ATG4A is a Novel Regulator of Mitophagy during Human Erythropoiesis

Autophagy is a highly conserved pathway that degrades and recycles intracellular components. While autophagy is activated in response to cellular stress, it also contributes to hematopoietic cell differentiation. Previous studies in mice lacking core autophagy proteins have identified mitochondrial clearance as a critical function of autophagy in erythroid differentiation and maturation. Impaired autophagy results in anemia, retained mitochondria and elevated levels of reactive oxygen species in mice. However, the kinetics and regulation of mitophagy in human erythropoiesis have not been investigated. To better understand the kinetics of mitophagy in human erythropoiesis we have developed a lentiviral mitophagy reporter (MT-Keima) and used it to monitor mitochondrial clearance during erythroid differentiation of primary human CD34⁺ hematopoietic stem and progenitor cells (HSPCs). Measurement of mitophagy during erythroid differentiation revealed active mitochondrial clearance during early and late stages of erythropoiesis culminating with total clearance of mitochondria at the terminal stages of differentiation. Gene expression analysis during human erythroid differentiation identified the upregulation of the core autophagy program including ATG4A encoding a cysteine protease, which was restricted to the erythroid lineage. Knockdown of ATG4A in primary human HSPCs significantly reduced mitophagy in early and late erythroid cells and resulted in increased mitochondrial mass in terminally differentiated reticulocytes. Furthermore decreased expression of ATG4A, but not its paralog ATG4B, reduced the (i) expansion of erythroid progenitors, (ii) total number of erythroblasts (iii) and significantly reduced enucleation relative to luciferase controls. Finally significantly fewer erythroid colonies were found in methylcellulose culture only in HSPCs with ATG4A KD when compared to luciferase controls, while numbers of myeloid colonies were preserved, supporting a role for ATG4A selectively in the human erythroid lineage. Together these results identify ATG4A as a novel erythroid-specific regulator of mitophagy and a new potential target for the therapeutic modulation of autophagy in human erythropoiesis.