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: 9470679
• 负责人: James Edward Laurel Dizon Asuncion
• 金额: $ 3.81万
• 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/9470679
• 关键词: Affect; Aggressive behavior; Alleles; Amination; 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.

摘要 单胺类神经递质，如多巴胺和5-羟色胺，在回路中调节快速突触传递 它调节着许多复杂的行为，包括攻击性、睡眠、注意力和情绪。在精神病学领域，许多人 治疗学以单胺系统为靶点，靶向调节和调节受体或转运体 单胺类神经传递。囊泡单胺转运体(VMAT)负责装载所有 进入突触小泡(SVS)和大致密核小泡(LDCV)的单胺类神经递质 分别介导突触和突触外释放。然而，每种类型的功能贡献 囊泡释放到电路的功能和行为尚不清楚。此前对果蝇的研究表明， 胺的释放量和位置可以通过C-末端运输结构域的突变来改变 果蝇VMAT(DVMAT)。在DVMAT零遗传背景下，几种电路和行为的功能 受到干扰，但被野生型和运输突变等位基因的转基因表达所拯救。然而， 有些行为并不是通过贩卖突变人来拯救的。导致DVMAT优先传输到 LDCV不能挽救产卵回路的功能。这表明产卵环路高度活跃。 对神经递质章鱼胺突触和突触外释放之间的微妙平衡敏感 (办公自动化)。我假设内源性DVMAT基因座的转移突变会导致电路 功能障碍，类似于基因拯救实验。为了进一步测试这一想法，我提议创造一种新的基因 使用CRISPR/Cas9改变内源基因中的贩运信号的DVMAT贩运模型。我的 初步数据表明，破坏DVMAT C末端表型的插入突变以前 实验。这一新的遗传模型将有助于研究突变对内源表达的影响。 水平，便于与用于电路分析的遗传和分子工具相结合，并代表了一种新的 用于基因筛选的平台，寻找DVMAT功能的新调节因子。我建议研究一下 果蝇产卵微回路中单胺释放的不同方式。我假设 胺释放方式的改变将导致肌肉收缩和节律性活动的模式 在不同于野生动物的目标器官中。这些拟议的研究将阐明新的机制 胺能信号转导与精神疾病新疗法研究与开发的新途径 精神错乱。