Interaction of endocytic vacuoles with cellular organelles as a trigger for the cell damage in acute pancreatitis.

Acute pancreatitis is a frequent and life-threatening inflammatory disease. Excessive alcohol intake and gallstones are important risk factors for this debilitating condition. The proportion of the population affected by this disease is increasing rapidly. In conditions of acute pancreatitis, digestive enzymes, which normally help to digest food in the intestine, become active inside the pancreas and this organ "digests" itself. Trypsin is a key digestive enzyme involved in this process. In normal healthy cells of the pancreas, a precursor of trypsin (trypsinogen) is packaged inside small granules (secretory granules). This precursor cannot breakdown proteins or cause damage to the cells. When the process of digestion is stimulated, trypsinogen is released from the cells as a result of secretion - fusion of the secretory granules containing trypsinogen with the surface membrane of the cell. Normally trypsinogen becomes activated only when it reaches intestine where trypsin fulfils its function of breaking down (digesting) dietary proteins. However when we recreated conditions that lead to acute pancreatitis, trypsinogen was activated inappropriately inside the cells of the pancreas. We think that this happens because not all trypsinogen is released from the cell during secretion and some of it is taken back into the cell in vesicles termed endocytic vacuoles (which somehow acquire the ability to activate trypsinogen and to form the active potentially damaging trypsin). We recently discovered that the endocytic vacuoles can rupture and release their contents into the interior of the cell. We also found that the vacuoles can fuse with the cell surface and deliver their contents into the extracellular space. These important unpublished observations form the basis of this application. We hypothesise that the rupture of the vacuoles and/or the fusion of vacuoles with the cell surface are essential for the cell damage that initiates acute pancreatitis (because these processes should allow trypsin to destroy vital intracellular and extracellular proteins). We would like to test this hypothesis. Another important and unresolved question is "How the trypsinogen is activated inside the endocytic vacuoles?" We propose that this happens because the trypsinogen-containing vacuoles interact with cell compartments (organelles) that are responsible for intracellular digestion. Intracellular digestion is needed to recycle the cell parts and proteins. Molecules that can break proteins are found in the compartments responsible for intracellular digestion and they can in principle activate trypsinogen. We are therefore planning to study the interaction of vesicles that retrieve trypsinogen into the cell with organelles responsible for cellular digestion (endosomes, lysosomes and autophagosomes). We will try to determine which of these organelles helps to activate trypsinogen and how the process of organelle interaction is initiated. Trypsinogen activation is not the only hallmark of cell injury. Other indicators include damage to the cellular compartment responsible for energy production (mitochondria) and damage to structures maintaining cell shape (cytoskeleton). We will investigate trypsinogen activation together with the other indicators of cell damage to determine the sequence of events leading to cell death in acute pancreatitis. Finally we will study how damage produced by trypsin in one cell can propagate and affect other cells in the organ. The understanding of the processes leading to activation of digestive enzymes will facilitate the development of the treatment against acute pancreatitis. To accelerate the development of the treatment all information, molecular probes and methodological innovations generated in the course of the project will feed directly into the translational research programme of drug discovery undertaken by the Liverpool NIHR Pancreas Biomedical Research Unit.

Acute pancreatitis (AP) is a severe, debilitating and sometimes fatal disease. Its incidence has escalated dramatically in recent years, however, no specific therapy exists. We have found that inducers of AP trigger formation of large endocytic vacuoles in pancreatic acinar cells within which trypsinogen activation occurs. Our preliminary experiments revealed rupture of these vacuoles and fusion of the vacuoles with the basal and lateral regions of the plasma membrane. In order to understand the pathophysiology of AP and develop translational approaches, we will investigate the mechanisms responsible for the intravacuolar trypsinogen activation, rupture of the vacuoles and fusion of the vacuoles with the plasma membrane. We will test if intravacuolar trypsinogen activation and the rupture of the vacuoles are important for the damage of acinar cells. We will determine if trypsinogen is activated in the vacuoles as a result of the interaction of the vacuoles with autophagosomes / lysosomes / endosomes and whether these organelle interactions are required for the rupture of the vacuoles. We will determine if the cells could be damaged as a result of the fusion of the vacuoles with basolateral membrane and release of trypsin into the extracellular milieu (experiments on undissociated pancreatic tissue will be essential for this part of the project). We hope to determine the sequence of events starting from aberrant signalling and organelle interactions to trypsinogen activation and cell damage. The project will involve confocal and two-photon microscopy of live pancreatic cells and tissues (the primary technical strength of our laboratory), and will be supplemented with immunocytochemistry, molecular biology, electrophysiology and electron microscopy. The project will be closely coordinated with the work of the Liverpool NIHR Pancreas Biomedical Research Unit so that any technical or conceptual developments could benefit the translational efforts of the Unit.

The MRC-funded pancreatic research group in Liverpool primarily concentrates on studies of fundamental cellular mechanisms relevant to the pathophysiology of acute pancreatitis (AP) and pancreatic cancer. The work of the group is closely co-ordinated with the Liverpool NIHR Pancreas Biomedical Research Unit, the only UK Research Unit focused on translational pancreatic research. The MRC funding will therefore underpin conceptual and technical developments in the group necessary for the translational efforts of NIHR unit. NIHR unit coordinates the efforts of clinicians, organic chemists, structural biologists, physiologists (our group) and pharmacologists of the University of Liverpool aiming to develop drugs against AP. In the framework of this project, all information about the interaction of endocytic vacuoles with cellular organelles, rupture of the vacuoles, fusion of the vacuoles with the plasma membrane as well as about intraorganellar and cytosolic trypsinogen activation will be immediately shared with the NIHR unit and could be used for the informed design of pharmacological approaches for the treatment of AP. Technical information about the measurements of formation / interaction of vacuoles, trypsinogen activation and release of trypsin from vacuoles in different cell and tissue preparations will be also shared with NIHR and could be utilised for development of procedures for testing new pharmacological compounds. The NIHR unit has established contacts with pharmaceutical companies interested in developing or testing compounds which could alleviate acute pancreatitis. The relevant conceptual knowledge and methodological expertise generated in the course of our project will be conveyed via the NIHR to collaborating pharmaceutical companies. Co-applicants are also actively involved in communicating the results of their research to the general public (see Pathways to Impact).
The advanced equipment (e.g. two-photon microscope) available in the pancreatic research group and technical expertise accumulated in the group (e.g. combined patch-clamp and confocal/two-photon microscopy, cloning and engineering fluorescently- tagged expression constructs and production of viruses necessary for efficient acinar cell transfection), will complement high throughput instruments and techniques established in the NIHR unit to create an optimal technology platform, necessary for both discovery and translation. Importantly, the pancreatic research group serves as a training base for MRC and Wellcome PhD students, as well as for clinical fellows undertaking research projects. Two MRC students are currently conducting research in our laboratory; two MRC students have recently successfully completed their studies and are now employed as post-doctoral scientists. Two clinical fellows are undertaking research projects in our laboratory. All PhD students are involved in collaboration with the NIHR unit (all are jointly supervised by members of the group and the unit). The MRC support is crucial for maintaining this training base; the equipment provided and supported by MRC is essential for the experimental work. The MRC support for the salary of an experienced postdoctoral scientist allows the maintenance of technical skills and transfer of expertise in advanced experimentation using primary cells and tissues to PhD students. The MRC support of the current grant application will therefore not only provide resources necessary for the specific project, aiming to characterise crucial early events of AP, but also enhance the collaborative efforts and the translational research of the Pancreas NIHR Unit, as well as strengthen the training base for PhD students with an interest in cell signalling, cell biology and pathophysiology of the pancreas.