Fig. 6: ALS motor axons show dysfunction in mitochondrial motility.
a Schematic of the mitochondrial motility assays using live fluorescence microscopy. Motor neuron progenitors were attached as neurospheres, followed by terminal differentiation and axon outgrowth. Mitochondria were labeled using TMRM. b–d Individual mitochondria become discernable at increasing magnifications (b, c), and were recorded in peripheral axons (d). Scale bars are as indicated, ranging from 500 µm down to 20 µm. e 6-min long timelapses were recorded at 0.5 Hz in 3,4 locations in at least 3 attached neurospheres from several independent differentiations per line (namely control n = 4, FUS KO n = 5, FUS R244C n = 3, FUS R495X n = 2, FUS P525L heterozygous n = 4, FUS P525L homozygous n = 5, TARDBP M337V n = 5). Traces were identified using TrackMate 7 with StarDist detector and simple LAP tracer and directional movement was analyzed in R using our package ‘mitotrackR’. f The proportions of stationary and motile mitochondria were calculated. The three axes represent these proportions in the three motility groups in ternary plots. Each bubble represents the number of mitochondria in individual recordings. The dashed lines indicate 50% thresholds in each motility group and the dotted lines are the anterograde-retrograde isoproportional lines (at which the anterograde and retrograde movement are equal). g The influence of the mutation in isogenic motor neurons on the mitochondrial motility (as the proportions of stationary, anterograde and retrograde mobile mitochondria) was assessed by Pearson’s χ2 test (one-sided, χ2 = 88192, df = 12, p < 2.2e-16). The association between isogenic mutants and mitochondrial motility is plotted. The sign of the residuals indicates the direction of the association, with attraction and repulsion colored as red and blue, respectively. The bubble size represents the contribution to the total χ2 score (squared residuals over χ2 statistic). h Mitochondria align across motor axons, such that each mitochondrion has a pair of nearest neighbors in the anterograde and retrograde direction. In the first frame of each recording from (f, g) the Euclidean distances between individual mitochondria as well as the distance from each mitochondrion to the pair of adjacent mitochondria was measured to then calculate the distance across the three mitochondria as well as the absolute difference between the pair of nearest neighbors in the opposing directions. The violin plots represent values for individual mitochondria, the colored dots the average value in each video, and the black dots and whiskers are median ± the confidence interval (α = 0.05). ANOVA was followed by post-hoc two-sided Wilcoxon rank sum test between mutant and control lines and the significance levels for the p-values are ns p > = 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001. The p-values for mitochondria spacing are 0.0159 (FUS KO), 0.0159 (FUS R244C), 0.0159 (FUS R495X), 0.0079 (FUS P525L heterozygous), 0.0025 (FUS P525L homozygous), and 0.0079 (TARDBP M337V). The p-values for the distances across adjacent mitochondria are 0.0159 (FUS KO), 0.0159 (FUS R244C), 0.0159 (FUS R495X), 0.0079 (FUS P525L heterozygous), 0.0025 (FUS P525L homozygous), and 0.0079 (TARDBP M337V). The p-values for the absolute difference are 0.0317 (FUS KO), 0.0317 (FUS R244C), 0.0159 (FUS R495X), 0.0079 (FUS P525L heterozygous), 0.0051 (FUS P525L homozygous), and 0.0079 (TARDBP M337V).
