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

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

Haemolysis and acanthocytosis in <i>ms/−</i> mice.

<p>(<b>A</b>) Serum of control (<i>+/−</i>) and mutant (<i>ms/−</i>) males obtained by centrifugation. (<b>B</b>) Peripheral blood smears of <i>ms/−</i> mice showing increased numbers of acanthocytes. High magnification images of a normal and an abnormally-shaped erythrocyte are shown (insets). Six animals from both the control and <i>ms/−</i> male groups were examined and one typical sample of each is shown. Bars correspond to 5 µm. (<b>C</b>) Plasma levels of haptoglobin (Hp) were assessed by western blotting as described in the Experimental section. The blot was reprobed with monoclonal anti-actin antibody as a loading control.</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

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

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