Figure 1.
Effect of DP-b99 on PTZ kindling induced seizures.
A- Seizure scores of PTZ-kindled vehicle- and DP-b99 treated mice during the course of the experiment (mean ± SE). Note the delay of seizures in DP-b99 treated group (n = 7 per group, repeated measures ANOVA:F (1, 10) = 5.6382, p<0.05). B – Schematic diagram of a hippocampal section showing suprapyramidal (SP) and infrapyramidal (IP) bundles of mossy fiber pathway arising from hilus of dentate gyrus (DG) to area of CA 3 pyramidal cells. Representative images of coronal sections immunostained for ZnT-3 (white) in CA3 hippocampal region of vehicle- and DP-b99 treated kindled animals. Vehicle treated animals demonstrated much longer IP length, indicating a robust mossy fibers sprouting (red asterisks). SR, stratum radiatum; SL, stratum lucidum; SP, stratum pyramidale; SO, stratum orient. Scale bar = 100 µm. C– Quantitative analysis of Zn-T3 immunofluorescence in vehicle- and DP-b99 treated kindled animals. ZN-T3 expression was evaluated in IP mossy fibers using the ratio of IP length to the length of SP (n = 4 animals for each group, mean ± SE; * - indicates p<0.05). D, E - Western blot analysis and quantification of β-dystroglycan cleavage product in the hippocampus of kindled animals (n = 4 per group, mean ± SE; * - indicates p<0.05).
Figure 2.
Enzymatic assay using DQ-gelatine.
400/ml of purified MMP-9 was incubated with DQ-gelatine and with DP-b99 of different concentrations (0.12 µM and 20 µM) at 37°C. Fluorescence was measured every minute. General MMP inhibitor GM6001 (25 µM) and specific Inhibitor I of MMP-9 and MMP-13 (5 µM) were used as a positive controls.
Figure 3.
DP-b99 diminishes neurodegenerative effect of kainate in hippocampal slice cultures.
A – Representative fluorescence photomicrographs of organotypic hippocampal slices (16 DIV) showing uptake of PI 24 h after various treatments: CTRL (untreated cultures); DP-b99 20 µM and 0.12 µM; KA (5 µM); KA+DP-b99 20 µM or 0.12 µM. B - Quantification done by ImageJ software and represented in arbitrary units of fluorescence shows a significant decrease in neuronal death in all groups treated with DP-b99 (n = 3, mean ± SE; * - p<0.05). C - Western blot analysis of β-dystroglycan cleavage. Organotypic hippocampal cultures were treated with 50 µM KA for 10 min. DP-b99 (0.12 µM or 20 µM) was added to the culture media 1 h before KA administration. D – Quantification of β-dystroglycan 30 kDa product of MMP-9 cleavage intensity from three independent experiments (n = 3, mean ± SE; * - p<0.05).
Figure 4.
DP-b99 prevents MMP-9 effect on dendritic spine morphology and F-actin arrangement.
A - Representative dendrites of GFP-transfected hippocampal neurons showing morphological changes in spines after indicated stimulations. Lower panels show the same dendrites following staining with Alexa Fluor 568 coupled to phalloidin (F-actin). Note that 1 h incubation of the cultures with MMP-9 (400 ng/ml) promotes dendritic spine elongation, whereas 1 h pretreatment with DP-b99 (0.12 µM) prevents the formation of these filopodia-like structures. Neither GM6001 nor DP-b99 on their own evoke spine elongation. B - Relative changes in spine length following 1 h stimulation with MMP-9 with or without DP-b99 or GM6001 pretreatment (n = 3, mean ± SE; *** - p<0.001). C - Fluorescence intensity of Alexa Fluor 568 coupled to phalloidin, calculated within spines vs. dendritic shaft (n = 3, mean ± SE; *** - p<0.001).