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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 on the laminin-induced co-clustering of ß-DG and AQP4.Primary astrocytes were treated for 7 h with 20 nM laminin and15 µM chloranil during the last 4 h. The concentration of chloranil varied from 0 (A,B,C), 6(D,E,F), 12(G,H,I), 25 (J,K,L), 50 (M,N, O) to 100 µM (P,Q,R). The cells were fixed and labelled for μ-DG (A, D, G, J, M and P) and AQP4 (B, E, H, K, N and Q). Clustered staining was quantified using confocal microscopy. Scale bar, 30 µm.
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pone-0017559-g004: Effect of chloranil on the laminin-induced co-clustering of ß-DG and AQP4.Primary astrocytes were treated for 7 h with 20 nM laminin and15 µM chloranil during the last 4 h. The concentration of chloranil varied from 0 (A,B,C), 6(D,E,F), 12(G,H,I), 25 (J,K,L), 50 (M,N, O) to 100 µM (P,Q,R). The cells were fixed and labelled for μ-DG (A, D, G, J, M and P) and AQP4 (B, E, H, K, N and Q). Clustered staining was quantified using confocal microscopy. Scale bar, 30 µm.

Mentions: To assess whether AQP4 clustering is also reduced in the presence of chloranil and flunarizine, ß-DG and AQP4 clusters were examined at higher resolution by laser confocal microscopy. The data show that chloranilsignificantly inhibits the laminin-induced AQP4 clustering and as for ß-DG, this inhibition is dose-dependent (Fig. 4). Similar results were found for flunarizine (data not shown).


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 on the laminin-induced co-clustering of ß-DG and AQP4.Primary astrocytes were treated for 7 h with 20 nM laminin and15 µM chloranil during the last 4 h. The concentration of chloranil varied from 0 (A,B,C), 6(D,E,F), 12(G,H,I), 25 (J,K,L), 50 (M,N, O) to 100 µM (P,Q,R). The cells were fixed and labelled for μ-DG (A, D, G, J, M and P) and AQP4 (B, E, H, K, N and Q). Clustered staining was quantified using confocal microscopy. Scale bar, 30 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0017559-g004: Effect of chloranil on the laminin-induced co-clustering of ß-DG and AQP4.Primary astrocytes were treated for 7 h with 20 nM laminin and15 µM chloranil during the last 4 h. The concentration of chloranil varied from 0 (A,B,C), 6(D,E,F), 12(G,H,I), 25 (J,K,L), 50 (M,N, O) to 100 µM (P,Q,R). The cells were fixed and labelled for μ-DG (A, D, G, J, M and P) and AQP4 (B, E, H, K, N and Q). Clustered staining was quantified using confocal microscopy. Scale bar, 30 µm.
Mentions: To assess whether AQP4 clustering is also reduced in the presence of chloranil and flunarizine, ß-DG and AQP4 clusters were examined at higher resolution by laser confocal microscopy. The data show that chloranilsignificantly inhibits the laminin-induced AQP4 clustering and as for ß-DG, this inhibition is dose-dependent (Fig. 4). Similar results were found for flunarizine (data not shown).

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