Fine-structural distribution of MMP-2 and MMP-9 activities in the rat skeletal muscle upon training: a study by high-resolution in situ zymography

Matrix metalloproteinases (MMPs) are key regulators of extracellular matrix remodeling, but have also important intracellular targets. The purpose of this study was to examine the activity and subcellular localization of the gelatinases MMP-2 and MMP-9 in skeletal muscle of control and physically trained rats. In control hind limb muscle, the activity of the gelatinases was barely detectable. In contrast, after 5 days of intense exercise, in Soleus (Sol), but not Extensor digitorum longus (EDL) muscle, significant upregulation of gelatinolytic activity in myofibers was observed mainly in the nuclei, as assessed by high resolution in situ zymography. The nuclei of quiescent satellite cells did not contain the activity. Within the myonuclei, the gelatinolytic activity colocalized with an activated RNA Polymerase II. Also in Sol, but not in EDL, there were few foci of mononuclear cells with strongly positive cytoplasm, associated with apparent necrotic myofibers. These cells were identified as activated satellite cells/myoblasts. No extracellular gelatinase activity was observed. Gel zymography combined with subcellular fractionation revealed training-related upregulation of active MMP-2 in the nuclear fraction, and increase of active MMP-9 in the cytoplasmic fraction of Sol. Using RT-PCR, selective increase in MMP-9 mRNA was observed. We conclude that training activates nuclear MMP-2, and increases expression and activity of cytoplasmic MMP-9 in Sol, but not in EDL. Our results suggest that the gelatinases are involved in muscle adaptation to training, and that MMP-2 may play a novel role in myonuclear functions

Forced Remyelination Promotes Axon Regeneration in a Rat Model of Spinal Cord Injury

Spinal cord injuries result in the loss of motor and sensory functions controlled by neurons located at the site of the lesion and below. We hypothesized that experimentally enhanced remyelination supports axon preservation and/or growth in the total spinal cord transection in rats. Multifocal demyelination was induced by injection of ethidium bromide (EB), either at the time of transection or twice during transection and at 5 days post-injury. We demonstrated that the number of oligodendrocyte progenitor cells (OPCs) significantly increased 14 days after demyelination. Most OPCs differentiated into mature oligodendrocytes by 60–90 dpi in double-EB-injected rats; however, most axons were remyelinated by Schwann cells. A significant number of axons passed the injury epicenter and entered the distant segments of the spinal cord in the double-EB-injected rats. Moreover, some serotoninergic fibers, not detected in control animals, grew caudally through the injury site. Behavioral tests performed at 60–90 dpi revealed significant improvement in locomotor function recovery in double-EB-injected rats, which was impaired by the blockade of serotonin receptors, confirming the important role of restored serotonergic fibers in functional recovery. Our findings indicate that enhanced remyelination per se, without substantial inhibition of glial scar formation, is an important component of spinal cord injury regeneration

DP-b99 modulates matrix metalloproteinase activity and neuronal plasticity.

DP-b99 is a membrane-activated chelator of zinc and calcium ions, recently proposed as a therapeutic agent. Matrix metalloproteinases (MMPs) are zinc-dependent extracellularly operating proteases that might contribute to synaptic plasticity, learning and memory under physiological conditions. In excessive amounts these enzymes contribute to a number of neuronal pathologies ranging from the stroke to neurodegeneration and epileptogenesis. In the present study, we report that DP-b99 delays onset and severity of PTZ-induced seizures in mice, as well as displays neuroprotective effect on kainate excitotoxicity in hippocampal organotypic slices and furthermore blocks morphological reorganization of the dendritic spines evoked by a major neuronal MMP, MMP-9. Taken together, our findings suggest that DP-b99 may inhibit neuronal plasticity driven by MMPs, in particular MMP-9, and thus may be considered as a therapeutic agent under conditions of aberrant plasticity, such as those subserving epileptogenesis

Enzymatic assay using DQ-gelatine.

<p>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.</p

DP-b99 diminishes neurodegenerative effect of kainate in hippocampal slice cultures.

<p><b>A</b> – 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>B -</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). <b>C -</b> 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. <b>D –</b> Quantification of β-dystroglycan 30 kDa product of MMP-9 cleavage intensity from three independent experiments (n = 3, mean ± SE; * - p<0.05).</p

Effect of DP-b99 on PTZ kindling induced seizures.

<p><b>A</b>- 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>B</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. <b>C</b>– 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). <b>D, E -</b> 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).</p