Fig. 4

Endogenous tPA controls the remodeling of PNNs through plasmin-dependent aggrecan cleavage. A,B MGE-derived WT interneuron cultures were treated at DIV14 with plasmin (200 nM), plasminogen (100 nM), tPA (10 and 200 nM), and processed 24h later for immunocytochemistry. A Representative images for PV (magenta) and WFA (yellow) stainings. B Quantification shows that plasmin, plasminogen, and plasminogen + tPA reduce WFA staining, whereas tPA has no effect. C Representative images of MGE-derived tPA Null interneuron cultures stained for PV (magenta) and WFA (yellow) 24h after treatment with plasmin (200 nM), plasminogen (100 nM), and tPA (10 nM). D WFA-positive area quantification reveals that plasmin induces the degradation of PNN in PV cells from tPA Null cultures whereas plasminogen fails to (mean ± sem; n=40 cells from 4 independent experiments). Kruskall-Wallis test followed by Dunn’s post hoc test for multiple comparisons; ###: p<0.001 (compared to control), ***: p<0.001 (compared to plasminogen + tPA). Scale bar: 10 μm. E–G Human aggrecan G1-G2 (156 nM) was incubated with ADAMTS4 (positive control), plasmin, plasminogen, tPA (7.8 nM for each protease), and aprotinin (78 nM) overnight. F,G Degradation products were visualized by Imperial blue staining. Like ADAMTS4, plasmin, and plasminogen + tPA can cleave aggrecan G1-G2 in vitro. Aprotinin reverses plasmin-dependent aggrecan degradation. N=4 independent experiments. One-way ANOVA, Fisher’s LSD post hoc test; #: p<0.05; ##: p<0.01; ###: p<0.001 (compared to Agg G1-G2). H Representative images of MGE-derived WT interneuron cultures treated with plasmin (100 nM), plasminogen (100 nM), and tPA (10 and 200 nM) and co-stained with aggrecan (cyan) and PV (magenta). I Quantitative analysis of aggrecan positive area shows that plasmin, plasminogen, and plasminogen + tPA reduce aggrecan staining whereas tPA treatment has no effect in WT interneurons (mean ± sem; n=40 cells from 4 independent experiments). Kruskall-Wallis test followed by Dunn’s post hoc test for multiple comparisons; ###: p<0.001 (compared to Control). Scale bar: 10 μm