Neddylation E1 obligatory subunit Nae1 is critical to neuromuscular junction development and maintenance

Rapsyn is a scaffold protein that is thought to anchor acetylcholine receptors at the neuromuscular junction (NMJ). We showed that it may be an E3 ligase that regulates NMJ development by neddylation. To obtain genetical evidence, we mutated Nae1 (APP-BP1), an obligatory subunit of the neddylation E1 enzyme specifically in muscle cells. The mutation decreased the stability of AChRα, reduced AChR clustering, and impaired NMJ development in Pax7-Cre;Nae1f/f mice (of either sex) and caused neonatal lethality. Moreover, while NMJs were normal in heterozygous mutant mice of Nae1 or rapsyn C366A (a knockin mutation that eliminates E3 ligase activity), double heterozygous mutant mice (of either sex) displayed NMJ deficits, indicative of a genetic interaction between Nae1 and rapsyn. These results provide genetic evidence for a role of neddylation in NMJ formation and support the notion that rapsyn serves as a neddylation E3 ligase in NMJ formation. Significance Statement Rapsyn possesses E3 ligase activity; a knockin mutation (C366A) that wipes out E3 ligase activity prevents NMJ formation in mice. But E3 ligases may also catalyze ubiquitination or sumoylation in addition to neddylation. This glaring gap hampers our understanding of how neddylation regulates NMJ formation. Here, we provided genetic evidence that neddylation is indeed essential. We generated muscle-specific mutant ( Pax7-Cre;Nae1f/f ) mice, investigated the impacts of heterozygous mutations of C366A and Nae1 alone and in combination and found that double heterozygous mutations caused NMJ deficits that were not observed in either heterozygous strain. These results were indicative of a genetic interaction between Nae1 and rapsyn, supporting the hypothesis that rapsyn serves as an E3 ligase to regulate NMJ formation via neddylation.