Fig. 1
From: Stathmin 1/2-triggered microtubule loss mediates Golgi fragmentation in mutant SOD1 motor neurons
Morphological and molecular Golgi alterations in motor neurons of mutant SOD1 mice. a. Confocal z-stacks (upper panels) show fragmentation of GM130-labeled Golgi membranes in lumbar motor neurons of 240-day-old mutant SOD1G85R and SOD1G93A mice as compared to non-transgenic mice and transgenic SOD1wt mice. Motor neurons are identified by expression of VaChT (vesicular acetylcholine transporter). 3D-modeling (lower panels) of GM130-labelled Golgi membranes in entire motor neurons confirms Golgi fragmentation. Scale bars 10 μm. b. Increased number of GM130-stained Golgi elements in mutant SOD1G85R and SOD1G93A motor neurons determined by 3D modeling of Golgi membranes in entire cells (mean ± sd, n = 12 motor neurons from three mice per genotype, *** p < 0.0001 by student’s t-test, unpaired. c. Decreased cross-sectional area of GM130-labeled Golgi area in mutant SOD1G85R and SOD1G93A motor neurons as compared to control motor neurons (mean ± sd, *** p < 0.001, n = 50 motor neurons from three mice per genotype, student's t test). See also Additional file 1: Figure S1. d. Percentage of motor neurons with GM130-labeled Golgi fragmentation at presymptomatic stage (mean ± sd, *** p < 0.001 by student’s t-test, n > 250 motor neurons from four mice per genotype were analyzed at presymptomatic stage corresponding to age 130 days (SOD1G93A, non Tg) or 180 days (SOD1G85R, SOD1wt). e. Electron microscopy of a lumbar motor neuron from a non-transgenic mouse aged 140 days showing a typical Golgi apparatus (upper panel) that is easily distinguished from unlinked, partially swollen and vesiculated Golgi profiles observed in mutant SOD1G93A motor neurons (arrows in lower panel, magnifications on the right). n: nucleus, m: mitochondria. Scale bars 500 nm (left panels), 200 nm (right panels). f. Western blots showing decreased levels of β-COP in mutant SOD1 mice, and normal levels of Sec23, GM130 and p115. Loading control β-actin. Shown is one representative blot per genotype out of four. The diagram below shows that β-COP levels (normalized to β-actin) are reduced to 25 ± 7.7 % and 42.5 ± 9.6 % of non Tg (mean ± sd, n = 4 per genotype, * p < 0.01 by Mann Whitney test). g. Subcellular fractionation of spinal cords. Western blot analyses show redistribution of GM130 in mutant SOD1 mice, as indicated by shift from its normal membrane localization in control and SODwt mice into fragmented membranes and vesicles in SOD1G85R and SOD1G93A mice and cytosol. P115 is not redistributed. Each blot is representative of three independant experiments on mice of the indicated genotypes. The diagram below shows densitometric determination of protein distribution (mean ± sd, n = 3 per genotype, * p < 0.01 by Mann Whitney test). h. Confocal imaging reveals accumulation of Golgi v-SNARE protein GS28 (upper panels) and GS15 (lower panels) in small vesicle-like punctae of motor neurons in mutant SOD1 mice, as compared to controls. Scale bars 10 μm. i. Western blots (upper two panels) demonstrating massively increased levels of Golgi v-SNAREs GS28 and GS15 in mutant SOD1G85R and SOD1G93A lumbar spinal cords, as compared to non-transgenic and SOD1wt spinal cords. Western blots (lower three panels) showing normal expression of the Golgi t-SNARE Syntaxin-5a and the endosomal v-SNARE Vti1a. β-actin indicates equal protein loading. Each blot is representative of three independant experiments on mice of the indicated genotypes. The diagrams show increased spinal cord protein levels of GS28 by 4 ± 1.6 (SOD1G85R) and by 3.7 ± 1.3 (SOD1G93A) fold and of GS15 by 3.7 ± 0.7 (SOD1G85R) and by 3.2 ± 1 (SOD1G93A) as compared to non Tg control (mean ± sd, * p < 0.001 by Mann Whitney test)