Astrocytic connexin43 phosphorylation contributes to seizure susceptibility after mild traumatic brain injury
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Abstract
Astrocytes play a crucial role in maintaining brain homeostasis through functional gap junctions (GJs) primarily formed by connexin43 (Cx43) in the cortical gray matter. 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. After mild TBI/concussion, we previously identified a subset of atypical astrocytes, which are correlated with the development of spontaneous seizures. These astrocytes exhibit reduced Cx43 expression and coupling. However, atypical astrocytes represent a relatively small subset of astrocytes within the cortical gray matter and previous studies suggest an overall increase of Cx43 protein after TBI. Additionally, Cx43 also has non-junctional and channel-independent functions, which include hemichannel communication with the extracellular milieu, cell adhesion, protein trafficking, protein-protein interactions, and intracellular signaling. In the present study, we set out to determine how mild TBI initiates alterations to Cx43 protein expression and localization, how they may be regulated, and whether they contribute to seizure susceptibility. We demonstrate remarkable heterogeneity of Cx43 protein levels from astrocyte to astrocyte. In accordance with our previous findings, a subset of astrocytes lost Cx43 expression, yet total cortical Cx43 protein increased. At the subcellular level, junctional Cx43 protein levels remained stable, while hemichannels and/or cytoplasmic Cx43 were increased. Phosphorylation of Cx43 at serine 368, a key regulatory site influencing GJ assembly and function, increased after mild TBI. Critically, Cx43 S368A mutant mice, lacking this phosphorylation, exhibited reduced susceptibility to pentylenetetrazol-induced seizures. 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 comprising astrocyte gap junctions which mediate astrocyte coupling into cellular networks, but it also has other non-junctional functions. Many pathologies present with altered Cx43 regulation. In this study, we assessed Cx43 alterations after mild traumatic brain injury (TBI) in a mouse model. We found that while some astrocytes lost Cx43 expression, other astrocytes had increased cytoplasmic and hemichannel Cx43. This increase correlated with an increase in phosphorylated Cx43 at serine 368. Cx43 S368A 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.
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Reply to the reviewers
Reviewer comment: *“The authors did not clarify whether the observed protection to PTZ-induced convulsions after mild TBI is due to the reduced size of gap junctions and/or increased activity in hemichannels.” And “The super-resolution imaging only assesses Cx43 gap junction plaque size and density but not the non-junctional portion of Cx43.” *
Response and planned revision: To determine whether seizure protection in Cx43 S368A mice is due to reduced gap junction plaque density or reduced hemichannel function, we will conduct solubility assays to assess the ratio of insoluble (junctional) to soluble (cytoplasmic/hemichannel) Cx43 in Cx43S368A and …
Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.
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Reply to the reviewers
Reviewer comment: *“The authors did not clarify whether the observed protection to PTZ-induced convulsions after mild TBI is due to the reduced size of gap junctions and/or increased activity in hemichannels.” And “The super-resolution imaging only assesses Cx43 gap junction plaque size and density but not the non-junctional portion of Cx43.” *
Response and planned revision: To determine whether seizure protection in Cx43 S368A mice is due to reduced gap junction plaque density or reduced hemichannel function, we will conduct solubility assays to assess the ratio of insoluble (junctional) to soluble (cytoplasmic/hemichannel) Cx43 in Cx43S368A and C57BL/6 control mice after TBI/sham (as in Fig. 2A-D currently only in C57BL/6 control mice). In parallel, we will perform EtBr uptake assays in acute brain slices from Cx43S368A and C57BL/6 control animals to assess hemichannel function.
Additionally, we will include super-resolution images without background subtraction, which show diffuse staining indicative of soluble Cx43. Of note, even at super-resolution individual gap junctions or hemichannels cannot be resolved. They appear as diffuse signal (currently not visible in our super-resolution images due to image deconvolution and background substration performed to isolate Cx43 plaques). Super-resolution imaging was used to count Cx43 gap junction plaque densities and size. Cx43 gap junction plaques are dense accruals of Cx43 immunostaining reminiscent functional and closed gap junctions. Complimentary experiments measured soluble (cytoplasmic Cx43 and hemichannels) and insoluble Cx43 (gap junctions) using biochemistry (Fig. 2A-D).
Reviewer comment: “The immunofluorescent images for Fig. 2E and Fig. 5 were not counterstained for astrocytes or cell membrane. How can the authors be sure that these are expressed by astrocytes and not other cells in the brain?”
Response and planned revision: Cx43 is predominantly expressed in astrocytes, with expression levels 10–100 times higher than in brain endothelial cells (e.g., Zhang et al., 2014; Vanlandewijck et al., Nature, 2018). As shown in Supplementary Fig. 2, our immunohistochemistry data reveal no overlap between Cx43 and endothelial cell markers, confirming that our staining protocol does not detect Cx43 in endothelial cells. Instead, the apparent localization of Cx43 along blood vessels reflects expression in astrocytic endfeet, which closely ensheath the vasculature. To further support this conclusion, we will conduct quantitative co-localization analyses of Cx43 with markers for neurons, microglia, oligodendrocytes, and NG2 glia in both Cx43S368A and C57BL/6 control mice. Additionally, we will include plots generated from publicly available single-cell RNA sequencing datasets to show that Cx43 mRNA is highly enriched in astrocytes and present at much lower levels in endothelial cells of the brain vasculature.
Reviewer comment* about developmental contributions to the phenotype of Cx43 S368A animals.*
Response: We cannot exclude a potential developmental component to the observed seizure protection in Cx43S368A mice. We included discussion of this possibility in the revised manuscript.
Reviewer comments* indicative of a lack of clarity around rationale and intent of specific experiments.*
Response: We thoroughly revised the Results section to explicitly state the rationale and purpose of each experiment. For example:
Reviewer comment: “The immunofluorescent images for Fig. 1D and E were taken at low resolution compared to the Cx43 puncta size. This does not allow accurate quantification of the Cx43 GJs or HCs.”
Response: The purpose of this experiment was to assess the heterogeneity of Cx43 expression (both junctional and non-junctional portions) with spatial resolution across a larger brain area. Complementary experiments here are quantification of protein amounts using western blot (Fig. 1B), quantification of junctional versus non-junctional Cx43 using the solubility assay and quantification of Cx43 plaques using super-resolution imaging (Fig. 2).
Reviewer comment: “TBI did not change Cx43 plaque size or density (Fig. 5). What was the rationale for examining the effects in the S368A mutant?”
__Response: We found an increase in phosphorylated Cx43 at ____S____368 after TBI and Cx43__S368A mutants are protected from seizures after administration of PTZ suggesting an important role for this specific Cx43 phosphorylation site in pathology. We discussed in the manuscript that “in cardiovascular infection/disease has demonstrated maintenance of gap junction coupling (Gy et al., 2011; Padget et al., 2024) while reduced hemichannel opening probability was reported (Hirschhäuser et al., 2021) in Cx43S368A mice”, suggesting that the protective phenotype is likely due to modification of either Cx43 gap junctions or hemichannels. However, functional consequences on Cx43 biology upon phosphorylation at S368 or lack thereof in the Cx43S368A mutant remain unexplored in the brain. Cx43 plaque size and density are reflective of Cx43 gap junctions and was therefore examined in Cx43S368A mice to reveal potential mechanism by which this mouse mutant is protected from seizures (even in the absence of TBI).
Reviewer comment: * “The IC50 for Tat-Gap19 for Cx43 HC is ~7 μM (Tocris). How can using it at 2 μM be effective?”*
Response: We reviewed our lab records and confirmed that 2 μM was a typographical error. The actual concentration used was 200 μM. This is consistent with the dose-response literature for astrocytes (e.g., Walrave et al., Glia 2018; Abudara et al., Front. Cell. Neurosci. 2014). We now included these references in the manuscript.
Reviewer comment: “Unclear whether mice in Fig. 4C received TBI.”
Response: We clarified that these mice were naïve, i.e. not subjected to TBI or sham procedures. This is now explicitly stated in both the Methods and the Results.
Reviewer comment: “CBX or Tat-Gap19 do not affect the phosphorylation state of Cx43.”
Response: We clarified that we used CBX and Tat-Gap19 as established gap junction and hemichannel blockers, irrespective of phosphorylation state. We now noted that Tat-GAP19 is a Cx43 mimetic peptide to specifically block Cx43 hemichannels.
Reviewer comment: “It is unclear whether the EtBr quantification in Fig. 3D is for S100β+ astrocytes.”
Response: We clarified that the quantification in Fig. 3D was performed exclusively in S100β+ astrocytes. Although neurons may take up EtBr under inflammatory conditions, they do not express Cx43 (as will be shown in Fig. 1 and Supplementary Data).
Reviewer comment: “I believe that the 'W.' in ref 'W. Chen et al., 2018' is unnecessary.”
Response: We will use the journal citation style implemented by a reference manager in the final version of the manuscript.
Reviewer request* to include two references related to phosphorylation and hemichannel permeability and the role of gap junctional coupling in epilepsy.*
Response: The PNAS reference was added to the manuscript.
That reduction in gap junctional communication is a relevant factor in epilepsy is discussed in the introduction where we also cite original literature of the authors of the proposed review article: “Many pathologies (Gajardo-Gómez et al., 2017; Masaki, 2015; Orellana et al., 2011; Sarrouilhe et al., 2017; Vis et al., 1998; Wang et al., 2018), including traumatic brain injury (TBI) (B. Chen et al., 2017; W. Chen et al., 2019; Wu et al., 2013; Xia et al., 2024) and acquired epilepsy (Bedner et al., 2015; Deshpande et al., 2017; Walrave et al., 2018) present with altered Cx43 regulation, and are often equated with GJ dysfunction.”
We feel that citing the original manuscripts more accurately reflect the current knowledge around the role of Cx43 in the context of epilepsy and other pathologies. Reader’s access to the original literature also highlights the gaps in knowledge more precisely that this manuscript seeks to close.
__Reviewer comment: __“I think the data of this manuscript is missing a control animal that would present all the compensation changes that occur during development that occur in mice carrying the mutated Cx43. Alternatively, a doable experiment would be the use of inducible KO/KI.”
Response: Previous studies investigating the role of Cx43 in neuronal excitability have primarily used full or conditional knockout models, as described in our introduction. Interestingly, these studies report that global deletion of Cx43 increases seizure susceptibility. However, such models eliminate all Cx43-dependent functions—both junctional and non-junctional—making it difficult to pinpoint the specific mechanisms underlying the observed effects. They do not distinguish whether increased excitability results from loss of gap junction coupling, disruption of hemichannel function, or depletion of cytoplasmic Cx43 signaling. In contrast, our current study does not aim to eliminate Cx43, but instead employs a targeted approach to interrogate the functional significance of a regulatory phosphorylation site, S368. This site is dynamically phosphorylated following TBI and has been previously associated—albeit only through correlative data—with seizure activity and other neuropathologies. By isolating the contribution of this post-translational modification while preserving overall Cx43 expression, our study provides novel mechanistic insight into how phosphorylation modulates Cx43 function and astrocyte-mediated regulation of brain excitability.
We appreciate the thoughtful suggestion to generate a conditional knock-in model to isolate developmental from acute effects of the Cx43 S368A mutation. However, the GJA1 gene locus is not amenable to this type of targeting (we explored this possibility with a . We also considered AAV-mediated CRISPR/dCas9 editing as an alternative, but current limitations in CNS transduction efficiency, promoter specificity, and guide RNA availability for precise point mutation insertion make this approach similarly unfeasible at this stage. Thus, while we acknowledge the developmental caveat (which we now discuss in the manuscript), the current manuscript provides novel and meaningful insight into the role of the Cx43S368 regulatory phosphorylation site in the context of astrocyte biology and seizure susceptibility and forms a strong foundation for future studies.
Thank you again for the opportunity to revise and strengthen our manuscript. We believe these planned experiments and clarifications address the reviewers' concerns in a thorough and scientifically rigorous manner.
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Referee #2
Evidence, reproducibility and clarity
This manuscript describes interesting findings on the effect of a Cx43 mutant that is not phosphorylated in Ser368. The authors did not clarify whether the observed protection to PTZ-induced convulsions after mild TBI is due to the reduced size of gap junctions and/or increased activity in hemichannels. A limitation of this work is that Cx43 S368A forms smaller gap junctions revealing an important phenotype change and therefore there is no appropriate control unless they generate a cell-specific inducible Cx43 KO.
In previous studies, it has been proposed that reduction in gap junctional communication is a relevant factor in …Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
This manuscript describes interesting findings on the effect of a Cx43 mutant that is not phosphorylated in Ser368. The authors did not clarify whether the observed protection to PTZ-induced convulsions after mild TBI is due to the reduced size of gap junctions and/or increased activity in hemichannels. A limitation of this work is that Cx43 S368A forms smaller gap junctions revealing an important phenotype change and therefore there is no appropriate control unless they generate a cell-specific inducible Cx43 KO.
In previous studies, it has been proposed that reduction in gap junctional communication is a relevant factor in epilepsy, which is not discussed (Please see doi: 10.3390/cells12121669) in the present manuscript. Also, Bao and collaborators have demonstrated that Cx43 hemichannels phosphorlated by PKC present reduced permeability to molecules but continuos permeable to smaller molecules (doi.org/10.1073/pnas.060315410). This is an important finding that should be mentioned in the intruduuction and considered in the discussion sections.Referee cross-commenting
Reviewer 1:
Dear Reviewer #2, The idea of performing control experiments in the point-mutant Cx43 or KO/KI mouse makes sense. If you think this is essential, then please enter it into your overall comments. However, performing this experiment will not be easily done within the one month revision time frame you proposed. Cheers.
Reviewer 2:
I think the data of this manuscript is missing a control animal that would present all the compensation changes that occur during development that occur in mice carrying the mutated Cx43. Alternatively, a doable experiment would be the use of inducible KO/KI. When comparing susceptibility to any drug it is very important to count with the best control possible. Otherwise, the results cannot be interpreted as cause-effect response.
Reviewer 1:
I agree with reviewer #2 that adding those two references will improve the ms. For the second ref mentioned, the doi link did not work; does reviewer #2 mean this ref: https://doi.org/10.1073/pnas.0603154104? "Change in permeant size selectivity by phosphorylation of connexin 43 gap-junctional hemichannels by PKC
Significance
If completed and/or interpreted carefully it could be relevant to enrich our knowledge on the importance of glial Cx43 in epilepsy.
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Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
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Referee #1
Evidence, reproducibility and clarity
Summary
Muñoz-Ballester et al. investigated the effects of TBI on Cx43 expression and function following TBI. They have examined the potential role of Cx43-containing gap junctions (GJs) and/or hemichannels (HCs), in their phosphorylated and unphosphorylated forms, in the mouse cortex. The experiments and hypotheses are simple and direct, but the results are not strong and generally correlative.
Major comments
- The immunofluorescent images for Fig. 1D and E were taken at low resolution compared to the Cx43 puncta size. This does not allow accurate quantification of the Cx43 GJs or HCs.
- The immunofluorescent images for Fig. 2E and Fig. …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
Summary
Muñoz-Ballester et al. investigated the effects of TBI on Cx43 expression and function following TBI. They have examined the potential role of Cx43-containing gap junctions (GJs) and/or hemichannels (HCs), in their phosphorylated and unphosphorylated forms, in the mouse cortex. The experiments and hypotheses are simple and direct, but the results are not strong and generally correlative.
Major comments
- The immunofluorescent images for Fig. 1D and E were taken at low resolution compared to the Cx43 puncta size. This does not allow accurate quantification of the Cx43 GJs or HCs.
- The immunofluorescent images for Fig. 2E and Fig. 5 (super resolution images) were not countered stained for astrocytes (e.g. S100β) or cell membrane. How can the authors be sure that these are expressed by astrocytes and not other cells in the brain? Also, even if countered stained for astrocytes, the punctae will indicate the total but not cell surface pool of Cx43, making it difficult to interpret the impact of surface GJs and HCs in TBI.
- The IC50 for Tat-Gap19 for Cx43 HC is ~7 μM (Tocris). How can using it at 2 μM be effective?
- TBI did not change Cx43 plaque size or density (Fig. 5). What was the rationale for examining the effects in the S368A mutant?
- CBX or Tat-Gap19 do not affect the phosphorylation state of Cx43.
Minor comments
- Fig 3D: it is unclear whether the quantification is for S100β+ astrocytes or not. Is there uptake of EtBr in neurons due to inflammatory effects?
- Fig. 4C: It is unclear whether these mice have received TBI or not.
- I believe that the "W." in ref "W. Chen et al., 2018" (p.30) is unnecessary.
Referee cross-commenting
Reviewer 1:
Dear Reviewer #2, The idea of performing control experiments in the point-mutant Cx43 or KO/KI mouse makes sense. If you think this is essential, then please enter it into your overall comments. However, performing this experiment will not be easily done within the one month revision time frame you proposed. Cheers.
Reviewer 2:
I think the data of this manuscript is missing a control animal that would present all the compensation changes that occur during development that occur in mice carrying the mutated Cx43. Alternatively, a doable experiment would be the use of inducible KO/KI. When comparing susceptibility to any drug it is very important to count with the best control possible. Otherwise, the results cannot be interpreted as cause-effect response.
Reviewer 1:
I agree with reviewer #2 that adding those two references will improve the ms. For the second ref mentioned, the doi link did not work; does reviewer #2 mean this ref: https://doi.org/10.1073/pnas.0603154104? "Change in permeant size selectivity by phosphorylation of connexin 43 gap-junctional hemichannels by PKC
Significance
The strength of this study is using a single-point mutant mouse of the Cx43 to assess the role of Cx43 phosphorylation in TBI-induced seizure susceptibility, pinpointing the molecular target. One limitation is that, while the S368A mutant directly addresses the seizure susceptibility issue, pharmacological treatments like CBX and Tat-Gap19 do not test the effects of phosphorylation. Another weakness is that the key mechanism underlying the effects of TBI on Cx43 is still unclear. This is because TBI does not change Cx43 plaque size (Fig. 5), it alters EtBr dye uptake in cells that may or may not be astrocytes (Fig. 3), and it changes Cx43 solubility, but this is correlative for GJs vs HCs. The overall idea of Cx43 contributing to seizures and TBI is interesting for the general neuroscience community. However, this study can use more direct experimentation to support its hypothesis.
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