Parkinson’s disease (PD) is a neurodegenerative disease characterized by impaired movement, muscular rigidity, resting tremor, bradykinesia, and postural instability (Przedborski, 2017). Along with nigrostriatal dopaminergic degeneration and cytoplasmic aggregate inclusion, persistent neuroinflammation is a neuropathological feature of PD and contributes to the progression of PD (Liu
Microglia are usually primed by pathological conditions, resulting in an amplified immune response to secondary inflammatory challenges (Perry and Teeling, 2013; Neher and Cunningham, 2019). The pathological significance of primed microglia both in aging and in neurodegenerative diseases has previously been studied (Perry and Holmes, 2014; Norden
The activity of IL-1β is particularly controlled by a cytosolic multimolecular complex termed ‘inflammasome,’ which contains nod-like receptor protein 3 (NLRP3), adaptor protein ASC, and pro-caspase-1 (Martinon
In the present study, we examined the effect of papaverine (PAP; 6,7-dimethoxy-1-veratryl-isoquinoline) in an MPTP-induced microglial priming mouse model challenged with lipopolysaccharide (LPS). PAP is a non-narcotic opium alkaloid isolated from
Adult male C57BL/6 mice (9 weeks of age) were purchased from Orient Bio Inc. (Seongnam, Korea), a branch of Charles River Laboratories. Mice were maintained at 21°C under a 12 h light:12 h dark cycle and had ad libitum access to water and rodent chow. Every effort was made to minimize animal suffering. All experiments were performed in accordance with the National Institutes of Health (NIH, Bethesda, MD, USA) and Ewha Womans University (Seoul, Korea) guidelines for laboratory animal care and use, and the study was approved by the Institutional Animal Care and Use Committee of the Medical School of Ewha Womans University (#EUM 20-022).
Mice were randomly divided into the following five groups (N=10-16): 1. CON (control); 2. MPTP; 3. LPS; 4. MPTP/LPS, MPTP+LPS; 5. MPTP/LPS+PAP, MPTP+LPS+papaverine. Mice were administered MPTP (20 mg/kg, i.p.), followed by 3 consecutive days of PAP administration (20 mg/kg, i.p.). Mice were administered LPS (0.5 mg/kg, i.p.) 1 d after the last treatment with PAP and were sacrificed 3 days after LPS administration. LPS and PAP were obtained from Sigma-Aldrich (St. Louis, MO, USA). MPTP was purchased from Tokyo Chemical Industry Co (Tokyo, Japan). A schematic of this procedure is shown in Fig. 1A.
Mice were anesthetized with sodium pentobarbital (80 mg/kg; Hanlim Pharm Co. Ltd., Seoul, Korea) to induce rapid and prolonged anesthesia. The mice were then perfused transcardially, their brains were removed, and 40 µm-thick sections were prepared using a cryotome. For immunohistochemistry (IHC) analysis, the sections were subjected to endogenous peroxidation inactivation with 3% hydrogen peroxide (H2O2), and non-specific binding was blocked with 4% bovine serum albumin (BSA). The sections were first incubated overnight with primary antibodies and were then incubated with biotinylated secondary antibodies for 1 h at 25°C, on the following day. The sections were subsequently incubated with avidin-biotin-horse radish peroxidase (HRP) complex reagent solution for 1.5 h (Vector Laboratories, Burlingame, CA, USA) and a peroxidase reaction was then performed using diaminobenzidine tetrahydrochloride (Vector Laboratories). For double immunofluorescence (IF) analysis, non-specific binding was blocked, and the sections were incubated with primary antibodies followed by fluorochrome-conjugated secondary antibodies. The tissue was then mounted with an antifade reagent (Vector Laboratories). Digital images of IHC and IF staining were captured using a Leica DM750 microscope (Leica Microsystems, Nussloch, Germany). Quantification was performed using ImageJ (NIH Image Engineering, Bethesda, MD, USA) and AxioVision (Carl Zeiss Microscopy GmbH, Jena, Germany). The following primary antibodies were used in this study: anti-tyrosine hydroxylase (anti-TH), anti-IL-1β, anti-NLRP3, anti-ASC antibodies from Cell Signaling Technology, Inc. (Danvers, MA, USA), anti-ionized calcium-binding adaptor molecule 1 (anti-Iba1) from Wako (Osaka, Japan), and biotinylated and fluorophore-conjugated secondary antibodies from Vector Laboratories. Anti-CD11b (OX42) was obtained from Bio-Rad (Hercules, CA, USA).
Tissues collected from SN were homogenized in ice-cold radioimmuno precipitation assay (RIPA) lysis buffer (10 mM Tris-Cl [pH 7.4], 300 mM NaCl, 1% Triton X-100, 0.1% sodium dodecyl sulfate [SDS], 0.1% sodium deoxycholate, 1 mM ethylenediaminetetraacetic acid) containing a protease inhibitor cocktail (Complete Mini, Roche, Mannheim, Germany). Subsequently, the samples were vortexed vigorously and incubated for 30 min at 4°C. The samples were then centrifuged at 20,000×g for 30 min, and the supernatant was collected. Protein samples (30-50 μg) were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and incubated with primary antibodies according to the manufacturer’ s instructions for dilution. The membranes were then thoroughly washed with TBST (tris-buffered saline and tween 20) and incubated with HRP-conjugated secondary antibodies (Bio-Rad; 1:3,000 dilution in TBST). Subsequently, the blots were developed using an enhanced chemiluminescence detection kit (Thermo Fisher Scientific, Waltham, MA, USA). For quantification, the density of specific target bands was normalized against β-actin using ImageJ software, version 1.37 (NIH). The following primary antibodies were used in this study: anti-IL-1β, anti-caspase-1, anti-NLRP3, anti-IkB, anti-p-IkB, anti-CREB, and anti-p-CREB from Cell Signaling Technology, Inc. Antibody against β-actin was purchased from Sigma-Aldrich.
Statistical analyses were performed using SPSS for Windows (version 18.0; SPSS Inc., Chicago, IL, USA). The differences among the groups were analyzed using a one-way analysis of variance (ANOVA). Post-hoc comparisons were conducted using the least significant difference (LSD) test. All values are presented as the mean ± standard error of the mean (SEM). A value of
We first established an
When we examined the effect of MPTP or LPS treatment on the survival of dopaminergic neurons, the number of TH+ dopaminergic cells did not change significantly compared with the control group (Fig. 2). However, the number of TH+ cells in the SN of the MPTP/LPS group was profoundly reduced compared to the other groups, including the CON, LPS, and MPTP groups; cell death was then recovered by PAP treatment (Fig. 2A, 2B; F4, 27=8.362,
Next, we examined the effect of MPTP and LPS on the expression of IL-1β, which plays a major role in microglial priming and the exacerbation of neuronal loss (Koprich
The changes in IL-1β expression prompted us to measure the expression of microglial NLRP3 to identify the involvement of inflammasomes. We found that NLRP3+ fluorescence intensities of the MPTP/LPS group were considerably augmented compared to the LPS or MPTP group, which was then reversed by PAP treatment (Fig. 4A-4C; F4, 15=26.75,
The number of ASC specks, a hallmark of inflammasome activation, in the LPS and MPTP group increased significantly compared to the CON group, while ASC specks of the MPTP/LPS group were markedly enhanced relative to the LPS or MPTP group (Fig. 5A-5C; F4, 23=13.21,
To further investigate the mechanism underlying inflammasome inhibition by PAP, we performed western blot analysis to determine the protein expression levels of NLRP3 inflammasome components and its related signaling molecules such as NF-κB and CREB. The results showed that total IL-1β protein levels (pro- and mature IL-1β) in the LPS or MPTP group increased significantly compared to the CON group. The cytokine levels in the MPTP/LPS group were higher than in the LPS or MPTP group, which was reversed by PAP treatment (Fig. 6A, 6B; F4, 15=25.21,
With regard to the signaling pathway, MPTP/LPS significantly increased NF-κB activity (p-IkB/total IkB), which was inhibited by PAP. However, there was no significant difference among the CON, LPS, and MPTP groups (Fig. 6C, 6D; F4, 15=22.58,
In the current study, we demonstrated that MPTP-induced microglial priming exacerbated nigrostriatal dopaminergic degeneration and amplified the inflammatory response to subsequent systemic inflammation induced by a sub-toxic dose of LPS. The priming effects of MPTP were alleviated by the administration of PAP. Thus, PAP inhibited microglial activation and dopaminergic neuronal cell death in MPTP/LPS-treated mice. PAP administration particularly suppressed NLRP3 inflammasome-dependent IL-1β processing, which potentially contributes to the anti-inflammatory and neuroprotective effects of PAP.
PAP, which is a selective PDE10 inhibitor, inhibits phosphodiesterase activity and increases the intracellular cAMP level (Zagorska
Another mechanism underlying the inhibition of NLRP3 inflammasome by PAP could possibly be NF-κB inhibition. In response to activation of receptors such as toll-like receptors (TLR2/4) and TNF receptors by priming signals, NF-κB is activated and upregulates the expression of inflammasome components such as NLRP3 and pro-IL1β/IL-18 (Seok
Previous studies have reported the therapeutic effects of PAP in various neuropathological conditions. PAP improved cognitive impairment in a Huntington’s disease mouse model by increasing GluA1 and CREB phosphorylation (Giralt
Our study reports for the first time the therapeutic effect of PAP in an MPTP-induced microglial priming model challenged with LPS, and the involvement of NLRP3 inflammasome in this mechanism. Considering that PAP is a non-addictive opium alkaloid with few side effects, it is a potential therapeutic candidate for PD and other neurodegenerative diseases that are associated with microglial priming and activation.
This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (2016R1A6A3A11930120, 2018R1A2B6003074 and 2020R1I1A1A01057922).
The authors have no conflict of interest to declare.