Animals. SNCA-KO mice (B6;129 × 1-Sncatm1Rosl/J), wild type mice (ICR/HaJ), and dbl-PAC-Tg(SNCAA53T);SNCA−/− mice were purchased from the Jackson Laboratory. SNCA-KO and dbl-PAC-Tg(SNCAA53T);SNCA−/− mice lack endogenous α-Syn expression in brain, while the latter has peripheral expression of the human A53T mutant form [12, 17]. The mice were maintained in a specific pathogen-free facility.
Lipopolysaccharide (LPS; Sigma, L2630) was dissolved in phosphate buffered saline (PBS; GIBCO, 10010023) and a dose of 3 mg/kg with a volume of 200 µl was given by intraperitoneal (i.p.) injections when mice (SNCA-KO) were 8 weeks old at t = 0 h, t = 6 h, and t = 24 h. For α-Syn injections, a dose of 5 mg/kg with a volume of 200 µl was given via tail vein intravenous injection (IV) following the last LPS injection and mice were sacrificed at t = 36 h. Proteins of mice brain were extracted by animal tissue protein extraction kit (Sangon Biotech, C500006).
For the Parkinson’s disease model, 15 4-month old dbl-PAC-Tg(SNCAA53T);SNCA−/− mice were randomly divided into three groups. This strain models early human Parkinson’s disease, showing a gastrointestinal dysfunction without major central nervous system pathology [17, 18]. Mice received intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP; Selleck Chemicals LLC, S4732) at 30 mg/kg/day (once daily for 5 successive days) [19–21], with or without IV injection of α-Syn at a dose of 5 mg/kg following each MPTP injection. Mice were sacrificed 12 h after the last injection. An equivalent volume of saline injection served as a control . For each mouse, the brain was cut in half along the sagittal plane. One part was fixed in 4% paraformaldehyde (PFA) for 24 h and dehydrated in 30% sucrose for 48 h before frozen section preparation, while the other part was used for protein extraction in substantia nigra (SN), striatum (ST), or cerebral cortex by animal tissue protein extraction kit (Sangon Biotech, C500006).
α-Syn monomer and aggregates preparation. α-Syn protein (HNAE, 12093) was diluted and identified according to the manufacturer’s instruction.
α-Syn aggregates were prepared according to a previously published protocol . Briefly, purified α-Syn was resuspended in water at a concentration of 1 mg/ml, incubated at 37℃ with agitation for 7 days to generate α-Syn aggregates. Isoforms were characterized by Western blot. Monomer preparations showed only a single band around 15 kDa (the expected molecular weight of monomeric α-Syn), while the oligomeric aggregates showed multiple bands ranging from 35 kDa to 135 kDa on the gel (Fig. s1a), consistent with previous studies [14, 24].
Primary microglia isolation. Primary microglia were generated from neonatal ICR mice and cultured as published [14, 25]. Primary microglia culture purity was assessed by staining for both the microglia marker iba-1 and the astrocyte marker glial fibrillary acidic protein (GFAP; for possible astroglial cell contamination) (Fig. s1b). The purity of microglia cultures was determined to be > 98% (Fig. s1c).
Microglia culture and treatment. Microglial cell line BV2, which was generated by infecting mouse primary microglia culture with retrovirus J2 carrying a v-raf/v-myc oncogene , were used in some experiments. BV2 secreted IL-1 and TNF-α following appropriate stimulation and retained most of morphological, phenotypical and functional properties as that of primary microglia. To confirm results, BV2 cells were used for some experiments before validation in primary cells or animals. Cell line or primary microglia were plated onto 6-well plate (Corning, 3516) at a density of 5 × 105 per well with F12/DMEM containing 10% fetal bovine serum (FBS) free of Penicillin-Streptomycin (PS), the cells were incubated at 5% CO2, 37℃ overnight, and then the media was replaced with F12/DMEM free of FBS and PS for further stimulation.
Stimulating factors included LPS (100 ng/ml) plus interferon γ (IFN-γ) (Peprotech, 315-05) (10 ng/ml), IL-4 (Peprotech, 214 − 14) (20 ng/ml) plus IL-13 (Peprotech, 210 − 13) (10 ng/ml), or physiological concentration of α-Syn (50 nM, 100 nM, 250 nM) for 6 h, 12 h or 24 h [14, 25]. The supernatants of media were collected and filtrated through 0.45 µm filter. Nitrate concentration was measured using Nitric Oxide Assay Kit (Beyotime, S0021). For mechanism related molecules detection, cells were stimulated for 12 h only.
For treatment of primary microglia with α-Syn oligomer, the procedure was the same and stimulation was for 12 h. Pre-treatment of microglia by α-Syn was conducted 2 h before oligomer stimulation. Longer pre-treatment (6 h, 12 h) by α-Syn (100 nM) was conducted to further investigate the monomeric α-Syn in attenuating pro-inflammatory effect of oligomeric α-Syn, before oligomeric α-Syn was added, monomeric α-Syn in culture system was washed out with PBS three times, and incubation of oligomeric α-Syn lasted for either another 6 h or 12 h.
For ERK phosphorylation induced by honokiol (sigma, H4914), primary microglia were treated with 10 µM and 20 µM honokiol for 90 min, either after or before treatment with α-Syn (100 nM) for 30 min.
Real-time PCR. Total RNA was isolated from microglia using TRIZOL reagent (Invitrogen, 15596-026) according to the manufacturer’s instruction. First strand cDNA was synthesized from about 2 µg total RNA using FastQuant RT Kit with gDNase (TIANGEN, KR106). 1 µl reverse-transcribed cDNA was used in real-time PCR with HieffTM qPCR SYBR® Green Master Mix(Low Rox)(YENSEA, 11203ES08). β-actin expression was used as a control for normalizing the amounts of cDNA. The primers used were as follows: iNOS, forward primer: ACCTTGTTCAGCTACGCCTT, reverse primer: CATTCCCAAATGTGCTTGTC. ARG-1, forward primer: CAACTCTTGGGAAGACAGCA, reverse primer: GTCAGTCCCTGGCTTATGGT. CD206, forward primer: TGATTGTTGATTGCCCACTT, reverse primer: AATCTGCAGGGTTGACATGA. CD16/32, forward primer: GGCTCATTGGACACAACAAC, reverse primer: TCCTATCAGCAGGCAGAATG β-actin, forward primer: TCCTCCCTGGAGAAGAGCTA, reverse primer: CGATAAAGGAAGGCTGGAAG. All results were analyzed using 2−△△Ctand were presented as mean s.e.m.
Enzyme-linked immunosorbent assay (ELISA). The supernatant of microglia stimulated with LPS + IFNγ, IL-4 + IL-13, α-Syn or control were collected and filtered with 0.45 µm filter, IL-10, TNF-α and IL-1β were examined by ELISA kit (Mouse-IL-10-ELISA-Kit, KE10008, Mouse-TNF-alpha-ELISA-Kit, KE10002, Mouse-IL-1-beta-ELISA-Kit,KE10003) according to the manufacturer’s instructions.
Fluorescence staining. Cells were plated overnight on microscopy grade petri dishes (Jet biofilm, BDD002035) at a density of 2 × 105/ml with F12/DMEM containing 10% FBS, and then media were replaced by F12/DMEM free of FBS and stimulated with LPS + IFN-γ or IL-4 + IL-13 or α-Syn for 12 h. Cells were stained with the first antibodies including Iba-1, (1:250, Wako, 019-19741), GFAP, (1:250, Abcam, ab68428), IL-1β, (1:250, Santa Cruz, sc-7884), ARG-1, (1:250, Santa Cruz, sc-271430), iNOS, (1:250, Abcam, ab-178945), NF-κB p65, (1:250, CST, 8242). On the next day, cells were stained with fluorescein-labeled antibodies including Alexa Fluor 594-conjugated goat ant-rabbit IgG (H + L), (1:500, Thermo Fisher, A-11037), Alexa Fluor 488-conjugated goat ant-mouse IgG (H + L), (1:500, Abcam, ab150117), Alexa Fluor 488-conjugated goat ant-rabbit IgG (H + L), (1:500, Abcam, ab150077) and DAPI. Images were acquired under confocal microscope (ZEISS, LSM710).
For frozen sections, the protocol used was similar to that of cell staining with the minimal difference that before nuclear staining, the frozen sections were immersed in 0.3% Sudan Black dissolved in 70% EtOH for 45 min to reduce auto-fluorescence. iNOS (1:250, Abcam, ab49999) and IL-1β (1:250, Santa Cruz, sc-52012) were also used at various points during parallel preparations.
For Parkinson’s disease model mice (dbl-PAC-Tg(SNCAA53T);SNCA−/−), cardiac perfusion was performed with 20 ml chilled PBS, then the brain was cut in half at the sagittal plane, with one part used for protein extraction and the other for frozen section preparation. Coronal sections of 30 µm starting at 2.46 mm from Bregma and ending at 4.04 mm from Bregma were collected serially and separated one section from the consecutive 120 µm for IHC or IF .
Immunohistochemistry and cytotoxicity assay. The supernatants of microglia stimulated with LPS + IFN-γ, α-Syn, oligomer, pre-α-Syn + oligomer, or control were collected and filtered through 0.45 µm filter (Millipore, R1EA), and stored at -80℃ until use. SH-SY5Y neurons were plated on coverslips at a density of 2 × 105/ml with DMEM/ F12 containing 10% FBS overnight, and then the media were changed with conditioned media from LPS + IFN-γ, α-Syn, oligomer, pre-α-Syn + oligomer (5 pg/ml and 400 pg/ml), or control stimulated microglia and co-cultured for 24 h. The procedure for staining dopaminergic neurons in SN with tyrosine hydroxylase (TH) (1:250, Millipore, AB152) and the HRP-labeled antibody (HRP-conjugated goat anti-rabbit IgG(H + L), 1:1000, Abcam, ab6721) and quantification of dopaminergic neurons were performed as previously published [14, 27]. F-actin of SH-SY5Y was stained with TRITC phalloidin (YEASEN, 40734ES75) and images were acquired using 20X magnification. To quantify neurite length, neurites were selected at the origin site at the neuronal cell body, then following along the longest neurite of each cell with the scale tool in Adobe Photoshop followed. Neurite lengths were normalized to control cells using segmented line in Image J (1.52a) software. Cytotoxicity was assessed using a CCK-8 kit according to the manufacturer’s instructions.
Western blotting. Proteins were extracted from microglia using cell lysis buffer (RIPA, HARVEYBIO, C1503; Halt Protease & Phosphatase Inhibitor Cocktail (100X), Thermo, 78444), and a BCA Protein Assay Kit (Applygen, P1511) according to the manufacturer’s instruction determined the concentration. Western blot was carried out as previously described. Antibodies used included iNOS ( 1:1000, Abcam, ab-178945), ARG-1 (1:1000, Santa Cruz, sc-271430), ERK1/2 (1:1000, Abcam, ab184699), p-ERK1/2 (1:1000, Abcam, ab76299), NF-κB (1:1000, CST, 8242), p-NF-κB (1:1000, CST, 3033), IKKα/β (1:1000, Abcam, ab178870), IKBα (1:1000, Abcam, ab32518), p-IKBα (1:1000, Abcam, ab133462), PPARγ (1:1000, Santa Cruz, sc-81152), GAPDH (1:1000, Trans, HC301), Histone H3 (1:1000, Abcam, ab201456). The secondary antibodies were HRP-conjugated goat anti-rabbit IgG (H + L) (1:5000, Biodragon, BF03008), HRP-conjugated goat anti-mouse IgG (H + L) (1:5000, Biodragon, BF03001).
For nuclear and cytoplasmic protein detection, ProteinExt Mammalian Nuclear and Cytoplasmic Protein Extraction Kit (Trans, DE201) was used according to the manufacturer’s instructions, and then western blot was carried out as above.
For α-Syn monomer and oligomer identification, 25 µg α-Syn monomer or oligomer was loaded onto an SDS-PAGE. Anti-alpha-synuclein (1:1000, Abcam, ab138501) and HRP-conjugated goat anti-rabbit IgG (H + L) (1:5000, Biodragon, BF03008) were used to recognize the target band.
The densitometry of western blot was calculated by Adobe Photoshop CC. GAPDH served as normalization control for total target proteins, and Histone H3 served as normalization control for nuclear proteins.
Co-IP. Microglia were plated on 10-cm dish with F12/DMEM containing 10% FBS at a 70%-80% confluent overnight, then media were changed to F12/DMEM free of FBS and stimulated with 250 nM α-Syn for 15 min, 30 min, 1 h, and 2 h. Cell protein was extracted by 1% NP-40 (Beyotime, ST366) in PBS containing 1% PIC (Pierce, 87786). Cells were lysed and collected into a 1.5 ml tube and centrifuged at 14000 rpm at 4℃ for 5 min. The supernatants of whole cell lysates were regarded as pre-IP or input. 10 µg mouse anti-human α-Syn antibody (Santa Cruz sc-12767) or mouse control IgG (abcam, ab18447) was incubated overnight with 1000 µg protein at 4℃ overnight. 100 µl Protein A + G Agarose (Beyotime, P2012) was washed twice with 200 µl 1% NP-40 in PBS, centrifuged at 14000 rpm, 4℃ for 2 min. The beads were added to protein and antibody reaction system and rocked at 4℃ for 6 h, collected by centrifugation at 14000 rpm, 4℃ for 2 min, then washed twice with 1% NP-40 in PBS. The protein-antibody complex was then loaded onto SDS-PAGE. Rabbit ERK (1:1000, abcam, ab184699) and rabbit anti-human α-Syn (1:1000, abcam, ab138501) were used to detect the specific protein band on PVDF membrane.
Statistical analysis. ANOVA analysis or t test was performed using GraphPad Prism 5. Data were shown as their mean s.e.m comparisons among groups. *p < 0.05 was considered a statistically significant difference.