Inactivation of aconitase enzymes contributes to the erythroid response to iron restriction 
. However, because iron deprivation simultaneously affects multiple signaling and metabolic pathways, the exact role of aconitases in erythropoietic regulation has remained unclear. The current studies have directly examined the role of aconitases during erythroid development by use of a specific active site inhibitor. Application of this approach has confirmed that aconitase activity potently regulates erythropoiesis in vivo and in vitro. In vivo, aconitase inhibition caused a rapid-onset hypoplastic anemia with slightly macrocytic, normochromic red cells (). These red cell features suggest that the anemia did not arise from impaired production of heme due to deficiency of succinyl CoA. Anemias caused by defective heme synthesis, such as those due to deficiencies of erythroid-specific δ-aminolevulinate synthase or ferrochelatase, are characteristically microcytic and hypochromic 
. The low reticulocyte counts combined with increased serum EPO levels () suggest that FA treatment interfered with red cell production, a conclusion further supported by the poor reticulocyte response to phenylhydrazine challenge seen in FA-treated animals (). Furthermore, in vivo and in vitro studies both showed impaired differentiation of erythroid progenitors associated with aconitase inhibition (, , ).
Precise definition of the isoform-specific contributions of aconitases to erythroid regulation will require development of new conditional knockout mice with individual and compound gene deletions. Several lines of evidence, however, suggest that mitochondrial aconitase plays a key role. Firstly, fluoroacetate acts predominantly as an inhibitor of mitochondrial aconitase, based on the location of its enzymatic conversion to fluorocitrate, the active inhibitor 
. Secondly, infusion of FA in mice lacking cytosolic aconitase (IRP1−/−) induced an anemia identical to that occurring in wild-type mice (Fig. S7
). Thirdly, IRP1−/− mice showed no hematologic defects in response to anemic challenge (). Fourthly, mitochondrial aconitase bound ERK more avidly than cytosolic aconitase and, unlike its cytosolic counterpart, displayed modulation of ERK binding by FA (). Several studies have previously demonstrated mitochondrial regulation of erythroid differentiation and proliferation. For example, the inhibition of mitochondrial biogenesis by chloramphenicol is associated with impaired erythropoiesis in vivo and in vitro 
. The ability of exogenous isocitrate to reverse in vitro differentiation defects caused by chloramphenicol has suggested that mitochondrial aconitase may participate in the regulation of erythropoiesis by mitochondrial function 
. Further supporting this notion, multiple murine models of impaired mitochondrial function all show a similar, distinctive erythroid maturation defect, i.e. an increase in CD71Intermediate
progenitors (R3 stage), closely resembling the defect seen with FA infusion ( and ) 
Among the various pathways downstream of EPO regulating erythroid development, only the ERK pathway displayed consistent modulation in response to aconitase inhibition. In particular, FA treatment diminished RSK T573 phosphorylation in erythroid progenitors subjected to either acute or chronic EPO stimulation (). The effects of aconitase inhibition on ERK signaling are likely to be biologically relevant because direct inhibition of ERK activation with the MEK inhibitor U0126 recapitulated several key features of the erythroid response to FA (). By contrast, our data suggest that the effects of FA were unrelated to changes in ATP, ROS, or mitochondrial membrane potential (). Why FA had no effect on these latter parameters may be related to the dose employed, to possible metabolic compensation by cytosolic aconitase, or to the dependence of early hematopoietic progenitors more on glycolysis than on oxidative phosphorylation.
The mechanism underlying the cross-talk between aconitase and ERK, while unclear, may relate to the assembly of protein complexes containing ERK and mitochondrial aconitase (). Interestingly, such complexes are phylogenetically conserved back to yeast and are regulated by aconitase activity. Previous studies have documented localization of ERK in mitochondria 
. Whether mitochondrial aconitase participates in an organelle-specific ERK scaffolding complex, as has been described for endosomes and Golgi 
, remains a subject of investigation. Nevertheless, the current studies provide a novel function for aconitase as a licensing factor for erythropoietin-dependent transduction of ERK signaling. This regulatory input could permit adjustment of ERK activity in accordance with aconitase function, which in turn is affected by iron availability, redox, and iron-sulfur cluster assembly pathways. In this manner, a variety of signals reflecting mitochondrial status potentially could be transmitted to the machinery driving proliferation and differentiation.
The ability to modulate red cell production with low dose aconitase inhibition may have therapeutic implications for polycythemia vera. Phlebotomy, the primary treatment for this disease, controls red cell mass via induction of iron deficiency, most likely acting through erythroid inactivation of aconitase. Attainment of iron deficiency may require numerous blood draws over a prolonged period and may be complicated by significant thrombocytosis 
. Repeated phlebotomy also poses a risk for phlebitis and infection. In a mouse model of JAK2 V617F-driven polycythemia, low dose aconitase inhibition rapidly normalized red cell counts in a lineage specific manner without causing anemia (, Table S2
). However, further studies are needed to determine the clinical feasibility of this approach. Fluoroacetate is a known toxic agent that targets the cardiovascular and central nervous systems in mice and humans. Reported lethal doses range from 0.5 to 19 mg/kg in mice and 2 to 10 mg/kg in humans 
. While a continuous FA infusion of 2 mg/kg/day corrects red cell counts in PV mice in the absence of overt symptoms, toxicology studies will be necessary to rule out adverse effects of both acute and chronic administration.