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A high throughput screen identifies chemical modulators of the laminin-induced clustering of dystroglycan and aquaporin-4 in primary astrocytes.

Noël G, Stevenson S, Moukhles H - PLoS ONE (2011)

Bottom Line: In the present study we used primary rat astrocyte cultures to screen a library of >3,500 chemicals and identified 6 drugs that inhibit the laminin-induced clustering of dystroglycan and AQP4.Detailed analysis of the inhibitory drug, chloranil, revealed that its inhibition of the clustering is due to the metalloproteinase-2-mediated ß-dystroglycan shedding and subsequent loss of laminin interaction with dystroglycan.Furthermore, chemical variants of chloranil induced a similar effect on ß-dystroglycan and this was prevented by the antioxidant N-acetylcysteine.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, Canada.

ABSTRACT

Background: Aquaporin-4 (AQP4) constitutes the principal water channel in the brain and is clustered at the perivascular astrocyte endfeet. This specific distribution of AQP4 plays a major role in maintaining water homeostasis in the brain. A growing body of evidence points to a role of the dystroglycan complex and its interaction with perivascular laminin in the clustering of AQP4 at perivascular astrocyte endfeet. Indeed, mice lacking components of this complex or in which laminin-dystroglycan interaction is disrupted show a delayed onset of brain edema due to a redistribution of AQP4 away from astrocyte endfeet. It is therefore important to identify inhibitory drugs of laminin-dependent AQP4 clustering which may prevent or reduce brain edema.

Methodology/principal findings: In the present study we used primary rat astrocyte cultures to screen a library of >3,500 chemicals and identified 6 drugs that inhibit the laminin-induced clustering of dystroglycan and AQP4. Detailed analysis of the inhibitory drug, chloranil, revealed that its inhibition of the clustering is due to the metalloproteinase-2-mediated ß-dystroglycan shedding and subsequent loss of laminin interaction with dystroglycan. Furthermore, chemical variants of chloranil induced a similar effect on ß-dystroglycan and this was prevented by the antioxidant N-acetylcysteine.

Conclusion/significance: These findings reveal the mechanism of action of chloranil in preventing the laminin-induced clustering of dystroglycan and AQP4 and validate the use of high-throughput screening as a tool to identify drugs that modulate AQP4 clustering and that could be tested in models of brain edema.

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Related in: MedlinePlus

Effect of chloranil and flunarizine on astrocyte survival and ß-dystroglycan, and AQP4 expression.A. Primary astrocytes were incubated for 4 h with 15 µM of active chemicals. Extracted proteins were loaded (30 µg/lane) and analyzed for ß-DG, syntrophin and AQP4 expression levels by western blot analysis. Note the 31 kDa band under the 42 kDa band corresponding to the cleaved form of ß-dystroglycan upon chloranil treatment. B. Primary astrocytes were incubated for 4 h with different concentrations of the active chemicals. Chemicals and media were washed away and the cells were assayed for cell viability by MTT assay.
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pone-0017559-g005: Effect of chloranil and flunarizine on astrocyte survival and ß-dystroglycan, and AQP4 expression.A. Primary astrocytes were incubated for 4 h with 15 µM of active chemicals. Extracted proteins were loaded (30 µg/lane) and analyzed for ß-DG, syntrophin and AQP4 expression levels by western blot analysis. Note the 31 kDa band under the 42 kDa band corresponding to the cleaved form of ß-dystroglycan upon chloranil treatment. B. Primary astrocytes were incubated for 4 h with different concentrations of the active chemicals. Chemicals and media were washed away and the cells were assayed for cell viability by MTT assay.

Mentions: To ensure that the reduction in ß-DG and AQP4 clustering was not due to either cytotoxicity or reduction in ß-DG or AQP4 expression levels, we assessed cell viability using the MTT assay andß-DG and AQP4levels by immunoblotting. Figure 5 shows that while ß-DGexpression level remains unchanged in the presence of flunarizine that ofAQP4is slightly decreased. Interestingly, chloranil causes the cell surface cleavage of full length 43 kDa ß-DG and consequent formation of the 31 kDa fragment of ß-DG (Fig. 5A). The size of this fragment is consistent with the cleavage of the short extracellular domain of ß-DG that has been reported to be mediated by metalloproteinases. We next investigated the effectof increasing concentrations of chloranil and flunarizine on astrocyte survival. Four hours following the treatment with chloranil and flunarizine, we subjected the astrocytes to the MTT cell viability assay and found that chloranil caused a slight reduction in the total number of actrocytes but only at a very high concentration (100 µM; Fig. 5B). These results demonstrate that the inhibition of ß-DG and AQP4 clustering observed with <100 µM chloranil is not due to adverse effects on cell survival (Fig. 3 and 4).


A high throughput screen identifies chemical modulators of the laminin-induced clustering of dystroglycan and aquaporin-4 in primary astrocytes.

Noël G, Stevenson S, Moukhles H - PLoS ONE (2011)

Effect of chloranil and flunarizine on astrocyte survival and ß-dystroglycan, and AQP4 expression.A. Primary astrocytes were incubated for 4 h with 15 µM of active chemicals. Extracted proteins were loaded (30 µg/lane) and analyzed for ß-DG, syntrophin and AQP4 expression levels by western blot analysis. Note the 31 kDa band under the 42 kDa band corresponding to the cleaved form of ß-dystroglycan upon chloranil treatment. B. Primary astrocytes were incubated for 4 h with different concentrations of the active chemicals. Chemicals and media were washed away and the cells were assayed for cell viability by MTT assay.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3049781&req=5

pone-0017559-g005: Effect of chloranil and flunarizine on astrocyte survival and ß-dystroglycan, and AQP4 expression.A. Primary astrocytes were incubated for 4 h with 15 µM of active chemicals. Extracted proteins were loaded (30 µg/lane) and analyzed for ß-DG, syntrophin and AQP4 expression levels by western blot analysis. Note the 31 kDa band under the 42 kDa band corresponding to the cleaved form of ß-dystroglycan upon chloranil treatment. B. Primary astrocytes were incubated for 4 h with different concentrations of the active chemicals. Chemicals and media were washed away and the cells were assayed for cell viability by MTT assay.
Mentions: To ensure that the reduction in ß-DG and AQP4 clustering was not due to either cytotoxicity or reduction in ß-DG or AQP4 expression levels, we assessed cell viability using the MTT assay andß-DG and AQP4levels by immunoblotting. Figure 5 shows that while ß-DGexpression level remains unchanged in the presence of flunarizine that ofAQP4is slightly decreased. Interestingly, chloranil causes the cell surface cleavage of full length 43 kDa ß-DG and consequent formation of the 31 kDa fragment of ß-DG (Fig. 5A). The size of this fragment is consistent with the cleavage of the short extracellular domain of ß-DG that has been reported to be mediated by metalloproteinases. We next investigated the effectof increasing concentrations of chloranil and flunarizine on astrocyte survival. Four hours following the treatment with chloranil and flunarizine, we subjected the astrocytes to the MTT cell viability assay and found that chloranil caused a slight reduction in the total number of actrocytes but only at a very high concentration (100 µM; Fig. 5B). These results demonstrate that the inhibition of ß-DG and AQP4 clustering observed with <100 µM chloranil is not due to adverse effects on cell survival (Fig. 3 and 4).

Bottom Line: In the present study we used primary rat astrocyte cultures to screen a library of >3,500 chemicals and identified 6 drugs that inhibit the laminin-induced clustering of dystroglycan and AQP4.Detailed analysis of the inhibitory drug, chloranil, revealed that its inhibition of the clustering is due to the metalloproteinase-2-mediated ß-dystroglycan shedding and subsequent loss of laminin interaction with dystroglycan.Furthermore, chemical variants of chloranil induced a similar effect on ß-dystroglycan and this was prevented by the antioxidant N-acetylcysteine.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, Canada.

ABSTRACT

Background: Aquaporin-4 (AQP4) constitutes the principal water channel in the brain and is clustered at the perivascular astrocyte endfeet. This specific distribution of AQP4 plays a major role in maintaining water homeostasis in the brain. A growing body of evidence points to a role of the dystroglycan complex and its interaction with perivascular laminin in the clustering of AQP4 at perivascular astrocyte endfeet. Indeed, mice lacking components of this complex or in which laminin-dystroglycan interaction is disrupted show a delayed onset of brain edema due to a redistribution of AQP4 away from astrocyte endfeet. It is therefore important to identify inhibitory drugs of laminin-dependent AQP4 clustering which may prevent or reduce brain edema.

Methodology/principal findings: In the present study we used primary rat astrocyte cultures to screen a library of >3,500 chemicals and identified 6 drugs that inhibit the laminin-induced clustering of dystroglycan and AQP4. Detailed analysis of the inhibitory drug, chloranil, revealed that its inhibition of the clustering is due to the metalloproteinase-2-mediated ß-dystroglycan shedding and subsequent loss of laminin interaction with dystroglycan. Furthermore, chemical variants of chloranil induced a similar effect on ß-dystroglycan and this was prevented by the antioxidant N-acetylcysteine.

Conclusion/significance: These findings reveal the mechanism of action of chloranil in preventing the laminin-induced clustering of dystroglycan and AQP4 and validate the use of high-throughput screening as a tool to identify drugs that modulate AQP4 clustering and that could be tested in models of brain edema.

Show MeSH
Related in: MedlinePlus