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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

Identification of modulators of ß-dystroglycan clustering using a high throughput screen of a library containing 3,594 drugs.A, B. Duplicate experiments (red and green)from three 96-well plates corresponding to approximately 200 drugs are represented. Drugs inducing a change greater than 1.6 fold in ß-DG clustering in laminin-treated compared to untreated astrocytes in both experiments (as circled in black) were considered effective and used for subsequent testing. C, D. Normalized fold differences in the number and area of the clusters plotted for each of the 3,594 compounds tested.
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pone-0017559-g002: Identification of modulators of ß-dystroglycan clustering using a high throughput screen of a library containing 3,594 drugs.A, B. Duplicate experiments (red and green)from three 96-well plates corresponding to approximately 200 drugs are represented. Drugs inducing a change greater than 1.6 fold in ß-DG clustering in laminin-treated compared to untreated astrocytes in both experiments (as circled in black) were considered effective and used for subsequent testing. C, D. Normalized fold differences in the number and area of the clusters plotted for each of the 3,594 compounds tested.

Mentions: A collection of 3,584 drugs and pharmaceutically active chemicals was tested at a concentration of 15 µM for the final 4 h of the 7 h laminin incubation. In addition to automatically reporting the number of clusters and their total area, the number of cells (i.e., number of nuclei) and the nuclear area were simultaneously measured. Chemicals causing 80% reduction in cell number were considered cytotoxic and were eliminated from our analysis. Compounds causing over 1.6-fold increase or decrease in the clustering ofß-DG without causing cell death were considered as active (Fig. 2 C and D). Examples of these compounds are illustrated in Figure 2A and Bin which data compiled from three 96-well plates corresponding to approximately 200 compounds are shown. Twelve active chemicals were initially identified as inhibitors of the clustering,but subsequent testing by dose-response proved that only 6 of these were true inhibitors inducing a reduction of ß-DG clustering ranging from 2.2 to 17.1-fold (Fig. 3 and data not shown). To our knowledge, none of these 6 compounds have been previously reported to reduce laminin-DG interaction or laminin-induced clustering of the DGC.


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)

Identification of modulators of ß-dystroglycan clustering using a high throughput screen of a library containing 3,594 drugs.A, B. Duplicate experiments (red and green)from three 96-well plates corresponding to approximately 200 drugs are represented. Drugs inducing a change greater than 1.6 fold in ß-DG clustering in laminin-treated compared to untreated astrocytes in both experiments (as circled in black) were considered effective and used for subsequent testing. C, D. Normalized fold differences in the number and area of the clusters plotted for each of the 3,594 compounds tested.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0017559-g002: Identification of modulators of ß-dystroglycan clustering using a high throughput screen of a library containing 3,594 drugs.A, B. Duplicate experiments (red and green)from three 96-well plates corresponding to approximately 200 drugs are represented. Drugs inducing a change greater than 1.6 fold in ß-DG clustering in laminin-treated compared to untreated astrocytes in both experiments (as circled in black) were considered effective and used for subsequent testing. C, D. Normalized fold differences in the number and area of the clusters plotted for each of the 3,594 compounds tested.
Mentions: A collection of 3,584 drugs and pharmaceutically active chemicals was tested at a concentration of 15 µM for the final 4 h of the 7 h laminin incubation. In addition to automatically reporting the number of clusters and their total area, the number of cells (i.e., number of nuclei) and the nuclear area were simultaneously measured. Chemicals causing 80% reduction in cell number were considered cytotoxic and were eliminated from our analysis. Compounds causing over 1.6-fold increase or decrease in the clustering ofß-DG without causing cell death were considered as active (Fig. 2 C and D). Examples of these compounds are illustrated in Figure 2A and Bin which data compiled from three 96-well plates corresponding to approximately 200 compounds are shown. Twelve active chemicals were initially identified as inhibitors of the clustering,but subsequent testing by dose-response proved that only 6 of these were true inhibitors inducing a reduction of ß-DG clustering ranging from 2.2 to 17.1-fold (Fig. 3 and data not shown). To our knowledge, none of these 6 compounds have been previously reported to reduce laminin-DG interaction or laminin-induced clustering of the DGC.

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