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.
Rac GTPases regulate the development of cortical/hippocampal GABAergic interneurons by affecting the early development and migration of GABAergic precursors. We have addressed the function of Rac1 and Rac3 proteins during the late maturation of hippocampal interneurons. We observed specific phenotypic differences between conditional Rac1 and full Rac3 knockout mice. Rac1 deletion caused greater generalized hyperactivity and cognitive impairment compared with Rac3 deletion. This phenotype matched with a more evident functional impairment of the inhibitory circuits in Rac1 mutants, showing higher excitability and reduced spontaneous inhibitory currents in the CA hippocampal pyramidal neurons. Morphological analysis confirmed a differential modification of the inhibitory circuits: deletion of either Rac caused a similar reduction of parvalbumin-positive inhibitory terminals in the pyramidal layer. Intriguingly, cannabinoid receptor-1-positive terminals were strongly increased only in the CA1 of Rac1-depleted mice. This increase may underlie the stronger electrophysiological defects in this mutant. Accordingly, incubation with an antagonist for cannabinoid receptors partially rescued the reduction of spontaneous inhibitory currents in the pyramidal cells of Rac1 mutants. Our results show that Rac1 and Rac3 have independent roles in the formation of GABAergic circuits, as highlighted by the differential effects of their deletion on the late maturation of specific populations of interneurons.
Background Experimental autoimmune encephalomyelitis (EAE) is a common animal model of multiple sclerosis (MS). C57BL/6 mice immunized with myelin oligodendrocyte glycoprotein exhibit chronic disease course, together with optic neuritis, consisting of demyelination/axonal loss of the optic nerve. Objectives To characterize functional and structural visual damages in two different phases of EAE: pre- and post-motor onset. Methods Visual alterations were detected with Visual Evoked Potential (VEP), Electroretinogram (ERG) and Optical Coherence Tomography (OCT). Optic nerve histology was performed at 7 (pre-motor onset) or 37 (post-motor onset) days post-immunization (dpi). Results At 7 dpi, optic nerve inflammation was similar in EAE eyes with and without VEP latency delay. Demyelination was detected in EAE eyes with latency delay (p < 0.0001), while axonal loss (p < 0.0001) and ERG b-wave amplitude (p = 0.004) were decreased in EAE eyes without latency delay compared to Healthy controls. At 37 dpi, functional and structural optic nerve damage were comparable between EAE groups, while a decrease of ERG amplitude and NGCC thickness were found in EAE eyes with VEP latency delay detected post-motor onset. Conclusions Thanks to non-invasive methods, we studied the visual system in a MS model, which could be useful for developing specific therapeutic strategies to target different disease phases.
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