Reference number : GC-2017-Q1-02485, amendment number : , agreement type : G, recipient legal name : Carréno, Sébastien (Université de Montréal), recipient country : CA, recipient province : nan, recipient city : Quebec|Québec, recipient postal code : nan, program name english : Discovery Grants Program - Individual, program name french : Programme de subventions à la découverte - individuelles, program purpose english : Autophagy, the process of self-eating, is evolutionarily conserved from yeast to mammals. This process leads to the lysosomal degradation of cytosolic components such as organelles, proteins or nucleic acids. Autophagy starts with the formation of a double-membrane sac that expends and forms the autophagosome that can engulf cytosolic components. The autophagosome then fuse with lysosomes that discharge their digestive enzymes to promote cargo degradation. Autophagy regulates the dynamic remodeling of subcellular compartments and participates to the control of several physiological processes such as embryogenesis, response to starvation, anti-tumorigenesis and anti-senescence. Therefore, the understanding of this important process is essential for both fundamental and biomedical research.
 
We conducted an in cellulo Drosophila screen on candidate genes controlling the phosphoinositide cycle and we found that the inositol 5-phosphatase dOCRL controls PI(4,5)P2 homeostasis (Ben El Kadhi et al., Current biology 2011). The majority of the PI(4,5)P2 is concentrated at the plasma membrane where it participates in nearly all events that involve the cell surface. We demonstrated that dOCRL is associated with endosomes and lysosomes and that it dephosphorylates PI(4,5)P2 on lysosomes to restrict this phosphoinositide at the plasma membrane. When dOCRL is knocked-down by RNAi, cells abnormally accumulate PI(4,5)P2 at the surface of giant lysosomes. In addition, we recently reported that PTEN activation promotes the hydrolysis of PI(4,5)P2, its own enzymatic product, by activating Phospholipase C (PLC) enzymes on endomembranes. We showed that this function can rescue dOCRL loss (Ben El Kadhi et al., in prep).
 
In our unpublished observations we showed that depletion of dOCRL leads to a defect in the autophagic flux. Lysosomes cannot longer fuse with autophagosome. Importantly we showed that activation PTEN activation can restore the autophagic flux in dOCRL depleted cells. We also discovered that this function was independent of PTEN enzymatic activity but was supported by a minimal chimera encompassing two of its non-enzymatic conserved domains. In addition we found that PTEN depletion promotes accumulation of Pi(4,5)P2 on endomembranes. 
 Besides its canonical role in autophagy activation by inhibiting the PI3K/Akt/mTORC1 pathway, our results suggest a novel role of PTEN in controlling the autophagic flux, independently of its enzymatic activity and through regulation of Pi(4,5)P2 levels on lysosomes.
 
To test this hypothesis we we will characterize the PTEN- PLC signaling pathway (aim 1) and we will characterize the role of this pathway on Pi(4,5)P2 homeostasis and autophagy (aim 2)., program purpose french : Autophagy, the process of self-eating, is evolutionarily conserved from yeast to mammals. This process leads to the lysosomal degradation of cytosolic components such as organelles, proteins or nucleic acids. Autophagy starts with the formation of a double-membrane sac that expends and forms the autophagosome that can engulf cytosolic components. The autophagosome then fuse with lysosomes that discharge their digestive enzymes to promote cargo degradation. Autophagy regulates the dynamic remodeling of subcellular compartments and participates to the control of several physiological processes such as embryogenesis, response to starvation, anti-tumorigenesis and anti-senescence. Therefore, the understanding of this important process is essential for both fundamental and biomedical research.
 
We conducted an in cellulo Drosophila screen on candidate genes controlling the phosphoinositide cycle and we found that the inositol 5-phosphatase dOCRL controls PI(4,5)P2 homeostasis (Ben El Kadhi et al., Current biology 2011). The majority of the PI(4,5)P2 is concentrated at the plasma membrane where it participates in nearly all events that involve the cell surface. We demonstrated that dOCRL is associated with endosomes and lysosomes and that it dephosphorylates PI(4,5)P2 on lysosomes to restrict this phosphoinositide at the plasma membrane. When dOCRL is knocked-down by RNAi, cells abnormally accumulate PI(4,5)P2 at the surface of giant lysosomes. In addition, we recently reported that PTEN activation promotes the hydrolysis of PI(4,5)P2, its own enzymatic product, by activating Phospholipase C (PLC) enzymes on endomembranes. We showed that this function can rescue dOCRL loss (Ben El Kadhi et al., in prep).
 
In our unpublished observations we showed that depletion of dOCRL leads to a defect in the autophagic flux. Lysosomes cannot longer fuse with autophagosome. Importantly we showed that activation PTEN activation can restore the autophagic flux in dOCRL depleted cells. We also discovered that this function was independent of PTEN enzymatic activity but was supported by a minimal chimera encompassing two of its non-enzymatic conserved domains. In addition we found that PTEN depletion promotes accumulation of Pi(4,5)P2 on endomembranes. 
 Besides its canonical role in autophagy activation by inhibiting the PI3K/Akt/mTORC1 pathway, our results suggest a novel role of PTEN in controlling the autophagic flux, independently of its enzymatic activity and through regulation of Pi(4,5)P2 levels on lysosomes.
 
To test this hypothesis we we will characterize the PTEN- PLC signaling pathway (aim 1) and we will characterize the role of this pathway on Pi(4,5)P2 homeostasis and autophagy (aim 2)., agreement title english : Role of PTEN during Pi(4,5)P2 homeostasis and autophagy, agreement title french : Role of PTEN during Pi(4,5)P2 homeostasis and autophagy, agreement number : RGPIN, agreement value : 130000.0, agreement start date : 2017-05-10, agreement end date : nan, description english : nan, description french : nan, expected results english : nan, expected results french : nan, additional information english : Grant or Award spanning more than one fiscal year. (2017-2018 to 2022-2023), additional information french : Subvention ou bourse octroyée s'appliquant à plus d'un exercice financier. (2017-2018 à 2022-2023), owner organization : nserc-crsng, owner organization title : Natural Sciences and Engineering Research Council of Canada | Conseil de recherches en sciences naturelles et en génie du Canada