Adhesion GPCR Cirl as a novel regulator of dopamine neurotransmission

粘附 GPCR Cirl 作为多巴胺神经传递的新型调节剂

• 批准号: 10401313
• 负责人: Lilian Coie
• 金额: $ 4.68万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10401313
• 关键词: Acute; Adhesions; Adult; Aggressive behavior; Arousal; Attention deficit hyperactivity disorder; Back; Basal Ganglia; Behavior; Biological Assay; Biological Models; Biosensor; Brain; CRISPR/Cas technology; Cations; Cocaine; Complement; Complex; Corpus striatum structure; Data; Dependence; Development; Disease; Disease model; Dopamine; Drosophila genus; Early-life trauma; Exposure to; Financial compensation; Fluorescence; Functional disorder; G-Protein-Coupled Receptors; Genes; Genetic Polymorphism; Goals; Homologous Gene; Hyperactivity; Knockout Mice; Ligands; Light; Link; Locomotion; Measures; Mediating; Motor; Motor Activity; Mus; Neurons; Phenocopy; Phenotype; Population; Predisposition; Prosencephalon; Protein Fragment; Rattus; Regulation; Research; Risk; Role; Signal Transduction; Single Nucleotide Polymorphism; Stimulant; Substance Use Disorder; Synapses; Temperature; Testing; Therapeutic; Zebrafish; calcium indicator; comorbidity; confocal imaging; density; dopamine transporter; dopaminergic neuron; fluorescence imaging; fly; imaging study; in vivo; in vivo imaging; interest; knock-down; locomotor control; mutant; neuropsychiatric disorder; neurotransmission; new therapeutic target; novel; novel therapeutics; null mutation; optogenetics; postsynaptic; postsynaptic neurons; receptor; relating to nervous system; response; selective expression; sensor; synaptic function; transmission process; trauma exposure; two-photon

PROJECT ABSTRACT/SUMARY Adhesion G-protein coupled receptors (GPCRs) are the second largest class of GPCRs yet their functions and ligands remain predominantly unidentified. Polymorphisms in the gene encoding the adhesion GPCR ADGRL3 have been associated with an increased risk for substance use disorder and attention deficit hyperactivity disorder in various linkage and association studies. Disrupting the function of the ADGRL3 homologs leads to hyperactivity in four different model systems – zebrafish, fruit flies, mice, and rats. In addition to hyperactivity, ADGRL3 knockout mice have higher dopamine levels in forebrain motor regions as well as increased sensitivity to the stimulant cocaine, which acts on the dopamine transporter. Together with dopamine’s established role in mediating locomotion, these findings suggest that ADGRL3 contributes to behavior by modulating dopamine signaling; however, a mechanistic link has yet to be established. The goal of this proposed research is to investigate the synaptic mechanisms that underlie ADGRL3 function in dopaminergic circuits using Drosophila as a model system. I have replicated the hyperactive phenotype in fruit flies that carry a null mutation for the ADGRL3 homolog, Cirl. To directly assay the role of Cirl in neurotransmission, I activated dopamine neurons acutely and found that Cirl null flies were much more sensitive to dopamine neurotransmission. Intriguingly, activating dopamine neurons in Cirl null flies throughout development rescued Cirl null hyperactivity to control levels, indicating that tonic dopamine neurotransmission during development may compensate for lack of Cirl function. To test whether Cirl functions in dopamine neurons to modulate activity, I reintroduced Cirl expression in dopamine neurons in the Cirl null background and instead found that this exacerbated the hyperactive phenotype. Thus, the hyperactivity seen in Cirl null mutants is likely not mediated by its absence from dopamine neurons, but rather in a separate population of neurons in which Cirl normally acts to reduce activity. My imaging studies have revealed that Cirl is expressed post-synaptically throughout the brain, and additionally localizes to the central complex which is involved in locomotion and motor planning and receives dense dopaminergic input. This proposal aims to first identify the dopamine neuron or group of neurons that induces hyperactivity when acutely activated, and identify their synaptic contacts in the central complex. Once I have identified this circuit, I will use optogenetics, in combination with in vivo 2-photon imaging of fluorescent biosensors to delineate the pre and postsynaptic changes in this circuit responsible for the hyperactive response to dopamine neuron activation during adulthood, as well as the reversal of this phenotype in Cirl null flies subjected to continuous dopamine neuron activation throughout development. These results will delineate how an adhesion GPCR modulates dopamine neurotransmission in vivo, and identify a novel therapeutic target for disorders caused by dysregulation of dopaminergic activity such as substance use disorder.

项目摘要/总结 粘附 G 蛋白偶联受体 (GPCR) 是第二大类 GPCR，但其功能和 配体仍然主要未被识别。编码粘附 GPCR ADGRL3 的基因多态性 与物质使用障碍和注意力缺陷多动风险增加有关 各种连锁和关联研究中的障碍。破坏 ADGRL3 同源物的功能会导致 四种不同模型系统（斑马鱼、果蝇、小鼠和大鼠）的过度活跃。除了多动症之外， ADGRL3 基因敲除小鼠的前脑运动区多巴胺水平更高，敏感性也更高 刺激性可卡因作用于多巴胺转运蛋白。连同多巴胺的既定作用 介导运动，这些发现表明 ADGRL3 通过调节多巴胺来促进行为 信号发送；然而，尚未建立机械联系。这项研究的目标是 使用以下方法研究多巴胺能回路中 ADGRL3 功能的突触机制 果蝇作为模型系统。我在携带 null 基因的果蝇中复制了过度活跃的表型 ADGRL3 同源物 Cirl 的突变。为了直接测定 Cirl 在神经传递中的作用，我激活了 敏锐地观察多巴胺神经元，发现 Cirl 无效果蝇对多巴胺更加敏感 神经传递。有趣的是，Cirl 果蝇在整个发育过程中激活多巴胺神经元被拯救 Cirl 无效过度活跃以控制水平，表明发育过程中强直的多巴胺神经传递 可以弥补Cirl功能的不足。为了测试 Cirl 是否在多巴胺神经元中发挥调节活动的作用， 我在 Cirl 空背景中重新引入了多巴胺神经元中的 Cirl 表达，结果却发现 加剧了过度活跃的表型。因此，Cirl 无效突变体中的过度活跃可能不是由介导的 因为它不存在于多巴胺神经元中，而是存在于一个单独的神经元群体中，Cirl 通常在该神经元群体中 采取减少活动的措施。我的影像学研究表明，Cirl 在整个突触后表达 大脑，还定位于参与运动和运动规划的中央复合体 接收密集的多巴胺能输入。该提案旨在首先识别多巴胺神经元或神经元组 急性激活时会引起过度活跃，并识别它们在中央复合体中的突触接触。 一旦我确定了这个电路，我将使用光遗传学，结合体内 2 光子成像 荧光生物传感器来描绘该电路中负责突触前和突触后的变化 成年期对多巴胺神经元激活的过度活跃反应，以及这种表型的逆转 在Cirl中，零果蝇在整个发育过程中受到持续的多巴胺神经元激活。这些结果将 描述粘附 GPCR 如何调节体内多巴胺神经传递，并确定一种新的 多巴胺能活性失调引起的疾病（例如物质使用障碍）的治疗目标。