Abstract
Accumulating evidence indicates that agrin, a heparan sulphate proteoglycan of the extracellular matrix, plays a role in the organization and maintenance of the blood-brain barrier. This evidence is based on the differential effects of agrin isoforms on the expression and distribution of the water channel protein, aquaporin-4 (AQP4), on the swelling capacity of cultured astrocytes of neonatal mice and on freeze-fracture data revealing an agrin-dependent clustering of orthogonal arrays of particles (OAPs), the structural equivalent of AQP4. Here, we show that the OAP density in agrin-null mice is dramatically decreased in comparison with wild-types, by using quantitative freeze-fracture analysis of astrocytic membranes. In contrast, anti-AQP4 immunohistochemistry has revealed that the immunoreactivity of the superficial astrocytic endfeet of the agrin-null mouse is comparable with that in wild-type mice. Moreover, in vitro, wild-type and agrin-null astrocytes cultured from mouse embryos at embryonic day 19.5 differ neither in AQP4 immunoreactivity, nor in OAP density in freeze-fracture replicas. Analyses of brain tissue samples and cultured astrocytes by reverse transcription with the polymerase chain reaction have not demonstrated any difference in the level of AQP4 mRNA between wild-type astrocytes and astrocytes from agrin-null mice. Furthermore, we have been unable to detect any difference in the swelling capacity between wild-type and agrin-null astrocytes. These results clearly demonstrate, for the first time, that agrin plays a pivotal role for the clustering of OAPs in the endfoot membranes of astrocytes, whereas the mere presence of AQP4 is not sufficient for OAP clustering.
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Acknowledgements
We thank Yeliz Donat-Krasnici, Ida Zeiher and Ria Knittel for skilful technical assistance in cell culturing, freeze-fracturing and microscopy. We gratefully acknowledge Natasha Tetkovic for expert mouse colony management and genotyping. We are also grateful to Dr. Markus Rüegg (Biozentrum, University of Basel, Switzerland) for providing us with the agrin-null mice and for critically reading the manuscript and to Dr. Hubert Kalbacher (Tübingen) for performing the enzyme-linked immunosorbent assay for agrin detection in the cell culture medium.
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This work was supported by the Deutsche Krebshilfe, Mildred-Scheel-Stiftung (grant no. 107686, to H.W.).
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Noell, S., Fallier-Becker, P., Deutsch, U. et al. Agrin defines polarized distribution of orthogonal arrays of particles in astrocytes. Cell Tissue Res 337, 185–195 (2009). https://doi.org/10.1007/s00441-009-0812-z
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DOI: https://doi.org/10.1007/s00441-009-0812-z