Dopamine Transporter Cell Surface Dynamics

多巴胺(DA)是一种主要的神经递质，具有多种生理影响，是运动所必需的， 工作记忆和奖励。在诱发释放后，DA细胞外半衰期由突触前决定 重摄取，由SLC6质膜DA转运体(DAT)介导。DAT是以下目标的主要目标 成瘾性和治疗性精神刺激剂，如苯丙胺、可卡因和哌醋甲酯(利他林)， 它们有效地抑制DA摄取，维持DA信号，并影响DA依赖的行为。DAT编码 变异与多种神经精神疾病有关，转基因小鼠的研究很清楚 证明多巴胺能信号和行为，以及精神刺激药效，对 DAT的表达水平。DAT在质膜上不是静态的，而是受制于强健的本构关系 内吞循环。此外，直接蛋白激酶C(PKC)的激活可迅速加速DAT的内化 并降低了DAT表面的可用性和功能。近二十年的努力已经阐明了许多 基础和PKC管制的DAT贩运的机制。然而，目前还不清楚DAT是怎样的 在真正的突触前DA终末中进行调节，DAT调节是否具有区域依赖性，以及 DAT贩运影响DA信令。此外，尚不清楚是否确定了贩运DAT的机制 在细胞系中，研究具有生理学意义。拟议研究的主要目标是确定 调节DAT表面表达的突触前机制，并测试DAT运输是否具有 对DA释放和清除的生理影响。具体地说，我们的目标是测试1)突触前GQ偶联受体激活是否以区域依赖的方式影响DAT表面的表达和功能，2) 纹状体内的逆行信号是否调节突触前DAT表面的表达和功能， 3)DAT转运功能障碍是否影响DA信号传导。这些假说源于强大的 初步数据显示纹状体DAT的区域特异性、GQ介导的双相DAT运输 突触前DAT中GQ偶联的M5和GI偶联的NOPR受体的终末及其可能的作用 贩卖人口。这些具有挑衅性的发现表明，纹状体内DAT的多种机制汇聚在一起。 调节DAT表面的可用性和功能。为了追寻这条调查路线，我们将利用各种 最先进的小鼠遗传工具，包括新开发的有条件和诱导的病毒方法 在DA神经元中实现shRNA介导的基因敲除以及化学发生和光发生 战略。体外纹状体切片研究将测量DAT表面表达的变化 突触前受体激活和快速扫描循环伏安法将测量DA的释放和DAT介导的 净空，并行。条件性转基因表达、基因切割和shRNA介导的基因敲除 将使用策略来定义影响小鼠内DAT交易的细胞自主机制 纹状体。从这些研究中收集的信息将使我们对DAT表面有更清晰的了解 原生突触前终末的动力学，以及DAT交易对脑内DA信号的影响 纹状体。此外，我们预计我们的发现将极大地影响未来旨在治疗DA相关疾病的策略，在这些策略中，操纵DAT功能可以提供治疗益处。

Dopamine (DA) is a major neurotransmitter with diverse physiological impact, and is required for movement, working memory, and reward. Following evoked release, DA extracellular half-life is determined by presynaptic reuptake, mediated by the SLC6 plasma membrane DA transporter (DAT). DAT is the primary target for addictive and therapeutic psychostimulants, such as amphetamine, cocaine and methylphenidate (Ritalin), which potently inhibit DA uptake, sustain DA signaling and impact DA-dependent behaviors. DAT coding variants are implicated in a variety of neuropsychiatric disorders, and transgenic mouse studies clearly demonstrate that DAergic signaling and behaviors, as well as psychostimulant efficacy, are highly sensitive to the level of DAT expression. DAT is not static at the plasma membrane, but is subject to robust constitutive endocytic recycling. Moreover, direct protein kinase C (PKC) activation rapidly accelerates DAT internalization and decreases DAT surface availability and function. Efforts over nearly two decades have elucidated many of the mechanisms underlying basal and PKC-regulated DAT trafficking. However, it remains unclear how DAT is regulated in bona fide presynaptic DA terminals, whether DAT regulation is region-dependent, and whether DAT trafficking impacts DA signaling. Moreover, it is unknown whether DAT trafficking mechanisms identified in cell line studies are physiologically relevant. The major goals of the proposed studies are to determine the presynaptic mechanisms that regulate DAT surface expression, and test whether DAT trafficking has physiological impact on DA release and clearance. Specifically, we aim to test 1) whether presynaptic Gq-coupled receptor activation impacts DAT surface expression and function in a region-dependent manner, 2) whether retrograde signaling within the striatum regulates presynaptic DAT surface expression and function, and 3) whether DAT trafficking dysfunction impacts DA signaling. These hypotheses stem from strong preliminary data that demonstrate region-specific, Gq-mediated, biphasic DAT trafficking in striatal DAergic terminals, and putative roles for the Gq-coupled M5 and Gi-couple NOPR receptors in presynaptic DAT trafficking. These provocative findings suggest that multiple mechanisms converge on DAT within the striatum to regulate DAT surface availability and function. To pursue this investigative line, we will leverage a variety of state-of-the-art mouse genetic tools, including a newly developed viral approach to conditionally and inducibly achieve shRNA-mediated gene knockdown in DA neurons, as well as chemogenetic and optogenetic strategies. Ex vivo striatal slice studies will measure changes in DAT surface expression in response to presynaptic receptor activation, and fast-scan cyclic voltammetry will measure DA release and DAT-mediated clearance, in parallel. Conditional transgene expression, gene ablation and shRNA-mediated knockdown strategies will be used to define the cell autonomous mechanisms that impact DAT trafficking within mouse striatum. The information gleaned from these studies will provide a clearer understanding of DAT surface dynamics in native presynaptic terminals, and the impact that DAT trafficking imposes on DA signaling in the striatum. Moreover, we anticipate that our findings will greatly impact future strategies aimed at treating DA-related disorders, in which manipulating DAT function could provide therapeutic benefit.