Distinct transcriptomic profile of satellite cells contributes to preservation of neuromuscular junctions in extraocular muscles of ALS mice

Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disorder characterized by progressive weakness of almost all skeletal muscles, whereas extraocular muscles (EOMs) are comparatively spared. While hindlimb and diaphragm muscles of end-stage SOD1G93A (G93A) mice (a familial ALS mouse model) exhibit severe denervation and depletion of Pax7+satellite cells (SCs), we found that the pool of SCs and the integrity of neuromuscular junctions (NMJs) are maintained in EOMs. In cell sorting profiles, SCs derived from hindlimb and diaphragm muscles of G93A mice exhibit denervation-related activation, whereas SCs from EOMs of G93A mice display spontaneous (non-denervation-related) activation, similar to SCs from wild-type mice. Specifically, cultured EOM SCs contain more abundant transcripts of axon guidance molecules, including Cxcl12, along with more sustainable renewability than the diaphragm and hindlimb counterparts under differentiation pressure. In neuromuscular co-culture assays, AAV-delivery of Cxcl12 to G93A-hindlimb SC-derived myotubes enhances motor neuron axon extension and innervation, recapitulating the innervation capacity of EOM SC-derived myotubes. G93A mice fed with sodium butyrate (NaBu) supplementation exhibited less NMJ loss in hindlimb and diaphragm muscles. Additionally, SCs derived from G93A hindlimb and diaphragm muscles displayed elevated expression of Cxcl12 and improved renewability following NaBu treatment in vitro. Thus, the NaBu-induced transcriptomic changes resembling the patterns of EOM SCs may contribute to the beneficial effects observed in G93A mice. More broadly, the distinct transcriptomic profile of EOM SCs may offer novel therapeutic targets to slow progressive neuromuscular functional decay in ALS and provide possible 'response biomarkers' in pre-clinical and clinical studies. Overall design: Skeletal muscles from different body regions exhibit different degree of vulerability to ALS, with hindlimb (HL) muscles like TA and EDL most severely affected and extraocular muscles (EOMs) least affected. We observed an association between the severity of NMJ degeneration and peri-NMJ SC depletion in HL, diaphragm (Dia) and EOMs in end-stage G93A mice. To investigate whether there is a mechanistic link between the two phenomena, we FACS-isolated HL, Dia, EOM SCs from end-stage G93A mice and their wildtype (WT) littermates to compare the transcriptome of SCs cultured under expansion and differentiation conditions using RNA-Seq. Additionally, we previously showed that feeding G93A mice with NaBu supplmenetation expanded their life span. Since NaBu is an HDAC inhibitor capable of changing the epigenetic landscape, we compared the transcriptome of G93A Dia, HL SCs with or without 3-day NaBu treatment.