Dopamine Transporter Cell Surface Dynamics

多巴胺（DA）是一种具有多种生理影响的主要神经递质，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神经递质决定。 再摄取，由SLC6质膜DA转运蛋白（DAT）介导。DAT是主要目标， 成瘾性和治疗性精神兴奋剂，如安非他明、可卡因和哌醋甲酯（利他林）， 其有效地抑制DA摄取、维持DA信号传导并影响DA依赖性行为。DAT编码 变异与多种神经精神疾病有关，转基因小鼠研究清楚地表明， 证明DA能信号传导和行为，以及精神兴奋剂的功效，是高度敏感的 DAT表达水平。DAT在质膜上不是静态的，而是受到稳健的组成性变化的影响。 内吞再循环此外，直接蛋白激酶C（PKC）激活迅速加速DAT内化 并降低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调节的DAT运输的机制。然而，目前还不清楚DAT是如何 调节真正的突触前DA末梢，DAT调节是否是区域依赖性的，以及是否 DAT运输影响DA信号传导。此外，尚不清楚DAT贩运机制是否确定 在细胞系研究中是生理相关的。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。具体来说，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性方式影响DAT表面表达和功能，2） 纹状体内的逆行信号传导是否调节突触前DAT表面表达和功能， DAT运输功能障碍是否影响DA信号传导。这些假设源于强大的 初步数据表明纹状体DA能神经元中区域特异性、Gq介导的双相DAT运输 Gq偶联的M5和Gi偶联的NOPR受体在突触前DAT中的作用 贩卖人口这些令人激动的发现表明，多种机制汇聚在纹状体内的DAT上 以调节DAT表面的可用性和功能。为了进行这一调查，我们将利用各种 最先进的小鼠遗传工具，包括一种新开发的病毒方法， 在DA神经元中实现shRNA介导的基因敲低，以及化学遗传学和光遗传学 战略布局离体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释放和DAT介导的 间隙，平行。条件性转基因表达、基因消除和shRNA介导的敲低 策略将用于定义影响小鼠内DAT运输的细胞自主机制 纹状体从这些研究中收集的信息将提供一个更清晰的理解DAT表面 动态的天然突触前末梢，以及DAT贩运对DA信号传导的影响， 纹状体此外，我们预计，我们的研究结果将极大地影响未来的策略，旨在治疗DA相关疾病，其中操纵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.