SGK1 inhibition in glia ameliorates pathologies and symptoms in Parkinson disease animal models
2021; Springer Nature; Volume: 13; Issue: 4 Linguagem: Inglês
10.15252/emmm.202013076
ISSN1757-4684
AutoresOh‐Chan Kwon, Jae‐Jin Song, Yunseon Yang, Seong‐Hoon Kim, Ji Young Kim, Min‐Jong Seok, Inhwa Hwang, Je‐Wook Yu, Jenisha Karmacharya, Han‐Joo Maeng, J. S. Kim, Eek‐hoon Jho, Seung Yeon Ko, Hyeon Son, Mi‐Yoon Chang, Sang‐Hun Lee,
Tópico(s)Neuroinflammation and Neurodegeneration Mechanisms
ResumoArticle1 March 2021Open Access Transparent process SGK1 inhibition in glia ameliorates pathologies and symptoms in Parkinson disease animal models Oh-Chan Kwon orcid.org/0000-0002-6433-4507 Department of Biochemistry and Molecular Biology, College of Medicine, Hanyang University, Seoul, Korea Hanyang Biomedical Research Institute, Hanyang University, Seoul, Korea Graduate School of Biomedical Science and Engineering, Hanyang University, SeoulThese authors contributed equally to this work Search for more papers by this author Jae-Jin Song Department of Biochemistry and Molecular Biology, College of Medicine, Hanyang University, Seoul, Korea Hanyang Biomedical Research Institute, Hanyang University, Seoul, KoreaThese authors contributed equally to this work Search for more papers by this author Yunseon Yang Department of Biochemistry and Molecular Biology, College of Medicine, Hanyang University, Seoul, Korea Hanyang Biomedical Research Institute, Hanyang University, Seoul, Korea Graduate School of Biomedical Science and Engineering, Hanyang University, SeoulThese authors contributed equally to this work Search for more papers by this author Seong-Hoon Kim Department of Biochemistry and Molecular Biology, College of Medicine, Hanyang University, Seoul, Korea Hanyang Biomedical Research Institute, Hanyang University, Seoul, Korea Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul Search for more papers by this author Ji Young Kim Department of Biochemistry and Molecular Biology, College of Medicine, Hanyang University, Seoul, Korea Hanyang Biomedical Research Institute, Hanyang University, Seoul, Korea Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul Search for more papers by this author Min-Jong Seok Department of Biochemistry and Molecular Biology, College of Medicine, Hanyang University, Seoul, Korea Hanyang Biomedical Research Institute, Hanyang University, Seoul, Korea Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul Search for more papers by this author Inhwa Hwang Korea Department of Microbiology and Immunology, Institute for Immunology and Immunological Diseases, Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, South Korea Search for more papers by this author Je-Wook Yu orcid.org/0000-0001-5943-4071 Korea Department of Microbiology and Immunology, Institute for Immunology and Immunological Diseases, Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, South Korea Search for more papers by this author Jenisha Karmacharya College of Pharmacy, Gachon University, Incheon, Korea Search for more papers by this author Han-Joo Maeng College of Pharmacy, Gachon University, Incheon, Korea Search for more papers by this author Jiyoung Kim Department of Life Science, University of Seoul, Seoul, Korea Search for more papers by this author Eek-hoon Jho orcid.org/0000-0003-2414-6234 Department of Life Science, University of Seoul, Seoul, Korea Search for more papers by this author Seung Yeon Ko Department of Biochemistry and Molecular Biology, College of Medicine, Hanyang University, Seoul, Korea Hanyang Biomedical Research Institute, Hanyang University, Seoul, Korea Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul Search for more papers by this author Hyeon Son Department of Biochemistry and Molecular Biology, College of Medicine, Hanyang University, Seoul, Korea Hanyang Biomedical Research Institute, Hanyang University, Seoul, Korea Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul Search for more papers by this author Mi-Yoon Chang Corresponding Author [email protected] Department of Biochemistry and Molecular Biology, College of Medicine, Hanyang University, Seoul, Korea Hanyang Biomedical Research Institute, Hanyang University, Seoul, Korea Search for more papers by this author Sang-Hun Lee Corresponding Author [email protected] orcid.org/0000-0001-7553-8188 Department of Biochemistry and Molecular Biology, College of Medicine, Hanyang University, Seoul, Korea Hanyang Biomedical Research Institute, Hanyang University, Seoul, Korea Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul Search for more papers by this author Oh-Chan Kwon orcid.org/0000-0002-6433-4507 Department of Biochemistry and Molecular Biology, College of Medicine, Hanyang University, Seoul, Korea Hanyang Biomedical Research Institute, Hanyang University, Seoul, Korea Graduate School of Biomedical Science and Engineering, Hanyang University, SeoulThese authors contributed equally to this work Search for more papers by this author Jae-Jin Song Department of Biochemistry and Molecular Biology, College of Medicine, Hanyang University, Seoul, Korea Hanyang Biomedical Research Institute, Hanyang University, Seoul, KoreaThese authors contributed equally to this work Search for more papers by this author Yunseon Yang Department of Biochemistry and Molecular Biology, College of Medicine, Hanyang University, Seoul, Korea Hanyang Biomedical Research Institute, Hanyang University, Seoul, Korea Graduate School of Biomedical Science and Engineering, Hanyang University, SeoulThese authors contributed equally to this work Search for more papers by this author Seong-Hoon Kim Department of Biochemistry and Molecular Biology, College of Medicine, Hanyang University, Seoul, Korea Hanyang Biomedical Research Institute, Hanyang University, Seoul, Korea Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul Search for more papers by this author Ji Young Kim Department of Biochemistry and Molecular Biology, College of Medicine, Hanyang University, Seoul, Korea Hanyang Biomedical Research Institute, Hanyang University, Seoul, Korea Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul Search for more papers by this author Min-Jong Seok Department of Biochemistry and Molecular Biology, College of Medicine, Hanyang University, Seoul, Korea Hanyang Biomedical Research Institute, Hanyang University, Seoul, Korea Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul Search for more papers by this author Inhwa Hwang Korea Department of Microbiology and Immunology, Institute for Immunology and Immunological Diseases, Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, South Korea Search for more papers by this author Je-Wook Yu orcid.org/0000-0001-5943-4071 Korea Department of Microbiology and Immunology, Institute for Immunology and Immunological Diseases, Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, South Korea Search for more papers by this author Jenisha Karmacharya College of Pharmacy, Gachon University, Incheon, Korea Search for more papers by this author Han-Joo Maeng College of Pharmacy, Gachon University, Incheon, Korea Search for more papers by this author Jiyoung Kim Department of Life Science, University of Seoul, Seoul, Korea Search for more papers by this author Eek-hoon Jho orcid.org/0000-0003-2414-6234 Department of Life Science, University of Seoul, Seoul, Korea Search for more papers by this author Seung Yeon Ko Department of Biochemistry and Molecular Biology, College of Medicine, Hanyang University, Seoul, Korea Hanyang Biomedical Research Institute, Hanyang University, Seoul, Korea Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul Search for more papers by this author Hyeon Son Department of Biochemistry and Molecular Biology, College of Medicine, Hanyang University, Seoul, Korea Hanyang Biomedical Research Institute, Hanyang University, Seoul, Korea Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul Search for more papers by this author Mi-Yoon Chang Corresponding Author [email protected] Department of Biochemistry and Molecular Biology, College of Medicine, Hanyang University, Seoul, Korea Hanyang Biomedical Research Institute, Hanyang University, Seoul, Korea Search for more papers by this author Sang-Hun Lee Corresponding Author [email protected] orcid.org/0000-0001-7553-8188 Department of Biochemistry and Molecular Biology, College of Medicine, Hanyang University, Seoul, Korea Hanyang Biomedical Research Institute, Hanyang University, Seoul, Korea Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul Search for more papers by this author Author Information Oh-Chan Kwon1,2,3, Jae-Jin Song1,2, Yunseon Yang1,2,3, Seong-Hoon Kim1,2,3, Ji Young Kim1,2,3, Min-Jong Seok1,2,3, Inhwa Hwang4, Je-Wook Yu4, Jenisha Karmacharya5, Han-Joo Maeng5, Jiyoung Kim6, Eek-hoon Jho6, Seung Yeon Ko1,2,3, Hyeon Son1,2,3, Mi-Yoon Chang *,1,2 and Sang-Hun Lee *,1,2,3 1Department of Biochemistry and Molecular Biology, College of Medicine, Hanyang University, Seoul, Korea 2Hanyang Biomedical Research Institute, Hanyang University, Seoul, Korea 3Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul 4Korea Department of Microbiology and Immunology, Institute for Immunology and Immunological Diseases, Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, South Korea 5College of Pharmacy, Gachon University, Incheon, Korea 6Department of Life Science, University of Seoul, Seoul, Korea *Corresponding author. Tel: +82 2 2220 0620; E-mail: [email protected] *Corresponding author. Tel: +82 2 2220 0625; Fax: +82 2 2220 2422; E-mail: [email protected] EMBO Mol Med (2021)13:e13076https://doi.org/10.15252/emmm.202013076 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Synopsis Pathogenic involvement of SGK1 has been implicated in various neurodegenerative disorders. In this study, we show that inhibition of SGK1 in glia treats Parkinson disease (PD) via suppressing glial inflammation and potentiating glial neurotrophic functions. SGK inhibition reduces neuroinflammation and protects dopamine neurons from degeneration. PD – associated behavioral deficits are improved in PD mice after SGK inhibition. Inhibition of SGK1 could be a useful strategy for treating PD and other neurodegenerative disorders. The paper explained Problem Neuronal cells have been the central cell type targeted for drug development in CNS disorders. However, effective disease-modifying therapies that rescue degenerating neurons have not yet been developed. It is mainly because astrocytes and microglia, brain-resident glia, 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. Results 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. 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 Parkinson disease (PD)-associated behavioral deficits in multiple in vitro and in vivo PD models. Impact Our results provide evidence that SGK1 inhibition in astrocytes and microglia can be therapeutically used in PD. Because the glial pathologies shown to be improved by SGK1 inhibition in this study commonly underlie other CNS disorders, SGK1 inhibition might eventually become a therapeutic intervention that is broadly applicable to a variety of neurodegenerative disorders and neurologic injuries. Introduction Parkinson disease (PD) is a common neurodegenerative disorder characterized by the degeneration of dopamine (DA) neurons in the substantia nigra (SN) of the midbrain and toxic intra-neuronal inclusion of misfolded synuclein alpha (Lewy bodies and neurites). Studies have shown that PD's pathologic features are caused by mitochondrial damage/dysfunction, endoplasmic reticulum stress, and defects in vesicular trafficking, synaptic recycling, and auto-lysosomal pathways in midbrain DA (mDA) neurons (Nguyen et al, 2019). Along with those intra-neuronal dysfunctions, persistent inflammation mediated primarily by neighboring glia commonly underlies PD and other neurodegenerative disorders (Yan et al, 2014; Guzman-Martinez et al, 2019). Astrocytes and microglia are brain-resident glial cells that typically play a homeostatic and neurotrophic role supporting neuronal cell survival and functioning and maintaining brain homeostasis in other ways (Horner & Palmer, 2003; Nedergaard et al, 2003; Molofsky et al, 2012). However, the naïve homeostatic properties of glia are compromised in disease contexts, and they instead establish harmful inflammatory brain environments by secreting inflammatory mediators (frequently annotated as M1 [microglia] and A1 [astrocyte] activation; Neumann et al, 2009; Liddelow et al, 2017a). The idea of polarizing pathologic M1(A1)-type glia back into their neurotrophic/homeostatic forms (M2 and A2 types) to treat neurodegenerative diseases for which there are no disease-modifying therapies has gained momentum (Hamby & Sofroniew, 2010). A previous study demonstrated that nuclear receptor subfamily 4 group A member 2 (NR4A2, also known as NURR1), originally known as a transcription factor specific for developing and adult mDA neurons, can also be expressed in astrocytes/microglia in response to toxic insults and that Nurr1-expressing glia protect neighboring mDA neurons by prohibiting the synthesis and release of pro-inflammatory cytokines from glial cells (Saijo et al, 2009). This Nurr1-mediated anti-inflammatory action in glia has been manifested in various brain disease contexts (Loppi et al, 2018; Popichak et al, 2018; Montarolo et al, 2019; Shao et al, 2019). Other studies have shown that forkhead box A2 (FOXA2; also known as HNF3B) is a potent cofactor that synergizes the anti-inflammatory role of Nurr1 in glia (Oh et al, 2015; Song et al, 2018). Studying intracellular anti-inflammatory pathways downstream of Nurr1 + Foxa2 in glia could be valuable for identifying reliable therapeutic targets to treat neurodegenerative disorders, including PD. Serum/ glucocorticoid related kinase 1 (SGK1) is a gene first identified as upregulated by serum and glucocorticoids in rat mammary tumor cells (Firestone et al, 2003). SGK1 expression is widely detected in the brain, and it is increased in pathologic conditions such as Rett syndrome (Nuber et al, 2005), Alzheimer disease (Chun et al, 2004; Lang et al, 2010; Zhang et al, 2018), multiple sclerosis (Wang et al, 2017), amyotrophic lateral sclerosis (Schoenebeck et al, 2005), and neuropathic pain (Geranton et al, 2007; Peng et al, 2013). The level of SGK1 is relatively higher in the midbrain than in other brain regions, and upregulation of SGK1 coincides with the onset of DA neuron death in a model of PD (Iwata et al, 2004; Stichel et al, 2005), collectively suggesting that SGK1 plays pathogenic roles in PD and other neurodegenerative disorders. However, SGK1 role in CNS is not clearly identified. In this study, we show that Sgk1 is a molecule that is negatively regulated by Nurr1 (N) and Foxa2 (F) in glial cells. We also show that glial inhibition of SGK1 mediates the anti-inflammatory functions of N + F. In addition to its anti-inflammatory effects, SGK1 inhibition in glia potentiates the neuroprotective functions of glia and prevents glial cell senescence, which has recently been reported as a critical pathologic feature of and therapeutic target in AD and PD (Bussian et al, 2018; Chinta et al, 2018). Using multiple in vitro and in vivo PD models, we show that silencing and pharmacological inhibition of SGK1 are effective therapeutic tools for treating PD. Results SGK1 inhibition in glia suppresses NFκB-mediated pro-inflammatory cytokine gene expression We and other research groups have previously shown that the midbrain factors N and F exert anti-inflammatory effects in glia by inhibiting the transcription of pro-inflammatory cytokines (Saijo et al, 2009; Oh et al, 2015; Song et al, 2018). In our microarray data (accession no. GSE145489), one of the genes that N and F most synergistically downregulated in glia cultured from mouse cortices was Sgk1 (reduced 9.3-fold by N and F in combination; Fig 1A and B). We further observed a similar reduction in SGK1 expression in N + F-expressing astrocytes derived from a mouse ventral midbrain (VM) in our RNA-sequencing data (RNA-seq; accession no. GSE106216; Fig 1C), which we validated in VM-derived glial cultures (GFAP+ astrocytes > 70%, IBA1+ microglia < 15%, exposure to H2O2, 250 µM, 4 h) using qPCR analyses (Fig 1D). SGK1 inhibition attracted our interest because SGK1 could regulate NFκB signal, the central intracellular pathway responsible for inflammatory cytokine transcription (Lawrence, 2009; Liu et al, 2017). Specifically, SGK1 is suggested to phosphorylate IκB kinase subunit beta (IKKB), which activates NFκB signaling by releasing NFκB from the inhibitory IκB-NFκB complex(Lang & Voelkl, 2013). Indeed, Western blot (WB) analyses showed that downregulating SGK1 proteins by forcing the expression of N and F in cultured VM-derived glial cells was followed by a decrease in phosphorylated (activated) IKKB and IκB (Fig 1E). Consequently, compared to the mock-transduced control glia, the higher proportion of the NFκB in N- and F-transduced glia was inactively trapped in the complex associated with IκB in cytosol (Fig 1F). The inactivation of NFκB signaling was further manifested by decreased levels of phosphorylated (activated) p65 (RELA: RELA proto-oncogene, NF-kB subunit), a major NFκB component, and decreased levels of RELA (p65) proteins trans-localized into the nucleus (Fig 1E). The N + F-mediated inactivation of NFκB signals in glia was recovered by the forced expression of SGK1 (Fig 1G), and the expression of pro-inflammatory cytokines reduced by N + F treatment returned to levels similar to those in the control glia (Fig 1H). NFκB activation induced by lipopolysaccharide (LPS, 1 mM, 24 h), a toll-like receptor 4 ligand, greatly subsided in cultured glial cells in the presence of a SGK1 inhibitor (GSK-650394) (Fig 1I). As expected, SGK1 inhibition using treatment with siRNA, sh-RNA, and specific inhibitors (EMD-638683, GSK-650394) consistently downregulated the key pro-inflammatory cytokines IL-1β, TNFα, IL-6, and iNOS (Fig 1J–M), whereas the overexpression of SGK1 had the opposite effect (Fig 1N). These findings collectively indicate that N + F-mediated anti-inflammatory functions in glia are substantially attained by blocking SGK1 signaling (Fig 1O), and thus, SGK1 inhibition could be used to treat neurodegenerative disorders in which neuroinflammation is a common underlying pathology. Figure 1. SGK1 inhibition is the key mediator for Nurr1 + Foxa2-induced anti-inflammatory function in glial cells A–D. Nurr1 (N) and Foxa2 (F) synergistically downregulate SGK1 expression in cultured glia. (A) Up- and downregulated genes in the microarray data for the cultured glia transduced with N + F (vs. mock-transduced control). Sgk1 is marked with an arrow in the top downregulated genes. (B–D) Effect of N and F on SGK1 expression in glial cells shown in the microarray (B), RNA-seq (C), and RT–PCR (D) analyses. Data in (B and C) represent values of fold change (FC) or read count (RC) with color intensities. n = 3. One-way ANOVA. Data are represented as mean ± SEM. Significantly different at P = 0.0493*, 0.0092**, 0.0009***. E–I. Downregulation of SGK1 is responsible for N + F function to inhibit NFκB-mediated pro-inflammatory cytokine expression. (E) WB analysis exhibiting N + F-induced inhibition of NFκB intracellular signaling in cultured VM-glia. The N + F-mediated downregulation of NFκB signaling is attained by inhibiting IκB phosphorylation (E) and thus blocking the release of NFκB from the NFκB-IκB inhibitory complex (F). Data are represented as mean ± SEM. n = 3 independent experiments; one-way ANOVA with Tukey-analysis. P = 0.015#, 0.002##, 1.33E-12### (sgk1), 0.036#, 0.00004## (p-IKKB). 0.00007### (p-IkBα). 0.005#, 2.11E-09### (p-p65). 1.93E-06### (p65 level in nucleus) in graph E. P = 6.44E-09*, 0.00004**, 4.45E-10*** in graph F. (G and H) N + F-mediated downregulation of NFκB signaling (phosphorylation of p65, p-p65, G) and pro-inflammatory cytokine expression (IL-1β, TNFα, H) were alleviated by forced SGK1 expression in the VM-glial cultures. Data are represented as mean ± SEM. n = 3 independent cultures; two-way ANOVA with Bonferroni post hoc analysis. Significantly different at P = 0.014#, 0.009*, 0.009**, 0.003+ (p-p65) in graph G and P = 1.79E-06*, 0.0003**, 0.004***, 0.0068#, 0.0392##, 0.0027### in graph H. (I) SGK1 inhibition blocks glial NFκB signaling. Data are represented as mean ± SEM. n = 4–6. One-way ANOVA. Significantly different at P = 0.002*, 0.00004**, 0.00001#, 3.01E-10##, 3.78E-06+, 1.59E-07++ in graph I. J–N. Glial pro-inflammatory cytokine expression regulated by SGK1 inhibition (J–M) and overexpression (N). Data are represented as mean ± SEM. n = 3 independent cultures; unpaired Student's t-test. Significantly different at P = 0.0041*, 0.0010#, 0.0010+, 0.0067## in graph J and P = 0.0002*, 0.0345#, 0.0023+, 0.0012**, 0.0001## in graph K and P = 0.0367*, 0.0042+, 0.0003** in graph L and P = 0.0001*, 0.0009#, 0.0002+, 0.0082** in graph M and P = 0.0367*, 0.0059#, 0.0005**, 0.0006## in graph N. O. Schematic summary to show how SGK1 inhibition mediates the N + F-induced anti-inflammatory action in glia. Download figure Download PowerPoint SGK1 inhibition-mediated transcriptome changes are associated with immune/inflammation reaction and glial activation/polarization The main purpose of this study was to test whether SGK1 inhibition in glial cells could be used to develop a therapy for PD. Therefore, glial cells were cultured from rodent VMs, which is primarily affected in PD. VM-glial cultures of GFAP+ astrocytes (60–70% of total cells) and Iba1+ microglia (5–15%, cf > 19.7 ± 1.7% counted in mouse adult midbrain) were used throughout this study unless otherwise noted because astroglia and microglia interact closely to prevent or exacerbate disease pathogenesis (Jha et al, 2019), and thus, the consequences of their interactions in mixed glia, not the isolated individual actions of astrocytes and microglia, are the target in developing therapeutic interventions for brain disease. To gain further insight into how SGK1 inhibition affects glial functions, we performed an RNA-seq analysis (GSE145490) in primary VM-derived glial cultures with and without the SGK1 inhibitor GSK-650394. Consistent with the SGK1 inhibition effects shown in Fig 1, genes downregulated by the SGK1 inhibitor (FPKM > 1, > 2.6 fold change [FC]) were found in the gene ontologies associated with "inflammatory reaction" and the "NFκB pathway" (highlighted in red in Fig 2A and B). Actually, 7 of the top 10 ontologies of the downregulated genes were related to inflammatory/immune reactions. The enrichment of differentially regulated genes (DEGs) in inflammatory/immune pathways was further confirmed by gene set enrichment analyses (Fig 2C). Furthermore, immune/inflammatory genes were the most significantly downregulated by the inhibitor treatment (Fig 2D and E). Using a 2-FC cutoff and 1% false discovery rate (FDR), 726 genes downregulated by the SGK1 inhibitor were also downregulated by N + F, and the overlapping genes were also found in the gene categories of immune/inflammation and the NFκB pathway (Appendix Fig S1A, C, E), further confirming that N + F inhibition of glial inflammation via the NFκB pathway is mediated by inhibiting SGK1 intracellular signals in glial cells. Figure 2. SGK1 inhibition-mediated transcriptome changes associated with immune/ inflammation reaction and microglial polarization In the RNA-seq data (GSE145490, normalized read count (RC) > 1, > 2.6-fold, 3,000 genes), genes downregulated in the VM-glia treated with the SGK1 inhibitor GSK-650394 vs. the vehicle (DMSO)-treated control were analyzed. A, B. Top gene ontologies (GOs) and KEGG pathways of the genes downregulated in VM-glia by the SGK1 inhibitor treatment. The purple bar indicates the number of genes under the designated GO term/KEGG pathway. The red bar indicates the p-value, and the negative log of the p-value (bottom) is plotted on the X-axis. C. Gene set enrichment analysis (GESA) for inflammatory and immune responses. D. Scatterplots for the differentially expressed genes (DEGs) highlighting the inflammatory/immune genes that are most significantly downregulated in the inhibitor-treated cultures. E. Heatmap for selected inflammatory genes in the SGK1 inhibitor-treated and inhibitor-untreated glial cultures. Data represent the RC values (inside box) and log2 [SGK1 inhibitor-treated/control] (color intensity). F. Microglia and astrocyte reactivation-specific marker expressions in the RNA-seq data. Download figure Download PowerPoint In the RNA-seq data, the expression of all the M1-specific markers reported (Ka et al, 2014) was downregulated by treatment with the SGK1 inhibitor. The expression of the M2 markers Il10, Ccl22, Egf, Fizz1, and Il2ra was upregulated, while five of them were downregulated (Fig 2F), indicating SGK1 inhibition establishes its own microglial transcriptome signature mainly by inhibiting M1 marker transcription. Out of 13 pan-reactive astrocyte markers (Liddelow et al, 2017b), 10 were upregulated in the glial cultures treated with the inhibitor. However, there was no clear A1–A2 trend in the astrocyte-specific marker expression patterns, indicating that SGK1 inhibition induces a distinct glial reactivity profile, as also shown in other recent studies (Marschallinger et al, 2020; Smith et al, 2020; Zhou et al, 2020). NLRP3-inflammasome- and CGAS-STING-mediated inflammatory pathways in glia are suppressed by SGK1 inhibition Downregulated inflammatory genes in the RNA-seq data included the key components of the NLR family pyrin domain containing 3 (NLRP3) inflammasome signal (Fig 3A), which is required for the activation of IL1B and IL18 and thus regarded as the archetypical molecular driver of inflammatory response (reviewed in ref. Afonina et al, 2017). The decreased mRNA expression of Nlrp3, PYD and CARD domain containing (Pycard, also known as Asc) apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and procaspase-1 was validated in independent cultures treated with the SGK1 inhibitor, sh-Sgk1, and si-Sgk1 (Fig 3B), indicating that SGK1 signals are involved in NRLP3 inflammasome activation in glial cells. Note that the transcriptional control of NLRP3 inflammasome components is considered rate limiting in the activation of the inflammasome (Huai et al, 2014; Ising et al, 2019). To determine whether SGK1 inhibition could indeed prevent the activation of the glial NRLP3 inflammasome pathway, VM-derived glial cells cultured in the presence or absence of the SGK1 inhibitor (for 4 days) were primed with LPS (0.25 µg/ml, 3 h), and then followed by ATP treatment (2.5 mM, 30 min). In the control cultures, the sequential LPS-ATP treatment robustly induced the activation of caspase1 and the secretion of IL1B, as determined by the detection of active caspase1 (p10) and mature/activated IL1B protein levels in the control cell culture supernatants (Fig 3C). By contrast,
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