Trafficking of the Drosophila Vesicular Monoamine Transporter

描述（由申请人提供）：囊泡单胺转运蛋白（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会导致类似抑郁症的行为状态。细胞内运输已被提出通过控制VMAT在两种类型的分泌囊泡的定位来调节VMAT：聚集在神经末梢活跃区的突触囊泡（SVs）和发挥神经调节作用的大致密核心囊泡（ldcv）。尽管在培养中已经确定了VMAT的转运基元，但在体内将其定位到SVs和ldcv所需的信号仍不清楚。此外，尚不清楚VMAT或任何其他神经递质转运体中的运输信号如何影响完整动物的定位或功能。我们建议用遗传生物黑腹果蝇来探索这些问题。我们描述了内源性dVMAT基因的突变，这为分析含有贩运突变体的转基因提供了有用的背景。我们还发现了两个剪接变体（DVMAT-A和B），它们包含不同的c端运输结构域和DVMAT-A的c端中体外内吞作用所需的基元。我们现在将确定这个基序和其他潜在的内吞作用信号如何促进DVMAT-A在体内定位到SVs。我们还将识别将DVMAT-A分类到ldcv所需的信号。为了确定dVMAT - a在体内的功能是否需要c端传输域，我们将比较dVMAT - a、dVMAT - b和c端截断对dVMAT突变体中依赖dVMAT - a功能的缺陷的修复能力。dVMAT突变还可以阻止表达dVMAT - b的视网膜下胶质细胞中的组胺储存，从而提供了一种研究这种不寻常亚型在体内功能的方法。我们将进行基因拯救实验，以确定DVMAT-B的c端是否需要其在体内发挥作用。为了进一步研究DVMAT-B中新的运输结构域如何在体内促进其功能，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相关的神经递质转运体是如何在体内调节的。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.