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.

The expression and function of gelatinolytic activity at the rat neuromuscular junction upon physical exercise.

The gelatinases MMP-9 and MMP-2 have been implicated in skeletal muscle adaptation to training; however, their specific role(s) in the different muscle types are only beginning to be unraveled. Recently, we found that treadmill running increased the activity and/or expression of these enzymes in myonuclei and in activated satellite cells of the soleus (Sol), but not extensor digitorum longus (EDL) muscles on the fifth day of training of adult rats. Here, we asked whether the gelatinases can be involved in physical exercise-induced adaptation of the neuromuscular compartment. To determine the subcellular localization of the gelatinolytic activity, we used high-resolution in situ zymography and immunofluorescence techniques. In both control and trained muscles, strong gelatinolytic activity was associated with myelin sheaths within intramuscular nerve twigs. In EDL, but not Sol, there was an increase in the gelatinolytic activity at the postsynaptic domain of the neuromuscular junction (NMJ). The increased activity was found within punctate structures situated in the vicinity of synaptic cleft of the NMJ, colocalizing with a marker of endoplasmic reticulum. Our results support the hypothesis that the gelatinolytic activity at the NMJ may be involved in NMJ plasticity.

Myosin VI in skeletal muscle: its localization in the sarcoplasmic reticulum, neuromuscular junction and muscle nuclei.

Myosin VI (MVI) is a unique unconventional motor moving backwards on actin filaments. In non-muscle cells, it is involved in cell migration, endocytosis and intracellular trafficking, actin cytoskeleton dynamics, and possibly in gene transcription. An important role for MVI in striated muscle functioning was suggested in a report showing that a point mutation (H236R) within the MVI gene was associated with cardiomyopathy (Mohiddin et al., J Med Genet 41:309-314, 2004). Here, we have addressed MVI function in striated muscle by examining its expression and distribution in rat hindlimb skeletal muscle. We found that MVI was present predominantly at the muscle fiber periphery, and it was also localized within muscle nuclei. Analysis of both the hindlimb and cardiac muscle longitudinal sections revealed ~3 µm striation pattern, corresponding to the sarcoplasmic reticulum. Moreover, MVI was detected in the sarcoplasmic reticulum fractions isolated from skeletal and cardiac muscle. The protein also localized to the postsynaptic region of the neuromuscular junction. In denervated muscle, the defined MVI distribution pattern was abolished and accompanied by significant increase in its amount in the muscle fibers. In addition, we have identified several novel potential MVI-binding partners, which seem to aid our observations that in striated muscle MVI could be involved in postsynaptic trafficking as well as in maintenance of and/or transport within the sarcoplasmic reticulum and non-sarcomeric cytoskeleton.

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.

Alpha--lipoic acid decreases DNA damage and oxidative stress induced by alcohol in the developing hippocampus and cerebellum of rat.

BACKGROUND/AIMS: The present study was initiated in order to investigate the protective effects of alpha-lipoic acid upon ethanol-induced DNA damage, lipid peroxidation and protein oxidation in the developing rat hippocampus and cerebellum. METHODS: Pregnant Wistar rats received ethanol with, or without lipoic acid from gestation day (GD) 7 throughout lactation. The changes in DNA damage, protein carbonyl, lipid hydroperoxide, catalase and superoxide dismutase activities were measured in the hippocampus and cerebellum of male offspring at the end of the lactation period. RESULTS: The results indicated that DNA damage, lipid peroxidation and protein oxidation in the hippocampus and cerebellum were significantly elevated in animals that received alcohol. However, the catalase and superoxide dismutase activity results showed patterns that differed from those of DNA damage, lipid peroxidation and protein oxidation. Lipoic acid treatment significantly decreased DNA damage compared with the group that were administered alcohol alone, and restored the elevated protein carbonyl and lipid hydroperoxide levels to the levels of the control group. CONCLUSIONS: Our findings confirm that oxidative stress and DNA damage occur in the developing hippocampus and cerebellum as a result of alcohol administration, and also suggest that lipoic acid has protective effects as an antioxidant against alcohol-induced disorders in the developing hippocampus and cerebellum.

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.