Loss of motoneuron-specific microRNA-218 causes systemic neuromuscular failure

Evidence is mounting that defective RNA metabolism is central to the pathogenesis of diseases affecting motoneurons (e.g. amyotrophic lateral sclerosis and spinal muscular atrophy). Yet, our understanding of motoneuron-specific gene regulatory pathways is largely limited to those mediated by transcription factors. Investigations into motoneuron-specific, RNA-mediated regulatory pathways (such as those involving microRNAs), may provide novel insights into potential pathogenic mechanisms. In this thesis, I identify a single microRNA (miR-218) that is both highly enriched and abundantly expressed in murine motoneurons. Using a combination of RNA sequencing and mouse genetics, I identify novel alternative promoters embedded within the Slit2/3 genes that contribute to miR-218’s specific expression in brainstem and spinal motoneurons. My most informative and exciting experiments derive from investigation of miR-218 knockout mice, generated by CRISPR-mediated multiplexed deletions of all four miR-218 alleles. Motoneurons in these mice exhibit dramatic neuromuscular synaptic failure, hyperexcitability, and cellular degeneration – the hallmarks of motoneuron diseases. Without miR-218, mice exhibit flaccid paralysis and neonatal death, firmly demonstrating that this microRNA is indispensable to motoneuron function and survival. How can a single, small non-coding RNA have such a fundamental importance to motoneuron gene regulation? Gene profiling wild type and knockout motoneurons uncovers an impressive network of hundreds of mRNAs that are under miR-218 mediated repression. Using differential expression and unbiased 3’UTR motif-enrichment analysis, I find that miR-218 target genes are expressed lower in motoneurons versus other subpopulations of spinal and cortical neurons. Moreover, I find that miR-218 doesn’t merely reinforce/potentiate target genes’ reduced expression (as has been suggested for microRNAs in general), but instead constitutively and independently drives the repression of its target network in motoneurons.In summary, this thesis (1) details the identification of one of the most dramatic examples of a neuronal subtype-specific microRNA in mammals, (2) establishes that loss of miR-218 results in neuromuscular failure and motoneuron degeneration, and (3) reveals that motoneurons use miR-218 to tune-down a genetic network expressed across other neuronal cell populations