Molecular mechanisms of dense-core vesicle release

致密核小泡分泌生长因子、肽类激素、神经肽和生物胺 在神经元和内分泌细胞中是一种受到严格调控的事件，它驱动着生理过程，如摄食、消化、 储能、哺乳、情绪、止痛。DCV释放受损与代谢和神经系统有关 糖尿病、饮食失调、抑郁症、药物成瘾和亨廷顿病等疾病。然而，分子 控制DCV释放的途径，特别是在神经和内分泌的电兴奋细胞中 系统，在很大程度上仍然没有定义。这项提议的目的是揭示调节DCV的分子机制 分泌物。我们的中心假设是，控制DCV释放的信号通路在不同的类别之间是不同的 细胞之间以及同一细胞内不同群体的DCV之间，根据它们的选择性表达和 贩运关键的，如尚未识别的调节分子。我们进一步假设，类似于小突触小泡， DCV的释放受到G蛋白偶联受体(GPCRs)神经调节信号的严格控制。我们的 创新的假说挑战了现有的只关注细胞内钙的主要研究范式 DCV释放的分子决定因素。不同释放机制的发现将提供新的理解 关于长期存在的与激发神经肽分泌相关的挑战的问题。我们将测试我们的 通过解决以下关键知识差距来提出假设：1)对神经活动模式和广度的理解 驱动不同神经元类DCV释放的细胞内钙浓度范围，2)和理解 神经调节生化信号如何调节释放的活性和/或钙的需求，3) 阐明能够执行这种新形式的神经调制串扰的内源性GPCRs，4)阐明 与包含不同细胞类别中不同货物的DCV相关联的不同蛋白质机械。建议数 研究建立在1)我们最近建立了几种监测速激肽和阿片类药物作用的分析方法的基础上 纹状体中的神经肽，2)我们最近发现的驱动内源性速激肽和 阿片类神经肽的释放，3)我们成功地开发了用于模拟的光激活多肽，从而 校准，内源性释放的时空方面，以及4)光学传感器的最新发展，报告 脑组织中的多肽释放。揭示管理DCV发布的一般原则将建立新的联系 细胞间和细胞内信号通路之间的关系，并揭示它们是如何在分子水平上整合的 许多通过DCV分泌来传递信息的生物系统。从长远来看，我们预计 已发现的独特信号通路可用于治疗代谢性疾病、心理障碍和 治疗神经退行性疾病和慢性疼痛，后者是解决阿片类药物危机的迫切需要。通过 揭示信号通路之间的新联系，这些信号通路是健康和健康中人类生理学的基础 这项工作的发现可能会影响到许多科学领域，包括癌症、心脏病学、发育、 胃肠病学和神经科学。 1

The secretion of growth factors, peptide hormones, neuropeptides and biogenic amines from dense-core vesicles (DCVs) in neurons and endocrine cells is a tightly-regulated event that drives physiological processes such as feeding, digestion, energy storage, lactation, emotion and analgesia. Compromised DCV release is implicated in metabolic and neurological disorders such as diabetes, eating disorders, depression, drug addiction, and Huntington’s disease. Yet the molecular pathways that govern the release of DCVs, particularly in electrically excitable cells of the nervous and endocrine systems, remain largely undefined. The objective of this proposal is to uncover molecular mechanisms that regulate DCV secretion. Our central hypothesis is that the signaling pathways that govern DCV release vary between different classes of cells, and between different populations of DCVs within the same cell, according to their selective expression and trafficking of key, as of yet unidentified regulatory molecules. We further posit that, similar to small synaptic vesicles, DCV release is tightly controlled by neuromodulatory signaling through G protein-coupled receptors (GPCRs). Our innovative hypothesis challenges the existing paradigm that focuses exclusively on intracellular calcium as the primary molecular determinant of DCV release. The discovery of diverse release mechanisms will provide a new understanding for long-standing questions surrounding the challenges associated with evoking neuropeptide secretion. We will test our hypothesis by addressing the following key knowledge gaps: 1) an understanding of the neural activity patterns and wide range of intracellular calcium concentrations that drive DCV release in different neuron classes, 2) and understanding of how neuromodulatory biochemical signaling can adjust the activity and/or calcium requirements for release, 3) elucidation of endogenous GPCRs that can carry out this novel form of neuromodulatory cross-talk, 4) elucidation of the diverse protein machineries associated with DCVs containing different cargoes in different cell classes. The proposed research builds on 1) our recent establishment of several assays for monitoring the actions of tachykinin and opioid neuropeptides in the striatum, 2) our recent discovery of diverse conditions for driving endogenous tachykinin and opioid neuropeptide release, 3) our successful development of photoactivatable peptides for mimicking, and thus calibrating, spatiotemporal aspects of endogenous release, and 4) the recent development of optical sensors that report peptide release in brain tissue. Uncovering the general principles that govern DCV release will establish new connections between intercellular and intracellular signaling pathways and reveal how they are integrated at the molecular level in numerous biological systems that transmit information via DCV secretion. In the long term, we anticipate that the unique signaling pathways uncovered can be exploited to treat metabolic diseases, psychological disorders and neurodegenerative disease, and for chronic pain, latter of which is urgently needed to address the Opioid Crisis. By uncovering new connections between signaling pathways that are fundamental to human physiology in both health and disease, the findings of this work will likely impact numerous scientific fields, including cancer, cardiology, development, gastroenterology, and neuroscience. 1