Macroautophagy or canonical autophagy, referred to as autophagy hereafter, delivers damaged proteins and organelles to lysosomes for degradation and plays important roles in maintaining tissue homeostasis by reducing tissue damage. During autophagy substrates are sequestered in doublemembraned autophagosomes within the cytoplasm which recruit LC3 to facilitate fusion with lysosomes.
A related pathway, called non-canonical autophagy, conjugates LC3 to single-membraned endosomes, phagosomes or lysosomes during the entry of extracellular material. Examples include engulfment of neighbouring cells by entosis, uptake of apoptotic cells, and micropinocytosis. In phagocytic cells, such as macrophages and dendritic cells, recruitment of LC3 to phagosomes occurs during pathogen uptake and is called LC3-associated phagocytosis (LAP).
The relative roles played by autophagy and non-canonical autophagy ‘in vivo’ are poorly understood. Studies on autophagy in mice have relied on inactivation of essential autophagy proteins, however these proteins are also required for non-canonical autophagy, and mouse models are defective in both pathways. The recruitment of LC3 to membranes during both, autophagy and non-canonical autophagy/ LAP, requires the E3 ligase-like activity of the ATG12–ATG5-ATG16L1 complex. The WD domain of ATG16L1 is required for non-canonical autophagy and LAP, but is not required for autophagy. This finding was employed to create mice defective in non-canonical autophagy and LAP by removing WD domain from the Atg16L1 gene. These mice (called E230) expressed the glutamate residues at 226 and 230 positions in the coiled-coil domain (CCD) of ATG16L1 protein required for binding with WIPI2, an interaction needed for autophagy. A second mouse model was generated that lacked the E230 residue required for autophagy and was called E226.
MEFs from E226 mice were defective in both autophagy and non-canonical autophagy. E226 mice showed growth defects and analysis of liver, kidney, muscle and brain showed accumulation of autophagy substrates p62and LC3. MEFs from the E230 mice were defective in non-canonical autophagy but could activate autophagy. The E230 mice survived postnatal starvation and grew normally. Liver, kidney, brain and muscle of E230 mice maintained levels of autophagy substrates p62 and LC3 that were the same as littermate controls. Tissue damage associated with loss of autophagy such as presence of p62 inclusions and inflammation were observed in tissue of E226 mice but were absent from E230 mice. This comparative study suggests that autophagy maintains tissue homeostasis in mice independently of the WD domain of ATG16L1required for non-canonical autophagy/LAP. A comparison of ATG5-ATG12-ATG16L1 complexes formed in liver and brain suggested that autophagy in the brain may be less dependent on strong interactions between WIPI2 and ATG16L1 than in liver. It is possible that E226 mice, which lack E230 required for strong WIPI2 binding, maintain autophagy in the brain to survive neonatal starvation
