Revealing Novel Mechanisms of Amphetamine Action in a Drosophila Model

描述（由申请人提供）：这个K 08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，成为一名独立的调查员，作为一名医生科学家，在精神兴奋剂滥用方面进行转化神经科学研究。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的基础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，以有效地确定相关的分子目标，并最终在啮齿动物模型的测试和验证。苍蝇提供了一个强大的模型系统，以研究精神兴奋剂信号的机制，因为它的优势，允许同时调查的生化途径在分子和行为水平。在解决我的培训差距，我的K 08培训目标是：1）发展苍蝇神经生物学和遗传学的专业知识，2）发展成像突触前和突触后多巴胺神经元信号传导的专业知识。作为这个奖项的建议，我将使用一个多学科的方法结合行为和影像学研究，以调查潜在的精神兴奋剂滥用的分子病理生理学，重点是安非他明（AMPH）的行动。虽然AMPH的主要作用位点已经被确定，但下游信号传导途径却知之甚少。我和我的同事已经证明，苍蝇幼虫对AMPH的反应是爬行更快，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（D2 R）。虽然突触后D2 R信号可能由G1 i/o依赖性和/或抑制蛋白依赖性/G1 i/o非依赖性（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，激酶Akt的行为作为一个中间体之间的几个信号分子下游的D2 R已知介导AMPH刺激的运动和了解其调节可能揭示AMPH作用的分子机制。我将测试的假设，AMPH刺激DA突触后神经元的苍蝇导致D2 R依赖的激活G1 I/O依赖和Arr依赖的信号通路。在这项工作中，我将解决2个具体目标：1）测试AMPH刺激的运动是否依赖于Arr依赖性和/或G1 i/o依赖性途径，使用RNAi敲低两种途径中的信号分子，和2）通过Akt生物传感器在完整的活苍蝇大脑中的多光子成像，以确定在体内对AMPH介导的突触后D2 R激活的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，并使我们转向急需的治疗和更好的刺激性病理生理学模型。 公共卫生相关性：滥用安非他明等精神兴奋剂药物是一个重大的公共卫生问题，会对健康造成广泛的不良后果，包括依赖、过量和死亡。尽管这些药物是上个世纪使用和滥用最多的药物之一，但我们对它们在大脑中作用的分子机制的认识仍然很不成熟。这个指导临床科学家研究职业发展奖（K 08）将使博士扎卡里弗赖贝格阐明兴奋剂的作用机制，如安非他明，因此打开大门，以新的治疗干预。

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.