p53 Inhibits the Direct Conversion of Fibroblasts into Motor Neurons

Spinal motor neurons coordinate all voluntary muscle movement. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the progressive damage of motor neurons, leading to movement loss, respiratory ailments, and death. Direct lineage conversion provides the fastest route to the production of patient-specific induced motor neurons (iMNs) for translational studies. However, the inefficiency of reprogramming currently limits the utility of this approach. Here, we show that upon p53 inhibition, reprogramming efficiency in both mouse somatic fibroblasts increases 10-fold. More strikingly, we see upwards of a 20-fold increase in the production of patient-specific iMNs upon p53 inhibition, leading to 40% of the starting fibroblasts becoming neurons. We have validated these iMNs to possess gene expression patterns and electrophysiology signatures typical of those seen in bona fide motor neurons. Furthermore, we demonstrate that iMNs generated under p53 inhibition form functional neuromuscular junctions with chick muscle upon introduction of an optimized, light-activated channelrhodopsin protein into our reprogramming system. Deeper analysis reveals that these iMNs possess more mature, complex morphological components and physiology, suggesting that p53 inhibition permits somatic cells to reach more complete conversion. Notably, it appears that p53 acts through an apoptosis-independent mechanism during the early stages of this reprogramming system to increase conversion efficiency. We have also shown that following p53 inhibition, there is a striking and early upregulation of neurogenic related genes, revealing that p53 potentially plays a potential role in neural lineage commitment. Our results demonstrate that p53 plays a role in neural lineage commitment and inhibition of this pathway allows the efficient production of subtype-specific neurons by direct lineage conversion that can be translated into patient-specific studies of neurodegenerative diseases.