Limits...
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.

Show MeSH

Related in: MedlinePlus

Effect of chloranil, 2,3-dichloro-5,6-dimethyl-(1,4)benzoquinone and benzoquinone on ß-dystroglycan shedding and laminin-mediated clustering.A. Primary astrocytes were incubated for 4 h with 15 µM of active chemicals. Protein extracts were loaded (30 µg/lane) and analyzed for ß-DG expression levels by western blot analysis. Note the 31 kDa band corresponding to the cleaved form of ß-DG upon chloranil as well as 2,3-dichloro-5,6-dimethyl-(1,4)benzoquinone or benzoquinone treatment. The chloranil induced shedding of ß-dystroglycan is prevented by co-incubation of the cells with 15 µM of chloranil and 1 mM of the ROS scavenger, N-Acetylcysteine (NAC). B, C. Primary astrocytes were treated for 7 h with 20 nM laminin and chloranil, 2,3-dichloro-5,6-dimethyl-(1,4)benzoquinone or benzoquinone during the last 4 h. The concentration of the compounds varied from 2.5 to 100 µM. Clustered staining was quantified using the automated microscopy assay.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3049781&req=5

pone-0017559-g008: Effect of chloranil, 2,3-dichloro-5,6-dimethyl-(1,4)benzoquinone and benzoquinone on ß-dystroglycan shedding and laminin-mediated clustering.A. Primary astrocytes were incubated for 4 h with 15 µM of active chemicals. Protein extracts were loaded (30 µg/lane) and analyzed for ß-DG expression levels by western blot analysis. Note the 31 kDa band corresponding to the cleaved form of ß-DG upon chloranil as well as 2,3-dichloro-5,6-dimethyl-(1,4)benzoquinone or benzoquinone treatment. The chloranil induced shedding of ß-dystroglycan is prevented by co-incubation of the cells with 15 µM of chloranil and 1 mM of the ROS scavenger, N-Acetylcysteine (NAC). B, C. Primary astrocytes were treated for 7 h with 20 nM laminin and chloranil, 2,3-dichloro-5,6-dimethyl-(1,4)benzoquinone or benzoquinone during the last 4 h. The concentration of the compounds varied from 2.5 to 100 µM. Clustered staining was quantified using the automated microscopy assay.

Mentions: Chloranil is a general electron acceptor that has a role in radical ion formation and has been shown to be most effective at producingreactive oxygen species (ROS) by accepting electrons from oxygen [38]. Based on this property of chloranil, its reported inability to interact or regulate collagenase activity [39] and the fact that metalloproteinases can be activated by ROS, we hypothesized that choranil inducesß-DG shedding by producing ROS that in turn activate specific metalloproteinases. To test this hypothesis,astrocytes were incubated either with chloranil alone or chloranil plus the ROS scavenger, N-acetyl-cysteine (NAC). Interestingly, NAC completely inhibited the proteolytic cleavage of ß-DG by chloranil (Fig. 8A).


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, 2,3-dichloro-5,6-dimethyl-(1,4)benzoquinone and benzoquinone on ß-dystroglycan shedding and laminin-mediated clustering.A. Primary astrocytes were incubated for 4 h with 15 µM of active chemicals. Protein extracts were loaded (30 µg/lane) and analyzed for ß-DG expression levels by western blot analysis. Note the 31 kDa band corresponding to the cleaved form of ß-DG upon chloranil as well as 2,3-dichloro-5,6-dimethyl-(1,4)benzoquinone or benzoquinone treatment. The chloranil induced shedding of ß-dystroglycan is prevented by co-incubation of the cells with 15 µM of chloranil and 1 mM of the ROS scavenger, N-Acetylcysteine (NAC). B, C. Primary astrocytes were treated for 7 h with 20 nM laminin and chloranil, 2,3-dichloro-5,6-dimethyl-(1,4)benzoquinone or benzoquinone during the last 4 h. The concentration of the compounds varied from 2.5 to 100 µM. Clustered staining was quantified using the automated microscopy assay.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0017559-g008: Effect of chloranil, 2,3-dichloro-5,6-dimethyl-(1,4)benzoquinone and benzoquinone on ß-dystroglycan shedding and laminin-mediated clustering.A. Primary astrocytes were incubated for 4 h with 15 µM of active chemicals. Protein extracts were loaded (30 µg/lane) and analyzed for ß-DG expression levels by western blot analysis. Note the 31 kDa band corresponding to the cleaved form of ß-DG upon chloranil as well as 2,3-dichloro-5,6-dimethyl-(1,4)benzoquinone or benzoquinone treatment. The chloranil induced shedding of ß-dystroglycan is prevented by co-incubation of the cells with 15 µM of chloranil and 1 mM of the ROS scavenger, N-Acetylcysteine (NAC). B, C. Primary astrocytes were treated for 7 h with 20 nM laminin and chloranil, 2,3-dichloro-5,6-dimethyl-(1,4)benzoquinone or benzoquinone during the last 4 h. The concentration of the compounds varied from 2.5 to 100 µM. Clustered staining was quantified using the automated microscopy assay.
Mentions: Chloranil is a general electron acceptor that has a role in radical ion formation and has been shown to be most effective at producingreactive oxygen species (ROS) by accepting electrons from oxygen [38]. Based on this property of chloranil, its reported inability to interact or regulate collagenase activity [39] and the fact that metalloproteinases can be activated by ROS, we hypothesized that choranil inducesß-DG shedding by producing ROS that in turn activate specific metalloproteinases. To test this hypothesis,astrocytes were incubated either with chloranil alone or chloranil plus the ROS scavenger, N-acetyl-cysteine (NAC). Interestingly, NAC completely inhibited the proteolytic cleavage of ß-DG by chloranil (Fig. 8A).

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