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(The FASEB Journal. 2006;20:2423-2424.)
© 2006 FASEB

Abnormalities in the pattern of AQP4 immunoreactivity

Jérôme Badaut1

Neurosurgery Research Group, Centre Hospitalier Universitaire Vaudois, Pav 3, CH-1011 Lausanne, Switzerland

1Correspondence: Neurosurgery Research Group, Centre Hospitalier Universitaire Vaudois, Pav 3, CH-1011 Lausanne, Switzerland. E-mail: jerome.badaut{at}chuv.ch

With regard to the paper by Meshorer et al. (1) , the authors address an interesting question concerning long-lasting consequences of cholinergic imbalance. To address this question, the authors used transgenic mice (TgS) with a neuronal overexpression of synaptic acetylcholinesterase, in which they observed a significant decrease in the baseline of the apparent water diffusion coefficient (ADC) measured by diffusion-weighted images (DWI). Molecular mechanisms were explored by cDNA microarrays, which showed significant modification in the expression level of transporters and ionic channels. The authors have pointed out a significant increase in the mRNA of AQP4 associated with an increase of AQP4 immunoreactivity (IR). AQP4 is a water channel and is supposed to be associated with water movements in the brain (2 , 3) . This direct observation of a relation between an increase in AQP4 and a decrease of the ADC in transgenic is very interesting and novel.

The authors compared AQP4 immunolabeling in TgS-transgenic mice to labeling in wild-type mice. Surprisingly, they observed AQP4 staining in cell bodies in the cortex and hippocampus of both mice, and the level of staining is higher in transgenic mice than in wild-type mice (1 ; see Fig.7, p. 918). This pattern of AQP4 labeling in the brain is not in agreement with previous AQP4 immunohistochemistry carried out by several groups and in different species (2 3 4 5 6 7 8 9 10) . Usually, AQP4 staining is observed on the astrocyte endfeet in proximity to the cerebral blood vessels and on the glia limitans (2 , 11 , 12) . This polarization of AQP4 on astrocyte endfeet is explained by the association between AQP4 and several anchorage proteins such as syntrophin and dystrophin (2 , 13 14 15) .

Meshorer et al. (1) used a monoclonal antibody produced by SEROTEC at a concentration of 1/100 on fixed mouse brain tissue. We have tested and compared AQP4 labeling obtained with the SEROTEC monoclonal AQP4-antibody (1/100) to a polyclonal AQP4-antibody (Chemicon; 1/100) on brain slices from ICR-CD1 mice perfused with 4% paraformaldehyde. The immunohistochemical procedure used (16 , 17) differs from that Meshorer et al. in that animals were perfused with 4% paraformaldehyde.

Using the antibody from SEROTEC, we observed AQP4-IR on the glia limitans (Fig. 1A , arrowheads) and on ependymal cells (arrows, Fig. 1A ). At higher magnification, a perivascular staining was visible, in agreement with previous reports (2 3 4 5 6 7 8 9 10) (Fig. 1B ). Double immunolabeling with the SEROTEC monoclonal anti-AQP4 and the polyclonal anti-AQP4. revealed that the labeling from both antibodies is colocalized (Fig. 1 C1, C2, C3, arrowheads). In our conditions therefore, AQP4 staining obtained with the SEROTEC monoclonal AQP4-antibody is on astrocyte endfeet, agreeing with previous reports (2 3 4 5 6 7 8 9 10) .


Figure 1
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  		Figure 1. A) AQP4 immunoreactivity in mouse brain using the SEROTEC monoclonal antibody against AQP4. AQP4-ir is observed on the glia limitans (arrowheads) and on ependymal cells (arrows). B) AQP4 staining around blood vessels in the cortex. C1) Double immunolabeling with monoclonal AQP4 and polyclonal AQP4 in the hippocampus. Superposition of C2 and C3 images shows colocalization of both antibodies (arrowheads). C2) AQP4 staining obtained with the SEROTEC monoclonal antibody (mAQP4,). C3) AQP4staining obtained with the CHEMICON polyclonal antibody (pAQP4, from). Arrowheads show the colocalization of both markers. B, C) Bar = 40 µm.

We believe that AQP4 labeling in the cell bodies of cells of the cortex and hippocampus (1 ; see Fig. 7, p. 918) is possibly artifactual and could be due to the procedure of fixation of the brain. Possibly, a classical perfusion with 4% paraformaldchyde could improve the labeling for AQP4.

REFERENCES

  1. Meshorer, E., Biton, I. E., Ben-Shaul, Y., Ben-Ari, S., Assaf, Y., Soreq, H., Cohen, Y. (2005) Chronic cholinergic imbalances promote brain diffusion and transport abnormalities. FASEB J. 19,910-922[Abstract/Free Full Text]
  2. Amiry-Moghaddam, M., Ottersen, O. P. (2003) The molecular basis of water transport in the brain. Nat. Rev. Neurosci. 4,991-1001[Medline]
  3. Badaut, J., Lasbennes, F., Magistretti, P. J., Regli, L. (2002) Aquaporins in brain: distribution, physiology, and pathophysiology. J. Cereb. Blood Flow Metab. 22,367-378[CrossRef][Medline]
  4. Badaut, J., Brunet, J. F., Grollimund, L., Hamou, M. F., Magistretti, P. J., Villemure, J. G., Regli, L. (2003) Aquaporin 1 and 4 expression after human acute subarachnoid hemorrhage. Acta Neurochir. Suppl. 86,495-498[Medline]
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  8. Manley, G. T., Fujimura, M., Ma, T., Noshita, N., Filiz, F., Bollen, A. W., Chan, P., Verkman, A. S. (2000) Aquaporin-4 deletion in mice reduces brain edema after acute water intoxication and ischemic stroke. Nat. Med. 6,159-163[CrossRef][Medline]
  9. Eid, T., Lee, T. S., Thomas, M. J., Amiry-Moghaddam, M., Bjornsen, L. P., Spencer, D. D., Agre, P., Ottersen, O. P., de Lanerolle, N. C. (2005) Loss of perivascular aquaporin 4 may underlie deficient water and K+ homeostasis in the human epileptogenic hippocampus. Proc. Natl. Acad. Sci. U. S. A. 102,1193-1198[Abstract/Free Full Text]
  10. Amiry-Moghaddam, M., Otsuka, T., Hurn, P. D., Traystman, R. J., Haug, F.-M., Froehner, S. C., Adams, M. E., Neely, J. D., Agre, P., Ottersen, O. P., Bhardwaj, A. (2003) An alpha-syntrophin-dependent pool of AQP4 in astroglial end-feet confers bidirectional water flow between blood and brain. Proc. Natl. Acad. Sci. U. S. A. 100,2106-2111[Abstract/Free Full Text]
  11. Badaut, J., Verbavatz, J. M., Freund-Mercier, M. J., Lasbennes, F. (2000) Presence of aquaporin-4 and muscarinic receptors in astrocytes and ependymal cells in rat brain: a clue to a common function?. Neurosci. Lett. 292,75-78[CrossRef][Medline]
  12. Badaut, J., Hirt, L., Granziera, C., Bogousslavsky, J., Magistretti, P. J., Regli, L. (2001) Astrocyte-specific expression of aquaporin-9 in mouse brain is increased after transient focal cerebral ischemia. J. Cereb. Blood Flow Metab. 21,477-482[Medline]
  13. Amiry-Moghaddam, M., Williamson, A., Palomba, M., Eid, T., de Lanerolle, N. C., Nagelhus, E. A., Adams, M. E., Froehner, S. C., Agre, P., Ottersen, O. P. (2003) Delayed K+ clearance associated with aquaporin-4 mislocalization: phenotypic defects in brains of alpha-syntrophin-null mice. Proc. Natl. Acad. Sci. U. S. A. 100,13615-13620[Abstract/Free Full Text]
  14. Vajda, Z., Pedersen, M., Fuchtbauer, E. M., Wertz, K., Stodkilde-Jorgensen, H., Sulyok, E., Doczi, T., Neely, J. D., Agre, P., Frokiaer, J., Nielsen, S. (2002) Delayed onset of brain edema and mislocalization of aquaporin-4 in dystrophin-null transgenic mice. Proc. Natl. Acad. Sci. U. S. A. 99,13131-13136[Abstract/Free Full Text]
  15. Vajda, Z., Pedersen, M., Doczi, T., Sulyok, E., Nielsen, S. (2004) Studies of mdx mice. Neuroscience 129,991-996[CrossRef]
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  17. Marlise de Castro Ribeiro, L. H., Julien Bogousslavsky, L. R., Badaut, J. (2006) Time course of aquaporin expression after transient focal cerebral ischemia in mice. J. Neurosci. Res. 83,1231-1240[CrossRef][Medline]

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