Astrocytic connexin43 phosphorylation contributes to seizure susceptibility after mild Traumatic Brain Injury.

Unlabelled: Astrocytes play a crucial role in maintaining brain homeostasis through functional gap junctions (GJs) primarily formed by connexin43 (Cx43). These GJs facilitate electrical and metabolic coupling between astrocytes, allowing the passage of ions, glucose, and metabolites. Dysregulation of Cx43 has been implicated in various pathologies, including traumatic brain injury (TBI) and acquired epilepsy. We previously identified a subset of atypical astrocytes after mild TBI that exhibit reduced Cx43 expression and coupling and are correlated with the development of spontaneous seizures. Given that mild TBI affects millions globally and can lead to long-term complications, including post-traumatic epilepsy, understanding the molecular events post-TBI is critical for developing therapeutic strategies. In the present study, we assessed the heterogeneity of Cx43 protein expression after mild TBI. In accordance with our previous findings, a subset of astrocytes lost Cx43 expression. As previously reported after TBI, we also found a significant increase in total Cx43 protein expression after mild TBI, predominantly in the soluble form, suggesting that while junctional Cx43 protein levels remained stable, hemichannels and cytoplasmic Cx43 were increased. We then investigated the phosphorylation of Cx43 at serine 368 after TBI, which is known to influence GJ assembly and function. Phosphorylation of Cx43 at serine 368 is elevated following TBI and Cx43 mutant mice, lacking this phosphorylation, exhibited reduced susceptibility to seizures induced by pentylenetetrazol (PTZ). These findings suggest that TBI-induced Cx43 phosphorylation enhances seizure susceptibility, while inhibiting this modification presents a potential therapeutic avenue for mitigating neuronal hyperexcitability and seizure development.

Significance Statement: Connexin43 (Cx43) is the main protein of astrocyte gap junctions, mediating astrocyte coupling of astrocytes into cellular networks, but has also other non-junctional functions. Many pathologies present with altered Cx43 regulation. In this study, we studied Cx43 alterations after mild traumatic brain injury (TBI) in a mouse model. We found that while some astrocyte lost Cx43 expression, a subset of astrocytes experienced an increase in cytoplasmic and hemichannel Cx43. This increase correlated with an increase in phosphorylated Cx43 at serine 368. Cx43 mutant mice, lacking this phosphorylation, exhibited reduced susceptibility to seizures induced by pentylenetetrazol (PTZ). These findings suggest that TBI-induced Cx43 phosphorylation enhances seizure susceptibility.