The Distribution, Regulation And Function Of Ferroportin 1 In Brain

Abnormally high level of iron in the brain has been demonstrated in a number of neurodegenerative disorders (NDs), such as Parkinson's disease (PD) and Alzheimer's disease (AD). Oxidative stress resulting from increased iron levels in the brain or defects in antioxidant defense mechanisms is widely believed to be associated with neuronal death in these disorders. However, a key question - why do iron levels increase abnormally in some regions of the brain? - has not been answered. Ferroportin 1(FPN1) is a newly discovered iron transport protein. It plays a key role in Fe2+ transport across the basal membrane of enterocytes in the gut. It has been suggested that this protein might have the same role in Fe2+ transport across the abluminal membrane of the blood-brain barrier as it works in enterocytes. However, the distribution of FPN1 in the brain has not been well studied, and whether FPN1 is expressed in the blood-brain barrier has not been well determined. In the present study, we investigated the distribution and expression of FPN1 in the brain by in situ hybridization, immunohisology and transfection, the effects of iron status, NO and age on FPN1 in neural cells by RT-PCR and western blot, the function of FPN1 in neural cells by scintillation counting.The results were as follows:As shown by in situ hybridization, FPN1 mRNA was located in the cytoplasm and distributed mainly in the cortex, hippocampus, compact part of substantia nigra, caudate putamen and medial habenular nucleus. The distinct cellular architecture of the cortex enabled us to determine some of the positive pyramidal cells with FPN1 mRNA staining. The FPN1 positive cells in substantia nigra were much more than that in other adjacent brain regions, and most of positive cells were distributed in the compact part. Within the field CA4 of hippocampus adjacent to dentate gyrus, most of positive cells were located at pyramidal layer. In caudate putamen, the positive neurons were around the nerve fiber.Immunohistochemical results showed that FPN1 protein was mostly distributed on the membrane, little in the cytoplasm and none in nucleus. The distribution of FPN1 protein in the brain regions was consistent with that of FPN1 mRNA. Within the cortex, all corticallayers had many positive cells except for the molecular layer. The cell body and primary processes of pyramidal cells were intensely immunoreactive. As detected by immunohistochemistry for the tyrosine hydroxylase, the dopaminergic neurons in compact part of substantia nigra were positive for FPNl staining, and FPNl protein was found within the cell body and primary proccess. Within the hippocampus, pyramidal cells in the pyramidal layer were immunopositive for FPNl. The strongest signal was detected in the neurons in the medial habenular nucleus of the brain.FPNl and TfR mRNA was expressed in the endothelial cells, as studied by RT-PCR. Furthermore, FPNl mRNA was more abundant in the endothelial cells than in the brain tissue minus the microvessels, but TfR mRNA was equally distributed in both of them. Immunohistochemical results showed FPNl was also present in the endothelium of blood vessels and in the ependymal cells of the brain.In cortex and striata, FPNl mRNA was up-regulated from 1 week to 9 weeks age but down-regulated from 9 weeks to 18 months age. In cortex and substantia nigra, FPNl protein was down-regulated from lweek to 18 months age. FPNl showed the highest expression in medial habenular nucleus, but no difference was found among three periods of age.To study the subcellular distribution of FPNl in the neural cell, the full-length FPNl ' was tagged with a green fluorescent protein (GFP). In the C6 cells transfected with pEGFP-FPNl, GFP-FPN1 fusion proteins translocated mostly on the membrane observed with scanning confocal laser microscopy. The result suggested that FPNl is located prominently on the cell membrane of C6 cells.The presence of TfR and FPNl mRNA in PC 12 cells was proved by RT-PCR. Exposure of PC 12 cells to iron donor FAC (100 U mol/L for 20 h) signi...