Trafficking of the Drosophila Vesicular Monoamine Transporter

果蝇囊泡单胺转运蛋白的贩运

• 批准号: 7808800
• 负责人: David Evan Krantz
• 金额: $ 30.54万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7808800
• 关键词: Adult; Anabolism; Animals; Behavioral; Biological Assay; Breeding; C-terminal; Cell membrane; Cells; Defect; Dense Core Vesicle; Development; Doctor of Medicine; Doctor of Philosophy; Drosophila genus; Drosophila melanogaster; Drug usage; Endocytosis; Endogenous depression; Fertility; Future; Genes; Genetic; Genetic Models; Histamine; Homologous Gene; In Vitro; Link; Mental Depression; Molecular Genetics; Mutation; Nerve; Nervous System Physiology; Nervous system structure; Neuroglia; Neurons; Neurotransmitters; Organism; Pharmaceutical Preparations; Protein Isoforms; Proteins; RNA Splicing; Recycling; Research Personnel; Reserpine; Role; Secretory Vesicles; Signal Transduction; Site; Sorting - Cell Movement; Synaptic Vesicles; Testing; Transgenes; Transgenic Organisms; Variant; Vesicle; fly; in vivo; monoamine; mutant; neuropsychiatry; novel; prevent; programs; research study; tool; trafficking; treatment strategy; vesicular monoamine transporter

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.

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神经递质的储存，药物利血平抑制VMAT可导致类似抑郁症的行为状态。已经提出细胞内运输通过控制VMAT定位于两种类型的分泌囊泡来调节VMAT：聚集在神经末梢的活性区的突触囊泡（SV）和执行神经调节作用的大致密核心囊泡（LDCV）。虽然VMAT的运输基序已经在培养中鉴定，但其在体内定位于SV和LDCV所需的信号仍不清楚。此外，还不知道VMAT或任何其他神经递质转运蛋白中的运输信号如何影响完整动物的定位或功能。我们建议使用模式遗传生物果蝇来探索这些问题。我们已经表征了内源性dVMAT基因中的突变，这为分析含有贩运突变体的转基因提供了有用的背景。我们还鉴定了两种剪接变体（DVMAT-A和B），其含有不同的C-末端运输结构域和体外内吞作用所需的DVMAT-A的C-末端中的基序。我们现在将确定这个基序和其他潜在的内吞信号如何有助于DVMAT-A在体内定位到SV。我们还将识别将DVMAT-A排序为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体内功能所必需的，我们将比较DVMAT-A、DVMAT-B和C-末端截短拯救依赖于DVMAT-A功能的dVMAT突变体中的缺陷的能力。dVMAT的突变还防止表达DVMAT-B的视网膜下神经胶质中的组胺储存，从而提供了一种在体内研究这种不寻常的同种型的功能的测定。我们将进行遗传拯救实验，以确定是否DVMAT-B的C-末端是其体内功能所必需的。为了进一步研究DVMAT-B中新的运输结构域如何有助于其体内功能，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蛋白在体内是如何调节的。这些结果可能有助于开发新的抑郁症治疗策略。