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：
8479301
负责人：
Haley E Melikian
金额：
$ 34.79万
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 2018-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8479301
关键词：
Amphetamines Antibodies Antidepressive Agents Attention deficit hyperactivity disorder Behavior Binding Binding Proteins Biochemical Biology Brain Bupropion Cell Line Cell membrane Cell surface Cells Chemicals Chronic Clathrin Cocaine Cognition Complex Conflict (Psychology)Corpus striatum structure Couples Data Dopamine Drug Addiction Dynamin Dynamin 2 Endocytosis Epidemic Future Gene Family Glean Goals Guanosine Triphosphate Phosphohydrolases Health Image Investigation Kinetics Knowledge Label Lead Life Ligands Link Measures Mediating Membrane Membrane Protein Traffic Membrane Proteins Methods Methylphenidate Molecular Mood Disorders Mouse Cell Line Movement Neurons Parkinson Disease Pathway interactions Pharmaceutical Preparations Physiological Process Protein Kinase C Proteins Psychostimulant dependence Psychotropic Drugs Recombinant Proteins Recycling Rewards Ritalin Role Schizophrenia Signal Transduction Slice Surface Synapses Techniques Testing Therapeutic Time Total Internal Reflection Fluorescent United States Wellbutrin addiction cellular imaging dopamine transporter drug efficacy expectation extracellular fluorophore gain of function genetic pedigree human disease inhibitor/antagonist innovation insight loss of function member mutant neuroblastoma cell neurotransmission novel novel therapeutic intervention presynaptic psychostimulant public health relevance reuptake small hairpin RNA small molecule stem trafficking uptake

项目摘要

DESCRIPTION (provided by applicant): Extracellular dopamine (DA) levels in the brain directly impact a variety of physiological functions, including movement, cognition and reward. Following synaptic release, DA availability is limited by presynaptic reuptake, mediated by the plasma membrane DA transporter (DAT). DAT is a member of the SLC6 carrier gene family and is the primary target for addictive and therapeutic psychostimulants, such as amphetamine, cocaine and methylphenidate (Ritalin) as well as for antidepressants, such as bupropion (Wellbutrin). These drugs potently inhibit DA uptake, and thereby increase extracellular DA concentrations, enhance neuronal signaling and significantly modulate DA-related behaviors. Thus, DAT activity and availability critically determine normal DA neurotransmission and psychoactive drug efficacy. DAT plasma membrane presentation is not static. Rather, DAT is dynamically shuttled to and from the plasma membrane by constitutive endocytic trafficking. Protein kinase C (PKC) activation and amphetamine (AMPH) exposure modulate DAT internalization and recycling rates, ultimately decreasing DAT surface availability. Recent studies in an ADHD pedigree have identified a DAT mutant that has altered DAT trafficking kinetics, both under basal and AMPH-stimulated conditions, and altered targeting to membrane raft microdomains, thereby implicating altered DAT trafficking in human disease. However, studies investigating DAT endocytic mechanisms have yielded conflicting results and DAT cell surface behavior is not well defined. The major goal of these studies is to directly examine DAT surface dynamics by TIRF microscopy in living cells and to elucidate the endocytic mechanisms that mediate basal and regulated DAT internalization. Specifically, we aim to (1) characterize the surface dynamics of wildtype and trafficking mutant DATs, (2) test the hypothesis that basal, PKC- and AMP-stimulated DAT endocytosis are clathrin-independent and Cdc42-dependent, and (3) test the hypothesis that DAT internalization is a dynamin-independent process. These hypotheses stem from strong preliminary data that demonstrate 1) DAT surface localization primarily to clathrin-independent foci, 2) dynamin-independent DAT trafficking and 3) Cdc42-dependent DAT endocytosis. For live TIRF imaging studies, we capitalize on a novel chemical biology approach that directly couples fluorophore to cell surface DAT. This innovative technique will facilitate the first direct examination of DAT surface dynamics without binding to bulky antibodies or inhibitory ligands. Complementary biochemical studies will be performed both in neuronal cell lines and mouse striatal slices, utilizing small molecule clathrin, dynamin and Cdc42 inhibitors to acutely perturb membrane trafficking, as well as standard shRNA and GTPase mutant approaches. The information gleaned from these studies will provide a clearer understanding of DAT surface dynamics and the mechanisms mediating DAT endocytosis, both for wildtype and trafficking mutants. We anticipate that our findings will greatly impact future strategies aimed at treating affective disorders and drug addiction. Moreover, the results will undoubtedly enhance our understanding of the molecular factors influencing DA availability in the brain.

描述（由申请人提供）：大脑中的细胞外多巴胺（DA）水平直接影响多种生理功能，包括运动、认知和奖励。突触释放后，DA 的可用性受到质膜 DA 转运蛋白 (DAT) 介导的突触前再摄取的限制。 DAT 是 SLC6 载体基因家族的成员，是成瘾和治疗性精神兴奋剂（如安非他明、可卡因和哌甲酯（利他林））以及抗抑郁药（如安非他酮（Wellbutrin））的主要靶点。这些药物可有效抑制 DA 摄取，从而增加细胞外 DA 浓度、增强神经元信号传导并显着调节 DA 相关行为。因此，DAT 活性和可用性关键决定正常的 DA 神经传递和精神活性药物的疗效。 DAT 质膜呈现不是静态的。相反，DAT 通过组成型内吞运输动态地往返于质膜。蛋白激酶 C (PKC) 激活和安非他明 (AMPH) 暴露可调节 DAT 内化和回收率，最终降低 DAT 表面可用性。最近针对 ADHD 谱系的研究发现了一种 DAT 突变体，该突变体在基础条件和 AMPH 刺激条件下都改变了 DAT 运输动力学，并改变了膜筏微区的靶向，从而暗示了人类疾病中 DAT 运输的改变。然而，研究 DAT 内吞机制的研究得出了相互矛盾的结果，并且 DAT 细胞表面行为尚未明确定义。这些研究的主要目标是通过 TIRF 显微镜直接检查活细胞中的 DAT 表面动力学，并阐明介导基础和调节 DAT 内化的内吞机制。具体来说，我们的目标是 (1) 表征野生型和运输突变型 DAT 的表面动力学，(2) 测试基础、PKC 和 AMP 刺激的 DAT 内吞作用不依赖网格蛋白且依赖 Cdc42 的假设，以及 (3) 测试DAT 内化是一个独立于动力的过程的假设。这些假设源于强有力的初步数据，这些数据证明了 1) DAT 表面定位主要位于不依赖于网格蛋白的病灶，2) 不依赖于动力的 DAT 运输和 3) 依赖于 Cdc42 的 DAT 内吞作用。对于实时 TIRF 成像研究，我们利用一种新颖的化学生物学方法，将荧光团直接耦合到细胞表面 DAT。这项创新技术将有助于首次直接检查 DAT 表面动力学，而无需与大体积抗体或抑制性配体结合。将在神经元细胞系和小鼠纹状体切片中进行补充生化研究，利用小分子网格蛋白、动力和 Cdc42 抑制剂来剧烈扰乱膜运输，以及标准 shRNA 和 GTPase 突变方法。从这些研究中收集的信息将为野生型和运输突变体的 DAT 表面动力学和介导 DAT 内吞作用的机制提供更清晰的了解。我们预计我们的发现将极大地影响未来治疗情感障碍和毒瘾的策略。此外，这些结果无疑将增强我们对影响大脑中DA可用性的分子因素的理解。