Based on the observation that microRNA (miRNA) 133b enhances regeneration after spinal cord injury in the adult zebrafish, we investigated whether this miRNA would be beneficial in a mammalian system in vitro and in vivo. We found that infection of cultured neurons with miR-133b promotes neurite outgrowth in vitro on an inhibitory substrate consisting of mixed chondroitin sulfate proteoglycans, when compared to infection with green fluorescent protein (GFP) for control. In vivo, viral infection of the injured adult mouse spinal cord at the time of injury at and in the vicinity of the lesion site enhanced expression of miR-133b. Measurements of locomotor recovery by Basso Mouse Scale (BMS) showed improvement of recovery starting at 4 weeks after injury and virus injection. This improvement was associated with downregulation of the expression levels of Ras homolog gene family member A (RhoA), chondroitin sulfate proteoglycans, and microglia/macrophage marker in the spinal cord as assayed 6 weeks after injury. Potential inhibitory molecules carrying consensus sequences for binding of miR-133b were identified in silico and verified in a reporter assay in vitro showing reductions in expression of RhoA, xylosyltransferase 1 (Xylt1), ephrin receptor A7 (Epha7), and purinergic receptor P2X ligand-gated ion channel 4 (P2RX4). These results encourage targeting miR-133 for therapy.
Keywords: Central nervous system; Inhibitory proteoglycans; Mouse; Regeneration; Spinal cord injury; miRNA.