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Agrin defines polarized distribution of orthogonal arrays of particles in astrocytes

Cell and Tissue Research volume 337, pages 185–195 (2009)Cite this article

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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References

  • Anders JJ, Brightman MW (1979) Assemblies of particles in the cell membranes of developing, mature and reactive astrocytes. J Neurocytol 8:777–795

    PubMed  Article  CAS  Google Scholar 

  • Bandtlow CE, Zimmermann DR (2000) Proteoglycans in the developing brain: new conconceptual insights for old proteins. Physiol Rev 80:1267–1290

    PubMed  CAS  Google Scholar 

  • Barber AJ, Lieth E (1997) Agrin accumulates in the brain microvascular basal lamina during development of the blood-brain barrier. Dev Dyn 208:62–74

    PubMed  Article  CAS  Google Scholar 

  • Berzin TM, Zipser BD, Rafii MS, Kuo-Leblanc V, Yancopoulos GD, Glass DJ, Fallon JR, Stopa EG (2000) Agrin and microvascular damage in Alzheimer's disease. Neurobiol Aging 21:349–355

    PubMed  Article  CAS  Google Scholar 

  • Beyer C, Raab H (1998) Nongenomic effects of oestrogen: embryonic mouse midbrain neurones respond with a rapid release of calcium from intracellular stores. Eur J Neurosci 10:255–262

    PubMed  Article  CAS  Google Scholar 

  • Bezakova G, Ruegg MA (2003) New insights into the roles of agrin. Nat Rev Mol Cell Biol 4:295–308

    PubMed  Article  CAS  Google Scholar 

  • Crane JM, Verkman AS (2009) Determinants of aquaporin-4 assembly in orthogonal arrays revealed by live-cell single-molecule fluorescence imaging. J Cell Sci 122:813–821

    PubMed  Article  CAS  Google Scholar 

  • Crane JM, VanHoek AN, Skach WR, Verkman AS (2008) Aquaporin-4 dynamics in orthogonal arrays in live cells visualized by quantum dot single particle tracking. Mol Biol Cell 19:3369–3378

    PubMed  Article  CAS  Google Scholar 

  • De Kok JB, Roelofs RW, Giesendorf BA, Pennings JL, Waas ET, Feuth T, Swinkels DW, Span PN (2005) Normalization of gene expression measurements in tumor tissues: comparison of 13 endogenous control genes. Lab Invest 85:154–159

    PubMed  Google Scholar 

  • Dermietzel R (1973) Visualization by freeze-fracturing of regular structures in glial cell membranes. Naturwissenschaften 60:208

    PubMed  Article  CAS  Google Scholar 

  • Frericks M, Esser C (2008) A toolbox of novel murine house-keeping genes identified by meta-analysis of large scale gene expression profiles. Biochim Biophys Acta 1779:830–837

    PubMed  CAS  Google Scholar 

  • Furman CS, Gorelick-Feldman DA, Davidson KG, Yasumura T, Neely JD, Agre P, Rash JE (2003) Aquaporin-4 square array assembly: opposing actions of M1 and M23 isoforms. Proc Natl Acad Sci USA 100:13609–13614

    PubMed  Article  CAS  Google Scholar 

  • Gautam M, Noakes PG, Moscoso L, Rupp F, Scheller RH, Merlie JP, Sanes JR (1996) Defective neuromuscular synaptogenesis in agrin-deficient mutant mice. Cell 85:525–535

    PubMed  Article  CAS  Google Scholar 

  • Gotow T, Hashimoto PH (1989) Developmental alterations in membrane organization of rat subpial astrocytes. J Neurocytol 18:731–747

    PubMed  Article  CAS  Google Scholar 

  • Hatton JD, Sang UH (1990) Orthogonal arrays are absent from the membranes of human glioblastoma tissues. Acta Anat 137:363–66

    PubMed  Article  CAS  Google Scholar 

  • Ihrie RA, Alvarez-Buylla A (2008) Cells in the astroglial lineare are neural stem cells. Cell Tissue Res 331:179–191

    PubMed  Article  Google Scholar 

  • Ivanova T, Karolczak M, Beyer C (2001) Estradiol stimulates the mitogen-activated protein kinase pathway in midbrain astroglia. Brain Res 889:264–269

    PubMed  Article  CAS  Google Scholar 

  • Ivanova T, Mendez P, Garcia-Segura LM, Beyer C (2002) Rapid stimulation of the P13-kinase/Akt signaling pathway in developing midbrain neurons by oestrogen. J Neuroendocrinol 14:73–79

    PubMed  Article  CAS  Google Scholar 

  • Ksiazek I, Burkhardt C, Lin S, Seddik R, Maj M, Bezakova G, Jucker M, Arber S, Caroni P, Sanes JR, Bettler B, Ruegg MA (2007) Synapse loss in cortex of agrin-deficient mice after genetic rescue of perinatal death. J Neurosci 27:7183–7195

    PubMed  Article  CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    PubMed  Article  CAS  Google Scholar 

  • Landis DMD, Reese TS (1981) Membrane structure in mammalian astrocytes: a review of freeze-fracture studies on adult, developing, reactive and cultured astrocytes. J Exp Biol 95:35–48

    PubMed  CAS  Google Scholar 

  • Lin W, Burgess RW, Dominguez B, Pfaff SL, Sanes JR, Lee K-F (2001) Distinct roles of nerve and muscle in postsynaptic differentiation of the neuromuscular synapse. Nature 410:1057–1064

    PubMed  Article  CAS  Google Scholar 

  • McMahan UJ (1990) The agrin hypothesis. Cold Spring Harb Symp Quant Biol 55:407–418

    PubMed  CAS  Google Scholar 

  • Moe SE, Sorbo JG, Siogaard R, Zeuthen T, Ottersen OP, Holen T (2008) New isoforms of rat aquaporin-4. Genomics 91:367–377

    PubMed  Article  CAS  Google Scholar 

  • Neuhaus J (1990) Orthogonal arrays of particles in astroglial cells: quantitative analysis of their density, size, and correlation with intramembranous particles. Glia 3:241–251

    PubMed  Article  CAS  Google Scholar 

  • Nicchia GP, Cogotzi L, Rossi A, Basco D, Brancaccio A, Svelto M, Frigeri A (2008) Expression of multiple AQP4 pools in the plasma membrane and their association with the dystrophin complex. J Neurochem doi:10.1111/j.1471-4159.2008.05302.x

    PubMed  Google Scholar 

  • Nico B, Frigeri A, Nicchia GP, Quondamatteo F, Herken R, Errede M, Ribatti D, Svelto M, Roncali L (2001) Role of aquaporin-4 water channel in the development and integrity of the blood-brain barrier. J Cell Sci 114:1297–1307

    PubMed  CAS  Google Scholar 

  • Nielsen S, Nagelhus EA, Amiry-Moghaddam M, Bourque C, Agre P, Ottersen OP (1997) Specialized membrane domains for water transport in glial cells: high-resolution immunogold cytochemistry of aquaporin-4 in rat brain. J Neurosci 17:171–180

    PubMed  CAS  Google Scholar 

  • Noell S, Fallier-Becker P, Beyer C, Kröger S, Mack AF, Wolburg H (2007) Effects of agrin on the expression and distribution of the water channel protein aquaporin-4 and volume regulation in cultured astrocytes. Eur J Neurosci 26:2109–2118

    PubMed  Article  Google Scholar 

  • Rascher G, Fischmann A, Kröger S, Duffner F, Grote E-H, Wolburg H (2002) Extracellular matrix and the blood-brain barrier in glioblastoma multiforme: spatial segregation of tenascin and agrin. Acta Neuropathol 104:85–91

    PubMed  Article  CAS  Google Scholar 

  • Rash JE, Yasumura T, Hudson CS, Agre P, Nielsen S (1998) Direct immunogold labeling of aquaporin-4 in square arrays of astrocyte and ependymocyte plasma membranes in rat brain and spinal cord. Proc Natl Acad Sci USA 95:11981–11986

    PubMed  Article  CAS  Google Scholar 

  • Reichenbach A (1989) Attemt to classify glial cells by means of their process specialization using the rabbit retinal Müller cell as an example of cytotopographic specialization of glial cell. Glia 2:250–259

    PubMed  Article  CAS  Google Scholar 

  • Rohlmann A, Gocht A, Wolburg H (1992) Reactive astrocytes in myelin-deficient rat optic nerve reveal an altered distribution of orthogonal arrays of particles (OAP). Glia 5:259–268

    PubMed  Article  CAS  Google Scholar 

  • Saadoun S, Papadopoulos MC, Davies DC, Krishna S, Bell BA (2002) Aquaporin-4 expression is increased in oedematous human brain tumours. J Neurol Neurosurg Psychiatry 72:262–265

    PubMed  Article  CAS  Google Scholar 

  • Schmechel DE, Rakic P (1979) A Golgi study of radial glial cells in developing monkey telencephalon: morphogenesis and transformation into astrocytes. Anat Embryol 156:115–152

    PubMed  Article  CAS  Google Scholar 

  • Silberstein C, Bouley R, Huang Y, Fang P, Pastor-Soler N, Brown D, Van Hoek AN (2004) Membrane organization and function of M1 and M23 isoforms of aquaporin-4 in epithelial cells. Am J Physiol Renal Physiol 287:F501–F511

    PubMed  Article  CAS  Google Scholar 

  • Simard M, Nedergaard M (2004) The neurobiology of glia in the context of water and ion homeostasis. Neuroscience 129:877–896

    PubMed  Article  CAS  Google Scholar 

  • Smith MA, Hilgenberg LGW (2002) Agrin in the CNS: a protein in search of a function? NeuroReport 13:1485–1495

    PubMed  Article  CAS  Google Scholar 

  • Sorbo JG, Moe SE, Ottersen OP, Holen T (2008) The molecular composition of square arrays. Biochemistry 47:2631–2637

    PubMed  Article  CAS  Google Scholar 

  • Stone DM, Nikolics K (1995) Tissue- and age-specific expression patterns of alterantively spliced agrin mRNA transcripts in embryonic rat suggest novel developmental roles. J Neurosci 15:6767–6778

    PubMed  CAS  Google Scholar 

  • Verbavatz J-M, Ma T, Gobin R, Verkman AS (1997) Absence of orthogonal arrays in kidney, brain and muscle from transgenic knockout mice lacking water channel aquaporin-4. J Cell Sci 110:2855–2860

    PubMed  CAS  Google Scholar 

  • Voigt T (1989) Development of glial cells in the cerebral wall of ferrets: direct tracing of their transformation from radial glia into astrocytes. J Comp Neurol 289:74–88

    PubMed  Article  CAS  Google Scholar 

  • Warth A, Kröger S, Wolburg H (2004) Redistribution of aquaporin-4 in human glioblastoma correlates with loss of agrin immunoreactivity from brain capillary basal laminae. Acta Neuropathol 107:311–318

    PubMed  Article  CAS  Google Scholar 

  • Wolburg H (1995) Orthogonal arrays of intramembranous particles. A review with special reference to astrocytes. J Brain Res 36:239–258

    CAS  Google Scholar 

  • Wolburg H (2006) The endothelial frontier. In: Dermietzel R, Spray DC, Nedergaard M (eds) Blood-brain interfaces: from ontogeny to artificial barriers. Wiley-VCH, Weinheim, pp 77–107

    Google Scholar 

  • Wolburg H, Warth A, Noell S, Mack AF (2008) Brain aquaporins and the blood-brain barrier in health and disease. In: Ribatti D, Nico B (eds) Recent advances in angiogenesis in central nervous system. Transworld Research Network, Kerala, pp 79–104

    Google Scholar 

  • Wolburg H, Noell S, Mack A, Wolburg-Buchholz K, Fallier-Becker P (2009) Brain endothelial cells and the glio-vascular complex. Cell Tissue Res 335:75–96

    PubMed  Article  Google Scholar 

  • Yang B, Brown D, Verkman AS (1996) The mercurial insensitive water channel (AQP-4) forms orthogonal arrays in stably transfected Chinese hamster ovary cells. J Biol Chem 271:4577–4580

    PubMed  Article  CAS  Google Scholar 

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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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Affiliations

  1. Institute of Pathology, University of Tübingen, Liebermeisterstraße 8, 72076, Tübingen, Germany

    Susan Noell, Petra Fallier-Becker & Hartwig Wolburg

  2. Theodor Kocher Institute, University of Bern, CH-3012, Bern, Switzerland

    Urban Deutsch

  3. Institute of Anatomy, University of Tübingen, 72076, Tübingen, Germany

    Andreas F. Mack

Authors
  1. Susan Noell
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  2. Petra Fallier-Becker
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  3. Urban Deutsch
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  4. Andreas F. Mack
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  5. Hartwig Wolburg
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Corresponding author

Correspondence to Hartwig Wolburg.

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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

Keywords

  • Aquaporin-4
  • Astrocytes
  • Blood-brain barrier
  • Freeze-fracturing
  • Mouse (Agrin-null mutation; C57BL/6)