Chapter 2 58 In control cultures, the network activity significantly increased after blocking GABA-A receptors. However, in 4H cultures treatment with GABA antagonists did not significantly change network activity, suggesting that there was less GABAergic signalling that could respond to antagonistic inhibition. Interestingly, the activity in 4H cultures did decrease after addition of GABA, which suggests a normal post-synaptic GABAergic response in 4H cells. The decreased number of inhibitory synapses could be caused by several mechanisms, e.g. reduced network maturation, a reduction in a specific subset of interneurons, less (active) inhibitory synapses per neuron, or an increase in the number excitatory synapses. Follow-up studies should give more insight into this. Also, as patient lines present differential changes, in line with the broad clinical presentation, an increased panel of patient lines would be advised. In light of our in vitro findings, it seems contradictory that GAD65/67 immunoreactivity is higher in 4H postmortem tissue. It could possibly be explained if only a subpopulation of interneurons is affected in 4H, as it is reported that interneuron subtypes have different levels of GAD65 (Kajita & Mushiake, 2021) or it can be indicative of axonal reorganization of remaining interneurons (Peng et al., 2013). Alternatively, the increased GAD65/67 expression does not reflect changes in the interneuron populations but is rather the consequence of hyperexcitability. Epilepsy is a feature in 4H and human post mortem studies on temporal lobe epilepsy have identified increased levels of GAD67 (Neder et al., 2002). In models of epilepsy, upon neuronal excitotoxicity and increased glutamate release, GAD65/67 is upregulated as compensatory mechanism to convert excess of glutamate into GABA (Esclapez & Houser, 1999). If the increase in GAD65/67 immunoreactivity is indeed a secondary effect in 4H this would explain why there is no change in the number of GABAergic cells in the iPSC-derived cultures. However, while postmortem analysis confirmed network changes in 4H, follow-up studies are warranted. Alterations in GABAergic activity may underlie 4H associated neurological signs like epilepsy, but also hypomyelination. It is currently unknown how POLR3 mutations cause hypomyelination. Coulombe et al. (Coulombe et al., 2021) hypothesized that hypomyelination in 4H is either caused by an unknown POLR3 target that plays a key role in myelin biogenesis, or that POLR3 mutations lead to a globally reduced transcription at a crucial milestone in oligodendrocyte development. We investigated an alternative hypothesis, where the altered myelination is secondary to neuronal dysfunction. Cortical (parvalbumin) interneurons in the human brain are myelinated (Micheva et al., 2016;
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