Myogenic Targets of Methylmercury Toxicity During Development

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Program in Toxicology, 2018.Methylmercury (MeHg) is a persistent and ubiquitous toxicant, through which the primary route of exposure is fish consumption. The mechanisms of MeHg toxicity and factors that contribute to MeHg susceptibility are not fully understood. MeHg has been studied primarily as a developmental neurotoxicant, with historic accidental poisonings and more recent epidemiological studies showing cognitive and motor deficits in children exposed in utero. Previously, motor deficits have been associated with damage to the neurons that control motor function. However, developing muscle has largely remained unexplored as a target of MeHg toxicity. Recent studies have pointed to the disruption of muscle development as a potential contributor to motor deficits associated with MeHg toxicity. Developmental exposures to MeHg in a Drosophila model have shown embryonic and pupal muscle disruption. In these studies, the Notch Receptor downstream transcriptional repressor, Enhancer-of-split mdelta (E(spl)mδ), was found to be both expressed in muscle and specifically upregulated by MeHg. Furthermore, Notch downstream genes involved in myoblast adhesion and fusion were associated with MeHg susceptibility. Therefore, I hypothesized that MeHg inhibits myotube development via the upregulation of Notch signaling. I tested this hypothesis with the Drosophila model and the mouse C2C12 myoblast model. The role of E(spl)mδ as muscle-specific mediator of MeHg toxicity was examined by using the GAL4-UAS system in Drosophila to modulate expression of E(spl)mδ in muscle, in combination with MeHg. To understand whether Notch signaling was also implicated in MeHg toxicity in a mammalian model, Notch target gene expression in response to MeHg was examined in C2C12 cells using quantitative PCR methods (RT-qPCR). MeHg disruption of muscle development was further explored using immunoctyochemistry and RT-qPCR to examine the processes of myotube formation and cell fate. In the Drosophila model, overexpression of E(spl)mδ partially mimicked a MeHg phenotype, while knockdown of E(spl)mδ in muscle protected against MeHg toxicity, suggesting that E(spl)mδ is a mediator in MeHg-induced toxicity in this tissue. In the C2C12 model, MeHg was shown to inhibit myotube formation via the inhibition of myoblast differentiation in a dose-dependent manner. Surprisingly, Notch signaling was downregulated with MeHg in this cell culture model, pointing to divergent mechanisms between these two models. However, MeHg also caused a robust downregulation of myogenin (MyoG), a transcription factor critical to myoblast differentiation. Evidence points to the downregulation of myogenin by MeHg resulting from negative impacts on mitochondria, a known target of MeHg. Overall, my results establish that developing muscle, specifically the underlying mechanisms of myoblast differentiation and myotube formation, is a direct target of MeHg toxicity. This research has broadened our understanding of targets beyond the nervous system, and gives evidence to myogenic targets contributing to motor deficits seen with MeHg exposure