Enhancing GABAergic Transmission Improves Locomotion in a Caenorhabditis elegans Model of Spinal Muscular Atrophy

Enhanced survival benefits in SMA worm models are mediated by the daf-2/daf-16 pathway. A, Knock-down daf-16 by RNAi feeding shortens lifespan of smn-1(ok355) mutant animals by 12.5% when compared to EV (p < 0.001, Log-rank test). B, smn-1(ok355);daf-2(e1370) mutant animals live three more days than smn-1(ok355) mutant animals, and this beneficial effect is suppressed by feeding RNAi targeting daf-16 (p < 0.001, Log-rank test). C, smn-1(rt248);daf-2(e1370) mutant animals live 15 d, and this beneficial effect is suppressed by feeding RNAi that targets all isoforms of daf-16 (p < 0.001, Log-rank test). Feeding RNAi that targets daf16a or daf16f isoforms also suppress the longevity of smn-1(rt248);daf-2(e1370) mutant animals although the effect is less robust than the pan daf-16 RNAi effect (p < 0.001, Log-rank test). D, Overexpression of daf-16 using TJ356 (daf-16::gfp) strain prolongs lifespan of smn-1(ok355) mutant animals by 43.8% when compared to smn-1(ok355) mutant animals (p < 0.001, Log-rank test).

Reduced daf-2 signaling rescues the body curvature of smn-1(ok355) animals during swimming. A, A trace of the head and tail trajectories for typical individuals from each genotype (e.g., N2, smn-1(ok355), daf-2(e1370) and smn-1(ok355);daf-2(e1370)). There is no qualitative difference between N2 versus the daf-2(e1370) mutant animals while the smn-1(ok355) mutant animals swim more slowly and beat less frequently than N2 worms. The smn-1(ok355);daf-2(e1370) mutant animals closely resemble N2 worms. B, Curvature kymographs of representative individuals of each genotype reveals a common pattern shared by N2, daf-2(e1370), and smn-1(ok355);daf-2(e1370) mutant animals. In contrast, smn-1(ok355) mutant animals exhibits relatively little curvature (white color) compared to other genotypes at a region from approximately the middle of the body to just before the tail, implicating a regional loss of neuromuscular function. CW, clockwise; CCW, counterclockwise. C, Quantification of regional body curvature in smn-1 mutant animals. We quantified the body curvature of animals with four genotypes (N2, smn-1(ok355), daf-2(e1370), and smn-1(ok355);daf-2(e1370)) and find that the curvature of each group of worms appears to have local maxima when averaged over time (for details, see Extended Data Fig. 3-1). For the N2 and daf-2(e1370) mutant animals, the mean normalized curvature decreases from head to tail. The daf-2(e1370) group shows a significant low curvature at the tail region compared to the N2 group. The smn-1(ok355) group has a distinct curvature pattern. Its curvature decreases rapidly after the worm’s mid-point and then increases sharply near the tail. This pattern corresponds to the region of low curvature observed in Figure 3B. In the daf-2 mutant background (smn-1(ok355);daf-2(e1370)), body curvature during swimming is normalized and appears similar to N2 and daf-2(e1370) mutant animals. This is quantitative evidence that the daf-2 mutant background rescues the neuromuscular defect seen in the midsection and tail of the smn-1(ok355) mutant animals. ns, no significance.

Statistical analysis of biomechanical properties (BMP). Bar graphs represent BMP algorithm calculated (A) length (mm), (B) speed (mm/s), (C) frequency (Hz), (D) propulsive force (nN), and (E) mechanical power (pW) for the indicated genotypes at L2 or late time points. Data shown are mean ± SD, ≥10 animals per genotype were recorded. Nonparametric Kruskal–Wallis test followed by Dunn’s multiple comparison test for significant; * p < 0.05, ** p < 0.01, ***p < 0.005, ****p < 0.001, ns, no significance. A, Genotypes are well matched for length at the two time points. B, Age-dependent increase in speed was shown in both N2 and daf-2(e1370) mutant animals but not in smn-1(ok355) or smn-1(ok355);daf-2(e1370) mutant animals. C, Beat frequency diminishes over time in the smn-1(ok355) mutant animals and this is suppressed in the smn-1(ok355);daf-2(e1370) mutant animals. D, Age-dependent increase in propulsive force is seen in N2 and daf-2(e1370) mutant animals; this is not seen in the smn-1(ok355) mutant animals, but is rescued in the smn-1(ok355);daf-2(e1370) mutant animals. E, Age-dependent increase in mechanical power force is seen in N2 and daf-2(e1370) mutant animals; this is not seen in the smn-1(ok355) animals but is rescued in the smn-1(ok355);daf-2(e1370) mutant animals.

Morphologic examination of presynapse and postsynapse in smn-1 mutant animals. A, top, Confocal images of GFP expression in the presynaptic terminal of GABAergic motor neurons [synaptobrevin::GFP driven by unc-25 (GAD) promoter] in L2 and Late (L2 + 3 d) animals (four genotypes: N2, smn-1(ok355), daf-2(e1370) and smn-1(ok355);daf-2(e1370)). Bottom, Confocal images of GFP expression in postsynaptic GABAergic neuromuscular synapses [UNC-49::GFP driven by unc-49 (GABA receptor) promoter] in L2 and Late (L2 + 3 d) animals. All images were obtained from same region of dorsal nerve cord of the C. elegans strains and each line of puncta images are derived from a single animal as a spatial raster plot.

Quantification of puncta analysis reveals GABAergic synaptic defects in smn-1 mutant animals are partially rescued by reduction of daf-2 activity. A, B, Quantification of puncta width in presynapse and postsynapse at L2 or late stage in different genotypes of worm. C, D, Quantification of puncta intensity in presynapse and postsynapse at L2 or late stage in different genotypes of worm. E, F, Quantification of puncta number in presynapse and postsynapse at L2 or late stage in different genotypes of worm. G, H, Quantification of gap between puncta in presynapse and postsynapse at L2 or late stage in different genotypes of worm. *p < 0.05; **p < 0.01; ***p < 0.005; ****p < 0.001; ns, no significance.

Loss of smn-1 influences sensitive to pyridostigmine and levamisole. A, B, L2 animals of genotype N2, smn-1(ok355) or smn-1;daf-2(e1370) were subjected to the levamisole or pyridostigmine sensitivity test. At this age, smn-1(ok355) and smn-1;daf-2(e1370) mutant animals resisted levamisole greater than N2 animals. C, D, Late stage (L2 + 3 d) animals of genotype N2, smn-1(ok355), or smn-1;daf-2(e1370) were also subjected to levamisole or pyridostigmine sensitivity test. At the late stage, smn-1(ok355) but not smn-1(ok355);daf-2(e1370) mutant animals are hypersensitive to levamisole. In addition, smn-1(ok355) and smn-1(ok355);daf-2(e1370) mutant animals show hypersensitivity to the pyridostigmine bromide although daf-2(e1370) mutant animals suppresses the degree of hypersensitivity conferred by smn-1(ok355) mutant animals. Data points represent the mean ± SEM percentage of animals paralyzed at 30-min intervals over a period of 6 h; **p < 0.001; ns, no significance, Log-rank test.

smn-1(ok355) mutant animals have an abnormal body curvature during swimming that is rectified by reduced UNC-49::GFP overexpression but unaffected by snf-11(ok156). A, The body curvature of animals with four genotypes (N2, smn-1(ok355), oxls22(UNC-49::GFP), smn-1(ok355);oxls22(UNC-49::GFP)) were quantified (for details, see Extended Data Fig. 8-1). For the N2 and oxls22(UNC-49::GFP) mutant animals, the mean normalized curvature decreases from 1 (by definition) to ∼0.5 and 0.4 (head to tail orientation), respectively. The smn-1(ok355) group has a fundamentally different shape, decreasing rapidly after the worm’s mid-point to 0.4, and then sharply increasing near the tail; this decrease in curvature corresponds to the region of low curvature observed in Figure 3B,C. In the oxls22(UNC-49::GFP) mutant background (smn-1(ok355);oxls22(UNC-49::GFP) body curvature during swimming is normalized and appears similar to N2 and oxls22(UNC-49::GFP) mutant animals. This result is very similar to observations noted above (Fig. 3C) on the effect of the daf-2 mutant background on the neuromuscular defect seen in the midsection and tail of the smn-1(ok355) mutant animals. B, The body curvature of animals with four genotypes (N2, smn-1(ok355), snf-11(ok156), smn-1(ok355);snf-11(ok156)) were quantified (for details, see Extended Data Fig. 8-2). For the N2 and snf-11(ok156) mutant animals, the mean normalized curvature decreases from 1 (by definition) to ∼0.5 and 0.4 (head to tail orientation), respectively. The smn-1(ok355) group has a fundamentally different shape, decreasing rapidly after the worm’s mid-point to 0.4, and then sharply increasing near the tail; this decrease in curvature corresponds to the region of low curvature observed in Figures 3B,C-7A. In the snf-11(ok156) mutant background, the body curvature of smn-1(ok355);snf-11(ok156) mutant animals during swimming remains abnormal and is indistinguishable from the swimming of smn-1(ok355) mutant animals. ns, no significance.