Ca2+ and secretory dynamics in salivary acinar cells

抽象的。 唾液是一种含有离子和大量蛋白质成分的水状液体，其分泌是唾液腺的主要功能。 唾液腺唾液分泌减少时，其重要性最为明显。通常，这发生在 自身免疫性疾病，干燥综合征（SS）。值得注意的是，在SS中，流体流量的减少发生在任何有害的流体流动之前。 由于免疫细胞浸润而发生的腺体形态变化。重要的是，这表明早期 在疾病中，刺激-分泌偶联机制的缺陷发生在任何腺体破坏之前。一项基本 然而，缺乏对SS中信号传导的最早改变的理解。为了模拟SS中的早期事件，我们使用了 干扰素基因刺激因子（STING）途径被激活的小鼠模型。这种普遍存在的途径， 在SS中被激活，是通过感知来自病毒产生的DNA的细胞质环状二核苷酸而启动的， 线粒体和垂死细胞导致1型干扰素反应，因此反映了SS疾病的关键特征。 在副交感神经输入之后，液体分泌的适当刺激绝对依赖于 刺激-分泌偶联机制的元件适当地定位和激活到细胞的不同结构域， 极化腺泡细胞我们的初步数据显示，STING激活后液体分泌减少约50%，但 矛盾的是，在表达遗传编码的Ca 2+指示剂的动物体内测量的腺泡细胞Ca 2+信号峰值 在腺泡细胞中显著增加。此外，虽然刺激的Ca 2+信号在生理上被限制在顶部， 在这种情况下，Ca 2+信号的空间特征在模型中被破坏。我们发现， 信号有助于最初的功能减退和最终的疾病进展。拟议的研究将 研究SS发作时导致功能减退的机制以及通路 与疾病进展有关。我们将使用互补但独立的方法来实现这些目标。 在具体目标1中，将使用单细胞RNA测序来鉴定唾液腺细胞中的基因和靶向通路。 研究人员将对涉及液体流动中断和疾病进展的人群进行评估，并将为所有后续研究提供信息。我们 将在蛋白质水平和功能测定中验证发现。同时，我们将探索有前途的候选人， 我们的初步功能数据表明，它的丰度、定位或活性 在SS疾病模型中被破坏。在可能的情况下，将在人体组织中验证靶点和途径， 我们在NIDCR的合作者。在具体的目标2中，我们将使用体内成像来定义参与细胞凋亡的机制。 在疾病模型中破坏Ca 2+信号。我们假设，在钙螯合的变化与钙离子 释放/流入机制将被揭示。我们假设改变的Ca 2+信号是一种代偿机制， 以响应最终有害的分泌减少。与这一观点相一致，线粒体形态是 在疾病模型中受到严重破坏。在具体目标3中，我们将研究线粒体Ca 2+处理， 生物能量学和活性氧物质的产生在疾病中改变。总的来说，我们的研究旨在提供 对SS发作时发生的事件的了解，最终应该为新的治疗干预提供靶点。

Abstract. Secretion of saliva, a watery fluid containing ions and a host of protein constituents, is the primary function of the major salivary glands. The importance of salivary fluid secretion is most palpable when it is reduced. Commonly, this occurs in the autoimmune disease, Sjogren’s syndrome (SS). Notably in SS, a decrease in fluid flow occurs prior to any deleterious morphological changes in the gland that occurs because of immune cell infiltration. Importantly, this indicates that early in disease, defects in the stimulus-secretion coupling machinery occur prior to any gland destruction. A fundamental understanding of the earliest alterations in signaling in SS are however lacking. To model early events in SS, we have used a mouse model where the Stimulator of Interferon Genes (STING) pathway is activated. This ubiquitous pathway, known to be activated in SS, is initiated by sensing cytoplasmic cyclic dinucleotides derived from DNA arising from virus, mitochondria and dying cells to result in a type -1 interferon response and thus mirrors key features of SS disease. Following parasympathetic nervous input, the appropriate stimulation of fluid secretion is absolutely dependent on the proper localization and activation of the elements of the stimulus-secretion coupling machinery to distinct domains of the polarized acinar cell. Our preliminary data show that fluid secretion is reduced ~50% following STING activation, but paradoxically the peak acinar cell Ca2+ signal measured in vivo in an animal expressing a genetically encoded Ca2+ indicator in acinar cells is markedly augmented. In addition, while stimulated Ca2+ signals are apically confined in physiological situations, the spatial characteristics of the Ca2+ signal are disrupted in the model. We posit that disruption of the Ca2+ signal contributes to both the initial hypofunction and ultimately the progression of disease. The proposed studies will investigate the mechanisms occurring at the onset of SS which result in hypofunction together with the pathways associated with progression of disease. We will use complimentary, but independent approaches to address these goals. In Specific aim 1, single cell RNA sequencing will be used to identify genes and target pathways in salivary gland cell populations involved in the disruption of fluid flow and progression of disease and will inform all subsequent studies. We will validate findings at the protein level and by functional assays. In tandem, we will explore promising candidates involved in fluid secretion suggested by our preliminary functional data whose abundance, localization or activity is disrupted in the SS disease models. Where possible targets and pathways will be validated in human tissues provided by our collaborators at NIDCR. In specific aim 2, we will use in vivo imaging to define mechanisms which are involved in the disruption of the Ca2+ signal in the disease models. We hypothesize that changes in Ca2+ sequestration together with Ca2+ release/influx mechanisms will be revealed. We hypothesize that the altered Ca2+ signals are a compensatory mechanism in response to reduced secretion that are ultimately detrimental. Consistent with this idea, mitochondrial morphology is severely disrupted in the disease model. In Specific aim 3, we will investigate how mitochondrial Ca2+ handling, bioenergetics and reactive oxygen species production are altered in disease. In total our studies are designed to provide insight in events occurring at the onset of SS which ultimately should provide targets for novel therapeutic intervention.