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Background <p>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).</p> Methods <p>Following a 48&#xa0;h treatment with ferric ammonium citrate (FAC, 250&#xa0;µM), MBEC protein abundance (P-gp, BCRP and claudin-5) and mRNA (<i>abcb1a</i>, <i>abcg2</i>, and <i>cldn5)</i> were assessed by western blotting and RT-qPCR, respectively. Protein function was evaluated by assessing transport of substrates <sup>3</sup>H-digoxin (P-gp), <sup>3</sup>H-prazosin (BCRP) and <sup>14</sup>C-sucrose (paracellular permeability). C57BL/6 mice received iron dextran (100&#xa0;mg/kg, intraperitoneally) over 4&#xa0;weeks, and MVEF protein abundance and iron levels (in&#xa0;MVEFs&#xa0;and plasma) were quantified via western blotting and inductively coupled plasma-mass spectrometry (ICP-MS), respectively.</p> Results <p>FAC treatment reduced P-gp protein by 50% and <i>abcb1a</i> mRNA by 43%, without affecting <sup>3</sup>H-digoxin transport. FAC did not alter BCRP protein or function, but decreased <i>abcg2</i> mRNA by 59%. FAC reduced claudin-5 protein and <i>cldn5</i> mRNA by 65% and 70%, respectively, resulting in a 200% increase in <sup>14</sup>C-sucrose permeability. <i>In vivo</i>, 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.</p> Conclusions <p>Iron overload modulates BBB transporters and junction proteins <i>in vitro</i>, highlighting potential implications for CNS drug delivery in neurodegenerative diseases.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年2月12日

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  • Pranav Runwal,
  • Jae Pyun,
  • Stephanie A. Newman,
  • Celeste Mawal,
  • Ashley I. Bush,
  • Liam M. Koehn,
  • Joseph A. Nicolazzo

摘要

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.