RP-4: Disruption of acinar cell NPC1/cholesterol pathway mediates alcoholic pancreatitis SUMMARY Alcoholic pancreatitis remains a disorder with significant morbidity and mortality, with no available treatments directed at the mechanisms of the disease. Thus, there is an urgent need to develop new approaches to reduce the disease severity. Recent data indicate that disordering of lysosomal/autophagy and mitochondrial pathways in acinar cells (the main cell type of the exocrine pancreas) drives pancreatitis, However the mechanisms of organellar dysfunctions in pancreatitis, especially alcoholic pancreatitis, remain poorly understood. Our recent study revealed that nonalcoholic pancreatitis in genetic and experimental models is associated with dysregulation of acinar cell cholesterol metabolism, which promotes pancreatitis responses such as trypsinogen activation, parenchymal necrosis, and inflammation. However, changes in acinar cell cholesterol homeostasis and their role in organellar dysfunctions and disease severity have not been studied in alcoholic pancreatitis. Our preliminary studies for this proposal, using mouse and cellular genetic and experimental models, indicate that the level of lysosomal membrane protein NPC1, a key mediator of cholesterol transport, decreases in alcoholic pancreatitis resulting in acinar cell cholesterol overload. We also found a dramatic decrease in pancreatic NPC1 in human pancreatitis. The data further indicate that NPC1 loss is caused by its misfolding in the endoplasmic reticulum (ER) followed by its degradation. The disruption of NPC1/cholesterol pathway promotes acinar cell lysosomal/autophagy and mitochondrial dysfunctions and worsens alcoholic pancreatitis in experimental models. Our preliminary studies indicate that pharmacologic enhancers of the folding chaperone HSP70 increase NPC1 stability and normalize cholesterol homeostasis and organelle functions. They suggest that a key downstream mechanism linking the disrupted NPC1/cholesterol pathway and organellar dysfunctions in alcoholic pancreatitis is hyperactivation of mTORC1 complex. This project will test the hypothesis that alcoholic pancreatitis is associated with loss of the key lysosomal cholesterol transporter NPC1 caused by its misfolding and subsequent ERAD-proteasomal degradation. This results in dysregulation of cholesterol homeostasis in acinar cells. The disrupted NPC1/cholesterol pathway promotes lysosomal/autophagy and mitochondrial dysfunctions via mTORC1 hyperactivation. Pharmacologic HSP70 enhancers normalize NPC1/cholesterol pathway, mTORC1 activity and organelle functions, and alleviate the severity of alcoholic pancreatitis. The proposed studies will establish NPC1/cholesterol pathway as an important clinically relevant modulator of disease severity; and identify molecular targets, namely NPC1, HSP70 and mTORC1, for new pharmacologic approaches to restore acinar cell cholesterol homeostasis and organellar functions and thus alleviat...