Chapter 2 56 DISCUSSION Although the genetic defects causing 4H leukodystrophy have been identified, our knowledge of underlying molecular mechanisms and the affected cellular subtypes is lacking. This study aimed to get more insight into the affected brain cell types and pathways. We started with an unbiased RNA sequencing screen on 4H and control fibroblasts, iPSCs and cerebellar cells. Although only a small number of genes were differentially regulated in all cell types, an interesting finding was the decreased expression of ARX in cerebellar cells of 4H patients. Considering the important role of ARX in cortical neuronal development and migration (Joseph et al., 2021; Kitamura et al., 2002; Marsh et al., 2016), we studied GAD65/67 expression in patient tissue and showed an increase in GAD65/67 immunoreactivity confirming interneuron changes in the cortex of 4H patients. In iPSCderived cortical neuron cultures, 4H neurons also showed decreased expression of ARX and affected interneuron development as measured by a decreased percentage of GABAergic synapses. No changes in the proportion of GABAergic cells were observed, suggesting the change in the number of synapses may be due to affected maturation or synapse formation in 4H neurons. The altered synaptic ratio has functional consequences, as it was correlated to an increased network activity. A decreased GABAergic signalling in 4H also became apparent after treatment with GABA antagonists. 4H neurons did not show changes in activity upon treatment with the GABA antagonists, while control cells showed a significantly increased activity after treatment. As ARX was reported to have important roles in the PV interneurons (Joseph et al., 2021), we tested whether specific interneuron lineages were affected in 4H. Indeed, QPCR analysis identified an increase in ERBB4, a marker for PV neurons, confirming other results that interneuron regulation may be affected in 4H. Together, interneurons are affected in 4H and we show decreased ARX expression in different neuronal subtypes using iPSC models. Cortical neuron cultures identified a decreased number of inhibitory synapses, increased network activity, and the results are specifically pointing to defects in PV neurons. We focused on the involvement of ARX in 4H leukodystrophy because of its role in cortical development and its association with other disorders. Loss of function mutations in ARX lead to pleiotropic disorders such as X-linked Lissencephaly with Ambiguous Genitalia (XLAG, OMIM 300215), Agenesis of the Corpus Callosum with Abnormal Genitalia (OMIM 30004), and Lissencephaly with cerebellar hypoplasia (Shoubridge et al., 2010). ARX impacts interneuron generation, development and migration (Lee et al., 2017) and loss of ARX expression in interneurons alters their excitability and causes epilepsy in mice (Joseph et
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