Astrocytic connexin43 phosphorylation contributes to seizure susceptibility after mild traumatic brain injury

Astrocytes, glial cells in the brain, play a crucial role in maintaining brain homeostasis through functional gap junctions (GJs) primarily comprising connexin43 (Cx43). These GJs facilitate electrical and metabolic coupling between astrocytes. Dysregulation of Cx43 has been implicated in various pathologies, including traumatic brain injury (TBI) and acquired epilepsy. After mild TBI/concussion, we previously identified a subset of atypical astrocytes that are associated with the development of spontaneous seizures. These astrocytes exhibit reduced Cx43 expression and coupling. However, previous studies have suggested an overall increase in Cx43 protein after TBI. Cx43 also has non-junctional and channel-independent functions including hemichannel communication with the extracellular milieu. In the present study, we set out to determine how mild TBI initiates alterations to Cx43 protein expression and localization, how these alterations are regulated, and whether they contribute to seizure susceptibility. We demonstrate remarkable heterogeneity of Cx43 protein levels across individual astrocytes. A subset of astrocytes displayed decreased Cx43 expression, yet total cortical Cx43 protein increased. Subcellularly, junctional Cx43 remained stable, while hemichannels and/or cytoplasmic Cx43 were increased. Phosphorylation of Cx43 at serine 368, a key regulatory site, increased after mild TBI. Critically, in Cx43^S368A mutant mice lacking this phosphorylation, the mild TBI-induced increase in soluble non-junctional Cx43 and increased hemichannel function did not occur. Furthermore, Cx43^S368A mutant mice exhibited reduced susceptibility to pentylenetetrazol-induced seizures, suggesting that TBI-induced Cx43^S368 phosphorylation enhances seizure susceptibility. We therefore anticipate that inhibiting this post-translational modification may represent a potential therapeutic strategy to mitigate neuronal hyperexcitability and seizure development.