Contribution of synaptic vesicle proteins to molecular mechanisms of amphetamine

描述（由申请人提供）：药物成瘾是一种以调节动机和奖励信号的大脑回路失调为特征的神经系统疾病。滥用药物会增加多巴胺（DA）在参与奖励处理的神经结构中的浓度；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（分别是伏隔核和背纹状体）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）和水疱单胺转运蛋白-2 （VMAT2）。DAT负责DA从细胞外空间再摄取到突触前末端。VMAT2将胞质DA转运到突触囊泡中。AMPH与DAT结合，导致DA转运逆转，并使DA从细胞质净流出到细胞外空间。AMPH在VMAT2的作用鲜为人知，并且是一个有争议的主题；然而，人们普遍认为AMPH与VMAT2结合会导致DA从突触囊泡外排到细胞质中。最近的研究表明，DAT和VMAT2通过突触囊泡膜蛋白突触回蛋白-3 （SYGR3）进行物理偶联。DAT-SYGR3-VMAT2复合体的形成是通过DAT正常摄取DA所必需的，它不仅取决于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，使用含有短发夹RNA （shRNA）的腺相关病毒载体（AAV）来敲除SYGR3。我们将使用这种病毒，通过分子、细胞和行为技术来确定去除SYGR3对amph诱导的DA逆向转运的影响。实验的设计是这样的，无论结果如何，我们将对AMPH作用的分子机制在多大程度上需要和/或干扰调节DA稳态的蛋白质之间的正常相互作用有更多的了解；此外，这些实验可能会增加我们对VMAT2在产生amph诱导的DA外排中的作用的理解。

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.