Physical inactivity and disuse are major contributors to age-related muscle loss. Denervation of skeletal muscle has been previously used as a model with which to investigate muscle atrophy following disuse. Although gene regulatory networks that control skeletal muscle atrophy after denervation have been established, the transcriptome in response to the recovery of muscle after disuse and the associated epigenetic mechanisms that may function to modulate gene expression during skeletal muscle atrophy or recovery have yet to be investigated. We report that silencing the tibialis anterior muscle in rats with tetrodotoxin (TTX)-administered to the common peroneal nerve-resulted in reductions in muscle mass of 7, 29, and 51% with corresponding reductions in muscle fiber cross-sectional area of 18, 42, and 69% after 3, 7, and 14 d of TTX, respectively. Of importance, 7 d of recovery, during which rodents resumed habitual physical activity, restored muscle mass from a reduction of 51% after 14 d TTX to a reduction of only 24% compared with sham control. Returning muscle mass to levels observed at 7 d TTX administration (29% reduction). Transcriptome-wide analysis demonstrated that 3714 genes were differentially expressed across all conditions at a significance of P £ 0.001 after disuse-induced atrophy. Of interest, after 7 d of recovery, the expression of genes that were most changed during TTX had returned to that of the sham control. The 20 most differentially expressed genes after microarray analysis were identified across all conditions and were cross-referenced with the most frequently occurring differentially expressed genes between conditions. This gene subset included myogenin (MyoG), Hdac4, Ampd3, Trim63 (MuRF1), and acetylcholine receptor subunit a1 (Chrna1). Transcript expression of these genes and Fboxo32 (MAFbx), because of its previously identified role in disuse atrophy together with Trim63 (MuRF1), were confirmed by real-time quantitative RT-PCR, and DNA methylation of their promoter regions was analyzed by PCR and pyrosequencing. MyoG, Trim63 (MuRF1), Fbxo32 (MAFbx), and Chrna1 demonstrated significantly decreased DNA methylation at key time points after disuseinduced atrophy that corresponded with significantly increased gene expression. Of importance, after TTX cessation and 7 d of recovery, there was a marked increase in the DNA methylation profiles of Trim63 (MuRF1) and Chrna1 back to control levels. This also corresponded with the return of gene expression in the recovery group back to baseline expression observed in sham-operated controls. To our knowledge, this is the first study to demonstrate that skeletal muscle atrophy in response to disuse is accompanied by dynamic epigenetic modifications that are associated with alterations in gene expression, and that these epigenetic modifications and gene expression profiles are reversible after skeletal muscle returns to normal

Administration of VP025 (Vasogen Inc.), a novel drug formulation based on phospholipid nanoparticles incorporating phosphatidylglycerol, has previously been shown to have a neuroprotective effect in the brain. We examined the effect of VP025 in a rat model of Parkinson's disease, the 6-hydroxydopamine (6-OHDA) lesion of the medial forebrain bundle. VP025 or phosphate-buffered saline (PBS) was administered to rats 14 days, 13 days and 1 day before the unilateral 6-OHDA lesion. Functional integrity of nigrostriatal dopaminergic neurons was assessed 7 and 21 days later by amphetamine-induced rotational testing and we observed that rotational counts were significantly less in rats that were pretreated with VP025 compared with PBS-pretreated 6-OHDA-lesioned rats. Neurochemical analysis at 10 and 28 days after lesion revealed that VP025 protected against a 6-OHDA-induced decrease in concentrations of striatal dopamine and its metabolites. Immunocytochemical studies of the ipsilateral substantia nigra showed that VP025 significantly inhibited 6-OHDA-induced loss of dopaminergic neurons. We also observed that increases in immunostaining for activated microglia and for activated p38 in dopaminergic neurons of 6-OHDA-lesioned rats were prevented by VP025. This study shows that VP025 has significant protective effects on the 6-OHDA-lesioned nigrostriatal pathway and may therefore have potential for the treatment of Parkinson's disease.

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We have measured, characterized, and localized calcium-dependent protein cross-linking activity in rat skeletal muscle, and in myotubes cultured independently or in coculture with spinal neurones, catalyzed by the enzyme tissue transglutaminase (tTG). The enzyme activity was present in both motor endplate and endplate-free zones of rat diaphragm muscle. tTG in the endplate zone was more tightly associated with the tissue. This form of association was absent in extracts of peripheral nerve. Cross-linking of endogenous proteins, as measured by the content of epsilon-(gamma-glutamyl)lysine isopeptide, was higher in the endplate than in the nonendplate zone. Cytosolic (C) and particulate (B) forms of tTG were separated by ion-exchange chromatography from both regions of the muscle. In the motor endplate zone, a higher proportion of tightly bound tTG was recovered as a separate (B1) particulate form. Km values for calcium activation of the three forms of tTG were in the range of 5-15 microM. Immunocytochemistry with polyclonal and monoclonal antibodies revealed the enzyme at motor endplates and at contacts between neurites of rat embryo spinal neurones and myotubes in primary cocultures. Appearance of the B1 transglutaminase could be induced by coculturing myotubes of the mouse C2C12 cell line with neurones. The results suggest that tTG is most concentrated and active at the motor endplate.

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This study examines the hypothesis that glutamate tonically suppresses the activity of the enzyme aromatic L-amino acid decarboxylase (AADC), and hence the biosynthesis of dopamine, to explain how antagonists of glutamate receptors might potentiale the motor actions of L-DOPA in animal models of Parkinson's disease. A variety of glutamate antagonists were therefore administered acutely to normal rats, which were sacrificed 30-60 min later and AADC activity assayed in the substantia nigra pars reticulata (SNr) and corpus striatum (CS). The NMDA receptor-ion channel antagonists MK 801, budipine, amantadine, memantine and dextromethorphan all caused a pronounced in creased in AADC activity, more especially in the SNr than CS. The NMDA glycine site antagonist (R)-HA 966 produced a modest increase in AADC activity in the CS but not SNr, whilst the NMDA polyamine site antagonist eliprodil, the NMDA competitive antagonist CGP 40116 and the AMPA antagonist NBQX were without effect. The results suggest that an increase in dopamine synthesis might contribute to the L-DOPA-facilitating actions of some glutamate antagonists.

Summary:Purpose: To evaluate the antiepileptic and neuroprotective properties of topiramate (TPM) alone and with coadministration of the N-methyl-D-aspartate (NMDA)-receptor antagonist budipine in a rat model of refractory status epilepticus.Methods: Male Sprague-Dawley rats had electrodes implanted into the perforant path and dentate granule cell layer of the hippocampus under halothane anesthesia. Approximately 1 week after surgery, the perforant path of each animal was electrically stimulated for 2 h to induce self-sustaining status epilepticus. Successfully stimulated rats were given intraperitoneally vehicle (n = 6), TPM (20-320 mg/kg; n = 28), budipine (10 mg/kg; n = 5), or budipine (10 mg/kg) and TPM (80 mg/kg; n = 6) 10 min after the end of the stimulation and monitored behaviorally and electroencephalographically for a further 3 h.The animals were killed 14 days later, and histopathology was assessed.Results: Neither budipine alone nor TPM at any dose terminated status epilepticus. Despite this, TPM resulted in various degrees of neuroprotection at doses between 40 and 320 mg/kg. Coadministration of budipine with TPM terminated the status epilepticus in all rats. This combination also significantly improved the behavioral profile and prevented status-induced cell death compared with control.Conclusions: Budipine and TPM are an effective drug combination in stopping self-sustained status epilepticus, and TPM alone was neuroprotective, despite the continuation of seizure activity.