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

• 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
• 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

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脑回路失调。滥用药物会增加与奖赏处理有关的神经结构中的多巴胺（DA）浓度;精神兴奋剂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，导致其靶点（分别是中脑背侧核和背侧纹状体）中的多巴胺浓度增加。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（VMAT 2）。DAT负责DA从细胞外间隙重新吸收到突触前末梢。VMAT 2将胞质DA转运到突触囊泡中。AMPH与DAT结合，导致DA转运逆转和DA从胞质溶胶到细胞外空间的净流出。AMPH对VMAT 2的作用较少被理解，并且是一个有争议的主题;然而，人们接受AMPH与VMAT 2的结合导致DA从突触囊泡流出到胞质溶胶中。最近的研究表明，DAT和VMAT 2是通过突触囊泡膜蛋白synaptogyrin-3（SYGR 3）物理耦合。这种DAT-SYGR 3-VMAT 2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SYGR 3的存在，还取决于功能性VMAT 2的存在。指导这项工作的假设是，AMPH对DAT和VMAT 2的作用也可能取决于SYGR 3介导的偶联，并且神经元暴露于AMPH可能导致SYGR 3介导的DAT和VMAT 2偶联的改变。这些假设将在多巴胺能神经元的原代培养物中进行测试，以及在成年大鼠体内，使用腺相关病毒载体（AAV）含有短发夹RNA（shRNA），旨在敲低SYGR 3。我们将使用这种病毒，以确定SYGR 3去除AMPH诱导的DA反向转运的影响，使用分子，细胞和行为技术。实验的设计，以便，无论结果如何，我们将有一个增加的程度上的理解，AMPH行动的分子机制需要和/或干扰正常的相互作用蛋白质之间的调节DA稳态;此外，这些实验可能会增加我们的理解VMAT 2在产生AMPH诱导DA流出的作用。