Early Nicotine Exposure Altered Larval Neuromuscular Function and Adult Germline Apoptosis via MiRNA-Dependent Mechanisms

Abstract

MicroRNAs fine tune the expression of target genes needed for homeostasis. Our pilot study showed that nicotine altered 17% miRNAs in C. elegans larvae. Here, we investigated the role of microRNAs in mediating nicotine-induced adolescent and adult-onset phenotypes: larval pharyngeal pumping, adult germ line apoptosis, and survival. Worms were treated with nicotine during the postembryonic stages with either 20µM or 20mM. For the pharyngeal pumping assay, videos were recorded for at least 15 worms/group to compute the mean pumps/sec. For adult apoptosis and lifespan assays, worms were washed off treatment before gametogenesis and allowed to grow till adulthood on nicotine-free media. Day 1 adults were stained with SYTO12 or acridine orange and apoptotic cells were counted for at least 50 gonads per treatment group. The remaining 200 worms were counted and transferred every 1-2 days until all worms died. Kaplan-Meier curves were used to compare the survival curves across treatment groups. Our data suggests that post-embryonic nicotine exposure inhibited pharyngeal pumping and increased germ cell apoptosis in WT, but not mutant worms. Due to the conservation of those signaling pathways, our results provide insights for miRNA-based treatment strategies for nicotine disorders in early and late stages.