Investigating neuron intrinsic defects in 4H and Globoid Leukodystrophy 81 3 INTRODUCTION Leukodystrophies, a group of inherited disorders characterized by brain white matter abnormalities, were for long viewed as disorders caused by glial dysfunctions. However, findings in recent years also point towards a role for neuronal defects in white matter pathology (Mar & Noetzel, 2010; van der Knaap & Bugiani, 2017). Whether the underlying mechanistic changes are disease-specific or more common among leukodystrophies is unclear. As insight into the mechanisms underlying neuronal dysfunction would facilitate the identification and development of new targeted therapies for leukodystrophies, we set out to study this. We investigated globoid cell leukodystrophy (GLD) and 4H leukodystrophy, two leukodystrophies with known neuronal involvement. GLD is characterized by neurodegenerative features such as axonal swellings, disrupted microtubule dynamics, and impaired axonal transport, as demonstrated in animal models like the twitcher mouse (Cantuti Castelvetri et al., 2013; Teixeira et al., 2014). These features are thought to result from the accumulation of psychosine, a hallmark of the disease. In 4H leukodystrophy, neuronal defects are less studied. Though initial evidence suggests changes in the inhibitory neuron population (Dooves et al., 2023). To investigate neurons in these leukodystrophies, we utilized an established human iPSCderived cortical model (Nadadhur et al., 2017) to assess intrinsic neuron properties such as neurite outgrowth, microtubule dynamics, and mitochondrial transport. In this co-culture system, we did not identify changes in the afore mentioned neuron properties. We hypothesized that healthy astrocytes potentially influence disease phenotypes, hence we assessed neuronal parameters in a neuron-mono culture model as well. Our analysis showed that 4H neuron mono-cultures show decreased neurite length, but only compared to GLD. Additionally, 4H neurons showed a decrease in motility of mitochondria over time, while for GLD and control neurons mitochondria motility increases. Besides this, GLD and 4H neurons did not display significant differences in neurite outgrowth, microtubule dynamics and axonal transport. Additionally, psychosine build-up was lacking in GLD neurons. On the other hand, mRNA sequencing analysis revealed that 4H neurons have a downregulation of genes involved in synaptic signalling and cortical development, alongside upregulation of genes related to developmental and ribosomal pathways. Pathway analysis further indicated potential dysregulation in Notch signalling and protein synthesis pathways in 4H neurons. Together, transcriptional alterations showed
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