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The Endo-Lysosomal System of Brain Endothelial Cells Is Influenced by Astrocytes In Vitro

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Abstract

Receptor- and adsorptive-mediated transport through brain endothelial cells (BEC) of the blood-brain barrier (BBB) involves a complex array of subcellular vesicular structures, the endo-lysosomal system. It consists of several types of vesicles, such as early, recycling, and late endosomes, retromer-positive structures, and lysosomes. Since this system is important for receptor-mediated transcytosis of drugs across brain capillaries, our aim was to characterise the endo-lysosomal system in BEC with emphasis on their interactions with astrocytes. We used primary porcine BEC in monoculture and in co-culture with primary rat astrocytes. The presence of astrocytes changed the intraendothelial vesicular network and significantly impacted vesicular number, morphology, and distribution. Additionally, gene set enrichment analysis revealed that 60 genes associated with vesicular trafficking showed altered expression in co-cultured BEC. Cytosolic proteins involved in subcellular trafficking were investigated to mark transport routes, such as RAB25 for transcytosis. Strikingly, the adaptor protein called AP1-μ1B, important for basolateral sorting in epithelial cells, was not expressed in BEC. Altogether, our data pin-point unique features of BEC trafficking network, essentially mapping the endo-lysosomal system of in vitro BBB models. Consequently, our findings constitute a valuable basis for planning the optimal route across the BBB when advancing drug delivery to the brain.

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Acknowledgements

Niels M. Kristiansen, Annemette B. Marnow, Mitra Shamsali, Anders Heuck, and Tibor Salzer are thanked for their expert technical assistance. Matthias Rommeswinkel, applicant specialist at Olympus Soft Imaging Solutions GmbH, is acknowledged for his technical support in Scan^R Image analysis software. We thank N. Joan Abbott for critical reading of the manuscript.

Funding

The work was supported by the Research Initiative on Brain Barriers and Drug Delivery funded by the Lundbeck foundation (Grant no. 2013-14113). KLH was supported by a grant from the Danish National Research Foundation (DNRF) (PUMPKIN DNRF85). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

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Authors and Affiliations

  1. Department of Biomedicine, Faculty of Health, Aarhus University, Ole Worms Allé 3, 8000, Aarhus, Denmark

    Andrea E. Toth, Piotr Siupka, Susanne T. Venø, Peder Madsen, Karin Lykke-Hartmann & Morten S. Nielsen

  2. Lundbeck Foundation, Research Initiative on Brain Barriers and Drug Delivery, Scherfigsvej 7, 2100, Copenhagen, Denmark

    Andrea E. Toth, Piotr Siupka, Susanne T. Venø, Louiza B. Thomsen, Torben Moos & Morten S. Nielsen

  3. Research Program for Molecular Neurology, Helsinki University, Haartmaninkatu 8, 00290, Helsinki, Finland

    Thomas J. P. Augustine & Hannes T. Lohi

  4. Laboratory of Neurobiology, Department of Health Science and Technology, Aalborg University, Fredrik Bajers Vej 3, 9220, Aalborg, Denmark

    Louiza B. Thomsen & Torben Moos

  5. Department of Clinical Genetics, Aarhus University Hospital, Brendstrupgårdsvej 21, 8200, Aarhus, Denmark

    Karin Lykke-Hartmann

  6. Department of Clinical Medicine, Faculty of Health, Aarhus University, Bartholins Alle 6, 8000, Aarhus, Denmark

    Karin Lykke-Hartmann

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  1. Andrea E. Toth
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  9. Karin Lykke-Hartmann
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  10. Morten S. Nielsen
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Correspondence to Morten S. Nielsen.

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Ethical Approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All animals were sacrificed following Danish legislation from the Danish Ministry of Environment and Food on slaughtering of animals (BEK no. 135 of 14/02/2014, European legislation identifier/eli/lta/2014/135). Rats were euthanized according to Danish legislation on animal experimentation (BEK number 12 of 07/01/2016, European legislation identifier/eli/lta/2016/12) and to the international guidelines on the ethical use of animals (European Communities Council Directive of 24 November 1986; 86/609/EEC). The ARRIVE guidelines have been followed.

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The authors declare no conflict of interest.

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Toth, A.E., Siupka, P., P. Augustine, T.J. et al. The Endo-Lysosomal System of Brain Endothelial Cells Is Influenced by Astrocytes In Vitro. Mol Neurobiol 55, 8522–8537 (2018). https://doi.org/10.1007/s12035-018-0988-x

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