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. 2017 Dec 1;10(12):1465–1480. doi: 10.1242/dmm.029736

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© 2017. Published by The Company of Biologists Ltd

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Fig. 2. — Lifespan and neuronal phenotypes in ATXN3 transgenics. (A) ATXN3-CAG89 worms had reduced lifespan compared with wild-type ATXN3-CAG10 or N2 worms [*P<0.05 and ****P<0.0001, respectively, by log-rank (Mantel–Cox) test; n=300-360]. ATXN3-CAG10 animals showed reduced lifespan compared with N2 wild-type worms [*P<0.05, by log-rank (Mantel–Cox) test; n=300-360]. The experiment was repeated three times. (B) CAG89 transgenics had a significantly higher paralysis phenotype compared with wild-type ATXN3-CAG10 transgenics [****P<0.0001, by log-rank (Mantel–Cox) test; n=270-300]. The experiment was repeated three times. (C) Cholinergic neuronal transmission was measured by determining the onset of paralysis induced by the cholinesterase inhibitor aldicarb. Adult day 1 unc-47(e307) and ATXN3-CAG89 mutant strains showed a higher hypersensitive phenotype in the presence of the aldicarb-induced paralysis compared with wild-type N2, ATXN3-CAG10 and unc-64(e246) worms [****P<0.0001 for unc-47(e307) mutant worms when compared with the controls, and ****P<0.0001, *P<0.05 and ****P<0.0001 for ATXN3-CAG89 mutants compared with the controls, by log-rank (Mantel–Cox) test; n=270-300]. Adult day 5 unc-47(e307) and ATXN3-CAG89 worms also showed a higher hypersensitive phenotype in presence of the aldicarb-induced paralysis compared with wild-type N2, ATXN3-CAG10 and unc-64(e246) worms [****P<0.0001, **P<0.01 and ****P<0.0001 for unc-47(e307) mutants, respectively, compared with the controls, and ****P<0.0001 for ATXN3-CAG89 mutants when compared with wild-type N2 and unc-64(e246) worms, by log-rank (Mantel–Cox) test; n=270-300]. A hypersensitive phenotype was observed at adult day 9 unc-47(e307) and ATXN3-CAG89 mutant strains when compared with wild-type N2, ATXN3-CAG10 and unc-64(e246) worms [****P<0.0001, **P<0.01 and ****P<0.0001 for unc-47(e307) mutant worms when compared with the controls, respectively, and **P<0.01 and ****P<0.0001 for ATXN3-CAG89 mutants when compared with the wild-type N2 and unc-64(e246) worms, by log-rank (Mantel–Cox) test; n=270-300]. The experiment was repeated three times. (D) Synchronized adult day 1, 5 and 9 worms were placed on NGM plates and photographed after 10 min of free movement. ATXN3-CAG89 mutant worms showed defects in motility when compared with wild-type N2 and ATXN3-CAG10 transgenic worms. (E,F) Representative photos of living, adult expressing wild-type unc-47p::mCherry, unc-47p::mCherry; ATXN3-CAG10 and unc-47p::mCherry; ATXN3-CAG89 transgenic worms at days 5 and 9 of adulthood. Images of the GABAergic motoneurons from entire unc-47p::mCherry and unc-47p::mCherry; ATXN3-CAG10 worms were taken. Image of an entire unc-47p::mCherry; ATXN3-CAG89 transgenic worm was taken and then zoomed in the panel on the right, representing a magnification of the area indicated. Increased incidences of gaps or breakages were observed in mutant unc-47p::mCherry; ATXN3-CAG89 transgenics compared with wild-type unc-47p::mCherry and unc-47p::mCherry; ATXN3-CAG10 controls. Quantification of neurodegeneration in transgenic worms at days 5 and 9 of adulthood. ATXN3-CAG89 transgenics showed a significant increase of neurodegeneration compared with unc-47p::mCherry and ATXN3-CAG10 controls (****P<0.0001 for day 5 of adulthood and ***P<0.001 for adult dav 9 worms, by Student's unpaired t-test; n=100 for each condition). These experiments were replicated three times.