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Regulatory mechanisms for iron transport across the blood-brain barrier
Accepted Manuscript Regulatory mechanisms for iron transport across the blood-brain barrier Kari A. Duck, Ian A. Simpson, James R. Connor PII:
S0006-291X(17)32061-2
DOI:
10.1016/j.bbrc.2017.10.083
Reference:
YBBRC 38700
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 11 October 2017 Accepted Date: 16 October 2017
Please cite this article as: K.A. Duck, I.A. Simpson, J.R. Connor, Regulatory mechanisms for iron transport across the blood-brain barrier, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.10.083. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Regulatory Mechanisms for Iron Transport across the BloodBrain Barrier Kari A. Duck1, Ian A. Simpson2, and James R. Connor1
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State Hershey Medical Center, Hershey, PA
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Correspondence should be sent to: James R. Connor, Ph.D. Distinguished Professor of Neurosurgery, Neural and Behavioral Sciences and Pediatrics Vice-Chair of Neurosurgery Director, Center for Aging and Neurodegenerative Diseases Penn State Hershey Medical Center 500 University Drive MC H110, C3830 Hershey, PA 17033 Telephone: 717-531-4541 Fax: 717-531-0091 Email: [email protected]
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Keywords: Blood-brain barrier, divalent metal transport 1, iron, endosome, transferrin, transferrin receptor
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Abbreviations: blood-brain barrier (BBB), transferrin (Tf), iron (Fe), iron-poor transferrin (apo-Tf), deferoxamine (DFO), divalent metal transporter 1 (DMT1), intravenous (IV), transferrin-bound iron (TBI), bovine retinal endothelial cell (BREC), cerebrospinal fluid (CSF), nitrilotriacetic acid (NTA), bovine brain microvascular endothelial cell (BBMVEC)
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Abstract Many critical metabolic functions in the brain require adequate and timely delivery of
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iron. However, most studies when considering brain iron uptake have ignored the iron
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requirements of the endothelial cells that form the blood-brain barrier (BBB).
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Moreover, current models of BBB iron transport do not address regional regulation of
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brain iron uptake or how neurons, when adapting to metabolic demands, can acquire
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more iron. In this study, we demonstrate that both iron-poor transferrin (apo-Tf) and
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the iron chelator, deferoxamine, stimulate release of iron from iron-loaded endothelial
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cells in an in vitro BBB model. The role of the endosomal divalent metal transporter 1
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(DMT1) in BBB iron acquisition and transport has been questioned. Here, we show
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that inhibition of DMT1 alters the transport of iron and Tf across the endothelial cells.
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These data support an endosome-mediated model of Tf-bound iron uptake into the
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brain and identifies mechanisms for local regional regulation of brain iron uptake.
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Moreover, our data provide an explanation for the disparity in the ratio of Tf to iron
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transport into the brain that has confounded the field.
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Introduction
Iron is an essential micronutrient that functions as a cofactor in key brain processes
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such as myelination and neurotransmitter synthesis [1]. Until recently, studies on the
uptake mechanism of iron into the brain considered the blood-brain barrier (BBB) as a passive conduit [2]. We have identified the endothelial cells of the BBB as a
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regulatory site for brain iron uptake. Identifying the signals and the underlying
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mechanism for this regulation are of considerable importance given the contribution
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of altered brain iron status observed in various neurodegenerative diseases [3-9] and
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the long-term effects of iron deficiency during development [10]. Understanding the
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mechanism(s) by which iron uptake into the brain is regulated is critical to
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determining the impact of intravenous (IV) iron supplements for treatment of
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neurological disorders such as restless legs syndrome [11, 12] or whether IV iron
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treatment can increase the risk of neurodegenerative diseases that are associated with
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excessive brain iron accumulation [3, 13]. Moreover, understanding the regulation of
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iron uptake mechanisms is relevant to the numerous attempts to use the brain’s iron
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delivery system to deliver therapeutic agents that have met with limited success [14].
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A key aspect to understanding the BBB as the regulatory site for brain iron
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acquisition must include a model wherein the iron needs of the endothelial cells are
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met. Until recently, the fact that mitochondrial-rich [15], ferritin-containing [16]
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endothelial cells required a mechanism for acquiring iron, and not just transporting it,
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had been overlooked. Moreover, the presence of the iron exporter, ferroportin, has
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been demonstrated in brain microvascular endothelial cells. Although its function has
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not been uniformly confirmed, the presence of this protein suggests a specific
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mechanism for iron release from the endothelial cells [17, 18]. In our previous study
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using bovine retinal endothelial cells (BREC), the presence of ferroportin was identified and its activity demonstrated by blocking iron release with hepcidin [2], a known inhibitor of ferroportin [19]. In this study, we identify factors that alter release and transport of iron across a model of the BBB.
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Materials and Methods Cell culture Bovine brain microvascular endothelial cells (BBMVEC) were
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cultured in complete growth medium (Cell Applications, Inc.). BBMVECs were
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grown to confluence on Costar transwell 0.4 µm porous filters (Corning) and then
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gently washed three times with 1X DPBS (Corning). The BBMVEC cells were
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then exposed to serum free media containing 138 nM hydrocortisone (Sigma
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Aldrich) for 72 hours to promote tight junction formation [20].
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Release assay BBMVEC in the transwell setup were loaded overnight with 10
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µCi/well of 59Fe-NTA complex. Wells were then washed three times with 1X
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DPBS (Corning) to remove iron remaining in the culture medium. Serum-free
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medium containing 70 kDa RITC-Dextran (Sigma Aldrich) was added to the
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apical chamber. The ability of 70 kDa RITC-Dextran to diffuse across the BBB
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serves as a measure of barrier integrity and enables correction for any leakage
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[21]. Serum-free medium containing 10 µM apo-Tf (Sigma Aldrich), 500 nM
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hepcidin (Peptides, Inc.), or 10 µM apo-Tf and 500 nM hepcidin was then added
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to the basal chamber. Where hepcidin was used, BBMVECs were treated with
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500 nM hepcidin overnight simultaneously with the 59Fe loading step. Cells were
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exposed to 100 uM deferoxamine (DFO) to compare the chelator effect to apo-Tf. Data were collected by taking 50 µL aliquots from the apical chamber at 0 and 4
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hours and the basal chamber at 0, 2, and 4 hours. Fluorescence (RITC excitation:
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555 nm, emission: 580 nm) was measured on a SpectraMax Gemini EM plate
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reader (Molecular Devices) and 59Fe was then measured on a Beckman Gamma
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4000 (Beckman Coulter).
105 Transport assay Tf flux across the BBB was measured by applying 10 µM 59Fe-
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AlexaFluor488-Tf to the apical chamber of our transwell model. 59Fe-
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AlexaFluor488-Tf was prepared as previously described [22]. Fresh media
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containing 10 µM 59Fe-AlexaFluor488-Tf and 70 kDa RITC-Dextran was added
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to the apical chamber at the beginning of the experiment. A DMT1 inhibitor (104
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nM XEN602, Xenon Pharmaceuticals [23]) was added to the apical chamber for 1
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hour prior to the addition of 59Fe-AlexaFluor488-Tf (zero time). Media aliquots
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were taken from the apical chamber at 0 and 6 hours. Basal chamber aliquots
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were taken at 2-hour intervals for 6 hours. Fluorescence (RITC excitation: 555
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nm, emission: 580 nm; Alexa Fluor488 excitation: 495 nM, emission 519 nm)
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was measured using a SpectraMax Gemini EM plate reader (Molecular Devices)
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and 59Fe was measured on a Beckman Gamma 4000 (Beckman Coulter). A
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Sephadex G-25 Quick Spin column was used as per the manufacturer’s
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instructions (Roche) to separate free iron from protein-bound iron. Tf flux (P0)
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was calculated from the fluorescence measurements as previously described [24].
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Protein expression assays Ferroportin protein expression was measured by
immunoblot as previously described [2]. Briefly, cells were RIPA lysis buffer
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containing 1X protease inhibitor cocktail (Sigma Aldrich). Ferroportin was
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detected with a ferroportin primary antibody (1:200, Alpha Diagnostics
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International) The membranes were visualized by adding Western Lightning Plus-
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ECL (Perkin Elmer) and imaged on a Fuji LAS-3000 Imaging System.
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Densitometry was performed using Multigauge software (Fuji Film). β-actin
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(1:3000, Sigma Aldrich) was used as a loading control. To control for cross-
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membrane variability, a common liver sample was used on each blot for
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normalization.
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Statistical Analyses Prism (GraphPad Software) software was used for all
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statistical analyses and data graphing. Data from three independent replicates
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were pooled and are expressed as mean ± standard error of the mean. Statistical
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differences between experimental groups were determined using an unpaired
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Student’s t test, one-way ANOVA and Bonferroni’s multiple comparisons test, or
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two-way ANOVA and Bonferroni’s multiple comparisons test. A level of
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significance of p < 0.05 was used for all differences evaluated.
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Results
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The first series of studies were designed to test the hypothesis that iron is released
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from brain endothelial cells and the release can be regulated. BBMVEC were loaded
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with 59Fe and exposed to hepcidin overnight. A baseline release of iron was observed over a four-hour period. There was no effect of hepcidin on the baseline release of
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(CSF side) of the BBMVEC resulted in a 140% increase in iron release within 2
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hours of exposure and persisted at 4 hours of exposure. The apo-Tf-stimulated release
Fe from the cells (Figure 1A). Exposure to 10 µM apo-Tf on the abluminal side
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was not blocked by hepcidin exposure (Figure 1B). Furthermore, the addition of the
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iron chelator DFO to the basal chamber also promoted release of iron (Figure 1C),
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which was also not inhibited by hepcidin (Figure 1D).
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The release of iron from endothelial cells is presumed to be via the iron export
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protein, ferroportin. Thus, we evaluated the expression of ferroportin in BBMVEC
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under the same experimental conditions of iron loading and exposure to apo-Tf and
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hepcidin. We demonstrate that ferroportin is expressed in the BBMVEC and this
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expression is decreased 38% by iron loading (Figure 2). The addition of hepcidin or
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apo-Tf had no effect on ferroportin expression when added alone to the iron-loaded
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endothelial cells but when added in combination, the levels of ferroportin were the
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same as control indicating that the combination of hepcidin and apo-Tf blocks the
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iron-induced decrease in ferroportin expression (Figure 2).
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To investigate the role DMT1 plays in the movement of systemic iron through the
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BBB and into the brain, we exposed the BBMVEC to the DMT1 inhibitor, XEN602.
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The presence of XEN602 had no effect on the amount of iron in the cell lysates
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(Figure 3A). The amount of bound iron released into the basal chamber (Figure 3B)
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was decreased 4-fold (p = 0.044) and the amount of unbound iron in the basal
chamber was decreased by 40% (Figure 3C, p = 0.012). The extent of Tf flux was
decreased by 59% in the presence of XEN602, (Figure 3D, p = 0.049).
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Discussion We previously reported that the BBB was capable of storing iron based on the
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presence of ferritin (for iron storage). Moreover, the presence of an iron regulatory
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protein in the BBB whose function is to regulate expression of ferritin, Tf receptor,
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and ferroportin clearly indicates the potential for intracellular iron-responsiveness by
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the BBB [2, 7, 16, 25-28]. More recently, we have directly demonstrated retention of
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iron in the endothelial cells of the brain suggesting that the BBB may function as an
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iron reservoir from which iron is released into the brain when signaled [22]. A key
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protein for the endocytic mechanism of uptake and release of iron into cells elsewhere
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in the body is DMT1. While the role of DMT1 is well-characterized in other systems,
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a role for DMT1in uptake of iron into the endothelial cell of the BBB remains
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unclear. The significance of DMT1 in cellular iron uptake was demonstrated in the
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Belgrade rat that has a mutation in DMT1, which is associated with decreased cellular
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iron uptake in enterocytes and reticulocytes resulting in microcytic anemia [29, 30].
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In normal mammalian cells, 60-80% of the iron bound to Tf is released into the cell
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during endocytosis with the remainder being released back into the incubation media
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[30, 31]. However, when similar experiments were conducted in reticulocytes from
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the Belgrade rat, the efficacy of this extraction decreased to 15%. Moreover, the
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failure to liberate the iron from the endosomes in the Belgrade reticulocytes
somewhat surprisingly resulted in its release into the media still bound to Tf and not reduced to Fe2+ [30].
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In the current study, we employed a DMT1 inhibitor, XEN602, to evaluate the role of
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DMT1 in iron and Tf transport at the BBB. The presence of the DMT1 inhibitor
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decreased Tf movement across the BBB and reduced both bound and free iron
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transported across the endothelial cells and released into the basal chamber (Figure
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3). These data are not only consistent with Tf-iron cycling in reticulocytes from
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Belgrade Rats [30] but also with the in vivo studies in the Belgrade rat where, despite
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high levels of circulating Tf, the Belgrade rats had an 85% reduction in free iron and
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Tf uptake into the brain compared to wild-type rats [32-34]. A similar reduction in
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free iron transport (≈80%) was observed when BRECs were exposed to NH4Cl,
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which prevents the acidification of the endosomes and the dissociation of iron from
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Tf; however, the levels of holo-Tf accumulation in the basal chamber appeared to be
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unaltered [24]. Recently, NHE9, a regulator of endosomal pH has been shown to
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significantly impact brain iron uptake and regulation within endothelial cells further
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emphasizing the role of endosomes in brain iron uptake [35].
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We have previously reported that the export of iron stored in the endothelial cells of
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the BBB is enhanced by the presence of apo-Tf and DFO as shown in this study; and
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by conditioned media from iron-deprived astrocytes and CSF derived from iron-
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deficient monkeys [2]. We assumed the mechanism for iron export from the endothelial cells involved export via ferroportin as we had previously demonstrated
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using bovine retinal endothelial cells (2) and as others have reported in the gut and for
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macrophages [36, 37]. Our present study revealed that ferroportin is present in bovine
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brain endothelial cells and demonstrated that iron loading decreases ferroportin
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expression as expected. However, the presence of hepcidin failed to block iron release
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from the endothelial cells either alone or in the presence of apo-Tf or the iron chelator
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DFO. Moreover, hepcidin did not alter the iron-loading induced decrease in
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ferroportin, although it did act synergistically with apo-Tf to increase ferroportin
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expression. This finding is different from our previous report using BRECs where
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hepcidin exposure caused a moderate decrease in 59Fe release [2], suggesting there
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are differences in ferroportin expression and the response to hepcidin between the
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retina and brain microvasculature. As ferroportin must be located in the cellular
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membrane to be functional as an exporter, the values measured in total cell lysate
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may not properly reflect the functional profile of the BBMVEC. There appears to be
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synergism between apo-Tf and hepcidin to overcome the iron induced decrease in
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ferroportin and maintain the ability of apo-Tf to continue to induce iron release from
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the endothelial cells, which is an observation for future study.
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In general, our data indicate transferrin-bound iron is first taken up into endosomes
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and upon their acidification, the iron is released via DMT1 into the cytoplasm where
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it is available to either bind to ferritin or be exported from the endothelial cell,
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presumably via ferroportin. Having discharged the iron in the endosome, the resultant
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apo-Tf in most cells undergoes exocytosis and is released back into the media.
However, in the polarized endothelium of the BBB the apo-Tf has the option of going
to either the luminal or abluminal membrane. In our previous study, we suggested
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that a certain proportion of apo-Tf was released from the abluminal membrane to be
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available to recombine with the iron released by ferroportin [2]. This is necessary in
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the model because an iron excess over fully-saturated Tf has been consistently
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reported in the CSF under normal conditions [27, 38], therefore a potential source of
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apo-Tf in the extracellular fluid must be identified. Although we cannot rule out a
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direct transcytotic mechanism, our data favor an endosomal-mediated model that
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releases vesicles containing Tf in which 60-80% of the Tf is apo and the remaining
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holo as reported in other cell types [30, 31, 39]. Based on the observed 3-4:1 disparity
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between the transport of iron and Tf, it would appear that the exocytosis of the Tf
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receptor with associated Tf (apo+holo) partitions between the luminal and abluminal
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membranes with a comparable 3:1 ratio.
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This study, in combination with our previous study, introduces the concept that apo-
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Tf and holo-Tf provide feedback to the endothelial cells that induce release (apo-Tf)
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or decrease release (holo-Tf) of iron from the endothelial cell. The mechanism for
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limiting the release of iron in response to holo-Tf could be via iron uptake back into
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the endothelial cell from the CSF, similar to the feedback mechanism reported in
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enterocytes [40, 41]. The idea of a feedback system in the brain for iron uptake is a
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novel concept in the central nervous system. The induction of iron release via apo-Tf
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(which can be mimicked by the iron chelator DFO) provides a new system not
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previously reported. The identification of the mechanism by which apo-Tf induces
iron release from endothelial cells, that may involve synergistic interaction with
hepcidin, is perhaps the critical step to understanding regional control of brain iron uptake.
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In conclusion, these data not only provide further support for a DMT1-dependent
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endocytic process mediating the passage of iron across the endothelial cells that
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constitute the BBB but addresses a long-observed enigma in the field; namely, the
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disproportionately greater transport of iron relative to Tf into the brain [24, 26, 27, 38,
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42-45]. We propose that the levels of apo-Tf and iron delivered to the brain are tightly
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regulated by the ratio of apo-Tf:holo-Tf in conjunction with hepcidin in the
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extracellular fluid of each brain region. Finally, these data also provide a potential
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explanation as to why attempts to harness the brain’s Tf-Tf receptor uptake system to
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deliver therapeutics has met with limited success due to the lack of appreciation of the
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importance of the endosomal route [14].
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Acknowledgements
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This work was supported by NIH R01 NS077678.
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Disclosure/Conflict of Interest
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The authors have no conflict of interest to disclose.
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[19] E. Nemeth, M.S. Tuttle, J. Powelson, M.B. Vaughn, A. Donovan, D.M. Ward, T. Ganz, J. Kaplan, Hepcidin Regulates Cellular Iron Efflux by Binding to Ferroportin and Inducing Its Internalization, Science, 306 (2004) 2090-2093. [20] D.A. Antonetti, E.B. Wolpert, L. DeMaio, N.S. Harhaj, R.C. Scaduto, Hydrocortisone decreases retinal endothelial cell water and solute flux coincident with increased content and decreased phosphorylation of occludin, Journal of neurochemistry, 80 (2002) 667-677. [21] G. Li, M.J. Simon, L.M. Cancel, Z.D. Shi, X. Ji, J.M. Tarbell, B. Morrison, B.M. Fu, Permeability of Endothelial and Astrocyte Cocultures: In Vitro Blood–Brain Barrier Models for Drug Delivery Studies, Annals of biomedical engineering, 38 (2010) 24992511. [22] K.A. Duck, E.B. Neely, I.A. Simpson, J.R. Connor, A role for sex and a common HFE gene variant in brain iron uptake, Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism, (2017) 271678x17701949. [23] J.A. Cadieux, Z. Zhang, M. Mattice, A. Brownlie-Cutts, J. Fu, L.G. Ratkay, R. Kwan, J. Thompson, J. Sanghara, J. Zhong, Y.P. Goldberg, Synthesis and biological evaluation of substituted pyrazoles as blockers of divalent metal transporter 1 (DMT1), Bioorganic & medicinal chemistry letters, 22 (2012) 90-95. [24] J.R. Burdo, D.A. Antonetti, E.B. Wolpert, J.R. Connor, Mechanisms and regulation of transferrin and iron transport in a model blood-brain barrier system, Neuroscience, 121 (2003) 883-890. [25] B.E. Enerson, L.R. Drewes, The rat blood-brain barrier transcriptome, Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism, 26 (2006) 959-973. [26] J.B. Fishman, J.B. Rubin, J.V. Handrahan, J.R. Connor, R.E. Fine, Receptor-mediated transcytosis of transferrin across the blood-brain barrier, Journal of Neuroscience Research, 18 (1987) 299-304. [27] T. Moos, E.H. Morgan, Transferrin and Transferrin Receptor Function in Brain Barrier Systems, Cellular and molecular neurobiology, 20 (2000) 77-95. [28] A.J. Siddappa, R.B. Rao, J.D. Wobken, K. Casperson, E.A. Leibold, J.R. Connor, M.K. Georgieff, Iron deficiency alters iron regulatory protein and iron transport protein expression in the perinatal rat brain, Pediatric research, 53 (2003) 800-807. [29] M. Garrick, D. Scott, S. Walpole, E. Finkelstein, J. Whitbred, S. Chopra, L. Trivikram, D. Mayes, D. Rhodes, K. Cabbagestalk, R. Oklu, A. Sadiq, B. Mascia, J. Hoke, L. Garrick, Iron supplementation moderates but does not cure the Belgrade anemia, Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine, 10 (1997) 65-76. [30] M.D. Garrick, K. Gniecko, Y. Liu, D.S. Cohan, L.M. Garrick, Transferrin and the transferrin cycle in Belgrade rat reticulocytes, Journal of Biological Chemistry, 268 (1993) 14867-14874. [31] A. Ciechanover, A.L. Schwartz, A. Dautry-Varsat, H.F. Lodish, Kinetics of internalization and recycling of transferrin and the transferrin receptor in a human
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Figure 1. Iron release from BBMVECs in a modeled BBB. The BBMVECs were loaded overnight with 59Fe and then exposed to the different treatments. A) 500 nM hepcidin treatment had no effect on baseline 59Fe release from BBMVEC. B) 10 uM apoTf significantly increased release into the basal chamber from BBMVEC. The apo-Tfinduced iron release was not altered by 500 nM hepcidin treatment. C) Apo-Tf and 100 uM DFO cause significant release of iron into the basal chamber over 4 hours. D) 100 uM DFO caused significant release of 59Fe from the BBMVEC, but this release was not blocked by 500 nM hepcidin treatment.
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Figure 2. Effect of treatment with Fe-NTA, apo-Tf, and hepcidin on ferroportin protein expression in BBMVECs. A) Representative western blot of ferroportin and βactin. B) There was a consistent 38% decrease in ferroportin when BBMVECs were treated overnight with 59Fe-NTA and this decrease was not affected by overnight exposure to 500 nM hepcidin. The decrease in ferroportin in response to 59Fe-NTA was eliminated when the cells were treated with a combination of overnight 59Fe-NTA with hepcidin followed by hepcidin with apo-Tf for 4 hours. Ferroportin was expressed as a ratio of the protein to β-actin. The ratio was normalized to a common liver sample to account for intra-experimental variance.
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Figure 3. Effect of a DMT1 inhibitor, XEN602, on iron transport across the BBB. The endothelial cells were first exposed to XEN602 for one hour followed by exposure to 59-Fe conjugated to AlexaFluor488-Tf. A) The total lysate-associated 59Fe was not altered by XEN602 treatment. B) XEN602 caused a significant decrease in protein-bound 59 Fe transported into the basal chamber (p = 0.044). C) Free 59Fe transport into the basal chamber was reduced in response to XEN602 treatment (p = 0.012). D) Tf flux into the basal chamber was reduced 59% by XEN602 treatment (p = 0.049).
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S0006-291X(17)32061-2
DOI:
10.1016/j.bbrc.2017.10.083
Reference:
YBBRC 38700
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 11 October 2017 Accepted Date: 16 October 2017
Please cite this article as: K.A. Duck, I.A. Simpson, J.R. Connor, Regulatory mechanisms for iron transport across the blood-brain barrier, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.10.083. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Regulatory Mechanisms for Iron Transport across the BloodBrain Barrier Kari A. Duck1, Ian A. Simpson2, and James R. Connor1
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State Hershey Medical Center, Hershey, PA
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4 Department of Neurosurgery, 2Department of Neural and Behavioral Sciences, Penn
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Correspondence should be sent to: James R. Connor, Ph.D. Distinguished Professor of Neurosurgery, Neural and Behavioral Sciences and Pediatrics Vice-Chair of Neurosurgery Director, Center for Aging and Neurodegenerative Diseases Penn State Hershey Medical Center 500 University Drive MC H110, C3830 Hershey, PA 17033 Telephone: 717-531-4541 Fax: 717-531-0091 Email: [email protected]
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Keywords: Blood-brain barrier, divalent metal transport 1, iron, endosome, transferrin, transferrin receptor
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Abbreviations: blood-brain barrier (BBB), transferrin (Tf), iron (Fe), iron-poor transferrin (apo-Tf), deferoxamine (DFO), divalent metal transporter 1 (DMT1), intravenous (IV), transferrin-bound iron (TBI), bovine retinal endothelial cell (BREC), cerebrospinal fluid (CSF), nitrilotriacetic acid (NTA), bovine brain microvascular endothelial cell (BBMVEC)
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Abstract Many critical metabolic functions in the brain require adequate and timely delivery of
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iron. However, most studies when considering brain iron uptake have ignored the iron
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requirements of the endothelial cells that form the blood-brain barrier (BBB).
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Moreover, current models of BBB iron transport do not address regional regulation of
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brain iron uptake or how neurons, when adapting to metabolic demands, can acquire
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more iron. In this study, we demonstrate that both iron-poor transferrin (apo-Tf) and
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the iron chelator, deferoxamine, stimulate release of iron from iron-loaded endothelial
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cells in an in vitro BBB model. The role of the endosomal divalent metal transporter 1
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(DMT1) in BBB iron acquisition and transport has been questioned. Here, we show
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that inhibition of DMT1 alters the transport of iron and Tf across the endothelial cells.
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These data support an endosome-mediated model of Tf-bound iron uptake into the
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brain and identifies mechanisms for local regional regulation of brain iron uptake.
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Moreover, our data provide an explanation for the disparity in the ratio of Tf to iron
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transport into the brain that has confounded the field.
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Introduction
Iron is an essential micronutrient that functions as a cofactor in key brain processes
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such as myelination and neurotransmitter synthesis [1]. Until recently, studies on the
uptake mechanism of iron into the brain considered the blood-brain barrier (BBB) as a passive conduit [2]. We have identified the endothelial cells of the BBB as a
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regulatory site for brain iron uptake. Identifying the signals and the underlying
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mechanism for this regulation are of considerable importance given the contribution
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of altered brain iron status observed in various neurodegenerative diseases [3-9] and
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the long-term effects of iron deficiency during development [10]. Understanding the
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mechanism(s) by which iron uptake into the brain is regulated is critical to
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determining the impact of intravenous (IV) iron supplements for treatment of
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neurological disorders such as restless legs syndrome [11, 12] or whether IV iron
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treatment can increase the risk of neurodegenerative diseases that are associated with
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excessive brain iron accumulation [3, 13]. Moreover, understanding the regulation of
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iron uptake mechanisms is relevant to the numerous attempts to use the brain’s iron
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delivery system to deliver therapeutic agents that have met with limited success [14].
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A key aspect to understanding the BBB as the regulatory site for brain iron
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acquisition must include a model wherein the iron needs of the endothelial cells are
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met. Until recently, the fact that mitochondrial-rich [15], ferritin-containing [16]
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endothelial cells required a mechanism for acquiring iron, and not just transporting it,
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had been overlooked. Moreover, the presence of the iron exporter, ferroportin, has
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been demonstrated in brain microvascular endothelial cells. Although its function has
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not been uniformly confirmed, the presence of this protein suggests a specific
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mechanism for iron release from the endothelial cells [17, 18]. In our previous study
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using bovine retinal endothelial cells (BREC), the presence of ferroportin was identified and its activity demonstrated by blocking iron release with hepcidin [2], a known inhibitor of ferroportin [19]. In this study, we identify factors that alter release and transport of iron across a model of the BBB.
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Materials and Methods Cell culture Bovine brain microvascular endothelial cells (BBMVEC) were
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cultured in complete growth medium (Cell Applications, Inc.). BBMVECs were
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grown to confluence on Costar transwell 0.4 µm porous filters (Corning) and then
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gently washed three times with 1X DPBS (Corning). The BBMVEC cells were
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then exposed to serum free media containing 138 nM hydrocortisone (Sigma
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Aldrich) for 72 hours to promote tight junction formation [20].
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Release assay BBMVEC in the transwell setup were loaded overnight with 10
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µCi/well of 59Fe-NTA complex. Wells were then washed three times with 1X
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DPBS (Corning) to remove iron remaining in the culture medium. Serum-free
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medium containing 70 kDa RITC-Dextran (Sigma Aldrich) was added to the
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apical chamber. The ability of 70 kDa RITC-Dextran to diffuse across the BBB
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serves as a measure of barrier integrity and enables correction for any leakage
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[21]. Serum-free medium containing 10 µM apo-Tf (Sigma Aldrich), 500 nM
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hepcidin (Peptides, Inc.), or 10 µM apo-Tf and 500 nM hepcidin was then added
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to the basal chamber. Where hepcidin was used, BBMVECs were treated with
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500 nM hepcidin overnight simultaneously with the 59Fe loading step. Cells were
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exposed to 100 uM deferoxamine (DFO) to compare the chelator effect to apo-Tf. Data were collected by taking 50 µL aliquots from the apical chamber at 0 and 4
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hours and the basal chamber at 0, 2, and 4 hours. Fluorescence (RITC excitation:
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555 nm, emission: 580 nm) was measured on a SpectraMax Gemini EM plate
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reader (Molecular Devices) and 59Fe was then measured on a Beckman Gamma
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4000 (Beckman Coulter).
105 Transport assay Tf flux across the BBB was measured by applying 10 µM 59Fe-
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AlexaFluor488-Tf to the apical chamber of our transwell model. 59Fe-
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AlexaFluor488-Tf was prepared as previously described [22]. Fresh media
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containing 10 µM 59Fe-AlexaFluor488-Tf and 70 kDa RITC-Dextran was added
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to the apical chamber at the beginning of the experiment. A DMT1 inhibitor (104
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nM XEN602, Xenon Pharmaceuticals [23]) was added to the apical chamber for 1
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hour prior to the addition of 59Fe-AlexaFluor488-Tf (zero time). Media aliquots
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were taken from the apical chamber at 0 and 6 hours. Basal chamber aliquots
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were taken at 2-hour intervals for 6 hours. Fluorescence (RITC excitation: 555
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nm, emission: 580 nm; Alexa Fluor488 excitation: 495 nM, emission 519 nm)
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was measured using a SpectraMax Gemini EM plate reader (Molecular Devices)
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and 59Fe was measured on a Beckman Gamma 4000 (Beckman Coulter). A
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Sephadex G-25 Quick Spin column was used as per the manufacturer’s
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instructions (Roche) to separate free iron from protein-bound iron. Tf flux (P0)
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was calculated from the fluorescence measurements as previously described [24].
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Protein expression assays Ferroportin protein expression was measured by
immunoblot as previously described [2]. Briefly, cells were RIPA lysis buffer
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containing 1X protease inhibitor cocktail (Sigma Aldrich). Ferroportin was
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detected with a ferroportin primary antibody (1:200, Alpha Diagnostics
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International) The membranes were visualized by adding Western Lightning Plus-
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ECL (Perkin Elmer) and imaged on a Fuji LAS-3000 Imaging System.
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Densitometry was performed using Multigauge software (Fuji Film). β-actin
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(1:3000, Sigma Aldrich) was used as a loading control. To control for cross-
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membrane variability, a common liver sample was used on each blot for
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normalization.
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Statistical Analyses Prism (GraphPad Software) software was used for all
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statistical analyses and data graphing. Data from three independent replicates
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were pooled and are expressed as mean ± standard error of the mean. Statistical
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differences between experimental groups were determined using an unpaired
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Student’s t test, one-way ANOVA and Bonferroni’s multiple comparisons test, or
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two-way ANOVA and Bonferroni’s multiple comparisons test. A level of
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significance of p < 0.05 was used for all differences evaluated.
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Results
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The first series of studies were designed to test the hypothesis that iron is released
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from brain endothelial cells and the release can be regulated. BBMVEC were loaded
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with 59Fe and exposed to hepcidin overnight. A baseline release of iron was observed over a four-hour period. There was no effect of hepcidin on the baseline release of
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(CSF side) of the BBMVEC resulted in a 140% increase in iron release within 2
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hours of exposure and persisted at 4 hours of exposure. The apo-Tf-stimulated release
Fe from the cells (Figure 1A). Exposure to 10 µM apo-Tf on the abluminal side
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was not blocked by hepcidin exposure (Figure 1B). Furthermore, the addition of the
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iron chelator DFO to the basal chamber also promoted release of iron (Figure 1C),
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which was also not inhibited by hepcidin (Figure 1D).
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The release of iron from endothelial cells is presumed to be via the iron export
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protein, ferroportin. Thus, we evaluated the expression of ferroportin in BBMVEC
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under the same experimental conditions of iron loading and exposure to apo-Tf and
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hepcidin. We demonstrate that ferroportin is expressed in the BBMVEC and this
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expression is decreased 38% by iron loading (Figure 2). The addition of hepcidin or
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apo-Tf had no effect on ferroportin expression when added alone to the iron-loaded
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endothelial cells but when added in combination, the levels of ferroportin were the
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same as control indicating that the combination of hepcidin and apo-Tf blocks the
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iron-induced decrease in ferroportin expression (Figure 2).
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To investigate the role DMT1 plays in the movement of systemic iron through the
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BBB and into the brain, we exposed the BBMVEC to the DMT1 inhibitor, XEN602.
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The presence of XEN602 had no effect on the amount of iron in the cell lysates
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(Figure 3A). The amount of bound iron released into the basal chamber (Figure 3B)
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was decreased 4-fold (p = 0.044) and the amount of unbound iron in the basal
chamber was decreased by 40% (Figure 3C, p = 0.012). The extent of Tf flux was
decreased by 59% in the presence of XEN602, (Figure 3D, p = 0.049).
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Discussion We previously reported that the BBB was capable of storing iron based on the
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presence of ferritin (for iron storage). Moreover, the presence of an iron regulatory
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protein in the BBB whose function is to regulate expression of ferritin, Tf receptor,
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and ferroportin clearly indicates the potential for intracellular iron-responsiveness by
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the BBB [2, 7, 16, 25-28]. More recently, we have directly demonstrated retention of
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iron in the endothelial cells of the brain suggesting that the BBB may function as an
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iron reservoir from which iron is released into the brain when signaled [22]. A key
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protein for the endocytic mechanism of uptake and release of iron into cells elsewhere
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in the body is DMT1. While the role of DMT1 is well-characterized in other systems,
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a role for DMT1in uptake of iron into the endothelial cell of the BBB remains
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unclear. The significance of DMT1 in cellular iron uptake was demonstrated in the
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Belgrade rat that has a mutation in DMT1, which is associated with decreased cellular
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iron uptake in enterocytes and reticulocytes resulting in microcytic anemia [29, 30].
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In normal mammalian cells, 60-80% of the iron bound to Tf is released into the cell
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during endocytosis with the remainder being released back into the incubation media
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[30, 31]. However, when similar experiments were conducted in reticulocytes from
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the Belgrade rat, the efficacy of this extraction decreased to 15%. Moreover, the
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failure to liberate the iron from the endosomes in the Belgrade reticulocytes
somewhat surprisingly resulted in its release into the media still bound to Tf and not reduced to Fe2+ [30].
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In the current study, we employed a DMT1 inhibitor, XEN602, to evaluate the role of
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DMT1 in iron and Tf transport at the BBB. The presence of the DMT1 inhibitor
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decreased Tf movement across the BBB and reduced both bound and free iron
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transported across the endothelial cells and released into the basal chamber (Figure
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3). These data are not only consistent with Tf-iron cycling in reticulocytes from
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Belgrade Rats [30] but also with the in vivo studies in the Belgrade rat where, despite
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high levels of circulating Tf, the Belgrade rats had an 85% reduction in free iron and
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Tf uptake into the brain compared to wild-type rats [32-34]. A similar reduction in
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free iron transport (≈80%) was observed when BRECs were exposed to NH4Cl,
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which prevents the acidification of the endosomes and the dissociation of iron from
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Tf; however, the levels of holo-Tf accumulation in the basal chamber appeared to be
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unaltered [24]. Recently, NHE9, a regulator of endosomal pH has been shown to
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significantly impact brain iron uptake and regulation within endothelial cells further
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emphasizing the role of endosomes in brain iron uptake [35].
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We have previously reported that the export of iron stored in the endothelial cells of
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the BBB is enhanced by the presence of apo-Tf and DFO as shown in this study; and
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by conditioned media from iron-deprived astrocytes and CSF derived from iron-
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deficient monkeys [2]. We assumed the mechanism for iron export from the endothelial cells involved export via ferroportin as we had previously demonstrated
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using bovine retinal endothelial cells (2) and as others have reported in the gut and for
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macrophages [36, 37]. Our present study revealed that ferroportin is present in bovine
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brain endothelial cells and demonstrated that iron loading decreases ferroportin
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expression as expected. However, the presence of hepcidin failed to block iron release
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from the endothelial cells either alone or in the presence of apo-Tf or the iron chelator
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DFO. Moreover, hepcidin did not alter the iron-loading induced decrease in
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ferroportin, although it did act synergistically with apo-Tf to increase ferroportin
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expression. This finding is different from our previous report using BRECs where
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hepcidin exposure caused a moderate decrease in 59Fe release [2], suggesting there
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are differences in ferroportin expression and the response to hepcidin between the
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retina and brain microvasculature. As ferroportin must be located in the cellular
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membrane to be functional as an exporter, the values measured in total cell lysate
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may not properly reflect the functional profile of the BBMVEC. There appears to be
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synergism between apo-Tf and hepcidin to overcome the iron induced decrease in
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ferroportin and maintain the ability of apo-Tf to continue to induce iron release from
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the endothelial cells, which is an observation for future study.
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In general, our data indicate transferrin-bound iron is first taken up into endosomes
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and upon their acidification, the iron is released via DMT1 into the cytoplasm where
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it is available to either bind to ferritin or be exported from the endothelial cell,
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presumably via ferroportin. Having discharged the iron in the endosome, the resultant
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apo-Tf in most cells undergoes exocytosis and is released back into the media.
However, in the polarized endothelium of the BBB the apo-Tf has the option of going
to either the luminal or abluminal membrane. In our previous study, we suggested
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that a certain proportion of apo-Tf was released from the abluminal membrane to be
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available to recombine with the iron released by ferroportin [2]. This is necessary in
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the model because an iron excess over fully-saturated Tf has been consistently
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reported in the CSF under normal conditions [27, 38], therefore a potential source of
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apo-Tf in the extracellular fluid must be identified. Although we cannot rule out a
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direct transcytotic mechanism, our data favor an endosomal-mediated model that
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releases vesicles containing Tf in which 60-80% of the Tf is apo and the remaining
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holo as reported in other cell types [30, 31, 39]. Based on the observed 3-4:1 disparity
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between the transport of iron and Tf, it would appear that the exocytosis of the Tf
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receptor with associated Tf (apo+holo) partitions between the luminal and abluminal
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membranes with a comparable 3:1 ratio.
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This study, in combination with our previous study, introduces the concept that apo-
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Tf and holo-Tf provide feedback to the endothelial cells that induce release (apo-Tf)
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or decrease release (holo-Tf) of iron from the endothelial cell. The mechanism for
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limiting the release of iron in response to holo-Tf could be via iron uptake back into
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the endothelial cell from the CSF, similar to the feedback mechanism reported in
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enterocytes [40, 41]. The idea of a feedback system in the brain for iron uptake is a
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novel concept in the central nervous system. The induction of iron release via apo-Tf
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(which can be mimicked by the iron chelator DFO) provides a new system not
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previously reported. The identification of the mechanism by which apo-Tf induces
iron release from endothelial cells, that may involve synergistic interaction with
hepcidin, is perhaps the critical step to understanding regional control of brain iron uptake.
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In conclusion, these data not only provide further support for a DMT1-dependent
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endocytic process mediating the passage of iron across the endothelial cells that
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constitute the BBB but addresses a long-observed enigma in the field; namely, the
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disproportionately greater transport of iron relative to Tf into the brain [24, 26, 27, 38,
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42-45]. We propose that the levels of apo-Tf and iron delivered to the brain are tightly
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regulated by the ratio of apo-Tf:holo-Tf in conjunction with hepcidin in the
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extracellular fluid of each brain region. Finally, these data also provide a potential
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explanation as to why attempts to harness the brain’s Tf-Tf receptor uptake system to
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deliver therapeutics has met with limited success due to the lack of appreciation of the
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importance of the endosomal route [14].
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Acknowledgements
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This work was supported by NIH R01 NS077678.
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Disclosure/Conflict of Interest
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The authors have no conflict of interest to disclose.
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Figure 1. Iron release from BBMVECs in a modeled BBB. The BBMVECs were loaded overnight with 59Fe and then exposed to the different treatments. A) 500 nM hepcidin treatment had no effect on baseline 59Fe release from BBMVEC. B) 10 uM apoTf significantly increased release into the basal chamber from BBMVEC. The apo-Tfinduced iron release was not altered by 500 nM hepcidin treatment. C) Apo-Tf and 100 uM DFO cause significant release of iron into the basal chamber over 4 hours. D) 100 uM DFO caused significant release of 59Fe from the BBMVEC, but this release was not blocked by 500 nM hepcidin treatment.
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Figure 2. Effect of treatment with Fe-NTA, apo-Tf, and hepcidin on ferroportin protein expression in BBMVECs. A) Representative western blot of ferroportin and βactin. B) There was a consistent 38% decrease in ferroportin when BBMVECs were treated overnight with 59Fe-NTA and this decrease was not affected by overnight exposure to 500 nM hepcidin. The decrease in ferroportin in response to 59Fe-NTA was eliminated when the cells were treated with a combination of overnight 59Fe-NTA with hepcidin followed by hepcidin with apo-Tf for 4 hours. Ferroportin was expressed as a ratio of the protein to β-actin. The ratio was normalized to a common liver sample to account for intra-experimental variance.
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Figure 3. Effect of a DMT1 inhibitor, XEN602, on iron transport across the BBB. The endothelial cells were first exposed to XEN602 for one hour followed by exposure to 59-Fe conjugated to AlexaFluor488-Tf. A) The total lysate-associated 59Fe was not altered by XEN602 treatment. B) XEN602 caused a significant decrease in protein-bound 59 Fe transported into the basal chamber (p = 0.044). C) Free 59Fe transport into the basal chamber was reduced in response to XEN602 treatment (p = 0.012). D) Tf flux into the basal chamber was reduced 59% by XEN602 treatment (p = 0.049).
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