LAP-like non-canonical autophagy in pancreatic acinar cells and its role in the pathophysiology of acute pancreatitis

Acute pancreatitis is a frequent inflammatory disease of pancreas for which there is currently no specific treatment. Understanding cellular and molecular mechanisms of this disease is essential for the development of therapy. The early events in the initiation of acute pancreatitis are associated with damage to pancreatic acinar cells. These cells secrete precursors of digestive enzymes (zymogens). In physiological conditions zymogens are activated in the intestine and participate in digestion. However, at the early stage of acute pancreatitis precursors of digestive enzymes are inappropriately activated inside the pancreas forming active proteases (enzymes which break down proteins) that damage the acinar cells. Trypsin is an important protease involved in this process. We consider that this happens since not all trypsinogen is released from the acinar cell during secretion and some is taken back into the cell in large vesicles termed endocytic vacuoles (which acquire the ability to activate trypsinogen and form the active trypsin).
The current project was initiated by our finding that endocytic vacuoles undergo an unusual form of autophagy. Autophagy is the process that allows a cell to degrade and reutilise cellular components. Formation of double membrane-bounded autophagosomes is a hallmark of well-characterised 'canonical' autophagy. Many proteins involved in this process are known and utilized to identify autophagosomes; LC3 is amongst such proteins. Recently, another degradative cellular mechanism involving LC3 was identified and termed LC3-associated phagocytosis (LAP).
The results of our preliminary experiments suggest that endocytic vacuoles in pancreatic acinar cells undergo non-canonical autophagy similar to LAP rather than canonical autophagy. This finding suggests a novel mechanism of injurious trypsinogen activation in pancreatic acinar cells. Following this unexpected finding we are planning to characterise the role of such LAP-like non-canonical autophagy in acute pancreatitis and to elucidate the molecular mechanism(s) involved in this process. The relevance of LAP-like non-canonical autophagy to intracellular trypsinogen activation and damage/death of pancreatic acinar cells will be a further focus of our study.
The project will involve collaborating laboratories from the University of Liverpool and University of East Anglia. Combined relevant expertise of the applicants includes: development of transgenic models for studies of LAP and autophagy, advanced optical and electron microscopy techniques, molecular biology techniques and considerable experience in evaluation of acute pancreatitis.

The project involves collaboration between laboratories from the University of Liverpool (UoL) and University of East Anglia (UEA) and is focused on characterising cellular mechanisms of acute pancreatitis.
Acute pancreatitis is a frequent inflammatory disease of pancreas for which there is currently no specific treatment. Elucidation of cellular mechanisms of this disease is paramount for the development of therapy. Abnormal pancreatic activation of trypsinogen and consequent damage of pancreatic acinar cells are important initiating factors of this disease. Exposure of the acinar cells to the inducers of acute pancreatitis results in formation of endocytic vacuoles. These organelles are formed as a consequence of abnormal compound exocytosis. The project is based on important findings made by participating laboratories: UoL group found that endocytic vacuoles are the sites of trypsinogen activation in pancreatic acinar cells, whilst UEA group recently characterised the mechanism of LC3-associated phagocytosis (LAP) and produced genetically-modified LAP-deficient mice. Collaboration between the groups has revealed that endocytic vacuoles undergo non-canonical autophagy similar in its properties to LAP. This suggests a novel mechanism of injurious trypsinogen activation in pancreatic acinar cells. Following this unexpected discovery we are planning to characterise the role of LAP-like non-canonical autophagy in experimental models of acute pancreatitis. Three such models, reflecting different aetiologies of human disease, are available in UoL. Another focus of the study will be on elucidating the molecular mechanism of LAP-like non-canonical autophagy and determining its role in death/damage of the acinar cells. In addition to experimental models of acute pancreatitis the project will involve confocal microscopy, correlative light-electron microscopy and standard cell and molecular biology techniques. We have relevant experience with all of these methods.

The study was initiated by collaboration between the research groups from the University of Liverpool (UoL) and University of East Anglia (UEA). The project will strengthen this collaboration and facilitate exchange of knowledge and technical expertise between the Universities.
The work of the pancreatic group at UoL is focussed on the elucidation of critical cellular mechanisms that underlie the pathophysiology of acute pancreatitis (AP), coupled with translational goals to define novel therapies. The "autodigestion" of the pancreas is the principal, characteristic feature of AP that has been recognised for more than a century, although the mechanism responsible has remained elusive. Our preliminary data indicate that a recently discovered process (LAP-like non-canonical autophagy) may be responsible for the premature digestive enzyme activation within the pancreatic acinar cell and therefore underpin the development of AP.
AP is one of the most common gastrointestinal diseases requiring hospitalization, the incidence of which has increased over recent decades. Currently there are ~25,000 new cases per year in the UK which imposes a heavy socio-economic burden on the NHS in patient care, costing ~£2.6 billion per year. There is a serious unmet need in AP, since no specific treatment is available. The results of our study will provide a greater understanding of important cellular changes that occur in AP; identification of critical mechanisms will have immediate impact on the design and development of novel protective agents for AP. This will benefit a team of clinicians, medicinal chemists and pharmacologists of the UoL currently engaged in the development of novel approaches for the treatment of AP. Since several pharmaceutical companies are also engaged in collaborative development of treatments for AP with the team at the UoL, knowledge exchange derived from this project may stimulate further interactions to identify suitable lead compounds that modulate LAP-like non-canonical autophagy.
In the longer term, major beneficiaries of this project will be the patients suffering from this painful, debilitating and sometimes fatal disease. The use of different AP models reflecting different aetiologies e.g. alcoholic AP, will inform about the influence of diet and lifestyle choices on the development of disease. A firm link between patients, scientists and clinicians has been established at Liverpool; the Liverpool Pancreas Patient and Public Involvement Group is an important voice for the sufferers of pancreatic disease and a valuable forum for engagement with the public. Research findings will be disseminated directly to this group throughout the elaboration of this project.
The UEA group has made important contributions to understanding molecular mechanisms of LAP. Furthermore, a valuable LAP deficient mouse model was generated in UEA, allowing systematic investigation of physiological and pathophysiological roles of LAP in different organs and systems. Since LAP is a relatively new phenomenon, the results of our study will have an important impact on the immediate research field focussed on the elucidation of its cellular mechanisms and physiological/pathophysiological roles. The results of our study will also be of benefit to researchers focussed on other exocrine secretory cell types, with potential relevance to understanding diseases such as Sjogren's syndrome.
Additionally, both UoL and UEA research groups serve as important training hubs for PhD students; during the last five years many MRC-, BBSRC- and Wellcome Trust-funded PhD students have successfully completed their training in our laboratories and have subsequently pursued scientific careers. The MRC funding of this project, including support of a postdoctoral scientist in UoL and a research assistant in UEA, would be invaluable for maintaining technical expertise and benefit knowledge transfer to PhD students undergoing training in our laboratories.