Prem Ponka

Dysregulation of Iron Homeostasis in the CNS Contributes to Disease Progression in a Mouse Model of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS), characterized by degeneration of spinal motor neurons, consists of sporadic and familial forms. One cause of familial ALS is missense mutations in the superoxide dismutase 1 (SOD1) gene. Iron accumulation occurs in the CNS of both forms of ALS; however, its contribution to the pathogenesis of ALS is not known. We examined the role of iron in a transgenic mouse line overexpressing the human SOD1G37R mutant. We show that multiple mechanisms may underlie the iron accumulation in neurons and glia in SOD1G37R transgenic mice. These include dysregulation of proteins involved in iron influx and sensing of intracellular iron; iron accumulation in ventral motor neurons secondary to blockage of anterograde axonal transport; and increased mitochondrial iron load in neurons and glia. We also show that treatment of SOD1G37R mice with an iron chelator extends life span by 5 weeks, accompanied by increased survival of spinal motor neurons and improved locomotor function. These data suggest that iron chelator therapy might be useful for the treatment of ALS.

Hereditary causes of disturbed iron homeostasis in the central nervous system.

Abstract: Iron is essential for oxidation‐reduction catalysis and bioenergetics; however, unless appropriately shielded, this metal plays a crucial role in the formation of toxic oxygen radicals that can attack all biological molecules. Organisms are equipped with specific proteins designed for iron acquisition, export and transport, and storage, as well as with sophisticated mechanisms that maintain the intracellular labile iron pool at an appropriate level. Despite these homeostatic mechanisms, organisms often face the threat of either iron deficiency or iron overload. This review describes several hereditary iron‐overloading conditions that are confined to the brain. Recently, a mutation in the L‐subunit of ferritin has been described that causes the formation of aberrant L‐ferritin with an altered C‐terminus. Individuals with this mutation in one allele of L‐ferritin have abnormal aggregates of ferritin and iron in the brain, primarily in the globus pallidus. Patients with this dominantly inherited late‐onset disease present with symptoms of extrapyramidal dysfunction. Mice with a targeted disruption of a gene for iron regulatory protein 2 (IRP2), a translational repressor of ferritin, misregulate iron metabolism in the intestinal mucosa and the central nervous system. Significant amounts of ferritin and iron accumulate in white matter tracts and nuclei, and adult IRP2‐deficient mice develop a movement disorder consisting of ataxia, bradykinesia, and tremor. Mutations in the frataxin gene are responsible for Friedreichs ataxia, the most common of the inherited ataxias. Frataxin appears to regulate mitochondrial iron‐sulfur cluster formation, and the neurologic and cardiac manifestations of Friedreichs ataxia are due to iron‐mediated mitochondrial toxicity. Patients with Hallervorden‐Spatz syndrome, an autosomal recessive, progressive neurodegenerative disorder, have mutations in a novel pantothenate kinase gene (PANK2). The cardinal feature of this extrapyramidal disease is pathologic iron accumulation in the globus pallidus. The defect in PANK2 is predicted to cause the accumulation of cysteine, which binds iron and causes oxidative stress in the iron‐rich globus pallidus. Finally, aceruloplasminemia is an autosomal recessive disorder of iron metabolism caused by loss‐of‐function mutations in ceruloplasmin gene that leads to misregulation of both systemic and central nervous system iron trafficking. Affected individuals suffer from extrapyramidal signs, cerebellar ataxia, progressive neurodegeneration of retina, and diabetes mellitus. Excessive iron depositions are found in the brain, liver, pancreas, and other parenchymal cells, but plasma iron concentrations are decreased. These conditions are not common, but awareness about them is important for differential diagnosis of various neurodegenerative disorders.

S-Nitrosylation of IRP2 Regulates Its Stability via the Ubiquitin-Proteasome Pathway

ABSTRACT Nitric oxide (NO) is an important signaling molecule that interacts with different targets depending on its redox state. NO can interact with thiol groups resulting in S-nitrosylation of proteins, but the functional implications of this modification are not yet fully understood. We have reported that treatment of RAW 264.7 cells with NO caused a decrease in levels of iron regulatory protein 2 (IRP2), which binds to iron-responsive elements present in untranslated regions of mRNAs for several proteins involved in iron metabolism. In this study, we show that NO causes S-nitrosylation of IRP2, both in vitro and in vivo, and this modification leads to IRP2 ubiquitination followed by its degradation in the proteasome. Moreover, mutation of one cysteine (C178S) prevents NO-mediated degradation of IRP2. Hence, S-nitrosylation is a novel signal for IRP2 degradation via the ubiquitin-proteasome pathway.

Ceruloplasmin Protects Injured Spinal Cord from Iron-Mediated Oxidative Damage

CNS injury-induced hemorrhage and tissue damage leads to excess iron, which can cause secondary degeneration. The mechanisms that handle this excess iron are not fully understood. We report that spinal cord contusion injury (SCI) in mice induces an “iron homeostatic response” that partially limits iron-catalyzed oxidative damage. We show that ceruloplasmin (Cp), a ferroxidase that oxidizes toxic ferrous iron, is important for this process. SCI in Cp-deficient mice demonstrates that Cp detoxifies and mobilizes iron and reduces secondary tissue degeneration and functional loss. Our results provide new insights into how astrocytes and macrophages handle iron after SCI. Importantly, we show that iron chelator treatment has a delayed effect in improving locomotor recovery between 3 and 6 weeks after SCI. These data reveal important aspects of the molecular control of CNS iron homeostasis after SCI and suggest that iron chelator therapy may improve functional recovery after CNS trauma and hemorrhagic stroke.

Role of nitric oxide in cellular iron metabolism

Iron regulatory proteins (IRP1 and IRP2) control the synthesis of transferrin receptors (TfR) and ferritin by binding to iron-responsive elements (IREs) which are located in the 3′ untranslated region (UTR) and the 5′ UTR of their respective mRNAs. Cellular iron levels affect binding of IRPs to IREs and consequently expression of TfR and ferritin. Moreover, NO•, a redox species of nitric oxide that interacts primarily with iron, can activate IRP1 RNA-binding activity resulting in an increase in TfR mRNA levels. We have shown that treatment of RAW 264.7 cells (a murine macrophage cell line) with NO+ (nitrosonium ion, which causes S-nitrosylation of thiol groups) resulted in a rapid decrease in RNA-binding of IRP2, followed by IRP2 degradation, and these changes were associated with a decrease in TfR mRNA levels. Moreover, we demonstrated that stimulation of RAW 264.7 cells with lipopolysaccharide (LPS) and interferon-γ (IFN-γ) increased IRP1 binding activity, whereas RNA-binding of IRP2 decreased and was followed by a degradation of this protein. Furthermore, the decrease of IRP2 binding/protein levels was associated with a decrease in TfR mRNA levels in LPS/IFN-γ-treated cells, and these changes were prevented by inhibitors of inducible nitric oxide synthase. These results suggest that NO+-mediated degradation of IRP2 plays a major role in iron metabolism during inflammation.

Non-heme Induction of Heme Oxygenase-1 Does Not Alter Cellular Iron Metabolism

The catabolism of heme is carried out by members of the heme oxygenase (HO) family. The products of heme catabolism by HO-1 are ferrous iron, biliverdin (subsequently converted to bilirubin), and carbon monoxide. In addition to its function in the recycling of hemoglobin iron, this microsomal enzyme has been shown to protect cells in various stress models. Implicit in the reports of HO-1 cytoprotection to date are its effects on the cellular handling of heme/iron. However, the limited amount of uncommitted heme in non-erythroid cells brings to question the source of substrate for this enzyme in non-hemolytic circumstances. In the present study, HO-1 was induced by either sodium arsenite (reactive oxygen species producer) or hemin or overexpressed in the murine macrophage-like cell line, RAW 264.7. Both of the inducers elicited an increase in active HO-1; however, only hemin exposure caused an increase in the synthesis rate of the iron storage protein, ferritin. This effect of hemin was the direct result of the liberation of iron from heme by HO. Cells stably overexpressing HO-1, although protected from oxidative stress, did not display elevated basal ferritin synthesis. However, these cells did exhibit an increase in ferritin synthesis, compared with untransfected controls, in response to hemin treatment, suggesting that heme levels, and not HO-1, limit cellular heme catabolism. Our results suggest that the protection of cells from oxidative insult afforded by HO-1 is not due to the catabolism of significant amounts of cellular heme as thought previously.

Nramp1 equips macrophages for efficient iron recycling

OBJECTIVE Natural resistance-associated macrophage protein 1 (Nramp1) is a divalent metal transporter expressed exclusively in phagocytic cells, such as macrophages and neutrophils. As macrophages are responsible for the engulfment and clearance of senescent red blood cells (RBC), we hypothesize that Nramp1 may participate in the recycling of iron acquired through phagocytosis. MATERIALS AND METHODS To test this hypothesis, we examined the contribution of Nramp1 expression to iron metabolism in macrophages loaded with iron via either hemin or opsonized RBC. RESULTS Western blot analysis indicated that Nramp1 protein levels increased with hemin, opsonized erythrocytes, or erythropoietin treatment. The pool of chelatable iron was also found to transiently increase following iron-loading with hemin or opsonized RBCs, with a greater increase observed in macrophages expressing Nramp1. Overexpression of Nramp1 was also found to result in a greater cellular release of (59)Fe following phagocytosis of (59)Fe-labeled reticulocytes. Expression of Nramp1 was associated with a twofold increase in heme oxygenase-1 (HO-1) levels in macrophages undergoing erythrophagocytosis. Nramp1-expressing macrophages were also found to phagocytose nearly twice as many RBC than their Nramp1-deficient counterparts. CONCLUSION The rapid and strong induction of Nramp1 during erythrophagocytosis, combined with its positive effects on (59)Fe-release, HO-1 induction and phagocytic ability, suggest that Nramp1 has a role in the recycling of hemoglobin-derived iron by macrophages.

Pyridoxal Isonicotinoyl hydrazone and its analogues

Iron is a precious metal for the organism because of its unsurpassed versatility as a biological catalyst. It is involved in a broad spectrum of essential biological functions such as oxygen transport (hemoglobin), electron transfer (mitochondrial heme and non-heme Fe proteins essential for energy production) and DNA synthesis (ribonucleotide reductase), to name just a few. However, the chemical properties of iron which allow this versatility also lead to the paradoxical situation that acquisition by the organism of an abundant element is exceedingly difficult. At pH 7.4 and physiological oxygen tension, the relatively soluble ferrous ion (Fe2+) is readily oxidized to ferric ion (Fe3+) which is susceptible to hydrolysis, forming virtually insoluble ferric hydroxides. The concentration of aquated Fe3+ (pH 7.4) cannot exceed 10-17 M. Moreover, unless bound to specific ligands, iron plays a key role in the formation of harmful oxygen radicals which ultimately cause oxidative damage to vital cell structures. Because of this virtual insolubility and potential toxicity, specialized mechanisms and molecules for the acquisition, transport, and storage of iron in a soluble nontoxic form have evolved to meet cellular and organismal iron requirements. In addition, organisms are equipped with sophisticated mechanisms that prevent the expansion of the catalytically active intracellular iron pool, while maintaining sufficient concentrations of the metal for metabolic use.1, 2, 3 However, despite these homeostatic mechanisms, organisms often face the threat of either iron deficiency or iron overload.

Iron targeting to mitochondria in erythroid cells.

Immature erythroid cells have an exceptionally high capacity to synthesize haem that is, at least in part, the result of the unique control of iron metabolism in these cells. In erythroid cells the vast majority of Fe released from endosomes must cross both the outer and the inner mitochondrial membranes to reach ferrochelatase, which inserts Fe into protoporphyrin IX. Based on the fact that Fe is specifically targeted into erythroid mitochondria, we have proposed that a transient mitochondria-endosome interaction is involved in Fe transfer to ferrochelatase [Ponka (1997) Blood 89, 1-25]. In this study, we examined whether the inhibition of endosome mobility within erythroid cells would decrease the rate of (59)Fe incorporation into haem. We found that, in reticulocytes, the myosin light-chain kinase inhibitor, wortmannin, and the calmodulin antagonist, W-7, caused significant inhibition of (59)Fe incorporation from (59)Fe-transferrin-labelled endosomes into haem. These results, together with confocal microscopy studies using transferrin and mitochondria labelled by distinct fluorescent markers, suggest that, in erythroid cells, endosome mobility, and perhaps their contact with mitochondria, plays an important role in a highly efficient utilization of iron for haem synthesis.

Iron regulatory protein‐independent regulation of ferritin synthesis by nitrogen monoxide

The discovery of iron‐responsive elements (IREs), along with the identification of iron regulatory proteins (IRP1, IRP2), has provided a molecular basis for our current understanding of the remarkable post‐transcriptional regulation of intracellular iron homeostasis. In iron‐depleted conditions, IRPs bind to IREs present in the 5′‐UTR of ferritin mRNA and the 3′‐UTR of transferrin receptor (TfR) mRNA. Such binding blocks the translation of ferritin, the iron storage protein, and stabilizes TfR mRNA, whereas the opposite scenario develops when iron in the intracellular transit pool is plentiful. Nitrogen monoxide (commonly designated nitric oxide; NO), a gaseous molecule involved in numerous functions, is known to affect cellular iron metabolism via the IRP/IRE system. We previously demonstrated that the oxidized form of NO, NO+, causes IRP2 degradation that is associated with an increase in ferritin synthesis [Kim, S & Ponka, P (2002) Proc Natl Acad Sci USA99, 12214–12219]. Here we report that sodium nitroprusside (SNP), an NO+ donor, causes a dramatic and rapid increase in ferritin synthesis that initially occurs without changes in the RNA‐binding activities of IRPs. Moreover, we demonstrate that the translational efficiency of ferritin mRNA is significantly higher in cells treated with SNP compared with those incubated with ferric ammonium citrate, an iron donor. Importantly, we also provide definitive evidence that the iron moiety of SNP is not responsible for such changes. These results indicate that SNP‐mediated increase in ferritin synthesis is, in part, due to an IRP‐independent and NO+‐dependent post‐transcriptional, regulatory mechanism.