physicians Public Health RNA Interference Receptor Activation Receptor Signaling Regulation Relative (related person)Reporting Research Research Personnel Rodent Model Scientist Signal Pathway Signal Transduction Signaling Molecule Site Speed Synapses Testing Time Training Validation Vertebrates Work arrestin 2 career dopamine transporter fly improved in vivo insight interdisciplinary approach knock-down molecular imaging monoamine neurotransmission novel novel strategies novel therapeutic intervention overdose death postsynaptic presynaptic protein expression psychostimulant response selective expression stimulant abuse tissue culture translational neuroscience

项目摘要

DESCRIPTION (provided by applicant): The overall goal of this K08 Mentored Clinical Scientist Career Development Award is to provide me with the mentored training to become an independent investigator as a physician-scientist pursuing translational neuroscience research in psychostimulant abuse. My specific career goals include application of genetic, behavioral and imaging tools towards development of an improved understanding of the basic neurobiology and synaptic signaling mechanisms underlying drug abuse and addiction. To accomplish this, I propose to use the fly Drosophila melanogaster as a model system to identify relevant molecular targets efficiently and for eventual testing and validation in rodent models. The fly provides a powerful model system to study mechanisms of psychostimulant signaling given its advantage of allowing concurrent investigation of biochemical pathways at molecular and behavioral levels. In addressing gaps in my training, my K08 training goals are: 1) to develop expertise in fly neurobiology and genetics, and 2) to develop expertise in imaging pre- and postsynaptic dopamine neuronal signaling. As proposed for this award, I will use a multidisciplinary approach combining behavioral and imaging studies to investigate the molecular pathophysiology underlying psychostimulant abuse, with a focus on amphetamine (AMPH) action. Though the primary sites of action for AMPH have been identified, the downstream signaling pathways are poorly understood. My colleagues and I have shown that fly larvae respond to AMPH by crawling faster and this is dependent both on presynaptic dopamine transporter and postsynaptic dopamine (DA) D2 receptors (D2R). While postsynaptic D2R signals may be mediated by G1i/o-dependent and/or arrestin-dependent/G1i/o-independent (Arr-dependent) downstream signaling pathways, it is not known whether AMPH action is dependent on one or both pathways. Importantly, the kinase Akt behaves as an intermediary between several signaling molecules downstream of D2R known to mediate AMPH-stimulated locomotion and understanding its regulation may shed light on molecular mechanisms of AMPH action. I will test the hypothesis that AMPH stimulation of DA postsynaptic neurons in the fly leads to D2R-dependent activation of both G1i/o-dependent and Arr-dependent signaling pathways. I will address 2 specific aims in this work: 1) to test whether AMPH-stimulated locomotion is dependent on the Arr-dependent and/or G1i/o-dependent pathways using RNAi knockdown of signaling molecules in both pathways, and 2) to determine in vivo effects on Akt activity of AMPH-mediated postsynaptic D2R activation via multiphoton imaging of the Akt biosensor within the intact living fly brain. These novel approaches and findings will facilitate further characterization of AMPH's molecular actions and move us toward critically needed treatments and to better models of stimulant pathophysiology.

描述（由申请人提供）：这一K08指导的临床科学家职业发展奖的总体目标是为我提供指导的培训，成为一名独立的研究者，成为一名医生 - 科学家，从事心理刺激虐待方面的转化神经科学研究。我的特定职业目标包括将遗传，行为和成像工具应用于对药物滥用和成瘾的基本神经生物学和突触信号传导机制的发展。为了实现这一目标，我建议使用果蝇果蝇（Fly Drosophila Melanogaster）作为模型系统，以有效地识别相关的分子靶标，并最终在啮齿动物模型中进行测试和验证。苍蝇为研究心理刺激信号传导的机制提供了强大的模型系统，鉴于其允许在分子和行为水平上同时研究生化途径的优势。在解决培训中的差距时，我的K08培训目标是：1）开发苍蝇神经生物学和遗传学方面的专业知识，以及2）在成像前和突触后多巴胺神经元信号方面发展专业知识。正如该奖项所提出的那样，我将采用一种多学科方法，结合行为和成像研究来研究心理刺激滥用的分子病理生理学，重点是苯丙胺（AMPH）作用。尽管已经确定了AMPH的主要作用部位，但对下游信号通路的了解很少。我和我的同事表明，蝇幼虫通过更快地爬行对AMPH做出反应，这既取决于突触前多巴胺转运蛋白和突触后多巴胺（DA）D2受体（D2R）。虽然突触后D2R信号可以由G1i/O依赖性和/或抑制蛋白依赖性/G1i/O-非依赖性（ARR依赖性）下游信号通路介导，但尚不知道AMPH的作用是否取决于一种或两种途径。重要的是，激酶AKT在已知介导Amph刺激的运动的D2R下游的几个信号分子之间表现为中介，并且理解其调节可能会揭示AMPH作用的分子机制。我将检验以下假设：苍蝇中AMPH刺激突触后神经元会导致d2R依赖性的G1i/O依赖性和依赖性信号传导途径的D2R依赖性激活。我将在这项工作中解决2个具体目标：1）测试使用AMPH刺激的运动是否取决于ARR依赖性和/或G1i/O依赖性途径，使用两个途径中的信号分子的RNAi敲低以及2）来确定体内对Akt介导的突触后D2R激活Akt活性的影响通过完整活蝇脑中Akt生物传感器的多光子成像。这些新颖的方法和发现将有助于进一步表征AMPH的分子作用，并使我们朝着迫切需要的治疗和更好的刺激性病理生理学模型迈进。