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系统的电兴奋细胞中， 在很大程度上是不确定的。该提案的目的是揭示调节DCV分泌的分子机制。 我们的中心假设是，控制DCV释放的信号通路在不同类型的细胞之间存在差异， 在同一细胞内不同DCV群体之间，根据它们的选择性表达和关键的运输， 尚未鉴定的调节分子。我们进一步证实，类似于小突触囊泡，DCV的释放是紧密的， 通过G蛋白偶联受体（GPCR）的神经调节信号控制。我们的创新假设 挑战了现有的范式，专注于细胞内钙作为主要的分子决定因素 DCV释放多种释放机制的发现将为长期存在的问题提供新的认识 围绕与唤起神经肽分泌相关的挑战。我们将测试我们的假设， 以下关键知识差距：1）了解神经活动模式和广泛的细胞内 在不同的神经元类别中驱动DCV释放的钙浓度，2）以及对神经调节如何 生物化学信号传导可以调节活性和/或释放的钙需求，3）阐明内源性GPCR 可以进行这种新形式的神经调节串扰，4）阐明不同的蛋白质机制 与包含不同单元类别中的不同货物的DCV相关联。这项研究的基础是：1）我们最近 建立了几种监测纹状体速激肽和阿片类神经肽作用的方法; 2）我们的 最近发现的驱动内源性速激肽和阿片样神经肽释放的不同条件，3）我们的 光活化肽的成功开发，用于模拟，从而校准，时空方面 内源性释放，以及4）报道脑组织中肽释放的光学传感器的最新发展。 揭示支配DCV释放的一般原则将在细胞间和细胞外之间建立新的联系。 细胞内信号通路并揭示它们如何在分子水平上整合到许多生物系统中 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 可用于治疗代谢性疾病、心理障碍和神经退行性疾病，以及慢性疼痛， 后者是解决阿片类药物危机的迫切需要。通过揭示信号之间的新联系 对于健康和疾病中的人类生理学至关重要的途径，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