Chapter 3 92 of clearance mechanism of psychosine becoming active at later stages. Possibly, psychosine might be secreted via extracellular vesicles or diluted through passive diffusion during regular media changes (Reiter et al., 2022). In accordance with this hypothesis, the line with the highest psychosine levels (KOX19) in mono-cultures is also the line with the highest level of psychosine at day 18 (10267 pmol/gram) so it could be that this line needs more time to go back to physiological psychosine levels. Interestingly, the KOX19 line also separated from all other samples in the PCA plots derived from mRNA bulk sequencing data, suggesting distinct transcriptomic profiles. However, this divergence did not translate into detectable differences in axon morphology and transport measurements. These findings highlight the significant influence of experimental conditions on psychosine dynamics, and related neuronal defects. Further investigation is needed to elucidate these mechanisms. It would be interesting to investigate psychosine levels in the media, pharmacologically block extracellular vesicle release, and track psychosine levels across multiple timepoints. Additionally, lipidomic profiling might be useful to better understand psychosine metabolism in models of GLD. Mitochondrial transport changes in 4H neurons Previously, we demonstrated that 4H neurons in co-culture with astrocytes exhibit neuronal abnormalities, including increased activity (Dooves et al., 2023). The absence of clear defects in the current study may be due to differences in the parameters assessed or a delayed onset of abnormalities, as prior findings were observed in cultures maintained for up to 14 weeks (Dooves et al., 2023). Nevertheless, a trend towards decreased mitochondrial transport over time in 4H mono-cultures was identified. Mitochondrial transport serves as proxy for axonal transport, a critical process for maintaining neuronal health. Axonal transport is important for delivery of newly synthesized macromolecules and organelles from the cell body to the synapse (anterograde transport) and the return of signalling endosomes and autophagosomes to the cell body for degradation (retrograde). Specifically, mitochondrial transport is dynamically regulated in response to changes in neuronal activity and various physiological and pathological states (MacAskill & Kittler, 2010; Sheng & Cai, 2012). Disrupted axonal transport, including mitochondrial trafficking, are implicated in several neurodegenerative diseases (Berth & Lloyd, 2023). These findings highlight the potential role of mitochondrial transport abnormalities in the pathology of 4H. Future investigations should focus on confirming whether mitochondrial dysfunction contributes to disease progression and on understanding the underlying molecular mechanisms.
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