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.

抽象的 单胺神经递质（例如多巴胺和5-羟色胺）调节电路中的快速突触传播 介导许多复杂的行为，包括侵略，睡眠，注意力和情绪。在精神病学中，许多人 治疗剂靶向单胺系统的受体或转运蛋白的介导和调节的转运蛋白 单胺神经传递。囊泡单胺转运蛋白（VMAT）负责加载全部 单胺神经递质分为突触囊泡（SVS）和大型致密囊泡（LDCV） 分别介导突触和突触外释放。但是，每种类型的功能贡献 囊泡释放到电路功能和行为尚不清楚。果蝇先前的研究已经证明 胺释放的数量和位点可以通过C末端贩运域的突变改变 果蝇VMAT（DVMAT）。在DVMAT无效的遗传背景中，几个电路和行为的功能 受到干扰，但被野生型和贩运突变等位基因的转基因表达挽救。然而， 某些行为不是通过贩运突变体挽救的。导致DVMAT优先流量到的突变 LDCV不挽救排卵电路的功能。这表明排卵电路高度高 对神经递质章鱼的突触和突触外释放之间的微妙平衡敏感 （OA）。我假设内源性DVMAT基因基因座中的运输突变将授予电路 功能障碍，类似于遗传救援实验。为了进一步测试这个想法，我建议创建一个新的遗传 DVMAT运输模型使用CRISPR/CAS9改变内源基因中的运输信号。我的 初步数据表明，破坏DVMAT C-末端的indel突变以前 实验。这种新型的遗传模型将有助于研究内源表达突变体的影响 水平，促进与遗传和分子工具进行电路分析的组合，并代表了一种新的 遗传筛选的平台，以找到DVMAT功能的新调节剂。我建议研究 果蝇果蝇中的单胺释放的不同模式。我假设 胺释放方式的改变将导致肌肉收缩和节奏活动的模式 在与野生型动物不同的目标器官中。这些提出的研究将阐明新机制 用于研究和开发精神病的新疗法的AMINEGIC信号和新途径 疾病。