A two-step model of autophagy: autophagosome formation, degradation and net turnover

Autophagy is a complex process that encompasses the enclosure of cytoplasmic debris or dysfunctional organelles in membranous vesicles, the autophagosomes, for their elimination in the lysosomes. A gold-standard method to assess its induction is the analysis of the autophagic flux using as a surrogate the expression of the microtubule-associated light chain protein 3 conjugated to phosphatidylethanolamine (LC3-II) by Western blot, in the presence of lysosomal inhibitors. Therefore, the current definition of autophagy flux actually puts the focus on the degradation stage of autophagy. In contrast, the most important autophagy controlling genes that have been identified in the last few years in fact target early stages of autophagosome formation. From a biological standpoint is therefore conceivable that autophagosome formation and degradation are independently regulated and we argue that both stages need to be systematically analyzed. Here, we propose a simple two-step model to understand changes in autophagosome formation and degradation using data from conventional LC3-II Western blot, and test it using two models of autophagy modulation in cultured microglia, the brain macrophages: rapamycin and the ULK1/2 inhibitor, MRT68921. Our model provides a comprehensive understanding of the autophagy process and will help to unravel the effect of genetic, pharmacological, and environmental manipulations on both the formation and degradation of autophagosomes. ### Competing Interest Statement The authors have declared no competing interest. * APh : Autophagosome ATG : Autophagy-related protein BAF : Bafilomycin A1 BCA : Bicinchoninic Acid DegR : Degradation Ratio DMEM : Dulbecco’s Modified Eagle Medium ECL : Enhanced Chemoluminescence EXP− : basal condition EXP+ : experimental condition FBS : Fetal Bovine Serum FormR : Formation Ratio GM-CSF : Granulocyte-Macrophage Colony Stimulating Factor LC3-II : microtubule-associated light chain protein 3 conjugated to phosphatidylethanolamine mTORC1 : Mechanistic Target of Rapamycin Complex 1 NetR : Net Ratio PD : Parkinson’s disease ss : steady-state TBS-T : Tris Buffered Saline containing 0.1% Tween 20 TFEB : transcription factor-EB ULK1/2 : unc-51-like kinases 1/2