Microglia P2Y6 receptor is related to Parkinson’s disease through neuroinflammatory process

In our study, 145 PD patients, 170 control subjects, and 30 possible multiple system atrophy (MSA) patients were recruited from the Department of Neurology, Ruijin Hospital affiliated to Shanghai Jiao Tong University School of Medicine. The clinical diagnosis of PD was established by two independent senior movement disorder specialists using the United Kingdom Parkinson’s Disease Society Brain Bank clinical diagnostic criteria [25]. The diagnosis of possible MSA was made according to the second consensus statement on the diagnosis of MSA [26]. The clinical features of the PD patients were collected, and PD symptoms were evaluated using the Unified Parkinson’s Disease Rating Scale (UPDRS) and the Hoehn and Yahr (H-Y) staging modified version. The study was approved by the Ethics Committee of Ruijin Hospital affiliated to Shanghai Jiao Tong University School of Medicine, and written informed consent was obtained from all participants. Venous whole blood samples (4–5 ml) were collected from the individuals of the three groups on an empty stomach in the morning and mixed thoroughly with EDTA to prevent coagulation. Blood samples were collected in tubes containing EDTA. Then, the peripheral blood mononuclear cells (PBMCs) were lysed with 1 ml TRIzol reagent, and the total RNA was harvested as described by the manufacturer’s protocol. Quantitative real-time PCR (qRT-PCR) was performed using 4 μg of total RNA as a template for the reverse transcription reaction using a TakaRa 1st-strand kit. Reactions were performed according to the manufacturer’s protocol. Complementary DNA (cDNA) synthesized from each sample was subjected to real-time PCR assays with specific primers and a SYBR@Premix Ex TaqTM qPCR SuperMix (Takara). The sequences of primers were as follows: human-P2Y6R: forward 5-GCT AAC TCT TGG CTC CCG AAC A-3, reverse 5-GCG GGA CGT GCA GAT CTG GGT A-3; GAPDH: forward 5-CGG AGT CAA CGG ATT TGG TCG TAT-3, reverse 5-AGC CTT CTC CAT GGT GGT GAA GAC-3. The two-step amplification protocol consisted of denaturation for 30 s at 95 °C and then followed by 40 cycles of 95 °C for 5 s and 60 °C for 60 s. This process was performed using a ABI7500 real-time PCR machine. The RNA quantities of target genes were calculated using the Ct method. The Ct values of P2Y6R amplification were normalized to those of GAPDH amplification.

Primary microglia were derived from male neonatal C57BL/6 mice (1-day old), using the “shaking off” method. Briefly, cerebral cortices were devoid of meninges and blood vessels, dissected in Hank’s salt (HBSS) and trypsinized with 0.25% trypsin-EDTA for 30 min at 37 °C. The mixed glial culture was incubated in DMEM/F-12 containing 10% fetal bovine serum (FBS) and 50 U/ml penicillin/streptomycin (PS) and seeded at 20 × 10⁶–25 × 10⁶ cells per 175 cm² flask coated with poly-l-ornithine (Gibco). After 2 weeks of culture, flasks were shaken and the media were collected and centrifuged at 1000 rpm for 5 min to obtain a pellet of microglia. Isolated microglia were allowed to attach to plates for 3 to 4 h, and unattached cells were removed by changing the medium. Microglial cultures were determined to be 95 to 98% pure, as assessed by staining for Iba-1.

BV-2 cells, SH-SY5Y cells, and N2a cells were routinely cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS and 100 U/ml PS. PC 12 cells were cultured in DMEM medium supplemented with 5% horse serum and 5% FBS. The cells were plated 24 h prior to transfection at a confluency of 70 to 80%. The cells were transfected with P2Y6R or control siRNA (scramble) by using Lipofectamine 2000 according to the manufacturer’s instructions. The P2Y6R siRNA sequences were as follows:

Cells were plated at a density of 3 × 10⁵ cells/well in six-well plates, as detailed above. Then, the cells were lysed with 1 ml TRIzol, and the total RNA was harvested as described by the manufacturer’s protocol. Reverse transcription polymerase chain reaction (RT-PCR) was performed using 4 μg of total RNA as a template for the reverse transcription reaction using a TakaRa 1st-strand kit. The cDNA was then used for PCR of the murine purinergic P2Y6R and cytokine genes using the following primers:

The PCR reaction protocol was as follows: denaturation at 95 °C for 5 min; followed by 25 cycles at 95 °C for 30 s; P2Y6R, iNOS, COX-2, IL-6, and TNF-α at 54 °C and MIP-2 and β-actin at 58 °C for 30 s, 72 °C for 30 s, and finally, extension at 72 °C for 5 min. The PCR products were separated by 2% agarose gel electrophoresis and visualized via ethidium bromide staining.

To examine the morphology change of primary microglia cells, we pretreated primary microglia cells with apyrase (1 U/ml) and MRS2578 (5 μM) for 30 min and then treated with LPS (100 ng/ul) for 12 h. The cells were fixed with a fresh formaldehyde solution for 20 min followed by permeabilization with 0.15% Triton X-100 in PBS. Cells were then incubated overnight at 4 °C with rabbit anti Iba1 antibody (019–19741, 1:200; Wako), followed by washing in PBS 3 × 10 min. Then, the slices were incubated with the Alexa Flour 594 goat anti-rabbit IgG secondary antibody (ab150080, 1:500; Sigma) for 1 h. Finally, cells were incubated with DAPI (10236276001, Roche; 1:20000) for 15 min. Images were analyzed with a phase contrast fluorescence microscope (Nickon Eclipse E400). Experiment was performed in triplicate.

Cells were washed in ice-cold phosphate-buffered saline (PBS) three times and lysed in RIPA lysis buffer (50 mM Tris–HCl, pH 8.0; 1% NP-40; 0.5% sodium deoxycholate; 150 mM NaCl; 0.1% SDS) containing protease and phosphatase inhibitor cocktails (Roche) and 1 mM phenyl-methylsulphonyl fluoride (PMSF). Protein content was determined using a Micro-BCA Protein Assay (Thermo Scientific). Equal amounts of protein (30 μg) were loaded per lane and separated by 10% SDS-PAGE. The proteins in the gels were transferred to Immobilon polyvinylidene difluoride (PVDF) membranes (Santa Cruz, CA, USA), and the membranes were subsequently blocked in TBST (10 mM Tris–HCl, pH = 8.0; 150 mM NaCl; 0.05% Tween-20) containing 5% nonfat milk. Anti-P2Y6 receptor polyclonal goat antibody (sc-15217, 1:1000, Santa Cruz), anti-phospho-p38 (#4511); anti-phospho-ERK1/2 (#4370), and anti-phospho-JNK (#4668) antibodies; the corresponding total-p38 (#9219), total-ERK1/2 (#9102), and total-JNK (#9528) antibodies; and anti-cleaved caspase-3 (#9661) antibody (Cell Signaling Technology) were used at a dilution of 1:1000 in 3% milk/TBST. The levels of iNOS and COX-2 were determined using anti-iNOS antibody (sc-69190, 1:500; Santa Cruz) and anti-COX-2 antibodies (160106, 1:800; Caymen Chemical). All the primary antibodies were incubated overnight at 4 °C. The immunoreactive bands were visualized using secondary antibodies conjugated to horseradish peroxidase (Santa Cruz, CA, USA) and chemiluminescent detection methods (Amersham Company). To confirm equal protein loading, the membranes were probed with antibodies recognizing the cytosolic protein, β-actin (A1978, 1:2500; Sigma-Aldrich). The data shown are representative of at least three separate experiments.

Cultured BV-2 cells were stimulated with and without 200 ng/ml LPS for 12 h, and the supernatant was then collected and cleared of cellular debris. Detection of UDP in the cell culture medium was determined by reverse-phase high-performance liquid chromatography (HPLC) (Varian Vista series) using an Agilent Zorbax 300SB-C18 column (300 A), as described previously [27]. Cell medium samples (200 μl) or standard samples were injected. The retention time of UDP released by the BV-2 cells was compared with standard samples, and the peaks were identified by comparing their retention times with those of co-eluted standards. Quantification was performed by integration of the peak areas and compared with those produced by standards of known concentrations prepared in the same solvent.

The conditioned medium collected from the BV-2 cells treated with LPS was evaluated for the production of MIP-2 cytokines using ELISA (BD Biosciences, San Jose, Calif.). ELISA was performed as recommended by the manufacturer. The optical density of each well was determined using a microplate reader at 405 nm, and the amounts of cytokines were calculated from the standard curve.

Cell viability was measured by an MTT assay. The neuronal cells (SH-SY5Y, N2a, and PC12 cells) were plated in 96-well plates. After treating with the conditioned medium separated from the BV-2 cells for 24 h, MTT solution at a final concentration of 5 mg/ml was added to each well, and the plates were incubated for another 4 h. One hundred fifty microliters of DMSO was then added to solubilize the MTT tetrazolium crystals. Finally, the optical density was determined at 570 nm.

We measured the levels of P2Y6R messenger RNA (mRNA) via qRT-PCR in 145 PD patients, 170 normal controls, and 30 possible MSA patients. The subjects’ demographics are summarized in Table 1. The results showed that the relative amount of P2Y6R mRNA in the PD patients was significantly higher than that in the healthy controls and MSA patients, while there was no difference between healthy controls and MSA patients (Fig. 1a). Furthermore, we compared the P2Y6R mRNA levels among different age groups. P2Y6R mRNA expression levels in PD patients in the <60, 60–69, and 70–79 age groups were approximately 3.9, 2.5, and 3.5 times greater compared to the respective age groups of the healthy controls (Fig. 1b). However, P2Y6R mRNA expression was not significantly different between PD patients and healthy controls older than 80. In addition, P2Y6R mRNA expression was uncorrelated with gender, age of disease onset, disease duration, H&Y stage, UPDRS score, and drug administration (data not shown). We also plotted the receiver operating characteristic curve (ROC curve) according to the expression levels of P2Y6R mRNA in both PD patients and healthy controls, with the cutoff point being the presence of disease or not. The area under the curve was 0.785 (95% CI = 0.734–0.838), the diagnostic cutoff point was 2.15, and the diagnostic sensitivity and specificity were 85.8 and 59.4% (Fig. 1c). Regarding PD patients and MSA patients, the area under the ROC curve was 0.792 (95% CI = 0.778–0.959), the diagnostic cutoff point was 2.77, and the diagnostic sensitivity and specificity were 76.4 and 93.7% (Fig. 1d).

We concluded that P2Y6R expression levels are elevated in PD patients and have no relationship with the severity of disease and that P2Y6R expression might be a stable diagnostic biomarker for PD.

We investigated the expression of P2Y6R in an activated microglia cell model. We found that incubation of BV-2 cells with LPS increased both mRNA and protein levels of P2Y6R in a time-dependent manner, and the highest levels of mRNA were reached at 6 h and of protein at 12 h (Fig. 2a, c). The trends in the expression of various inflammatory cytokines (TNF-α, iNOS, IL-6, COX-2, and MIP-2 mRNA) were accord with the level of P2Y6R in LPS stimulated BV-2 cells. The relative amounts of TNF-α, iNOS, IL-6, COX-2, and MIP-2 mRNA were increased (Fig. 2c). LPS stimulation for 6 and 12 h also significantly upregulated the protein expression levels of COX-2 and iNOS (Fig. 2b). These findings suggest that P2Y6R expression was upregulated after microglia activation.

Because we have demonstrated that the change in P2Y6R expression was coordinated with inflammatory responses, we supposed that P2Y6R might play an important role in the LPS-induced production of inflammatory cytokines. UDP is known to be a specific agonist of the P2Y6 purinergic receptor, while LPS is not the direct agonist of P2Y6R. An obvious sine qua non of this hypothesis is that microglia can release UDP in response to LPS stimulation. We therefore investigated whether LPS increased the release of extracellular UDP from microglia. Cultured BV-2 cells were stimulated with and without LPS, and the supernatant was then collected for nucleotide assay based on HPLC. The amount of UDP in the LPS-treated supernatant was significantly higher than that in the untreated supernatant (Fig. 3a).

To examine the role of UDP/P2Y6R signaling in activated microglia-induced cytokine production, we pretreated the BV-2 cells with apyrase (1 U/ml) and MRS2578 (5 μM) for 30 min. Apyrase can hydrolyze tri- and di-phosphate nucleotides and therefore abrogate both ATP and UDP signaling [28, 29]. MRS2578 is the specific irreversible P2Y6R antagonist which could completely blocked P2Y6R [30, 31]. We found both apyrase and MRS2578 reduced MIP-2 mRNA expression levels induced by LPS stimulation (Fig. 3b). ELISA was also performed to quantify the impact of apyrase and MRS2578 on MIP-2 secretion. Both apyrase and MRS2578 significantly decreased the amount of MIP-2 released into the medium (Fig. 3c). To further validate the role of UDP/P2Y6R signaling in activated microglia-induced cytokine production, we turned to primary microglia cells. The morphology of microglia cells in the control group most frequently showed an ovoid shape. Primary microglia cells showed significant change in morphology after LPS treatment. Most of the LPS-stimulated microglia cells had a large and flat shape with other cells presenting a ramified morphology, while pretreated primary microglia cells with apyrase and MRS2578 can inhibit activation of the microglia as was seen by typically resembling morphology that seen in the control group (Fig. 3d). Confirming these results, we found that blocking UDP/P2Y6R signaling by apyrase and MRS2578 can reduce the inflammatory cytokines (TNF-α, iNOS, IL-6, COX-2, and MIP-2 mRNA) production in primary microglia cells induced by LPS (Fig. 3e).

To complement the experiments with antisense oligonucleotides, we also transfected BV-2 cells with P2Y6R siRNA to knock down P2Y6R expression. The interfering efficacy of P2Y6R mRNA and protein expression was confirmed by RT-PCR and western blot, respectively. We chose the siRNA with the best interference efficiency for further studies (Fig. 4a). Interestingly, we found that knocking down P2Y6R reduced the protein expression levels of iNOS and COX-2 in BV-2 cells induced by LPS (Fig. 4b) and the inflammatory cytokine production was also downregulated (Fig. 4c). These findings suggested that inhibition of UDP/P2Y6R signaling could partly prevent LPS-induced neuroinflammation.

To further investigate the effect of P2Y6R in microglia on neuronal cells viability, conditioned medium from LPS-stimulated BV-2 cells was added to different neuronal cells (SH-SY5Y, N2a, and PC12 cells). We transfected the BV-2 cells with P2Y6R siRNA and then incubated the neuronal cells with the supernatant from BV-2 cells treated with LPS. The cell viability and the degree of apoptosis of the neuronal cells were then measured based on MTT assay and the expression of caspase-3. The results showed that the supernatant of the LPS-treated BV-2 cells induced a decrease in cell viability (Fig. 5a) and an increase in caspase-3 expression levels in different neuronal cells (SH-SY5Y, N2a, and PC12 cells) compared with the cells exposed to normal medium (Fig. 5b). However, the decreased cell viability (Fig. 5a) and increased caspase-3 expression (Fig. 5b) were reversed by knocking down P2Y6R. This approach indicated that P2Y6R in microglia contributed to the cell death of neuronal cells and that knocking down P2Y6R could increase cell viability and decrease apoptosis of neuronal cells caused by the cytotoxicity of LPS-stimulated BV-2 cells.

Previous studies have shown that P2Y receptor signaling is mainly through mitogen-activated protein kinases (MAPK) [31, 32]. To explore the mechanism by which P2Y6R is involved in neuroinflammation process, we investigated the levels of MAPK, including the extracellular signal-regulated protein kinase (ERK1/2), p38, and the jun-NH2-kinase (JNK). We found that P2Y6R activation induced by its specific agonist UDP caused strong and rapid ERK1/2 phosphorylation (Fig. 6a). However, UDP had no effect on p38 and JNK expression, whereas LPS could activate all of the MAPK pathways (data not shown). In addition, the involvement of MAPK pathways in P2Y6R activation was also examined using a pharmacological approach. We investigated the impact of specific inhibitors for ERK1/2 (U0126), p38 (SB203580), and JNK1/2 (SP600125) on MIP-2 mRNA expression, COX-2 protein expression, and MIP-2 secretion. Inhibition of the ERK1/2 pathway by U0126 applied 30 min before UDP stimulation resulted in a significant decrease in COX-2 protein and MIP-2 mRNA expression (Fig. 6b). However, no significant inhibition of MIP-2 transcript expression was observed in SB203580- and SP600125-pretreated cells (data not shown). Similarly, pretreatment of the BV-2 cells with U0126 but not SB203580 or SP600125 before UDP stimulation abolished the release of MIP2 measured by ELISA (Fig. 6c). These results suggest that ERK1/2 activation is important in the regulation of P2Y6R cytotoxicity.