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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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Hx Expression in brain. (A) Western blotting analysis on cell extracts from E. coli expressing the recombinant Hx or the unrelated recombinant protein TTC3 (left) and on serum of a wild-type mouse and of an Hx- mouse (right) assayed with the monoclonal antibody 3D6/E12. The antibody recognizes the recombinant Hx of 42 kD (corresponding to amino acids 81–459 of mouse Hx) and a single band of 57 kD in wild-type serum, but not in Hx- serum. (B) Western blotting analysis on brain extracts of a wild-type mouse and an Hx- mouse assayed with the monoclonal antibody 3D6/E12. Hx is detected only in wild-type sample. (C) Histochemical β-galactosidase detection in the brain of an Hx- mouse. Tissues were stained with X-Gal and counterstained with nuclear fast red as reported in ‘Materials and methods’. β-galactosidase activity is shown in lateral (a) and third (b) ventricles. β-galactosidase activity is restricted to ependymal cells lining the ventricular system (arrows), whereas no blue staining is detected in the epithelial cells of the choroid plexus (arrowhead). Bar = 250 μm. The insets in ‘a’ and ‘b’ show ependymal cells, stained in blue, at higher magnification (bar = 100 μm).
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fig01: Hx Expression in brain. (A) Western blotting analysis on cell extracts from E. coli expressing the recombinant Hx or the unrelated recombinant protein TTC3 (left) and on serum of a wild-type mouse and of an Hx- mouse (right) assayed with the monoclonal antibody 3D6/E12. The antibody recognizes the recombinant Hx of 42 kD (corresponding to amino acids 81–459 of mouse Hx) and a single band of 57 kD in wild-type serum, but not in Hx- serum. (B) Western blotting analysis on brain extracts of a wild-type mouse and an Hx- mouse assayed with the monoclonal antibody 3D6/E12. Hx is detected only in wild-type sample. (C) Histochemical β-galactosidase detection in the brain of an Hx- mouse. Tissues were stained with X-Gal and counterstained with nuclear fast red as reported in ‘Materials and methods’. β-galactosidase activity is shown in lateral (a) and third (b) ventricles. β-galactosidase activity is restricted to ependymal cells lining the ventricular system (arrows), whereas no blue staining is detected in the epithelial cells of the choroid plexus (arrowhead). Bar = 250 μm. The insets in ‘a’ and ‘b’ show ependymal cells, stained in blue, at higher magnification (bar = 100 μm).

Mentions: As several authors have reported Hx expression in the central nervous system [17, 18, 23], and Hx promoter activity was found in several brain regions in mice [20, 21], we analysed Hx expression in the mouse brain. To this end, we used the monoclonal antibody 3D6/E12 raised against a recombinant Hx lacking the first 80 amino acids of the native protein. As shown in Fig. 1A, this antibody recognizes the recombinant Hx of 42 kD in E. coli extracts and the mouse protein of 57 kD in the serum of wild-type animals. Western blotting analysis of total brain homogenates from 2-month-old mice, transcardially perfused with PBS, showed Hx expression in wild-type mice, and, as expected, not in Hx- animals (Fig. 1B).


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)

Hx Expression in brain. (A) Western blotting analysis on cell extracts from E. coli expressing the recombinant Hx or the unrelated recombinant protein TTC3 (left) and on serum of a wild-type mouse and of an Hx- mouse (right) assayed with the monoclonal antibody 3D6/E12. The antibody recognizes the recombinant Hx of 42 kD (corresponding to amino acids 81–459 of mouse Hx) and a single band of 57 kD in wild-type serum, but not in Hx- serum. (B) Western blotting analysis on brain extracts of a wild-type mouse and an Hx- mouse assayed with the monoclonal antibody 3D6/E12. Hx is detected only in wild-type sample. (C) Histochemical β-galactosidase detection in the brain of an Hx- mouse. Tissues were stained with X-Gal and counterstained with nuclear fast red as reported in ‘Materials and methods’. β-galactosidase activity is shown in lateral (a) and third (b) ventricles. β-galactosidase activity is restricted to ependymal cells lining the ventricular system (arrows), whereas no blue staining is detected in the epithelial cells of the choroid plexus (arrowhead). Bar = 250 μm. The insets in ‘a’ and ‘b’ show ependymal cells, stained in blue, at higher magnification (bar = 100 μm).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4496126&req=5

fig01: Hx Expression in brain. (A) Western blotting analysis on cell extracts from E. coli expressing the recombinant Hx or the unrelated recombinant protein TTC3 (left) and on serum of a wild-type mouse and of an Hx- mouse (right) assayed with the monoclonal antibody 3D6/E12. The antibody recognizes the recombinant Hx of 42 kD (corresponding to amino acids 81–459 of mouse Hx) and a single band of 57 kD in wild-type serum, but not in Hx- serum. (B) Western blotting analysis on brain extracts of a wild-type mouse and an Hx- mouse assayed with the monoclonal antibody 3D6/E12. Hx is detected only in wild-type sample. (C) Histochemical β-galactosidase detection in the brain of an Hx- mouse. Tissues were stained with X-Gal and counterstained with nuclear fast red as reported in ‘Materials and methods’. β-galactosidase activity is shown in lateral (a) and third (b) ventricles. β-galactosidase activity is restricted to ependymal cells lining the ventricular system (arrows), whereas no blue staining is detected in the epithelial cells of the choroid plexus (arrowhead). Bar = 250 μm. The insets in ‘a’ and ‘b’ show ependymal cells, stained in blue, at higher magnification (bar = 100 μm).
Mentions: As several authors have reported Hx expression in the central nervous system [17, 18, 23], and Hx promoter activity was found in several brain regions in mice [20, 21], we analysed Hx expression in the mouse brain. To this end, we used the monoclonal antibody 3D6/E12 raised against a recombinant Hx lacking the first 80 amino acids of the native protein. As shown in Fig. 1A, this antibody recognizes the recombinant Hx of 42 kD in E. coli extracts and the mouse protein of 57 kD in the serum of wild-type animals. Western blotting analysis of total brain homogenates from 2-month-old mice, transcardially perfused with PBS, showed Hx expression in wild-type mice, and, as expected, not in Hx- animals (Fig. 1B).

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