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Haemopexin affects iron distribution and ferritin expression in mouse brain.

Morello N, Tonoli E, Logrand F, Fiorito V, Fagoonee S, Turco E, Silengo L, Vercelli A, Altruda F, Tolosano E - J. Cell. Mol. Med. (2009)

Bottom Line: Hx protein has been found in the sciatic nerve, skeletal muscle, retina, brain and cerebrospinal fluid (CSF).However, a strong reduction in the number of ferritin-positive cells was observed in the cerebral cortex of Hx- animals.These data demonstrate that Hx plays an important role in controlling iron distribution within brain, thus suggesting its involvement in iron-related neurodegenerative diseases.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biotechnology Center, University of Torino, Torino, Italy.

ABSTRACT
Haemopexin (Hx) is an acute phase plasma glycoprotein, mainly produced by the liver and released into plasma where it binds heme with high affinity and delivers it to the liver. This system provides protection against free heme-mediated oxidative stress, limits access by pathogens to heme and contributes to iron homeostasis by recycling heme iron. Hx protein has been found in the sciatic nerve, skeletal muscle, retina, brain and cerebrospinal fluid (CSF). Recently, a comparative proteomic analysis has shown an increase of Hx in CSF from patients with Alzheimer's disease, thus suggesting its involvement in heme detoxification in brain. Here, we report that Hx is synthesised in brain by the ventricular ependymal cells. To verify whether Hx is involved in heme scavenging in brain, and consequently, in the control of iron level, iron deposits and ferritin expression were analysed in cerebral regions known for iron accumulation. We show a twofold increase in the number of iron-loaded oligodendrocytes in the basal ganglia and thalamus of Hx- mice compared to wild-type controls. Interestingly, there was no increase in H- and L-ferritin expression in these regions. This condition is common to several human neurological disorders such as Alzheimer's disease and Parkinson's disease in which iron loading is not associated with an adequate increase in ferritin expression. However, a strong reduction in the number of ferritin-positive cells was observed in the cerebral cortex of Hx- animals. Consistent with increased iron deposits and inadequate ferritin expression, malondialdehyde level and Cu-Zn superoxide dismutase-1 expression were higher in the brain of Hx- mice than in that of wild-type controls. These data demonstrate that Hx plays an important role in controlling iron distribution within brain, thus suggesting its involvement in iron-related neurodegenerative diseases.

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Decreased expression of H- and L-Ft in Hx- brain. (A) Western blotting analysis of H- and L-Ft expression in total brain extracts of four wild-type and four Hx- mice. A representative experiment for each protein is shown. Band intensities were measured by densitometry and normalized to actin expression (AU: arbitrary unit). Densitometry data represent mean ± S.E.M.; n= 4 for each genotype. *=P < 0.05; **=P < 0.01. Results shown are representative of at least three independent experiments. (B) Reconstruction with Neurolucida/ Neuroexplorer system of two serial brain sections of an Hx- mouse and a wild-type mouse labelled with anti H- (left) and L- Ft (right) antibodies, respectively. We marked in red the positive cells in the cerebral cortex, called region ‘a’, and in green the positive cells in the basal ganglia and thalamus, called region ‘b’. Note the reduction in the number of H+ and L-Ft+ cells in the region ‘a’ of Hx- mice. (C) Quantification of H+ (left) and L-Ft+ (right) cells shown in (B). Data represent mean ± S.E.M., n= 3 mice for each genotype. *=P < 0.05.
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fig04: Decreased expression of H- and L-Ft in Hx- brain. (A) Western blotting analysis of H- and L-Ft expression in total brain extracts of four wild-type and four Hx- mice. A representative experiment for each protein is shown. Band intensities were measured by densitometry and normalized to actin expression (AU: arbitrary unit). Densitometry data represent mean ± S.E.M.; n= 4 for each genotype. *=P < 0.05; **=P < 0.01. Results shown are representative of at least three independent experiments. (B) Reconstruction with Neurolucida/ Neuroexplorer system of two serial brain sections of an Hx- mouse and a wild-type mouse labelled with anti H- (left) and L- Ft (right) antibodies, respectively. We marked in red the positive cells in the cerebral cortex, called region ‘a’, and in green the positive cells in the basal ganglia and thalamus, called region ‘b’. Note the reduction in the number of H+ and L-Ft+ cells in the region ‘a’ of Hx- mice. (C) Quantification of H+ (left) and L-Ft+ (right) cells shown in (B). Data represent mean ± S.E.M., n= 3 mice for each genotype. *=P < 0.05.

Mentions: Ferritin expression was analysed, firstly, by Western blotting on extracts from whole brain. We observed a lower expression level of H- and L-Ft in Hx- brains than in wild-type controls (Fig. 4A). This was further confirmed by ELISA (wild-type H-Ft: 884.5 ± 8.5 ng/mg versus Hx- H-Ft: 753.5 ± 7.5 ng/mg, P < 0.01; wild-type L-Ft: 13.45 ± 0.25 ng/mg total protein versus Hx- L-Ft: 12.05 ± 0.15 ng/mg, P < 0.05). Then, ferritin expression was analysed by immunohistochemistry on serial sections encompassing the same regions analysed for iron deposits. In the basal ganglia and in the thalamus no differences in the number of H+- or L-Ft+ cells were observed between Hx- and wild-type mice. On the other hand, we noted a decrease in the number of both H+ and L-Ft+ cells in the cortex of Hx- mice compared to that of wild-type animals (Fig. 4B). To quantify this difference, H+ and L-Ft+ cells in the cortex (called region ‘a’) and in the basal ganglia and thalamus (called region ‘b’) were counted. There was a decrease of about 40% in the number of both H+ and L-Ft+ cells in the region ‘a’ of Hx- mice, whereas no differences were detected in the region ‘b’ (Fig. 4C). The decrease in ferritin expression only in the cortex was further confirmed by Western blotting analysis on extracts from regions ‘a’ and ‘b’ (not shown).


Haemopexin affects iron distribution and ferritin expression in mouse brain.

Morello N, Tonoli E, Logrand F, Fiorito V, Fagoonee S, Turco E, Silengo L, Vercelli A, Altruda F, Tolosano E - J. Cell. Mol. Med. (2009)

Decreased expression of H- and L-Ft in Hx- brain. (A) Western blotting analysis of H- and L-Ft expression in total brain extracts of four wild-type and four Hx- mice. A representative experiment for each protein is shown. Band intensities were measured by densitometry and normalized to actin expression (AU: arbitrary unit). Densitometry data represent mean ± S.E.M.; n= 4 for each genotype. *=P < 0.05; **=P < 0.01. Results shown are representative of at least three independent experiments. (B) Reconstruction with Neurolucida/ Neuroexplorer system of two serial brain sections of an Hx- mouse and a wild-type mouse labelled with anti H- (left) and L- Ft (right) antibodies, respectively. We marked in red the positive cells in the cerebral cortex, called region ‘a’, and in green the positive cells in the basal ganglia and thalamus, called region ‘b’. Note the reduction in the number of H+ and L-Ft+ cells in the region ‘a’ of Hx- mice. (C) Quantification of H+ (left) and L-Ft+ (right) cells shown in (B). Data represent mean ± S.E.M., n= 3 mice for each genotype. *=P < 0.05.
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fig04: Decreased expression of H- and L-Ft in Hx- brain. (A) Western blotting analysis of H- and L-Ft expression in total brain extracts of four wild-type and four Hx- mice. A representative experiment for each protein is shown. Band intensities were measured by densitometry and normalized to actin expression (AU: arbitrary unit). Densitometry data represent mean ± S.E.M.; n= 4 for each genotype. *=P < 0.05; **=P < 0.01. Results shown are representative of at least three independent experiments. (B) Reconstruction with Neurolucida/ Neuroexplorer system of two serial brain sections of an Hx- mouse and a wild-type mouse labelled with anti H- (left) and L- Ft (right) antibodies, respectively. We marked in red the positive cells in the cerebral cortex, called region ‘a’, and in green the positive cells in the basal ganglia and thalamus, called region ‘b’. Note the reduction in the number of H+ and L-Ft+ cells in the region ‘a’ of Hx- mice. (C) Quantification of H+ (left) and L-Ft+ (right) cells shown in (B). Data represent mean ± S.E.M., n= 3 mice for each genotype. *=P < 0.05.
Mentions: Ferritin expression was analysed, firstly, by Western blotting on extracts from whole brain. We observed a lower expression level of H- and L-Ft in Hx- brains than in wild-type controls (Fig. 4A). This was further confirmed by ELISA (wild-type H-Ft: 884.5 ± 8.5 ng/mg versus Hx- H-Ft: 753.5 ± 7.5 ng/mg, P < 0.01; wild-type L-Ft: 13.45 ± 0.25 ng/mg total protein versus Hx- L-Ft: 12.05 ± 0.15 ng/mg, P < 0.05). Then, ferritin expression was analysed by immunohistochemistry on serial sections encompassing the same regions analysed for iron deposits. In the basal ganglia and in the thalamus no differences in the number of H+- or L-Ft+ cells were observed between Hx- and wild-type mice. On the other hand, we noted a decrease in the number of both H+ and L-Ft+ cells in the cortex of Hx- mice compared to that of wild-type animals (Fig. 4B). To quantify this difference, H+ and L-Ft+ cells in the cortex (called region ‘a’) and in the basal ganglia and thalamus (called region ‘b’) were counted. There was a decrease of about 40% in the number of both H+ and L-Ft+ cells in the region ‘a’ of Hx- mice, whereas no differences were detected in the region ‘b’ (Fig. 4C). The decrease in ferritin expression only in the cortex was further confirmed by Western blotting analysis on extracts from regions ‘a’ and ‘b’ (not shown).

Bottom Line: Hx protein has been found in the sciatic nerve, skeletal muscle, retina, brain and cerebrospinal fluid (CSF).However, a strong reduction in the number of ferritin-positive cells was observed in the cerebral cortex of Hx- animals.These data demonstrate that Hx plays an important role in controlling iron distribution within brain, thus suggesting its involvement in iron-related neurodegenerative diseases.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biotechnology Center, University of Torino, Torino, Italy.

ABSTRACT
Haemopexin (Hx) is an acute phase plasma glycoprotein, mainly produced by the liver and released into plasma where it binds heme with high affinity and delivers it to the liver. This system provides protection against free heme-mediated oxidative stress, limits access by pathogens to heme and contributes to iron homeostasis by recycling heme iron. Hx protein has been found in the sciatic nerve, skeletal muscle, retina, brain and cerebrospinal fluid (CSF). Recently, a comparative proteomic analysis has shown an increase of Hx in CSF from patients with Alzheimer's disease, thus suggesting its involvement in heme detoxification in brain. Here, we report that Hx is synthesised in brain by the ventricular ependymal cells. To verify whether Hx is involved in heme scavenging in brain, and consequently, in the control of iron level, iron deposits and ferritin expression were analysed in cerebral regions known for iron accumulation. We show a twofold increase in the number of iron-loaded oligodendrocytes in the basal ganglia and thalamus of Hx- mice compared to wild-type controls. Interestingly, there was no increase in H- and L-ferritin expression in these regions. This condition is common to several human neurological disorders such as Alzheimer's disease and Parkinson's disease in which iron loading is not associated with an adequate increase in ferritin expression. However, a strong reduction in the number of ferritin-positive cells was observed in the cerebral cortex of Hx- animals. Consistent with increased iron deposits and inadequate ferritin expression, malondialdehyde level and Cu-Zn superoxide dismutase-1 expression were higher in the brain of Hx- mice than in that of wild-type controls. These data demonstrate that Hx plays an important role in controlling iron distribution within brain, thus suggesting its involvement in iron-related neurodegenerative diseases.

Show MeSH
Related in: MedlinePlus