Evaluation of potential synergistic action of a combined treatment with alpha-methyl-prednisolone and taurine on the mdx mouse model of Duchenne muscular dystrophy

Aims: Glucocorticoids are the sole drugs clinically used in Duchenne muscular dystrophy, in spite of the relevant side effects. Combination of glucocorticoids with synergistic drugs may be one strategy to lower doses and control side effects, meanwhile providing wider control of the complex pathology. This study is a preclinical evaluation of the effect of a combined treatment of amethyl-prednisolone (PDN) with taurine, a safe aminoacid with positive effects on some pathology-related events. Methods: PDN (1 mg/kg/day i.p.) and taurine (1 g/kg/day orally) were administered either alone or in combination, for 4-8 weeks to male dystrophic mdx mice chronically exercised on a treadmill. Effects were assessed in vivo and ex vivo with a variety of methodological approaches. Results: In vivo, each treatment significantly increased fore limb strength, a marked synergistic effect being observed with the combination PDN + taurine. Ex vivo, PDN + taurine completely restored the mechanical threshold, an electrophysiological index of calcium homeostasis, of extensor digitorum longus myofibres and the benefit was greater than for PDN alone. In parallel, the overactivity of voltage-independent cation channels in dystrophic myofibres was reduced. No effects were observed on plasma levels of creatine kinase, while lactate dehydrogenase was decreased by taurine and, to a minor extent, by PDN + taurine. A similar histology profile was observed in PDN and PDN + taurine-treated muscles. PDN + taurine significantly increased taurine level in fast-twitch muscle and brain, by high-pressure liquid chromatography analysis. Conclusions: The combination PDN + taurine has additive actions on in vivo and ex vivo functional end points, with less evident advantages on histopathology and biochemical markers of the disease

Effects of prednisolone on the dystrophin-associated proteins in the blood-brain barrier and skeletal muscle of dystrophic mdx mice.

The mdx mouse, the most widely used animal model of Duchenne muscular dystrophy (DMD), develops a seriously impaired blood-brain barrier (BBB). As glucocorticoids are used clinically to delay the progression of DMD, we evaluated the effects of chronic treatment with α-methyl-prednisolone (PDN) on the expression of structural proteins and markers in the brain and skeletal muscle of the mdx mouse. We analyzed the immunocytochemical and biochemical expression of four BBB markers, including endothelial ZO-1 and occludin, desmin in pericytes, and glial fibrillary acidic protein (GFAP) in glial cells, and the expression of the short dystrophin isoform Dp 71, the dystrophin-associated proteins (DAPs), and aquaporin-4 (AQP4) and α-β dystroglycan (DG) in the brain. We evaluated the BBB integrity of mdx and PDN-treated mdx mice by means of intravascular injection of horseradish peroxidase (HRP). The expression of DAPs was also assessed in gastrocnemius muscles and correlated with utrophin expression, and laminin content was measured in the muscle and brain. PDN treatment induced a significant increase in the mRNA and protein content of the BBB markers; a reduction in the phosphorylation of occludin in the brain and of AQP4/β DG in both tissues; an increase of Dp71 protein content; and an increase of both mRNA and protein levels of the AQP4/α-β DG complex. The latter was associated with enhanced laminin and utrophin in the muscle. The HRP assay demonstrated functional restoration of the BBB in the PDN-treated mdx mice. Specifically, mdx mice showed extensive perivascular labeling due to escape of the marker, while HRP was exclusively intravascular in the PDN-treated mice and the controls. These data illustrate for the first time that PDN reverses the BBB alterations in the mdx mouse and re-establishes the proper expression and phosphorylation of β-DG in both the BBB and skeletal muscle. Further, PDN partially protects against muscle damage. The reduction in AQP4 and occludin phosphorylation, coupled with their anchoring to glial and endothelial membranes in PDN-treated mice, suggests that the drug may target the glial and endothelial cells. Our results suggest a novel mechanism for PDN action on cerebral and muscular function, restoring the link between DAPs and the extracellular matrix, most likely through protein kinase inactivation