Genomic stress drives activation of interferon signaling and innate immune pathways during SMA Type II myoblasts differentiation

Abstract

Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by loss of the SMN1 gene. The SMN protein is ubiquitously expressed and has several roles in the cell, including the regulation of RNA metabolism and genome stability. Although SMA is primarily considered a motor neuron disease, because of its ubiquitous expression, loss of SMN leads to systemic pathological consequences, including in skeletal muscle. To better understand the cell-intrinsic effects of SMN loss on myoblast differentiation, we characterized three human SMA Type II myoblast cell lines and controls in vitro. We observed impaired myogenic differentiation, including reduced fusion index and nuclei alignment. RNA sequencing analysis on the SMA myoblasts revealed activation of DNA damage pathways and innate immune activation, confirmed by the presence of 53BP1 foci and increased phosphorylation of the H2AX histones, as well as accumulation of R-loop structures and an increase of single-stranded DNA in the cytoplasm. Resolution of R-loops and DNA damage is known to cause the presence of immunogenic DNA: RNA species in the cytoplasm, activating sterile inflammation pathways. Treating control myoblasts with double-stranded DNA or a cGAS agonist phenocopied the differentiation defects in SMA myotubes. Conversely, treating SMA myoblasts with a STING inhibitor improved fusion. In summary, our results demonstrate that SMN loss exerts a cell-intrinsic impairment in muscle precursor cells, suggesting that the observed muscle dysfunction in Type II patients is not only a consequence of impaired innervation but also of the sterile inflammation caused by the genomic instability after SMN loss.