Dopamine Transporter Cell Surface Dynamics

多巴胺转运蛋白细胞表面动力学

基本信息

批准号：
9902390
负责人：
Haley E Melikian
金额：
$ 44.26万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-05-01 至 2023-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9902390
关键词：
Ablation Acute Amphetamines Antidepressive Agents Anxiety Attention deficit hyperactivity disorder Behavior Binding Brain Bypass Cell Line Cell membrane Cell surface Cells Cocaine Code Complex Corpus striatum structure Coupled Data Disease Dopamine Endocytosis Epidemic Functional disorder Future Genes Genetic Glean Goals Guanosine Triphosphate Phosphohydrolases Half-Life Health In Situ Investigation Knockout Mice Knowledge Lead Light Locomotion Measures Mediating Mood Disorders Movement Mus Muscarinic Acetylcholine Receptor Neurotransmitters Nociception Opioid Parkinsonian Disorders Pathway interactions Patients Periodicity Pharmacology Phorbol Esters Physiological Presynaptic Receptors Presynaptic Terminals Protein Kinase C Psychostimulant dependence Receptor Activation Recycling Regulation Reporting Rewards Ritalin Role Scanning Shapes Short-Term Memory Signal Transduction Slice Stimulus Surface Testing Therapeutic Transgenic Mice United States Variant Viral designer receptors exclusively activated by designer drugs dopamine transporter dopaminergic neuron drug seeking behavior expectation extracellular genetic approach genetic manipulation infancy knock-down mouse genetics neuropsychiatric disorder neurotransmission novel novel therapeutic intervention optogenetics presynaptic psychostimulant receptor response reuptake small hairpin RNA stem tool trafficking transgene expression transmission process uptake

项目摘要

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.

多巴胺（DA）是具有不同生理影响的主要神经递质，并且是运动所必需的工作记忆和奖励。诱发释放后，DA细胞外半衰期由突触前确定重新摄取，由SLC6质膜DA转运蛋白（DAT）介导。 DAT是成瘾性和治疗性精神刺激剂，例如苯丙胺，可卡因和哌醋甲酯（Ritalin），Ritalin），，有效抑制DA的吸收，维持DA信号传导并影响DA依赖性行为。 DAT编码变体与多种神经精神疾病有关，而转基因小鼠研究清楚证明DAERGIC信号传导和行为以及精神刺激性功效对 DAT表达式的水平。 DAT在质膜上不是静态的，而是受强大的本构态。内吞回收。此外，直接蛋白激酶C（PKC）激活迅速加速了DAT内在化并降低DAT表面可用性和功能。近二十年来的努力阐明了许多基础和PKC调节的基础机制进行了贩运。但是，目前尚不清楚如何在真正的突触前DA终端中进行调节，DAT调节是否取决于区域，以及是否存在 DAT运输会影响DA信号。此外，尚不清楚dat贩运机制是否确定在细胞系中，研究在生理上是相关的。拟议研究的主要目标是确定调节DAT表达表达的突触前机制，并测试DAT运输是否具有生理对DA释放和清除率的影响。具体而言，我们旨在测试1）突触前GQ耦合受体激活是否以区域依赖性方式影响DAT表达和功能，2）纹状体内的逆行信号是否调节突触前的DAT表达和功能， 3）DAT运输功能障碍是否会影响DA信号传导。这些假设源于强者初步数据证明了区域特异性，GQ介导的双相dat纹状体dagricking GQ耦合的M5和GI-couple NOPR受体在突触前DAT中的终端和推定角色贩运。这些挑衅性的发现表明，多种机制在纹状体内收敛调节DAT表面可用性和功能。为了追求这一调查线，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达到DA神经元中的shRNA介导的基因敲低，以及化学遗传学和光遗传学策略。离体纹状体切片研究将根据响应于突触前受体激活和快速扫描的循环伏安法将测量DA释放并介导清除，并行。条件转基因表达，基因消融和shRNA介导的敲低将使用策略来定义影响鼠标内DAT运输的细胞自主机制纹状体。这些研究收集的信息将对DAT表面有更清晰的了解本地突触前终端的动力学以及DAT运输对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旨在治疗DA相关疾病的未来策略，在该策略中，操纵DAT功能可以提供治疗益处。