Single-nuclei sequencing of skeletal muscle reveals subsynaptic-specific transcripts involved in neuromuscular junction maintenance

Abstract

AbstractThe neuromuscular junction (NMJ) is the synapse formed between motor neurons and skeletal muscle fibers. Its stability relies on the continued expression of genes in a subset of myonuclei, called NMJ myonuclei. Here, we use single-nuclei RNA-sequencing (snRNA-seq) to identify numerous undescribed NMJ-specific transcripts. To elucidate how the NMJ transcriptome is regulated, we also performed snRNA-seq on sciatic nerve transected, botulinum toxin injected andMuskknockout muscles. These data show that NMJ gene expression is not only driven by agrin-Lrp4/MuSK signaling, but is also affected by electrical activity and trophic factors other than agrin. By selecting three previously undescribed NMJ genesEtv4,Lrtm1andPdzrn4, we further characterize novel contributors to NMJ stability and function. AAV-mediated overexpression and AAV-CRISPR/Cas9-mediated knockout show thatEtv4is sufficient to upregulate expression of ∼50% of the NMJ genes in non-synaptic myonuclei, while muscle-specific knockout ofPdzrn4induces NMJ fragmentation. Further investigation ofPdzrn4revealed that it localizes to the Golgi apparatus and interacts with MuSK protein. Collectively, our data provide a rich resource of NMJ transcripts, highlight the importance of ETS transcription factors at the NMJ and suggest a novel pathway for NMJ post-translational modifications.