Mice Overexpressing Both Non-Mutated Human SOD1 and Mutated SOD1G93A Genes: A Competent Experimental Model for Studying Iron Metabolism in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by degeneration and loss of motor neurons in the spinal cord, brainstem and motor cortex. Up to 10% of ALS cases are inherited (familial, fALS) and associated with mutations, frequently in the superoxide dismutase 1 (SOD1) gene. Rodent transgenic models of ALS are often used to elucidate a complex pathogenesis of this disease. Of importance, both ALS patients and animals carrying mutated human SOD1 gene show symptoms of oxidative stress and iron metabolism misregulation. The aim of our study was to characterize changes in iron metabolism in one of the most commonly used models of ALS – transgenic mice overexpressing human mutated SOD1G93A gene. We analyzed the expression of iron-related genes in asymptomatic, 2-month old and symptomatic, 4-month old SOD1G93A mice. In parallel, respective age-matched mice overexpressing human non-mutated SOD1 transgene and control mice were analyzed. We demonstrate that the overexpression of both SOD1 and SOD1G93A genes account for a substantial increase in SOD1 protein levels and activity in selected tissues and that not all the changes in iron metabolism genes expression are specific for the overexpression of the mutated form of SOD1

Urinary hepcidin levels in iron-deficient and iron-supplemented piglets correlate with hepcidin hepatic mRNA and serum levels and with body iron status

Among livestock, domestic pig (Sus scrofa) is a species, in which iron metabolism has been most intensively examined during last decade. The obvious reason for studying the regulation of iron homeostasis especially in young pigs is neonatal iron deficiency anemia commonly occurring in these animals. Moreover, supplementation of essentially all commercially reared piglets with iron entails a need for monitoring the efficacy of this routine practice followed in the swine industry for several decades. Since the discovery of hepcidin many studies confirmed its role as key regulator of iron metabolism and pointed out the assessment of its concentrations in biological fluids as diagnostic tool for iron-related disorder. Here we demonstrate that urine hepcidin-25 levels measured by a combination of weak cation exchange chromatography and time-of-flight mass spectrometry (WCX-TOF MS) are highly correlated with mRNA hepcidin expression in the liver and plasma hepcidin-25 concentrations in anemic and iron-supplemented 28-day old piglets. We also found a high correlation between urine hepcidin level and hepatic non-heme iron content. Our results show that similarly to previously described transgenic mouse models of iron disorders, young pigs constitute a convenient animal model to explore accuracy and relationship between indicators for assessing systemic iron status

Dietary iron deficiency anemia

Erytropoeza jest procesem biologicznym o największym zapotrzebowaniu na żelazo, które jest niezbędne do syntezy hemu w komórkach prekursorowych erytrocytów i dlatego jej prawidłowy przebieg jest szczególnie wrażliwy na niedobór tego mikroelementu. Żywieniowy niedobór żelaza jest głównym powodem występowania niedokrwistości (anemii) u ludzi. Niska zawartość żelaza w diecie prowadzi do wyczerpania zapasów tego mikroelemntu w organizmie, następstwem czego jest rozwój anemii na tle niedoboru żelaza. Ten typ anemii występuje najczęściej wśród społeczeństw krajów rozwijających się. Głównymi grupami ryzyka występowania anemii na tle niedoboru żelaza są niemowlęta, dzieci, dorastająca młodzież oraz kobiety ciężarne i kobiety w okresie laktacji. Diagnoza anemii na tle niedoboru żelaza wymaga przeprowadzenia u pacjentów analizy parametrów hematologicznych we krwi. Powinna również obejmować analizę parametrów biochemicznych żelaza oraz poziomu ferrytyny w surowicy. W ostatnich latach wskazuje się na nowe parametry, które mogą okazać się pomocne dla lekarzy w diagnozowaniu niedokrwistości na tle niedoboru żelaza.Erythropoiesis is the biological process that consumes the highest amount of body iron for heme synthesis in erythrocyte percursors. Iron deficiency anemia (IDA) is the most frequent form of anemia in humans worldwide caused by deficiency of dietary iron. IDA develops as a result of depleted iron stores. IDA is more common in developing countries, with infants, children, adolescents, pregnant and lactating women being at a significantly higher risk for this condition. To reach a definitive diagnosis of IDA, in addition to performing analysis of blood hematological parameters, iron serum parametres and ferritin level should be measured. In recent years, new parameters have been developed to help physicians in the diagnosis of IDA

Iron metabolism - state of the art 2014

Żelazo jest biometalem występującym w dwóch głównych stopniach utlenienia - Fe(II) i Fe(III). O wykorzystaniu żelaza przez organizmy żywe zadecydowała szeroka rozpiętość potencjału oksydoredukcyjnego tego metalu, możliwa dzięki zmiennym interakcjom z wiążącymi go ligandami oraz udział w reakcjach przeniesienia elektronu. Żelazo występuje w centrach aktywnych wielu enzymów katalizujących różnorodne reakcje, stanowiące podłoże kluczowych procesów metabolicznych takich jak fosforylacja oksydacyjna, synteza DNA, obróbka micro RNA, transport tlenu. Z drugiej strony, żelazo jest toksyczne poprzez udział w reakcji Fentona, w której powstaje rodnik wodorotlenkowy, utleniacz niszczący struktury komórkowe. Komórkowa homeostaza żelaza polega na dostarczeniu tego metalu do podstawowych procesów biochemicznych, w których uczestniczy oraz na ograniczeniu jego udziału w reakcji Fentona. Obrót żelaza w komórce pozostaje głównie pod kontrolą cytoplazmatycznych białek IRP1 i IRP2 wiążących się z RNA, które koordynują syntezę białek uczestniczących w komórkowym transporcie żelaza, jego magazynowaniu i metabolicznym użyciu. Ogólnoustrojowa równowaga żelaza opiera się w dużej mierze na osi regulatorowej pomiędzy hepcydyną, peptydem syntetyzowanym głównie w hepatocytach oraz ferroportyną, białkiem transportującym żelazo z komórek. Funkcjonowanie tej osi zapewnia prawidłową dystrybucję i obrót żelaza między absorpcyjnymi enterocytami, makrofagami układu siateczkowo-śródbłonkowego oraz prekursorami czerwonych krwinek. Artykuł podsumowuje najważniejsze odkrycia z ostatnich 15 lat, które okazały się kluczowe dla zrozumienia homeostazy żelaza.Iron is biometal, existing in two main oxidation states, i.e. Fe(II)/Fe(III). The extensive range of redox potential available to this metal by varying its interactions with coordinating ligands, as well as its capacity to participate in one-electron transfer reactions, are the reasons why iron is essential for almost all living organisms. Iron is found in the active sites of a large number of enzymes that catalyze diverse redox reactions underlying fundamental metabolic processes, including respiratory oxidation, DNA synthesis, microRNA processing and oxygen transport. On the other hand, iron is toxic due to its capacity to catalyze via Fenton reaction the production of hydroxyl radical, a highly destructive oxidant. Cellular iron homeostasis consists in providing iron for a variety of biochemical processes and in limiting iron availability for Fenton reaction. Cellular iron homeostasis is mainly controlled by the iron regulatory proteins (IRP1 and IRP2) - two cytoplasmic RNA-binding proteins involved in the mechanisms that coordinate the synthesis of a number of key proteins responsible for cellular iron transport, storage and utilization. Systemic iron balance is largely based on a regulatory axis between the liver-derived peptide hepcidin and the iron exporter ferroportin proved to be fundamental for the coordination of iron fluctuations in the body and its distribution among the main sites of iron metabolism such as absorptive enterocytes, reticuloendothelial macrophages, hepatocytes and erythroid precursors of red blood cells. The article briefly resumes main discoveries within last 15 years, critical for the understanding iron homeostasis

Haemolysis and perturbations in the systemic iron metabolism of suckling, copper-deficient mosaic mutant mice - an animal model of Menkes disease.

The biological interaction between copper and iron is best exemplified by the decreased activity of multicopper ferroxidases under conditions of copper deficiency that limits the availability of iron for erythropoiesis. However, little is known about how copper deficiency affects iron homeostasis through alteration of the activity of other copper-containing proteins, not directly connected with iron metabolism, such as superoxide dismutase 1 (SOD1). This antioxidant enzyme scavenges the superoxide anion, a reactive oxygen species contributing to the toxicity of iron via the Fenton reaction. Here, we analyzed changes in the systemic iron metabolism using an animal model of Menkes disease: copper-deficient mosaic mutant mice with dysfunction of the ATP7A copper transporter. We found that the erythrocytes of these mutants are copper-deficient, display decreased SOD1 activity/expression and have cell membrane abnormalities. In consequence, the mosaic mice show evidence of haemolysis accompanied by haptoglobin-dependent elimination of haemoglobin (Hb) from the circulation, as well as the induction of haem oxygenase 1 (HO1) in the liver and kidney. Moreover, the hepcidin-ferroportin regulatory axis is strongly affected in mosaic mice. These findings indicate that haemolysis is an additional pathogenic factor in a mouse model of Menkes diseases and provides evidence of a new indirect connection between copper deficiency and iron metabolism

Decreased copper content and activity/expression of SOD1 in circulating erythrocytes from <i>ms/−</i> mice.

<p>(<b>A</b>) Decreased copper content in erythrocytes of <i>ms/−</i> mice. Values are expressed as the means ± S.D. for erythrocyte samples obtained from 15 control (<i>+/−</i>) and 15 mutant (<i>ms/−</i>) males. (<b>B</b>) <i>left-hand panel</i>, the activity of SOD was measured after resolution by gel electrophoresis using the Nitroblue Tetrazolium (NBT)/riboflavin method as described in the Experimental section. The analyses were performed using erythrocyte total extracts obtained from <i>ms/−</i> and <i>+/−</i> males and representative results are shown. (<b>C</b>) <i>left-hand panel</i>, SOD1 levels in erythrocytes were analyzed by western blotting as described in the Experimental section. The analyses were performed using erythrocyte total extracts obtained from <i>ms/−</i> and control (<i>+/−</i>) males, and representative results are shown. The blot was reprobed with monoclonal anti-actin antibody as a loading control. (<b>B,C</b>) <i>right-hand panels</i>, the intensity of the SOD bands was quantified with a molecular Imager using Quantity One software (Bio-Rad) and is plotted in arbitrary units to present activity (<b>B</b>) and protein level (<b>C</b>). Results are expressed as the mean ± S.D. for 5 mice of both the <i>ms/−</i> and <i>+/−</i> genotypes. Significant differences are indicated (* – P<0.05; ** – P<0.01).</p

Hepatic and renal iron status in control (+/−) and mutant (<i>ms/−</i>) mice.

<p>Non-haem hepatic (<b>A</b>) and renal (<b>C</b>) iron content was measured as described in the Experimental section. Values are expressed as the means ± S.D. for both liver and kidney samples obtained from 15 mice of each genotype. L-ferritin levels in hepatic (<b>B</b>) and renal (<b>D</b>) cytosolic protein extracts (50 µg/lane) were assessed by western blot analysis <i>left-hand panels</i>. The blots were reprobed with polyclonal anti-human actin antibody as a loading control. (<b>B</b>) and (<b>D</b>) <i>right-hand panels</i> Immunolabelled ferritin bands from four mice were quantified using a Molecular Imager and ferritin protein levels (means ± S.D.) are plotted in arbitrary units. ** – P<0.01.</p

Increased expression of haem oxygenase 1 (HO1) in kidneys of <i>ms/−</i> mice.

<p>(<b>A</b>) Real-time quantitative PCR analysis of renal HO1 mRNA expression. The histogram displays HO1 mRNA levels in arbitrary units (means ± S.D., n = 6). (<b>B</b>) <i>left-hand panel</i> Western blot analysis of HO1 protein levels in hepatic membrane fractions prepared from <i>+/−</i> and <i>ms/−</i> males. The blot was reprobed with polyclonal anti-human actin antibody as a loading control. <i>right-hand panel</i> Immunolabelled HO1 bands from six mice were quantified using a Molecular Imager and HO1 protein levels (means ± S.D.) are plotted in arbitrary units. ** – P<0.01. (<b>C</b>) <i>left-hand panel</i> Immunofluorescent staining of HO1 in <i>+/−</i> and <i>ms/−</i> kidneys analyzed by confocal microscopy. RN – renal tubules; RG – renal glomeruli. <i>middle panel</i> Transmitted light image shows the structure of glomeruli and tubules as well as the presence of a large lesion (asterisk) in the kidney of a mutant. <i>right-hand panel</i> To confirm the specificity of HO1 detection, kidney sections of <i>+/−</i> and <i>ms/−</i> males were incubated with only the secondary antibody. No HO1 staining was detected in these negative controls. Nuclei were counterstained with DAPI. Bars correspond to 50 µm.</p

Correlation between decreased ferroportin (Fpn) protein level and increased hepcidin (Hepc) mRNA expression in the liver of <i>ms/−</i> males.

<p>(<b>A</b>) Immunofluorescent staining of Fpn in <i>+/−</i> and <i>ms/−</i> males. <i>top panel</i> Livers analyzed by confocal microscopy. <i>middle panel</i> Tissue morphology observed in transmitted light. <i>bottom panel</i> To confirm the specificity of Fpn detection, liver sections of <i>+/−</i> and <i>ms/−</i> males were incubated with only the secondary antibody. No Fpn staining was detected in these negative controls. Nuclei were counterstained with DAPI. Bars correspond to 50 µm. (<b>B</b>) Colocalization (<i>bottom panel</i>) of Fpn (red channel) and F4/80, a macrophage marker (green channel) in liver from <i>+/−</i> and <i>ms/−</i> males analyzed by confocal microscopy. Nuclei were counterstained with DAPI. Bars correspond to 50 µm. (<b>C</b>) Real-time quantitative PCR analysis of hepatic Hepc mRNA expression in <i>+/−</i> and <i>ms/−</i> males. The histogram displays Hepc mRNA levels in arbitrary units (means ± S.D., n = 6). Significant difference is indicated (** – P<0.01).</p

Increased expression of haem oxygenase 1 (HO1) in Kupffer cells of <i>ms/−</i> mice.

<p>(<b>A</b>) Real-time quantitative PCR analysis of hepatic HO1 mRNA expression. The histogram displays HO1 mRNA levels in arbitrary units (means ± S.D., n = 6). (<b>B</b>) <i>left-hand panel</i> Western blot analysis of HO1 protein levels in hepatic membrane fractions prepared from <i>+/−</i> and <i>ms/−</i> males. The blot was reprobed with polyclonal anti-human actin antibody as a loading control. <i>right-hand panel</i> Immunolabelled HO1 bands from six mice were quantified using a Molecular Imager and HO1 protein levels (means ± S.D.) are plotted in arbitrary units. * – P<0.05. (<b>C</b>) <i>top panel</i> Immunofluorescent staining of HO1 in <i>+/−</i> and <i>ms/−</i> livers analyzed by confocal microscopy. <i>middle panel</i> Tissue morphology observed in transmitted light. <i>bottom panel</i> To confirm the specificity of Fpn detection, liver sections of <i>+/−</i> and <i>ms/−</i> males were incubated with only the secondary antibody. No HO1 staining was detected in these negative controls. Nuclei were counterstained with DAPI. Bars correspond to 50 µm. (<b>D</b>) Colocalization (<i>bottom panel</i>) of HO1 (red channel) and F4/80, a macrophage marker (green channel) in the livers of <i>+/</i>− and <i>ms/−</i> males analyzed by confocal microscopy. Nuclei were counterstained with DAPI. Bars correspond to 50 µm.</p