CELLULAR DISTRIBUTION OF IRON IN THE BRAIN OF THE BELGRADE RAT J. R. BURDO,* J. MARTIN,* S. L. MENZIES,* K. G. DOLAN,² M. A. ROMANO,² R. J. FLETCHER,² M. D. GARRICK,²‡ L. M. GARRICK²§ and J. R. CONNOR* k *Department of Neuroscience and Anatomy, Pennsylvania State University, Hershey Medical Center, 500 University Drive, Hershey, PA 17033, U.S.A. Departments of ²Biochemistry, ‡Pediatrics and §Medicine, SUNY at Buffalo, Buffalo, NY 14214, U.S.A. Abstract —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.  1999 IBRO. Published by Elsevier Science Ltd. Key words: Divalent Metal Transporter 1, Belgrade rat, iron staining, tanycytes, neurons, glia. Iron is essential for normal neurological function. Biochemi- cal processes in the brain that are dependent on iron include neurotransmitter synthesis, myelin production and main- tenance, and basic cell functions such as energy production. 1 Regional specialization of function in the brain and the presence of a blood–brain barrier (BBB) present unique chal- lenges to the study of iron transport in this organ. Understand- ing iron transport into and within the brain is necessary to elucidate the underlying pathological conditions in common neurodegenerative disorders such as Parkinson’s and Alzhei- mer’s diseases, in which iron accumulates abnormally. 20 Iron transport in mammals is thought to be a process largely dependent on the movement of the transferrin–iron complex (Tf–Fe). 19 This complex binds to transferrin receptors (TfRs) on the extracellular membrane of cells, resulting in endo- cytosis of the Tf–Fe–TfR complex. The endosome is then acidified, and the iron atoms disassociate from Tf. The iron atoms must then be translocated out of the endosome to be used by the cell. The mechanism by which this transloca- tion takes place involves Divalent Metal Transporter 1 (DMT1). 11,13 DMT1, also known as DCT1 (divalent cation transporter 1) and Nramp2 (natural resistance-associated macrophage protein 2), is believed to be a proton symporter, transporting one atom of Fe 2+ and one atom of H + in the same direction. 15 Evidence also implicates DMT1 in uptake of iron from the duodenal lumen into enterocytes. 10,15,23 The microcytic anemic (mk/mk) mouse 10 and the Belgrade (b/b) rat 11 have identical mutations in DMT1, a glycine-to- arginine substitution at amino acid 185 (G185R). In both species, this mutation leads to a hypochromic, microcytic anemia. The phenotype of the b/b rat is probably the result of less iron reaching the mitochondria 12 and more Tf-bound iron being recycled from the endosome to the extracellular space than normal. 13 The DMT1 mutation presents a unique opportunity to examine iron transport into the rat brain. At the level of the vasculature endothelial cell, there is conflicting evidence as to whether the Tf–Fe complex is translocated across the BBB as a whole or if the iron disassociates from Tf within the endothelial cell to be transported alone into the brain. 6,9,16,25–27 A previous study has shown that both iron and Tf uptake into the two-week-old b/b brain is reduced. 8 Because neurons 17,21,28,30 and glia in culture 29 possess TfRs capable of endocytosing the Tf–Fe complex, we determined the cellular distribution of iron in the Belgrade rat brain. We hypothesized that iron staining will be diminished in the cells of the b/b brain if they rely on DMT1 for iron transport from the endosome. In the present study, we investigated the iron status of b/b brains and found that there is indeed less detect- able iron in the brains of these animals than in heterozygote ( +/b) controls. EXPERIMENTAL PROCEDURES Animals The Buffalo colony of Belgrade rats derives from the original colony, where the mutation arose on an albino rat believed to be of the Wistar strain. The colony was transmitted to Buffalo via the Center for Disease Control in Atlanta, where the rats had been crossbred to Wistar rats. Recently, we began breeding b/b rats to Fisher F-344 ( +/ +) rats to map the gene. 11 The animals used in this study were the N 8 generation, products of crosses between a b/b male × +/b females; thus, both +/b rats and their b/b littermates had 68.4% Fisher background and 31.6% Wistar background. To improve the Brain iron in Belgrade rats 1189 1189 Neuroscience Vol. 93, No. 3, pp. 1189–1196, 1999 Copyright  1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/99 $20.00+0.00 PII: S0306-4522(99)00207-9 Pergamon kTo whom correspondence should be addressed. Tel.: +1-717-531-8650; fax: +1-717-531-5184. E-mail address: jrc3@psu.edu (J. R. Connor) Abbreviations: BBB, blood–brain barrier; DMT1, Divalent Metal Trans- porter 1; G185R, glycine-to-arginine substitution at amino acid 185; Tf, transferrin; Tf–Fe, transferrin–iron complex; TfR, transferrin receptor.