Cortical interneuron development is affected in 4H leukodystrophy 57 2 al., 2021; Marsh et al., 2016). In animal models it was shown that Arx acts with FoxA2 to regulate expression of Shh (Cho et al., 2014). SHH plays important roles in the development of granule cells (and thereby cerebellar volume); oligodendrocytes (and thereby myelination); jaw and teeth; and the eyes (Dahmane & Ruiz i Altaba, 1999; Francis-West et al., 1998; Hardcastle et al., 1998; Kahn et al., 2017; Nery et al., 2001; Ortega et al., 2013; Prykhozhij, 2010). It is possible that POLR3 disorders, and 4H in particular, involve ARXrelated pathway defects, where altered ARX expression drives the clinical phenotype through an ARX to SHH pathway. Although downregulation of ARX may have significant impact on neuronal development, we could not confirm the SHH pathway as a therapeutic target for 4H in our disease model. Neurons were treated with SAG starting from day 18 to day 56 of differentiation. At day 18, the neuronal batches undergo quality control and are frozen for later use. It is possible that treatment starting from day 18 is too late and some of the developmental alterations following a decreased expression of ARX and a dysregulated SHH pathway have already taken place. However, this would mean that targeting the SHH pathway is not a viable option for patients, who are generally diagnosed postnatally. Another explanation may be that the SAG concentration was not high enough to activate the SHH pathway, although the expression of SHH target GLI1 was increased after SAG treatment. It is possible that the affected development of cortical interneurons is caused by mechanisms that do not involve ARX. In this study we were able to show a correlation between ARX levels and interneuron generation, but additional studies are needed to show a causal effect between ARX and 4H interneuron development. Additionally, ARX works on SHH-independent pathways. For example ARX may affect histone demethylation through KDM5C (Poeta et al., 2013; Poeta et al., 2021) which has been implicated in neurodevelopmental disorders (Vallianatos et al., 2020). In conclusion, SAG treatment did not revert the neuronal phenotype in our cortical 4H cultures and following studies should reveal whether a changed treatment protocol or different target would have more beneficial effects. The percentage of GABAergic synapses was significantly decreased in 4H cultures, suggesting an alteration in the synaptic balance. Indeed, network activity measurements showed a higher activity in most 4H cultures, although results did not reach statistical significance. Interestingly, there was a significant correlation between the percentage of GABAergic synapses and the amount of bursts and network burst, showing that synaptic balance changes also have consequences for functional network behaviour. This was also confirmed by the measurement of network activity after treatment with GABA antagonists.
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