Cortical interneuron development is affected in 4H leukodystrophy 51 2 4H neurons show robust myelination in vitro A subset of human interneurons, mainly the parvalbumin neurons, is myelinated (Stedehouder et al., 2017) and one of the major characteristics of 4H is hypomyelination. Therefore, we decided to analyse oligodendrocyte maturation and myelination on human neuron-oligodendrocyte co-cultures. We altered a previously published protocol for neuron-OPC culture (Dooves et al., 2019) to generate a myelinating co-culture. Indeed, after 40 days of co-culture we observed robust generation of mature oligodendrocytes (defined as MBP+ cells) and myelination, shown by overlap between MBP and NF200 staining (Fig. 6AB). The presence of myelin was confirmed by electron microscopy, which showed compacted myelin sheets around neurites (Fig. 6C). Next, the myelinating cultures were applied to 4H cell lines. Cortical neurons of 4H or control patients were co-cultured with control glial cells. After 40 days of co-culture cells were fixed and stained for neuron (NF200) and oligodendrocyte markers (OLIG2, MBP). In both 4H and control cultures oligodendrocytes matured into MBP+ cells and showed myelination of neurites (Fig. 6D). On average there was no statistically significant change in the generation of oligodendrocyte lineage cells in 4H cultures, although three of six patient lines showed a decrease in the percentage of OLIG2+ cells compared to controls (control 52.2 ± 2.74; 4H 37.9 ± 19.29, Fig. 6E). In 4H cultures, the percentage of mature oligodendrocytes (MBP+/OLIG2+ cells) was about 35% for cultures with control and 4H lines, but the variation between 4H lines was high (36.8 ± 15.97), while maturation was quite stable between control lines (34.5 ± 5.50, Fig. 6F). Myelination was analysed on immunostainings by measuring the percentage of NF200+ neurites that show co-localization with MBP. No changes in the percentage of myelinated neurites were observed between cultures with 4H and control neurons (control 34.7 ± 4.85; 4H 45.1 ± 8.05, Fig. 6G). While this neuron-glia culture set-up provides a novel tool to study myelination defects in brain diseases, we could not identify changes in 4H. Targeting the SHH pathway does not improve 4H interneuron phenotypes ARX is an important regulator of SHH gradients during development (Cho et al., 2014), suggesting that the decreased expression of ARX in 4H might affect interneuron development through the SHH pathway. We tested the prospects for targeting the SHH pathway in 4H in the cortical neuronal cultures. In a pilot study it was established that twice a week treatment with SHH pathway agonist SAG at 100 nM from day 18 onwards increased the percentage of GABAergic synapses on a 4H line, without any (negative) effects on a control line (Supplementary Fig. 2A).
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