Elucidating the Role of DLC1 Isoform 1 in Human Motor Neuron Development and Spinal Muscular Atrophy

Abstract

Spinal muscular atrophy (SMA) is a motor neuron (MN) disease caused by loss of the ubiquitously expressed Survival Motor Neuron (SMN) spliceosome protein, resulting in selective degeneration of spinal MNs but the mechanism underlying the specific loss of MNs remains unknown. A previous report showed that Deleted in Liver Cancer 1 (DLC1) is the most down-regulated gene in MNs derived from a SMA patient but its roles in MN development and SMA pathogenesis remain to be elucidated. Here, we detected a gradual increase in DLC1-i1 expression level as human embryonic stem cells differentiated into MN lineage. Knockdown (KD) of DLC1-i1 led to a reduction in MN formation, axonal outgrowth and increase apoptosis, whereas overexpression of DLC1-i1 promoted MN differentiation with extensive neurite projections. Furthermore, SMN KD not only caused MN loss but also intron retention of DLC1-i1, resulting in downregulation of DLC1-i1 expression. We also confirmed decreased levels of DLC1-i1 in spinal MNs differentiated from SMA patients’ urine derived induced pluripotent stem cells (iPSCs) compared with healthy individuals, as well as in spinal cords of SMA postnatal mice compared with that of wild-type (WT) mice. To further reveal the molecular underpinnings of SMA, we generated neuromuscular organoids from WT and SMA patients derived-iPSCs for disease modelling and single cell RNA-seq. More importantly, SMA mice injected with DLC1-i1-AAV PHP.eB showed better locomotion ability compared with mice injected with GFP-AAV. Altogether, our results indicate that DLC1-i1 functions downstream of SMN to direct axonal outgrowth during MN differentiation and may serve as a new therapeutic target for the treatment of SMA.