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Expression of Iron-Related Proteins at the Neurovascular Unit Supports Reduction and Reoxidation of Iron for Transport Through the Blood-Brain Barrier

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Abstract

The mechanisms for iron transport through the blood-brain barrier (BBB) remain a controversy. We analyzed for expression of mRNA and proteins involved in oxidation and transport of iron in isolated brain capillaries from dietary normal, iron-deficient, and iron-reverted rats. The expression was also investigated in isolated rat brain endothelial cells (RBECs) and in immortalized rat brain endothelial (RBE4) cells grown as monoculture or in hanging culture inserts with defined BBB properties. Transferrin receptor 1, ferrireductases Steap 2 and 3, divalent metal transporter 1 (DMT1), ferroportin, soluble and glycosylphosphatidylinositol (GPI)-anchored ceruloplasmin, and hephaestin were all expressed in brain capillaries in vivo and in isolated RBECs and RBE4 cells. Gene expression of DMT1, ferroportin, and soluble and GPI-anchored ceruloplasmin were significantly higher in isolated RBECs with induced BBB properties. Primary pericytes and astrocytes both expressed ceruloplasmin and hephaestin, and RBECs, pericytes, and astrocytes all exhibited ferrous oxidase activity. The coherent protein expression of these genes was demonstrated by immunocytochemistry. The data show that brain endothelial cells provide the machinery for receptor-mediated uptake of ferric iron-containing transferrin. Ferric iron can then undergo reduction to ferrous iron by ferrireductases inside endosomes followed by DMT1-mediated pumping into the cytosol and subsequently cellular export by ferroportin. The expression of soluble ceruloplasmin by brain endothelial cells, pericytes, and astrocytes that together form the neurovascular unit (NVU) provides the ferroxidase activity necessary to reoxidize ferrous iron once released inside the brain.

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Abbreviations

ACM:

Astrocyte-conditioned medium

α-SMA:

Alpha-smooth muscle actin

BBB:

Blood-brain barrier

bFGF:

Basic fibroblast growth factor

BSA:

Bovine serum albumin

CNS:

Central nervous system

DAPI:

4′,6-Diamidino-2-phenylindole dihydrochloride

Dcytb:

Duodenal cytochrome b

DMT1:

Divalent metal transporter 1

GFAP:

Glial fibrillary acidic protein

GPI:

Glycophosphatidylinositol

MTP1:

Metal transporter protein 1

NVU:

Neurovascular unit

PBS:

Phosphate-buffered saline

PDGFRβ:

Platelet derived growth factor receptor β

RBE4:

Immortalized rat brain endothelial cell line

RBECs:

Rat brain endothelial cells

TEER:

Trans-endothelial electrical resistance

Tf:

Transferrin

Steap:

Six-transmembrane epithelial antigen of prostate

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Acknowledgments

The Research Initiative on Blood-Brain Barrier and Drug Delivery founded by the Lundbeck foundation (Grant no. 2013-14113), The Danish Innovation Fund (Grant no. 014-2011-5), and Fonden til Lægevidenskabens Fremme are all thanked for generous support. We thank Merete Fredsgaard, Mathias Buhl Blæsild and Hanne Krone Nielsen, Aalborg University, Denmark for excellent technical assistance.

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Authors and Affiliations

  1. Laboratory of Neurobiology, Biomedicine Group, Department of Health Science and Technology, Aalborg University, Fr. Bajers Vej 3B, 1.216, 9220, Aalborg, Denmark

    Annette Burkhart, Tina Skjørringe, Kasper Bendix Johnsen, Louiza Bohn Thomsen & Torben Moos

  2. Department of Biomedicine, Aarhus University, Aarhus, Denmark

    Piotr Siupka & Morten Schallburg Nielsen

  3. Pharmacosmos A/S, Holbæk, Denmark

    Lars Lykke Thomsen

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  1. Annette Burkhart
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  2. Tina Skjørringe
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Correspondence to Annette Burkhart.

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Burkhart, A., Skjørringe, T., Johnsen, K.B. et al. Expression of Iron-Related Proteins at the Neurovascular Unit Supports Reduction and Reoxidation of Iron for Transport Through the Blood-Brain Barrier. Mol Neurobiol 53, 7237–7253 (2016). https://doi.org/10.1007/s12035-015-9582-7

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