A Novel Model for Brain Iron Uptake: Introducing the Concept of Regulation

Neurologic disorders such as Alzheimer's, Parkinson's disease, and Restless Legs Syndrome involve a loss of brain iron homeostasis. Moreover, iron deficiency is the most prevalent nutritional concern worldwide with many associated cognitive and neural ramifications. Therefore, understanding the mechanisms by which iron enters the brain and how those processes are regulated addresses significant global health issues. The existing paradigm assumes that the endothelial cells (ECs) forming the blood—brain barrier (BBB) serve as a simple conduit for transport of transferrin-bound iron. This concept is a significant oversimplification, at minimum failing to account for the iron needs of the ECs. Using an in vivo model of brain iron deficiency, the Belgrade rat, we show the distribution of transferrin receptors in brain microvasculature is altered in luminal, intracellular, and abluminal membranes dependent on brain iron status. We used a cell culture model of the BBB to show the presence of factors that influence iron release in non-human primate cerebrospinal fluid and conditioned media from astrocytes; specifically apo-transferrin and hepcidin were found to increase and decrease iron release, respectively. These data have been integrated into an interactive model where BBB ECs are central in the regulation of cerebral iron metabolism.

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Hepcidin regulates ferroportin expression and intracellular iron homeostasis of erythroblasts

Blood ◽

10.1182/blood-2011-01-330241 ◽

2011 ◽

Vol 118 (10) ◽

pp. 2868-2877 ◽

Cited By ~ 53

Author(s):

De-Liang Zhang ◽

Thomas Senecal ◽

Manik C. Ghosh ◽

Hayden Ollivierre-Wilson ◽

Tiffany Tu ◽

...

Keyword(s):

Iron Deficiency ◽

Iron Homeostasis ◽

Fine Tuning ◽

Deficiency Anemia ◽

Iron Release ◽

Intracellular Iron ◽

Subsequent Increase ◽

A Cell ◽

Export Protein

Abstract The iron-regulatory hormone, hepcidin, regulates systemic iron homeostasis by interacting with the iron export protein ferroportin (FPN1) to adjust iron absorption in enterocytes, iron recycling through reticuloendothelial macrophages, and iron release from storage in hepatocytes. We previously demonstrated that FPN1 was highly expressed in erythroblasts, a cell type that consumes most of the serum iron for use in hemoglobin synthesis. Herein, we have demonstrated that FPN1 localizes to the plasma membrane of erythroblasts, and hepcidin treatment leads to decreased expression of FPN1 and a subsequent increase in intracellular iron concentrations in both erythroblast cell lines and primary erythroblasts. Moreover, injection of exogenous hepcidin decreased FPN1 expression in BM erythroblasts in vivo, whereas iron depletion and associated hepcidin reduction led to increased FPN1 expression in erythroblasts. Taken together, hepcidin decreased FPN1 expression and increased intracellular iron availability of erythroblasts. We hypothesize that FPN1 expression in erythroblasts allows fine-tuning of systemic iron utilization to ensure that erythropoiesis is partially suppressed when nonerythropoietic tissues risk developing iron deficiency. Our results may explain why iron deficiency anemia is the most pronounced early manifestation of mammalian iron deficiency.

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CBIO-10. REDUCED IRON EXPORT FUNCTIONS IN A CELL INTRINSIC MANNER TO DRIVE GLIOBLASTOMA GROWTH

Neuro-Oncology ◽

10.1093/neuonc/noaa215.070 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii17-ii17

Author(s):

Katie Troike ◽

Erin Mulkearns-Hubert ◽

Daniel Silver ◽

James Connor ◽

Justin Lathia

Keyword(s):

Tumor Cells ◽

Iron Uptake ◽

Iron Homeostasis ◽

Iron Status ◽

Tumor Grade ◽

Hfe Gene ◽

Iron Release ◽

Female Patients ◽

Cellular Iron

Abstract Glioblastoma (GBM), the most common primary malignant brain tumor in adults, is characterized by invasive growth and poor prognosis. Iron is a critical regulator of many cellular processes, and GBM tumor cells have been shown to modulate expression of iron-associated proteins to enhance iron uptake from the surrounding microenvironment, driving tumor initiation and growth. While iron uptake has been the central focus of previous investigations, additional mechanisms of iron regulation, such as compensatory iron efflux, have not been explored in the context of GBM. The hemochromatosis (HFE) gene encodes a transmembrane glycoprotein that aids in iron homeostasis by limiting cellular iron release, resulting in a sequestration phenotype. We find that HFE is upregulated in GBM tumors compared to non-tumor brain and that expression of HFE increases with tumor grade. Furthermore, HFE mRNA expression is associated with significantly reduced survival specifically in female patients with GBM. Based on these findings, we hypothesize that GBM tumor cells upregulate HFE expression to augment cellular iron loading and drive proliferation, ultimately leading to reduced survival of female patients. To test this hypothesis, we generated Hfe knockdown and overexpressing mouse glioma cell lines. We observed significant alterations in the expression of several iron handling genes with Hfe knockdown or overexpression, suggesting global disruption of iron homeostasis. Additionally, we show that knockdown of Hfe in these cells increases apoptosis and leads to a significant impairment of tumor growth in vivo. These findings support the hypothesis that Hfe is a critical regulator of cellular iron status and contributes to tumor aggression. Future work will include further exploration of the mechanisms that contribute to these phenotypes as well as interactions with the tumor microenvironment. Elucidating the mechanisms by which iron effulx contributes to GBM may inform the development of next-generation targeted therapies.

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A Novel in Vivo Model for Assessing the Impact of Geophagic Earth on Iron Status

Nutrients ◽

10.3390/nu8060362 ◽

2016 ◽

Vol 8 (6) ◽

pp. 362 ◽

Cited By ~ 5

Author(s):

Gretchen Seim ◽

Elad Tako ◽

Cedric Ahn ◽

Raymond Glahn ◽

Sera Young

Keyword(s):

Iron Status ◽

In Vivo Model ◽

The Impact

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An essential cell-autonomous role for hepcidin in cardiac iron homeostasis

eLife ◽

10.7554/elife.19804 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 63

Author(s):

Samira Lakhal-Littleton ◽

Magda Wolna ◽

Yu Jin Chung ◽

Helen C Christian ◽

Lisa C Heather ◽

...

Keyword(s):

Iron Deficiency ◽

Iron Overload ◽

Iron Homeostasis ◽

Iron Absorption ◽

Metabolic Dysfunction ◽

Cardiac Iron ◽

Cardiac Iron Overload ◽

A Cell ◽

The Brain ◽

Specific Deletion

Hepcidin is the master regulator of systemic iron homeostasis. Derived primarily from the liver, it inhibits the iron exporter ferroportin in the gut and spleen, the sites of iron absorption and recycling respectively. Recently, we demonstrated that ferroportin is also found in cardiomyocytes, and that its cardiac-specific deletion leads to fatal cardiac iron overload. Hepcidin is also expressed in cardiomyocytes, where its function remains unknown. To define the function of cardiomyocyte hepcidin, we generated mice with cardiomyocyte-specific deletion of hepcidin, or knock-in of hepcidin-resistant ferroportin. We find that while both models maintain normal systemic iron homeostasis, they nonetheless develop fatal contractile and metabolic dysfunction as a consequence of cardiomyocyte iron deficiency. These findings are the first demonstration of a cell-autonomous role for hepcidin in iron homeostasis. They raise the possibility that such function may also be important in other tissues that express both hepcidin and ferroportin, such as the kidney and the brain.

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Response of Monocyte Iron Regulatory Protein Activity to Inflammation: Abnormal Behavior in Genetic Hemochromatosis

Blood ◽

10.1182/blood.v91.7.2565 ◽

1998 ◽

Vol 91 (7) ◽

pp. 2565-2572 ◽

Cited By ~ 45

Author(s):

Stefania Recalcati ◽

Roberta Pometta ◽

Sonia Levi ◽

Dario Conte ◽

Gaetano Cairo

Keyword(s):

Iron Homeostasis ◽

Iron Status ◽

Regulatory Protein ◽

Abnormal Behavior ◽

Iron Regulatory Protein ◽

Iron Release ◽

Protein Activity ◽

Intracellular Iron ◽

Ifn Γ ◽

Genetic Hemochromatosis

Abstract In genetic hemochromatosis (GH), iron overload affects mainly parenchymal cells, whereas little iron is found in reticuloendothelial (RE) cells. We previously found that RE cells from GH patients had an inappropriately high activity of iron regulatory protein (IRP), the key regulator of intracellular iron homeostasis. Elevated IRP should reflect a reduction of the iron pool, possibly because of a failure to retain iron. A defect in iron handling by RE cells that results in a lack of feedback regulation of intestinal absorption might be the basic abnormality in GH. To further investigate the capacity of iron retention in RE cells of GH patients, we used inflammation as a model system as it is characterized by a block of iron release from macrophages. We analyzed the iron status of RE cells by assaying IRP activity and ferritin content after 4, 8, and 24 hours of incubation with lipopolysaccharide (LPS) and interferon-γ (IFN-γ). RNA-bandshift assays showed that in monocytes and macrophages from 16 control subjects, IRP activity was transiently elevated 4 hours after treatment with LPS and IFN-γ but remarkably downregulated thereafter. Treatment with NO donors produced the same effects whereas an inducible Nitric Oxide Synthase (iNOS) inhibitor prevented them, which suggests that the NO pathway was involved. Decreased IRP activity was also found in monocytes from eight patients with inflammation. Interestingly, no late decrease of IRP activity was detected in cytokine-treated RE cells from 12 GH patients. Ferritin content was increased 24 hours after treatment in monocytes from normal subjects but not in monocytes from GH patients. The lack of downregulation of IRP activity under inflammatory conditions seems to confirm that the control of iron release from RE cells is defective in GH.

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Iron Status in Mice Carrying a Targeted Disruption of Lactoferrin

Molecular and Cellular Biology ◽

10.1128/mcb.23.1.178-185.2003 ◽

2003 ◽

Vol 23 (1) ◽

pp. 178-185 ◽

Cited By ~ 69

Author(s):

Pauline P. Ward ◽

Marisela Mendoza-Meneses ◽

Grainne A. Cunningham ◽

Orla M. Conneely

Keyword(s):

Iron Homeostasis ◽

Iron Status ◽

Transferrin Saturation ◽

Basal Diet ◽

Postnatal Period ◽

Homologous Gene ◽

Physiological Functions ◽

Adult Mice ◽

Exact Function

ABSTRACT Lactoferrin is a member of the transferrin family of iron-binding glycoproteins present in milk, mucosal secretions, and the secondary granules of neutrophils. While several physiological functions have been proposed for lactoferrin, including the regulation of intestinal iron uptake, the exact function of this protein in vivo remains to be established. To directly assess the physiological functions of lactoferrin, we have generated lactoferrin knockout (LFKO−/−) mice by homologous gene targeting. LFKO−/− mice are viable and fertile, develop normally, and display no overt abnormalities. A comparison of the iron status of suckling offspring from LFKO−/− intercrosses and from wild-type (WT) intercrosses showed that lactoferrin is not essential for iron delivery during the postnatal period. Further, analysis of adult mice on a basal or a high-iron diet revealed no differences in transferrin saturation or tissue iron stores between WT and LFKO−/− mice on either diet, although the serum iron levels were slightly elevated in LFKO-/- mice on the basal diet. Consistent with the relatively normal iron status, in situ hybridization analysis demonstrated that lactoferrin is not expressed in the postnatal or adult intestine. Collectively, these results support the conclusion that lactoferrin does not play a major role in the regulation of iron homeostasis.

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Furin-mediated release of soluble hemojuvelin: a new link between hypoxia and iron homeostasis

Blood ◽

10.1182/blood-2007-07-100677 ◽

2008 ◽

Vol 111 (2) ◽

pp. 924-931 ◽

Cited By ~ 190

Author(s):

Laura Silvestri ◽

Alessia Pagani ◽

Clara Camaschella

Keyword(s):

Iron Deficiency ◽

Bone Morphogenetic Proteins ◽

Skeletal Muscles ◽

Iron Homeostasis ◽

Bmp Signaling ◽

Specific Mechanism ◽

Furin Cleavage ◽

In Cells

The liver peptide hepcidin regulates iron absorption and recycling. Hemojuvelin (HJV) has a key role in hepcidin regulation, and its inactivation causes severe iron overload both in humans and in mice. Membrane HJV (m-HJV) acts as a coreceptor for bone morphogenetic proteins (BMPs), whereas soluble HJV (s-HJV) may down-regulate hepcidin in a competitive way interfering with BMP signaling. s-HJV is decreased by iron in vitro and increased by iron deficiency in vivo. However, the mechanisms regulating the 2 HJV isoforms remain unclear. Here we show that s-HJV originates from a furin cleavage at position 332–335. s-HJV is reduced in the cleavage mutant R335Q as well as in cells treated with a furin inhibitor, and increased in cells overexpressing exogenous furin, but not in cells overexpressing an inactive furin variant. Furin is up-regulated by iron deficiency and hypoxia in association with the stabilization of HIF-1α. Increased s-HJV in response to HIF-1α occurs during differentiation of murine muscle cells expressing endogenous Hjv. Our data are relevant to the mechanisms that relate iron metabolism to the hypoxic response. The release of s-HJV might be a tissue-specific mechanism, signaling the local iron requests of hypoxic skeletal muscles independently of the oxygen status of the liver.

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Iron Deficiency and Iron Excess Differently Affect Dendritic Architecture of Pyramidal Neurons in the Hippocampus of Piglets

Journal of Nutrition ◽

10.1093/jn/nxaa326 ◽

2020 ◽

Vol 151 (1) ◽

pp. 235-244

Author(s):

Vivian Perng ◽

Chong Li ◽

Carolyn R Klocke ◽

Shya E Navazesh ◽

Danna K Pinneles ◽

...

Keyword(s):

Iron Deficiency ◽

Hippocampal Neurons ◽

Iron Homeostasis ◽

Pyramidal Neurons ◽

Iron Status ◽

Duodenal Mucosa ◽

Deficiency Anemia ◽

Final Body Weight ◽

Iron Regulatory Proteins ◽

Dendritic Complexity

ABSTRACT Background Both iron deficiency and overload may adversely affect neurodevelopment. Objectives The study assessed how changes in early-life iron status affect iron homeostasis and cytoarchitecture of hippocampal neurons in a piglet model. Methods On postnatal day (PD) 1, 30 Hampshire × Yorkshire crossbreed piglets (n = 15/sex) were stratified by sex and litter and randomly assigned to experimental groups receiving low (L-Fe), adequate (A-Fe), or high (H-Fe) levels of iron supplement during the pre- (PD1–21) and postweaning periods (PD22–35). Pigs in the L-Fe, A-Fe, and H-Fe groups orally received 0, 1, and 30 mg Fe · kg weight−1 · d−1 preweaning and were fed a diet containing 30, 125, and 1000 mg Fe/kg postweaning, respectively. Heme indexes were analyzed weekly, and gene and protein expressions of iron regulatory proteins in duodenal mucosa, liver, and hippocampus were analyzed through qRT-PCR and western blot, respectively, on PD35. Hippocampal neurons stained using the Golgi-Cox method were traced and their dendritic arbors reconstructed in 3-D using Neurolucida. Dendritic complexity was quantified using Sholl and branch order analyses. Results Pigs in the L-Fe group developed iron deficiency anemia (hemoglobin = 8.2 g/dL, hematocrit = 20.1%) on PD35 and became stunted during week 5 with lower final body weight than H-Fe group pigs (6.6 compared with 9.6 kg, P < 0.05). In comparison with A-Fe, H-Fe increased hippocampal ferritin expression by 38% and L-Fe decreased its expression by 52% (P < 0.05), suggesting altered hippocampal iron stores. Pigs in the H-Fe group had greater dendritic complexity in CA1/3 pyramidal neurons than L-Fe group pigs as shown by more dendritic intersections with Sholl rings (P ≤ 0.04) and a greater number of dendrites (P ≤ 0.016). Conclusions In piglets, the developing hippocampus is susceptible to perturbations by dietary iron, with deficiency and overload differentially affecting dendritic arborization.

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Ferroportin deficiency in erythroid cells causes serum iron deficiency and promotes hemolysis due to oxidative stress

Blood ◽

10.1182/blood-2018-04-842997 ◽

2018 ◽

Vol 132 (19) ◽

pp. 2078-2087 ◽

Cited By ~ 14

Author(s):

De-Liang Zhang ◽

Manik C. Ghosh ◽

Hayden Ollivierre ◽

Yan Li ◽

Tracey A. Rouault

Keyword(s):

Oxidative Stress ◽

Iron Deficiency ◽

Iron Overload ◽

Serum Iron ◽

Iron Homeostasis ◽

Erythroid Cells ◽

Loss Of Function ◽

Hepatic Cells ◽

Tissue Iron

Abstract Ferroportin (FPN), the only known vertebrate iron exporter, transports iron from intestinal, splenic, and hepatic cells into the blood to provide iron to other tissues and cells in vivo. Most of the circulating iron is consumed by erythroid cells to synthesize hemoglobin. Here we found that erythroid cells not only consumed large amounts of iron, but also returned significant amounts of iron to the blood. Erythroblast-specific Fpn knockout (Fpn KO) mice developed lower serum iron levels in conjunction with tissue iron overload and increased FPN expression in spleen and liver without changing hepcidin levels. Our results also showed that Fpn KO mice, which suffer from mild hemolytic anemia, were sensitive to phenylhydrazine-induced oxidative stress but were able to tolerate iron deficiency upon exposure to a low-iron diet and phlebotomy, supporting that the anemia of Fpn KO mice resulted from erythrocytic iron overload and resulting oxidative injury rather than a red blood cell (RBC) production defect. Moreover, we found that the mean corpuscular volume (MCV) values of gain-of-function FPN mutation patients were positively associated with serum transferrin saturations, whereas MCVs of loss-of-function FPN mutation patients were not, supporting that erythroblasts donate iron to blood through FPN in response to serum iron levels. Our results indicate that FPN of erythroid cells plays an unexpectedly essential role in maintaining systemic iron homeostasis and protecting RBCs from oxidative stress, providing insight into the pathophysiology of FPN diseases.

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Sensing of Liver Iron Content Requires Cell-Cell Communication between Hepatocytes and Liver Sinusoidal Endothelial Cells

Blood ◽

10.1182/blood-2019-125586 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 432-432

Author(s):

Silvia Colucci ◽

Sandro Altamura ◽

Matthias Hentze ◽

Martina U. Muckenthaler

Keyword(s):

Endothelial Cells ◽

Mrna Expression ◽

Iron Content ◽

Iron Homeostasis ◽

Iron Status ◽

Molecular Signature ◽

Iron Release ◽

Liver Sinusoidal Endothelial Cells ◽

Intracellular Iron ◽

Sinusoidal Endothelial Cells

The liver stores iron and senses systemic and tissue iron availability. Hepatocytes control iron homeostasis by producing the peptide hormone hepcidin that controls dietary iron absorption and iron release from intracellular stores. Recent data challenged the exclusive role of hepatocytes in controlling iron levels. Indeed, liver sinusoidal endothelial cells (LSECs) increase BMP2 and BMP6 levels in response to iron, which control hepcidin expression in a paracrine manner. However the molecular mechanism(s) of how BMPs respond to iron levels remain unknown. We established primary murine LSEC cultures and exposed these to iron sources. Unexpectedly, BMP2 mRNA expression is strongly reduced by iron treatment, while BMP6 levels are only mildly increased. This finding suggests that intracellular iron content cannot directly activate BMP2 transcription and only slightly contribute to BMP6 upregulation in LSEC cultures. However, if LSECs are co-cultured with iron-loaded primary hepatocytes the expression of BMP2 and BMP6 is increased and the fold induction of BMP6 is greater compared to LSECs cultured alone, suggesting that the iron status of hepatocytes instructs the LSEC BMP response. These data are supported by findings in a genetic mouse model of iron overload (Slc40a1C326S/C326S). Hepatocytes isolated from Slc40a1C326S/C326S mice display an iron-loaded molecular signature and the expected low mRNA expression of Transferrin Receptor 1 (Tfr1). By contrast, LSECs show high expression of Tfr1, indicating intracellular iron deficiency. Despite this, hepatic BMP levels are increased, suggesting that BMP2 and BMP6 expression are directly related to the intracellular iron content of hepatocytes but not LSECs. RNA-sequencing of isolated hepatic cell populations is ongoing to identify putative hepatocyte regulators involved in the iron-mediated BMP2 and BMP6 regulation. Disclosures Muckenthaler: Silence Therapeutics: Consultancy; Novartis: Research Funding.

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