Revealing Novel Mechanisms of Amphetamine Action in a Drosophila Model

描述（由申请人提供）：K08指导临床科学家职业发展奖的总体目标是为我提供指导培训，使我成为一名独立的研究者，作为一名从事精神兴奋剂滥用转化神经科学研究的内科科学家。我的具体职业目标包括应用遗传、行为和成像工具，以提高对药物滥用和成瘾的基本神经生物学和突触信号机制的理解。为了实现这一目标，我建议使用果蝇黑腹果蝇作为模型系统，有效地识别相关的分子靶点，并最终在啮齿动物模型中进行测试和验证。苍蝇提供了一个强大的模型系统来研究精神兴奋剂信号传导机制，因为它的优势是允许在分子和行为水平上同时研究生化途径。为了解决我训练中的差距，我的K08训练目标是：1)发展苍蝇神经生物学和遗传学方面的专业知识，2)发展突触前和突触后多巴胺神经元信号成像方面的专业知识。为了获得这个奖项，我将采用多学科方法结合行为和成像研究来研究精神兴奋剂滥用的分子病理生理学，重点是安非他明（AMPH）的作用。虽然已经确定了AMPH的主要作用位点，但对其下游信号通路知之甚少。我和我的同事已经证明，果蝇幼虫对AMPH的反应是爬得更快，这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信号可能由G1i/o依赖性和/或抑制蛋白依赖性/G1i/o依赖性（arr依赖性）下游信号通路介导，但尚不清楚AMPH作用是否依赖于一条或两条通路。重要的是，Akt激酶作为D2R下游几个信号分子之间的中介，介导AMPH刺激的运动，了解其调控可能有助于阐明AMPH作用的分子机制。我将验证AMPH刺激果蝇DA突触后神经元导致G1i/o依赖性和arr依赖性信号通路的d2r依赖性激活的假设。在这项工作中，我将解决2个具体目标：1)通过RNAi敲除两种通路中的信号分子，测试amph刺激的运动是否依赖于arr依赖性和/或G1i/o依赖性通路；2)通过完整活蝇脑内Akt生物传感器的多光子成像，确定amph介导的突触后D2R激活对Akt活性的体内影响。这些新方法和发现将有助于进一步表征AMPH的分子作用，并将我们推向迫切需要的治疗方法和更好的兴奋剂病理生理模型。

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. PUBLIC HEALTH RELEVANCE: Abuse of psychostimulant drugs such as amphetamine represents a major public health problem with a broad array of adverse health consequences, including dependence, overdose and death. Although these drugs have been some of the most used and abused substances for the last century, our knowledge of their molecular mechanisms of action in the brain remains rudimentary. This Mentored Clinical Scientist Research Career Development Award (K08) will enable Dr. Zachary Freyberg to elucidate mechanisms of action for stimulants such as amphetamine and therefore open the door to novel therapeutic interventions.