Ogt-mediated O-GlcNAcylation inhibits astrocytes activation through modulating NF-κB signaling pathway

Abstract

Previous studies have shown that Ogt-mediated O-GlcNAcylation is essential for neuronal development and function. However, the function of O-GlcNAc transferase (Ogt) and O-GlcNAcylation in astrocytes remains largely unknown. Here we show that Ogt deficiency induces inflammatory activation of astrocytes in vivo and in vitro, and impairs cognitive function of mice. The restoration of O-GlcNAcylation via GlcNAc supplementation inhibits the activation of astrocytes, inflammation and improves the impaired cognitive function of Ogt deficient mice. Mechanistically, Ogt interacts with NF-κB p65 and catalyzes the O-GlcNAcylation of NF-κB p65 in astrocytes. Ogt deficiency induces the activation of NF-κB signaling pathway by promoting Gsk3β binding. Moreover, Ogt depletion induces the activation of astrocytes derived from human induced pluripotent stem cells. The restoration of O-GlcNAcylation inhibits the activation of astrocytes, inflammation and reduces Aβ plaque of AD mice in vitro and in vivo. Collectively, our study reveals a critical function of Ogt-mediated O-GlcNAcylation in astrocytes through regulating NF-κB signaling pathway.

Fig. 1

Ogt deficiency leads to activation of astrocytes in vitro and in vivo. a, b Representative images of GFAP immunostaining (a) and quantification results showed that Ogt deficiency increased the average area of adult astrocytes (b). Scale bar, 50 μm. n = 10 astrocytes were picked up per animal and 30 cells in total were analyzed per group. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; unpaired Student’s t-test. c, d Western blot (WB) assay (c) and quantification results (d) showed that the protein level of GFAP significantly increased in cKO astrocytes compared with Ctrl astrocytes. Astrocytes isolated from 2 to 3 adult mice were pooled together and regarded as n = 1 in the present study. n = 3 independent experiments. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; unpaired Student’s t-test. All the original images of western blot assays can be found in Additional file 10: Fig. S10. e–g qRT-PCR results show that mRNA levels of pan reactive (e) and A1 specific (f) markers increased in Ogt deficient adult astrocytes, and A2 specific markers decreased (g). n = 3 independent experiments. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; unpaired Student’s t-test. h Representative images of GFAP and Iba1 immunostaining. Scale bar, 100 μm. The right panels showed the higher magnification of the area marked with white frame in left panel images. Scale bar (right panels, 20 μm). i–l Quantification results showed that the level of GFAP fluorescence intensity (i) and the number of GFAP⁺ cells (j) significantly increased in the hippocampus region of cKO mice compared to Ctrl mice. n = 4 mice per genotype. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; unpaired Student’s t-test. k–n three-dimension (3D) analysis of astrocytic morphology showed the increase of the total length (k), the number of processes (l), the process volume (m) and total process area (n) of astrocytes from the hippocampus of adult cKO mice compared with Ctrl mice. n = 10 astrocytes were picked up per animal and 40 cells in total were analyzed per group. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; unpaired Student’s t-test. o 3D Sholl analysis showed the increased process arbor complexity of astrocytes in the hippocampus of cKO mice compared with Ctrl mice. n = 10 astrocytes were picked up per animal and 40 cells in total were analyzed per group. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; two-way ANOVA followed by Sidak’s multiple comparisons test, F(_{1, 5304)} = 2258. p qRT-PCR results showed that mRNA level of GFAP increased in the hippocampal tissues of cKO mice compared to Ctrl mice. n = 5 mice per genotype. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; unpaired Student’s t-test. q–s WB assay (q) and quantification results showed that the protein level of GFAP (r) increased in the hippocampal tissues of cKO mice compared to Ctrl mice, but the level of Iba1 (s) showed no difference between Ctrl and cKO mice. n = 6 mice per genotype. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; unpaired Student’s t-test

Fig. 2

Ogt deficiency induces inflammatory response of astrocytes. a Representative images of GFAP and IL-1β immunostaining with cultured adult Ctrl and cKO astrocytes in vitro. Scale bar, 50 μm. b, c qRT-PCR results showed that mRNA levels of IL-1β (b) and TNF-α (c) increased in cKO astrocytes. n = 3 independent experiments. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; unpaired Student’s t-test. d–f WB assay (d) and quantification results showed that protein levels of IL-1β (e) and TNF-α (f) significantly increased in adult cKO astrocytes in vitro. n = 3 independent experiments. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; unpaired Student’s t-test. g Representative images of GFAP and IL-1β immunostaining in the hippocampus of brains of Ctrl and cKO mice. Scale bar, 20 μm. h, i qRT-PCR results showed that mRNA levels of IL-1β (h) and TNF-α (i) increased in the hippocampal tissues of cKO mice compared to Ctrl mice. n = 5 mice per genotype. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; unpaired Student’s t-test. j–l WB assay (j) and quantification results showed that the IL-1β (k) and TNF-α (l) protein levels significantly increased in the hippocampal tissues of cKO mice compared to Ctrl mice. n = 5 mice per genotype. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; unpaired Student’s t-test. m, n ELISA assay results show that the levels of IL-1β (m) and TNF-α (n) increased in the hippocampal tissue supernatants of cKO mice compared to Ctrl mice. n = 6 mice per genotype. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; unpaired Student’s t-test. o–q WB assay (o) and quantification results showed that the treatment with Ogt inhibitor (Ogt-I) OSMI-4 reduced the level of O-GlcNAcylation in adult astrocytes (p), but increased the level of GFAP (q) compared to Ctrl group. n = 3 independent experiments. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; unpaired Student’s t-test. r Representative images of GFAP and IL-1β immunostaining of adult astrocytes treated with PBS (Ctrl) and Ogt-I (20 μM for 72 h), respectively. Scale bar, 50 μm. s–u WB assay (s) and quantification results show that the treatment with Ogt inhibitor (Ogt-I) OSMI-4 significantly increased the levels of IL-1β (t) and TNF-α (u) of adult astrocytes in vitro. n = 3 independent experiments. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; unpaired Student’s t-test

Fig. 3

Ogt deficient astrocytes impair hippocampal neurons and mice cognition. a Representative images of neurons with Golgi staining in CA1 region of Ctrl and cKO mice. Scale bar, 10 μm. b Representative images of Golgi-stained dendritic spines of second-order segment in the CA1 region of Ctrl and cKO mice. Scale bar, 1 μm. c–e Sholl analysis showed the overall decrease in the number of dendritic intersections per radius (c) and the number of dendrites per cell (d) and total length of dendrites (e) in the Golgi-stained neurons from the CA1 region of cKO mice compared with Ctrl mice. n = 15 neurons from 3 mice per group were analyzed. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; two-way ANOVA analysis followed by Sidak’s multiple-comparison test for c, F_{(1, 1400)} = 153.7; unpaired Student’s t-test for d, e. f Quantification results show the decreased dendritic spine density of cKO mice compared to that of Ctrl mice. Spine density was calculated by dividing the number of spines with the length of dendrite. n = 24 neurons from 3 mice per group were analyzed. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; unpaired Student’s t-test. g The escape latency during the training period of Ctrl and cKO adult mice. cKO mice required longer time to reach the platform starting from the first day of the 4-day training period. Ctrl/cKO, n = 10 mice. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; two-way ANOVA analysis followed by Sidak’s multiple-comparison test, F_{(3, 72)} = 23.06. h–j During the probe test, cKO mice required longer time to reach the platform (h), decreased time in target quadrant (i), and decreased numbers of crossing the platform (j). Ctrl mice, n = 10; cKO mice, n = 10. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; unpaired Student’s t-test for h, j and two-way ANOVA analysis followed by Sidak’s multiple-comparison test for i, F_{(3, 72)} = 30.62. k The percentage of spontaneous alternations in cKO mice was lower than that in Ctrl mice during the Y maze spontaneous alternation task test. Ctrl, n = 8 mice; cKO, n = 8 mice. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; unpaired Student’s t-test. l Representative heatmap shows the distribution of exploring time of Ctrl and cKO mice during the testing trial in Y maze spatial novelty preference test. m–o cKO mice showed the decreased percentage of exploring time (m), the number of entries (n) and distance (o) in the novel arm compared to those in Ctrl mice during the Y maze spatial novelty preference test. n = 8 mice for Ctrl/cKO group. Values represent mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001; two-way ANOVA analysis followed by Sidak’s multiple-comparison test, F_{(2, 42)} = 67.41 for m, F_{(2, 42)} = 15.92 for n, F_{(2, 42)} = 10.84 for o. p In the passive avoidance task test, cKO mice displayed shorter latency to enter the dark compartment than that of Ctrl mice during the retention test while Ctrl and cKO mice showed no difference of the latency during the training session. n = 8 mice for Ctrl/cKO group. Values represent mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001; two-way ANOVA analysis followed by Sidak’s multiple-comparison test, F_{(1, 28)} = 456.2

Fig. 4

Restoration of O-GlcNAcylation inhibits reactivation and inflammation of cKO astrocytes in vitro and in vivo. a ELISA assay results showed that the level of UDP-GlcNAc significantly decreased in the supernatants of cKO astrocytes compared with Ctrl astrocytes, and the GlcNAc replenishment significantly increased UDP-GlcNAc level of cKO astrocytes. n = 4 independent experiments. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; One-way ANOVA analysis followed by Tukey’s multiple-comparison test, F_{(2, 9)} = 53.04. b Representative images of GFAP and IL-1β immunostaining with adult Ctrl and cKO astrocytes treated with PBS (Ctrl and cKO) and O-GlcNAcylation substrate GlcNAc (20 μM, cKO + GlcNAc) for 72 h, respectively. Scale bar, 50 μm. c–g WB assay (c) and quantification results showed that GlcNAc supplementation significantly restored the decreased level of O-GlcNAcylation (d), and reduced the levels of GFAP (e), IL-1β (f) and TNF-α (g) of cKO astrocytes in vitro. n = 3 independent experiments. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA analysis followed by Tukey’s multiple-comparison test, F_{(2, 6)} = 59.32 for d, F_{(2, 6)} = 69.6 for e, F_{(2, 6)} = 480.6 for f, F_{(2, 6)} = 788.7 for g. h–j ELISA result showed that GlcNAc supplementation significantly increased the level of UDP-GlcNAc (h), but reduced the levels of of IL-1β (i) and TNF-α (j) in the supernatants of adult cKO astrocytes. n = 3 independent experiments. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA analysis followed by Tukey’s multiple-comparison test, F_{(3, 8)} = 15.49 for h, F_{(3, 8)} = 19.37 for i, F_{(3, 8)} = 19.08 for j. k Representative images of GFAP and O-GlcNAcylation immunostaining with the brain sections of adult Ctrl and cKO mice treated with saline (Ctrl + saline, cKO + saline) and GlcNAc (Ctrl + GlcNAc, cKO + GlcNAc), respectively. Scale bar, 50 μm. l Quantification results of O-GlcNAcylation immunostaining in k showed that GlcNAc supplementation significantly increased the immunostaining intensity of O-GlcNAcylation in the brain of cKO mice. n = 4 mice per group. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA analysis followed by Tukey’s multiple-comparison test, F_{(3, 12)} = 36.08. m–p WB assay (m) and quantification results showed that GlcNAc supplementation significantly reduced the protein levels of GFAP (n), IL-1β (o) and TNF-α (p) in the hippocampal tissues of cKO mice compared with saline-treated cKO mice. n = 3 per group. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA analysis followed by Tukey’s multiple-comparison test, F_{(3, 8)} = 65.8 for n, F_{(3, 8)} = 65.61 for o, F_{(3, 8)} = 17.5 for p

Fig. 5

Restoration of O-GlcNAcylation improves behavioral deficits of Ogt deficient mice. a Representative images of the explored path (upper) and spent time (lower, heatmap) of novel object recognition task test of Ctrl and cKO mice treated with saline (Ctrl + saline, cKO + saline) and GlcNAc (Ctrl + GlcNAc, cKO + GlcNAc) during the testing trial, respectively. Heatmap image of each animal was shown in Additional file 5: Fig. S5e. b The time (seconds) spending in exploring the novel and old object of Ctrl and cKO adult mice treated with saline and GlcNAc during the testing trial. Compared to Ctrl mice, cKO mice spent less time exploring the novel object. cKO mice treated with GlcNAc displayed increased time exploring the novel object compared to cKO mice treated with saline during the testing trial. n = 6 mice per group. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; two-way ANOVA analysis followed by Sidak’s multiple-comparison test, F_{(3, 40)} = 4.342. c Y maze spontaneous alternation task test results showed that cKO mice treated with GlcNAc displayed an increased percentage of spontaneous alternations compared to cKO mice treated with saline. n = 6 mice per group. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA analysis followed by Tukey’s multiple-comparison test, F_{(3, 20)} = 14.94. d Representative travelled path and heatmap images showing the exploring path (upper) and spent time (lower, heat map) of Ctrl and cKO mice treated with saline and GlcNAc during Y maze spatial novelty preference test. Heatmap image for each animal was shown in supplemental Fig. 6c. e–g cKO mice treated with GlcNAc showed a higher percentage of exploring time (e), increased number of entries (f) and distance (g) in the novel arm compared to those of cKO mice treated with saline during Y maze spatial novelty preference test. n = 6 mice per group. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; two-way ANOVA analysis followed by Sidak’s multiple-comparison test, F_{(6, 60)} = 11.13 for e, F_{(6, 60)} = 6.752 for f, F_{(6, 60)} = 4.623 for g. h The escape latency of Ctrl and cKO mice treated with saline and GlcNAc during the training period in Morris water maze test. cKO mice with the GlcNAc administration showed the decreased time to reach the platform of during 4-day training period compared to cKO mice treated with saline. n = 6 mice per group. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; two-way ANOVA analysis followed by Sidak’s multiple-comparison test, F_{(3, 80)} = 14.31. i–k cKO mice treated with GlcNAc exhibited the reduced latency (i), increased time in target quadrant (j) and increased numbers of crossing the platform (k) compared to saline-treated cKO mice during the probe trial of Morris water maze test. n = 6 mice per group. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA analysis followed by Tukey’s multiple-comparison test, F_{(3, 20)} = 4.668 for i; F_{(3, 20)} = 3.493 for k; two-way ANOVA analysis followed by Sidak’s multiple-comparison test for j, F_{(3, 40)} = 7.787

Fig. 6

Ogt catalyzes O-GlcNAcylation of NF-κB on S384 and inhibits the activation of NF-κB signaling pathway. a, b Reciprocal IP-WB analysis showed that Ogt readily precipitated p65 (a), and vice versa in astrocytes (b). c, d Reciprocal IP-WB analysis showed that O-GlcNAcylation precipitated p65 (c), and vice versa in astrocytes (d). e Click-iT reaction results revealed that p65 was modified with O-GlcNAcylation. f–h WB assay showed that under Ogt knock down (KD) significantly decreased the levels of Ogt and O-GlcNAcylation, but significantly increased the level of p-p65 in N2a cells (f, Input part). IP with HA (HA-p65) antibody followed by WB assays (f, IP part) showed that O-GlcNAcylation levels of exogenous p65 (g) were significantly decreased, and the level of p-p65 (h) was significantly increased under Ogt KD condition. n = 3 independent experiments. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; unpaired Student’s t-test. i–k WB assay showed that ectopic expression of Ogt (Ogt-Flag, OE-Ogt) and significantly increased the levels of Ogt and O-GlcNAcylation without affecting the levels of total p65 and p-p65 in N2a cells (i, Input part). IP with HA (HA-p65) antibody followed by WB assays (i) showed that the O-GlcNAcylation level of p65 (p65-HA) (j, IP) significantly increased, but the level of p-p65 (k) significantly decreased. n = 3 independent experiments. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; unpaired Student’s t-test. l–n IP followed by WB assays showed that Ogt KD promoted the binding of Gsk3β to p-65. n = 3 independent experiments. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; unpaired Student’s t-test. o–q IP followed by WB assays showed that mutation of S384A significantly increased the binding of Gsk3β to p-65 compared to other O-GlcNAcylation sites. n = 3 independent experiments. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA analysis followed by Tukey’s multiple-comparison test, F_{(4, 10)} = 47.49 for p, F_{(4, 10)} = 42.31 for q

Fig. 7

Ogt depletion reduces the level of O-GlcNAcylation and induces activation of astrocytes derived from iPSCs. a qRT-PCR results showed that Ogt depletion significantly reduced the level of Ogt in astrocytes derived from iPSCs. n = 3 independent experiments. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; unpaired Student’s t-test. b–d qRT-PCR results showed that Ogt depletion significantly enhanced the level of Gfap (b), IL-1β (c) and TNF-α (d) in astrocytes derived from iPSCs. n = 3 independent experiments. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; unpaired Student’s t-test. e–g WB assay (e) and quantification results showed that Ogt depletion significantly enhanced the level of Ogt (f) and O-GlcNAcylation (g) in astrocytes derived from iPSCs. n = 3 independent experiments. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; unpaired Student’s t-test. h–j WB assay (h) and quantification results showed that Ogt depletion significantly enhanced the levels of of IL-1β (i) and TNF-α (j) in astrocytes derived from iPSCs. n = 3 independent experiments. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; unpaired Student’s t-test. k–n WB assay and quantification results showed that Ogt depletion did not affect the level of cytoplasmic p65 in astrocytes derived from iPSCs (k, l), but significantly increased the level of nuclear p-p65 (m, n). n = 4 independent experiments. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; unpaired Student’s t-test

Fig. 8

Restoration of O-GlcNAcylation inhibits astrocytes reactivation of AD model mice. a–d Immunostaining (a) and quantification results showed that GlcNAc administration significantly reduced GFAP immunostaining intensity (b), Aβ plaque area (c) and the number of GFAP⁺Aβ⁺ cells (d) in the hippocampus region of AD mice compared to Ctrl mice, respectively. 4–5 sections were picked up per animal and n = 5 mice per group. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA analysis followed by Tukey’s multiple-comparison test, F_{(2, 12)} = 8.772 for b, F_{(2, 12)} = 75.82 for c and F_{(2, 12)} = 172.7 for d. e ELISA assay results show that the GlcNAc supplementation increased the levels of UDP-GlcNAc in the supernatants of hippocampal tissues of GlcNAc-treated AD mice compared with saline-treated AD mice. n = 4 mice per group. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA analysis followed by Tukey’s multiple-comparison test, F_{(2, 9)} = 16.13. f–j WB assay (f) and quantification results showed that GlcNAc supplementation significantly restored the level of O-GlcNAcylation (g), and decreased the levels of GFAP (h), IL-1β (i) and TNF-α (j) of AD astrocytes compared to PBS-treated AD cells. n = 3 independent experiments. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA analysis followed by Tukey’s multiple-comparison test, F_{(2, 6)} = 17.08 for (g), F_{(2, 6)} = 24.8 for h, F_{(2, 6)} = 108.5 for i, F_{(2, 6)} = 102.3 for j. k–m ELISA assay results showed that the GlcNAc supplementation increased the level of UDP-GlcNAc (k), and reduced the levels of IL-1β (l) and TNF-α (m) in the supernatants of cultured AD astrocytes compared with PBS-treated AD astrocytes. n = 4 independent experiments for k and n = 3 independent experiments for l, m. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA analysis followed by Tukey’s multiple-comparison test, F_{(2, 9)} = 12.46 for k, F_{(2, 6)} = 100.2 for l, F_{(2, 6)} = 327.7 for m. n Representative images of GFAP and p65 immunostaining with Ctrl and AD adult astrocytes treated with PBS (Ctrl and AD) and GlcNAc (20 μM, AD + GlcNAc) for 72 h, respectively. Scale bar, 50 μm. o Representative images of GFAP and p-p65 immunostaining with Ctrl and AD adult astrocytes treated with PBS (Ctrl and AD) and GlcNAc (20 μM, AD + GlcNAc) for 72 h, respectively. Scale bar, 50 μm. p–r WB assay (p) and quantification results showed that the GlcNAc supplementation did not affect the level of total p65 (q), but significantly increased the protein level of p-p65 (r) in AD astrocytes. n = 3 independent experiments. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA analysis followed by Tukey’s multiple-comparison test, F_{(2, 6)} = 0.6736 for q, F_{(2, 6)} = 100.1 for r. s–u WB assay (s) and quantification results showed that GlcNAc supplementation significantly restored the level of cytoplasmic p65 (t) and reduced the level of p-p65 in nucleus (u). n = 3 independent experiments. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA analysis followed by Tukey’s multiple-comparison test, F_{(2, 6)} = 23.04 for t, F_{(2, 6)} = 63.16 for u