Synergistic neuroprotective effects of lithium and valproic acid or other histone deacetylase inhibitors in neurons: roles of glycogen synthase kinase-3 inhibition

Abstract

Lithium and valproic acid (VPA) are two primary drugs used to treat bipolar mood disorder and have frequently been used in combination to treat bipolar patients resistant to monotherapy with either drug. Lithium, a glycogen synthase kinase-3 (GSK-3) inhibitor, and VPA, a histone deacetylase (HDAC) inhibitor, have neuroprotective effects. The present study was undertaken to demonstrate synergistic neuroprotective effects when both drugs were coadministered. Pretreatment of aging cerebellar granule cells with lithium or VPA alone provided little or no neuroprotection against glutamate-induced cell death. However, copresence of both drugs resulted in complete blockade of glutamate excitotoxicity. Combined treatment with lithium and VPA potentiated serine phosphorylation of GSK-3 alpha and beta isoforms and inhibition of GSK-3 enzyme activity. Transfection with GSK-3alpha small interfering RNA (siRNA) and/or GSK-3beta siRNA mimicked the ability of lithium to induce synergistic protection with VPA. HDAC1 siRNA or other HDAC inhibitors (phenylbutyrate, sodium butyrate or trichostatin A) also caused synergistic neuroprotection together with lithium. Moreover, combination of lithium and HDAC inhibitors potentiated beta-catenin-dependent, Lef/Tcf-mediated transcriptional activity. An additive increase in GSK-3 serine phosphorylation was also observed in mice chronically treated with lithium and VPA. Together, for the first time, our results demonstrate synergistic neuroprotective effects of lithium and HDAC inhibitors and suggest that GSK-3 inhibition is a likely molecular target for the synergistic neuroprotection. Our results may have implications for the combined use of lithium and VPA in treating bipolar disorder. Additionally, combined use of both drugs may be warranted for clinical trials to treat glutamate-related neurodegenerative diseases.

Figures

Figure 1.

Neuroprotection of lithium or VPA in young and aging CGCs. A, CGCs were treated with indicated doses of LiCl (0.5–3 mm) for 6 d beginning from either 1 or 6 DIV and then exposed to 50 μm glutamate for 24 h. Cell viability was quantified by MTT assay and expressed as means ± SEM of percentage of vehicle-treated control from six independent cultures. B, The experimental conditions are as described in A except that the treatment drug was VPA (0.1–0.8 mm). C, CGCs were treated with LiCl (3 mm) or VPA (0.4 mm) for 6 d beginning from 1 DIV and then exposed to glutamate (Glut) in a broad concentration range (50–800 μm) for 24 h. Cell viability was quantified by MTT assay and expressed as means ± SEM of percentage of vehicle-treated control from four independent cultures. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the untreated control.

Figure 2.

Synergistic neuroprotective effects of lithium and VPA in aging CGC cultures. A, B, CGCs at 6 DIV were continuously treated with 0.8 mm VPA in the presence of 0.5–3.0 mm LiCl (A) or 3 mm LiCl in the presence of 0.2–1.0 mm VPA (B). Cells were stimulated with 50 μm glutamate at 12 DIV and assayed for cell viability by MTT assay 24 h later. Data are means ± SEM from five independent cultures. Note that maximal synergism was observed with 0.8 mm VPA and 3 mm LiCl. C, CGCs at 6 DIV were pretreated with LiCl (3 mm), VPA (0.8 mm), or in combination for 6 d before glutamate (Glut; 50 μm) treatment for 24 h. Data of LDH released into the medium are expressed as means ± SEM of percentage of total LDH for three independent cultures. ***p < 0.001. D, Combined lithium and VPA treatment blocked glutamate-induced p53 upregulation. CGCs at 6 DIV were pretreated with LiCl (3 mm), VPA (0.8 mm), or both for 6 d before glutamate (50 μm) treatment for 24 h. Cells were then harvested for Western blotting of p53. Shown are results from a typical experiment that was repeated three times. Arrows, Note that glutamate-induced p53 upregulation was unaffected by lithium or VPA alone but was blocked by their cotreatment.

Figure 3.

Morphological assessments of lithium–VPA synergism. A, Calcein-AM staining. CGCs at 6 DIV were pretreated with 3 mm LiCl, 0.8 mm VPA, or both in combination for 6 d before glutamate (Glut) exposure for 24 h. Cells were stained with calcein-AM and examined under an inverted fluorescence microscope. Scale bar, 30 μm. B, MTT staining. Experimental conditions are as described above, except that cells were stained with MTT. Photomicrographs were taken using an inverted light microscope. C, Hoechst dye nuclear staining. CGCs were treated as described above. After exposure to glutamate for 24 h, cells were fixed with 4% formaldehyde and then stained with Hoechst dye 33258 (5 μg/ml in PBS) for 5 min at 4°C. Photomicrographs were taken using an inverted UV illumination microscope. Scale bar, 10 μm.

Figure 4.

VPA potentiates lithium-induced serine phosphorylation of GSK-3α and β isoforms. CGCs at 6 DIV were treated with 3 mm LiCl and/or 0.8 mm VPA for 30 min (A), 24 h (B), or 72 h (C). Cells were harvested for Western blotting analysis of levels of phospho-GSK-3αSer21and phospho-GSK-3βSer9. β-Actin was used as a loading control. Typical Western blots are shown at the panels, and quantified results (means ± SEM of percentage of control from four independent experiments) are at the right. *p < 0.05, **p < 0.01, ***p < 0.001 between the indicated groups.

Figure 5.

Combined treatment with lithium and VPA potentiates inhibition of GSK-3 activity. CGCs at 6 DIV were treated with 3 mm LiCl, 0.8 mm VPA, or a combination of the two for 24 h. Cells were harvested for Western blotting of levels of p-TauSer400, p-TauThr205, total Tau, and GAPDH (as a loading control). Typical blots are shown in A, and quantified results (means ± SEM from three independent experiments) are in B. *p < 0.05, **p < 0.01 between the indicated groups. Note that p-TauSer400, p-TauThr205, and total Tau appeared as doublets in the Western blots, which likely reflects differential degrees of glycosylation (Liu et al., 2002). C, Cortical neurons at 6 DIV were treated with 3 mm LiCl, 0.8 mm VPA, or their combination for 24 h. Cells were harvested for Western blotting analysis of p-GSK-3αSer21, p-GSK-3βSer9, and p-TauSer400. D, Cell lysates prepared from CGCs at 7 DIV were immunoprecipitated with anti-GSK-3β antibody. The immunocomplex was then assayed for GSK-3β enzymatic activity in the absence or presence of LiCl (3 mm), VPA (0.8 mm), or a combination of both. The data are means ± SEM of percentage of control from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 between the indicated groups.

Figure 6.

Effects of lithium treatment on levels of phospho-GSK-3βSer9 and glutamate excitotoxicity in aging CGCs. CGCs at 3, 6, or 13 DIV were treated with vehicle or 3 mm LiCl for 30 min and then harvested for Western blotting of phospho-GSK-3βSer9, using β-actin as the loading control. Typical blots are shown in A, and quantified results (means ± SEM from 4 independent cultures) are shown in B. **p < 0.01 between the indicated groups. Note that basal and lithium-stimulated levels of phospho-GSK-3βSer9 were diminished with successive days in culture. Cells at 6 DIV were also treated with 1–9 mm LiCl for 30 min followed by determination of phospho-GSK-3βSer9 levels using β-actin as the loading control. Typical blots are shown in C, and quantified results (means ± SEM from four independent cultures) are shown in D. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the vehicle-treated control. E, For examining the effects of lithium on glutamate-induced excitotoxicity, cells at 6 DIV were treated with 1–9 mm LiCl for 6 d followed by exposure to 50 μm glutamate for 24 h. Cell viability was then determined by MTT assay and expressed as means ± SEM of percentage of vehicle-treated control from three independent cultures. *p < 0.05 compared with the vehicle-treated control. Note that the optimal concentration of lithium for inducing an increase in phospho-GSK-3βSer9 and reducing glutamate-induced cell death remained at 3 mm.

Figure 7.

Effects of rat GSK-3 isoform-specific siRNAs and GSK-3β plasmids on glutamate-induced excitotoxicity. Cells were transfected with 100 nm siRNA specific to either GSK-3α (siGSK-3α; αP1269) or GSK-3β (siGSK-3β; βP555) before plating by electroporation. Scrambled siRNA (siScramble) at a corresponding concentration was used as the transfection control. Western blotting revealed that protein levels of both GSK-3α and GSK-3β were markedly decreased 6 d after transfection with a mixture of siRNA for GSK-3α and siRNA for GSK-3β (siGSK-3α + β) (A). Transfected cells were also treated with 0.8 mm VPA or its vehicle from 6 to 12 DIV and then exposed to 50 μm glutamate (Glut) for 24 h (B). Cell viability was measured by MTT assay and expressed as means ± SEM of percentage of vehicle-treated control from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 between the indicated groups. Note that transfection with siGSK-3α and/or siGSK-3β was neuroprotective only in the VPA-treated groups. In another experiment (C), CGCs were transfected at the time of plating with a plasmid of wild-type GSK-3α (wt-GSK-3α), GSK-3α dominant-negative mutant (dn-GSK3α-pMT2-KR), wild-type GSK-3β (wt-GSK-3β), or GSK-3β dominant-negative mutant (dn-GSK-3β-K85R or dn-GSK-3β-R96A). Cells were then treated with a combination of LiCl (3 mm) and VPA (0.8 mm) or vehicle from 6 to 12 DIV, followed by 24 h exposure to glutamate (50 μm). Cell viability determined by MTT assay was expressed as means ± SEM from three independent experiments. Note that neuroprotection elicited by lithium and VPA was attenuated by wild-type GSK-3α or GSK-3β but not their dominant-negative mutants.

Figure 8.

HDAC1-specific siRNA and HDAC inhibitors PB and SB mimic VPA-induced synergy in neuroprotection. Cells were transfected with 100 nm siRNA specific to rat HDAC1 isoform (siHDAC1) before plating by electroporation. Scrambled siRNA (siScramble) was used as the transfection control. Western blotting revealed that protein levels of HDAC1 were attenuated 6 d after transfection with siHDAC1, whereas the levels of β-actin were unchanged (A). Transfected cells were also treated with 3 mm LiCl or its vehicle from 6 to 12 DIV and then exposed to 50 μm glutamate (Glut) for 24 h (B). Cell viability was measured by MTT assay and expressed as means ± SEM of percentage of vehicle-treated control from six independent cultures. Note that siHDAC1 was neuroprotective only in the group treated with lithium. CGCs at 6 DIV were pretreated for 6 d with 3 mm LiCl in the absence or presence of 1 mm PB (C) or 1 mm SB (D) before treatment with 50 μm glutamate for 24 h. Cell viability was quantified by MTT assay and expressed as means ± SEM of percentage of vehicle-treated control from four independent cultures. Cells at 6 DIV were also treated with lithium in conjunction with PB in (E) or SB (F) for 24 h and then harvested for Western blotting analysis of pGSK-3αSer21/βSer9 and β-actin (used as a loading control). G, Quantified results of GSK-3α and GSK-3β serine phosphorylation in CGCs treated with lithium and/or PB. H, Quantified results of GSK-3α and GSK-3β serine phosphorylation in CGCs treated with lithium and/or SB. Data are means ± SEM of percentage of vehicle control from three independent experiments.

Figure 9.

Combined treatment with TSA and lithium also induces synergy in neuroprotection and GSK-3 serine phosphorylation. A, CGCs at 6 DIV were pretreated with 3 mm LiCl and/or 20 nm TSA for 6 d and then exposed to 50 μm glutamate for 24 h. Cell viability was quantified by MTT assay and expressed as means ± SEM of percentage of vehicle-treated control from five independent cultures. B, C, CGCs at 6 DIV were treated with 3 mm LiCl and/or 20 nm TSA for 24 h and then harvested for Western blotting of GSK-3αSer21/βSer9 and β-actin (used as a loading control). Quantified results of GSK-3 serine phosphorylation are means ± SEM of percentage of vehicle control from three independent experiments.

Figure 10.

Combined treatment with LiCl and VPA potentiates Lef/Tcf-dependent transcriptional activity and nuclear β-catenin protein level in CGCs. A, CGCs were transfected with Lef-OT reporter construct before plating, using an Amaxa Nucleofector. At 6 DIV, CGCs were treated with 3 mm LiCl and/or the indicated concentrations of VPA for 24 h. Cells were then lysed for assay of Lef/Tcf-dependent luciferase activity. Data are means ± SEM of fold increase relative to the empty vector control from three independent experiments. B, CGCs at 6 DIV were treated with 3 mm LiCl, 0.8 mm VPA, or their combination for 24 h. Cells were then harvested, and nuclear proteins were prepared for Western blotting analysis of β-catenin. GAPDH was used as a loading control.

Figure 11.

Cotreatment with lithium and HDAC inhibitors induces synergy in Lef/Tcf-dependent transcriptional activation in HEK 293 cells. The OT cell line was derived from HEK 293 cells and stably transfected with a reporter containing three wild-type Lef binding sites regulating the luciferase expression. A, OT cells were incubated with the indicated concentrations of LiCl in the range of 2–20 mm for 24 h. B–D, OT cells were incubated with VPA, PB, or TSA at the indicated concentrations in the absence or presence of LiCl at doses shown in A. After 24 h incubation, cells were harvested for luciferase activity assay. Data are means ± SEM of fold increase relative to untreated control from three independent experiments.

Figure 12.

Effects of HDAC inhibitors in the absence or presence of lithium on histone acetylation. CGCs at 6 DIV were treated with 3 mm LiCl in the absence or presence of 0.8 mm VPA (A), 1 mm SB (B), 1 mm PB (C), or 20 nm TSA (D) for 24 h. Cells were harvested for Western blotting analysis of acetylated histone-H3 (AH3) using antibodies directed against both Lys9 and Lys14 acetylation or only Lys9 or Lys14 acetylation for A and B, and only Lys9 and Lys14 coacetylations were measured for C and D. β-Actin protein levels were used as the loading control.

Figure 13.

Effects of chronic treatment of mice with lithium and/or VPA on levels of phospho-GSK-3βSer9 in brains. CD-1 mice were treated with chow containing lithium carbonate (3 g/kg) and/or VPA (25 g/kg) for 20 d. Animals were killed, and brains were removed for Western blotting analyses of levels of p-GSK-3βSer9 and β-actin (used as the loading control) in the frontal cortex (A) and cerebellum (B). Each lane represents the result of an individual animal. C, D, Quantification of results of p-GSK-3βSer9 levels in the frontal cortex and cerebellum. Data are means ± SEM of six animals in each group. *p < 0.05, **p < 0.01, ***p < 0.001 between the indicated groups.