Rationale:
Epilepsy involves persistent transcriptional dysregulation across brain cell types. Previous research from our laboratory identified the JAK/STAT pathway – particularly STAT3 – as a principal regulator of gene changes in acute epileptogenesis as well as chronic human epilepsy. Targeting the robust JAK/STAT activation observed in the days following status epilepticus (SE) with the FDA-approved JAK inhibitor CP690550 (CP) did not alter long-term disease burden. However, brief treatment with CP in chronic epilepsy led to durable seizure suppression, reversal of cognitive deficits, and alleviation of histopathological changes. The molecular basis for this temporal difference in therapeutic efficacy is unknown.
Methods:
We performed single-nucleus RNA sequencing on hippocampal tissue under three conditions: naïve age-matched controls, chronic epilepsy (12wks. post-SE), and chronic epilepsy with CP (2 weeks daily treatment). To dissect acute versus chronic transcriptional dynamics post-SE, we analyzed single-cell RNAseq data from hippocampi harvested at 4 days post-saline and -kainate. Differentially expressed genes (DEGs) were identified across all cell populations. Transcriptional rescue following CP was assessed via two complementary approaches: (1) clustering DEGs within each cell type and classifying them as rescued or not rescued (gene-centric analysis), and (2) evaluating how these DEGs collectively influence cell-level transcriptional states, measured by Euclidean distances between PCA-derived centroids across experimental conditions (cell-centric analysis). MAGIC analysis identified drivers of rescued versus non-rescued gene clusters. Ontological analysis provided functional heuristics describing rescued and non-rescued genes across cell types.
Results:
Both acute epileptogenesis and chronic epilepsy induced robust gene expression changes across cell populations. In concordance with previously reported results that acute CP treatment did not alter disease, bulk RNAseq data from animals treated with CP acutely post-SE showed marginal rescue of genes altered by SE. However, chronic CP treatment rescued a substantial fraction of DEGs, especially in dentate granule cells and astrocytes. Notably, rescued clusters were enriched for STAT3-target genes. Preliminary analyses suggest rescue of both up- and down-regulated genes across diverse cell types. Ongoing analyses aim to delineate cell-type-specific transcriptional programs distinguishing the acute versus chronic period.
Conclusions:
JAK/STAT pathway inhibition with CP rescues transcriptional dysregulation observed in chronic epilepsy across cell types, demonstrating profound therapeutic effect at the molecular level. These findings highlight CP as a promising candidate for reversing pathology in chronic epilepsy. Future studies will focus on 1) identifying cell populations resistant to CP rescue, providing a foundation for combinatorial therapeutic strategies, and 2) expanding the analysis to include time points months after CP treatment to identify genes and cell types which remain enduringly rescued after drug withdrawal.
Funding:
This work was supported by CURE, Lily’s Fund, and NIH grants R01NS108756 and R21NS093364.