Transcriptomic disruption and functional hypoactivity in DYT-SGCE MGE-patterned inhibitory neurons
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ABSTRACT:
PROVIDER: PRJNA1180093 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR31174136_1.fastq.gz | Fastqsanger.gz | |||
| SRR31174136_2.fastq.gz | Fastqsanger.gz | |||
| SRR31174137_1.fastq.gz | Fastqsanger.gz | |||
| SRR31174137_2.fastq.gz | Fastqsanger.gz | |||
| SRR31174138_1.fastq.gz | Fastqsanger.gz |
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Project description: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 gangliacerebello- 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 patientderived 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 SGCEmutation 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 SGCEmutation 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
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Project description:The Dlx homeodomain transcription factors are implicated in regulating the function of inhibitory GABAergic interneurons; therefore understanding their functions will provide insights into disorders such as epilepsy, mental retardation and autism. Identifying genes that are downstream of Dlx1/2 function and are relevant for the differentiation and survival of GABAergic interneurons. During embryonic development, cortical GABAergic interneurons are generated in the proliferative zone of the medial ganglionic eminence (MGE), from where they migrate to reach their final positions in the cortex. The differentiation of these interneuron precursors is dependent on Dlx genes, as shown by Dlx1/Dlx2 double mutants, which have a block in GABAergic cell differentiation and in cell migration. When interneuron progenitors are isolated from the mutant MGE and growth in culture, they are able to proceed along their differentiation program. However, mutant cells growth in vitro show defects in cell morphogenesis and increased cell apoptosis. We hypothesize that Dlx transcription factors regulate important aspects of GABAergic neuron differentiation such as the formation and growth of axon and dendrites, and the formation of inhibitory synapses. We generated E15.5 mouse embryos that are Dlx1/2 -/- or Dlx1/2 +/?. Genotype was confirmed by PCR. A total of 8 litters were used. For each experiment, we pooled tissue from at least 6 different embryos of the same genotype. We dissected the ventricular and subventricular zones of the MGE (rostral part). This area contains ~1 million of progenitor cells per embryo. We isolated total RNA using the Stratagene RNA Miniprep kit (these samples are called MGE+/ and MGE-/- in our proposal). In addition, we used the same area (ventricular and subventricular zones of the rostral MGE) to perform primary neuronal cultures. Cells were maintained 3 days in vitro. After that, we isolated total RNA using the Stratagene RNA Miniprep kit (samples called primary cells+/ and primary cells-/- in our proposal). We would like to perform gene expression comparison between: 1) MGE+/ and MGE-/-, and 2) primary cells+/ and primary cells-/-.
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Project description:To identify the activity-induced gene expression programs in inhibitory and excitatory neurons, we analyzed RNA extracted from cultured E14 mouse MGE- and CTX-derived neurons (DIV 10) after these cultures were membrane-depolarized for 0, 1 and 6 hrs with 55mM extracellular KCl. To identify the gene programs regulated in these cells by the activity-induced early-response transcription factor Npas4, we repeated the same experiment in the MGE- and CTX-cultures lacking Npas4 (Npas4-KO). Littermate mouse E14 MGE- or CTX-derived neurons (WT or KO for Npas4) were cultured for 9 days, quieted overnight with TTX and AP-5 and then membrane-depolarized for 0, 1 or 6 hours by raising the extracellular KCl-concentration to 55mM. RNA was then extracted and analyzed using Affymetrix GeneChip Mouse Expression Set 430 2.0 microarray platform.
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