Role of VPS4A and ESCRT-III in terminal erythropoiesis

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Project Summary Congenital dyserythropoietic anemias (CDAs) are a heterogeneous group of genetic disorders characterized by ineffective erythropoiesis, hemolysis, and bi- or multi-nucleated erythroblasts in the bone marrow. The mechanisms of erythroid dysfunction in CDAs are incompletely understood; studying the molecular defects in these diseases can reveal critical pathways in terminal erythropoiesis. We have identified mutations in VPS4A as a novel cause of CDA in three unrelated patients with a syndrome of dyserythropoiesis, hemolytic anemia, and neurodevelopmental delay, pointing to the importance of Vacuolar Protein Sorting 4A (VPS4A) for terminal erythropoiesis and normal red blood cell survival. However, the mechanisms of its action need further investigation. VPS4A is an ATPase that has been shown in yeast and in vitro cell cultures to participate with the Endosomal Sorting Complex Required for Transport (ESCRT)-III in endosomal vesicle trafficking, viral budding, and the abscission step of cytokinesis. It is required for budding of endosomal vesicles into multivesicular bodies, a critical step in the pathway for the sorting, recycling, and removal of transmembrane protein receptors. We have shown for the first time a human disease in patients with dominant negative mutations in the ATPase domain of VPS4A, resulting in dyserythropoiesis with erythroblasts connected by cytoplasmic bridges consistent with cytokinesis failure at the abscission step and reticulocytes with evidence of altered trafficking of the transferrin receptor (TfR1/CD71). We hypothesize that VPS4A is essential for terminal erythropoiesis and that loss of function results in dyserythropoiesis through a combination of cytokinesis failure and endolysosomal defects. The goal of this work is to define the molecular mechanisms by which these VPS4A variants disturb erythropoiesis, and to elucidate the roles of VPS4A and the ESCRT machinery in human and murine terminal erythropoiesis. We aim to model the erythroid defects due to VPS4 in vivo using a transgenic mouse model with erythroid-targeted expression of a known dominant-negative VPS4 mutant (VPS4EQ), enabling studies on the pathogenic mechanisms and natural history of the disease (Aim 1). Using human iPSCs, we will investigate the contributions of cytokinesis failure and iron trafficking defects to the dyserythropoiesis observed in these individuals using normal, patient-derived, and VPS4AEQ CRISPR gene-edited iPSCs (Aim 2). These studies will demonstrate the role of VPS4A and ESCRT-III machinery as an essential molecular pathway for erythropoiesis.