Age-dependent and iron-independent expression of two mRNA isoforms of divalent metal transporter 1 in rat brain
Introduction
The DMT1, also known as Nramp2 or DCT1, is a newly discovered proton-coupled metal-ion transport protein. It was first identified on the basis of its homology to Nramp1 in 1995 [14]. In 1997, two groups [10], [16] independently identified DMT1 as the first mammalian transmembrane iron transporter. There are at least two different splice forms of DMT1 [13], [16], [19]. One splice form, called DMT1(+IRE) mRNA, contains an iron-responsive element (IRE) in the 3′-UTR and encodes a 561 amino-acid protein. Another splice form, designated DMT1(−IRE) mRNA, does not contain a classical IRE and encodes a 568 amino-acid protein [16], [24]. DMT1 is widely expressed [10], [16]. At cellular level, DMT1 can express on the endosomal membrane and acts to export iron from the endosome into cytoplasm of the cell [15], [35], [40] in addition to express on the plasma membrane. This implies that DMT1 is important in both transferrin-bound and non-transferrin-bound iron uptake and transport.
In the brain, DMT1 mRNA plus protein is consistently found in neurons and epithelial cells of the choroid plexus and presents at moderate levels in the substantia nigra [3], [5], [16], [23]. The cellular localization and functional characterization suggest that DMT1 might play a role in physiological iron transport in the brain [4], [16], [26], [47]. In the neurons of the substantial nigra in Parkinson's disease (PD), DMT1 is moderately high expressed that coincidentally correlates to the iron abnormally deposition in the same area [2]. Therefore, disruption of DMT1 expression may be involved in the increased iron accumulation in PD. In addition to PD, abnormally high level of iron in the brain has also been demonstrated in other neurodegenerative disorders [1], [21], [31], [39]. The oxidative stress induced by the increased iron has been widely believed to be involved in the cascade of events leading to neuronal death in these disorders [22], [25], [31], [34], [36]. Due to the suggested importance of DMT1 in brain iron metabolism and the putative contribution in the development of some neurodegenerative disorders, we investigated DMT1(+IRE) and (−IRE) mRNAs’ expression and protein synthesis in the cortex, hippocampus, striatum and substantia nigra of the rats with different ages or fed with high- or low-iron diet for 6-weeks in this study. Results showed that DMT1 expression in these brain regions is age-dependent but iron-independent. The lack of response of DMT1 to iron status in the brain of adult rats suggests that the IRE of brain DMT1 mRNA might be not really iron-responsive and that DMT1-mediated iron transport might be not the rate–limiting step in brain iron uptake although it might play a critical role in brain cell iron uptake.
Section snippets
Materials
Unless otherwise stated, all chemicals were purchased from Sigma Chemical Co. (St. Louis. MO, USA). Taq DNA polymerase was obtained from Gibco BRL (Gaithersbury, MD, USA), and Advantage™ RT-f was from QIAGEN (Valencia, CA, USA). SDS, acrylamide, bisacrylamide and agarose were purchased from Bio-Rad Laboratories (Richmond, CA, USA), and the antibodies against DMT1(+IRE) or (−IRE) from ADI (ADI, San Antonio, TX, USA). Iron standard (1 mg iron/ml) molecular weight standards were obtained from Alpha
Effect of age and iron diets on biochemical indicators and brain iron
The findings from the rats with different age showed that the development has a significant effect on brain iron contents (Table 1). Total iron concentrations in the cortex, striatum and substantia nigra were significantly higher in the PNW3 rats than those in the PNW1 rats, while in the hippocampus, a significant increase in iron content was found in the PNW9 rats as compared with the PNW1 rats. Although iron in some brain regions increased with the age after PNW3, there were no significant
Discussion
One of the major objectives of the present study was to investigate the effect of dietary iron on iron contents as well as DMT1 expression in different brain regions, including the cortex, hippocampus, striatum, and substantia nigra of adult rats. Our result showed that treatment with a low- or high-iron diet for 6-weeks could alter brain iron levels. It is in agreement with the finding reported by Pinero et al. [30], they demonstrated that long-term iron deficiency and iron excess result in
Acknowledgements
The studies were supported by Competitive Earmarked Grants of The Hong Kong Research Grants Council (PolyU5270/01M/B-Q445) and The Hong Kong Polytechnic University Research Grants (G-YX14, G-YD78, A-PD92, G-T616, A-PC98, G-T856 and A-PC23).
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