Chapter 3 82 abnormalities in 4H neuron population and provided insights into underlying molecular pathways, emphasizing the need for further studies to elucidate neuron-specific vulnerabilities in leukodystrophies. Importantly, this report highlights the need for careful selection of in vitro models to investigate the neuronal aspects of leukodystrophies, as demonstrated by the lack of psychosine build-up in GLD cultures. RESULTS Neurite outgrowth, microtubule dynamics and mitochondrial transport unaffected by 4H and GLD in co-culture To investigate neuronal features in leukodystrophies we used an established human iPSCderived cortical neuron culture containing both glutamatergic and GABAergic neurons that were matured in presence of rat astrocytes and transduced with EB3-GFP LV at DIV20 (Fig. 1A). At day 21, bright-field imaging showed dense neurite growth in all samples, including controls and leukodystrophy models (Fig. 1B). The neurite length of EB3-GFP transduced neurons was analysed after fixation at DIV21 (Fig. 1B-C) and showed that the cumulative neurite length ranged from 150 to 1019 µm per neuron. Although there was some variation in mean neurite length for cell lines, there were no significant differences in mean neurite length among control, 4H and GLD (Fig. 1D1-2). Similarly, axon length was measured, the shortest axon was 46 µm and the longest 438 µm. No significant differences in mean axon length were observed among the phenotypes (Fig. 1E). To study microtubule dynamics, we investigated EB3 comet density and velocity in growthcones of iPSC-derived neurons. The 4H lines, in particular hvs201, showed high mean comet density compared to the others (Fig. 1F1); however the difference between control, 4H and GLD were not significant (H(2) = 5.088, p = 0.0571, Fig. 1F2). In the EB3 comet speed we also did not identify a difference between controls and any of the leukodystrophies (Fig. 1G). To study mitochondrial motility, we quantified the percentage of stationary and motile mitochondria by live-cell imaging (Fig. 1H1). This revealed no significant differences observed between control, 4H, and GLD groups (Fig. 1H2). Mitochondrial transport direction was further analysed, and again no significant differences in the proportions of mitochondria moving anterograde, retrograde, or in both directions were identified (Fig. 1I1I4). Noteworthy for GLD, the GALC KO line had particularly low percentage of mitochondria moving towards both directions (Fig. 1I2).
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