Effect of dGlyRS overexpression on protein synthesis and peripheral neuropathy phenotypes of PN-GlyRS Drosophila models. (A), dGlyRS mRNA levels as determined by qPCR on whole third instar larvae that ubiquitously (actin5C-GAL4) express a dGlyRS transgene. Data are shown as percentage (%) of driver-only control larvae. n = 5 samples per genotype, with each sample consisting of five larvae; ***P< 0.005 by unpaired t-test with Welch's correction. (Band C), Representative images (B) and quantification (C) of relative translation rate (% of driver-only control) as determined by FUNCAT in motor neurons (OK371-GAL4) of larvae expressing E71G, G240R, or G526R hGlyRS (2x: two transgene copies), in the presence or absence of Drosophila GlyRS (dGlyRS) co-overexpression. n = 10–16 animals per genotype; *P< 0.05; ***P< 0.005 by Brown-Forsythe and Welch ANOVA with Dunnett's T3 multiple comparisons test. Scale bar: 10 μm. (D), Climbing speed in a negative geotaxis assay of 7-day-old female flies expressing hGlyRS transgenes in motor neurons (OK371-GAL4), in the presence or absence of dGlyRS co-overexpression. n = 11–21 groups of 10 flies per genotype; *P< 0.05; ***P< 0.0005 by one-way ANOVA with Sidak’s multiple comparisons test. (E) Percentage of larvae with innervated muscle 24. hGlyRS transgenes were expressed in motor neurons (OK371-GAL4), in the presence or absence of dGlyRS co-overexpression. n = 14–26 animals per genotype; ***P< 0.005 by Fisher’s exact test. (Fand G), Representative images (F) and quantification (G) of neuromuscular synapse length on distal muscle 8 of larvae expressing hGlyRS transgenes in motor neurons (OK371-GAL4), in the presence or absence of dGlyRS co-overexpression. n = 17–24 animals per genotype; *P< 0.05; ***P< 0.0001 by one-way ANOVA with Sidak’s multiple comparisons test. Scale bar: 50 μm. (Hand I), Representative images (H) and quantification (I) of dendritic coverage (% of driver-only control) of class IV multidendritic sensory neurons in the larval body wall upon selective expression of hGlyRS transgenes in these neurons (ppk-GAL4), in the presence or absence of dGlyRS co-overexpression. n = 10–16 animals per genotype; *P< 0.05; **P< 0.01; ***P< 0.0001 by Brown-Forsythe and Welch ANOVA with Dunnett's T3 multiple comparisons test. Scale bar: 100 μm.