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

Abstract

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.

Keywords: Drosophila; Methylmercury; Myotoxicity; Neuromuscular development; Nrf2 signaling.

Fig. 1. Endogenous CncC signaling in DIOMs.

a A schematic of the pupal body plan. The brain and ventral nerve cord (VNC) are located in the head and extend into the thorax. The indirect flight muscles (IFMs) are located dorsally in the thorax. The dorsal internal oblique muscles (DIOMs) form parallel configurations in A1-A5 abdominal segments. b Expression of the ARE-GFP reporter in the DIOMs from stage p4 through p7 (~10–48h APF) (Also see Movie S1). Hours after puparium formation (APF) indicated at the bottom of each image. c Expression of Cnc-GAL4>UAS-RFP at stage p7.

Fig. 2. An in vivo ARE activity reporter is responsive to CncC and Keap1 expression.

a A schematic for inducing gene expression using the ARE-GFP; Mef2-GAL4, Tub-GAL80^TS strain. b Representative images of non-induced and induced 48h APF pupae. Top row is of age-matched, temperature control pupa of each genotype. The bottom row is of pupa after a 36-hour 29°C induction starting at head eversion (HE). c Representative Western blot of GFP and actin protein from whole pupa protein extracts from each genotype and temperature. The Canton S (CS) wild-type strain acts as a negative control for GFP protein. d Quantification of GFP band intensities. CncC and Keap1 experimental groups are normalized to respective w1118 temperature controls (n = 3, mean ± s.d.m., One-way ANOVA with Tukey’s HSD, p<0.05, significant differences indicated by different letter labels). Comparison of 29°C w1118 and CncC^RNAi band intensities by Student’s t test is statistically significant (**p<0.01).

Fig. 3. MeHg induces DIOM myospheres and activates the CncC pathway.

a Representative image of DIOM morphology in ARE-GFP pupa after larval MeHg exposure. b Quantification of myospheres in most medial DIOMs in A2-A5 (denoted by the white asterisks) in ARE-GFP pupa upon larval MeHg exposure (n ≥ 22 abdomens/treatment, Kruskal-Wallis test with Dunn’s post-hoc for multiple comparisons, p<0.05, significant differences indicated by different letter labels). c Eclosion ability of ARE-GFP pupa upon larval MeHg exposure (n = 3, 450 flies/treatment, mean ± s.d.m., Z-test between each treatment group to 0 μM MeHg, ****p<0.0001). d Mean nuclei GFP fluorescence intensity (arbitrary units) of DIOMs upon larval MeHg exposure (100 individual nuclei/abdomen, n ≥ 5 abdomens/treatment). e Representative Western blot of GFP and actin protein bands from whole ARE-GFP pupa protein extracts upon larval MeHg exposure. The Canton S (CS) wild-type strain is a negative control for GFP protein. Quantification of GFP protein band intensities (normalized to actin) is represented as a bar graph (n = 3, d, e mean ± s.d.m., One-way ANOVA with Tukey’s HSD, p<0.05, significant differences indicated by different letter labels). f Gene expression by qPCR of CncC and CncC-regulated genes in Canton S pupa after larval exposure to 0, 5, or 15 μM MeHg (n = 3, mean ± s.d.m., One-way ANOVA with Tukey’s HSD, *p<0.05, **p<0.01).

Fig. 4. Muscle-specific CncC signaling modulation of MeHg effects on eclosion ability and DIOM morphology.

a, b Effect of muscle-specific CncC overexpression (Mef2>CncC) and knockdown (Mef2>CncC^RNAi) on eclosion. c, d Effect of Keap1 knockdown (>Keap1^RNAi) and overexpression (>Keap1) on eclosion compared to control (Mef2>w1118) (a-d n = 3, 450 flies/genotype/treatment, mean ± s.d.m., Z-test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). e Representative images of MeHg effect on DIOM morphology without (>w1118 control) or with knockdown (>Keap1^RNAi) or overexpression (>Keap1) of Keap1. Number of myospheres originating from the most medial DIOMs in the A2-A5 abdominal segments (indicated by white asterisks) were quantified: (f) 0 μM, (g) 5 μM, and (h) 10 μM MeHg (f-h n ≥ 20 abdomens/genotype/treatment, mean ± s.d.m., Kruskal-Wallis test with Dunn’s post-hoc for multiple comparisons, p<0.05, significant differences indicated by different letter labels).

Fig. 5. The DIOMs are not required for eclosion.

a Representative images of 45h APF (~p7) DIOMs morphology with control (CncC RFP>w1118) and CncC expression (CncC RFP>CncC) directed by the CncC Gal4 driver. b The number of myospheres originating from the most medial DIOMs in the A2-A5 abdominal segments (indicated by white asterisks) were quantified (n = 20 abdomens/genotype, mean ± s.d.m., Mann-Whitney test, ****p<0.0001). c Eclosion ability (n = 3, 450 flies/genotype, mean ± s.d.m, Z test).

Fig. 6. Muscle-specific CncC signaling modulation of MeHg effects on IFM morphology.

a Representative images of MeHg effects on IFM morphology with either no change (>w1118), knockdown (>Keap1^RNAi), or overexpression (>Keap1) of Keap1 with the Mef2-RFP-GAL4 driver. b-d Number of myospheres in each thorax were quantified at indicated MeHg treatment (n ≥ 20/genotype/treatment, mean ± s.d.m., Kruskal-Wallis test with Dunn’s post-hoc for multiple comparisons, p<0.05, significant differences indicated by different letter labels). e Representative images of IFM morphology with either no change (>w1118), knockdown (>Keap1^RNAi), or overexpression (>Keap1) of Keap1 with the MHC-RFP-GAL4 driver. f-h Number of myospheres in each thorax were quantified at indicated MeHg treatment (n ≥ 15/genotype/treatment, mean ± s.d.m., Kruskal-Wallis test with Dunn’s post-hoc for multiple comparisons, p<0.05, significant differences indicated by different letter labels).

Fig. 7. Neural-specific CncC signaling modulation of MeHg effects on eclosion.

a Expression domain of the Elav(III)-GAL4 driver. b Expression domain of Elav(I)-GAL4 driver. c-f Eclosion ability with CncC overexpression (>CncC) or Keap1 knockdown (>Keap1^RNAi) under control of the Elav(III)-GAL4 driver (c, d) or Elav(I)-GAL4 driver (e, f) (n = 3, 450 flies/genotype/treatment, mean ± s.d.m., Z-test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Fig. 8. Glutamatergic neuron-specific CncC signaling modulation of MeHg effects on eclosion.

a, b Expression pattern of the Elav(III)-GAL4 and OK371-GAL4 drivers (ventral view). c, d Eclosion ability with OK371-GAL4 driven CncC overexpression (>CncC) and knockdown (>CncC^RNAi) compared to control (>w1118). e, f Eclosion ability with OK371-GAL4 driven Keap1 knockdown (>Keap1^RNAi) and overexpression (>Keap1) compared to control (>w1118) (c-f n = 3, 450 flies/genotype/treatment, mean ± s.d.m., Z-test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). g Representative images of MeHg effects on IFM morphology with either no change (>w1118), knockdown (>Keap1^RNAi), or overexpression (>Keap1) of Keap1 in glutamatergic neurons. Number of missing dorsoventral muscles (DVMs, indicated by white asterisks) and dorsal lateral muscles (DLMs, indicated by white arrows) in each thorax were quantified at indicated MeHg treatment (h-j n ≥ 20/genotype/treatment, mean ± s.d.m., Kruskal-Wallis test with Dunn’s post-hoc for multiple comparisons within same muscle group, p<0.05, significant differences indicated by different letter labels).