Transcriptomic disruption and hypoactivity in DYT-SGCE medial ganglionic eminence-patterned inhibitory neurons

Myoclonus dystonia is a Mendelian inherited, childhood-onset dystonic disorder, caused by mutations in the autosomal dominantly inherited gene SGCE, and in which both motor and psychiatric phenotypes are observed. Results from murine and in vivo human studies suggest that dystonia is caused by disruption to neuronal networks, in particular the basal ganglia-cerebello-thalamo-cortical circuit. Work focused on the cortical component implicates disruption to neuronal excitatory-inhibitory balance as being a key contributor to the observed phenotypes. Our previous work, focused on cortical excitatory glutamatergic neurons, demonstrated a hyperexcitable phenotype and more complex dendritic arborization in an in vitro model of myoclonus dystonia. In contrast, human electrophysiological studies have suggested that it is the loss of inhibitory tone in this region that contributes to the overall hyperkinesis. To explore this further, we have evaluated the impact of SGCE mutations on medial ganglionic eminence-derived inhibitory GABAergic neurons using the same patient-derived induced pluripotent and gene-edited embryonic stem cell lines, comparing each with their isogenic wild-type control. Differentiation towards inhibitory interneurons demonstrated no significant differences in either early stage (NKX2.1 and FOXG1) or late stage (GAD67 and GABA) developmental markers. Single-cell RNA sequencing also confirmed evidence of markers consistent with medial ganglionic eminence-derived GABAergic neurons and, when compared with two publicly available human fetal ganglionic eminence transcriptomic datasets, confirmed that the cells generated resembled those found in vivo. Further analysis of these data demonstrated transcriptomic dysregulation in genes related to axonal organization, synaptic signalling and action potential generation in the SGCE-mutation-positive neurons. Subsequent characterization of dendritic morphology found SGCE-mutation-positive neurons to have shorter branches, fewer higher-order branches and reduced branching complexity, in comparison to their wild-type controls. Functional analyses using Ca2+ imaging and multi-electrode array approaches to examine network activity identified significantly lower calcium responses to GABA and reduced spike and burst frequencies in the SGCE-mutation-carrying lines, in comparison to their isogenic controls. Reduced activity was also observed in single-cell patch-clamp studies, with fewer neurons firing action potential trains, coupled with fewer spontaneous postsynaptic currents, in comparison to controls. Collectively, this work indicates lower neuronal inhibitory activity and complexity of the dendritic arbor in the context of SGCE mutations, further contributing to the disruption of neuronal excitatory-inhibitory balance in motor circuits and potentially underlying the observed clinical hyperkinetic phenotype. These changes might also represent common characteristics across the wider dystonia spectrum, with potential for future target identification with amenability to therapeutic intervention.