Cortical interneuron development is affected in 4H leukodystrophy 59 2 Stedehouder et al., 2017), and myelination can be regulated by GABA receptor activity on oligodendrocytes (Pudasaini et al., 2022). To study whether the decreased interneuron generation in 4H cortical cultures also affected myelination a new co-culture protocol was developed. This co-culture of human neurons and glial cells show robust maturation of oligodendrocytes and myelination of human axons, a challenging phenomenon to study in vitro. No differences in oligodendrocyte maturation or myelination were observed between cultures with control or 4H neurons. This could suggest that the hypomyelination observed in 4H patients is caused by an oligodendrocyte intrinsic effect, rather than mediated through affected neurons. However, it is also possible that our culture set-up was not sensitive enough to pick up changes in myelination for 4H. The neurons used for the coculture consist of a mix of GABAergic and glutamatergic neurons, so a myelination defect on interneurons may be masked by normal myelination of glutamatergic neurons. Additionally, PV neurons mature during late development and not all neurons in the presented co-culture system showed myelinated processes. Although the novel neuron-glia co-culture can measure myelination in vitro, further improvement of these model systems may be necessary to identify mechanisms underlying hypomyelination in 4H leukodystrophy. We aimed to identify the interneuron subtype that was most affected in 4H cultures. We were not able to identify a significant decrease in any of the five major cortical interneurons populations (Yu et al., 2021). Instead, we found a significantly increased expression of ERBB4, an early marker for PV interneurons. There are a few possible explanations for the discrepancy between the decrease in GABAergic synapses and the increase in ERBB4 expression. It is possible that interneuron generation in 4H is normal, but that interneurons are affected during later development or maturation stages. This is consistent with the finding that there were no changes in the number of GAD65/67+ cells in 4H cultures. As ERBB4 is an early marker for PV interneurons, it is also possible that the increase reflects a lack of maturation, rather than an actual increase in PV neurons. A defect in maturation would be consistent with the sustained DLX2 expression, which decreased in control cells between day 30 and day 56, but not in 4H cells. Unfortunately, PV interneurons mature late in cortical development (late gestation/postnatal (Cao et al., 1996; Fung et al., 2010; Ulfig, 2002)), and we were not able to measure PVALB expression in our neuronal cultures. PV interneurons are the largest group of GABAergic neurons in the cortex. They are fast spiking interneurons with a high metabolic demand, which makes them vulnerable for injury (Ruden et al., 2021). Interestingly, the development of SST and PV interneurons is depending on SHH levels
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