Investigating neuron intrinsic defects in 4H and Globoid Leukodystrophy 91 3 DISCUSSION To investigate neuronal properties in leukodystrophies, this study explored 4H and GLD neuronal phenotypes using patient iPSC-based cortical neuron cultures in presence and absence of rat astrocytes. In mono-culture, 4H neurons had shorter neurites compared to GLD and control neurons, though only reached statistical significance in comparison to GLD. Additionally, 4H neurons showed a decrease in mitochondrial transport between DIV2 and 7 in cultures, while control (and GLD) neuron cultures presented an increase in mitochondrial transport. In addition, transcriptomic analysis in 4H neurons revealed downregulation of synaptic and cortical development genes and upregulation of developmental and ribosomal pathways, suggesting potential mechanisms underlying 4H pathology and underscoring the potential of this model to study 4H specific mechanisms. In GLD cultures, psychosine accumulation, a hallmark of GLD, was not detected in co-culture and only moderately present in the neuron-mono cultures. When rat astrocytes were present in the GLD neuron culture, no significant differences in neurite outgrowth, microtubule dynamics, or mitochondrial transport were observed between groups. Transcriptomic results for GLD were also inconclusive, reinforcing the limitations of these models for studying GLD disease mechanisms. Missing psychosine accumulation in GLD cultures We did not observe psychosine accumulation in GLD patient neurons when co-cultured with rodent astrocytes. This suggests that astrocytes may influence metabolic processes in ways that limit the manifestation of leukodystrophy-related neuronal defects. This is in line with the observation that GLD iPSCs grown on healthy donor feeder layers also lacked psychosine build-up. Additionally, it is well-established that astrocytes can present neuroprotective functions (Belanger & Magistretti, 2009). Interestingly, the absence of phenotypic changes in the co-cultures suggests that effective targeting of astrocytes might be a therapeutic strategy for mitigating neuronal defects in GLD. In contrast, co-culture conditions with healthy rat astrocytes in models of 4H have revealed abnormalities in the GABAergic population (Dooves et al., 2023), implying that the lack of phenotypes in GLD cocultures may be specific to the pathology of the leukodystrophy subtype. This emphasized that we need to optimize in vitro models to adequately captures disease-specific changes. Surprisingly, psychosine did not robustly accumulate in neuron-mono cultures either, except for line KOX19, which showed slightly elevated levels (999 pmol/gram). Considering that accumulation was detected at the neuronal progenitor stage, there is likely some kind
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