WNKs regulate mouse behavior and alter central nervous system glucose uptake and insulin signaling

Reviewer #1 (Public review):

Summary:

The study by Akita B. Jaykumar et al. explored an interesting and relevant hypothesis whether serine/threonine With-No-lysine (K) kinases (WNK)-1, -2, -3, and -4 engage in insulin-dependent glucose transporter-4 (GLUT4) signaling in the murine central nervous system. The authors especially focused on the hippocampus as this brain region exhibits high expression of insulin and GLUT4. Additionally, disrupted glucose metabolism in the hippocampus has been associated with anxiety disorders, while impaired WNK signaling has been linked to hypertension, learning disabilities, psychiatric disorders or Alzheimer's disease. The study took advantage of selective pan-WNK inhibitor WNK 643 as the main tool to manipulate WNK 1-4 activity both in vivo by daily, per-oral drug administration to wild-type mice, and in vitro by treating either adult murine brain synaptosomes, hippocampal slices, primary cortical cultures, and human cell lines (HEK293, SH-SY5Y). Using a battery of standard behavior paradigms such as open field test, elevated plus maze test, and fear conditioning, the authors convincingly demonstrate that the inhibition of WNK1-4 results in behavior changes, especially in enhanced learning and memory of WNK643-treated mice. To shed light on the underlying molecular mechanism, the authors implemented multiple biochemical approaches including immunoprecipitation, glucose-uptake assay, surface biotylination assay, immunoblotting, and immunofluorescence. The data suggest that simultaneous insulin stimulation and WNK1-4 inhibition results in increased glucose uptake and the activity of insulin's downstream effectors, phosphorylated Akt and phosphorylated AS160. Moreover, the authors demonstrate that insulin treatment enhances the physical interaction of the WNK effector OSR1/SPAK with Akt substrate AS160. As a result, combined treatment with insulin and the WNK643 inhibitor synergistically increases the targeting of GLUT4 to the plasma membrane. Collectively, these data strongly support the initial hypothesis that neuronal insulin- and WNK-dependent pathways do interact and engage in cognitive functions.

In response to our initial comments, the authors mildly revised the manuscript, which did not improve the weaknesses to a sufficient level. Our follow-up comments are labeled under "Revisions 1".

Strengths:

The insulin-dependent signaling in the central nervous system is relatively understudied. This explorative study delves into several interesting and clinically relevant possibilities, examining how insulin-dependent signaling and its crosstalk with WNK kinases might affect brain circuits involved in memory formation and/or anxiety. Therefore, these findings might inspire follow-up studies performed in disease models for disorders that exhibit impaired glucose metabolism, deficient memory, or anxiety, such as Diabetes mellitus, Alzheimer's disease, or most of psychiatric disorders.

The graphical presentation of the figures is of high quality, which helps the reader to obtain a good overview and to easily understand the experimental design, results, and conclusions.

The behavioral studies are well conducted and provide valuable insights into the role of WNK kinases in glucose metabolism and their effect on learning and memory. Additionally, the authors evaluate the levels of basal and induced anxiety in Figures 1 and 2, enhancing our understanding of how WNK signaling might engage in cognitive function and anxiety-like behavior, particularly in the context of altered glucose metabolism.

The data presented in Figures 3 and 4 are notably valuable and robust. The authors effectively utilize a variety of in vivo and in vitro models, combining different treatments in a clear manner. The experimental design is well-controlled, efficiently communicated, and well-executed, providing the reader with clear objectives and conclusions. Overall, these data represent particularly solid and reproducible evidence on the enhanced glucose uptake, GLUT4 targeting, and downstream effectors' activation upon insulin and WNK/OSR1 signaling crosstalk.

Weaknesses:

(1) The study used a WNK643 inhibitor as the only tool to manipulate WNK1-4 activity. This inhibitor seems selective; however, it has been reported that it exhibits different efficiency in inhibiting the individual WNK kinases among each other (e.g. PMID: 31017050, PMID: 36712947). Additionally, the authors do not analyze nor report the expression profiles or activity levels of WNK1, WNK2, WNK3, and WNK4 within the relevant brain regions (i.e. hippocampus, cortex, amygdala). Combined, these weaknesses raise concerns about the direct involvement of WNK kinases within the selected brain regions and behavior circuits. It would be beneficial if the authors provided gene profiling for WNK1, 2, 3, and -4 (e.g. using Allen brain atlas). To confirm the observations, the authors should either add results from using other WNK inhibitors or, preferentially, analyze knock-down or knock-out animals/tissue targeting the single kinases.

Revisions 1: The authors added Fig. S1A during the revisions to show expression of Wnt1-4. While the expression data from humans is interesting, the experimental part of the study is performed in mice. It would be more informative for the authors to add expression profiles from mice or overview the expression pattern with suitable references in the introduction to address this point. The authors did not add data from knock down or knockout tissue targeting the single kinases.

(2) The authors do not report any data on whether the global inhibition of WNKs affects insulin levels as such. Since the authors demonstrate the synergistic effect of simultaneous insulin treatment and WNK1-4 inhibition, such data are missing.

Revisions 1: The authors added Fig. S5A to address this point. It is appreciated that authors performed the needed experiment. Unfortunately, no significant change was found, therefore, the authors still cannot conclude that they demonstrate a synergistic effect of simultaneous insulin treatment and WNT1-4 inhibition. It is a missed opportunity that the authors did not measure insulin in the CSF or tissue lysate to support the data.

(3) The study discovered that the Sortilin receptor binds to OSR1, leading the authors to speculate that Sortilin may be involved in the insulin-dependent GLUT4 surface trafficking. The authors conclude in the result section that "WNK/OSR1/SPAK influences insulin-sensitive GLUT4 trafficking by balancing GLUT4 sequestration in the TGN via regulation of Sortilin with GLUT4 release from these vesicles upon insulin stimulation via regulation of AS160." However, the authors do not provide any evidence supporting Sortilin's involvement in such regulation, thus, this conclusion should be removed from the section. Accordingly, the first paragraph of the discussion should be also rephrased or removed.

Revisions 1: The authors added Fig. 5M-N to address this point. The new experiment is appreciated. However, the authors still do not show that sortilin is involved in insulin or WNK-dependent GLUT4 trafficking in their set up since the authors do not demonstrate any changes in GLUT4 sorting or binding. The conclusions should therefore be rephrased or included purely in the discussion. Moreover, the discussion was not adjusted either, leading to over interpretation based on the available data.

(4) The background relevant to Figure 5, as well as the results and conclusions presented in Figure 5 are quite challenging to follow due to the lack of a clear introduction to the signaling pathways. Consequently, understanding the conclusions drawn from the data is also difficult. It would be beneficial if the authors addressed this issue with either reformulations or additional sections in the introduction. Furthermore, the pulldown experiments in this figure lack some of the necessary controls.

Revisions 1: The Authors insufficiently addressed this point during the revisions and did not rewrite the introduction as suggested.

(5) The authors lack proper independent loading controls (e.g. GAPDH levels) in their immunoblots throughout the paper, and thus their quantifications lack this important normalization step. The authors also did not add knock-out or knock-down controls in their co-IPs. This is disappointing since these improvements were central and suggested during the revision process.

(6) The schemes that represent only hypotheses (Fig. 1K, 4A) are unnecessary and confusing and thus should be omitted or placed at the end of each figure if the conclusions align.

(7) Low-quality images, such as Fig. 5H should be replaced with high-resolution photos, moved to the supplementary, or omitted.

Reviewer #2 (Public review):

This study by Jaykumar and colleagues seeks to expand the field's appreciation of insulin responses in the brain, specifically by implicating WNK kinase function in various neuronal responses, ranging from behavioral / memory changes to GLUT4 trafficking to the cell surface with subsequent glucose uptake. This revised study is now comprehensive and presents a logical and reasonably documented cascade of molecular interactions responsible in part for GLUT4 trafficking under the regulation of WKK and insulin. Additional data allow the authors to dissect a plausible WNK/OSR1/SPAK-sortilin pathway for the modulation of GLUT4 trafficking, in part by capitalizing on a overlay of various techniques and systems. The data - much of it in vivo or ex vivo - showing a potential role for WNK function in brain glucose utilization remains a compelling part of the story, with the dissection of the signaling cascade and a potential role for sortilin in mediating WNK function via effects on GLUT4 cellular localization now more convincing.

Initially, the group shows that oral WNK463 treatment - an inhibitor of WNKs broadly - in mice augments a number of memory readouts. These findings fit within the context of the overall story the authors present: that WNK function is critical to brain glucose utilization, which impacts learning. Multiple approaches are used to show that WNK463 treatment, i.e. inhibition of WNKs, increases glucose uptake, including labeled 2-deoxyglucose uptake in vivo in the brain and in isolated synaptosome, and uptake in ex vivo hippocampal slices. These findings are solid and consistent. With the exception of some relatively minor comments regarding the data presentation made to the authors and now fully addressed, the findings showing that WNK463 treatment increases GLUT4-mediated glucose uptake and surface localization of GLUT4 are reasonable, with the hippocampal slice data being particularly relevant.

While the details of the WNK signaling cascade is dense, in the revised application one clearly appreciates the molecular interrogation and interactions the group is dissecting, supported by the use of multiple models. With the additional findings, these systems and the data now reinforce each other, presenting a strongly documented overall story.

A limitation of the study with the initial submission was the authors' reliance upon a single pharmacological tool (WNK463) to inhibit WNK kinases. WNK463 apparently has substantial specificity for WNKs and WNK463 treatment lessened OSR1 phosphorylation (a WNK substrate). Nevertheless, the cohesiveness of the findings in terms of the broader pathway engagement (GLUT4 trafficking, glucose uptake) is consistent with the author's proposed mechanisms and conclusions. The authors have additionally addressed this concern in the revised manuscript with more information supporting the specificity of WNK463 as well as the multiple approaches to confirm the effect of WNK463 on the WNK signaling pathway of interest.

The final few paragraphs of the discussion that weave the author's findings into the field more broadly, including Sortilin function and neurological disorders, are appreciated. Additional clarity in the Methods section is also helpful.

Author response:

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

Joint Public Review:

Summary:

The major issues are the need for more information concerning WNK expression in brain regions and additional confirmation of the role of sortilin on WNT signaling. There is a lack of sufficient evidence supporting sortilin's involvement in insulin- and WNK-dependent GLUT4 regulation. The recommendation is to examine what WNK kinase is selectively expressed in the region of interest and then explore its engagement with the sortilin and GLUT4 pathways. Further identification of components of the WNK/OSr1/SPAK-sortilin pathway that regulate GLUT4 in brain slices or primary neurons will be helpful in confirming the results. The use of knock-down or knock-out models would be helpful to explore the direct interaction of the pathways. Immortalized and primary cells also represent useful models.

Together our results indicate that one or more WNK family members regulate insulin sensitivity. As all WNK family members are expressed in relevant brain regions, whether the results are due to actions of a single WNK family member or more likely due to their combined impact will be an important question to ask in the future.

There are multiple publications describing how sortilin is involved in insulin-dependent Glut4 trafficking; thus, we did not further address that issue. We have added data on an additional action of WNK463 which indicates that it can block association of OSR1 with sortilin. While these results do not delve further into how sortilin works, they support the conclusion that WNK/OSR1/SPAK can influence insulin-dependent glucose transport via distinct cellular events (AS160, sortilin, Akt) which are WNK463 sensitive.

Altogether we added 12 new panels of data from new and previously performed experiments and we modified 3 existing subfigures in response to comments.

Weaknesses:

(1) The study used a WNK643 inhibitor as the only tool to manipulate WNK1-4 activity. This inhibitor seems selective; however, it has been reported that it exhibits different efficiency in inhibiting the individual WNK kinases among each other (e.g. PMID: 31017050, PMID: 36712947). Additionally, the authors do not analyze nor report the expression profiles or activity levels of WNK1, WNK2, WNK3, and WNK4 within the relevant brain regions (i.e. hippocampus, cortex, amygdala). Combined, these weaknesses raise concerns about the direct involvement of WNK kinases within the selected brain regions and behavior circuits. It would be beneficial if the authors provided gene profiling for WNK1, 2, 3, and -4 (e.g. using Allen brain atlas). To confirm the observations, the authors should either add results from using other WNK inhibitors or, preferentially, analyze knock-down or knock-out animals/tissue targeting the single kinases.

Thank you for the excellent suggestion to include mRNA data for the four WNKs. We have included a supplementary figure showing expression of WNK1-4 mRNAs in prefrontal cortex and the hippocampus curated from the Allen Brain Atlas. As per the Allen Brain Atlas, all four WNKs are detected in these regions with WNK4 mRNA the most highly expressed followed by WNK2, WNK3 and then WNK1 (Figure S1A).

With regard to the use of WNK463, we continue to use WNK463 because we have examined its actions in cell lines that only express WNK1, e.g. A549 (Haman Center lung cancer RNA-seq data), and in A549 with WNK1 deleted using CRISPR in which we saw no effects of WNK463 on several assays we use for WNK1 including suppression of autophagy. WNK463 was reported in the literature to inhibit only the four WNKs out of more than 400 kinases tested, indicating more selectivity than many small molecules used to target other enzymes. In other cell lines, we also use WNK1 knockdown which replicates the effect of WNK463 (Figure S7A-D). However, in SHSY5Y cells, WNK1 knockdown did not replicate the effect of WNK463 on pAKT levels (Figure S7E-F), suggesting a cooperativity among other WNK family members in neuronal cells. This makes WNK463 an ideal tool to test our hypotheses in this study as it targets all 4 WNKs (WNK1-4).

(2) The authors do not report any data on whether the global inhibition of WNKs affects insulin levels. Since the authors wish to demonstrate the synergistic effect of simultaneous insulin treatment and WNK1-4 inhibition, such data are missing.

Thank you for this comment. To obtain this information, we treated C57BL/6J mice with WNK463 for 3 days once daily at a dose of 6 mg/kg and then fasted overnight. Plasma insulin levels were measured. Results showed that the plasma insulin levels trended upwards in the WNK463 treated animals compared to the vehicle treated groups but failed to reach any statistical significance. We have now included these data in supplementary figure S5A.

The study discovered that the Sortilin receptor binds to OSR1, leading the authors to speculate that Sortilin may be involved in the insulin-dependent GLUT4 surface trafficking. However, the authors do not provide any evidence supporting Sortilin's involvement in insulin- or WNKdependent GLUT4 trafficking. Thus, this conclusion should be qualified, rephrased, or additional data included.

Work from several groups have shown that sortilin is involved in insulin-dependent GLUT4 trafficking, for example [9-11,135-139] as we noted in the manuscript. We now show that WNK463 blocks co-immunoprecipitation of Flag-tagged sortilin with endogenous OSR1 in HEK293T cells. This result supports our model for WNK/OSR1/SPAK- insulin mediated regulation of sortilin. We included these data in figures 5M, 5N.

Minor issues:

(1) The method and result sections lack information regarding the gender and age of mice used in the behavioral experiments. This information should be added.

Thank you for pointing this out. We apologize for the omission. The requested information has now been added in the methods section.

(2) The authors present an analysis of relative protein levels in Figure 1B and Figure 4B, however, the original immunoblots (?) are not included in the study. These data should be added to provide complete and transparent evidence for the analysis.

Thank you for this request. The blots have now been included in the supplementary figure S2A and Figure 4B, respectively.

(3) The basis for Figure 3A needs to be explained and supported with suitable references either in the background or in the result section.

Thank you for pointing this out. Figure 3A has been moved to Figure 3H as it represents the model summary of the data presented in Figure 3. Other figure numbers have been changed accordingly. This figure 3A (now 3H) and the model diagram of Figure 5 (now Figure 5O) are now cited in the Discussion, where the results are considered in detail.

(4) Figure 4E should be labeled as 'Primary cortical neurons' for clarity, as the major focus is on the hippocampus. To increase consistency, the authors should consider performing the same experiment on hippocampal cultures or explaining using cortical neurons.

Thank you for the suggestion. Figure 4E (now 4F) has been labelled as Primary cortical neurons for clarity. The major focus of this study is to understand the regulation of WNKmediated regulation of insulin signaling in the areas of the brain that are insulin sensitive such as the hippocampus and the prefrontal cortex. Therefore, we included cortical neurons to test this hypothesis.

(5) Figure 5B: The use of whole brain extracts is inconsistent with the rest of the study, especially considering the indication of differing insulin activity in selected brain regions. The authors should explain why they could not use only hippocampal tissue.

In this manuscript, we are trying to test our hypothesis in insulin-sensitive neuronal cells which includes, but not limited to, the hippocampus. Figure 5B used whole brain extracts, which contain brain regions that are insulin-sensitive as well as insulin-insensitive regions, to show the association between OSR1 and AS160. However, this observation was replicated in the insulin-sensitive SH-SY5Y cell model suggesting that association of OSR1 and AS160 is modulated in the presence of insulin as shown in Figure 5B, 5C. We added data from SH-SY5Y cells showing effects of WNK463. These data support the concept that this is an interaction that is modulated by WNKs and will occur as long as both OSR1/SPAK and AS160 are expressed.

(6) Figure 5B-C - Knock-out or knock-down condition should be included in the co-IP experiment. This is especially straightforward to generate in the SH-SY5Y cells. Moreover, these figures lack loading controls.

If we understand correctly, the issue with regard to including knockdown conditions stems from the issues raised regarding specificity of the antibody which we have addressed in point 10 below. We have now included input blots for both AS160 and OSR1 which serve as the loading control for the IP experiment in figure 5B and 5C.

(7) Figure 5C-D - A condition with WNK463 inhibition alone is missing. This condition is necessary for evaluating the effects of WNK643 inhibition with and without insulin stimulation.

Thank you for this observation. We have now added the data for that condition. The aim of this experiment in Figure 5C (now 5B and 5C) is to show that insulin is important to facilitate interaction between OSR1 and AS160 in differentiated SHSY5Y cells and the effect of WNK463 to diminish this insulin-dependent interaction. With only WNK463, there was minimal interaction between AS160 and OSR1 as now shown in Figure 5B, 5C.

(8) Figure 5G - This figure shows the overexpression of plasmids in HEK cells, however, it lacks samples that overexpress the plasmid individually (single expression). Such data should be added, especially when the addition of the blocking peptide does not fully disable the interaction between AS160 and SPAK. Additionally, this figure also lacks a loading control, which is essential for validating the results.

Thank you for this comment. Figure 5G (now Figure 5F, 5G) is an in vitro IP in which we have mixed a purified Flag-SPAK fragment residues 50-545 with a lysate from cells expressing Myc-AS160 (residues 193-446). This is essentially an in vitro IP; because it is not an IP experiment from cell lysates where we overexpressed these plasmids which would require a loading control. The lysates were divided in half and one half did not receive the blocking peptide while the other half did, creating a control. From our experience, this blocking peptide does not completely block interactions between SPAK/OSR1 and NKCC2 fragments which are well-characterized interacting partners [a]. The reason for the partial block in interactions could also be attributed to the multivalent nature of interaction between these proteins. This confusion in our methodology used has been noted and we have tried to explain it with more clarity in the methods, results and the figure legend section. Our Commun. Biol. paper [134] that describes this assay and uses it extensively is now available online.

(a) Piechotta K, Lu J, Delpire E. Cation chloride cotransporters interact with the stressrelated kinases Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress response 1 (OSR1) J Biol Chem. 2002;277:50812–50819. doi: 10.1074/jbc.M208108200.

(9) Figure 5J, L - These figures are missing negative controls. The authors should add Sortilin knock-down or knock-out conditions for the immunoprecipitation experiments. Also, the figures lack loading controls. Moreover, the labeling "Control" should be specified, as it is unclear what this condition represents.

Thank you for noting the lack of clarity in the controls provided. Controls in Figure 5J and 5L refer to IgG Control which serves as the negative control in this case. This has now been specified in the figures (and added Figures 5M and 5N, as well). The issue with OSR1 and sortilin antibody specificity and cross-reaction has been addressed in point 10.

(10) Figure 5I - The fluorescent signals for the individual channels of OSR1 and Sortilin appear identical (even within the background signal). This raises concerns about potential antibody cross-reaction. One potential solution would be to include additional stainings with different antibodies and perform staining of each protein alone to ensure the specificity of the colocalization.

Thank you for pointing this out and giving us an opportunity to provide better images that will address the issues raised regarding antibody cross-reaction and antibody specificity. We realize that the images that we originally provided appeared to show all the puncta colocalize which could give rise to the concern about potential antibody cross-reaction. We have replaced them with more appropriate representative images that clearly show some selected regions of common staining as well as regions where there is no overlap.

(11) Figures 5D, 5F, 5H, 5L, 5M: These analyses should be first normalized to the loading control such as GAPDH.

In Figure 5F (now 5E), the analysis has been normalized to the total AS160 protein levels. Because we are reporting changes in pAS160 protein, normalizing it to the total AS160 gives a better idea about the changes in the phosphorylated AS160 form compared to the whole protein and this is more appropriate compared to other loading controls such as GAPDH.

In Figure 5H (now Figure 5G), the analysis is an in vitro IP assay using purified protein fragments. Therefore, using GAPDH as a control is not applicable in this case. Please refer to our response to comment 8 for details.

In Figures 5L, 5M and 5D (now 5K, 5L, 5C) shown, the IP proteins have been normalized to the input protein levels serving as a loading control for the IP experiment.

(12) Figure 5K: The significance/meaning of the red star is unclear. It should be explained in the figure legend.

Thank you for the opportunity to enhance the readability of our manuscript. The meaning of red star denotes the condition in the yeast two-hybrid assay which shows the binding of CCT of OSR1 with C-terminus of sortilin. This has now been clarified in the figure legend.

(13) Differences in WNK643 dosage and administration periods can affect the results. There is a lack of explanation with regard to the divergent WNK643 treatments of mice across different behavior conditions of fear conditioning, the novel object test, and the elevated plus maze test. This should be considered.

Thank you for pointing out that the explanation regarding the WNK463 dosage and times are unclear. WNK463 was dosed 3 days before the start of the behavior experiment daily at a dose of 6 mg/kg and continued throughout the test protocol. This is the same protocol used for all experiments. The text describing the protocol has been reworded with more clarity on dosage and times in methods and result section.