Deletion of a non-canonical regulatory sequence causes loss of Scn1a expression and epileptic phenotypes in mice
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ABSTRACT:
PROVIDER: PRJNA716688 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR14054506.fastq.gz | Fastqsanger.gz | |||
| SRR14054507.fastq.gz | Fastqsanger.gz | |||
| SRR14054508.fastq.gz | Fastqsanger.gz | |||
| SRR14054509.fastq.gz | Fastqsanger.gz | |||
| SRR14054510_1.fastq.gz | Fastqsanger.gz |
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Project description:Background: Genes with multiple co-active promoters appear common in brain, yet little is known about functional requirements for these potentially redundant genomic regulatory elements. SCN1A, which encodes the NaV1.1 sodium channel alpha subunit, is one such gene with two co-active promoters. Mutations in SCN1A are associated with epilepsy, including Dravet Syndrome (DS). The majority of DS patients harbor coding mutations causing SCN1A haploinsufficiency, however putative causal non-coding promoter mutations have been identified. Methods: To determine the functional role of one of these potentially redundant Scn1a promoters, we focused on the non-coding Scn1a 1b regulatory region, previously described as a non-canonical alternative transcriptional start site. We bred a transgenic mouse line with deletion of the extended evolutionarily-conserved 1b non-coding interval and characterized changes in gene and protein expression, and assessed seizure activity and alteration in behavior. Results: Mice harboring a deletion of the 1b non-coding interval exhibited surprisingly severe reductions of Scn1a and NaV1.1 expression throughout the brain. This was accompanied by electroencephalographic and thermal-evoked seizures, and some behavioral deficits. Conclusions: This work contributes to functional dissection of the regulatory wiring of a major epilepsy risk gene, SCN1A. We identified the 1b region as a critical disease-relevant regulatory element and provide evidence
2021-03-26 | GSE169481 | GEO
Project description:Background: Genes with multiple co-active promoters appear common in brain, yet little is known about functional requirements for these potentially redundant genomic regulatory elements. SCN1A, which encodes the NaV1.1 sodium channel alpha subunit, is one such gene with two co-active promoters. Mutations in SCN1A are associated with epilepsy, including Dravet Syndrome (DS). The majority of DS patients harbor coding mutations causing SCN1A haploinsufficiency, however putative causal non-coding promoter mutations have been identified. Methods: To determine the functional role of one of these potentially redundant Scn1a promoters, we focused on the non-coding Scn1a 1b regulatory region, previously described as a non-canonical alternative transcriptional start site. We bred a transgenic mouse line with deletion of the extended evolutionarily-conserved 1b non-coding interval and characterized changes in gene and protein expression, and assessed seizure activity and alteration in behavior. Results: Mice harboring a deletion of the 1b non-coding interval exhibited surprisingly severe reductions of Scn1a and NaV1.1 expression throughout the brain. This was accompanied by electroencephalographic and thermal-evoked seizures, and some behavioral deficits. Conclusions: This work contributes to functional dissection of the regulatory wiring of a major epilepsy risk gene, SCN1A. We identified the 1b region as a critical disease-relevant regulatory element and provide evidence
2021-03-26 | GSE169485 | GEO
Project description:Deletion of a non-canonical regulatory sequence causes loss of Scn1a expression and epileptic phenotypes in mice II
| PRJNA716683 | ENA
Project description:Dravet syndrome (DS) is a severe epileptic encephalopathy caused by heterozygous loss-of-function mutations in the SCN1A gene, indicating a haploinsufficient genetic mechanism underlining this pathology. Here, we tested whether dCas9-mediated Scn1a gene activation could rescue Scn1a haploinsufficiency and restore physiological levels of its gene product, the Nav1.1 voltage-gated sodium channel. We screeened sgRNAs for their ability to stimulate Scn1a gene transcription in association with the dCas9 activation system. Interestingly, we identified one single sgRNA able to significantly increase Scn1a gene expression levels in cell lines as well as in primary neurons, with high specificity. Accordingly, levels of Nav1.1 protein were sufficiently augmented to potentiate firing ability of wild-type immature GABAergic interneurons. A similar effect in activating the Scn1a transcription was elicited in Dravet GABAergic interneurons rescuing their dysfunctional properties. To determine whether this approach could have therapeutic effect, we packaged adeno-associated viruses with the Scn1a-dCas9 activation system and showed their ability to ameliorate the febrile epileptic crises in DS mice. Our results pave the way for exploiting the dCas9-based gene activation as an effective and targeted approach to DS and other similar disorders resulting from altered gene dosage.
2019-03-05 | GSE111436 | GEO
Project description:BackgroundGenes with multiple co-active promoters appear common in brain, yet little is known about functional requirements for these potentially redundant genomic regulatory elements. SCN1A, which encodes the NaV1.1 sodium channel alpha subunit, is one such gene with two co-active promoters. Mutations in SCN1A are associated with epilepsy, including Dravet syndrome (DS). The majority of DS patients harbor coding mutations causing SCN1A haploinsufficiency; however, putative causal non-coding promoter mutations have been identified.MethodsTo determine the functional role of one of these potentially redundant Scn1a promoters, we focused on the non-coding Scn1a 1b regulatory region, previously described as a non-canonical alternative transcriptional start site. We generated a transgenic mouse line with deletion of the extended evolutionarily conserved 1b non-coding interval and characterized changes in gene and protein expression, and assessed seizure activity and alterations in behavior.ResultsMice harboring a deletion of the 1b non-coding interval exhibited surprisingly severe reductions of Scn1a and NaV1.1 expression throughout the brain. This was accompanied by electroencephalographic and thermal-evoked seizures, and behavioral deficits.ConclusionsThis work contributes to functional dissection of the regulatory wiring of a major epilepsy risk gene, SCN1A. We identified the 1b region as a critical disease-relevant regulatory element and provide evidence that non-canonical and seemingly redundant promoters can have essential function.
| S-EPMC8080386 | biostudies-literature
Project description:In this study we recapitulated in the mouse, an SCN1A mutation found in a human Dravet syndrome (DS) patient. The targeted mutation, NC_000068.7:g.66293870C>G (GRCm38.p6) lies in a highly conserved alternate poison exon (20N) of the mouse Scn1a. We performed molecular and behavioral analysis of Scn1a +/- mice and Scn1a +/+ littermate controls. We found that the mutation causes Scn1a mRNA and protein levels to be reduced by about 50% in brain compared to control mice. In addition, the Scn1a +/- mice exhibit behavioral phenotypes seen in previous DS model mice models. We performed qPCR and RNA-seq analysis of the brains of four Scn1a +/- mice and four Scn1a +/+ littermate controls. There was a ~50% reduction in Scn1a RNA-seq counts in Scn1a +/- mice. Our data provides evidence that the mutation causes increased inclusion of the poison exon 20N in Scn1a transcripts leading to nonsense mediated decay and reduction in protein levels.
2020-08-01 | GSE153461 | GEO
Project description:Dravet Syndrome (DS) is a severe epileptic encephalopathy caused primarily by haploinsufficiency of the SCN1A gene. While different therapeutic strategies based on the upregulation of the healthy gene copy are being developed, repetitive seizures might lead to endurable and hardly treatable neural deficits. In fact, whether and to which extent this severe pathology is reversible after symptom onset remains unknown and the lack of this knowledge prevents the development of more effective therapies. We generated a novel Scn1a conditional knock-in mouse model (Scn1aStop) where Scn1a expression could be re-activated on-demand during the mouse lifetime. Scn1a gene disruption leads to the development of seizures, often associated with SUDEP and behavioral alterations including hyperactivity, social interaction deficits and cognitive impairment starting from the second/third week of age. However, we showed that Scn1a gene reconstitution when symptoms were already manifested (P30) led to a complete rescue of both spontaneous and thermic inducible seizures and marked amelioration of behavioral abnormalities. Normalization of firing of hippocampal parvalbumin interneurons was observed after reactivation of the Scn1a mutant allele. We also identified dramatic gene expression alterations associated with astrogliosis in DS mice, rescued by Scn1a gene expression normalization at P30. Interestingly, regaining of Nav1.1 physiological levels rescued epileptic seizures also in adult DS mice (P90) after months of repetitive seizure events. These results uncover the principles governing the reversibility of a severe epileptic encephalopathy as DS and they are central for accelerating therapeutic translation, providing key insights on the timing of delivery of the disease modifying therapies in DS.
2021-11-29 | GSE171191 | GEO
Project description:Pathogenic heterozygous missense mutations in the DNM1 gene result in a novel form of epileptic encephalopathy. DNM1 encodes for the large GTPase dynamin-1, an enzyme with an obligatory role in the endocytosis of synaptic vesicles (SVs) at mammalian nerve terminals. Pathogenic DNM1 mutations cluster within regions required for its essential GTPase activity, implicating disruption of this enzyme activity as being central to epileptic encephalopathy. We reveal that the most prevalent pathogenic mutation in the GTPase domain of DNM1, R237W, disrupts dynamin-1 enzyme activity and SV endocytosis when overexpressed in central neurons. To determine how this dominant-negative heterozygous mutant impacted cell, circuit and behaviour when expressed from its endogenous locus, we generated a mouse carrying the R237W mutation. Neurons isolated from heterozygous mice displayed dysfunctional SV endocytosis, which translated into altered excitatory neurotransmission and seizure-like phenotypes. Importantly, these phenotypes were corrected at the cell, circuit and in vivo level by the drug, BMS-204352, which accelerates SV endocytosis in wild-type neurons. This study therefore provides the first direct link between dysfunctional SV endocytosis and epilepsy, and importantly reveals that SV endocytosis is a viable therapeutic route for monogenic intractable epilepsies.
2023-08-14 | PXD039667 | Pride
Project description:dCas9-based Scn1a gene activation restores inhibitory interneuron excitability and restrains epileptic crises in Dravet syndrome mice
| PRJNA436996 | ENA
Project description:Inherited mutations of calcium ion channels exhibit neurological defects, such as epilepsy, ataxia, and migraine, and these phenotypes are shared among humans and mouse models. Absence epilepsy and ataxic phenotypes are present in the calcium channelopathy mutants stargazer (stg-gamma2 subunit), tottering (tg-alpha1 subunit), and lethargic (lh-beta4 subunit). These mutations of high-voltage-activated (HVA) calcium channel subunits initiate increases in membrane excitability of low-voltage-activated (LVA) calcium channels in thalamic neurons, thus enhancing LVA currents. Elevated LVA currents, produced from T-type calcium channels, induce rhythmic thalamocortical burst firing and spike-wave seizures. The changes in gene expression originating from the different mutations result in similar T-type channel function; however, the network of gene modifications causing altered molecular plasticity and the emergence of pathological phenotypes remain unknown. We would like to understand how loss of 3 different subunits of a calcium ion channel differentially alter gene expression in the brain. Characterizing the gene expression profiles of the mutant stg, tg, and lh mice will provide evidence of the epileptic and ataxic mechanisms, which may identify potential therapeutic targets. We will compare the gene expression profiles among adult mutant stg, tg, and lh mice, which display the ataxic and epileptic phenotypes, along with their wildtype, litter-mate controls, in the cerebellum and brain (without cerebellum). We propose that the neuronal dysfunction associated with the ataxic and epileptic phenotypes develops from a common network of interacting gene alterations within the mutant stg, tg, and lh brain. The changes dictated by the initial mutations leading to the ataxic and epileptic phenotypes are hypothesized to be localized to the cerebellum and the remaining areas of the brain, including the thalamus and cortex, respectively. Adult mutant stg, tg, and lh mice, between 2 and 5 months of age, along with wildtype, litter-mate controls, will be sacrificed. Two brain regions responsible for two phenotypes will be examined. The cerebellum (ataxia) will be dissected from remainder of the forebrain (generalized epilepsy). Each tissue set will be collected in triplicate (3 separate mice per set) to account for biological variance, and total RNA isolated via standard Trizol procedure (Invitrogen) and purified with the RNeasy cleanup kit (Qiagen). RNA samples will be stored at -80 C until sent for analysis using the Affymetrix GeneChip Mouse Genome 430 2.0 whole genome array.
2008-06-14 | E-GEOD-6275 | biostudies-arrayexpress