Fig. 2
Poly(GR) causes axonal defects without TBPH pathology. a (GR)80 expression leads to the accumulation of multilamellar bodies (MLBs) and axonal degeneration in Drosophila. Transmission electron microscopy (EM) analysis of the brain in 3-week-old control and (GR)80 flies. Red arrowheads indicate MLBs. Scale bars: 1 μm (central brain), 0.5 μm (ventral ganglion). b Number of MLBs per neuron in the brain of 3-week-old flies. n = 55 neurons from three control flies and n = 95 neurons from three (GR)80 flies were counted in a blinded manner. ***p < 0.001 (Chi-square analysis). c EM analysis of the brain of 3-week-old control and (GR)80 flies. Red boxes in i and ii are enlarged in i′ and ii′. d EM analysis of ventral ganglions of 3-week-old control and (GR)80 flies. Asterisks indicate the axons without vesicles and filaments in iii and iv. Scale bars: 2 μm in i and ii, 0.5 μm in i′ and ii′. e Quantification of axonal degeneration in the brain and ventral ganglion. Swollen axons, axons with vacuoles and damaged mitochondria and gaps in the neurophile are quantified as degenerative axon (n = 3 flies per genotype quantified in a blinded manner). Values are mean ± s.e.m. *p < 0.05 (Student’s t test). f Immunostaining for cleaved caspase-3 (red) in ELAV-positive brain neurons (green) of 21-day-old control and (GR)80 flies. Arrowheads indicate apoptotic cells. Scale bar: 5 μm. g Number of neurons positive for cleaved caspase-3 in 21- and 25-day-old control and (GR)80 central brains. n = 6 brains per genotype. Dots represent the percentage of average cleaved caspase-3-positive neurons out of 200–250 neurons in each fly. Values are mean ± s.e.m. *p < 0.05 (Wilcoxon signed-rank test). h Survival curve of (GR)80 flies expressing GFP or p35. The p value was determined by log-rank test. i Immunostaining of ELAV-positive brain neurons (green) for TBPH (red) in 21-day-old control and (GR)80 flies. j Western blot analysis of soluble and insoluble proteins in the brains of 3-week-old control and (GR)80 flies. k Quantification of western blot experiments (n = 3 independent blots). Soluble: p = 0.4345, Insoluble: p = 0.4852 (Welch’s t test). Source data are provided as a Source Data file.
