Investigating neuron intrinsic defects in 4H and Globoid Leukodystrophy 83 3 Lastly, we investigated mitochondria transport length and velocity. Although, for GLD lines the distance and velocity of mitochondria movement was lower than controls for the KO line and higher compared to controls for the KI line, we did not observe a disease specific phenotype (Fig. 1J-K). In conclusion, besides some subtle trends, we did not identify any significant disease specific phenotypes in 4H or GLD neurons in this co-culture set-up. No Psychosine Accumulation in GLD Co-Cultures Based on earlier findings, we expected altered neurite outgrowth, disrupted microtubule dynamics and impaired axonal transport in GLD neurons (Cantuti Castelvetri et al., 2013; Teixeira et al., 2014). As enzyme deficiencies in GLD result in psychosine accumulations, a key disease feature of GLD, we analysed psychosine levels in our cultures at day 21. Surprisingly, psychosine levels were elevated in iPSCs, and in neuron differentiation products but not in co-cultures i.e., levels in GLD co-cultures were similar to those observed in control samples (Fig. 1L). Additionally, no indication of other disturbances in glycosphingolipid metabolism were found (Supplementary Fig. 1A-C). In summary, no distinct neuronal phenotypes were identified in 4H and GLD neurons based on neurite outgrowth, microtubule dynamics, or mitochondrial transport at 21 days of coculture. Additionally, psychosine accumulation was not observed in GLD neurons under these conditions. Distinct mitochondrial dynamics and minimal psychosine accumulation in neuron-mono cultures of leukodystrophies We hypothesized that presence of healthy rat astrocytes in the iPSC-derived neuron cultures might prevent psychosine accumulation in GLD neurons, potentially masking GLD-specific neuronal features. Therefore, we implemented a neuron-mono culture. To allow for a direct comparison of unique GLD and 4H neuronal phenotypes, we used this setting to model 4H as well. By examining two timepoints, we aimed to capture any dynamic changes in neuronal functioning that might emerge over time (Fig. 2A). We assessed the same neuronal parameters as we did for the co-cultures.
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