Project Summary/Abstract The erythroid iron restriction response underlies two of the most common types of anemia: anemia of chronic disease and inflammation (ACDI) and iron deficiency anemia (IDA). These anemias confer a major global burden of morbidity and mortality, with no optimal therapies yet available for ACDI. This lineage-specific progenitor response to nutrient deprivation involves loss of isocitrate production and can be reversed by providing exogenous isocitrate in vitro and in vivo. We have recently developed an oral formulation showing sustained efficacy in murine ACDI using a clinically feasible dosing regimen. Results from the last funding period identified the molecular basis for erythropoietin (Epo) resistance associated with iron restriction, a major clinical problem (J. Exp. Med. 2018). In essence, iron deprivation caused a failure in cell surface delivery of the Epo receptor (EpoR) and associated factors (Scrib and TfR2). This abnormality was reversed by isocitrate treatment which restored Epo responsiveness. More recently, we have discovered that iron and isocitrate modulate Golgi integrity in an erythroid lineage-specific manner. Furthermore, erythroid iron restriction induced an early and sustained disruption of the microtubule cytoskeleton, a structure known to be critical for Golgi maintenance. Isocitrate treatment did not prevent the initial microtubule disruption but promoted its reassembly at later time points. Our studies of patient specimens, as well as prior published reports, support the clinical relevance of this microtubule response. We then mined a comprehensive proteomic dataset on staged human erythroid progenitors to identify features that might contribute to the microtubule instability. At all stages, erythroid cells manifested a striking deficiency of stabilizing microtubule-associated proteins (MAPs), such as the ubiquitous MAP4, and abundantly expressed the microtubule destabilizer, Stathmin 1 (STMN1). We therefore postulated the existence of a non-canonical, iron-regulated, stabilizing MAP and examined ferritin heavy chain (FTH1), known to possess microtubule bundling activity and be controlled by iron. Erythroid iron restriction caused a prompt and potent FTH1 decline due to proteolytic and non-proteolytic mechanisms, the latter likely involving IRP translational repression. Isocitrate rescued FTH1 levels but did not prevent early proteolysis; a likely target in its rescue is IRP1, known to be regulated by isocitrate and to participate in the erythroid iron restriction response. Importantly, lentiviral knockdown of FTH1 disrupted microtubules and impaired differentiation in a manner similar to iron restriction. Vesicular and protein transport may occur through either microtubule-dependent or –independent mechanisms. Notably, we discovered a strong and specific interaction of endogenous FTH1 with EpoR in erythroid cells, implicating FTH1 in microtubule recruitment of receptor vesicles. The proposed expe...