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 toge...