Trafficking and Regulation of Monoamine Transporters

描述(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，是成瘾精神刺激剂可卡因、安非他明和MDMA(“摇头丸”)以及治疗剂：哌醋甲酯(“利他林”)和布维酮(“威布曲林”，“Zyban”)的分子靶标。这些药物有效地抑制单胺转运，显著提高细胞外单胺水平，并增强下游信号转导。因此，转运体细胞表面的可用性对于正常的突触传递和精神活性药物的疗效都是至关重要的。大量证据表明，单胺转运体可以动态地往返于质膜。此外，苯丙胺暴露和蛋白激酶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的可用性。尽管受管制的转运体贩运得到了很好的记录，但管理转运体调节和贩运的细胞和分子机制并没有明确的定义。鉴于药物转运体阻断对突触传递的显著影响，转运体隔离很可能显著影响下游神经元信号转导。此外，载体可获得性的调节肯定会对精神活性药物的疗效产生重大影响。这个项目的主要目标是阐明急性转运蛋白调节和贩运的细胞和分子机制。具体地说，我们假设(1)刹车机制控制结构性的和PKC刺激的多巴胺转运体(DAT)内吞作用；(2)内吞后DAT转运不同于经典的转运途径；(3)苯丙胺和PKC诱导的DAT封存在机械上是不同的。这些假说建立在强有力的初步数据基础上，显示了结构性和PKC调节的DAT内化所需的不同的内吞基序，新的DAT内吞后转运特性和苯丙胺诱导的DAT封存的PKC非依赖性特性。我们将使用定点突变来定义组成性、PKC和苯丙胺调节的DAT内化和循环信号。最先进的活体细胞成像方法将被用来确定管理基础和受调控的DAT内吞运输的机制。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。这些办法将全面了解受管制的运输工具贩运的基础机制。我们预计我们的结果将对未来的成瘾和情感障碍的治疗策略产生重大影响。此外，研究结果将极大地提高我们对大脑中单胺可获得性和信号传递因素的理解。与公共健康相关：精神疾病和精神刺激性成瘾在美国是日益严重的问题，在过去几年里几乎达到了流行的程度。尽管对这些情况背后的机制进行了大量调查，但我们的知识仍然存在很大差距。目前的项目将调查大脑中精神刺激剂和抗抑郁药的主要靶点，期望即将到来的信息可能会导致治疗这些疾病的新的治疗方法。

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.