Reducing the Excess Activin Signaling Rescues Muscle Degeneration in Myotonic Dystrophy Type 2 Drosophila Model

Abstract

Expanded non-coding RNA repeats of CCUG are the underlying genetic causes for myotonic dystrophy type 2 (DM2). There is an urgent need for effective medications and potential drug targets that may alleviate the progression of the disease. In this study, 3140 small-molecule drugs from FDA-approved libraries were screened through lethality and locomotion phenotypes using a DM2 Drosophila model expressing 720 CCTG repeats in the muscle. We identified ten effective drugs that improved survival and locomotor activity of DM2 flies, including four that share the same predicted targets in the TGF-β pathway. The pathway comprises two major branches, the Activin and BMP pathways, which play critical and complex roles in skeletal development, maintenance of homeostasis, and regeneration. The Drosophila model recapitulates pathological features of muscle degeneration in DM2, displaying shortened lifespan, a decline in climbing ability, and progressive muscle degeneration. Increased levels of p-smad3 in response to activin signaling were observed in DM2 flies. Decreased levels of activin signaling using additional specific inhibitors or genetic method ameliorated climbing defects, crushed thoraxes, structure, and organization of muscle fibers. Our results demonstrate that a decrease in activin signaling is sufficient to rescue muscle degeneration and is, therefore, a potential therapeutic target for DM2.

Keywords: Drosophila; TGF-β pathway; chemical screen; myotonic dystrophy type 2.

Figure 1

The studied drugs rescued lethality and motor function in the DM2 fly model. (A) The four compounds targeting the TGF-β pathway rescued the lethality of 24B-GAL4 > (CCTG)₇₂₀. (B) Upon drug treatment separately, climbing ability of Mef2-GAL4 > (CCTG)₇₂₀ measured at day 5. (C) IFM transversal sections of Mef2-GAL4 > (CCTG)₇₂₀ with H&E (hematoxylin and eosin) staining at day 5. Images were taken at 100× magnification with a Leica DM5000. (D) Quantitative analysis of muscle areas of Mef2-GAL4 > (CCTG)₇₂₀ with the administration of the drug. All statistical models were tested using one-way ANOVA, * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 2

Flies expressing CCTG repeats in the muscle at adult stage show shorter lifespan, declined climbing ability, and muscle degeneration. (A) Flies expressing 720 CCTG repeats in adult muscle have a significantly shorter lifespan in comparison to those expressing 16 CCTG repeats. (B) Climbing ability of flies measured at days 5 and 15. (C) IFM transversal sections of flies with H&E (hematoxylin and eosin) staining at days 5 and 15. The flies of (CCTG)₇₂₀ displayed muscle degeneration. Images were taken at 100× magnification with a Leica DM5000. (D) Quantitative analyses of muscle areas. (E) Confocal images of the myofiber of IFM in 15-day-old male flies showed increased cleaved caspase 3 in the muscle of (CCTG)₇₂₀. Cleaved caspase 3 (green), DAPI (blue). Bar equals 50 μm. All statistical models were tested using two-way ANOVA, *** p < 0.001.

Figure 3

P-smd3 is increased when expressing CCTG repeats in the adult muscle of flies. (A) Confocal images of the myofiber of IFM in 15-day-old male flies at 19 °C (the expression of CCTG repeats was suppressed) or 29 °C (the expression of CCTG repeats was activated. P-smad3 in the nucleus marked with red arrows. Increased p-smad3 in adult muscle cell nucleus of (CCTG)₇₂₀ was detected compared to (CCTG)₁₆ at 29 °C. p-smad3 (gray), and DAPI (blue). Bar equals 20 μm. (B) Western blotting was used to measure p-smad3 levels in DM2 flies that were increased compared with the control flies at 29 °C. (C) Bar graphs show changes in the quantitative analysis of relative smad3 phosphorylation level after (CCTG)₇₂₀ expression. Data from three independent experiments are shown as means ± SEM (p-values were determined by two-way ANOVA, *** p < 0.001).

Figure 4

Reducing the activin signaling could improve climbing ability and crushed thoraxes of DM2 flies. (A,B) Climbing ability of flies measured at day 15. Attenuating key molecules, med or smox, from the activin signaling ameliorated climbing defects in (CCTG₎₇₂₀ flies (A). (CCTG)₇₂₀ flies treated with specific inhibitors of activin signaling, the concentrations of SB431542 and SIS3: 60 µM, 20 µM (B). (C,D) IFM transversal sections of flies with H&E (hematoxylin and eosin) staining at day 15. Images were taken at 100× magnification with a Leica DM5000. Reducing med or smox could suppress crushed thoraxes in (CCTG)₇₂₀ flies (C). (CCTG)₇₂₀ flies treated with SB431542 or SIS3. The concentrations of SB431542 and SIS3: 60 µM, 20 µM (D). (E) Quantitative analyses of muscle areas of (CCTG)₇₂₀ flies crossed with med or smox RNAi lines (15 days after eclosion). (F) Quantitative analyses of muscle areas of (CCTG)₇₂₀ flies exposed to SB431542 or SIS3 (15 days after eclosion). The concentrations of SB431542 and SIS3 were 60 µM and 20 µM, respectively. All statistical models were tested using one-way ANOVA, *** p < 0.001.

Figure 5

Reducing the activin signaling could rescue myofiber defects of DM2 flies. (A,B) Confocal images of the myofiber of 15-day-old male flies. DAPI (blue) and phalloidin (red). Bar equals 10 μm. Attenuating med or smox ameliorated myofiber defects in (CCTG)₇₂₀ flies (A). (CCTG)₇₂₀ flies exposed to SB431542 and SIS3. The concentrations of SB431542 and SIS3 were 60 µM and 20 µM, respectively. Attenuating med or smox ameliorated myofiber defects in (CCTG)₇₂₀ flies (B).