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 3 H-digoxin (P-gp), 3 H-prazosin (BCRP) and 14 C-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 3 H-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 14 C-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.

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