Contribution of synaptic vesicle proteins to molecular mechanisms of amphetamine

突触小泡蛋白对安非他明分子机制的贡献

基本信息

批准号：
8592217
负责人：
Kathleen Marie Salerno
金额：
$ 4.58万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
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：
2013-09-01 至 2014-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8592217
关键词：
Acute Addictive Behavior Adult Affect Amphetamines Behavior Behavior Disorders Behavioral Bilateral Binding Brain Cell Line Cell membrane Clinical Complex Corpus striatum structure Coupled Couples Coupling Cytoplasm Cytosol Data Development Dopamine Dopaminergic Cell Dorsal Drug Addiction Excision Exposure to Extracellular Space Functional disorder Future Homeostasis In Vitro Injection of therapeutic agent Knowledge Laboratories Lead Locomotion Measures Mediating Membrane Proteins Microdialysis Midbrain structure Modeling Molecular Molecular Target Motivation Neurons Nucleus Accumbens Outcome Pathway interactions Peptides Pharmaceutical Preparations Presynaptic Terminals Proteins Public Health Rattus Recycling Regulation Research Rewards Role Scanning Signal Transduction Structure Substantia nigra structure Synaptic Cleft Synaptic Vesicles Techniques Testing Ventral Striatum Ventral Tegmental Area Virus Work addiction adeno-associated viral vector analog base behavioral sensitization design dopamine transporter dopaminergic neuron drug of abuse extracellular in vivo insight knock-down monoamine motivated behavior nervous system disorder nigrostriatal pathway protein protein interaction psychostimulant public health relevance relating to nervous system research study response reuptake reward processing small hairpin RNA social stimulant abuse synaptogyrin therapy design uptake vesicular monoamine transporter 2

项目摘要

DESCRIPTION (provided by applicant): Drug addiction is a neurological disorder characterized by dysregulation of brain circuits which regulate motivation and reward signaling. Drugs of abuse increase dopamine (DA) concentrations in neural structures involved with reward processing; psychostimulant drugs do so by acting on dopaminergic neurons in the ventral tegmental area (VTA) and substantia nigra (SN), resulting in increased dopamine concentrations in their targets (respectively, the nucleus accumbens and dorsal striatum). The psychostimulant amphetamine (AMPH) acts at monoamine transporters, including the plasma membrane dopamine transporter (DAT) and the vesicular monoamine transporter-2 (VMAT2). DAT is responsible for DA reuptake from the extracellular space into the presynaptic terminal. VMAT2 transports cytosolic DA into synaptic vesicles. AMPH binds to DAT causing a reversal of DA transport and a net efflux of DA from the cytosol to the extracellular space. The actions of AMPH at VMAT2 are less-understood and a subject of controversy; however, it is accepted that binding of AMPH to VMAT2 results in efflux of DA from synaptic vesicles into the cytosol. Recent findings demonstrate that DAT and VMAT2 are physically coupled via the synaptic vesicle membrane protein synaptogyrin-3 (SYGR3). The formation of this DAT-SYGR3-VMAT2 complex is required for normal uptake of DA through DAT, and depends not only on the presence of SYGR3, but also on the presence of functional VMAT2. The hypotheses directing this work is that the action of AMPH on DAT and VMAT2 may also depend on SYGR3-mediated coupling, and that exposure of neurons to AMPH may result in alterations in SYGR3- mediated coupling of DAT and VMAT2. These hypotheses will be tested in primary cultures of dopaminergic neurons, as well as in adult rats in vivo, using an adeno-associated viral vector (AAV) containing a short hairpin RNA (shRNA) designed to knock down SYGR3. We will use this virus to determine the effects of SYGR3 removal on AMPH-induced reverse transport of DA using molecular, cellular, and behavioral techniques. Experiments are designed so that, regardless of the outcome, we will have an increased understanding of the extent to which the molecular mechanisms of AMPH action require and/or interfere with normal interactions between proteins regulating DA homeostasis; furthermore, these experiments may increase our understanding of the role of VMAT2 in generating AMPH-induced DA efflux.

描述（由申请人提供）：药物成瘾是一种神经系统疾病，其特征是调节动机和奖励信号的大脑回路失调。滥用药物会增加参与奖励处理的神经结构中的多巴胺 (DA) 浓度；精神兴奋药物通过作用于腹侧被盖区（VTA）和黑质（SN）的多巴胺能神经元，导致其目标（分别是伏隔核和背侧纹状体）的多巴胺浓度增加。精神兴奋剂安非他明 (AMPH) 作用于单胺转运蛋白，包括质膜多巴胺转运蛋白 (DAT) 和囊泡单胺转运蛋白 2 (VMAT2)。 DAT 负责将 DA 从细胞外空间重新摄取到突触前末端。 VMAT2 将胞质 DA 转运到突触小泡中。 AMPH 与 DAT 结合，导致 DA 转运逆转以及 DA 从细胞质净流出至细胞外空间。 AMPH 在 VMAT2 中的作用鲜为人知，并且是一个有争议的话题；然而，人们普遍认为 AMPH 与 VMAT2 的结合会导致 DA 从突触小泡流出到细胞质中。最近的研究结果表明，DAT 和 VMAT2 通过突触小泡膜蛋白 synaptogyrin-3 (SYGR3) 进行物理耦合。这种 DAT-SYGR3-VMAT2 复合物的形成是通过 DAT 正常摄取 DA 所必需的，并且不仅取决于 SYGR3 的存在，还取决于功能性 VMAT2 的存在。指导这项工作的假设是 AMPH 对 DAT 和 VMAT2 的作用也可能依赖于 SYGR3 介导的偶联，并且神经元暴露于 AMPH 可能会导致 SYGR3 介导的 DAT 和 VMAT2 偶联的改变。这些假设将在多巴胺能神经元的原代培养物以及成年大鼠体内进行测试，使用含有旨在敲除 SYGR3 的短发夹 RNA (shRNA) 的腺相关病毒载体 (AAV)。我们将使用该病毒，通过分子、细胞和行为技术来确定 SYGR3 去除对 AMPH 诱导的 DA 反向转运的影响。实验的设计目的是，无论结果如何，我们都将更好地了解 AMPH 作用的分子机制需要和/或干扰调节 DA 稳态的蛋白质之间的正常相互作用的程度；此外，这些实验可能会增加我们对 VMAT2 在产生 AMPH 诱导的 DA 流出中的作用的理解。