Revealing Novel Mechanisms of Amphetamine Action in a Drosophila Model

描述(由申请者提供)：本次K08临床科学家导师职业发展奖的总体目标是为我提供导师培训，使我成为一名独立的调查员，作为一名医生-科学家，从事精神刺激性虐待的翻译神经科学研究。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背后的基本神经生物学和突触信号机制的理解。为了实现这一点，我建议使用果蝇作为一个模型系统，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。苍蝇提供了一个强大的模型系统来研究心理刺激信号的机制，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。为了解决我在培训中的不足，我的K08培训目标是：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。根据这个奖项的提议，我将使用行为和成像研究相结合的多学科方法来研究精神刺激剂滥用背后的分子病理生理学，重点是苯丙胺(AMPH)的作用。虽然AMPH的主要作用部位已经确定，但下游的信号通路却知之甚少。我和我的同事们已经证明，苍蝇幼虫对AMPH的反应是爬行得更快，这取决于突触前多巴胺转运体和突触后多巴胺D2受体(D2R)。虽然突触后D2R信号可能由G1i/o依赖和/或Arrestin/G1i/o非依赖(ARR依赖)的下游信号通路介导，但AMPH的作用是依赖于一条还是两条途径尚不清楚。重要的是，激酶Akt在D2R下游的几个信号分子之间起中介作用，已知这些信号分子介导Amph刺激的运动，了解其调节可能有助于阐明Amph作用的分子机制。我将测试这一假设，即AMPH刺激苍蝇DA突触后神经元导致依赖于D2R的G1i/o依赖和ARR依赖的信号通路的激活。我将针对这项工作中的两个具体目标：1)使用RNAi敲除这两条通路中的信号分子来测试Amph刺激的运动是否依赖于ARR依赖和/或G1i/o依赖的通路；2)通过对完整的苍蝇脑内Akt生物传感器的多光子成像，在体内确定Amph介导的突触后D2R激活对Akt活性的影响。这些新的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学模型。

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.