Ferric Ammonium Citrate Reduces Claudin-5 Abundance and Function in Primary Mouse Brain Endothelial Cells

Abstract

Background: Iron overload is implicated in many neurodegenerative diseases, where there is also blood-brain barrier (BBB) dysfunction. As there is a growing interest in the role of iron in modulating key BBB proteins, this study assessed the effect of iron on the expression and function of P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and claudin-5 in primary mouse brain endothelial cells (MBECs) and their abundance in mouse brain microvessel-enriched membrane fractions (MVEFs).

Methods: Following a 48 h treatment with ferric ammonium citrate (FAC, 250 µM), MBEC protein abundance (P-gp, BCRP and claudin-5) and mRNA (abcb1a, abcg2, and cldn5) were assessed by western blotting and RT-qPCR, respectively. Protein function was evaluated by assessing transport of substrates ³H-digoxin (P-gp), ³H-prazosin (BCRP) and ¹⁴C-sucrose (paracellular permeability). C57BL/6 mice received iron dextran (100 mg/kg, intraperitoneally) over 4 weeks, and MVEF protein abundance and iron levels (in MVEFs and plasma) were quantified via western blotting and inductively coupled plasma-mass spectrometry (ICP-MS), respectively.

Results: FAC treatment reduced P-gp protein by 50% and abcb1a mRNA by 43%, without affecting ³H-digoxin transport. FAC did not alter BCRP protein or function, but decreased abcg2 mRNA by 59%. FAC reduced claudin-5 protein and cldn5 mRNA by 65% and 70%, respectively, resulting in a 200% increase in ¹⁴C-sucrose permeability. In vivo, iron dextran treatment significantly elevated plasma iron levels (2.2-fold) but did not affect brain MVEF iron content or alter P-gp, BCRP or claudin-5 protein abundance.

Conclusions: Iron overload modulates BBB transporters and junction proteins in vitro, highlighting potential implications for CNS drug delivery in neurodegenerative diseases.

Keywords: P-glycoprotein; blood–brain barrier; breast cancer resistance protein; claudin-5; iron overload.

Fig. 1

Viability of MBECs following a 48 h treatment with 1, 10, 50 or 250 µM of FAC. A vehicle control (CTRL), constituting 0.5% v/v Milli-Q water in media, was used and 5% v/v DMSO in media was included as a positive control for cell death. Data are presented as mean ± SEM (n = 3). **p < 0.01 relative to vehicle (CTRL) treated MBECs, as assessed by a one-way ANOVA followed by post-hoc Dunnett’s test.

Fig. 2

Representative western blots and total protein (left) and mean fold change in protein of interest normalized to total protein (right) demonstrating protein abundance in MBECs for (a) P-gp, (b) BCRP, and (c) claudin-5 and (d) mRNA transcripts of abcb1a, abcg2 and cldn5, following treatment with 250 µM of FAC or vehicle (CTRL) for 48 h. Data are presented as mean ± SEM (n = 4) expressed as relative fold-change of respective CTRL, **p < 0.01, ***p < 0.001, and ns = not significant p > 0.05, when compared with CTRL as assessed by an unpaired Student’s t-test.

Fig. 3

Bi-directional transport profiles (abluminal-to-luminal and luminal-to-abluminal) of (a) ³H-digoxin and (c) ³H-prazosin across MBECs treated with 250 µM of FAC or vehicle (CTRL) for 48 h. The luminal-to-abluminal and abluminal-to-luminal P_app values of (b) ³H-digoxin and (d) ³H-prazosin are presented. Data are presented as mean ± SEM (n = 4), ***p < 0.001 and ns = not significant when compared with vehicle (CTRL) treated MBECs, as assessed by a two-way ANOVA followed by post-hoc Tukey’s test.

Fig. 4

(A) Effect of FAC treatment on TEER values of MBECs, (b) bi-directional transport profiles (abluminal-to-luminal and luminal-to-abluminal) of ¹⁴C-sucrose in MBECs treated with 250 µM of FAC or vehicle (CTRL) for 48 h, and (c) the luminal-to-abluminal and abluminal-to-luminal P_app values of ¹⁴- sucrose are presented. Data are presented as mean ± SEM (n = 4), ****p < 0.0001 and ns = not significant when compared with vehicle-treated MBECs as assessed by an unpaired Student’s t test and, a two-way ANOVA followed by post-hoc Tukey’s test.

Fig. 5

Representative western blots and total protein (left) and mean fold change in protein normalized to total protein (right) demonstrating abundance of (a) P-gp, (b) BCRP and (c) claudin-5 in membrane fractions of brain MVEFs obtained from C57BL/6 mice treated with 100 mg/kg treatment of iron dextran or vehicle (0.9% saline) (CTRL) every 3 days for 4 weeks. Data are presented as mean ± SEM (n = 6), ns = not significant when compared with vehicle treated mice, as assessed by an unpaired Student’s t-test.

Fig. 6

Iron concentration in a) plasma and b) cytosolic fraction of brain MVEFs following iron dextran administration. Mice were treated with 100 mg/kg of iron dextran or vehicle (0.9% saline) (CTRL) every 3 days for 4 weeks. Data are presented as mean ± SEM (n = 6–21), ****p < 0.0001 and ns = not significant when compared with vehicle-treated mice, as assessed by an unpaired Student’s t-test.