Abstracts

Rationale: Scn1a+/- mice recapitulate key features of Dravet syndrome, including seizures provoked by hyperthermia, unprovoked seizures, and high risk of SUDEP. Penetrance and severity of Dravet phenotypes is dependent on the strain background. Scn1a+/- mice on 129S6/SvEvTac (S6.Scn1a+/-) have no overt phenotype and normal lifespan, while Scn1a+/- mice on the [S6xC57BL/6J] F1 strain (F1.Scn1a+/-) exhibit spontaneous seizures and premature lethality. We previously performed bulk hippocampal RNA-seq of Scn1a+/- and WT mice on the protective S6 and permissive [S6xB6] F1 strains and found few differences in gene expression between genotypes within the same strain prior to seizure onset. In contrast, F1.Scn1a+/- mice with active seizures had differential expression of many genes associated with neuroprotection and astrogliosis compared to F1.WT or S6.Scn1a+/- mice. However, bulk RNA-seq analysis did not allow analysis at the level of specific cellular subpopulations. In the current study, we use single nucleus RNA sequencing (snRNAseq) to examine changes in gene expression and cell composition at the single cell level.

Methods: S6.Scn1a+/- is maintained on the S6 strain (Taconic). S6.Scn1a+/- mice were crossed with C57BL/6J (Jackson Labs) to generate F1.Scn1a+/- mice and wild-type (WT) littermates (F1.WT). Scn1a+/- mice were video-monitored for spontaneous seizures at P22-P24 and were stratified by presence or absence of seizures prior to tissue collection. At P24, hippocampi were isolated and snap frozen. Single nuclei were isolated and single nucleus 3' v3 libraries were prepared and sequenced in the NuSeq Core. Raw sequence reads were processed using the 10x Genomics Cell Ranger pipeline with a pre-mRNA reference for alignment. Subsequent analyses used the R packages Seurat (4.3.0) and DeSeq2 (1.34.0).

Results:
Consistent with our prior bulk RNA-seq study, there were few differences in gene expression between genotypes within the same strain in the absence of seizures. For example, F1.WT and F1.KO mice without seizures had only 10 differentially expressed genes (DEG) limited to 4 neuron clusters. In contrast, relative to F1.WT mice, F1.KO with seizures (range: 2-20 seizures) had over 1800 DEGs across 10 neuron clusters. The most dramatic changes in gene expression were in dentate granule cells, with no DEGs between genotypes in the absence of seizures and >1100 DEGs following seizures. In addition, there were evident shifts in granule cell populations following seizures, with enrichment of Penk-expressing dentate granule cells that may signify recruitment of semilunar granule cells. Seizure activity also resulted in 222 DEGs in astrocytes, as well as shifts in astrocyte subpopulations with enrichment of type A2 reactive astrocytes.

Conclusions:
Prior to seizure onset, there were minimal changes in gene expression and cell proportions between Scn1a^+/- and WT on the 129 or F1 strains. Following seizure onset, F1.Scn1a.^+/- mice exhibited changes cell proportions and gene expression profiles within dentate granule cell and astrocyte clusters.

Funding:
Supported by NINDS/NIH grant R01 NS084959 (to JAK)