Cortical interneuron development is affected in 4H leukodystrophy 53 2 PCR analysis confirmed upregulation of SHH target GLI1 in 4H cells after treatment with 100 nM SAG for 2 weeks (Supplementary Fig. 2B-C). As such, all 4H and iPSC lines were treated with 100 nM SAG from day 18 until the end point of the experiment at day 56. Morphology analysis of neurons showed that SAG treatment affected the dendritic complexity by decreasing the number of dendritic segments, extremities and dendritic length per cell in control cells (segments vehicle 6.34 ± 1.07, SAG 4.29 ± 1.12 t(6) = 2.481, P = 0.024; extremities vehicle 3.34 ± 0.39, SAG 2.36 ± 0.46, t(6) = 3.176, P = 0.010; length vehicle 283 ± 25, SAG 193 ± 17, t(6) = 3.194, P = 0.019; Fig. 7A-B). In 4H cells, no significant effect of SAG treatment on neuronal morphology was observed. A paired samples test comparing vehicle and SAG treatment showed that SAG treatment significantly increased the percentage of VGAT+ synapses compared to vehicle treatment (vehicle 31.3 ± 10.19, SAG 34.6 ± 9.93, t(20) = -2.125, P = 0.046). However, the increase was small and no significant improvement in the 4H lines specifically was observed (Fig. 7C-D). Although SAG treatment was able to increase expression of SHH target GLI1, it was not able to improve 4H interneuron development in vitro. 4H mutations affect parvalbumin interneuron lineage Next, we aimed to identify whether specific cortical interneuron subtypes are affected in 4H. Primers targeted the five major human interneuron subtypes: somatostatin (SST), PV, VIP, ID2 and NDNF neurons (Yu et al., 2021). The expression of NEUN was decreased in 4H cultures at day 30, although the difference was not significant (control 1.27 ± 0.82, 4H 0.41 ± 0.25, t(8) = 2.171, P = 0.062, Fig. 8A). However, since a difference in the number of neurons may affect the relative expression of GABAergic markers when only corrected for a housekeeping gene, all other markers were corrected for expression of both EIF4G2 and NEUN. DLX2 is important for development of cortical interneurons and downregulated upon maturation of neurons (Achim et al., 2014). Indeed, in control cells the DLX2 expression decreased between day 30 and day 56, which was less pronounced in 4H cells (control day 30 1.98 ± 1.90, day 56 0.31 ± 0.12; 4H day 30 2.06 ± 1.01, day 56 1.61 ± 1.70; Fig. 8B). Despite the decreased number of GABAergic synapses in 4H cultures, markers for specific interneuron subtypes were not significantly decreased in 4H cultures (Fig. 8C-H). In contrast, the expression of ERBB4, a marker for PV neurons, was significantly increased in 4H neurons compared to controls (day 30 control 2.28 ± 3.47, 4H 21.45 ± 19.51, Z = -2.132, P = 0.038; day 56 control 0.42 ± 0.09, 4H 2.50 ± 2.76, t(6) = -2.710, P = 0.035, Fig. 8H). This suggests that the generation of specific interneuron subtypes is not impaired in 4H, but rather the development or maturation of interneurons may be affected.
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