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Dystroglycan controls dendritic morphogenesis of hippocampal neurons in vitro.

Bijata M, Wlodarczyk J, Figiel I - Front Cell Neurosci (2015)

Bottom Line: The structural changes were associated with activation of the guanosine triphosphatase Cdc42.This effect was abolished in neurons that overexpressed a β-DG mutant that was defective in MMP-9-mediated cleavage.Altogether, our results indicate that DG controls dendritic arborization in vitro in MMP-9-dependent manner.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biophysics, Department of Molecular and Cellular Neurobiology, Nencki Institute of Experimental Biology Warsaw, Poland.

ABSTRACT
Dendritic outgrowth and arborization are important for establishing neural circuit formation. To date, little information exists about the involvement of the extracellular matrix (ECM) and its cellular receptors in these processes. In our studies, we focus on the role of dystroglycan (DG), a cell adhesion molecule that links ECM components to the actin cytoskeleton, in dendritic development and branching. Using a lentiviral vector to deliver short-hairpin RNA (shRNA) that specifically silences DG in cultured hippocampal neurons, we found that DG knockdown exerted an inhibitory effect on dendritic tree growth and arborization. The structural changes were associated with activation of the guanosine triphosphatase Cdc42. The overexpression of DG promoted dendritic length and branching. Furthermore, exposure of the cultures to autoactivating matrix metalloproteinase-9 (aaMMP-9), a β-DG-cleaving protease, decreased the complexity of dendritic arbors. This effect was abolished in neurons that overexpressed a β-DG mutant that was defective in MMP-9-mediated cleavage. Altogether, our results indicate that DG controls dendritic arborization in vitro in MMP-9-dependent manner.

No MeSH data available.


Related in: MedlinePlus

Matrix metalloproteinase-9 dependent cleavage of β-DG is important for dendritic development. (A) Immunoblot analysis of β-DG and GFP expression in protein lysates from HEK 293 cells transfected with either OE β-DG-GFP (WT) or OE β-DG-MUT-GFP (MUT) plasmids and then treated with aaMMP-9. β-actin served as a loading control. (B) Representative images of neurons used for the morphometric analysis. The cells were co-transfected with an RFP-coding vector to visualize the exact morphology. (C) Sholl analysis of hippocampal neurons treated as indicated (ncell = 50 ± 4). In control cultures (without aaMMP-9), no statistically significant differences were found between DG-overexpressed neurons (transfected with either OE DG-GFP or OE β-DG-GFP) and neurons transfected with the OE β-DG-MUT-GFP vector. In cultures treated with aaMMP-9, at distances of 30–140 μm from the cell body, the differences between DG-overexpressed neurons (OE DG-GFP and OE β-DG-GFP) and neurons that overexpressed mutated β-DG (OE β-DG-MUT-GFP) were statistically significant (p < 0.02). (D) Total dendritic length for neurons treated as indicated. All of the experiments were performed in triplicate. The data are expressed as mean ± SEM. ∗∗∗p < 0.001 (Student’s t-test).
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Figure 4: Matrix metalloproteinase-9 dependent cleavage of β-DG is important for dendritic development. (A) Immunoblot analysis of β-DG and GFP expression in protein lysates from HEK 293 cells transfected with either OE β-DG-GFP (WT) or OE β-DG-MUT-GFP (MUT) plasmids and then treated with aaMMP-9. β-actin served as a loading control. (B) Representative images of neurons used for the morphometric analysis. The cells were co-transfected with an RFP-coding vector to visualize the exact morphology. (C) Sholl analysis of hippocampal neurons treated as indicated (ncell = 50 ± 4). In control cultures (without aaMMP-9), no statistically significant differences were found between DG-overexpressed neurons (transfected with either OE DG-GFP or OE β-DG-GFP) and neurons transfected with the OE β-DG-MUT-GFP vector. In cultures treated with aaMMP-9, at distances of 30–140 μm from the cell body, the differences between DG-overexpressed neurons (OE DG-GFP and OE β-DG-GFP) and neurons that overexpressed mutated β-DG (OE β-DG-MUT-GFP) were statistically significant (p < 0.02). (D) Total dendritic length for neurons treated as indicated. All of the experiments were performed in triplicate. The data are expressed as mean ± SEM. ∗∗∗p < 0.001 (Student’s t-test).

Mentions: We found that the silencing of DG and enhanced activity of MMP-9 exert similar effects on dendritic morphology. To test whether this similarity arises from the fact that MMP-9 inactivates DG by proteolytic processing, we modified the protease cleavage site in the OE β-DG-GFP vector using site-directed mutagenesis. The OE β-DG-MUT-GFP vector was introduced into HEK 293 cells, and its resistance to cleavage by MMP-9 was determined. We performed Western blot using an anti-GFP antibody to verify the cleavage of overexpressed β-DG and anti-β-DG to verify the cleavage of endogenous β-DG. As shown in Figure 4A, the mutation prevented the cleavage of β-DG by MMP-9 compared with wild type β-DG.


Dystroglycan controls dendritic morphogenesis of hippocampal neurons in vitro.

Bijata M, Wlodarczyk J, Figiel I - Front Cell Neurosci (2015)

Matrix metalloproteinase-9 dependent cleavage of β-DG is important for dendritic development. (A) Immunoblot analysis of β-DG and GFP expression in protein lysates from HEK 293 cells transfected with either OE β-DG-GFP (WT) or OE β-DG-MUT-GFP (MUT) plasmids and then treated with aaMMP-9. β-actin served as a loading control. (B) Representative images of neurons used for the morphometric analysis. The cells were co-transfected with an RFP-coding vector to visualize the exact morphology. (C) Sholl analysis of hippocampal neurons treated as indicated (ncell = 50 ± 4). In control cultures (without aaMMP-9), no statistically significant differences were found between DG-overexpressed neurons (transfected with either OE DG-GFP or OE β-DG-GFP) and neurons transfected with the OE β-DG-MUT-GFP vector. In cultures treated with aaMMP-9, at distances of 30–140 μm from the cell body, the differences between DG-overexpressed neurons (OE DG-GFP and OE β-DG-GFP) and neurons that overexpressed mutated β-DG (OE β-DG-MUT-GFP) were statistically significant (p < 0.02). (D) Total dendritic length for neurons treated as indicated. All of the experiments were performed in triplicate. The data are expressed as mean ± SEM. ∗∗∗p < 0.001 (Student’s t-test).
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Related In: Results  -  Collection

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Figure 4: Matrix metalloproteinase-9 dependent cleavage of β-DG is important for dendritic development. (A) Immunoblot analysis of β-DG and GFP expression in protein lysates from HEK 293 cells transfected with either OE β-DG-GFP (WT) or OE β-DG-MUT-GFP (MUT) plasmids and then treated with aaMMP-9. β-actin served as a loading control. (B) Representative images of neurons used for the morphometric analysis. The cells were co-transfected with an RFP-coding vector to visualize the exact morphology. (C) Sholl analysis of hippocampal neurons treated as indicated (ncell = 50 ± 4). In control cultures (without aaMMP-9), no statistically significant differences were found between DG-overexpressed neurons (transfected with either OE DG-GFP or OE β-DG-GFP) and neurons transfected with the OE β-DG-MUT-GFP vector. In cultures treated with aaMMP-9, at distances of 30–140 μm from the cell body, the differences between DG-overexpressed neurons (OE DG-GFP and OE β-DG-GFP) and neurons that overexpressed mutated β-DG (OE β-DG-MUT-GFP) were statistically significant (p < 0.02). (D) Total dendritic length for neurons treated as indicated. All of the experiments were performed in triplicate. The data are expressed as mean ± SEM. ∗∗∗p < 0.001 (Student’s t-test).
Mentions: We found that the silencing of DG and enhanced activity of MMP-9 exert similar effects on dendritic morphology. To test whether this similarity arises from the fact that MMP-9 inactivates DG by proteolytic processing, we modified the protease cleavage site in the OE β-DG-GFP vector using site-directed mutagenesis. The OE β-DG-MUT-GFP vector was introduced into HEK 293 cells, and its resistance to cleavage by MMP-9 was determined. We performed Western blot using an anti-GFP antibody to verify the cleavage of overexpressed β-DG and anti-β-DG to verify the cleavage of endogenous β-DG. As shown in Figure 4A, the mutation prevented the cleavage of β-DG by MMP-9 compared with wild type β-DG.

Bottom Line: The structural changes were associated with activation of the guanosine triphosphatase Cdc42.This effect was abolished in neurons that overexpressed a β-DG mutant that was defective in MMP-9-mediated cleavage.Altogether, our results indicate that DG controls dendritic arborization in vitro in MMP-9-dependent manner.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biophysics, Department of Molecular and Cellular Neurobiology, Nencki Institute of Experimental Biology Warsaw, Poland.

ABSTRACT
Dendritic outgrowth and arborization are important for establishing neural circuit formation. To date, little information exists about the involvement of the extracellular matrix (ECM) and its cellular receptors in these processes. In our studies, we focus on the role of dystroglycan (DG), a cell adhesion molecule that links ECM components to the actin cytoskeleton, in dendritic development and branching. Using a lentiviral vector to deliver short-hairpin RNA (shRNA) that specifically silences DG in cultured hippocampal neurons, we found that DG knockdown exerted an inhibitory effect on dendritic tree growth and arborization. The structural changes were associated with activation of the guanosine triphosphatase Cdc42. The overexpression of DG promoted dendritic length and branching. Furthermore, exposure of the cultures to autoactivating matrix metalloproteinase-9 (aaMMP-9), a β-DG-cleaving protease, decreased the complexity of dendritic arbors. This effect was abolished in neurons that overexpressed a β-DG mutant that was defective in MMP-9-mediated cleavage. Altogether, our results indicate that DG controls dendritic arborization in vitro in MMP-9-dependent manner.

No MeSH data available.


Related in: MedlinePlus