Investigating neuron intrinsic defects in 4H and Globoid Leukodystrophy 85 3 ◀ Figure 1: Lack of leukodystrophy-specific neuronal defects in co-cultures. A) Schematic representation of co-culture set-up with rat astrocytes. B) Immunofluorescent images of βIII-tubulin staining in control (ND1.2), 4H (hvs201) and GLD (KI H4) neurons showing dense neurite networks. Scale bar = 100 µm. C) Representative images of EB3-GFP transduced neurons (control, 4H and GLD) used for quantification of neurite and axonal lengths. Graphs displaying D) Neurite length, E) Axon length, F) EB3 comet density and G) EB3 Comet Velocity. Scatter plots represent individual observations, with horizontal lines showing the mean for each cell line. Bar graphs summarize the group mean for either Control (ctrl), 4H or Globoid (GLD) Leukodystrophy with individual dots for cell line means. Mitochondrial (Mito.) H) Motility and I) Direction are depicted by bar graphs for each line with error bars displaying the standard deviation, as well as bar graphs summarizing the mean for controls, 4H or GLD with dots for cell line means. J) Distance and K) Speed of mitochondria are depicted by open bars for retrograde travelling mitochondria and open for anterograde travelling mitochondria. Each dot represents a mean for a cell line. L) Psychosine concentrations (pmol/gram) measured in iPSCs, DIV0 neurons, rat astrocytes, and co-cultures at DIV21 for control and GLD samples. DIV = days in vitro. Over time, between 3 and 8 days in vitro (DIV), neurite length increased in all but one GLD line. While on the contrary another GLD line, KI H4, presents a very steep increase in neurite length over time (Fig. 2B1). ANOVA showed a significant difference between neurite length of control, 4H and GLD combined (F(2)=5.657, p = 0.0416, Supplementary Table III). Post-hoc multiple comparisons showed that the difference occurred between 4H and GLD (Mean difference -73.57, P = 0.0355; Fig. 2B2 and Supplementary Fig. 2A). Similarly, the longest neurite, axon length increased over time, with no observable differences between controls and any of the leukodystrophies (Fig. 2C, Supplementary Fig. 2B). Of note, the GLD 5.2 line was the only line that displayed a decreased mean neurite length and axon length at DIV3 compared to DIV8. The microtubule dynamics, specifically EB3 comet density, seemed to be slightly lower in both leukodystrophies compared to control, but this difference was not statistically significant (Fig. 2D, Supplementary Fig. 2C). Also, no differences were observed in EB3 comet velocity across control and leukodystrophy groups (Fig. 2E, Supplementary Fig. 2D). To note, ANOVA on EB3 comet velocity was near the significance threshold (p = 0.07, Supplementary Table III), post-hoc multiple comparisons showed that this was driven by a difference between 4H and GLD cultures. Mitochondrial transport analysis in the neuron-mono cultures revealed an increase in the percentage of motile mitochondria over time, between DIV2 and DIV7, in both control and GLD lines, while 4H lines showed a reduction in motile mitochondria over time (Fig. 2F, Supplementary Fig. 2E). In 4H, overall mitochondrial motility was reduced (Fig. 2G,
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