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：
9012058
负责人：
Haley E Melikian
金额：
$ 35.24万
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/9012058
关键词：
Amphetamines Antibodies Antidepressive Agents Attention deficit hyperactivity disorder Behavior Binding Binding Proteins Biochemical Biology Biosynthetic Proteins 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 Recycling Rewards Ritalin Role Schizophrenia Signal Transduction Slice Surface Synapses Techniques Testing Therapeutic Time Total Internal Reflection Fluorescent United States 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 reuptake small hairpin RNA small molecule small molecule inhibitor 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）水平直接影响各种生理功能，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刺激剂的主要目标，例如苯丙胺，可卡因和甲基苯甲酸酯（Ritalin），以及抗抑郁药，例如安单肽（wellbutrin）。这些药物有效抑制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与DA相关的行为。因此，DAT活动和可用性批判性地确定正常的DA神经传递和精神活性药物功效。 DAT质膜表现不是静态的。相反，DAT通过本构内吞贩运动态从质膜往返于质膜。蛋白激酶C（PKC）激活和苯丙胺（AMPH）暴露调节DAT内在化和回收率，最终降低了DAT表面的可用性。在ADHD谱系中的最新研究确定了一个DAT突变体，该突变体在基础和Amph刺激的条件下改变了DAT运输动力学，并改变了针对膜筏微区域的靶向，从而暗示了人类疾病中的DAT运输改变。但是，研究DAT内吞机制的研究产生了矛盾的结果，并且DAT细胞表面行为尚未很好地定义。这些研究的主要目的是直接检查活细胞中TIRF显微镜的DAT表面动态，并阐明介导基础和调节DAT内在化的内吞机制。具体而言，我们的目的是（1）表征野生型和运输突变体DAT的表面动力学，（2）检验以下假设：基础，PKC和AMP刺激的DAT内吞作用是与网状蛋白无关的，并且依赖性CDC42依赖性，以及（3）测试（3） DAT内部化是一个独立的过程的假设。这些假设源于强大的初步数据，这些数据证明了1）DAT表面定位主要是与网格蛋白独立的焦点，2）独立于动态蛋白依赖性的DAT运输和3）CDC42依赖性DAT内吞作用。对于实时TIRF成像研究，我们利用了一种新型的化学生物学方法，该方法将荧光团直接与细胞表面DAT结合在一起。这种创新的技术将有助于首次直接检查DAT表面动力学，而无需与笨重的抗体或抑制配体结合。互补的生化研究将在神经元细胞系和小鼠纹状体切片中进行，利用小分子网格蛋白，Dynamin和Cdc42抑制剂以及标准的shRNA和GTPase突变剂方法。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旨在治疗情感障碍和吸毒成瘾的未来策略。此外，结果无疑将增强我们对影响大脑可用性的分子因素的理解。