Transplantation of GABAergic interneurons (INs) can provide long-term functional benefits in animal models of epilepsy and other neurological disorders. Whereas GABAergic INs can be differentiated from embryonic stem cells, alternative sources of GABAergic INs may be more tractable for disease modeling and transplantation. We identified five factors (Foxg1, Sox2, Ascl1, Dlx5, and Lhx6) that convert mouse fibroblasts into induced GABAergic INs (iGABA-INs) possessing molecular signatures of telencephalic INs. Factor overexpression activates transcriptional networks required for GABAergic fate specification. iGABA-INs display progressively maturing firing patterns comparable to cortical INs, form functional synapses, and release GABA. Importantly, iGABA-INs survive and mature upon being grafted into mouse hippocampus. Optogenetic stimulation demonstrated functional integration of grafted iGABA-INs into host circuitry, triggering inhibition of host granule neuron activity. These five factors also converted human cells into functional GABAergic INs. These properties suggest that iGABA-INs have potential for disease modeling and cell-based therapeutic approaches to neurological disorders.

SummaryDirect cell reprogramming enables direct conversion of fibroblasts into functional neurons and oligodendrocytes using a minimal set of cell-lineage-specific transcription factors. This approach is rapid and simple, generating the cell types of interest in one step. However, it remains unknown whether this technology can be applied to convert fibroblasts into astrocytes, the third neural lineage. Astrocytes play crucial roles in neuronal homeostasis, and their dysfunctions contribute to the origin and progression of multiple human diseases. Herein, we carried out a screening using several transcription factors involved in defining the astroglial cell fate and identified NFIA, NFIB, and SOX9 to be sufficient to convert with high efficiency embryonic and postnatal mouse fibroblasts into astrocytes (iAstrocytes). We proved both by gene-expression profiling and functional tests that iAstrocytes are comparable to native brain astrocytes. This protocol can be then employed to generate functional iAstrocytes for a wide range of experimental applications.

Dravet syndrome (DS) is a severe epileptic encephalopathy caused mainly by heterozygous loss-of-function mutations of the SCN1A gene, indicating haploinsufficiency as the pathogenic mechanism. Here, we tested whether catalytically dead Cas9 (dCas9)-mediated Scn1a gene activation can rescue Scn1a haploinsufficiency in a mouse DS model and restore physiological levels of its gene product, the Na v 1.1 voltage-gated sodium channel. We screened single guide RNAs (sgRNAs) for their ability to stimulate Scn1a transcription in association with the dCas9 activation system. We identified a specific sgRNA that increases Scn1a gene expression levels in cell lines and primary neurons with high specificity. Na v 1.1 protein levels were augmented, as was the ability of wild-type immature GABAergic interneurons to fire action potentials. A similar enhancement of Scn1a transcription was achieved in mature DS interneurons, thus rescuing their ability to fire. To test the therapeutic potential of this approach, we delivered the Scn1a-dCas9 activation system to DS pups using adeno-associated viruses. Parvalbumin interneurons recover their firing ability and febrile seizures were significantly attenuated. Our results pave the way for exploiting dCas9-based gene activation as an effective and targeted approach in DS and other disorders resulting from altered gene dosage.

Intrinsic homeostasis enables neuronal circuits to maintain activity levels within an appropriate range by modulating neuronal voltage-gated conductances, but the signalling pathways involved in this process are largely unknown. We characterized the process of intrinsic homeostasis induced by sustained electrical activity in cultured hippocampal neurons based on the activation of the Repressor Element-1 Silencing Transcription Factor/ Neuron-Restrictive Silencer Factor (REST/NRSF). We showed that 4-aminopyridine-induced hyperactivity enhances the expression of REST/NRSF, which in turn, reduces the expression of voltage-gated Na þ channels, thereby decreasing the neuronal Na þ current density.This mechanism plays an important role in the downregulation of the firing activity at the single-cell level, reestablishing a physiological spiking activity in the entire neuronal network. Conversely, interfering with REST/ NRSF expression impaired this homeostatic response. Our results identify REST/NRSF as a critical factor linking neuronal activity to the activation of intrinsic homeostasis and restoring a physiological level of activity in the entire neuronal network.