Trafficking of the Drosophila Vesicular Monoamine Transporter

描述(申请人提供)：囊泡单胺转运体(VMAT)负责神经系统中所有单胺类神经递质的储存，药物利血平抑制VMAT会导致类似抑郁的行为状态。细胞内转运被认为是通过控制VMAT定位于两种类型的分泌小泡来调节的：聚集在神经末梢活动区的突触小泡(SVS)和发挥神经调节作用的大致密核心小泡(LDCV)。尽管VMAT的贩运基序已经在培养中被确定，但其在体内定位到SVS和LDCV所需的信号仍不清楚。此外，VMAT或任何其他神经递质转运体中的贩运信号如何影响完整动物的定位或功能尚不清楚。我们建议使用模式遗传有机体黑腹果蝇来探索这些问题。我们已经鉴定了内源性dVMAT基因的突变，这为分析含有运输突变的转基因提供了有用的背景。我们还鉴定了两个剪接变异体(DVMAT-A和B)，它们含有不同的C-末端运输结构域和体外内吞作用所需的DVMAT-A的C-末端基序。我们现在将确定这个基序和其他潜在的内吞信号如何在体内将DVMAT-A定位到SVS。我们还将确定将DVMAT-A分类为LDCV所需的信号。为了确定DVMAT-A的C末端运输结构域是否是其在体内发挥功能所必需的，我们将比较DVMAT-A、DVMAT-B和C末端截断对依赖于DVMAT-A功能的dVMAT突变体的缺陷的修复能力。DVMAT的突变也阻止了表达DVMAT-B的视网膜下胶质细胞中组胺的储存，从而为研究这种不寻常的异构体在体内的功能提供了一种分析方法。我们将进行遗传救援实验，以确定DVMAT-B的C末端是否为其在体内发挥作用所必需的。为了进一步研究DVMAT-B中新的运输结构域在体内对其功能的贡献，我们将确定其在视网膜下胶质细胞中的亚细胞定位。这些实验将有助于确定与神经精神疾病有关的神经递质转运体在体内是如何调节的。这一结果可能有助于开发新的抑郁症治疗策略。

DESCRIPTION (provided by applicant): The vesicular monoamine transporter (VMAT) is responsible for the storage of all monoamine neurotransmitters in the nervous system and inhibition of VMAT by the drug reserpine causes a behavioral state resembling depression. Intracellular trafficking has been proposed to regulate VMAT by controlling its localization to two types of secretory vesicles: synaptic vesicles (SVs) that cluster at the active zone of nerve terminals, and large dense core vesicles (LDCVs) that perform a neuromodulatory role. Although trafficking motifs for VMAT have been identified in culture, the signals required for its localization to SVs and LDCVs in vivo remain unclear. Furthermore, it is not known how trafficking signals in either VMAT or any other neurotransmitter transporter effects either localization or function in an intact animal. We propose to use the model genetic organism Drosophila melanogaster to explore these questions. We have characterized a mutation in the endogenous dVMAT gene, which provides a useful background for the analysis of transgenes containing trafficking mutants. We also have identified two splice variants (DVMAT-A and B) that contain divergent C-terminal trafficking domains and a motif in the C-terminus of DVMAT-A required for endocytosis in vitro. We will now determine how this motif and other potential endocytosis signals contribute to the localization of DVMAT-A to SVs in vivo. We also will identify signals required to sort DVMAT-A to LDCVs. To determine whether the C-terminal trafficking domain of DVMAT-A is required for its function in vivo, we will compare the ability of DVMAT-A, DVMAT-B and a C-terminal truncation to rescue defects in dVMAT mutants that depend on the function of DVMAT-A. Mutation of dVMAT also prevents histamine storage in subretinal glia that express DVMAT-B, thus providing an assay to study the function of this unusual isoform in vivo. We will perform genetic rescue experiments to determine whether the C-terminus of DVMAT-B is required for its function in vivo. To further investigate how the novel trafficking domain in DVMAT-B contributes to its function in vivo, we will determine its subcellular localization in the subretinal glia. These experiments will help determine how a neurotransmitter transporter linked to neuropsychiatric illness is regulated in vivo. The results may aid the development of novel treatment strategies for depression.