Chapter 2 60 (Flandin et al., 2011; Tyson et al., 2015), and deletion of Arx in PV neurons led to increased neuronal activity and altered synaptic properties in a mouse model (Joseph et al., 2021). PV interneurons play a crucial role in spike timing of glutamatergic neurons, and abnormalities in PV signalling have been implicated in epilepsy, occasionally seen in 4H patients. For example, optogenetic stimulation of PV neurons can induce ictal events in mice (de Curtis & Avoli, 2016) probably due to a depolarization block (Calin et al., 2021). Overall, our data suggests that PV interneurons may be specifically affected in 4H and should be further studied. To conclude, transcriptome analysis of cerebellar cells differentiated from patient iPSCs revealed ARX as a potentially important regulator of 4H brain pathomechanisms. Indeed, a decreased expression of ARX was confirmed in cortical neuron cultures, which showed affected generation of GABAergic synapses consistent with known effects of ARX on interneuron development. The decrease in GABAergic synapses correlated to increased neuronal network activity. QPCR analysis revealed alterations in ERBB4 expression, suggesting the PV interneurons might be of particular interest for 4H. Although more research is needed, this study provides a first insight into specific brain cell types and pathomechanisms affected in 4H leukodystrophy. ACKNOWLEDGEMENTS The authors would like to thank Kyoko Watanabe and Danielle Posthuma for analysing RNA sequencing data; Jan van Weering, Joke Wortel and Rien Dekker for assistance with electron microscopy; Brendan Lodder for assistance with RNA isolation; and the INF department for the MEA inhibitor compounds NIW is member of the European Reference Network for Rare Neurological Disorders (ERN-RND), project ID 739510. FUNDING This study was funded by the European Leukodystrophy Association (ELA 2018-011C3A). COMPETING INTERESTS The authors report no competing interests.
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