Contribution of synaptic vesicle proteins to molecular mechanisms of amphetamine

描述(申请人提供)：毒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。滥用药物增加了与奖赏加工有关的神经结构中的多巴胺(DA)浓度；精神刺激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，导致其靶区(分别是伏隔核和背侧纹状体)中的多巴胺浓度增加。精神刺激剂苯丙胺(AMPH)作用于单胺转运体，包括质膜多巴胺转运体(DAT)和囊泡单胺转运体2(VMAT2)。DAT负责将DA从细胞外间隙重新摄取到突触前终末。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，DA从胞浆净流出到细胞外间隙。Amph在VMAT2的作用知之甚少，也是一个有争议的主题；然而，Amph与VMAT2的结合导致DA从突触小泡流出到胞浆中，这是公认的。最近的研究表明，DAT和VMAT2是通过突触小泡膜蛋白突触素-3(SYGR3)进行物理偶联的。这种DAT-SYGR3-VMAT2复合体的形成是通过DAT正常摄取DA所必需的，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，神经元暴露于AMPH可能导致SYGR3介导的DAT和VMAT2偶联的改变。这些假说将在多巴胺能神经元的原代培养中以及体内成年大鼠身上进行测试，使用腺相关病毒载体(AAV)，该载体含有旨在击倒SYGR3的短发夹状RNA(ShRNA)。我们将使用这种病毒，通过分子、细胞和行为技术来确定SYGR3的去除对Amph诱导的DA反向运输的影响。实验的设计是这样的，无论结果如何，我们都将进一步了解AMPH作用的分子机制需要和/或干扰调节DA动态平衡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外流中的作用的理解。

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.