A C. elegans genome-wide RNAi screen for altered levamisole sensitivity identifies genes required for muscle function.

Timothy Chaya,Michael Clupper,Elaine N Miller,Kirsten Kervin,Andy Lam,Jessica E Tanis,Shrey Patel,Erin M Smith,A J M Walhout

doi:10.1093/g3journal/jkab047

Abstract

At the neuromuscular junction (NMJ), postsynaptic ionotropic acetylcholine receptors (AChRs) transduce a chemical signal released from a cholinergic motor neuron into an electrical signal to induce muscle contraction. To identify regulators of postsynaptic function, we conducted a genome-wide RNAi screen for genes required for proper response to levamisole, a pharmacological agonist of ionotropic L-AChRs at the Caenorhabditis elegans NMJ. A total of 117 gene knockdowns were found to cause levamisole hypersensitivity, while 18 resulted in levamisole resistance. Our screen identified conserved genes important for muscle function including some that are mutated in congenital myasthenic syndrome, congenital muscular dystrophy, congenital myopathy, myotonic dystrophy, and mitochondrial myopathy. Of the genes found in the screen, we further investigated those predicted to play a role in endocytosis of cell surface receptors. Loss of the Epsin homolog epn-1 caused levamisole hypersensitivity and had opposing effects on the levels of postsynaptic L-AChRs and GABAA receptors, resulting in increased and decreased abundance, respectively. We also examined other genes that resulted in a levamisole-hypersensitive phenotype when knocked down including gas-1, which functions in Complex I of the mitochondrial electron transport chain. Consistent with altered ATP synthesis impacting levamisole response, treatment of wild-type animals with levamisole resulted in L-AChR–dependent depletion of ATP levels. These results suggest that the paralytic effects of levamisole ultimately lead to metabolic exhaustion.

Highlights

Acetylcholine (ACh) released from motor neurons activates postsynaptic ionotropic ACh receptors (AChRs), resulting in an electrical signal that leads to muscle contraction.Disruption of cholinergic signaling at the neuromuscular junction (NMJ) is the underlying cause of severe muscle weakness observed in individuals with congenital myasthenic syndromes (CMS) and the autoimmune syndrome Myasthenia gravis (Engel et al.2015)
Altered time to levamisole-induced paralysis can be used to identify genes that impact the balance of postsynaptic excitatory and inhibitory signaling
While levamisole resistant mutants have been isolated, mutations in genes required for both viability and levamisole response could not be identified in the forward genetic screens, and there has never been a genetic screen for levamisole

Summary

Introduction

Acetylcholine (ACh) released from motor neurons activates postsynaptic ionotropic ACh receptors (AChRs), resulting in an electrical signal that leads to muscle contraction.Disruption of cholinergic signaling at the neuromuscular junction (NMJ) is the underlying cause of severe muscle weakness observed in individuals with congenital myasthenic syndromes (CMS) and the autoimmune syndrome Myasthenia gravis (Engel et al.2015). Clinical features of some congenital myopathies and muscular dystrophies resemble CMS despite different genetic causes and muscle histopathology (Mahjneh et al 2013; Rodríguez Cruz et al 2014; Montagnese et al 2017). This suggests that characterizing mechanisms which regulate postsynaptic cholinergic signaling could provide insight into multiple neuromuscular disorders. A forward genetic screen for mutants with strong levamisole resistance identified mutations in 12 different genes encoding subunits of the L-AChR as well as proteins required for L-AChR trafficking, clustering, and muscle contraction (Lewis et al.1980; Gally et al 2004; Eimer et al 2007; Gendrel et al 2009). Levamisole resistant mutants have been isolated in a forward genetic screen for animals that initially exhibit paralysis in response to levamisole, but subsequently adapt and regain motility (Rapti et al 2011; Boulin et al 2012; Richard et al 2013; D’alessandro et al 2018)

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Results

Conclusion