Trafficking and Regulation of Monoamine Transporters

描述（由申请人提供）：突触前单胺再摄取是影响神经元外单胺水平和终止突触传递的主要因素。SL6载体基因家族中的单胺转运体介导再摄取，是成瘾性精神兴奋剂可卡因、安非他明和MDMA（“摇头丸”）以及治疗药物哌甲酯（“利他林”）和安非他酮（“安非他酮”、“Zyban”）的分子靶点。这些药物能有效抑制单胺转运，显著提高细胞外单胺水平并增强下游信号。因此，转运细胞表面可用性对正常突触传递和精神活性药物疗效都至关重要。大量证据表明，单胺转运蛋白动态地往返于质膜。此外，安非他明暴露和蛋白激酶C （PKC）激活都通过调节转运体运输动力学来改变转运体表面可用性。虽然有充分的文献记载转运蛋白的调控，但调控转运蛋白调控和转运的细胞和分子机制并没有明确定义。鉴于转运蛋白阻断对突触传递的显著作用，转运蛋白隔离很可能显著影响下游神经元信号传导。此外，转运体可用性的调节肯定会对精神活性药物的疗效产生重大影响。本项目的主要目标是阐明介导急性转运蛋白调控和转运的细胞和分子机制。具体来说，我们假设：(1)一种制动机制控制构成型和pkc刺激的多巴胺转运体（DAT）内吞；(2)内吞后数据贩运不同于经典贩运途径；(3)安非他明和pkc诱导的数据隔离在机制上是不同的。这些假设是基于强有力的初步数据，这些数据显示了构成型和pkc调节的DAT内化所需的独特内吞基序，新的DAT内吞后运输特性以及安非他明诱导的DAT隔离的pkc独立特性。我们将使用位点定向诱变来定义构成型、PKC型和安非他明调节的DAT内化和再循环信号。最先进的活细胞成像方法将用于定义控制基础和调节的DAT内吞贩运的机制。最后，分子和蛋白质组学方法将定义DAT内吞运输所需的DAT相互作用蛋白。这些方法将全面了解受管制的运输者贩运的基本机制。我们期望我们的结果将显著影响未来的成瘾和情感障碍的治疗策略。此外，这些结果将大大提高我们对单胺可用性和大脑信号传导的因素的理解。公共卫生相关性：在美国，精神疾病和精神兴奋剂成瘾问题日益严重，在过去几年中几乎达到流行病的程度。尽管对这些疾病背后的机制进行了大量的调查，但我们的知识仍有很大的差距。目前的项目将调查精神兴奋剂和抗抑郁药在大脑中的主要靶点，期望即将到来的信息可能导致治疗这些疾病的新治疗方法。

DESCRIPTION (provided by applicant): Presynaptic monoamine reuptake is a major factor that influences extraneuronal monoamine levels and terminates synaptic transmission. Monoamine transporters within the SL6 carrier gene family mediate reuptake and are the molecular targets for the addictive psychostimulants cocaine, amphetamines, and MDMA ("ecstasy"), as well as for the therapeutic agents: methylphenidate ("Ritalin") and buproprion ("Wellbutrin", "Zyban"). These agents potently inhibit monoamine transport, significantly elevating extracellular monoamine levels and enhancing downstream signaling. Thus, transporter cell surface availability is paramount to both normal synaptic transmission and psychoactive drug efficacy. Abundant evidence demonstrates that monoamine transporters dynamically traffic to and from the plasma membrane. Moreover, both amphetamine exposure and protein kinase C (PKC) activation change transporter surface availability by modulating transporter trafficking kinetics. Although regulated transporter trafficking is well documented, the cellular and molecular mechanisms governing transporter regulation and trafficking are not clearly defined. Given the pronounced effect pharmacological transporter blockade exerts on synaptic transmission, transporter sequestration is highly likely to significantly effect downstream neuronal signaling. Moreover, modulation of transporter availability is certain to have significant impact on the efficacy of psychoactive drugs. The major goals of this project are to elucidate the cellular and molecular mechanisms mediating acute transporter regulation and trafficking. Specifically, we hypothesize that (1) A brake mechanism controls constitutive and PKC-stimulated dopamine transporter (DAT) endocytosis; (2) Post-endocytic DAT trafficking differs from classical trafficking pathways; and (3) Amphetamine- and PKC-induced DAT sequestration are mechanistically distinct. These hypotheses are based on strong preliminary data demonstrating distinct endocytic motifs required for constitutive and PKC-regulated DAT internalization, novel DAT post-endocytic trafficking properties and PKC-independent properties of amphetamine-induced DAT sequestration. We will use site- directed mutagenesis to define constitutive, PKC-, and amphetamine-regulated DAT internalization and recycling signals. State-of-the-art live cellular imaging approaches will be used to define the mechanisms governing basal and regulated DAT endocytic trafficking. Finally, molecular and proteonomic approaches will define DAT-interacting proteins required for DAT endocytic trafficking. These approaches will provide a comprehensive picture of the mechanisms underlying regulated transporter trafficking. We expect our results will significantly impact future addiction and affective disorder treatment strategies. Moreover, the outcomes will greatly improve our understanding of the factors contributing to monoamine availability and signaling in the brain. PUBLIC HEALTH RELEVANCE: Mental illness and psychostimulant addiction are growing problems in the United States, reaching near epidemic proportions over the past several years. Despite numerous investigations into the mechanisms underlying these conditions, there are still large gaps in our knowledge. The current project will investigate the major targets in the brain for both psychostimulants and antidepressants, with the expectation that the forthcoming information may lead to novel therapeutic approaches for treating these disorders.