Sandro Altamura

Iron toxicity in diseases of aging: Alzheimer's disease, Parkinson's disease and atherosclerosis.

Excess free iron generates oxidative stress that hallmarks diseases of aging. The observation that patients with Alzheimers disease or Parkinsons disease show a dramatic increase in their brain iron content has opened the possibility that disturbances in brain iron homeostasis may contribute to the pathogenesis of these disorders. While the reason for iron accumulation is unknown, iron localization correlates with the production of reactive oxygen species in those areas of the brain that are prone to neurodegeneration. A role for iron is also proposed in atherosclerosis, a further frequent disorder of aging. We will review experimental evidences for an involvement of iron in these diseases and discuss some mouse models with impairment in iron-related genes that may be useful to study the role of iron in these disorders.

The liver-specific microRNA miR-122 controls systemic iron homeostasis in mice

Systemic iron homeostasis is mainly controlled by the liver through synthesis of the peptide hormone hepcidin (encoded by Hamp), the key regulator of duodenal iron absorption and macrophage iron release. Here we show that the liver-specific microRNA miR-122 is important for regulating Hamp mRNA expression and tissue iron levels. Efficient and specific depletion of miR-122 by injection of a locked-nucleic-acid-modified (LNA-modified) anti-miR into WT mice caused systemic iron deficiency, characterized by reduced plasma and liver iron levels, mildly impaired hematopoiesis, and increased extramedullary erythropoiesis in the spleen. Moreover, miR-122 inhibition increased the amount of mRNA transcribed by genes that control systemic iron levels, such as hemochromatosis (Hfe), hemojuvelin (Hjv), bone morphogenetic protein receptor type 1A (Bmpr1a), and Hamp. Importantly, miR-122 directly targeted the 3′ untranslated region of 2 mRNAs that encode activators of hepcidin expression, Hfe and Hjv. These data help to explain the increased Hamp mRNA levels and subsequent iron deficiency in mice with reduced miR-122 levels and establish a direct mechanistic link between miR-122 and the regulation of systemic iron metabolism.

Bone morphogenetic protein (BMP)-responsive elements located in the proximal and distal hepcidin promoter are critical for its response to HJV/BMP/SMAD

The hemochromatosis proteins HFE, transferrin receptor 2 (TfR2) and hemojuvelin (HJV, HFE2) positively control expression of the major iron regulatory hormone hepcidin. HJV is a bone morphogenetic protein (BMP) co-receptor that enhances the cellular response to BMP cytokines via the phosphorylation of SMAD proteins. In this study, we show that two highly conserved and sequence-identical BMP-responsive elements located at positions −84/−79 (BMP-RE1) and −2,255/−2,250 (BMP-RE2) of the human hepcidin promoter are critical for both the basal hepcidin mRNA expression and the hepcidin response to BMP-2 and BMP-6. While BMP-RE1 and BMP-RE2 show additive effects in responding to HJV-mediated BMP signals, only BMP-RE1 that is located in close proximity to a previously identified STAT-binding site is important for the hepcidin response to IL-6. These data identify a missing link between the HJV/BMP signaling pathways and hepcidin transcription, and further define the connection between inflammation and BMP-dependent hepcidin promoter activation. As such, they provide important new information furthering our understanding of disorders of iron metabolism and the anemia of inflammation.

A novel inflammatory pathway mediating rapid hepcidin-independent hypoferremia

Regulation of iron metabolism and innate immunity are tightly interlinked. The acute phase response to infection and inflammation induces alterations in iron homeostasis that reduce iron supplies to pathogens. The iron hormone hepcidin is activated by such stimuli causing degradation of the iron exporter ferroportin and reduced iron release from macrophages, suggesting that hepcidin is the crucial effector of inflammatory hypoferremia. Here, we report the discovery of an acute inflammatory condition that is mediated by Toll-like receptors 2 and 6 (TLR2 and TLR6) and which induces hypoferremia in mice injected with TLR ligands. Stimulation of TLR2/TLR6 triggers profound decreases in ferroportin messenger RNA and protein expression in bone marrow-derived macrophages, liver, and spleen of mice without changing hepcidin expression. Furthermore, C326S ferroportin mutant mice with a disrupted hepcidin/ferroportin regulatory circuitry respond to injection of the TLR2/6 ligands FSL1 or PAM3CSK4 by ferroportin downregulation and a reduction of serum iron levels. Our findings challenge the prevailing role of hepcidin in hypoferremia and suggest that rapid hepcidin-independent ferroportin downregulation in the major sites of iron recycling may represent a first-line response to restrict iron access for numerous pathogens.

Myc depletion induces a pluripotent dormant state mimicking diapause

Summary Mouse embryonic stem cells (ESCs) are maintained in a naive ground state of pluripotency in the presence of MEK and GSK3 inhibitors. Here, we show that ground-state ESCs express low Myc levels. Deletion of both c-myc and N-myc (dKO) or pharmacological inhibition of Myc activity strongly decreases transcription, splicing, and protein synthesis, leading to proliferation arrest. This process is reversible and occurs without affecting pluripotency, suggesting that Myc-depleted stem cells enter a state of dormancy similar to embryonic diapause. Indeed, c-Myc is depleted in diapaused blastocysts, and the differential expression signatures of dKO ESCs and diapaused epiblasts are remarkably similar. Following Myc inhibition, pre-implantation blastocysts enter biosynthetic dormancy but can progress through their normal developmental program after transfer into pseudo-pregnant recipients. Our study shows that Myc controls the biosynthetic machinery of stem cells without affecting their potency, thus regulating their entry and exit from the dormant state.

Growth differentiation factor 15 in patients with congenital dyserythropoietic anaemia (CDA) type II

Congenital dyserythropoietic anaemias (CDAs) are heterogeneous, hereditary disorders hallmarked by ineffective erythropoiesis and tissue iron overload. Growth differentiation factor 15 (GDF15) was suggested to mediate iron overload in iron-loading anaemias, such as the thalassaemias and CDAI by suppressing hepcidin, the key regulator of iron absorption. Here, we show that serum GDF15 concentrations are elevated in subjects with CDAI and CDAII. Despite similar disease characteristics, CDAI patients present with significantly higher GDF15 concentrations compared to CDAII patients. Hepcidin concentrations are inappropriately low in CDAII patients considering the severe hepatic iron overload associated with this disorder. GDF15 significantly correlates with the degree of anaemia (Hb), the response of erythropoiesis (reticulocyte index) as well as with iron availability in the serum (transferrin saturation). The observation that GDF15 is elevated in CDAII patients is consistent with the proposal that GDF15 is among the erythroid factors down-regulating hepcidin and contributing to iron overload in conditions of dyserythropoiesis.

A novel TMPRSS6 mutation that prevents protease auto-activation causes IRIDA

IRIDA (iron-refractory iron-deficiency anaemia) is a rare autosomal-recessive disorder hallmarked by hypochromic microcytic anaemia, low transferrin saturation and high levels of the iron-regulated hormone hepcidin. The disease is caused by mutations in the transmembrane serine protease TMPRSS6 (transmembrane protease serine 6) that prevent inactivation of HJV (haemojuvelin), an activator of hepcidin transcription. In the present paper, we describe a patient with IRIDA who carries a novel mutation (Y141C) in the SEA domain of the TMPRSS6 gene. Functional characterization of the TMPRSS6(Y141C) mutant protein in cultured cells showed that it localizes to similar subcellular compartments as wild-type TMPRSS6 and binds HJV, but fails to auto-catalytically activate itself. As a consequence, hepcidin mRNA expression is increased, causing the clinical symptoms observed in this IRIDA patient. The present study provides important mechanistic insight into how TMPRSS6 is activated.

Pegylated interferon‐α induced hypoferremia is associated with the immediate response to treatment in hepatitis C

Pegylated interferon‐α (PEG‐IFN‐α) forms an integral part of the current treatment for hepatitis C virus (HCV) infection. PEG‐IFN‐α suppresses HCV production by augmenting the innate antiviral immune response. Recent studies have reported the induction of hepcidin, the iron regulatory hormone, by IFN‐α in vitro. As hepcidin plays an important role in innate immunity, we hypothesized that this finding may be of clinical relevance to HCV and investigated the changes in iron homeostasis during the first 24 hours of treatment. Blood samples were obtained from HCV patients immediately prior to and 6, 12, and 24 hours following the first dose of PEG‐IFN‐α/ribavirin (RBV). Samples were analyzed for hepcidin, cytokine, iron levels, and HCV viral load, and hepcidin messenger RNA (mRNA) expression was quantified in peripheral blood mononuclear cells. Hepcidin induction by IFN‐α was further analyzed in cell culture. In HCV patients a single dose of PEG‐IFN‐α/RBV resulted in a significant increase in serum hepcidin, peaking at 12 hours, coinciding with a 50% reduction in serum iron and transferrin saturation over the 24‐hour period. Patients with a ≥2 log decline in HCV viral load over the first 24 hours had significantly lower SI and TS levels at 12 and 24 hours. Moreover, 24‐hour SI levels were an independent predictor of the immediate HCV viral decline, an indicator of ultimate treatment outcome. In cell culture, a direct induction of hepcidin by IFN‐α was seen, controlled by the STAT3 transcription factor. Conclusion: Hepcidin induction occurs following the initiation of PEG‐IFN‐α treatment for HCV, and is mediated by way of STAT3 signaling. The subsequent hypoferremia was greatest in those with the most significant decline in viral load, identifying systemic iron withdrawal as a marker of immediate interferon‐α efficacy in HCV patients. (HEPATOLOGY 2012)

SELDI-TOF MS detection of urinary hepcidin.

Hepcidin is a 25-residue hepatic peptide that regulates iron absorption from the diet and tissue iron distribution. Inappropriately low Hepcidin expression is implicated in the pathogenesis of hereditary hemochromatosis and iron-loading anemias, like the thalassemias. Increased hepcidin expression mediates iron retention in the anemias of inflammation and plays a pathogenic role in iron-refractory iron-deficiency anemia (IRIDA). Because of its clinical importance, Hepcidin is expected to be a useful biomarker for diagnosis and management of iron-related disorders. So far an ELISA for human hepcidin and SELDI-TOF-MS based approaches have been applied to monitor urinary and/or serum hepcidin levels. Here we report a modified protocol for SELDI-TOF based detection of human, urinary hepcidin. We show that CM10 Proteinchips are superior to NP20 Proteinchips commonly used in previously reported protocols to sensitively and accurately detect urinary hepcidin. Application of this modified hepcidin assay accurately detects increased hepcidin levels in the urine of sepsis patients.

Human L-ferritin deficiency is characterized by idiopathic generalized seizures and atypical restless leg syndrome

Human L-ferritin deficiency causes reduced cellular iron availability and increased ROS production with enhanced oxidized proteins, which results in idiopathic generalized seizures and atypical restless leg syndrome.