Cortical interneuron development is affected in 4H leukodystrophy 45 2 To confirm that cortical interneurons are affected in primary brain tissue of 4H patients, we studied patient tissue for expression of GAD65/67, a pan GABAergic marker widely used to visualize all inhibitory neurons. Patient tissue showed elevated immunoreactivity for GAD65/67 compared to control, strengthening the hypothesis that cortical interneuron populations are affected in 4H (control 6% of 1315 cells; 4H patient 1 42% of 787 cells; 4H patient 2 36% of 1656 cells; Fig. 3D-E). As patient tissue is extremely scarce and iPSC-based model systems present differential gene expression in 4H cells, we continued functional analysis in iPSC-derived cortical cultures. The amounts of GABAergic and glutamatergic cells were analysed with staining for GAD65/67 at 5, 10 and 14 weeks post plating of day 18 cortical neurons. No changes in the percentages of GABAergic or glutamatergic cells were observed in 4H cultures (Fig. 3F). To test whether there are differences in neuronal maturity, the neurons were stained for CTIP2, which is expressed in interneurons and projection neurons during fetal development (Chen et al., 2008; Saito et al., 2011). There was an increase in CTIP2+ cells in 4H neurons at 14 weeks post plating, which was significant for the interneuron portion (i.e. CTIP2+GAD65/67+ cells; control 13.3 ± 2.32, 4H 26.2 ± 13.59, Z = -2.13, P = .033, Fig. 3G), but not for the glutamatergic portion (i.e. CTIP2+GAD65/67- cells; control 6.2 ± 2.71, 4H 9.35 ± 4.19). The decrease in ARX expression, increased GAD65/67 immunoreactivity in 4H patient tissue and sustained CTIP2 positivity in iPSC-derived GABAergic cells together suggests altered interneuron development in 4H. 4H neurons show a decreased generation of GABAergic synapses To investigate how cortical neuron development is affected in 4H, cortical neuronal cultures were matured and analysed for morphology changes (Fig. 4). Morphology of 4H and control neurons was analysed on dendritic (MAP2) and axonal (NF-200) staining with automated morphology analysis using Columbus software (Perkin Elmer). The measured parameters included axonal and dendritic length, branching level, and number of extremities and roots. No significant change in any parameter for neuronal morphology was observed between 4H and control neurons (Fig. 4A-C).
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