PROJECT SUMMARY/ABSTRACT Amyotrophic lateral sclerosis (ALS) is a relatively-rare disease that leads to motor neuron degeneration in 1:50,000 people in the US. Since mutations in ~50 different genes have been linked to ALS, there is the daunting possibility that treatments will require the development of many separate therapies. Consequently, identification of the shared pathways and key nodal points affected in multiple forms of sporadic and familial ALS could provide greater impact for the overall ALS community. In this regard much effort is currently focused on pathways relevant to protein-stasis, autophagy and cell stress, since protein aggregates are a common feature of ALS. This proposal takes a complimentary approach by examining the mechanistic role played by an essential motor neuron-specific microRNA (miR-218) that is affected by ALS. Gene expression studies to identify microRNAs dysregulated in sporadic and familial ALS consistently detect downregulation of miR-218. A recent analysis of ALS patients found a cohort with mutations in miR-218, suggesting insufficient miR-218 levels/activity may be a risk factor for the disease. Because many ALS-linked genes affect RNA metabolism and microRNA processing complexes, it is hypothesized that miR-218 activity is downregulated in many types of ALS leading to a pattern of gene dysregulation that fails to sustain motor neurons. Conversely, it is predicted that ectopic miR-218 may restore proper gene expression in motor neurons and counteract ALS. While previous studies have established that miR-218 controls motor neuron connectivity in embryos, the goal of this grant is to identify the gene networks controlled by miR-218 in adult motor neurons using genetics to decrease (aim 1) and elevate (aim 2) miR-218 with precise spatiotemporal control. A floxed- miR-218 allele was created and following Cre-mediated deletion in adult motor neurons it was found that neuromuscular defects arose - indicating miR-218 is a critical regulatory molecule in mature motor neurons. In Aim 1 miR-218 will be conditionally deleted in adult mouse motor neurons and next generation RNA sequencing from single-nuclei will be used to (1a) uncover the miR-218 gene network in adult motor neurons, and (1b) cross- correlate miR-218-regulated genes with dysregulated genes in mouse models of ALS. In Aim 2, miR-218 will be conditionally (ectopically) expressed in mice to (2a) define non-cell-autonomous effects of mir-218, and (2b) model baseline levels of miR-218 in motor neurons that would be tolerable for potential ALS-therapies. These studies will pave the way for future experiments to directly test whether miR-218 can be used to attenuate ALS. To make this possible an independent but complementary R03 was submitted (see complimentary application) to allow others with ALS-models to explore this promising possibility with our miR-218 reagents. This R21 grant is an important step toward understanding the mechanism-of-action of miR-218...