Valproic acid and other histone deacetylase inhibitors induce microglial apoptosis and attenuate lipopolysaccharide-induced dopaminergic neurotoxicity

Abstract

Valproic acid (VPA), a widely prescribed drug for seizures and bipolar disorder, has been shown to be an inhibitor of histone deacetylase (HDAC). Our previous study has demonstrated that VPA pretreatment reduces lipopolysaccharide (LPS)-induced dopaminergic (DA) neurotoxicity through the inhibition of microglia over-activation. The aim of this study was to determine the mechanism underlying VPA-induced attenuation of microglia over-activation using rodent primary neuron/glia or enriched glia cultures. Other histone deacetylase inhibitors (HDACIs) were compared with VPA for their effects on microglial activity. We found that VPA induced apoptosis of microglia cells in a time- and concentration-dependent manner. VPA-treated microglial cells showed typical apoptotic hallmarks including phosphatidylserine externalization, chromatin condensation and DNA fragmentation. Further studies revealed that trichostatin A (TSA) and sodium butyrate (SB), two structurally dissimilar HDACIs, also induced microglial apoptosis. The apoptosis of microglia was accompanied by the disruption of mitochondrial membrane potential and the enhancement of acetylation levels of the histone H3 protein. Moreover, pretreatment with SB or TSA caused a robust decrease in LPS-induced pro-inflammatory responses and protected DA neurons from damage in mesencephalic neuron-glia cultures. Taken together, our results shed light on a novel mechanism whereby HDACIs induce neuroprotection and underscore the potential utility of HDACIs in preventing inflammation-related neurodegenerative disorders such as Parkinson's disease.

Figures

Fig. 1

VPA treatment triggers apoptosis in primary rat microglial cultures. Enriched rat microglial cells were treated with various concentrations of VPA for 24 h. A: Cell viability was determined by the MTT analysis. The percentages of viable cells from triplicate determinations are shown as mean ± SEM compared with vehicle control, B: Externalization of PS in microglia exposed to various concentrations of VPA for 12 h or 24 h, as determined using PE-conjugated annexin V followed by flow cytometry analysis. The percentages of apoptotic cells from five independent experiments are shown as mean ± SEM. # p< 0.05, * p<0.001 compared with vehicle control.

Fig. 2

VPA induces DNA fragmentation and morphological changes in microglia-enriched culture. A: DNA fragmentation in cells treated with 0.6 or 1.2 mM of VPA for 24 h was analyzed by agarose gel electrophoresis. The left lane (M) represents the molecular weight marker (100 bp). B: Enriched rat microglial cells treated with vehicle or 0.6 mM VPA for 24 h. The cell morphology was observed using light microscope.

Fig. 3

VPA induces DNA damage in microglia-enriched culture: A TUNEL study. The VPA-treated microglia were fixed with 4% paraformaldehyde followed by TUNEL assays (red) and DAPI staining (blue). Merge: merged images of TUNEL assay and DAPI staining. The photographic figures are from a representative experiment. Arrow: TUNEL-positive cells.

Fig. 4

Treatment with HDACIs induces microglial apoptosis. The enriched microglial cells were treated with 50 nM of TSA or 1.2 mM of SB for 24 h. A: The cell viability was determined by the MTT analysis and the percentages of viable cells from triplicate determinations are shown as mean ± SEM. B: The apoptotic cells were identified using PE-conjugated annexin V followed by flow cytometry analysis at 12 h and 24 h after TSA or SB treatment. The dot plots are from a representative experiment at 24 h (upper panel), and the percentages of apoptotic cells are shown as mean ± SEM from three independent experiments (lower panel). # p< 0.05, * p<0.001 compared with vehicle control.

Fig. 5

Treatment with HDACIs causes a decrease in MTP in the microglial cells. The microglial cell cultures were treated with the indicated concentrations of VPA, TSA, or SB for 24 h. The loss of MTP (ΔΨm) was determined by rhodamine 123 staining. The histograms are from a representative experiment (upper panel). A decrease in rhodamine 123 fluorescence (shift to the left) indicates a decrease in MTP. The percentages of cells with ΔΨm (cells to the left of the white line) are shown as means ± SEM from three independent experiments (lower panel). # p< 0.05, * p<0.001 compared with vehicle control.

Fig. 6

HDACIs increase histone H3 acetylation in the microglial cells. The levels of histone H3 acetylation in the microglial cells treated with 1.2 mM VPA, 50 nM TSA, or 1.2 mM SB for 12 h were analyzed by flow cytometry using specific acetyl-histone H3 antibody conjugated with FITC and PI staining for DNA content as described in the Method. The dot plot (upper panel) of DNA content versus FITC fluorescence for each sample was used to calculate the percentage of histone H3-acetylated cells (green cells). The percentages of histone H3-acetylated cells (lower panel) are shown as mean ± SEM from four independent experiments done in triplicate. *p < 0.001, compared with vehicle control.

Fig. 7

HDACIs failed to change the cell cycle in the microglial cells. The microglial cells were treated with 1.2 mM VPA, 50 nM TSA, or 1.2 mM SB for 24 h and stained with PI. DNA content was analyzed by flow cytometry. The percentages of cells in the sub-G1, G1, S, and G2-M phases of the cell cycle from experiments done in duplicate are shown as mean ± SEM. # p<0.05, compared with vehicle control.

Fig. 8

HDACIs attenuate LPS-induced production of microglial pro-inflammatory factors and reduce microglial dopamingeric neurotoxicity. Mesencephalic neuron-glia cultures were pretreated with 1.2 mM SB or 50 nM TSA for 24 h prior to stimulation with 10 ng/ml LPS. A: The release of TNF-α into the supernatant was determined 3 h post-LPS treatment. B: The level of nitrite in the supernatant, an indicator of NO production, was determined 24 h after LPS treatment. Results of four independent experiments done in triplicate are shown as means ± SEM. * p<0.001 compared with cultures treated with LPS alone. C: Seven days after LPS stimulation, the viability of DA neurons was assessed by DA uptake assays. The ratios of DA uptake capacity from cultures treated with LPS to those without LPS in the same treatment groups. The results of four independent experiments in triplicate are shown as means ± SEM. * p<0.001 compared with LPS alone. D: The morphology of tyrosine hydroxylase-immunoractive neurons. The figures shown are from a representative experiment.