IBs had a clear vesicular ultrastructure suggesting that

IBs had a clear vesicular ultrastructure, suggesting that a large part of the iron-containing mitochondrial remnants might be cleared via normal autophagy processes during final reticulocyte maturation [21]. IB formation was followed by characteristic changes in RBC morphology, with irregularly shaped RBCs and a schistocyte-like cell morphology in a subfraction of erythrocytes. These poikilocytes are thought to be secondary to incomplete autophagic clearance of mitochondrial remnants by the spleen because removal of siderotic bodies from erythrocytes in the spleen is known to result in fragmented RBCs [22], and erythrocytes with siderotic bodies are frequently observed after splenectomy 23, 24. A compensatory downregulation of TfR-1 expression on reticulocytes was shown after GlyT1 inhibition, indicating reduced iron incorporation into erythroblasts and thus reduced availability of functional iron. Downregulation of TfR-1 on erythroblasts and reduced erythroblast iron incorporation has also been reported in GlyT1−/− mice [16]. These findings Quetiapine Fumarate with human data and an animal model considered relevant for human sideroblastic anemia in which TfR-1 expression is significantly upregulated 25, 26. Although IBs are formed by mitochondrial excess of iron, the weak increase in free PPIX observed with GlyT1 inhibition may indicate at the same time a small net deficit of available iron within the erythroblasts, resulting in protoporphyrin not being transformed into heme by ferrochelatase. The most likely explanation is that of a slight asynchrony between the shutoff of TfR-1 synthesis/iron uptake and the reduction in ALA and protoporphyrin synthesis due to reduced availability of glycine. It is possible that reduced heme synthesis results in an iron-replete signaling within the mitochondria [27], which decreases TfR-1 synthesis and iron uptake but at the same time may result in transient upregulation of ALAS2, producing a small excess synthesis of protoporphyrin above the available iron. Such a paradoxical coexistence of mitochondrial iron accumulation and PPIX increase has also been reported for disorders with a distinct IB pathoetiology, such as refractory anemia with ring sideroblasts [28] and X-linked sideroblastic anemia with ataxia [6]. An iron-deplete signal would be furthermore consistent with the observed mild thrombocytosis because elevated platelet counts are typically found in iron deficiency anemia, although the molecular activation pathways are unknown [29]. Even though the IBs found in this study showed ultrastructural similarities with IBs of sideroblastic anemia, their rapid and marked formation within 24 hours, their restriction predominantly to reticulocytes, and in particular, the absence of pathological bone marrow sideroblasts render the bitopertin-induced IBs pathogenetically distinct from those associated with acquired forms of sideroblastic anemia. Why IBs are formed under these experimental conditions is incompletely understood, but it can be hypothesized that it is the inability to adapt with an adequate reduction in iron incorporation to the reduced heme biosynthesis, resulting in accumulation of iron in the mitochondria. To date, IBs have not been detected in cynomolgus monkeys and humans treated with bitopertin (unpublished data), pointing to the well-known differences in iron regulation and homeostasis within mammalian species. Significant transcriptome differences during human and murine terminal erythroid differentiation were reported recently [30] and are consistent with older studies showing that, for example, the final heme synthesis rate is higher in rat than in human reticulocytes [31]. Therefore, reduction in heme synthesis induced by inhibition of GlyT1 might cause a higher fraction of remaining uncommitted mitochondrial iron in rat reticulocytes; however, a final proof of this hypothesis is missing. Although GlyT1 inhibition disturbed Hb synthesis, systemic iron homeostasis remained unaffected. The significant increase in the iron-stained areas, predominantly in the spleen, associated with moderate increases in serum ferritin was observed in treated rats, indicating iron retention. This is due to a shift in iron from the Hb/RBC compartment to the iron storage compartment, also reported for various forms of hyporegenerative anemias [32]. Importantly, critical organs for tissue iron accumulation, such as the heart and pancreas, were negative for iron/hemosiderin even when rats were treated with bitopertin for 2 years.