The phosphodiesterase 10 inhibitor papaverine exerts anti-inflammatory and neuroprotective effects via the PKA signaling pathway in neuroinflammation and Parkinson's disease mouse models

Abstract

Background: Neuroinflammation plays a pivotal role in the pathogenesis of Parkinson's disease (PD). Thus, the development of agents that can control neuroinflammation has been suggested as a promising therapeutic strategy for PD. In the present study, we investigated whether the phosphodiesterase (PDE) 10 inhibitor has anti-inflammatory and neuroprotective effects in neuroinflammation and PD mouse models.

Methods: Papaverine (PAP) was utilized as a selective inhibitor of PDE10. The effects of PAP on the expression of pro-inflammatory molecules were examined in lipopolysaccharide (LPS)-stimulated BV2 microglial cells by ELISA, RT-PCR, and Western blot analysis. The effects of PAP on transcription factors were analyzed by the electrophoretic mobility shift assay, the reporter gene assay, and Western blot analysis. Microglial activation and the expression of proinflammatory molecules were measured in the LPS- or MPTP-injected mouse brains by immunohistochemistry and RT-PCR analysis. The effect of PAP on dopaminergic neuronal cell death and neurotrophic factors were determined by immunohistochemistry and Western blot analysis. To assess mouse locomotor activity, rotarod and pole tests were performed in MPTP-injected mice.

Results: PAP inhibited the production of nitric oxide and proinflammatory cytokines in LPS-stimulated microglia by modulating various inflammatory signals. In addition, PAP elevated intracellular cAMP levels and CREB phosphorylation. Treatment with H89, a PKA inhibitor, reversed the anti-inflammatory effects of PAP, suggesting the critical role of PKA signaling in the anti-inflammatory effects of PAP. We verified the anti-inflammatory effects of PAP in the brains of mice with LPS-induced systemic inflammation. PAP suppressed microglial activation and proinflammatory gene expression in the brains of these mice, and these effects were reversed by H89 treatment. We further examined the effects of PAP on MPTP-injected PD model mice. MPTP-induced dopaminergic neuronal cell death and impaired locomotor activity were recovered by PAP. In addition, PAP suppressed microglial activation and proinflammatory mediators in the brains of MPTP-injected mice.

Conclusions: PAP has strong anti-inflammatory and neuroprotective effects and thus may be a potential candidate for treating neuroinflammatory disorders such as PD.

Keywords: Microglia; Neuroinflammation; Neuroprotection; PDE10 inhibitor; PKA signaling; Papaverine; Parkinson’s disease.

Fig. 1

Effect of PAP on NO and inflammatory molecules in LPS-stimulated BV2 microglia. a BV2 cells were pretreated with PAP for 1 h and incubated with LPS (100 ng/mL). After incubation for 16 h, the supernatants were obtained and the levels of nitrite, TNF-α, IL-1β, and IL-10 were measured. b, c BV2 cells were pretreated with PAP for 1 h and incubated with LPS for 6 h. Western blot analysis (b) and RT-PCR (c) were performed to measure the expression of inflammatory molecules. Representative gels are shown in the left panel, and the quantification of three independent experiments is shown in the right panel. Data are shown as the mean ± SEM of three independent experiments. *p < 0.05 vs. control group; ^#p < 0.05 vs. LPS-treated samples. d BV2 cells were transfected with PDE10-specific or control siRNA. Downregulation of PDE10 expression by PDE10 siRNA was confirmed by Western blot analysis. e The cells transfected with PDE10 siRNA or control siRNA were treated with LPS for 16 h. Then, the amounts of NO, TNF-α, IL-1β, and IL-10 released into the media were determined. ^#p < 0.05 vs. control siRNA-transfected cells in the presence of LPS

Fig. 2

Effect of PAP on MAPKs, Akt, and NF-κB/AP-1 activities in LPS-stimulated BV2 cells. a Cell lysates were prepared from BV2 cells treated with LPS for 30 min in the absence or presence of PAP, and Western blotting was performed to determine the effect of PAP on MAPKs and Akt activity. Quantification data are shown in the right panels (n = 3). Levels of the phosphorylated forms of MAPKs and Akt were normalized to the total forms and expressed as fold changes vs. untreated control samples, which were arbitrarily set to 1. b EMSA for NF-κB and AP-1 was performed using nuclear extracts prepared from BV2 cells treated with PAP in the presence of LPS for 3 h. c Transient transfection analysis of [κB]₃-luc, and AP-1-luc reporter gene activity. Data are shown as the mean ± SEM of three independent experiments. ^#p < 0.05 vs. LPS-treated samples

Fig. 3

PAP reduced ROS production via suppression of a NADPH oxidase subunit and upregulation of Nrf2/ARE signaling. a BV2 cells were treated with PAP 1 h prior to LPS stimulation for 16 h, and intracellular ROS level was measured by the DCF-DA method. b RT-PCR to assess mRNA expression of NADPH oxidase subunits (p47^phox, gp91^phox, p67^phox, and p22^phox) in BV2 cells. c Western blot analysis to assess phosphorylation of the p47^phox subunit. BV2 cells were treated with PAP 1 h followed by LPS (100 ng/mL, 30 min) and then subjected to immunoblot analysis using antibodies against phospho-p47^phox. d EMSA to assess Nrf2 DNA binding activity. e Transient transfection analysis of ARE-luc reporter gene activity. Data are shown as the mean ± SEM of three independent experiments. *p < 0.05 vs. control group; ^#p < 0.05 vs. LPS-treated group

Fig. 4

PAP enhanced PKA/CREB signaling, and H89 reversed the anti-inflammatory effects of PAP in LPS-stimulated BV2 cells. a Intracellular cAMP level was measured in BV2 cells treated with PAP in the presence or absence of LPS for 30 min. b Western blot analysis to assess the phosphorylated and total forms of CREB using the same cell lysates as a. c EMSA to assess CREB DNA binding activity. d Transient transfection analysis of CRE-luc reporter gene activity. e Western blot to detect the nuclear translocation of CREB. f Cells were pretreated with H89 for 30 min, then treated with PAP for 1 h followed by LPS for 16 h. The production of NO, TNF-α, IL-1β, IL-10, and ROS was measured. Data are shown as the mean ± SEM of three independent experiments. *p < 0.05, control vs. LPS-treated group; ^#p < 0.05, LPS vs. LPS+PAP-treated group; ^##p < 0.05, LPS+PAP vs. LPS+PAP+H89 group

Fig. 5

PAP increased PPARγ activity, which also depends on PKA signaling. a Transient transfection analysis of PPRE-luc reporter gene activity. b BV2 cells were transfected with PPAR-γ-specific or control siRNA, and treated with LPS in the absence or presence of PAP (30 μM) for 16 h. Then, the production of NO, ROS, and cytokines was measured. ^#p < 0.05 vs. LPS-treated samples; ^##p < 0.05 vs. control siRNA-transfected cells in the presence of LPS+PAP. c, d BV2 cells transfected with the reporter plasmid (ARE-luc, and PPRE-luc) were pretreated with H89 for 30 min. They were then treated by PAP for 1 h followed by LPS for 6 h. A luciferase assay was performed to measure the reporter gene activities of PPRE (c) and ARE (d). Data are shown as the mean ± SEM of three independent experiments. *p < 0.05, control vs. LPS-treated group; ^#p < 0.05, LPS vs. LPS+PAP-treated group; ^##p < 0.05, LPS+PAP vs. LPS+PAP+H89 group

Fig. 6

Effects of PAP on microglial activation and the mRNA expression of inflammatory markers in the brains of LPS-injected mice. a, b Immunohistochemical staining for Iba-1 and quantification of the number of Iba-1-positive microglia 3 days after LPS injection (each group n = 4–5). Microglial activation in the cortex, hippocampus, and substantia nigra of LPS-injected mice was reduced by PAP (30 mg/kg), and this was reversed by H89 treatment. Representative images (a) and the quantification of data (b) are shown. Scale bars, 100 μm. c, d Effects of PAP on the mRNA levels of iNOS, cytokines, microglial activation markers (TLR2, TLR4), and proinflammatory MMPs (MMP-3, MMP-8) in the cortices of LPS-injected mice (each group n = 4). Representative gels (c) and quantification data (d) are shown. e, f Effect of H89 on PAP-mediated suppression of proinflammatory gene expression in LPS-injected mouse brains. *p < 0.05, control vs. LPS-treated group; ^#p < 0.05, LPS vs. LPS+PAP-treated group; ^##p < 0.05, LPS+PAP vs. LPS+PAP+H89 group

Fig. 7

Effect of PAP on dopaminergic neuronal cell death, microglial activation, and the expression of inflammatory genes in the brains of MPTP-injected mice. a Representative images of TH-positive neuronal cells in the substantia nigra and striatum (each group n = 4–5). Quantitative analysis was performed by measuring the number of TH-positive cells in the substantia nigra, and the optical density of TH-positive fibers in the striatum (right panel). b Representative images of Iba-1-positive microglial cells in the substantia nigra (each group n = 4–5). c Real-time PCR analysis was performed to examine the expression of iNOS and cytokines in the substantia nigra (each group n = 4). *p < 0.05 vs. control group; ^#p < 0.05 vs. MPTP group

Fig. 8

Effect of PAP/H89 on locomotor activity and the expression of TH and PGC-1α in the brains of MPTP-injected mice. a A schematic of the experimental procedure. Mice were injected with PAP (30 mg/kg, i.p.) every day for 3 days before MPTP injection. H89 was injected (1 mg/kg, i.p.) 1 h prior to every PAP injection. Mice were sacrificed 7 days following MPTP injection, and histological and biochemical analyses were performed. b, c Rotarod and pole tests were performed 1 and 6 days after MPTP injection, respectively (each group n = 12–14). d, e Immunostaining results showing TH and PGC-1α expression in the substantia nigra (each group n = 6–7). The white arrows indicate TH-positive cells with PGC-1α in their nucleus. f Quantification of TH and/or PGC-1α- positive cells in the substantia nigra pars compacta. g The protein extracts from the substantia nigra of each group were subjected to Western blot analysis using TH or PGC-1α antibodies (each group n = 5). Representative blots are provided in the upper panel, and quantification of the Western blot data is shown in bottom panel. *p < 0.05, control vs. MPTP-treated group; ^#p < 0.05, MPTP vs. MPTP+PAP-treated group; ^##p < 0.05, MPTP+PAP vs. MPTP+PAP+H89 group

Fig. 9

Effect of PAP/H89 on p-CREB level and its downstream neuroprotective factors in the brains of MPTP-injected mice. a Immunostaining results showing TH and p-CREB expression in the substantia nigra (each group n = 6–7). The white arrows indicate cells positive for both TH and p-CREB. b Quantification of p-CREB and/or TH-positive cells in the substantia nigra. c, d IHC results for GDNF staining in the substantia nigra (c), and quantification data from the substantia nigra pars compacta and substantia nigra region (each group n = 6–7). e The protein extracts from the substantia nigra of each group were subjected to Western blot analysis using p-CREB, GDNF, Bcl2, and BDNF antibodies (each group n = 5), and representative blots are provided. f Quantification of Western blot data. *p < 0.05, control vs. MPTP-treated group; ^#p < 0.05, MPTP vs. MPTP+PAP-treated group; ^##p < 0.05, MPTP+PAP vs. MPTP+PAP+H89 group

Fig. 10

Effect of H89 on PAP-mediated microglial inactivation and CREB phosphorylation in the brains of MPTP-injected mice. a Immunohistochemical staining for Iba-1 and quantification of the number of Iba-1-positive microglia in the substantia nigra of MPTP-injected mice (each group n = 6–7). b The p-CREB levels in microglia were measured by immunofluorescence staining. The white arrows indicate cells positive for both OX42 and p-CREB. *p < 0.05, control vs. MPTP-treated group; ^#p < 0.05, MPTP vs. MPTP+PAP-treated group; ^##p < 0.05, MPTP+PAP vs. MPTP+PAP+H89 group