Molecular mechanisms of dense-core vesicle release

密核囊泡分泌生长因子、肽类激素、神经肽和生物胺 在神经元和内分泌细胞中，是一种受到严格调节的事件，它驱动着诸如进食，消化， 能量储存、哺乳、情感和镇痛。受损的DCV释放涉及代谢和神经系统 疾病如糖尿病、饮食失调、抑郁症、药物成瘾和亨廷顿氏病。然而， 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不确定。该提案的目的是揭示调节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类别之间存在差异 以及同一细胞内的不同DCV群体之间，根据它们的选择性表达和 贩运关键的，尚未识别的调节分子。我们进一步证实，类似于小突触囊泡， DCV的释放受到通过G蛋白偶联受体（GPCR）的神经调节信号的严格控制。我们 创新的假说挑战了现有的范式，专注于细胞内钙作为主要的 DCV释放的分子决定因素。不同释放机制的发现将提供一个新的认识 对于长期存在的问题，围绕与唤起神经肽分泌相关的挑战。我们将测试我们的 通过解决以下关键知识差距的假设：1）了解神经活动模式和广泛的 在不同神经元类别中驱动DCV释放的细胞内钙浓度范围，2）以及对 神经调节生物化学信号传导如何调节活性和/或释放的钙需求，3） 阐明了可以进行这种新形式的神经调节性串扰的内源性GPCR，4）阐明了神经调节性串扰的内源性GPCR。 与含有不同细胞类别中的不同货物的DCV相关的不同蛋白质机器。拟议 研究建立在1）我们最近建立的几种用于监测速激肽和阿片样物质的作用的测定方法上 2）我们最近发现的驱动内源性速激肽的不同条件， 阿片类神经肽释放，3）我们成功开发了用于模拟的光活化肽，因此 校准，内源性释放的时空方面，和4）最近发展的光学传感器，报告 脑组织中的肽释放。揭示管理DCV发布的一般原则将建立新的连接 细胞间和细胞内信号通路之间的联系，并揭示它们如何在分子水平上整合， 许多生物系统通过DCV分泌传递信息。从长远来看，我们预计， 发现的独特信号通路可用于治疗代谢疾病、心理障碍和 神经退行性疾病和慢性疼痛，后者是迫切需要解决阿片类药物危机。通过 揭示信号通路之间的新联系，这些信号通路对人类健康和 疾病，这项工作的发现可能会影响许多科学领域，包括癌症，心脏病，发展， 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