Latent effects of early-life methylmercury exposure on motor function in Drosophila.

The developmental toxicant, methylmercury (MeHg), can elicit motor deficits that last well into adulthood. Recent studies using Drosophila showed that the developing musculature is sensitive to high doses of MeHg, where a larval feeding paradigm resulted in compromised myotendinous junction (MTJ) formation during development, by a mechanism involving the NG2 homologue, kon-tiki (kon). Low-dose exposures to MeHg that do not produce muscle pathology during development, nevertheless result in impaired flight behavior later in adult life. The present study evaluated the potential for relatively low-dose exposure to produce latent adult muscle pathology and motor impairments, as assayed by climbing and flight, as well as to evaluate molecular mechanisms that may contribute to motor deficits. Wildtype larvae were fed 0, 2, 2.5, or 5 µM MeHg laden food until eclosion. The effect of 5 µM MeHg on MTJ-related gene expression during pupal development was assessed via quantitative RT-qPCR analysis. Upon eclosion, adults were transferred to standard food bottles for 4, 11, or 30 days prior to motor testing. Survivorship (%) was determined from a subset of 200 flies per treatment. Average climbing speed (cm/s) was quantified 4-days post-eclosion (PE). Flight ability was assayed 11- or 30-days PE by measuring landing height (cm) of flies dropped into an adhesive-lined vertical column. In parallel, total body mercury was measured to estimate the influence of residual MeHg at the time of motor testing. Muscle morphology was assessed using immuno-fluorescence microscopy. Exposure to 5uM MeHg significantly reduced climbing speed, and flight ability 4 and 11 - days PE, respectively. While age-related flight deficits were seen in each sex, flight deficits due to MeHg persisted to 30-day PE timepoints exclusively in males. Expression of kon was upregulated across the window of pupal development essential to establishing adult MTJ. However, experimentally restricting the induction of comparable levels of kon to muscle during the same periods did not recapitulate the flight deficits, indicating that muscle-specific induction of kon alone is not sufficient to contribute to latent flight impairments. Adult flight muscle morphology of 11-day PE flies treated with 5 µM MeHg was indistinct from controls, implying muscle structure is not grossly perturbed to impair flight. Collectively, the current data suggest that developmental exposure to 5 µM MeHg reduces flight ability in each sex at 11 day-PE and that latent deficits at 30-day PE are male-specific. It remains to be determined whether the developing MTJ of Drosophila is a sensitive target of MeHg, and whether or not kon acts in conjunction with additional MTJ factors to constitute a MeHg target.

Neuroligin-1 Is a Mediator of Methylmercury Neuromuscular Toxicity.

Methylmercury (MeHg) is a developmental toxicant capable of eliciting neurocognitive and neuromuscular deficits in children with in utero exposure. Previous research in Drosophila melanogaster uncovered that developmental MeHg exposure simultaneously targets the developing musculature and innervating motor neuron in the embryo, along with identifying Drosophila neuroligin 1 (nlg1) as a gene associated with developmental MeHg sensitivity. Nlg1 and its transsynaptic partner neurexin 1 (Nrx1) are critical for axonal arborization and NMJ maturation. We investigated the effects of MeHg exposure on indirect flight muscle (IFM) morphogenesis, innervation, and function via flight assays and monitored the expression of NMJ-associated genes to characterize the role of Nlg1 mediating the neuromuscular toxicity of MeHg. Developmental MeHg exposure reduced the innervation of the IFMs, which corresponded with reduced flight ability. In addition, nlg1 expression was selectively reduced during early metamorphosis, whereas a subsequent increase was observed in other NMJ-associated genes, including nrx1, in late metamorphosis. Developmental MeHg exposure also resulted in persistent reduced expression of most nlg and nrx genes during the first 11 days of adulthood. Transgenic modulation of nlg1 and nrx1 revealed that developing muscle is particularly sensitive to nlg1 levels, especially during the 20-36-h window of metamorphosis with reduced nlg1 expression resulting in adult flight deficits. Muscle-specific overexpression of nlg1 partially rescued MeHg-induced deficits in eclosion and flight. We identified Nlg1 as a muscle-specific, NMJ structural component that can mediate MeHg neuromuscular toxicity resulting from early life exposure.

Tissue-specific Nrf2 signaling protects against methylmercury toxicity in Drosophila neuromuscular development.

Methylmercury (MeHg) can elicit cognitive and motor deficits due to its developmental neuro- and myotoxic properties. While previous work has demonstrated that Nrf2 antioxidant signaling protects from MeHg toxicity, in vivo tissue-specific studies are lacking. In Drosophila, MeHg exposure shows greatest developmental toxicity in the pupal stage resulting in failed eclosion (emergence of adults) and an accompanying 'myosphere' phenotype in indirect flight muscles (IFMs). To delineate tissue-specific contributions to MeHg-induced motor deficits, we investigated the potential of Nrf2 signaling in either muscles or neurons to moderate MeHg toxicity. Larva were exposed to various concentrations of MeHg (0-20 µM in food) in combination with genetic modulation of the Nrf2 homolog cap-n-collar C (CncC), or its negative regulator Keap1. Eclosion behavior was evaluated in parallel with the morphology of two muscle groups, the thoracic IFMs and the abdominal dorsal internal oblique muscles (DIOMs). CncC signaling activity was reported with an antioxidant response element construct (ARE-GFP). We observed that DIOMs are distinguished by elevated endogenous ARE-GFP expression, which is only transiently seen in the IFMs. Dose-dependent MeHg reductions in eclosion behavior parallel formation of myospheres in the DIOMs and IFMs, while also increasing ARE-GFP expression in the DIOMs. Modulating CncC signaling via muscle-specific Keap1 knockdown and upregulation gives a rescue and exacerbation, respectively, of MeHg effects on eclosion and myospheres. Interestingly, muscle-specific CncC upregulation and knockdown both induce lethality. In contrast, neuron-specific upregulation of CncC, as well as Keap1 knockdown, rescued MeHg effects on eclosion and myospheres. Our findings indicate that enhanced CncC signaling localized to either muscles or neurons is sufficient to rescue muscle development and neuromuscular function from a MeHg insult. Additionally, there may be distinct roles for CncC signaling in myo-morphogenesis.

Methylmercury myotoxicity targets formation of the myotendinous junction.

Methylmercury (MeHg) is a ubiquitous environmental contaminant and developmental toxicant known to cause a variety of persistent motor and cognitive deficits. While previous research has focused predominantly on neurotoxic MeHg effects, emerging evidence points to a myotoxic role whereby MeHg induces defects in muscle development and maintenance. A genome wide association study for developmental sensitivity to MeHg in Drosophila has revealed several conserved muscle morphogenesis candidate genes that function in an array of processes from myoblast migration and fusion to myotendinous junction (MTJ) formation and myofibrillogenesis. Here, we investigated candidates for a role in mediating MeHg disruption of muscle development by evaluating morphological and functional phenotypes of the indirect flight muscles (IFMs) in pupal and adult flies following 0, 5, 10, and 15 µM MeHg exposure via feeding at the larval stage. Developmental MeHg exposure induced a dose-dependent increase in muscle detachments (myospheres) within dorsal bundles of the IFMs, which paralleled reductions eclosion and adult flight behaviors. These effects were selectively phenocopied by altered expression of kon-tiki (kon), a chondroitin sulfate proteoglycan 4/NG2 homologue and a central component of MTJ formation. MeHg elevated kon transcript expression at a crucial window of IFM development and transgene overexpression of kon could also phenocopy myosphere phenotypes and eclosion and flight deficits. Finally, the myosphere phenotype resulting from 10 µM MeHg was partially rescued in a background of reduced kon expression using a targeted RNAi approach. Our findings implicate a component of the MTJ as a MeHg toxicity target which broaden the understanding of how motor deficits can emerge from early life MeHg exposure.