Investigating neuron intrinsic defects in 4H and Globoid Leukodystrophy 95 3 + 1x Pen/Strep. Astrocytes of passage 3 were frozen (medium with 10% DMSO) and used for experiments. Frozen rat astrocytes were quickly thawed in a water bath and plated at a density of 4k per cm2 on geltrex coated 4 well-IBIDIs (ibiTreat, 80426). When confluent, 25k neurons per chamber (which equals to 10k neurons per cm2) were plated on top of the rat astrocytes in NB medium with 20 ng/ml BDNF + 10 ng/ml GDNF + 10 ng/ml IGF1 + 1 μM cAMP. Twice a week half medium changes were performed, with supplements for the entire volume. On DIV6 and DIV10, 1 day before the refreshments, 2 µm of AraC was added, to inhibit proliferation. The next day, half media refreshments were performed until DIV20. Neural Maturation Mono-Culture Frozen day 18 neurons were quickly thawed in the water bath and plated at a density of 10k neurons per cm2 on PLO/Laminin coated 4 well-IBIDIs (ibiTreat) in STEMdiff™ forebrain neuron maturation medium. Full media refreshments were performed twice a week. Analysis of Sphingolipid Metabolism In order to analyse cultures on GLD associated deficiencies in sphingolipid metabolism, 1 million neurons were plated at a density of 10k neurons/cm2 according to previously described co-culture or mono-culture protocols. They were maintained for either 21 days (co-culture) or 7 days (mono-culture). Cells were collected using a cell scraper and pelleted by centrifugation. Supernatant was removed and cell pellets were stored and shipped at - 80°C for analysis of glucosylceramide (GlcCer), galactosylceramide (GalCer), glucosylsphingosine (LysoGlcCer) and galactosylsphingosine (LysoGalCer, psychosine) at the Core Facility Metabolomics of the Amsterdam UMC, location AMC, the Netherlands. For iPSC, neuron and rat samples frozen cells (approximately 1 million) were quickly thawed in a water bath and frozen pellets were shipped for analysis. Live-cell Imaging for Microtubule Dynamics For the analysis of MT dynamics in the growth cone, we applied a previously described protocol (Sousa et al., 2024). Briefly, neurons were transduced (at DIV20 for co-cultures, or DIV1 and 6 for mono-cultures) with lentivirus expressing CMV-EB3-GFP (27000 transducing unit (TU)/well, plasmid from F. Polleux, Columbia University, USA). 24 hours after, timelapse recordings were performed in phenol-free media or forebrain maturation media supplemented as mentioned above, at 37°C and 5% CO2, on a Leica SP8 microscope (Leica Microsystems) and Leica Application Suite X (LAS X) software (version 3.5.7.23225, RRID:SCR_013673) with a HC FLUOTAR L 25x/0.95 W objective. For quantification of EB3
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