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. 2011 Oct;44(1):142-51.
doi: 10.1016/j.nbd.2011.06.016. Epub 2011 Jul 2.

Activated microglia decrease histone acetylation and Nrf2-inducible anti-oxidant defence in astrocytes: restoring effects of inhibitors of HDACs, p38 MAPK and GSK3β (VSports最新版本)

Fernando Correa  1 , Carina Mallard, Michael Nilsson, Mats Sandberg
Affiliations

Activated microglia decrease histone acetylation and Nrf2-inducible anti-oxidant defence in astrocytes: restoring effects of inhibitors of HDACs, p38 MAPK and GSK3β (VSports最新版本)

Fernando Correa et al. Neurobiol Dis. 2011 Oct.

Abstract

Histone deacetylase (HDAC) inhibitors have promising neuroprotective and anti-inflammatory properties although the exact mechanisms are unclear. We have earlier showed that factors from lipopolysaccharide (LPS)-activated microglia can down-regulate the astroglial nuclear factor-erythroid 2-related factor 2 (Nrf2)-inducible anti-oxidant defence. Here we have evaluated whether histone modification and activation of GSK3β are involved in these negative effects of microglia. Microglia were cultured for 24 h in serum-free culture medium to achieve microglia-conditioned medium from non-activated cells (MCM(0)) or activated with 10 ng/mL of LPS to produce MCM(10). Astrocyte-rich cultures treated with MCM(10) showed a time-dependent (0-72 h) increase in astroglial HDAC activity that correlated with lower levels of acetylation of histones H3 and H4 and decreased levels of the transcription factor Nrf2 and γ-glutamyl cysteine ligase modulatory subunit (γGCL-M) protein levels. The HDAC inhibitors valproic acid (VPA) and trichostatin-A (TSA) elevated the histone acetylation levels, restored the Nrf2-inducible anti-oxidant defence and conferred protection from oxidative stress-induced (H(2)O(2)) death in astrocyte-rich cultures exposed to MCM(10) VSports手机版. Inhibitors of GSK3β (lithium) and p38 MAPK (SB203580) signaling pathways restored the depressed histone acetylation and Nrf2-related transcription whereas an inhibitor of Akt (Ly294002) caused a further decrease in Nrf2-related transcription. In conclusion, the study shows that well tolerated drugs such as VPA and lithium can restore an inflammatory induced depression in the Nrf2-inducible antioxidant defence, possibly via normalised histone acetylation levels. .

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Fig. 1
Fig. 1
(A) Histone deacetylase (HDAC) activity in astrocyte-rich cultures exposed to control conditions (see Material and methods for details), conditioned-medium from unstimulated microglia (MCM0) or conditioned-medium from 10 ng/mL LPS-stimulated microglia (MCM10). HDAC activity started to increase already after 24 h and was elevated 72 h in cells treated with MCM10. HDAC activity remained elevated at 72 h post-treatment whereas in the case of MCM0 and control conditions there were no changes. Results are mean ± SEM. Statistics: *p<0.05 vs same time-point control. (B) Exposure of astrocyte-rich cultures to MCM10 for 24 h induced a decrease in the acetylation levels of histone H3 (acetyl-H3) with a concomitant increase in the methylation levels of this histone (tri-methyl-H3) when compared to control conditions. No changes in the acetylation levels of histone H4 (acetyl-H4) were observed. A representative blot is shown. (C) Densitometric analysis of acetyl-H4, acetyl-H3 and tri-methyl-H3 protein expression in astrocyte-rich cultures exposed for 24 h to control conditions or MCM10. Data are plotted as ratio of the protein of interest/tubulin obtained in each condition. Statistics: *p<0.05 vs control; ***p<0.005 vs control. (D) Exposure of astrocyte-rich cultures to MCM10 for 72 h induced a decrease in the acetylation levels of histone H3 (acetyl-H3) and histone H4 (acetyl-H4) followed by an increase in the methylation levels of this histone (tri-methyl-H3) when compared to control conditions. A representative blot is shown. (E) Densitometric analysis of acetyl-H4, acetyl-H3 and tri-methyl-H3 protein expression in astrocyte-rich cultures exposed for 72 h to control conditions or MCM10. Data are plotted as ratio of the protein of interest/tubulin obtained in each condition. Statistics: *p<0.05 vs control; **p<0.01 vs control; ***p<0.005 vs control.
Fig. 2
Fig. 2
(A) Treatment with the HDAC inhibitor valproic acid (VPA; 1 mM) for 24 h induced an increase in the acetylation levels of histones H3 and H4 in astrocyte-rich cultures. A representative blot is shown. (B) Densitometric analysis of acetyl-H4 and acetyl-H3 protein expression in astrocyte-rich cultures exposed for 24 h to control conditions or VPA 1 mM. Data are plotted as ratio of the protein of interest/tubulin obtained in each condition. Statistics: *p<0.05 vs control; ***p<0.005 vs control. (C) Treatment with VPA (1 mM) for 24 h restored the levels of Nrf2 and γGCL-M that were down-regulated by the exposure to MCM10. A representative blot is shown. (D) Densitometric analysis of Nrf2 and γGCL-M protein expression in astrocyte-rich cultures exposed for 24 h to control conditions or MCM10 in the presence or absence of VPA (1 mM). Data are plotted as ratio of the protein of interest/tubulin obtained in each condition. Statistics: **p<0.01 vs control; #p<0.05 vs MCM10; ##p<0.01 vs MCM10. (E) Treatment with the HDAC inhibitor trichostatin-A (TSA; 10 nM) for 24 h induced an increase in the acetylation levels of histone H3 and histone H4. A representative blot is shown. (F) Densitometric analysis of acetyl-H4 and acetyl-H3 protein expression in astrocyte-rich cultures exposed for 24 h to control conditions or TSA (10 nM). Data are plotted as ratio of the protein of interest/tubulin obtained in each condition. Statistics: *p<0.05 vs control. (G) Treatment with TSA (10 nM) for 24 h restored the levels of Nrf2 and γGCL-M that were down-regulated by the exposure to MCM10. A representative blot is shown. (H) Densitometric analysis of Nrf2 and γGCL-M protein expression in astrocyte-rich cultures exposed for 24 h to control conditions or MCM10 in the presence or absence of TSA (10 nM). Data are plotted as ratio of the protein of interest/tubulin obtained in each condition. Statistics: *p<0.05 vs control; ##p<0.01 vs MCM10.
Fig. 3
Fig. 3
(A) Cells were exposed for 24 h to control conditions or MCM10 in the presence or absence of VPA (1 mM). Twenty-four hours later, cells were exposed to hydrogen peroxide (250 µM) for 3 h and cell death was assessed by measuring the LDH released into the medium. Exposure to MCM10 rendered astrocyte-rich cultures more susceptible to oxidative stress induced by hydrogen peroxide showing higher levels of cell loss. This effect was reversed by the treatment with VPA (1 mM). The results shown are the mean ± SEM. Statistics: **p<0.01 vs control; ###p<0.005 vs MCM10. (B) Cells were exposed for 24 h to control conditions or MCM10 in the presence or absence of TSA (10 nM). Twenty-four hours later, cells were exposed to hydrogen peroxide (250 µM) for 3 h and cell death was assessed by measuring the LDH released into the bathing medium. Exposure to MCM10 rendered astrocyte-rich cultures more susceptible to oxidative stress induced by hydrogen peroxide showing higher levels of cell loss. This effect was reversed by the treatment with TSA 10 nM. The results shown are the mean ± SEM. Statistics: *p<0.05 vs control; ###p<0.005 vs MCM10.
Fig. 4
Fig. 4
(A) Treatment with the p38 MAPK inhibitor SB203580 (20 µM) for 24 h reversed the negative effects of MCM10 on the acetylation levels of histone H3. No changes were observed in the acetylation levels of histone H4. A representative blot is shown. (B) Densitometric analysis of acetyl-H4 and acetyl-H3 protein expression in astrocyte-rich cultures exposed for 24 h to control conditions or MCM10 in the presence or absence of SB203580 (20 µM). Data are plotted as ratio of the protein of interest/tubulin obtained in each condition. Statistics: **p<0.01 vs control; ##p<0.01 vs MCM10. (C) Treatment with the GSK3β inhibitor lithium chloride (LiCl; 5 mM) for 24 h reversed the effects of MCM10 on the acetylation levels of histone H3. No changes were observed in the acetylation levels of histone H4. A representative blot is shown. (B) Densitometric analysis of acetyl-H4 and acetyl-H3 protein expression in astrocyte-rich cultures exposed for 24 h to control conditions or MCM10 in the presence or absence of LiCl (5 mM). Data are plotted as ratio of the protein of interest/tubulin obtained in each condition. Statistics: ***p<0.005 vs control; ###p<0.005 vs MCM10.
Fig. 5
Fig. 5
(A) Astrocyte-rich cultures were transiently transfected for 24 h with the ARE reporter gene vector along with a Renilla luciferase expression vector. Transiently transfected cells were treated for 24 h with MCM10 in the presence or absence of the Akt inhibitor Ly294002 (10 µM). MCM10 induced a reduced ARE-related transcription activity, expressed by a lower luciferase activity. This effect was enhanced when the Akt signalling pathway was inhibited. Statistics: **p<0.01 vs control; ***p<0.005 vs control. (B) Transiently transfected cells were treated for 24 h with MCM10 in the presence or absence of the GSK3β inhibitor LiCl (5 mM). Inhibition of GSK3β signalling pathway was able to fully reverse the effects of MCM10 on the ARE-related transcription activity. Statistics: *p<0.05 vs control; ###p<0.005 vs MCM10. (C) Transiently transfected cells were treated for 24 h with MCM10 in the presence or absence of the p38 MAPK inhibitor SB203580 (20 µM). Inhibition of p38 MAPK signalling pathway fully reversed the effects of MCM10 on the ARE promoter activity. Statistics: *p<0.05 vs control; ###p<0.005 vs MCM10. (D) Transiently transfected cells were treated for 24 h with MCM10 in the presence or absence of the GSK3β inhibitor LiCl (5 mM), the p38 MAPK inhibitor SB203580 (20 µM) or the two inhibitors combined. Inhibition of both GSK3β and p38 MAPK signalling pathways had additive effects and was able to fully reverse the effects of MCM10 on the ARE promoter activity. Statistics: *p<0.05 vs control; ###p<0.005 vs MCM10; +++p<0.05 vs MCM10 + SB203580 or MCM10 + LiCl.
Fig. 6
Fig. 6
(A) Treatment with the HDAC inhibitor valproic acid (VPA; 1 mM) for 72 h induced an increase in the acetylation levels of histones H3 and H4. A representative blot is shown. (B) Densitometric analysis of acetyl-H4 and acetyl-H3 protein expression in astrocyte-rich cultures exposed for 72 h to control conditions or VPA (1 mM). Data are plotted as ratio of the protein of interest/tubulin obtained in each condition. Statistics: *p<0.05 vs control; ***p<0.005 vs control. (C) Treatment with VPA (1 mM) for 72 h restored the levels of Nrf2 and γGCL-M that were down-regulated by the exposure to MCM10. A representative blot is shown. (D) Densitometric analysis of Nrf2 and γGCL-M protein expression in astrocyte-rich cultures exposed for 72 h to control conditions or MCM10 in the presence or absence of VPA (1 mM). Data are plotted as ratio of the protein of interest/tubulin obtained in each condition. Statistics: *p<0.05 vs control; ***p<0.005 vs control; #p<0.05 vs MCM10; ##p<0.01 vs MCM10. (E) Treatment with the HDAC inhibitor trichostatin-A (TSA; 10 nM) for 72 h increased the acetylation levels of histone H3 and histone H4. A representative blot is shown. (F) Densitometric analysis of Acetyl-H4 and Acetyl-H3 protein expression in astrocyte-rich cultures exposed for 72 h to control conditions or TSA (10 nM). Data are plotted as ratio of the protein of interest/tubulin obtained in each condition. Statistics: *p<0.05 vs control. (G) Treatment with TSA (10 nM) for 72 h restored the levels of Nrf2 and γGCL-M that were down-regulated by the exposure to MCM10. A representative blot is shown. (H) Densitometric analysis of Nrf2 and γGCL-M protein expression in astrocyte-rich cultures exposed for 72 h to control conditions or MCM10 in the presence or absence of TSA (10 nM). Data are plotted as ratio of the protein of interest/tubulin obtained in each condition. Statistics: *p<0.05 vs control; **p<0.01 vs control; #p<0.05 vs MCM10; ##p<0.01 vs MCM10.
Fig. 7
Fig. 7
(A) Cells were exposed for 72 h to control conditions or MCM10 in the presence or absence of VPA (1 mM). Next, cells were exposed to hydrogen peroxide (250 µM) for 3 h and cell death was assessed by measuring the LDH released into the bathing medium. Long-term exposure to MCM10 rendered astrocyte-rich cultures more susceptible to oxidative stress induced by hydrogen peroxide, showing higher levels of cell loss. This effect was partially reversed by the treatment with VPA (1 mM). The results shown are the mean ± SEM. Statistics: ***p<0.005 vs control; ###p<0.005 vs MCM10. (B) Cells were exposed for 72 h to control conditions or MCM10 in the presence or absence of TSA (10 nM). Next, cells were exposed to hydrogen peroxide (250 µM) for 3 h and cell death was assessed by measuring the LDH released into the bathing medium. Prolonged exposure to MCM10 rendered astrocyte-rich cultures more susceptible to oxidative stress induced by hydrogen peroxide, showing higher levels of cell loss. This effect was partially reversed by the treatment with TSA (10 nM). The results shown are the mean ± SEM. Statistics: ***p<0.005 vs control; ###p<0.005 vs MCM10.
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