Vesicular monoamine transporter trafficking affects mode of neurotransmitter release and microcircuit function

囊泡单胺转运蛋白运输影响神经递质释放模式和微电路功能

• 批准号: 9765409
• 负责人: James Edward Laurel Dizon Asuncion
• 金额: $ 4.5万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-19 至 2020-09-18
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9765409
• 关键词: Affect; Aggressive behavior; Alleles; Amines; Animals; Antidepressive Agents; Attention; Behavior; Behavioral; C-terminal; CRISPR/Cas technology; Calcium; Cell membrane; Cells; Complex; DNA; Data; Dense Core Vesicle; Dopamine; Drosophila genus; Drosophila melanogaster; Equilibrium; Functional disorder; Genes; Genetic; Genetic Models; Genetic Screening; Glutamates; Image; Imaging Techniques; Invertebrates; Laboratories; Link; Logic; Mediating; Membrane Transport Proteins; Mental Depression; Mental disorders; Modeling; Molecular; Molecular Genetics; Mood Disorders; Moods; Muscle; Muscle Contraction; Mutation; Nerve; Nervous system structure; Neuroanatomy; Neurons; Neurotransmitters; Octopamine; Organ; Other Genetics; Oviposition; Pathway interactions; Pattern; Periodicity; Pharmaceutical Preparations; Pharmacology; Phenocopy; Process; Psychiatry; Secretory Vesicles; Serotonin; Signal Transduction; Site; Sleep; Synapses; Synaptic Transmission; Synaptic Vesicles; System; Testing; Therapeutic; Transgenes; Transgenic Organisms; experimental study; extracellular; functional restoration; insertion/deletion mutation; monoamine; mutant; neuroregulation; neurotransmission; neurotransmitter release; neurotransmitter reuptake; noradrenaline transporter; novel; novel therapeutics; null mutation; optogenetics; protein transport; receptor; repaired; research and development; synaptic function; therapeutic target; tool; trafficking; vesicular monoamine transporter; vesicular release

Abstract Monoamine neurotransmitters, such as dopamine and serotonin, modulate fast synaptic transmission in circuits that mediate many complex behaviors including aggression, sleep, attention, and mood. In psychiatry, many therapeutics target monoamine systems at either the receptors or transporters that mediate and regulate monoamine neurotransmission. The vesicular monoamine transporter (VMAT) is responsible for loading all monoamine neurotransmitters into both synaptic vesicles (SVs) and large dense-core vesicles (LDCVs), which mediate synaptic and extrasynaptic release, respectively. However, the functional contribution of each type of vesicular release to circuit function and behavior is unknown. Previous studies in Drosophila have demonstrated that the amount and site of amine release can be altered by mutations in the C-terminal trafficking domain of Drosophila VMAT (DVMAT). In a DVMAT null genetic background, the function of several circuits and behaviors are perturbed, but are rescued by transgenic expression of wild-type and trafficking mutant alleles. However, some behaviors are not rescued by trafficking mutants. Mutations that cause DVMAT to preferentially traffic to LDCVs do not rescue function of the oviposition circuit. This suggests that the oviposition circuit is highly sensitive to the delicate balance between synaptic and extrasynaptic release of the neurotransmitter octopamine (OA). I hypothesize that trafficking mutations in the endogenous DVMAT gene locus will confer circuit dysfunction, resembling genetic rescue experiments. To further test this idea, I propose to create a new genetic model of DVMAT trafficking using CRISPR/Cas9 to alter trafficking signals in the endogenous gene. My preliminary data demonstrate that indel mutations that disrupt the DVMAT C-terminus phenocopy previous experiments. This novel genetic model will be useful to study the effects of mutants at endogenous expression levels, facilitates combinations with genetic and molecular tools for circuit analysis, and represents a new platform for genetic screens to find novel regulators of DVMAT function. I propose to study the contributions of the different modes of monoamine release in the Drosophila melanogaster oviposition microcircuit. I hypothesize that alteration of the mode of amine release will result in patterns of muscular contractions and rhythmic activity in target organs that will differ from wild-type animals. These proposed studies will elucidate novel mechanisms of aminergic signaling and new avenues for the research and development of new therapeutics for psychiatric disorders.

抽象的 单胺神经递质，如多巴胺和血清素，调节回路中的快速突触传递 它调节许多复杂的行为，包括攻击性、睡眠、注意力和情绪。在精神病学领域，许多 治疗以介导和调节受体或转运蛋白的单胺系统为目标 单胺神经传递。囊泡单胺转运蛋白（VMAT）负责装载所有 单胺神经递质进入突触小泡（SV）和大致密核心小泡（LDCV）， 分别介导突触和突触外释放。然而，每种类型的功能贡献 囊泡释放对电路功能和行为的影响尚不清楚。先前对果蝇的研究表明 胺释放的量和位点可以通过 C 端运输结构域的突变来改变 果蝇 VMAT (DVMAT)。在 DVMAT 无效遗传背景下，几个回路和行为的功能 受到干扰，但通过野生型和运输突变等位基因的转基因表达而得到拯救。然而， 有些行为并不能通过贩卖突变体来挽救。导致 DVMAT 优先流量的突变 LDCV 不具有产卵回路的救援功能。这表明产卵回路高度 对神经递质章鱼胺的突触和突触外释放之间的微妙平衡敏感 （办公自动化）。我假设内源 DVMAT 基因座中的运输突变将赋予电路 功能障碍，类似于基因拯救实验。为了进一步测试这个想法，我建议创建一个新的基因 使用 CRISPR/Cas9 改变内源基因中的运输信号的 DVMAT 运输模型。我的 初步数据表明，破坏 DVMAT C 末端表型的 indel 突变 实验。这种新颖的遗传模型将有助于研究突变体在内源表达方面的影响 水平，促进与遗传和分子工具的组合进行电路分析，并代表了一种新的 基因筛选平台，寻找 DVMAT 功能的新型调节因子。我建议研究以下人员的贡献 果蝇产卵微电路中单胺释放的不同模式。我假设 胺释放模式的改变将导致肌肉收缩和节律活动的模式 与野生型动物不同的靶器官。这些拟议的研究将阐明新的机制 胺能信号传导的研究和精神科新疗法研究和开发的新途径 失调。