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 SGCE gene, and in which both motor and psychiatric phenotypes are observed. Results from murine and in vivo human studies suggest dystonia is caused by disruption to neuronal networks, and 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 in the observed phenotypes. Our previous work, focused on cortical excitatory glutamatergic neurons, demonstrated a hyperexcitable phenotype and more complex dendritic arborisation in an in vitro model of Myoclonus Dystonia. By 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 to their isogenic wild-type control. Differentiation towards inhibitory interneurons demonstrated no significant differences in neither early (NKX2.1, FOXG1), nor late stage (GAD67, GABA), developmental markers. Single-cell RNA sequencing additionally confirmed evidence of markers consistent with Medial Ganglionic Eminence-derived GABAergic neurons, and when compared to two publicly available human foetal ganglionic eminence transcriptomic datasets, confirmed that the cells generated resembled those found in vivo. Further analysis of this data demonstrated transcriptomic dysregulation in genes related to axonal organization, synaptic signalling and action potential generation in the SGCE-mutation positive neurons. Subsequent characterisation of dendritic morphology found SGCE-mutation positive neurons to have shorter branches, fewer higher order branches and reduced branching complexity, compared to their wild-type controls. Functional analyses using Ca2+ imaging and MEA approaches to examine network activity identified significantly lower calcium responses to GABA and reduced spike and burst frequencies in the SGCE-mutation carrying lines, compared 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 post-synaptic currents, compared 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 may also represent common characteristics across the wider dystonia spectrum, with potential for future target identification with amenability to therapeutic intervention.