Development and iron-dependent expression of hephaestin in different brain regions of rats.

It has been suggested that Hephaestin (Heph), a newly discovered ceruloplasmin homologue, is necessary for iron egress from the enterocytes into circulation via interacting with ferroportin1 (FP1). Based on the putative function of Heph, and the similarity between the process of iron transport in the enterocytes and that in the blood-brain barrier (BBB) cells, it has also been proposed that Heph plays a similar role in exporting iron from the BBB cells and other brain cells as it works in the enterocytes via interacting with FP1. The existence of FP1 in the brain has been demonstrated. In this study, we investigated Heph expression and effects of development and iron in the cortex, hippocampus, striatum, and substantia nigra. The data demonstrated that all the four regions we examined have the ability to express Heph mRNA and protein. The findings also showed that both the development and iron status have a significant effect on Heph expression and the effects of iron status are regionally specific. It was also suggested that Heph expression is probably regulated at the transcriptional level by the development and iron in these brain regions. These findings, together with other published data, support a putative role of Heph in the iron metabolism in the brain.

Ceruloplasmin expression and its role in iron transport in C6 cells.

Ceruloplasmin (CP) is essential for brain iron homeostasis. However, little is known about the effect of iron on CP expression in the brain. Also, the role of CP in brain iron transport has not been well determined. In this study, we investigated the effects of iron on CP expression and the role of CP in iron transport in the C6 rat glioma cells. Our data showed that treatment of the cells with iron (cell iron overload) or iron chelators (cell iron deficiency) did not induce a significant change in the expression of CP mRNA. However, western blotting analysis demonstrated that cell iron overload induced a significant decrease in CP protein content in the cells and that treatment with iron chelators led to a significant increase in CP protein level in the cells. These findings suggest a translational regulation of CP expression by iron in the cells. We also examined the effects of CP on iron transport in the cells. We found that glycosylphosphatidylinositol-anchored CP did not have any impact on iron uptake by normal iron or iron-deficient cells nor on iron release from normal iron or iron-sufficient cells. However, low concentrations of soluble CP (2-8 microg/ml) increased iron uptake by iron-deficient C6 glioma cells, while the same concentrations of CP had no effect on iron uptake by normal iron cells and iron release from normal iron and iron-sufficient cells. The possible reason for the difference between our results in vitro and those obtained from in vivo studies was discussed.

Role of soluble ceruloplasmin in iron uptake by midbrain and hippocampus neurons.

Ceruloplasmin (CP) is essential for brain iron homeostasis. However, its precise function in brain iron transport has not been definitely determined. In this study, we investigated the effects of soluble CP on iron influx and efflux in primary neuronal culture from the midbrain (the substantia nigra and striatum) and the hippocampus. Our data showed that low concentrations of CP (2, 4, 8 microg/ml) can promote iron influx into iron-deficient neurons, but not the neurons with normal iron status. The same concentrations of CP had no effect on iron efflux from iron-sufficient and normal-iron neurons. Contrary to our expectation, we did not find any regional difference in the effects of CP on iron influx as well as efflux in neurons. The changes in quenching (iron influx) and also dequenching (iron efflux) of intracellular fluorescence, induced by the addition of CP with iron, in the midbrain neurons were no different from those in the hippocampus neurons. The data showed that soluble CP has a role in iron uptake by iron-deficient brain neurons under our experimental conditions. The physiological significance of the results forms the focus for future work.

Increased divalent metal transporter 1 expression might be associated with the neurotoxicity of L-DOPA.

Based on the available data, we speculated that changes in brain iron metabolism induced by L-DOPA might be associated with the neurotoxicity of L-DOPA. To investigate this possibility, the effects of L-DOPA on the expression of iron influx proteins [transferrin receptor (TfR) and divalent metal transporter 1 (DMT1)], iron efflux protein (ferroportin 1), and iron uptake in C6 glioma cells were determined in this study using Northern blot and Western blot analysis and the calcein method. The findings showed that treatment of C6 cells with different concentrations of L-DOPA (0-100 microM) did not affect the expression of mRNA and protein of TfR and DMT1 with iron-responsive element (+IRE) and protein of ferroportin 1. However, a significant increase in the expression of DMT1(-IRE) mRNA and protein was found in cells treated, respectively, with 10 and 30 microM L-DOPA (mRNA) and 1, 5, 10 and 30 microM L-DOPA (protein). The increase in DMT(-IRE) protein induced by L-DOPA treatment was in parallel with the increase in DMT(-IRE) mRNA. The levels of DMT1(-IRE) mRNA and protein peaked in the cells treated with 10 microM L-DOPA and then decreased progressively with increasing concentrations of L-DOPA. Further study demonstrated that treatment of the cells with 10 microM L-DOPA induced a significant increase in ferrous uptake by C6 glioma cells. The findings suggested that the increased DMT1(-IRE) expression might be partly associated with the neurotoxicity of L-DOPA. Clinical relevance of the findings needs to be investigated further.