Characterizing the Role of RNA-Binding Protein Human Antigen R in the Neuromuscular Junction in Duchenne Muscular Dystrophy

Abstract

Duchenne muscular dystrophy (DMD) is a progressive X-linked disorder caused by mutations in the dystrophin gene, leading to severe skeletal muscle fibre degeneration and neuromuscular junction (NMJ) abnormalities, including fragmentation and disorganization of the post-synaptic apparatus. Human Antigen R (HuR) is an RNA-binding protein involved in post-transcriptional regulation of transcripts associated with muscle differentiation and endplate function. However, its role in maintaining the structure of the post-synaptic apparatus at the NMJ remains unclear. This project investigated HuR's contribution to dystrophic endplate organization in the mdx mouse model of DMD. First, we confirmed significant post-synaptic disorganization in the mdx muscle relative to wild-type (WT) controls. Additionally, HuR protein expression was elevated (~3-fold) across multiple fast- and slow-twitch mdx muscles from 4-12 weeks, with smaller, non-significant increases observed at 24+ weeks. To further characterize HuR localization, subcellular fractionation was performed in the tibialis anterior (TA) muscle at 11 weeks, a time point representative of the increased HuR expression observed across the multiple muscles and ages we examined. Subcellular fractionation demonstrated significantly increased nuclear HuR protein levels in mdx muscle compared to WT controls (p<0.001). Consistent with these findings, immunofluorescence analyses also revealed a strong trend towards increased nuclear HuR abundance in mdx muscle fibres relative to WT controls in the TA when all ages were combined (p=0.054). Given these findings, we knocked down HuR expression in vivo using recombinant adeno-associated virus-mediated short hairpin RNA delivery through intramuscular injections in the TA muscles of mdx mice. HuR protein expression was reduced by approximately 60% relative to shLuc-treated controls, restoring HuR levels to those observed in WT muscle (p<0.01). This led to a partial improvement in post-synaptic organization, as evidenced by increased acetylcholine receptor compactness compared with shLuc-treated mdx muscle (p<0.05) and untreated mdx muscle (p<0.0001). Finally, the pan-histone deacetylase inhibitor Vorinostat was evaluated as a pharmacological approach to assess changes in HuR expression, localization, and potential effects on post-synaptic morphology. However, Vorinostat did not significantly increase acetylated tubulin levels, suggesting that adequate HDAC inhibition may not have been achieved, which limited our ability to assess its effects on HuR expression and post-synaptic organization. Overall, HuR protein expression is dysregulated in dystrophic muscle across multiple ages and muscle types, while reducing HuR expression partially improved post-synaptic membrane organization, supporting a role for HuR in the molecular mechanisms underlying endplate abnormalities in DMD.