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Background Autophagosome, the central organelle in autophagy process, can assemble via canonical pathway mediated by LC3-II, the lipidated form of autophagy-related protein LC3/ATG8, or noncanonical pathway mediated by the small GTPase Rab9. Canonical autophagy is essential for exocrine pancreas homeostasis, and its disordering initiates and drives pancreatitis. The involvement of noncanonical autophagy has not been explored. We examine the role of Rab9 in pancreatic autophagy and pancreatitis severity. Methods We measured the effect of Rab9 on parameters of autophagy and pancreatitis responses using transgenic mice overexpressing Rab9 (Rab9TG) and adenoviral transduction of acinar cells. Effect of canonical autophagy on Rab9 was assessed in ATG5-deficient acinar cells. Results Pancreatic levels of Rab9 and its membrane-bound (active) form decreased in rodent pancreatitis models and in human disease. Rab9 overexpression stimulated noncanonical and inhibited canonical/LC3-mediated autophagosome formation in acinar cells through up-regulation of ATG4B, the cysteine protease that delipidates LC3-II. Conversely, ATG5 deficiency caused Rab9 increase in acinar cells. Inhibition of canonical autophagy in Rab9TG pancreas was associated with accumulation of Rab9-positive vacuoles containing markers of mitochondria, protein aggregates, and trans-Golgi. The shift to the noncanonical pathway caused pancreatitis-like damage in acinar cells and aggravated experimental pancreatitis. Conclusions The results show that Rab9 regulates pancreatic autophagy and indicate a mutually antagonistic relationship between the canonical/LC3-mediated and noncanonical/Rab9-mediated autophagy pathways in pancreatitis. Noncanonical autophagy fails to substitute for its canonical counterpart in protecting against pancreatitis. Thus, Rab9 decrease in experimental and human pancreatitis is a protective response to sustain canonical autophagy and alleviate disease severity., Graphical abstract

Background & Aims: The pathogenic mechanism of pancreatitis is poorly understood. Recent evidence implicates defective autophagy in pancreatitis responses; however, the pathways mediating impaired autophagy in pancreas remain largely unknown. Here, we investigate the role of lysosome associated membrane proteins (LAMPs) in pancreatitis. Methods: We analyzed changes in LAMPs in experimental models and human pancreatitis, and the underlying mechanisms: LAMP deglycosylation and degradation. LAMP cleavage by cathepsin B (CatB) was analyzed by mass spectrometry. We used mice deficient in LAMP-2 to assess its role in pancreatitis. Results: Pancreatic levels of LAMP-1 and LAMP-2 greatly decrease across various pancreatitis models and in human disease. Pancreatitis does not trigger the LAMPsâ bulk deglycosylation but induces their degradation via CatB-mediated cleavage of the LAMP molecule close to the boundary between luminal and transmembrane domains. LAMP-2 null mice spontaneously develop pancreatitis that begins with acinar cell vacuolization due to impaired autophagic flux, and progresses to severe pancreas damage characterized by trypsinogen activation, macrophage-driven inflammation, and acinar cell death. LAMP-2 deficiency causes a decrease in pancreatic digestive enzymes content, and stimulates the basal and inhibits cholecystokinin-induced amylase secretion by acinar cells. The effects of LAMP-2 knockout and acute cerulein pancreatitis overlap, which corroborates the pathogenic role of LAMP decrease in experimental pancreatitis models. Conclusions: The results indicate a critical role for LAMPs, particularly LAMP-2, in maintaining pancreatic acinar cell homeostasis and provide evidence that defective lysosomal function, resulting in impaired autophagy, leads to pancreatitis. Mice with LAMP-2 deficiency present a novel genetic model of human pancreatitis caused by lysosomal/autophagic dysfunction. Keywords: Amylase Secretion, Autophagy, Cathepsin B, Cerulein