Kevin G. Dolan

Distribution of divalent metal transporter 1 and metal transport protein 1 in the normal and Belgrade rat.

Iron accumulation in the brain occurs in a number of neurodegenerative diseases. Two new iron transport proteins have been identified that may help elucidate the mechanism of abnormal iron accumulation. The Divalent Metal Transporter 1 (DMT1), is responsible for iron uptake from the gut and transport from endosomes. The Metal Transport Protein 1 (MTP1) promotes iron export. In this study we determined the cellular and regional expression of these two transporters in the brains of normal adult and Belgrade rats. Belgrade rats have a defect in DMT1 that is associated with lower levels of iron in the brain. In the normal rat, DMT1 expression is highest in neurons in the striatum, cerebellum, thalamus, ependymal cells lining the third ventricle, and vascular cells throughout the brain. The staining in the ependymal cells and endothelial cells suggests that DMT1 has an important role in iron transport into the brain. In Belgrade rats, there is generalized decrease in immunodetectable DMT1 compared to normal rats except in the ependymal cells. This decrease in immunoreactivity, however, was absent on immunoblots. The immunoblot analysis indicates that this protein did not upregulate to compensate for the chronic defect in iron transport. MTP1 staining is found in most brain regions. MTP1 expression in the brain is robust in pyramidal neurons of the cerebral cortex but is not detected in the vascular endothelial cells and ependymal cells. MTP1 staining in Belgrade rats was decreased compared to normal, but similar to DMT1 this decrease was not corroborated by immunoblotting. These results indicate that DMT1 and MTP1 are involved in brain iron transport and this involvement is regionally and cellularly specific. J Neurosci. Res. 66:1198–1207, 2001.

Cellular distribution of iron in the brain of the Belgrade rat.

In this study, we investigated the cellular distribution of iron in the brain of Belgrade rats. These rats have a mutation in Divalent Metal Transporter 1, which has been implicated in iron transport from endosomes. The Belgrade rats have iron-positive pyramidal neurons, but these are fewer in number and less intensely stained than in controls. In the white matter, iron is normally present in patches of intensely iron-stained oligodendrocytes and myelin, but there is dramatically less iron staining in the Belgrade rat. Those oligodendrocytes that stained for iron did so strongly and were associated with blood vessels. Astrocytic iron staining was seen in the cerebral cortex for both normal rats and Belgrade rats, but the iron-stained astrocytes were less numerous in the mutants. Iron staining in tanycytes, modified astrocytes coursing from the third ventricle to the hypothalamus, was not affected in the Belgrade rat, but was affected by diet. The results of this study indicate that Divalent Metal Transporter 1 is important to iron transport in the brain. Iron is essential in the brain for basic metabolic processes such as heme formation, neurotransmitter production and ATP synthesis. Excess brain iron is associated with a number of common neurodegenerative diseases. Consequently, elucidating the mechanisms of brain iron delivery is critical for understanding the role of iron in pathological conditions.

Non‐transferrin‐bound iron uptake in Belgrade and normal rat erythroid cells

Belgrade (b) rats have an autosomal recessive, microcytic, hypochromic anemia. Transferrin (Tf)‐dependent iron uptake is defective because of a mutation in DMT1 (Nramp2), blocking endosomal iron efflux. This experiment of nature permits the present study to address whether the mutation also affects non‐Tf‐bound iron (NTBI) uptake and to use NTBI uptake compared to Tf‐Fe utilization to increase understanding of the phenotype of the b mutation. The distribution of 59Fe2+ into intact erythroid cells and cytosolic, stromal, heme, and nonheme fractions was different after NTBI uptake vs. Tf‐Fe uptake, with the former exhibiting less iron into heme but more into stromal and nonheme fractions. Both reticulocytes and erythrocytes exhibit NTBI uptake. Only reticulocytes had heme incorporation after NTBI uptake. Properly normalized, incorporation into b/b heme was ∼20% of +/b, a decrease similar to that for Tf‐Fe utilization. NTBI uptake into heme was inhibited by bafilomycin A1, concanamycin, NH4Cl, or chloroquine, consistent with the endosomal location of the transporter; cellular uptake was uninhibited. NTBI uptake was unaffected after removal of Tf receptors by Pronase or depletion of endogenous Tf. Concentration dependence revealed that NTBI uptake into cells, cytosol, stroma, and the nonheme fraction had an apparent low affinity for iron; heme incorporation behaved like a high‐affinity process, as did an expression assay for DMT1. DMT1 serves in both apparent high‐affinity NTBI membrane transport and the exit of iron from the endosome during Tf delivery of iron in rat reticulocytes; the low‐affinity membrane transporter, however, exhibits little dependence on DMT1. J. Cell. Physiol. 178:349–358, 1999.

Effect of the iron chelator desferrioxamine on manganese‐induced toxicity of rat pheochromocytoma (PC12) cells

Alterations in iron levels are likely to influence the biological actions of Mn in PC12 cells, because both metals are transported via the divalent metal transporter 1 (DMT1; also Nramp2 or DCT1). Studies were performed to determine the effect of the iron chelator desferrioxamine (DfO) on Mn‐induced PC12 cell death and neuronal differentiation. Cell death almost doubled when PC12 cells were exposed for 24 hr to both DfO (10 μM) and Mn (0.3 mM) as opposed to Mn alone. DfO also stimulated Mn‐induced neuronal differentiation by enhancing the phosphorylation of both ERK1 and 2 and also attenuated the increase in caspase 3‐like activity induced by 0.3 mM Mn by approximately 50%, indicating that caspase activation, as reported previously, does not contribute to Mn‐induced PC12 cell death. DfO also affected Mn‐induced suppression of mitochondrial function as indicated by an additional 16% loss of ATP formation in PC12 cells cotreated with 0.3 mM Mn. Because sequestration of iron by DfO would be expected to lead to increased transport of Mn, studies were performed to determine whether iron inhibited Mn transport in PC12 cells. Iron inhibited 54Mn transport with an IC50 of approximately 20 μM. In addition, coincubation of DfO with Mn in PC12 cells resulted in increased expression of both the iron response element‐positive and the iron response element‐negative forms of DMT1. Taken together, these results demonstrate that iron status is likely to have a direct effect on the uptake and biological actions of Mn and probably other divalent metals that are transported by DMT1.

Microscopic R2* mapping of reduced brain iron in the Belgrade rat.

R2* mapping has recently been used to detect iron overload in patients with movement disorders. We demonstrate here that this technique can also be used to detect reduced brain iron, as in the case of a missense mutation in the iron‐transporting protein divalent metal transporter 1. Surprisingly, we found that the same brain regions are affected (ie, the globus pallidus, substantia nigra, and cerebellar dentate nucleus); this suggests a much more extensive role for these structures in regulating overall brain iron homeostasis. Therefore, for the clinical monitoring of movement disorders for which normal brain iron homeostasis (either overload or reduction) may be implicated, R2* mapping appears to be well‐suited.