Unravelling mechanisms causing astrocytic uncoupling in epilepsy

Abstract

The dynamic regulation of connexins throughout its life cycle has profound impact on the level of gap junctional coupling in astrocytes. Astrocytic uncoupling in epilepsy can be viewed in the context of modifications in their connexins. The present work sheds a light on seizure-induced alterations in connexins associated with uncoupling in astrocytes. This study unequivocally proves that post seizure uncoupling in astrocytes is not caused by the scarcity of connexins. In fact, in hippocampal specimens from epilepsy patients and in the mouse model of epilepsy 3 months post injection, an increase in the total Cx43 protein levels was found. This increase in the total Cx43 levels, however did not result in the increase in plasma membrane Cx43 levels indicating impaired translocation of this protein. All these features of Cx43 expression and distribution were also observed in the mouse model of epilepsy at the 3 months’ time point highlighting its clinical relevance. At this time point, a significant change in the phosphorylation of Cx43 was detected. Remarkably, as early as 4 h after kainate injection, altered Cx43 phosphorylation, indicated by a shift of Cx43 bands towards P2, was noticed. This phosphorylation of Cx34 was further characterized in detail with the help of mass-spectrometry reported for the first time in an epilepsy model. In total, 14 Cx43 phosphorylated sites were detected of which, phosphorylated T290 and Y301 have not been described before. Phosphorylation at S257, S262, S328 and S330 was found to be differentially regulated after kainate-induced seizures. Phosphorylation at S262 could be associated with astrocytic uncoupling in the ipsilateral hippocampus. Interestingly, this phosphorylation could also be induced by TNFα and IL-1β and could be blocked by XPRO1595, a blocker of the soluble form of TNFα. Altogether, the present study provides vital insights into the mechanisms of astrocytic uncoupling in epilepsy. These can be utilized to devise strategies to prevent astrocytic uncoupling in experimental and clinical epilepsies.