Chapter 7 214 DISCUSSION The aim of this thesis was to advance the understanding of 4H leukodystrophy by identifying the key cell types and molecular mechanisms implicated in the disease. Results presented in this thesis highlight that intrinsic neuronal deficits are a defining feature of 4H leukodystrophy, independent of the surrounding glial environment. The use of diverse model systems — ranging from 2D cultures to advanced 3D spheroid and co-culture models — generated an extensive dataset that provides unique insights into cell-specific vulnerabilities, molecular alterations, and addressed the interplay of neuronal, glial, and microglial elements in 4H pathology, creating a foundation for future investigations and therapeutic strategies. 1. Neuron intrinsic defects in 4H Our investigations revealed that ARX expression was downregulated in cerebellar differentiation products derived from 4H patient samples. ARX has a known involvement in interneuron generation, development and migration (Lee et al., 2017). Additionally loss of ARX expression alters interneuron excitability and causes epilepsy in mice (Joseph et al., 2021; Marsh et al., 2016). Therefore, ARX dysregulation implicated potential interneuron involvement in 4H. In cortical neuron cultures containing both glutamatergic neurons and interneurons, we indeed identified abnormalities in GABAergic synapse formation and neuronal activity. Further investigations explored intrinsic neuronal dysfunction in 4H through assessments of neurite outgrowth, axonal transport, and microtubule dynamics in various in vitro models. While these parameters showed no significant differences between 4H and control samples, reduced neurite outgrowth in 4H mono-cultures compared to another leukodystrophy, Globoid Cell Leukodystrophy, was evident. Transcriptomic analyses provided additional insights, identifying downregulation of synapse formation and cortical development pathways, which collectively underscore that neuronal deficits in 4H are intrinsic and not secondary to external factors (Chapter 3). While these results point to neuronal involvement in 4H pathomechanisms, we still do not understand how. Here I will discuss if specific interneuron subclasses are more affected and how this could be studied further: How could Shh pathway dysfunctions lead to brain abnormalities in 4H? What other molecular pathways interact with Shh signalling that could contribute to 4H pathogenesis?
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