Single Nuclear RNA Sequencing Reveals Activation of Neuroinflammation Within the Pre‐Bötzinger Complex Following Repeated Seizures

Abstract

Epilepsy is one of the most common neurological diseases in the world. Treatment with anti-epileptic drugs (AEDs) prevents recurrent seizures in ~70% of patients with epilepsy, but the remaining 30% of patients with refractory epilepsies continue to experience seizures. The consequences of repeated seizures include but are not limited to memory impairments and a high risk of post-ictal cardiorespiratory failure, and Sudden Unexpected Death in Epilepsy (SUDEP). Fundamental gaps exist in understanding how repeated seizures disrupt the vital cardiorespiratory control system and neural networks facilitating memory formation in refractory epilepsy. A factor commonly identified in many neurological conditions is neuroinflammation, which may support beneficial health functions but is dysregulated in neurological disease. Key cells within the CNS that mediate beneficial and/or pathological neuroinflammation are resident microglia and astrocytes, which have been shown to be dysfunctional in human epilepsy and alter neuronal function. However, it is not known what mechanistic role they play in contributing to epilepsy-related memory impairment, impairment in cardiorespiratory control, or increased SUDEP risk. Here, we tested the hypothesis that repeated seizures lead to activation of neuroinflammation mediated by microglia and astrocytes in CNS regions of memory and ventilatory control, leading to a progressive decline in neurologic function. 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 (SSkcnj16-/) show that repeated sound-induced seizures lead to a progressively more severe post-ictal suppression of breathing frequency and heart rate, and impaired memory. Single nuclear RNA sequencing of pre-Bötzinger Complex/nucleus ambiguous cells was performed from rats exposed to 0, 3, 7, or 10 seizures to measure cell type-specific gene expression changes induced by seizures. Bioinformatic analyses of transcriptomic changes in microglia, neuronal, and glial cells within these regions point to activation of specific inflammatory pathways following repeated seizures such as IL-1Beta signaling and Toll-like Receptor signaling. Identifying neuroinflammatory signals/pathways induced by repeated seizures within distinct neural circuits will enhance understanding of pathophysiological consequences of uncontrolled seizures in patients with refractory epilepsy and hold the potential for identifying new therapeutic targets aimed at improving memory and cardiorespiratory function to reduce the risk of SUDEP.