SGK1 inhibition in glia ameliorates pathologies and symptoms in Parkinson disease animal models

Abstract Astrocytes and microglia are brain‐resident glia that can establish harmful inflammatory environments in disease contexts and thereby contribute to the progression of neuronal loss in neurodegenerative disorders. Correcting the diseased properties of glia is therefore an appealing strategy for treating brain diseases. Previous studies have shown that serum/ glucocorticoid related kinase 1 (SGK1) is upregulated in the brains of patients with various neurodegenerative disorders, suggesting its involvement in the pathogenesis of those diseases. In this study, we show that inhibiting glial SGK1 corrects the pro‐inflammatory properties of glia by suppressing the intracellular NFκB‐, NLRP3‐inflammasome‐, and CGAS‐STING‐mediated inflammatory pathways. Furthermore, SGK1 inhibition potentiated glial activity to scavenge glutamate toxicity and prevented glial cell senescence and mitochondrial damage, which have recently been reported as critical pathologic features of and therapeutic targets in Parkinson disease (PD) and Alzheimer disease (AD). Along with those anti‐inflammatory/neurotrophic functions, silencing and pharmacological inhibition of SGK1 protected midbrain dopamine neurons from degeneration and cured pathologic synuclein alpha (SNCA) aggregation and PD‐associated behavioral deficits in multiple in vitro and in vivo PD models. Collectively, these findings suggest that SGK1 inhibition could be a useful strategy for treating PD and other neurodegenerative disorders that share the common pathology of glia‐mediated neuroinflammation.

30th Jul 2020 1st Editorial Decision 30th Jul 2020 Dear Prof. Lee, Thank you for the submission of your manuscript to EMBO Molecular Medicine. We have now received feedback from the three reviewers who agreed to evaluate your manuscript. As you will see from the reports below, the referees acknowledge the interest of the study but also raise serious concerns that should be addressed in a major revision. Particular attention should be given to identifying the cell type specific function of SGK1 inhibition. In our opinion, the mechanism of Nurr1/Foxa2-dependent regulation of Sgk1, although relevant, is not a focus of this study and should be addressed only in writing.
Addressing the reviewers' concerns in full will be necessary for further considering the manuscript in our journal, and acceptance of the manuscript will entail a second round of review. EMBO Molecular Medicine encourages a single round of revision only and therefore, acceptance or rejection of the manuscript will depend on the completeness of your responses included in the next, final version of the manuscript. For this reason, and to save you from any frustrations in the end, I would strongly advise against returning an incomplete revision.
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I look forward to receiving your revised manuscript.
Yours sincerely, Zeljko Durdevic ***** Reviewer's comments ***** Referee #1 (Remarks for Author): The manuscript submitted by Kwon et al investigated the effects of Sgk1 inhibition on glia with proinflammatory properties and the therapeutic potentials of Sgk1 inhibition using different in vivo models of Parkinson's disease. Their results showed that inhibition of Sgk1 suppressed KFkB-, NLRP3 inflammasome-and cGAS-STING-mediated inflammatory pathways in the culture system of mixed astrocyte and microglia. In addition, Sgk1 inhibition attenuated glial cell senescence and glial Sgk1 in Parkinson disease models. On the mechanistic front, the authors show an impressive amount of in vitro and in vivo data demonstrating that glial Sgk1 is a key target inhibited by Nurr1/Foxa2 and that inhibition of Sgk1 (by pharmacological or siRNA strategies) suppress proinflammatory pathways and neurotoxic pathways. On the functional/therapeutic front, the authors show that Sgk1 inhibition increases mDA neuronal survival in multiple in vitro models and improves neuronal survival, nigrostriatal projection, and motor performance in two independent PD mouse models. The experiments are well designed and nicely performed. Findings are logically and clearly presented. Overall, this is a very thorough and solid study with a focus of high biological and translational interest. Some revisions and clarifications are recommended.
Overall 1) Recommend using HGNO gene symbols for abbreviations, examples, SNCA for alpha-synuclein, TBK1 for Tank binding kinase 1 (P8, L12). 2) Current trend uses the non-possessive form for Parkinson disease and Alzheimer disease. 3) Unify the symbol for the control treatment group, such as "Cont", throughout all the figure panels. Currently a mixture of terms/symbols were used, creating confusion and ambiguity (e.g., Cont, Cont(-), DMSO, -, _, etc). 4) Provide some text clarification on the specificity of Sgk1 inhibitors used in this study based on published information. 5) It would be nice if the authors can discuss the potential shared or distinct effects of microglial and astrocytic Sgk1 on neurons.
Specific 1) P2, L18: Change to ".... that share the common pathology of glia-mediated neuroinflammation." 2) Fig. 1C: Explain RC. 3) Fig. 1E: Place individual "total protein" panels right under their respective "phosphorylated protein" panels. 4) Fig. 1O: Revise the diagram to better illustrate the action of phosphorylated IKKbeta in phosphorylating IkB and releasing NF-kB for nuclear translocation. The old diagram shows no connection of the nuclear translocated NF-kB to the one in the NF-kB/IkB complex. 5) Fig. 2F: Did the authors draw the graph? If not, I recommend they re-draw it for i) copy right issue, and ii) leave out the molecules not directly relevant to this study. 6) Fig. 4F: What is the topographical relationship between the insets relative to the whole pictures? Same, different, or a small part? 7) P8, L12: TBK1 instead of phosphor-TANK 8) P11, L7: Delete "AD".  Fig. 6B, 6C, and 6F. 12) P13, L4: Typo "pathological aggregation". 13) P13, L16: Change "neurodegeneration" to "oxidative and inflammatory damage" 14) P13, L25: Typo "neuroprotective/pro-inflammatory" 15) P14, L29: Behavioral deficits improved beginning 4 weeks (instead of 3 weeks) after the start of Sgk1 inhibitor treatment. 16) Define the time window when locomotor activity tests were performed in Fig. 7D, 7O, and 8G. 17) Fig. 9F, 9G: Draw precise lines to indicate which animals showed statistical differences. It is not clear to me whether the horizontal lines with changing positions from panel to panel indicate all 3 or just 1-2 of the animals. 18) Fig. 9 Legend (P42, L29 and 30): Typos -it should be (G) instead of (J) 19) Fig. 9 legends (P42, L31): Define how many qPCR repeats for each bar, representing one animal each.

Referee #3 (Remarks for Author):
Neuroinflammation is tightly associated with PD pathogenesis and disease progress. The manuscript entitled "Sgk1 inhibition in glia ameliorates pathologies and symptoms of Parkinson's disease" by Kwon et al. show that Sgk1 has a role in inflammation in both microglia and astrocytes. Inhibition of Sgk1 mediates the anti-inflammatory effects of Nurr1 and Foxa2. Inhibition of Sgk1 also protects DA neurons from α-synuclein(PFF)-induced damage and improves animal behaviors. The study described new findings for understanding the role of Sgk1 in inflammation in association with PD. It is an interesting study. However, there are some weaknesses that should be addressed.
Major points: 1. Fig.1, 1D and 1E showed that Nurr1 or Foxa2 alone decreased Sgk1 expressions. In protein level, the decrease of Sgk1 by Nurr1 or Foxa2 alone is similar to that by the combination of Nurr1 and Foxa2, but the phosphorylations of IκB and p65 are different among these groups. Also, fig 1E needs to be quantified. 2. Fig. 4, the treatment with hydrogen peroxide is not a way to produce superoxide that is detected by mitoSox. Even in the data sheet of reagent mitoSox, the hydrogen peroxide does not induce mitochondrial superoxide. 3. Fig. 5B, it lacks controls. Treatment of co-cultures with GSK650394 could not exclude the effects of inhibitor on neurons. 4. Fig. 6 and 8, the efficiency of Sgk1 knockdown is not presented by immunoblots or real-time PCR. 5. Fig. 8 A, the amount of microglia is surprisingly high in non-treated brain. 6. Fig. 8, the shRNA with CMV promoter to Sgk1 is not neuronal specific, it still can not rule out the effects on neurons.
Minor point: 1. The title is not correctly described as the study used animal model but not PD patients. It should clearly address in PD animal models. 2. There are many typo errors that should be corrected.

Authors' responses to the editor's comments
The editor's comment:

Particular attention should be given to identifying the cell type specific function of SGK1 inhibition.
Authors' response: In the revised paper, we added the data regarding the cell type specific expressions of Sgk1 and functions of Sgk1 inhibition as follow: 1) Sgk1 expressions in neurons, astrocytes, and microglia in cultures (Suppl. Fig. S6A) and in vivo midbrain (Suppl. Fig. S8C).

In our opinion, the mechanism of Nurr1/Foxa2-dependent regulation of Sgk1, although relevant, is not a focus of this study and should be addressed only in writing.
Authors' response: We very much appreciate this appropriate point and considerate suggestion. Based on this suggestion, we speculated on several possible mechanisms for Nurr1+Foxa2-mediated regulation of Sgk1 expression in the Discussion section (p.20, line 4p.20, line 22).

Authors' responses to the reviewers' comments
Referee #1 : *Portions revised in the text based on the reviewer's points are highlighted in red.

General Summary: The manuscript submitted by Kwon et al investigated the effects of Sgk1 inhibition on glia with pro-inflammatory properties and the therapeutic potentials of Sgk1 inhibition using different in vivo models of Parkinson's disease. Their results showed that inhibition of Sgk1 suppressed KFkB-, NLRP3 inflammasome-and cGAS-STING-mediated inflammatory pathways in the culture system of mixed astrocyte and microglia. In addition, Sgk1 inhibition attenuated glial cell senescence and mitochondria damage by enhancing glutamate uptake. Lastly, the author found Sgk1 inhibition ameliorated PD-associated behavior and pathology in two PD mouse models. Overall the authors presented substantial data on the roles of Sgk1 in multiple major cellular pathways in vitro, and the beneficial effect on the inhibition of Sgk1 in PD pathology. However, the data seem a bit too diffuse to form a cohesive story and certain critical questions are not addressed.
Authors' response: We are very grateful for the reviewer's positive comments and suggestions to help enhance the impact of our study. In response to the points and suggestions raised by the reviewer, we have substantially revised the paper. Our point-by-point responses to the reviewer's comments are as follows: We understand the reviewer's criticism that our study seems a bit too diffuse to form a cohesive story, because we addressed multiple pathogenic manifestations cured by glial Sgk1 inhibition such as inflammation, oxidative and mitochondrial stress, glial cell senescence, αsynucleinopathy, and mDA neuron degeneration. However, as explained in the Discussion section of this manuscript (p.20, line 24p.21, line 21 in the revised manuscript), we would say that those pathologic phenomena are closely linked and thus amelioration of the group of inflammatory sequelae was triggered by a single treatment of Sgk1 inhibition in glia. To help this study be more comprehensive, the links between the pathogenic pathways regulated by Sgk1 inhibition are summarized in a schematic representation ( Supplementary Fig. S9 in the revised manuscript).

Extending their previous work (Oh et al. 2016), in this manuscript the authors propose that Nurr1 and Foxa2 are the major regulators of SGK. SGK1 transcription is controlled by a wide variety of inputs including cell stress, hormones, cytosolic Ca2+ activity, Insulin, growth factors, and a number of major pathways such as PI3K, mTORC2, and PDKHowever, the mechanisms regarding Nurr1 and Foxa2's regulation of SGK1 -direct or indirect -are not addressed.
Authors' response: How Nurr1+Foxa2 regulates Sgk1 expression is intriguing but was not addressed in this study. Due to the complexity in the possible Sgk1 regulatory mechanisms by Nurr1+Foxa2 as described above, we think that the postulated mechanism could not easily be tested within the limited time frame of this paper's revision. In addition, we are afraid to say that the mechanism of Nurr1/Foxa2-dependent regulation of Sgk1, although relevant, is not a focus of this study, and will be addressed in a separate study.

It remains unclear which major mechanism of SGK1 in vitro contributes to effects on PD pathology in vivo.
Authors' response: Because all the glial pathogenic pathways regulated by Sgk1 inhibition are closely linked, it is hard to identify which is the major contributing mechanism. We believe that summation of the pathologic pathways mediated by glial Sgk1 inhibition, but not by a single pathology, is responsible for the amelioration of PD pathologies. However, based on our findings, we think that downregulation of the NFkB-mediated inflammatory pathway is positioned at the most upstream point that allows it to initiate and trigger the other therapeutic effects of Sgk1 inhibition. This explanation is provided in the Discussion section of the revised text (p.20, line 24; p.21, line 21 and schematized in Supplementary Fig. S9).

In vivo experiments (Figures 7-9) using pharmacological and viral manipulation of SGK1 inhibitors do not inform cell-type-specific function.
Authors' response: As suggested by this reviewer's points 4 and 5, we have carried out celltype-specific effects experiments to investigate Sgk1 inhibition in pure neuron, astrocyte, and microglia cultures in the revised paper. Expectedly, Sgk1 inhibition in either the astrocytic or microglial cultures resulted in neurotrophic effects that prevented neuronal (mDA neuronal) degeneration (the astrocytic effect was more robust than that of the microglia effect) ( Supplementary Fig. S7 and p.15. line 22p.16. line 8. ). Furthermore, sh-Sgk1-treated astrocytes and microglia cooperated to exert more potent neuroprotective effects. By contrast, Sgk1 inhibition in neuronal cultures non-significantly altered neuronal (mDA neuronal) viability (Supplementary. Fig. S6B and p.15, line 5 -20 in the revised manuscript). Based on the in vitro findings, it is believed that pharmacological inhibition and silencing of Sgk1 in astrocytes and microglia additively/synergistically exerted therapeutic functions in the PD animal models.

The culture system in this manuscripts is a mixture of astrocytes and microglia. Is the ratio of the two cells comparable to that in the brain? Also, the author did not distinguish the roles of Sgk1 in astrocytes and microglia in such an experimental system.
Authors' response: The ratio of astrocytes and microglia in the brain varies very much depending on reports, detection methods, and brain regions. Thus, we counted the glial cells and the ratio in the midbrain of adult mice (microglia:19.7±1.7 % of total astrocyte+microglia). The data is now shown in p. 6, line 10). ) We appreciate this appropriate point. As described in our response to point 3, cell-typespecific effects of Sgk1 inhibition were carried out in pure astrocyte and microglia cultures. The results suggest astrocyte-and microglia-specific effects of Sgk1 inhibition that act to protect (mDA) neurons from toxic insult. These results have been described in the revised manuscript ( Supplementary Fig. S7 and p.15. line 22p.16. line 8).

According to Figure 8A, the infection efficiency of shSgk1 AAV9 is comparable in neurons and astrocytes: 1. Is it possible the beneficial effects of shSgk1 come from neurons? 2. What is the effects of shSgk1 in astrocytes in the PD models.
Authors' response: Our response to this point is same as that in points 3 and 4.

What is the expression pattern of Sgk1 in both normal brain and PD models? Is it enriched in certain CNS cell types?
Authors' response: Based on the reviewer point, the following data are shown in the revised text.
Based on these findings, we concluded that Sgk1 expression increases in the SN of PD mice and the increased Sgk1 expression was ubiquitously detected without a cell-type specificity (p.17. lines 13-16).

In the in vivo interventions (Figures 7-9) the authors do not show SGK1 downregulation after pharmacological and viral interventions. These would be useful controls.
Authors' response: We appreciate this appropriate point. The data demonstrating total and cell-type-specific Sgk1 expressions that were downregulated in sh-Sgk1-treated mouse midbrain are now shown in Fig. 8D and E of the revised manuscript

For shSkg1 treatment, the efficiency of Skg1 knockdown should be shown with different cell markers (Astrocyte, microglia, and neuron)
Authors' response: Our response to this point is the same as that for minor comment 1.

There are a number of western blots missing quantification throughout the manuscript.
Authors' response: Based on the reviewer's comment, all the western blot data were quantified throughout the manuscript.

Some figure legends need to be clarified.(e.g. Fig9 Fa dn G: what are the three bars of control or treatment group?)
Authors' response: We are very sorry for the missing information in Fig. 9F and G and the incorrect description in the figure legend. The errors have been corrected in the Figure and its legend (p.46, lines 11-12).
In addition we have carefully check all the figures and their legends.

Referee #2 :
*Portions revised in the text based on the reviewer's points are highlighted in red.

General Summary: This work by Kwon et al reports the underlying mechanism and therapeutic outcome of inhibiting glial Sgk1 in Parkinson disease models. On the mechanistic front, the authors show an impressive amount of in vitro and in vivo data demonstrating that glial Sgk1 is a key target inhibited by Nurr1/Foxa2 and that inhibition of Sgk1 (by pharmacological or siRNA strategies) suppress proinflammatory pathways and neurotoxic pathways. On the functional/therapeutic front, the authors show that Sgk1 inhibition increases mDA neuronal survival in multiple in vitro models and improves neuronal survival, nigrostriatal projection, and motor performance in two independent PD mouse models. The experiments are well designed and nicely performed. Findings are logically and clearly presented. Overall, this is a very thorough and solid study with a focus of high biological and translational interest. Some revisions and clarifications are recommended.
Authors' response: We are very grateful for the reviewer's positive comments and suggestions to help enhance the impact of our study. In response to the points and suggestions raised by the reviewer, we have substantially revised the paper. Our point-by-point responses to the reviewer's comments are as follows:

Overall 1) Recommend using HGNC gene symbols for abbreviations, examples, SNCA for alphasynuclein, TBK1 for Tank binding kinase 1 (P8, L12).
Authors' response: As recommended, gene symbols were changed to HGNC format throughout the text.

2) Current trend uses the non-possessive form for Parkinson disease and Alzheimer disease.
Authors' response: We appreciate instruction on how to use current descriptive forms. The disease terms have been changed to the non-possessive form throughout the text. ambiguity (e.g., Cont, Cont(-), DMSO, -, _, etc).

Authors' response:
We appreciate the reviewer for carefully checking our manuscript and suggesting correction of the inconsistent terms and symbols in the figures. We have corrected the inconsistencies throughout the figures.

4) Provide some text clarification on the specificity of Sgk1 inhibitors used in this study based on published information.
Authors' response: The specificity for the Sgk1 inhibitors is shown on p.23, line 30; p.23, line 31 of the revised text.

5) It would be nice if the authors can discuss the potential shared or distinct effects of microglial and astrocytic Sgk1 on neurons.
Authors' response: We appreciate this appropriate suggestion. A similar comment was made by the other reviewer. Thus, we have carried out cell-type-specific effects experiments that investigated Sgk1 inhibition in pure astrocyte and microglia cultures in the revised paper. Expectedly, Sgk1 inhibition in either astrocytic or microglial cultures resulted in neurotrophic effects that prevented neuronal (mDA neuronal) degeneration (the astrocytic effect was more robust than the microglia effect). Furthermore, sh-Sgk1-treated astrocytes and microglia cooperated with each other to exert more potent neuroprotective effects. The data are described in Supplementary Fig. S7 and p. 16. line 1-8 of the revised manuscript.

2) Fig. 1C: Explain RC.
Authors' response: We are sorry for the missing information. The full name of FC (fold change) and RC (read count) were inserted in Fig. 1B and C and the legend of the revised manuscript (p.40, line 9).

3) Fig. 1E: Place individual "total protein" panels right under their respective "phosphorylated protein" panels.
Authors' response: We rearranged the WB panels as suggested.

4) Fig. 1O: Revise the diagram to better illustrate the action of phosphorylated IKKbeta in phosphorylating IkB and releasing NF-kB for nuclear translocation. The old diagram shows no connection of the nuclear translocated NF-kB to the one in the NF-kB/IkB complex.\ 1st Revision -Editorial Decision
24th Dec 2020 Dear Prof. Lee, Thank you for the submission of your revised manuscript to EMBO Molecular Medicine. I am pleased to inform you that we will be able to accept your manuscript pending the following final amendments: 1) With approaching holidays and the end of the year we encountered high number of submissions, so that our data editors were not able to process all received manuscripts before the holiday season. Therefore, we will send you the document with data editor's suggestions after the holidays and as soon as our data editors process your manuscript. Please do not submit your revised manuscript before we send you the file with data editor's suggestions. Thank you for your understanding.
2) In the main manuscript file, please do the following: -Add up to 5 keywords.
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-Add callouts for Fig 5F and Fig 7T. There is a callout for Fig 7V but

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Animal numbers were kept at a minimum to repeatedly observe a result. Appropriateness of the animal sizes were evaluated by IACUC at Hanyang university.
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