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

    eLife logo

    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.

Reviewed by

Read the full article See related articles

Discuss this preprint

Start a discussion What are Sciety discussions?

Listed in

Log in to save this article

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 (E Cl ) and seizures via increased KCC2 activity. However, increased KCC2 activity alone should not affect E Cl whose determinants are more complex. We determined the net effects of WNK-SPAK/OSR1 pathway inhibitor WNK463 on E Cl 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. Thus, mutual NKCC1 inhibition and KCC2 activation via the WNK-SPAK/OSR1 pathway exert powerful anti-ictal effects by facilitating [Cl - ] i extrusion during ILDs.

Article activity feed

  1. 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.

  2. 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.

  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 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.

  4. 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.

  5. Version published to 10.64898/2026.06.10.731153 on bioRxiv