Single-nuclear RNA sequencing reveals sex-specific differences in cell composition and recruitment of neuroinflammatory pathways after repeated seizures within the NA/pre-Bötzinger Complex region

Abstract

Epilepsy affects millions worldwide, but a significant portion suffers from uncontrollable epilepsy. Repeated seizures have many consequences, including a high risk of post-ictal cardiorespiratory failure and Sudden Unexpected Death in Epilepsy (SUDEP). Major risk factors for SUDEP include biological sex and the occurrence of generalized tonic-clonic seizures (GTCSs). How repeated seizures lead to cardiorespiratory dysfunction remains unknown. It was reported that a key factor in many neurological diseases is neuroinflammation, which is mediated mostly by microglia and astrocytes that become dysfunctional. Mechanistically, questions remain how they affect neuronal function in epilepsy and contribute to cardiorespiratory dysfunction and increased SUDEP risk. Here we tested the hypothesis that repeated seizures lead to differential neuroinflammatory activation after repeated seizures in CNS regions of ventilatory control in a sex-specific manner. Preliminary data in our novel rat model with genetic mutations in Kcnj16, a gene encoding an inwardly-rectifying potassium channel Kir5.1 in Dahl salt-sensitive rat (SS kcnj16-/- ) show that repeated sound-induced seizures lead to progressively more severe post-ictal suppression of breathing frequency and heart rate and increased mortality in males. Single-nuclear RNA sequencing of Nucleus Ambiguous/pre-Bötzinger Complex (NA/preBötC) cells (roughly 80,000 and 40,000) was performed in tissues from male and female SS kcnj16-/- rats (n=3/group) exposed to 0, 3, 7, or 10 seizures to measure cell type-specific gene expression changes induced by seizures. Bioinformatic analyses of transcriptomic changes across sex revealed the identification of 19 unique cell types in males and 21 unique cell types in females in this brainstem region. Differentially expressed genes (DEGs; Seurat) in both males and females showed significant ( p<0.05) recruitment of various inflammatory and stress-related pathways (e.g. CXCR4 signaling, Immunogenic Cell Death Signaling). Data integration across sex enabled sex-specific comparisons across days of the seizure (around 120,000 cells). Importantly, males show significant upregulation in DEGs with seizures in mTOR signaling and Extracellular Neutrophil Trap Signaling when compared to females. Importantly, this dataset helps characterize at a genetic and cell-type specific manner, sex differences in a crucial region not characterized before in the literature. Also, these data show clear sex differences in the cellular milieu and transcriptomic profiles in the NA/preBötC region before and after repeated seizures, suggesting different neuroinflammatory pathways may play a larger role in males than females. Understanding sex differences and how they may be playing a role in neuroinflammaton after repeated seizures may provide novel insights into identifying new therapeutic drugs aimed at reducing the risk of SUDEP in males and, more broadly, those suffering from uncontrolled epilepsy. Supported by HL122358, HL160122 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.