Developmental Changes in the Expression of Iron Regulatory Proteins and Iron Transport Proteins in the Perinatal Rat Brain Asha Jyothi M. Siddappa, 1 Raghavendra B. Rao, 1,2 Jane D. Wobken, 1 Elizabeth A. Leibold, 3 James R. Connor, 4 and Michael K. Georgieff 1,2 * 1 Division of Neonatology, Department of Pediatrics, University of Minnesota, Minneapolis, Minnesota 2 Center For Neurobehavioral Development, University of Minnesota, Minneapolis, Minnesota 3 Department of Medicine, Eccles Program in Human Molecular Biology and Genetics, University of Utah, Salt Lake City, Utah 4 Department of Neuroscience and Anatomy, Pennsylvania State University College of Medicine, Hershey, Pennsylvania The perinatal brain requires a tightly regulated iron trans- port system. Iron regulatory proteins (IRPs) 1 and 2 are cytosolic proteins that regulate the stability of mRNA for the two major cellular iron transporters, transferrin recep- tor (TfR) and divalent metal transporter-1 (DMT-1). We studied the localization of IRPs, their change in expres- sion during perinatal development, and their relationship to TfR and DMT-1 in rat brain between postnatal days (PND) 5 and 15. Twelve-micron frozen coronal sections of fixed brain tissue were obtained from iron-sufficient Sprague-Dawley rat pups on PND 5, 10, and 15, and were visualized at 20 to 1,000 light microscopy for diaminobenzidine activity after incubation with specific primary IRP-1, IRP-2, DMT-1, and TfR antibodies and a universal biotinylated secondary and tertiary antibody system. IRP and transport protein expression increased in parallel over time. IRP1, IRP2, and DMT-1 were par- tially expressed in the choroid plexus epithelial cells at PND 5 and 10, and fully expressed at PND 15. The cerebral blood vessels and ependymal cells strongly ex- pressed IRP1, IRP2, and DMT-1 as early as PND 5. Substantive TfR staining was not seen in the choroid plexus or ependyma until PND 15. Glial and neuronal expression of IRP1, IRP2, DMT-1, and TfR in cortex, hippocampal subareas and striatum increased over time, but showed variability in cell number and intensity of expression based on brain region, cell type, and age. These developmental changes in IRP and transporter expression suggest potentially different time periods of brain structure vulnerability to iron deficiency or iron overload. © 2002 Wiley-Liss, Inc. Key words: iron; transferrin receptor; divalent metal transporter-1; regulation; brain Iron is required for optimal neuronal growth and function during development. Iron forms an important component of enzymes and proteins involved in neuro- transmitter synthesis, neuronal oxidative metabolism, my- elin formation, and DNA synthesis (Beard et al., 1993). The first 16 postnatal days in the rat are characterized by a high rate of cell proliferation followed by a period of rapid myelination (Rice and Barone, 2000), during which time the brain sustains a high metabolic rate. Iron requirements parallel this rapid rate of development (Connor, 1994). Iron deficiency during mammalian brain development results in long-term impairment in brain function (Larkin and Rao, 1990; Lozoff et al., 2000), while iron overload results in lethal oxidizing agents, potentially leading to the peroxidation of lipid rich cell membranes (Connor and Menzies, 1995). The developing brain requires an elegant system of iron transport to maintain homeostasis between facilitating iron transport for important cellular functions, reducing its exposure to the potentially toxic effects of iron. The brain acquires iron from diferric transferrin in the plasma via receptor-mediated endocytosis of trans- ferrin receptors (TfR) present on the capillary vascular endothelial cells and choroid plexus epithelial cells (Moos and Morgan, 2000; Burdo and Connor, 2002). The re- cently cloned divalent metal transporter-1 (DMT-1) has also been identified in the cytoplasm of mature neurons, oligodendrocytes, astrocytes, and ependymal epithelium, where it likely functions as a ferrous iron transporter from the endosome into the cytosol for utilization (Gruenheid et al. 1999; Burdo et al., 2001). DMT-1 has also been Contract grant sponsor: National Institutes of Health; Contract grant num- bers: HD-29421, GM 45201, NS 34280. *Correspondence to: Michael K. Georgieff, M.D., University of Minne- sota, Mayo Mail Code 39, 420 Delaware Street SE, Minneapolis, MN 55455. E-mail: georg001@umn.edu Received 29 October 2001; Revised 19 February 2002; Accepted 21 February 2002 Published online 1 May 2002 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/jnr.10246 Journal of Neuroscience Research 68:761–775 (2002) © 2002 Wiley-Liss, Inc.