Alternative splicing and gene expression defects in motorneurons derived from DM1 hiPSCs and MBNL knockouts hiPSCs generated by CRISPR/Cas9.

Myotonic dystrophy type I (DM1) is the most frequent neuromuscular disease (NMD) in adults and only symptomatic treatments have been proposed to patients. DM1 has long been defined by muscular defects but several studies suggested the involvement of the neuromuscular junction (NMJ). In this aim, our purpose raised the question about the pathological contribution of the motoneurons (MNs) in DM1 physiopathology. By using a micropatterned 96-well plate as a co-culture platform, we generated a functional humanized system in 11 days which associates hiPS-derived MNs and human primary skeletal muscle cells. To focus only on DM1 motoneurons-dependent phenotypes, we generated hybrid systems using wild-type skeletal muscle cells combined with mutated hiPS-derived MNs. In this context, we have identified several alterations affecting synaptic vesicular trafficking, AChR clustering and communication between pre- and post-synaptic compartments. More interestingly, hiPS-derived MNs depleted for MBNL1 and MBNL2 reproduced alterations at the pre- and post-synaptic levels. These experiments suggested that the functional defects associated to MNs can be directly attributed to the MBNL family proteins. These findings may be clinically relevant to open new therapeutic approaches taking into account the alterations affecting DM1 motoneurons. Overall design: 12 samples were analyzed with 3 replicates per condition: 3 WT hiPSC-derived motorneurones (control), 3 Myotonic Dystrophy type 1 (DM1) hiPSC-derived motorneurones, 3 MBNL2(-/-) hiPSC-derived motorneurones and 3 MBNL1(-/-); MBNL2(-/-) (DKO) hiPSC-derived motorneurones.