AC. elegansgenome-wide RNAi screen for altered levamisole sensitivity identifies genes required for muscle function

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.

An updatedC. elegansnuclear body muscle transcriptome for studies in muscle formation and function

“…The physiology of its body muscle shares a high degree of similarity to the skeletal muscle of vertebrates with several mutually conserved genes (Lecroisey et al 2007; Gieseler et al 2017; Hrach et al 2020; Ellwood et al 2021b). In addition, C. elegans is a popular choice for studying disorders affecting humans, including muscular dystrophy (Hrach et al 2020; Chaya et al 2021; Ellwood et al 2021a), since many human disease genes when introduced to worms phenocopy the symptoms (Markaki and Tavernarakis 2020; Natale et al 2020; Chandler et al 2021; Yue et al 2021).…”

“…In addition, over 200 proteins have been identified in C. elegans to be essential for the assembly, maintenance, and function of the sarcomere, many of which have human orthologs [2]. C. elegans is also a popular choice for studying disorders affecting humans, including muscular dystrophy [3][4][5], since many human disease genes, when introduced in worms phenocopy the symptoms [6][7][8][9].…”

Patient-specific variants of NFU1/NFU-1 disrupt cholinergic signaling in a model of multiple mitochondrial dysfunctions syndrome 1

“…Notably, a recent RNAi screen determined that knockdown of genes involved in ATP synthesis pathways result in hypersensitivity to levamisole ( Chaya et al, 2021 ). These results indicate that disruption of mitochondrial metabolism in body wall muscle is sufficient to induce levamisole hypersensitivity.…”

Vitamin B12 impacts amyloid beta-induced proteotoxicity by regulating the methionine/S-adenosylmethionine cycle

“…The supernatant was collected and moved to a fresh tube. ATP quantitation was performed with the ATP Bioluminescence Assay Kit CLS II (Roche Diagnostics) using a Glomax Multi-Detection System reader as described ( Chaya et al, 2021 ). ATP was normalized to protein content measured with the Pierce BCA protein assay kit (ThermoFisher Scientific).…”