Elsevier

Neuroscience

Volume 138, Issue 1, 2006, Pages 159-169
Neuroscience

Molecular neuroscience
Differential, strain-specific cellular and subcellular distribution of multidrug transporters in murine choroid plexus and blood–brain barrier

https://doi.org/10.1016/j.neuroscience.2005.11.011Get rights and content

Abstract

Multidrug transporters of the ATP-binding cassette family play an important role in regulating drug distribution and efflux in the brain, owing to their selective distribution in microvessels and choroid plexus. Their expression pattern and cellular distribution remain controversial, in part due to technical difficulties in localizing these membrane proteins in closely associated cells, such as endothelial cells and astrocytic end-feet at the blood–brain barrier. Here, we used high-resolution immunofluorescence staining with cell-type specific markers to investigate the distribution of major ATP-binding cassette transporters in mouse brain. We report that four ATP-binding cassette transporters, Mdr1, Mrp1, Mrp2 and Mrp5 can be detected in brain endothelial cells, forming three distinct layers, with Mdr1 and Mrp5 being located on the luminal side, Mrp1 on the abluminal (basal) side, and Mrp2 in between. Mrp3 and Mdr3 were undetectable. In choroid plexus, only Mrp1, Mrp2 and Mrp3 were detected, again with a differential distribution. Mrp1 was targeted basolaterally in epithelial cells, Mrp2 was restricted to endothelial cells, and Mrp3 was co-localized with zonula occludens-1 at tight junctions. Analysis of Mdr1a0/0 and Mrp10/0 mice, generated in the FVB strain, revealed no major alteration in expression of the remaining transporters. An unexpected strain difference was unraveled, with wildtype FVB mice selectively lacking Mrp2 protein in brain, but not liver. In conclusion, these results indicate that ATP-binding cassette transporters provide multiple penetration barriers in the blood–brain barrier and choroid plexus, with a selective cellular and subcellular distribution, emphasizing their potential role for drug resistance in neurological disorders, such as epilepsy.

Section snippets

Animals

Adult mice were purchased from commercial suppliers: C57BL/6 (Jackson Laboratories, Bar Harbor, ME, USA), SVJ (Institute for Laboratory Animal Sciences, University of Zurich), Swiss (Harlan, Horst, The Netherlands), and FVB wild type, Mdr1a0/0, Mrp10/0 inbred strains (Taconic Farms, Hallingore, Denmark). Generation and characterization of Mrp20/0 mice will be described elsewhere (Vlaming et al., in preparation). All mice were housed in a 12-h light/dark cycle with free access to food and water.

Characterization of antibodies

The distribution of ABC-transporters in the BBB and choroid plexus was analyzed in sections prepared from fresh frozen tissue and weakly fixed by immersion in a low concentration of paraformaldehyde. This procedure was selected because pilot experiments in perfusion-fixed tissue resulted in a low signal-to-noise ratio and unspecific staining of numerous organelles (not shown).

At first, commercially available monoclonal antibodies raised against human-specific sequences were tested. Good results

Discussion

Numerous studies have investigated the expression and distribution of ABC-transporters at the BBB and in the choroid plexus, with partially controversial findings that might reflect discrepancies in mRNA and protein expression, species specificity, and technical differences. Our results show that a strong signal-to-noise ratio can be achieved for immunofluorescence staining in weakly fixed tissue, a prerequisite for high resolution imaging using laser scanning microscopy. Using this approach,

Acknowledgments

We are grateful to Corinne Sidler and Franziska Parpan for outstanding technical assistance. This study was supported by the Swiss National Science Foundation (National Center of Competence in Research “Neural plasticity and repair”) and the Dutch Cancer Society, grant NKI 2003–2940.

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