First Responders to Hyperosmotic Stress in Murine Astrocytes: Connexin 43 Gap Junctions Are Subject to an Immediate Ultrastructural Reorganization.

Anja Beckmann,Alexander Grißmer,Alice Ferdinand,Carola Meier,Johanna Recktenwald

doi:10.3390/biology10121307

Abstract

Simple SummaryGap junctions are intercellular channels that provide the means for direct transport of small molecules, ions, and water between connected cells. With these functions, gap junctions are essential for the maintenance of astrocytic homeostasis and of particular importance in the context of pathophysiological disbalances. These include the hyperosmolar hyperglycemic syndrome or the pathology after brain trauma. We demonstrate that short-term hyperosmolarity reduces intercellular communication via gap junctions. These functional changes coincide with the transformation of gap junction ultrastructure as evidenced by freeze-fracture replica immunolabeling and transmission electron microscopy. The hyperosmolarity-induced immediate changes in the ultrastructural assembly of connexons, the protein constituents of gap junction channels, have not been described in astrocytes before and are revealing the coherence of structure and function in gap junctions. Phosphorylation of Connexin 43, the main gap junction protein in astrocytes, at amino acid 368 (Serine) might link the two.In a short-term model of hyperosmotic stress, primary murine astrocytes were stimulated with a hyperosmolar sucrose solution for five minutes. Astrocytic gap junctions, which are mainly composed of Connexin (Cx) 43, displayed immediate ultrastructural changes, demonstrated by freeze–fracture replica immunogold labeling: their area, perimeter, and distance of intramembrane particles increased, whereas particle numbers per area decreased. Ultrastructural changes were, however, not accompanied by changes in Cx43 mRNA expression. In contrast, transcription of the gap junction regulator zonula occludens (ZO) protein 1 significantly increased, whereas its protein expression was unaffected. Phosphorylation of Serine (S) 368 of the Cx43 C–terminus has previously been associated with gap junction disassembly and reduction in gap junction communication. Hyperosmolar sucrose treatment led to enhanced phosphorylation of Cx43S368 and was accompanied by inhibition of gap junctional intercellular communication, demonstrated by a scrape loading-dye transfer assay. Taken together, Cx43 gap junctions are fast reacting elements in response to hyperosmolar challenges and can therefore be considered as one of the first responders to hyperosmolarity. In this process, phosphorylation of Cx43S368 was associated with disassembly of gap junctions and inhibition of their function. Thus, modulation of the gap junction assembly might represent a target in the treatment of brain edema or trauma.

Highlights

Gap junctional intercellular communication (GJIC) within the astroglial syncytium is the main component for brain homeostasis, for instance by enabling the spatial buffering of potassium [1]
As we found all three forms of possible fracture faces of gap junctions, i.e., (I) gap junctions containing a fracture step between E- and P-face (Figure 1A,D), (II) gap junctions consistent of P–faces only (Figure 1B,E), or (III) gap junctions comprising E-faces only (Figure 1C,F), we reasoned that short-time hyperosmolarity did not generally cause the disconnection of gap junctions at cell-to-cell contact sides
As Cx43 is the only connexin protein expressed in cultured astrocytes [50], we chose to investigate the effects of hyperosmolarity on Cx43 in this study

Summary

Introduction

Gap junctional intercellular communication (GJIC) within the astroglial syncytium is the main component for brain homeostasis, for instance by enabling the spatial buffering of potassium [1]. Cell membranes of the two connected cells converge to a distance of 2–3 nm, which is considered the structural equivalent for GJIC [2]. Besides their localization at astrocyte–astrocyte or at astrocyte–oligodendrocyte contact sites and in the vicinity of synaptic glomeruli [3,4], another important position of astrocytic gap junctions is in proximity to astrocyte end–feet and the blood–brain barrier, to the integrity of which they are likely to contribute [5,6]. Connexin (Cx) 26, Cx30, and Cx43 were shown to play a role in brain homeostasis, Cx43 is considered the main gap junction protein in astrocytes. A number of in vivo and in vitro studies, using pharmacological gap junction inhibition or the deletion or overexpression of Cx43, confirmed the relevance of Cx43 for astrocytic well-being [7,8,9]

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