WNK-SPAK/OSR1 signaling pathway facilitates ictal activity via reduced neuronal chloride extrusion rate

  1. Volodymyr I Dzhala
  2. Fu Hung Shiu
  3. Negah Rahmati
  4. Aloe Carroll
  5. Reagan Bae
  6. Kristopher T Kahle
  7. Kevin J Staley

  • Curated by eLife

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    eLife Assessment

    This study presents an important investigation of how inhibition of the WNK-SPAK/OSR1 pathway influences neuronal chloride homeostasis and seizure-like activity in organotypic hippocampal slice cultures. The evidence supporting the principal mechanistic conclusions is solid, although the proposed regulation of chloride transporters is inferred rather than directly demonstrated in this model. The work will be of interest to researchers studying chloride homeostasis, inhibitory neurotransmission, and epilepsy.

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Abstract

Seizures upregulate Na+-K+-2Cl- (NKCC1)-mediated Cl− influx and downregulate K+-Cl-(KCC2)-mediated Cl- efflux via the WNK-SPAK/OSR1 kinases, leading to cytoplasmic chloride ([Cl-]i) accumulation, reduced GABAergic inhibition and anticonvulsant failure. Early studies found that inhibiting WNK-kinase reduced baseline [Cl-]i (ECl) and seizures via increased KCC2 activity. However, increased KCC2 activity alone should not affect ECl whose determinants are more complex. We determined the net effects of WNK-SPAK/OSR1 pathway inhibitor WNK463 on ECl and [Cl-]i transients during spontaneous ictal-like discharges (ILDs). We found that WNK463 reduced interictal [Cl-]i but did not change baseline [Cl-]i measured in the presence of TTX. WNK463 enhanced neuronal Cl- extrusion during and after ILDs, before abolishing ILDs. Pharmacological inhibition and targeted siRNA silencing demonstrated that the anti-ictal effects of WNK463 involved both NKCC1 and KCC2. Our data support a mechanism whereby mutual NKCC1 inhibition and KCC2 activation via the WNK-SPAK/OSR1 pathway exert powerful anti-ictal effects by facilitating [Cl-]i extrusion during ILDs.

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  1. eLife

    eLife Assessment

    This study presents an important investigation of how inhibition of the WNK-SPAK/OSR1 pathway influences neuronal chloride homeostasis and seizure-like activity in organotypic hippocampal slice cultures. The evidence supporting the principal mechanistic conclusions is solid, although the proposed regulation of chloride transporters is inferred rather than directly demonstrated in this model. The work will be of interest to researchers studying chloride homeostasis, inhibitory neurotransmission, and epilepsy.

  2. eLife

    Reviewer #1 (Public review):

    Summary:

    Epileptogenesis involves deficits in GABAergic inhibition mediated by chloride-permeable GABA(A) channels, which are caused by elevated intracellular chloride. These changes in chloride homeostasis result from increased activity of the NKCC1 chloride importer and loss of function of the KCC2 chloride extruder. The authors used organotypic hippocampal slice preparations from mice expressing the genetically encoded chloride indicator Clomeleon as a model of posttraumatic epileptogenesis to show that these changes are mediated by the WNK-SPAK/OSR1 pathway. Blockade of the WNK-SPAK/OSR1 pathway with WNK463 alleviated epileptic activity. These findings provide important insights into the mechanisms of epileptogenesis and open new avenues for treating epilepsy.

    Strengths:

    The conclusions are well supported by the detailed pharmacological analysis.

    Comments on revised version.

    The authors addressed all the recommendations in their revised manuscript.

  3. eLife

    Reviewer #2 (Public review):

    Summary:

    The authors investigate whether inhibition of the WNK-SPAK/OSR1 pathway using the allosteric inhibitor WNK463 improves neuronal chloride homeostasis and suppresses epileptiform activity in organotypic hippocampal slice cultures. Using SuperClomeleon imaging combined with extracellular field recordings, they demonstrate that WNK463 accelerates recovery of intracellular chloride following chloride loading, reduces interictal chloride accumulation, and progressively suppresses recurrent ictal-like discharges. Pharmacological inhibition and siRNA-mediated knockdown of NKCC1 and KCC2 are then used to investigate the contribution of these transporters to the anti-ictal effects of WNK463.

    Strengths:

    The study addresses an important question in the field of chloride homeostasis and epilepsy and combines complementary experimental approaches. In particular, the distinction between baseline chloride measured in the presence of TTX and activity-dependent interictal chloride accumulation provides a useful conceptual framework for interpreting previous studies of WNK-SPAK inhibition. The imaging, electrophysiological, and pharmacological data are internally consistent and support the conclusion that WNK463 alters chloride dynamics and substantially suppresses ictal-like activity in this model.

    Weaknesses:

    The revised manuscript addresses several concerns raised in my initial review. The authors have broadened the discussion of KCC2 dysfunction and added further context on therapeutic targeting, as well as a useful discussion of the limitations of the organotypic hippocampal slice model.

    My main remaining concern is the mechanistic interpretation of the data. The authors have appropriately softened several statements and now acknowledge that membrane potential and the GABA reversal potential were not directly measured. However, strong mechanistic conclusions remain in the revised manuscript, including statements that the anti-ictal effects of WNK463 are "primarily mediated" by reduced NKCC1 and enhanced KCC2 transport activity and are "mainly due to" NKCC1 inhibition and KCC2 activation. These conclusions go beyond what is directly demonstrated, as WNK463-induced regulation of NKCC1 and KCC2 was not directly measured in this preparation. Lee et al. (2022) provides important support for this mechanism, demonstrating reduced KCC2-T1007 phosphorylation and enhanced KCC2-dependent chloride extrusion following WNK463 treatment. However, these findings were obtained using different experimental preparations, including cultured cortical neurons and an acute 4-AP seizure model. The current experiments therefore support a contribution of NKCC1 and KCC2, but do not directly establish coordinated regulation of these transporters as the primary mechanism underlying seizure suppression. This is also relevant to the simultaneous NKCC1/KCC2 inhibition experiments, where the overall anti-ictal effect of WNK463 is reduced but not completely abolished.

    The statistical reporting has also been improved, but the experimental unit remains unclear for analyses involving multiple cells recorded from individual slices. The authors clarify the use of repeated-measures analyses, but it remains unclear whether individual cells or slices were treated as independent observations and how multiple cells from the same slice were accounted for statistically.

  4. eLife

    Author response:

    The following is the authors’ response to the original reviews.

    eLife Assessment:

    This study presents a valuable investigation of how inhibition of the WNK-SPAK/OSR1 pathway influences neuronal chloride homeostasis and seizure-like activity in organotypic hippocampal slice cultures. The evidence supporting the principal mechanistic conclusions is incomplete because several proposed mechanisms are inferred from changes in chloride dynamics rather than directly demonstrated. The work will be of interest to researchers studying chloride homeostasis, inhibitory neurotransmission, and epilepsy.

    We appreciate the thoughtful reviews and assessment. Additional data analysis has been performed, and the paper has been carefully modified to address each concern, as described below.

    Public Reviews:

    Reviewer #1 (Public review):

    Summary:

    The authors used extracellular field potential recordings and two-photon imaging to monitor neuronal network activity and intracellular chloride concentration ([Cl-]i) in organotypic hippocampal slices from mice expressing the genetically encoded chloride fluorophore Clomeleon. These slices were used as a model of acute traumatic brain injury and epileptogenesis in vitro. The study provides evidence that blocking the WNK-SPAK/OSR1 pathway with WNK463 alleviates epileptic activity, and that this anticonvulsant effect involves suppression of the chloride loader NKCC1 and enhancement of chloride extrusion via KCC2. Overall, this is a solid study with a comprehensive pharmacological analysis.

    Strengths:

    The conclusions are well supported by the detailed pharmacological analysis.

    Weaknesses:

    The only weakness I see is the absence of cellular-level electrophysiology, which precludes interpretation of the imaging data in the context of GABA action polarity.

    The referee correctly points out that we did not perform cellular-level electrophysiological measurements. Before the advent of transgenic ratiometric chloride-sensitive fluorophores, we conducted detailed studies of neuronal cation-chloride transport using electrophysiological measures (Staley and Proctor, 1999). We did not measure membrane potential in the current study because this data is not needed for characterization of cation-chloride membrane cotransport, which is electroneutral. Electroneutral processes are not affected by membrane potential, and electroneutral processes in turn do not alter the membrane potential. We did not measure membrane GABA currents because the polarity of GABA currents was clearly demonstrated by our fluorometric measures of the corresponding increases or decreases in cytoplasmic chloride concentration (Figure 2). We have added the following sentence to the Discussion (Page 16): “In this fluorometric study, we did not measure RMP and EGABA directly, but rather inferred the impact of electroneutral cation-chloride transport on these parameters from the effects on [Cl-]i (Figure 2) and seizure activity (Figures 1, 3-9)”.

    Reviewer #2 (Public review):

    Summary:

    The authors investigate whether inhibition of the WNK-SPAK/OSR1 pathway using the allosteric inhibitor WNK463 improves neuronal chloride homeostasis and suppresses epileptiform activity in organotypic hippocampal slice cultures. Using Super Clomeleon imaging combined with extracellular field recordings, they demonstrate that WNK463 accelerates recovery of intracellular chloride following chloride loading, reduces interictal chloride accumulation, and progressively suppresses recurrent ictal-like discharges. Pharmacological inhibition and siRNA-mediated knockdown of NKCC1 and KCC2 are then used to investigate the contribution of these transporters to the anti-ictal effects of WNK463.

    Strengths:

    The study addresses an important question in the field of chloride homeostasis and epilepsy and combines complementary experimental approaches. In particular, the distinction between baseline chloride measured in the presence of TTX and activity-dependent interictal chloride accumulation provides a useful conceptual framework for interpreting previous studies of WNK-SPAK inhibition. The imaging, electrophysiological, and pharmacological data are internally consistent and support the conclusion that WNK463 alters chloride dynamics and substantially suppresses ictal-like activity in this model.

    Weaknesses:

    The principal limitation of the manuscript is that several mechanistic conclusions extend beyond the experimental observations. Throughout the results and discussion, the authors interpret the observed changes in intracellular chloride dynamics as evidence of enhanced CCC-mediated chloride extrusion, while the pharmacological and siRNA-mediated experiments are interpreted as supporting coordinated NKCC1 inhibition and KCC2 activation, ultimately leading to restoration of GABAergic inhibition and negative shifts in EGABA. While these interpretations are plausible and consistent with the data, they remain inferential because transporter phosphorylation or activity, EGABA, and inhibitory synaptic function were not directly assessed in the current study. Moreover, although the pharmacological and knockdown experiments support a contribution of NKCC1 and KCC2 to the actions of WNK463, they do not definitively establish coordinated modulation of both transporters as the primary mechanism underlying seizure suppression. These mechanistic conclusions should therefore be presented more cautiously.

    The referee correctly points that we did not definitively establish the link between coordinated modulation of NKCC1 and KCC2 transporter activity, changes in EGABA and GABA function to the anti-ictal action of WNK463. Indeed we have an entire section of results dedicated to the anti-ictal effects of WNK463 that persist after block of NKCC1 and KCC2. The primarily biochemical study by Lee et al (2022) demonstrated that the WNK-SPAK/OSR1 inhibitor WNK463 reduced KCC2-T1007 phosphorylation and EGABA in cultured cortical neurons challenged with high 30 mM [Cl-]i in vitro, and limited KA-induced status epilepticus in vivo. We are studying the same system with different pharmacological, electrophysiological, optogenetic and morphological tools during spontaneous ictal-like epileptiform discharges in organotypic hippocampal slices in vitro. We prominently cite the previous study and clearly report new findings and demonstrate underlying mechanisms. We have also modified the language surrounding our conclusions (e.g. last sentence of abstract, and last sentence of first paragraph of Discussion).

    The manuscript would also benefit from broader contextualization within the current literature. The introduction largely focuses on previous work from the authors' group and provides a relatively narrow overview of chloride homeostasis in epilepsy. In particular, the discussion would benefit from broader consideration of studies examining KCC2 dysfunction in human epilepsy and experimental models, alternative mechanisms regulating KCC2 activity following seizures, and recent therapeutic strategies targeting KCC2.

    We agree. We have added the following paragraph to the Discussion (p. 16-17): “The KCC2 protein is a potassium-chloride co-transporter that maintains low [Cl-]i (Rivera et al., 1999). KCC2 dysfunction has been implicated in epilepsy across human and experimental studies. Reduced KCC2 expression and impaired chloride extrusion have been demonstrated in resected tissue from patients with temporal lobe epilepsy, and pathogenic variants in SLC12A5 further support a causal role for impaired KCC2 function in human epilepsy (Huberfeld et al., 2007; Puskarjov et al., 2014). Experimental seizures can suppress KCC2 through several mechanisms in addition to WNK-SPAK/OSR1 signaling, including BDNF-TrkB-dependent downregulation, dephosphorylation of the stabilizing Ser940 site with subsequent internalization, and calpain-mediated proteolysis (Rivera et al., 2004; Silayeva et al., 2015; Puskarjov et al., 2012). These convergent mechanisms suggest that loss of KCC2 function may both result from and amplify seizure activity by weakening GABAergic inhibition. Accordingly, KCC2 has emerged as a therapeutic target: strategies include modulation of regulatory phosphorylation, enhancement of KCC2 expression, and direct pharmacological potentiation. Notably, the KCC2 activator OV350 enhances chloride extrusion and suppresses experimental seizures, including benzodiazepine-refractory status epilepticus, providing proof-of-concept for direct KCC2 activation as an antiseizure strategy (Jarvis et al., 2023).”

    Finally, although the authors appropriately acknowledge that the experiments were performed exclusively in vitro, the discussion could more explicitly address the limitations of the organotypic hippocampal slice model, including how culture-induced network reorganization and spontaneous epileptiform activity may influence chloride homeostasis and the extent to which these findings generalize to traumatic brain injury and chronic epilepsy in vivo. In addition, the statistical analysis would benefit from clarification regarding the experimental unit and the treatment of repeated measurements.

    Thank you. We addressed the limitation of organotypic hippocampal slices in vitro in the Limitation and Future Directions paragraph (p. 18-19): “Our experiments were conducted in organotypic hippocampal slices in vitro. Advantages of this preparation include homogenous injury and consistent epileptogenesis. This provides excellent control of the extracellular environment and imaging but does not replicate the in vivo vascular supply and maintenance of ionic homeostasis. While we find that increased WNK-SPAK/OSR1-mediated KCC2 activity via enhanced Cl- extrusion rates robustly reduces seizure activity in vitro, it is possible that in vivo, increased KCC2 activity could lead to extracellular potassium accumulations (Viitanen et al., 2010) that would limit Cl- extrusion independently of the velocity of KCC2 transport (Thompson et al., 1988; Staley and Proctor, 1999). Thus, it will be important to assess these findings in vivo.”

    We provided better clarification regarding the treatment of repeated measurements (p. 22): “Experimental data were analyzed using a repeated measures ANOVA for two types of study design. Studies that investigated either (1) changes in mean (median) parameters over three or more time points or consecutive time intervals; or (2) differences in mean (median) parameters under three or more different conditions”.

    Reviewer #3 (Public review):

    Summary:

    Dzhala and colleagues present findings from organotypic slice cultures suggesting that simultaneous modulation of the complementary cation-chloride cotransporters NKCC1 and KCC2 through inhibition of the WNK-SPAK/OSR1 pathway reduces seizure-like activity. The manuscript is generally well written, and the data support the conclusion that WNK463 exerts robust anti-ictal effects in this model. However, several issues should be addressed to strengthen the mechanistic interpretation and statistical rigor of the study, and improve confidence in the conclusions.

    Strengths:

    (1) The study addresses an important mechanistic question by investigating how inhibition of the WNK-SPAK/OSR1 pathway with WNK463 influences seizure activity and neuronal chloride homeostasis.

    (2) The experimental design is logical and comprehensive, progressing from characterization of chloride dynamics to pharmacological and genetic interrogation of the underlying mechanism using multiple complementary approaches, including pharmacological inhibition, siRNA-mediated knockdown, electrophysiology, and chloride imaging.

    (3) The combination of simultaneous extracellular electrophysiology and two-photon chloride imaging provides complementary functional and mechanistic information and represents a major technical strength of the study.

    (4) The TTX experiments elegantly distinguish activity-dependent chloride accumulation from resting intracellular chloride concentration, substantially strengthening the central mechanistic conclusions.

    Weaknesses:

    (1) The mechanistic conclusions regarding KCC2 activation and NKCC1 inhibition are stronger than the data directly support. Throughout the manuscript, the authors conclude that WNK463 activates KCC2 and inhibits NKCC1. Although this interpretation is consistent with the established biology of the WNK-SPAK/OSR1 pathway, the evidence presented here is indirect. Specifically, the authors infer KCC2 activation and NKCC1 inhibition from the observation that pharmacological inhibition or siRNA-mediated knockdown of these transporters alters the effects of WNK463, together with measurements of chloride dynamics. While these findings are compatible with a KCC2- and NKCC1-dependent mechanism, they do not directly establish that WNK463 regulates either transporter. Direct evidence would require measurements of transporter activity, phosphorylation state, membrane expression, or other biochemical indices of transporter regulation. I therefore recommend that the authors both temper the mechanistic language throughout the manuscript and explicitly acknowledge in the Discussion that the proposed regulation of KCC2 and NKCC1 is inferred from indirect evidence rather than directly demonstrated in the present study.

    We agree. We modified the key conclusory sentences in the Abstract and Discussion as described in the response to referee 2. The referee correctly points that we did not directly establish the link between the action of WNK463 and coordinated activation of KCC2 and inhibition NKCC1. We highlighted the established link in the discussion (p. 17-18): “NKCC1 and KCC2 are rapidly stimulated and inhibited, respectively, by direct phosphorylation mediated by the Cl--sensitive WNK-SPAK/OSR1 complex (Alessi et al., 2014). The WNK family senses changes in the [Cl-]i, cell volume, extracellular osmolarity and transduces this information to CCCs (Shekarabi et al., 2017). WNKs stimulate the kinases SPAK and OSR1, which directly phosphorylate and stimulate NKCC1 or inhibit KCC2 (Kahle et al., 2013), providing compensatory and reciprocal regulation between transporters activity to maintain chloride homeostasis and neuronal excitability (Hartmann and Nothwang, 2022). Conversely, inhibiting the WNK-SPAK/OSR1 pathway might be an especially potent strategy to reduce neuronal Cl- elevation and facilitate neuronal Cl- extrusion by coincident NKCC1 inhibition and KCC2 activation…” Consistently with the established biology of the WNK-SPAK/OSR1 pathway we found that (p.15): “…WNK463 improved [Cl-]i homeostasis, progressively enhanced postictal [Cl-]i extrusion rates, reduced ictal duration and abolished recurrent ILDs. Our pharmacological investigations suggest that the powerful anti-ictal effects of WNK463 are mediated by reduced NKCC1 and enhanced KCC2 transport activity...”

    (2) The conclusions drawn from the siRNA-mediated knockdown experiments should be interpreted more cautiously. First, it is unclear whether silencing NKCC1 or KCC2 induced compensatory changes in the expression or function of the complementary cotransporter. Given the well-established interplay between NKCC1 and KCC2 in regulating intracellular chloride homeostasis, compensatory adaptations could influence the interpretation of these experiments and should be addressed or acknowledged as a limitation. Second, the sample size for the siRNA experiments appears relatively small. It is unclear how many independent animals contributed slices to each experimental group, making it difficult to assess the degree of biological replication. In addition, effect sizes are not reported. Clarifying the number of biological replicates and reporting effect sizes would improve the rigor of the statistical analysis and increase confidence in these findings.

    We agree and now address this in the discussion (p. 17-18): “WNKs stimulate the kinases SPAK and OSR1, which directly phosphorylate and stimulate NKCC1 or inhibit KCC2 (Kahle et al., 2013), providing compensatory and reciprocal regulation between transporters activity to maintain chloride homeostasis and neuronal excitability (Hartmann and Nothwang, 2022). ”

    We agree that the sample size for the siRNA electrophysiological experiments is relatively small (n=3-4 slices). We now clarify the number of mice were used per each experimental group (p.19). We provide the n (number of slices) and the number of paired cells in the Results and for every experimental conditions.

    (3) The final pharmacological experiments require clarification, as the conclusions appear internally inconsistent. Earlier experiments suggest that the anticonvulsant effects of WNK463 depend on coordinated regulation of both NKCC1 and KCC2. However, in the final experiment, the authors state that simultaneous pharmacological inhibition of NKCC1 and KCC2 does not prevent the anticonvulsant effects of WNK463. In contrast, the accompanying statistical analysis indicates that combined transporter inhibition significantly reduces the effect of WNK463 relative to control conditions. These interpretations appear inconsistent and make it difficult to determine the extent to which the anticonvulsant action of WNK463 depends on NKCC1 and KCC2. The authors should clarify whether simultaneous inhibition of both transporters completely abolishes, partially attenuates, or merely reduces the magnitude of the WNK463 response, and revise the text accordingly. If the effect is only partially attenuated, alternative mechanisms contributing to the anticonvulsant actions of WNK463 should also be considered and discussed.

    We agree. Our data demonstrated that WNK463 had no effect in the presence of high concentration of bumetanide (500 µM) that nonspecifically blocks CCCs (NKCC1 and KCC2-3) (Figure 6). In contrast (p. 15): ”Simultaneous inhibition of NKCC1 and KCC2 by the combination of bumetanide (10 µM) and VU0463271 (1 µM) significantly reduced the anti-ictal effects of WNK463 (Figure 9).” The conclusory sentence in the Results indicates (p.15): “These results suggest that the anti-ictal effects of WNK-SPAK/OSR1 inhibitor WNK463 are mainly due to downstream interactions with NKCC1 inhibition and KCC2 activation, with additional effects due to interactions with an unidentified Cl- transporter (e.g. KCC3) or exchanger, or channel.”

    (4) The statistical analysis and reporting require further attention. First, median values should not be reported with standard deviations, as standard deviation describes variability around the mean rather than the median. For non-normally distributed data, the authors should report median values together with an appropriate measure of variability, such as the interquartile range (25th-75th percentile) or another suitable summary. Second, in several instances, ANOVA results are reported using only a single degree of freedom value (e.g., page 6, DF = 53). This is incomplete, as an F statistic is defined by two degrees of freedom: the numerator degrees of freedom (between-group variability) and the denominator degrees of freedom (within-group variability). Reporting statistical results using standard notation (F(df_between, df_within) = F statistic, p = value) would improve clarity and allow proper interpretation of the analyses.

    Thank you. Group measures are expressed now as mean ± standard deviation (SD) or median (25%-75%) as indicated. In a one-way RM ANOVA, (DF) represents the between-groups degree of freedom, and (F) represents the F-statistic and reported as F(DFtreatment, DFresidual)=value. In the Friedman RM ANOVA on Ranks, the chi-square statistic was used to assess the differences between the groups. The Tukey Test or Holm-Sidak Test was used for all pairwise comparisons of the responses to the different treatment groups. Please see Statistical Analysis in the Methods section (p.23-24).

    Recommendations for the authors:

    Reviewer #1 (Recommendations for the authors):

    I have only minor comments/suggestions.

    (1) While [Cl-]i and ECl are informative parameters, interpreting the GABA driving force and determining whether depolarizing/excitatory GABA actions during seizures or interictal states are alleviated by treatment requires knowledge of the membrane potential and its dynamics during epileptic events. I would recommend including this information if available. Gramicidin perforated-patch recordings (ideally) would substantially strengthen the conclusions, at least for a critical subset of experiments.

    Please see our response above. We have added the following sentence to the discussion (page 16): “In this fluorometric study, we did not measure RMP and EGABA directly, but rather inferred the impact of electroneutral cation-chloride transport on these parameters from the effects on [Cl-]i (Figure 2) and seizure activity (Figures 1, 3-9)”.

    (2) Did the authors observe any signs of cell shrinkage in response to WNK463, or transient cellular swelling during interictal-like discharges (ILDs) and its prevention by WNK463? Given that cytotoxic edema is typically associated with increased tissue light transparency and shrinkage of the extracellular space (e.g., as measured with TMA⁺), this reviewer wonders whether such approaches could be used as a proxy for cell swelling. Tissue edema, which manifests as increased tissue volume, could also be assessed by measuring slice thickness.

    These are excellent ideas and cell volume is an area of interest for us, but we would argue that assessment of volume effects are outside the scope of the current 9-figure study. We observed cell volume (transverse area) increases during prolonged ictal-like discharges. This effect was prevented by WNK463 during suppression of ictal-like discharges. However, we did not provide the volume analysis in this paper.

    (3) The paper should be corrected for spelling and grammar (e.g., Figure 2 legend: "(b) Linier regression analysis revealed that... " - probably should be "Linear... "?).

    Thank you. Corrected.

    Reviewer #2 (Recommendations for the authors):

    (1) Temper mechanistic statements throughout the manuscript by clearly distinguishing experimental observations from inferred mechanisms and by explicitly framing conclusions regarding coordinated NKCC1/KCC2 modulation, chloride extrusion, EGABA, and restoration of GABAergic inhibition as interpretations supported by, rather than directly demonstrated by, the current data.

    We have softened the key conclusory sentences in the Abstract and Discussion as described above. In the introduction, we highlighted the potential of both NKCC1 and KCC2 targeting for control of seizures (p. 2): “Previous studies have demonstrated that inhibition of NKCC1 reduced [Cl-]i in injured neurons, enhanced GABAergic inhibition and the efficacy of anticonvulsants (Pond et al., 2006; Dzhala et al., 2008; Dzhala et al., 2024; Nardou et al., 2011; Cleary et al., 2013; Sivakumaran and Maguire, 2016; Soul et al., 2021). Conversely, enhancement of KCC2 activity improved [Cl-]i extrusion and control of seizures (Gagnon et al., 2013; Moore et al., 2018; Dzhala and Staley, 2021).”

    In the discussion we highlighted the potential of coordinated NKCC1 and KCC2 modulation (p. 17-18): “NKCC1 and KCC2 are rapidly stimulated and inhibited, respectively, by direct phosphorylation mediated by the Cl--sensitive WNK-SPAK/OSR1 complex (Alessi et al., 2014). The WNK family senses changes in the [Cl-]i, cell volume, extracellular osmolarity and transduces this information to CCCs (Shekarabi et al., 2017). WNKs stimulate the kinases SPAK and OSR1, which directly phosphorylate and stimulate NKCC1 or inhibit KCC2 (Kahle et al., 2013). Conversely, inhibiting the WNK-SPAK/OSR1 pathway might be an especially potent strategy to reduce neuronal Cl- elevation and facilitate neuronal Cl- extrusion by coincident NKCC1 inhibition and KCC2 activation…”

    (2) Expand the introduction and discussion to provide a more balanced overview of the current literature on chloride homeostasis in epilepsy, including alternative mechanisms regulating KCC2 dysfunction and recent therapeutic strategies targeting KCC2.

    In the introduction, we highlighted the potential of both NKCC1 and KCC2 targeting for control of seizures (p. 2): “Previous studies have demonstrated that inhibition of NKCC1 reduced [Cl-]i in injured neurons, enhanced GABAergic inhibition and the efficacy of anticonvulsants (Pond et al., 2006; Dzhala et al., 2008; Dzhala et al., 2024; Nardou et al., 2011; Cleary et al., 2013; Sivakumaran and Maguire, 2016; Soul et al., 2021). Conversely, enhancement of KCC2 activity improved [Cl-]i extrusion and control of seizures (Gagnon et al., 2013; Moore et al., 2018; Dzhala and Staley, 2021).” We did not provide additional references to comply with the word limit. We have added a paragraph and citations (pp. 16-17) to the Discussion to address this issue.

    (3) Discuss more explicitly the limitations of the organotypic hippocampal slice model when extrapolating the findings to traumatic brain injury and chronic epilepsy in vivo.

    Please see our response above. We discussed the limitation of the organotypic hippocampal slice model in the Limitations and future directions section (p. 18-19): “Our experiments were conducted in organotypic hippocampal slices in vitro. Advantages of this preparation include homogenous injury and consistent epileptogenesis (Berdichevsky et al., 2012). This provides excellent control of the extracellular environment and imaging but does not replicate the in vivo vascular supply and maintenance of ionic homeostasis. While we find that increased WNK-SPAK/OSR1-mediated KCC2 activity via enhanced Cl- extrusion rates robustly reduces seizure activity in vitro, it is possible that in vivo, increased KCC2 activity could lead to extracellular potassium accumulations (Viitanen et al., 2010) that would limit Cl- extrusion independently of the velocity of KCC2 transport (Thompson et al., 1988; Staley and Proctor, 1999). Thus, it will be important to assess these findings in vivo.”

    (4) Clarify how repeated measurements from individual cells and slices were handled in the statistical analyses, including the experimental unit for each analysis.

    We clarified in the methods (p.22): “Experimental data were analyzed using a repeated measures ANOVA for two types of study design. Studies that investigated either (1) changes in mean (or median) parameters over three or more time points or consecutive time intervals); or (2) differences in mean (or median) parameters under three or more different conditions”.

    Reviewer #3 (Recommendations for the authors):

    (1) The manuscript would also benefit from including statements regarding randomization, blinding (beyond the immunohistochemistry analysis), and any inclusion or exclusion criteria, as these details are increasingly expected to improve transparency and reproducibility.

    We agree. Images were blinded for the immunohistochemistry analysis (p. 21) and quantitative analysis of intracellular chloride changes (p. 23). Cells that did not respond to GABA/L-glutamate puff applications (Figure 2) and chloride transients that failed to fit an exponential decay function (p. 5) were excluded from the analysis. All other data generated or analyzed during this study were included in the manuscript.

    (2) The duration of baseline recordings prior to drug application should be specified in the Methods.

    Thank you. This information included in the Method section (p.19): “Slices were stabilized for 10-20 min in ACSF prior recordings and/or imaging”.

    (3) Some wording could be refined for readability. For example, "insignificantly reduced " on pages 8 and 14 would be more appropriately stated as "did not significantly reduce. "

    Thank you. Corrected.

    (4) The first sentence of the Introduction refers to "anticonvulsant (AED)-resistant seizures. " I suspect the authors intended "antiepileptic drug (AED)-resistant seizures, " as the current wording does not match the acronym.

    Thank you. Corrected.

  5. Version published to 10.7554/elife.112181.2 on eLife
  6. Version published to 10.7554/elife.112181 on eLife
  7. Version published to 10.64898/2026.06.10.731153v3 on bioRxiv
  8. Version published to 10.64898/2026.06.10.731153v2 on bioRxiv
  9. Version published to 10.7554/elife.112181.1 on eLife
  10. eLife

    eLife Assessment

    This study presents a valuable investigation of how inhibition of the WNK-SPAK/OSR1 pathway influences neuronal chloride homeostasis and seizure-like activity in organotypic hippocampal slice cultures. The evidence supporting the principal mechanistic conclusions is incomplete because several proposed mechanisms are inferred from changes in chloride dynamics rather than directly demonstrated. The work will be of interest to researchers studying chloride homeostasis, inhibitory neurotransmission, and epilepsy.

  11. eLife

    Reviewer #1 (Public review):

    Summary:

    The authors used extracellular field potential recordings and two-photon imaging to monitor neuronal network activity and intracellular chloride concentration ([Cl-]i) in organotypic hippocampal slices from mice expressing the genetically encoded chloride fluorophore Clomeleon. These slices were used as a model of acute traumatic brain injury and epileptogenesis in vitro. The study provides evidence that blocking the WNK-SPAK/OSR1 pathway with WNK463 alleviates epileptic activity, and that this anticonvulsant effect involves suppression of the chloride loader NKCC1 and enhancement of chloride extrusion via KCC2. Overall, this is a solid study with a comprehensive pharmacological analysis.

    Strengths:

    The conclusions are well supported by the detailed pharmacological analysis.

    Weaknesses:

    The only weakness I see is the absence of cellular-level electrophysiology, which precludes interpretation of the imaging data in the context of GABA action polarity.

  12. eLife

    Reviewer #2 (Public review):

    Summary:

    The authors investigate whether inhibition of the WNK-SPAK/OSR1 pathway using the allosteric inhibitor WNK463 improves neuronal chloride homeostasis and suppresses epileptiform activity in organotypic hippocampal slice cultures. Using Super Clomeleon imaging combined with extracellular field recordings, they demonstrate that WNK463 accelerates recovery of intracellular chloride following chloride loading, reduces interictal chloride accumulation, and progressively suppresses recurrent ictal-like discharges. Pharmacological inhibition and siRNA-mediated knockdown of NKCC1 and KCC2 are then used to investigate the contribution of these transporters to the anti-ictal effects of WNK463.

    Strengths:

    The study addresses an important question in the field of chloride homeostasis and epilepsy and combines complementary experimental approaches. In particular, the distinction between baseline chloride measured in the presence of TTX and activity-dependent interictal chloride accumulation provides a useful conceptual framework for interpreting previous studies of WNK-SPAK inhibition. The imaging, electrophysiological, and pharmacological data are internally consistent and support the conclusion that WNK463 alters chloride dynamics and substantially suppresses ictal-like activity in this model.

    Weaknesses:

    The principal limitation of the manuscript is that several mechanistic conclusions extend beyond the experimental observations. Throughout the results and discussion, the authors interpret the observed changes in intracellular chloride dynamics as evidence of enhanced CCC-mediated chloride extrusion, while the pharmacological and siRNA-mediated experiments are interpreted as supporting coordinated NKCC1 inhibition and KCC2 activation, ultimately leading to restoration of GABAergic inhibition and negative shifts in EGABA. While these interpretations are plausible and consistent with the data, they remain inferential because transporter phosphorylation or activity, EGABA, and inhibitory synaptic function were not directly assessed in the current study. Moreover, although the pharmacological and knockdown experiments support a contribution of NKCC1 and KCC2 to the actions of WNK463, they do not definitively establish coordinated modulation of both transporters as the primary mechanism underlying seizure suppression. These mechanistic conclusions should therefore be presented more cautiously.

    The manuscript would also benefit from broader contextualization within the current literature. The introduction largely focuses on previous work from the authors' group and provides a relatively narrow overview of chloride homeostasis in epilepsy. In particular, the discussion would benefit from broader consideration of studies examining KCC2 dysfunction in human epilepsy and experimental models, alternative mechanisms regulating KCC2 activity following seizures, and recent therapeutic strategies targeting KCC2.

    Finally, although the authors appropriately acknowledge that the experiments were performed exclusively in vitro, the discussion could more explicitly address the limitations of the organotypic hippocampal slice model, including how culture-induced network reorganization and spontaneous epileptiform activity may influence chloride homeostasis and the extent to which these findings generalize to traumatic brain injury and chronic epilepsy in vivo. In addition, the statistical analysis would benefit from clarification regarding the experimental unit and the treatment of repeated measurements.

  13. eLife

    Reviewer #3 (Public review):

    Summary:

    Dzhala and colleagues present findings from organotypic slice cultures suggesting that simultaneous modulation of the complementary cation-chloride cotransporters NKCC1 and KCC2 through inhibition of the WNK-SPAK/OSR1 pathway reduces seizure-like activity. The manuscript is generally well written, and the data support the conclusion that WNK463 exerts robust anti-ictal effects in this model. However, several issues should be addressed to strengthen the mechanistic interpretation and statistical rigor of the study, and improve confidence in the conclusions.

    Strengths:

    (1) The study addresses an important mechanistic question by investigating how inhibition of the WNK-SPAK/OSR1 pathway with WNK463 influences seizure activity and neuronal chloride homeostasis.

    (2) The experimental design is logical and comprehensive, progressing from characterization of chloride dynamics to pharmacological and genetic interrogation of the underlying mechanism using multiple complementary approaches, including pharmacological inhibition, siRNA-mediated knockdown, electrophysiology, and chloride imaging.

    (3) The combination of simultaneous extracellular electrophysiology and two-photon chloride imaging provides complementary functional and mechanistic information and represents a major technical strength of the study.

    (4) The TTX experiments elegantly distinguish activity-dependent chloride accumulation from resting intracellular chloride concentration, substantially strengthening the central mechanistic conclusions.

    Weaknesses:

    (1) The mechanistic conclusions regarding KCC2 activation and NKCC1 inhibition are stronger than the data directly support. Throughout the manuscript, the authors conclude that WNK463 activates KCC2 and inhibits NKCC1. Although this interpretation is consistent with the established biology of the WNK-SPAK/OSR1 pathway, the evidence presented here is indirect. Specifically, the authors infer KCC2 activation and NKCC1 inhibition from the observation that pharmacological inhibition or siRNA-mediated knockdown of these transporters alters the effects of WNK463, together with measurements of chloride dynamics. While these findings are compatible with a KCC2- and NKCC1-dependent mechanism, they do not directly establish that WNK463 regulates either transporter. Direct evidence would require measurements of transporter activity, phosphorylation state, membrane expression, or other biochemical indices of transporter regulation. I therefore recommend that the authors both temper the mechanistic language throughout the manuscript and explicitly acknowledge in the Discussion that the proposed regulation of KCC2 and NKCC1 is inferred from indirect evidence rather than directly demonstrated in the present study.

    (2) The conclusions drawn from the siRNA-mediated knockdown experiments should be interpreted more cautiously. First, it is unclear whether silencing NKCC1 or KCC2 induced compensatory changes in the expression or function of the complementary cotransporter. Given the well-established interplay between NKCC1 and KCC2 in regulating intracellular chloride homeostasis, compensatory adaptations could influence the interpretation of these experiments and should be addressed or acknowledged as a limitation. Second, the sample size for the siRNA experiments appears relatively small. It is unclear how many independent animals contributed slices to each experimental group, making it difficult to assess the degree of biological replication. In addition, effect sizes are not reported. Clarifying the number of biological replicates and reporting effect sizes would improve the rigor of the statistical analysis and increase confidence in these findings.

    (3) The final pharmacological experiments require clarification, as the conclusions appear internally inconsistent. Earlier experiments suggest that the anticonvulsant effects of WNK463 depend on coordinated regulation of both NKCC1 and KCC2. However, in the final experiment, the authors state that simultaneous pharmacological inhibition of NKCC1 and KCC2 does not prevent the anticonvulsant effects of WNK463. In contrast, the accompanying statistical analysis indicates that combined transporter inhibition significantly reduces the effect of WNK463 relative to control conditions. These interpretations appear inconsistent and make it difficult to determine the extent to which the anticonvulsant action of WNK463 depends on NKCC1 and KCC2. The authors should clarify whether simultaneous inhibition of both transporters completely abolishes, partially attenuates, or merely reduces the magnitude of the WNK463 response, and revise the text accordingly. If the effect is only partially attenuated, alternative mechanisms contributing to the anticonvulsant actions of WNK463 should also be considered and discussed.

    (4) The statistical analysis and reporting require further attention. First, median values should not be reported with standard deviations, as standard deviation describes variability around the mean rather than the median. For non-normally distributed data, the authors should report median values together with an appropriate measure of variability, such as the interquartile range (25th-75th percentile) or another suitable summary. Second, in several instances, ANOVA results are reported using only a single degree of freedom value (e.g., page 6, DF = 53). This is incomplete, as an F statistic is defined by two degrees of freedom: the numerator degrees of freedom (between-group variability) and the denominator degrees of freedom (within-group variability). Reporting statistical results using standard notation (F(df_between, df_within) = F statistic, p = value) would improve clarity and allow proper interpretation of the analyses.

  14. Mentioned by @biorxiv-neursci.bsky.social on Bluesky

    WNK-SPAK/OSR1 signaling pathway facilitates ictal activity via reduced neuronal chloride extrusion rate https://www.biorxiv.org/content/10.64898/2026.06.10.731153v1

  15. Mentioned by @biorxivpreprint.bsky.social on Bluesky

    WNK-SPAK/OSR1 signaling pathway facilitates ictal activity via reduced neuronal chloride extrusion rate https://www.biorxiv.org/content/10.64898/2026.06.10.731153v1

  16. Version published to 10.64898/2026.06.10.731153v1 on bioRxiv