Iron is an indispensable element in mammals. Maintaining proper iron concentration in our body is of great significance because iron, in forms of heme, iron-sulfur cluster (Fe-S), or iron itself as important cofactors, are involved in multiple biochemical pathways, including hemoglobin synthesis and mitochondrial respiratory chain (Hentze et al., 2004; Darshan et al., 2010; Ganz and Nemeth, 2012; Rouault, 2013). For this reason, mammals have developed sophisticated mechanisms to maintain proper iron concentration in the body: (1) systemic iron homeostasis is maintained by hepcidin-ferroportin (hepcidin-FPN1, encoded by HAMP and SCL40A1) axis (Nemeth et al., 2004; Ganz and Nemeth, 2011); (2) cellular iron homeostasis is mediated by iron regulatory proteins (IRPs, IRP1 and IRP2, also called ACO1 and IREB2) through IRP-IRE (iron responsive element) system (Rouault, 2006; Wallander et al., 2006; Muckenthaler et al., 2008; Rouault, 2013). More tissue specific strategies have also been developed, e. g. ferritinophagy to regulate erythropoiesis (Mancias et al., 2015). These ways function and interplay to fine-tune iron levels in the body (Zhang et al., 2014).
IRP1 and IRP2 are both iron-regulatory RNA binding proteins that regulate the expression of a series of iron-related genes at the post-transcriptional levels (Hentze et al., 2010; Anderson et al., 2012). Under conditions of iron deficiency, the IRE in the target mRNA can be recognized and bound by IRPs, but the consequence of IRP binding depends on the position of the IRE on the mRNA of the target genes. If the IRE is in the 5'-untranslated region (UTR) of the target mRNA, the binding of IRPs may inhibit the translation of the genes, including L- and H-ferritin and FPN1; if the IRE is in the 3'-UTR, the binding of IRPs may stabilize the mRNA, such as that of transferrin receptor 1 (TfR1) (Casey et al., 1988; Müllner et al., 1989) and divalent metal transporter 1 (DMT1) (Tybl et al., 2020). When cellular iron is sufficient, IRP1 binds to a [4Fe-4S] cluster, therefore, gains aconitase activity and loses the ability to bind IRE, whereas IRP2 is removed by iron and oxygen-mediated proteasome degradation (Salahudeen et al., 2009; Vashisht et al., 2009) to avoid the excessive iron uptake and to promptly stores excess intracellular iron and/or export excess iron.
Studies in animal models have shown that IRPs also play an important role in the regulation of systemic iron homeostasis. It has been reported that Irp2−/− mice suffer from microcytic anemia (Cooperman et al., 2005; Galy et al., 2005), neurologic defects (LaVaute et al., 2001; Jeong et al., 2011) and diabetes (Santos et al., 2020). The cause of these symptoms are considered to be lack of functional iron in erythroblast progenitors, the cells in central nervous system (CNS), and β cells of Irp2−/− mice. The patient with bi-allelic loss-of-function variants in the gene iron responsive element binding protein 2 (IREB2) leading to an absence of IRP2 also presented neurological and haematological features (Costain et al., 2019), similar, but much more severely, to the observation in Irp2−/− mice. The symptoms could be caused by the deficiency of Fe-S biogenesis, which further compromised the mitochondrial quality (Li et al., 2018; Li et al., 2019).
Recently, we found that mitochondrial dysfunction was closely associated with the reduced expression of a number of genes that are involved in Fe-S biogenesis and mitochondrial respiratory chain (Li et al., 2018). The further investigation revealed that Irp2 may function as a key to switch the metabolism between aerobic glycolysis and oxidative phosphorylation (OXPHOS), mediated by upregulation of hypoxia-inducible factor subunits Hif1α and Hif2α in mouse embryonic fibroblasts (MEFs) (Li et al., 2019). HIF1 and HIF2 are two important transcription factors that can regulate the expression of a series of genes. Active HIF is a heterodimer, composed of α subunit (HIF1α or Hif2α) and β subunit (HIF1β, also called ARNT) and can bind hypoxia responsive element (HRE), which is a very important mechanism in intestinal iron absorption (Mastrogiannaki et al., 2009; Shah et al., 2009) and under other conditions, such as cancer (Keith et al., 2011) and ischemia (Kapitsinou et al., 2014; Barteczek et al., 2017). IRP1 can bind to the IRE in the 5'-UTR of HIF2α mRNA to regulate HIF2α at the post-transcriptional level (Sanchez et al., 2007; Zimmer et al., 2008). In Irp1−/− mice, elevated Hif2α up-regulates erythropoietin (EPO), causing the mice to develop polycythemia and pulmonary hypertension (Anderson et al., 2013; Ghosh et al., 2013; Wilkinson and Pantopoulos, 2013). Interestingly, in Irp2−/− MEFs we found that Hif1α and Hif2α were both up-regulated, which switches the metabolism type from oxidative phosphorylation to glycolysis (Li et al., 2019). Inhibition of both Hif1α and Hif2α reversed the energy metabolism.
In this study, we confirmed the elevated Hif2α, not Hif1α, in Irp2−/− mice. The upregulated glycolytic pathway-related proteins were also observed and associated with the enhanced glycolysis, while down-regulated frataxin (Fxn) and iron–sulfur cluster scaffold protein (IscU), respectively, were observed and associated with deficiency of iron-sulfur clusters. Consequently, the expression of electron transmit chain (ETC) subunits was reduced and OXPHOS was weakened. After the inhibition of Hif2α by PT-2385, the energy metabolism was shifted from glycolysis to OXPHOS in Irp2−/− mice, the histological and behavioral indicators were restored, and neurodegenerative symptoms were alleviated. Our results indicate that the neurodegenerative disorder induced by the loss of Irp2 is, at least partially, mediated by the upregulated Hif2α.