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Requirement of functional ryanodine receptor type 3 for astrocyte migration ¹

MARINA MATYASH², VITALI MATYASH², CHRISTIANE NOLTE, VINCENZO SORRENTINO^*, and HELMUT KETTENMANN³

Max-Delbrück-Center for Molecular Medicine, Cellular Neuroscience, D-13092 Berlin, Germany;
^* Molecular Medicine Section, Department of Neuroscience, University of Siena, Italy; and
Instituto San Raffaele, I-20132 Milano, Italy

³Correspondence: Cellular Neuroscience, Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Strasse 10, D-13092 Berlin, FRG. E-mail: hketten{at}mdc-berlin.de

SPECIFIC AIMS

Astrocyte motility plays an important role in the response of the brain to injury and during regeneration. The objective of the present study was to examine the expression of ryanodine receptors, intracellular Ca²⁺ release channels, in astrocytes and their involvement in controlling migration and chemotaxis in this highly abundant cell type of the central nervous system.

PRINCIPAL FINDINGS

1. Astrocytes in culture and acutely isolated from the brain express ryanodine receptor type 3 gene
To determine which mRNAs of ryanodine receptors (ryrs) types are expressed by astrocytes, we isolated RNA from astrocytic cultures and performed RT-PCR analysis. Only ryr3 expression was detected, but not ryr1 or ryr2 (Fig. 1 ). To confirm the specificity of ryr3 RT-PCR-generated products, these were subjected to the restriction enzyme digestion. As controls, we used mRNA from whole brain and muscle. Immunolabeling with RyR3-antibodies showed a labeling which was distributed all over the cytoplasm, forming a fine network, similar to the pattern revealed by labeling of endoplasmic reticulum.

View larger version (43K): [in this window] [in a new window]   Figure 1. Expression of ryanodine receptor 3 gene by astrocytes. A) RNA from mouse brain (br) and from cultured mouse astrocytes (astr) was reversely transcribed and cDNA was amplified by PCR using ryrs gene-specific primers, forward 1 and reversed 1 (F1/R1). All three ryrs were expressed in the brain, however only ryr3 was expressed in cultured astrocytes (band corresponds to 540 bp). M, molecular weight marker. B) Restriction digestion with Bgl II of the ryr3 PCR-products. Agarose gel shows the 540 bp band (uncut, left) and the 320 bp and 220 bp DNA fragments after incubation with Bgl II (right). C) The schematic drawing illustrates the localization of ryrs gene-specific primers within cDNA template. For conventional PCR, primer pairs F1/R1 or F1/R2 were used, for nested-PCR a sample was amplified with the F1/R1 primer pair, then an aliquot of the reaction was reamplified with F1/R2 primer pair. D) Phase contrast image of acutely isolated cells from the EGFP/GFAP transgenic mouse and corresponding picture of EGFP fluorescence (E). The arrowhead points to the cell identified as astrocyte due to its EGFP label. Bar: 10 µm. F) cDNA from EGFP-positive acutely isolated astrocytes (EGFP-astr) was amplified by nested-PCR, while cDNA derived from cultured astrocytes (astr) was amplified with F1/R2 primer pair by conventional PCR. PCR products only for RyR3 were detected; bands correspond to 307 bp.

To test for the expression of RyRs in brain astrocytes in situ, we used astrocytes acutely isolated from the mouse brain. We obtained a purified population of astrocytes by sorting brain cells from a transgenic animal in which all astrocytes express the enhanced green fluorescence protein. Only ryr3 was expressed in acutely isolated astrocytes.

2. Ryanodine receptor activation triggers an elevation of intracellular Ca²⁺ in cultured and freshly isolated astrocytes
To test for the presence of functional ryanodine receptors in astrocytes, we examined the effect of ryanodine receptor agonists on the level of cytoplasmic Ca²⁺ by imaging with the Ca²⁺-sensitive dye Fura-2/AM. The ryanodine receptor agonist, 4-chloro-m-cresol (4-CmC), elicited Ca²⁺ responses in all investigated astrocytes. The EC₅₀ was determined at 1.5 mM, while the response showed a plateau at concentrations exceeding 2 mM. The blockade of 4-CmC responses by the high ryanodine concentration further substantiated that 4-CmC activates ryanodine receptors in astrocytes. Acutely isolated astrocytes responded to 4-CmC with an increase in intracellular Ca²⁺ level, indicating that astrocytes in the brain express functional ryanodine receptors.

3. Astrocyte migration is impaired after pharmacological blockade of ryanodine receptors
We used two in vitro assays, a wound healing model and a chemotaxis assay, to study mechanisms which control astrocyte motility. We found that antagonizing concentrations of ryanodine impair the ability of astrocytes to migrate into a cell-free area. Differences between control and ryanodine-treated cultures became clearly visible after 10 h post injury. This observation indicates that the migratory activity of astrocytes is impaired, thus implying a role of ryanodine receptors in regulation of astrocyte migration. By varying the time of ryanodine application we found the initial phase is not a critical period, but that RyRs must be functional during the entire period of cell migration.

4. Astrocytes from RyR3 knockout mice display a reduced migratory activity
We studied the migration of astrocytes isolated from RyR3 knockout mice (RyR3^-/-), in which the RyR3 gene was disrupted thus generating a nonfunctional receptor. In both motility assays, the migratory ability of RyR3^-/- astrocytes was strongly impaired vs. wild-type cells. Moreover, 200 µM ryanodine did not have any inhibitory effect on the astrocyte migration from the RyR3^-/- mice (Fig. 2 ). We conclude that functional ryanodine receptors are required for normal astrocyte motility.

View larger version (73K): [in this window] [in a new window]   Figure 2. Scar cell-free areas remain larger in RyR3 knockout astrocyte monolayers than in wild-type astrocyte monolayers. The confluent monolayer of astrocytes was scratched. Cells were fixed and stained with fluorescent dye DiOC6(3). Images of fixed size (180x103 µm2) were taken. Control indicates cell growth after wounding in normal culture medium, whereas ryanodine indicates that medium was supplemented with 200 µM drug. Images containing the injured area are shown immediately (A, D) or 20 h after wounding (B, C, E, F), either for astrocytes isolated from wild-type (RyR3+/+) (A–C) or from RyR3 knockout (RyR-/-) mice (D–F). Bar: 100 µm. G) Scar cell-free area was measured as described. Cell-free area in wild-type or RyR3-/- astrocyte monolayers measured immediately or 20 h after wounding. Cells were either grown in normal medium or in medium supplemented with 200 µM ryanodine. Bars represent the mean ± SD of cell-free areas from four independent experiments. * P < 0.001, compare to wild-type, ** P = 0.3, compare to RyR3 knockout cells without ryanodine.

5. RyR3 control astrocyte motility but not chemotaxis
To test whether ryanodine receptors also influence chemotaxis, we used the chemokine MIP-1 which is a well established chemoattractant for astrocytes. While ryanodine reduced the basal motility, it did not affect the MIP-1-stimulated chemotactic response. MIP-1 increased the number of transmigrating cells to 30% in control and to 25% in the presence of ryanodine. We conclude that ryanodine receptors are thus not a prerequisite for the chemotactic response induced by MIP-1.

CONCLUSIONS

In the present study, we provide evidence that astrocytes express only one member of the ryanodine receptor family, namely RyR3. The message for ryr3 was derived from astrocytes: (1) the astrocytic cultures were, with a low percentage, contaminated by microglial cells or fibroblasts, however, fibroblasts have been described to express only ryr1, and we did not detect ryr3 expression in purified microglial cells. (2) In additional experiments we harvested GFAP-positive astrocytes acutely isolated from the brain of transgenic mouse and performed RT-PCR analyses. In the brain, all three RyR receptor types are expressed. Considering the abundance of astrocytes in the brain tissue, they may represent the major source for RyR3 in the brain. Since astrocytes can be easily isolated from the brain and kept in culture, these cells are a convenient model to assess the biological role of ryanodine receptor 3.

In the adult brain astrocyte migration occurs in response to brain trauma. Astrocytes migrate into the damaged region of the brain and subsequently proliferate in the brain tissue surrounding the damaged region. We have employed the cell monolayer wound healing model, an established in vitro system to test for involvement of ryanodine receptors in astrocyte migration. We conclude that the presence of functional ryanodine receptors is important for cell locomotion: (1) ryanodine at 200 µM strongly impaired astrocyte migration. (2) In RyR3 knockout mice astrocyte migration is strongly decreased compared with wild-type cells and it is not affected by blocking concentration of ryanodine. These results were confirmed using the Boyden chamber, a model for chemotaxis and motility. In contrast, functional ryanodine receptors are not a prerequisite for MIP-1-induced chemotaxis. This indicates that RyR3 is important for controlling motile activity, but not the chemotaxis behavior of astrocytes (Fig. 3 ).

View larger version (11K): [in this window] [in a new window]   Figure 3.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0380fje; to cite this article, use FASEB J. (November 14, 2001) 10.1096/fj.01-0380fje

² Present address: Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, D-01307, Dresden, Germany.

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This Article Full Text (PDF) All Versions of this Article: 16/1/84 01-0380fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by MATYASH, M. Articles by KETTENMANN, H. Search for Related Content PubMed PubMed Citation Articles by MATYASH, M. Articles by KETTENMANN, H.