Cell culture and treatment

Primary microglial cultures were prepared as previously reported [15]. In brief, cortical tissues were dissected from neonatal pups (P1–P3), digested and filtered through a 70-µm strainer. After centrifugation, the pellets were suspended in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum (FBS; Gibco, Gaithersburg, MD, USA). The cells were plated into T75 flasks, and the medium was changed every other day. Upon reaching confluency, microglia were harvested by shaking the flasks orbitally at 180 rpm for 2 h and seeded into culture dishes. After attachment for approximately 2 h, the microglia were gently rinsed with prewarmed phosphate-buffered saline (PBS) and cultured in fresh medium for further experiments. BV2 cells, MEFs, and HEK293 cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin in an incubator with 5% CO2. For in vitro studies, microglia were treated with α-Syn PFF or lipopolysaccharide (L2880, Sigma, USA). The ULK1 activator BL-918 (provided by Prof. Liang Ou-Yang, Sichuan University) and the Toll-like receptor 4 (TLR4) inhibitor TAK-242 (HY-11109, MCE, USA) were added 30 min before α-syn PFF or LPS treatment.

Quantitative polymerase chain reaction (qPCR)

Total RNA was extracted and reverse transcribed via a cDNA synthesis kit (R323; Vazyme, China) according to the manufacturer’s instructions. qPCR was performed via SYBR Green PCR Master Mix (Q711, Vazyme, China) with the specific primers listed in Table 1 via the 7500 Real-Time PCR system (Applied Biosystems). The mRNA levels of the target genes were calculated via the 2−△△Ct method and normalized to those of the housekeeping gene Gapdh.

Table 1 Sequences of the primers used for the qPCR analysis of gene expressionWestern blot and antibodies

Cells and tissues were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors as previously described [12]. Protein concentrations were determined with a BCA assay kit (20201ES90, Yeasen, China). Proteins were separated by SDS‒PAGE and transferred onto polyvinylidene difluoride membranes (ISEQ00010, Millipore, USA). The membranes were blocked with 5% (w/v) nonfat milk and incubated with primary antibodies against the proteins of interest at 4 °C overnight. Next, the membranes were washed and incubated with an appropriate secondary antibody (1:5000, 115-035-003/111-035-003, Jackson ImmunoResearch Lab, USA) at room temperature (RT) for 1 h. The protein bands were visualized via the ECL reagent (P10300, NCM Biotech, China) on a ChemiDoc XRS + System (Bio-Rad, USA). Densitometric analysis was performed via ImageJ software.

The primary antibodies used were as follows: anti-ULK1 (1:500, 8054 S, CST, USA), anti-LC3 (1:2000, NB100–2220, Novus, USA), anti-tyrosine hydroxylase (anti-TH, 1:1000, T1299, USA), anti-SQSTM1/p62 (1:1000, P0067, Sigma, USA), anti-p-STAT1Y701 (1:500, AP0054, ABclonal, China), anti-STAT1 (1:500, 14994, CST, USA), anti-α-Syn (1:500, AB138501, Abcam, USA), anti-ACTB (1:5000, A3854, Sigma, USA) and anti-GAPDH (1:5000, 60004-1-IG, Proteintech, China).

α‑Syn PFF preparation

α-Syn PFFs were generated as previously described [16]. In brief, Escherichia coli BL21 (DE3) cells were transformed with the pET-28b (+) plasmid encoding human α-Syn, incubated in LB medium and treated with 1 mM isopropyl β-D-thiogalactoside to induce α-Syn expression. Bacteria were harvested by centrifugation at 5000 rpm for 5 min and resuspended in Tris buffer (150 mM Tris at pH 8.0, 10 mM EDTA, 150 mM NaCl), followed by sonication and centrifugation at 13,000 rpm for 15 min. The resulting supernatant was collected for α-Syn purification. Endotoxin was depleted via a ToxinEraser kit (L00338, GenScript, China). α-Syn monomers were diluted to 5 mg/ml and shaken at 1,000 rpm in an Eppendorf Thermomixer for 7 days to facilitate α-Syn aggregate formation. For the experiments, α-Syn aggregates were diluted to the desired concentration and sonicated for 30 s (0.5 s on/off) at 10% power immediately prior to use. α-Syn PFF was visualized via transmission electron microscopy. The seeding capability of α-yn PFFs was validated by testing α-syn phosphorylation at S129 (pS129-α-Syn) in primary neurons (Supplementary Fig. 1e).

Animals and stereotaxic surgery

C57BL/6 mice (6–8 weeks old, male) were purchased from Shanghai Laboratory Animal Center (Shanghai, China). To generate mice with microglial Stat1 ablation, Cx3cr1CreERT2 mice (kindly gifted by Prof. Jian Cheng, Soochow University) were crossed with Stat1fl/fl mice (GemPharmatech, Nanjing, China), followed by genotyping. All 6-week-old male Stat1fl/fl; Cx3cr1CreERT2/+ (described as Stat1cKO hereafter) mice and their littermates (Stat1fl/fl) were intraperitoneally injected with 75 mg/kg/day tamoxifen (Sigma, T5648) for 5 days to induce Cre expression and recombination. Tlr4−/− mice were obtained from the Model Animal Research Center (MARC) of Nanjing University. The mice were maintained at the Laboratory Animal Center of Suzhou Medical College at Soochow University and housed in a specific pathogen-free facility under a 12 h light‒dark cycle at 21 ± 2 °C, with food and tap water ad libitum. All procedures involving mice were performed in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of Soochow University (ECSU-201600014).

α-Syn-based PD models were established via two different protocols: recombinant adeno-associated virus (AAV)-mediated α-Syn overexpression (OE) and PFF injection into the striatum via a stereotaxic apparatus. For surgery, all the mice were anesthetized with 2.5% avertin. To induce α-Syn OE, 1.5 µl of AAV2/9-hα-Syn (1.26E + 12 v.g., Obio, Shanghai) or AAV2/9-Vector was bilaterally delivered into the SN, with coordinates at anteroposterior (AP) -3.0 mm, mediolateral (ML) ± 1.25 mm, and dorsoventral (DV) + 4.5 mm relative to the bregma as previously described [12]. The needle was left in place for 5 min before being retracted slowly. To model α-Syn spreading in PD, α-Syn PFF (5 µg) was bilaterally injected into the striatum at the following coordinates: AP + 0.2 mm, ML ± 2.0 mm, and DV + 3.2 mm, as previously described [17, 18]. For controls, an equal volume of PBS was injected.

Microglia isolation from adult mice

Microglia were acutely isolated from adult mice as previously reported [12]. Briefly, the mice were anesthetized and perfused with chilled PBS. The brains were dissected and homogenized in serum-free DMEM. The homogenates were filtered through a 70-µm strainer followed by centrifugation at 1000×g for 10 min at 4 °C. Microglia were then isolated via density gradient centrifugation. Specifically, the pellets were suspended in 22% Percoll and centrifuged to remove myelin fragments. The resulting pellets were resuspended in 37% Percoll solution. Next, a density gradient was added to a 15-ml centrifuge tube and centrifuged at 1000×g for 40 min with no brake. Microglia were carefully collected at 70–37% confluence and resuspended in 3 volumes of Hank’s balanced salt solution, followed by centrifugation at 800×g for 10 min at 4 °C. The purity of the isolated microglia (gated on CD11bHighCD45intermediate and then CX3CR1+) was assessed via flow cytometry, which revealed that the purity reached > 90% (Supplementary Fig. 2).

Immunostaining

The mice were anesthetized and sequentially perfused with saline and 4% paraformaldehyde (PFA) for fixation. The brains were dehydrated in 10-30% sucrose solution and cut into 18 μm thick sections via a cryostat (Leica, German). For immunofluorescence staining, the sections were blocked in 5% BSA with 0.25% Triton X-100 for 1 h at RT and incubated with primary antibodies overnight at 4 °C. After being rinsed three times in PBS, the sections were incubated with Alexa Fluor 488- or 555-conjugated secondary antibodies (1:500, Thermo Fisher) in the dark for 1 h. Next, the sections were mounted onto slides and stained with DAPI. Images were taken under a confocal microscope (LSM700; Carl Zeiss, Germany). For immunohistochemical staining, the sections were soaked in 3% hydrogen peroxide for 10 min to reduce endogenous peroxidase activity before blocking. The sections were finally incubated with a horseradish peroxidase-conjugated secondary antibody. Staining was detected via a DAB kit (GK500705, Gene Tech). The following primary antibodies were used: anti-ULK1 (1:200, 8054 S, CST, USA), anti-IBA1 (1:1000, 019–19741, Wako, Japan), anti-NLRP3 (1:200, 15101, CST, USA), anti-TH (1:1000, T1299, Sigma, USA), anti-p-STAT1Y701 (1:200, AP0054, ABclonal, China), and anti-GFAP (1:1000, z033429-2, Dako, Denmark).

For cell staining, cells grown on coverslips were fixed with 4% PFA for 10 min, rinsed twice with PBS, and then permeabilized in 0.25% Triton X-100/PBS for 5 min. Next, the cells were blocked in PBS with 5% FBS for 10 min, incubated with anti-p-STAT1Y701 and anti-IBA1 antibodies at 4 °C overnight, and then with Alexa Fluor 488- and 555-conjugated secondary antibodies for 1 h at RT. Coverslips were mounted with Fluoroshield medium containing DAPI. Images were acquired via confocal microscopy.

TH+ neurons in the SNpc were quantified by an experienced researcher who was blinded to the experimental groups. After adjusting the threshold and carefully marking the borders of the SN, TH+ neurons with only a visible nucleus were counted by ImageJ via the Cell Counter plugin. Every fifth section throughout the SN was collected, and at least four sections were incorporated into the counting system for each mouse. The total number of TH+ neurons in the SN of the hemisphere was estimated by multiplying the counted number by five since every fifth section was used for analysis. For the mean fluorescent intensity (MFI) analysis, a threshold was selected with image/adjustment tools to achieve the desired range of intensity values for each experiment. Once determined, this threshold was applied to all the images. After exclusion of the background, the selected area in the signal intensity range of the threshold was measured via the measurement option under the Analyze/Measure menu. The Manders’ overlap coefficient was used to measure the colocalization of microglia with ULK1, NLRP3 or p-STAT1Y701. Microglia were counted with the Analyze Particles function. At least three sections per mouse were included for analysis.

ULK1 mRNA quantification in peripheral blood mononuclear cells (PBMCs)

To quantify the ULK1 mRNA level in PBMCs, 5 ml of blood was collected from each individual via the median cubital vein and stored in an EDTA anticoagulant tube (BD Bioscience, USA) at -80 °C within 1 h after collection. Total RNA was extracted via a PAX-gene Blood RNA kit and reverse transcribed into cDNA. qPCR was performed as described above, using the primers for the human ULK1 gene listed in Table 1.

The study was approved by the Ethics Committee of the Second Affiliated Hospital of Soochow University in China, and written informed consent was obtained from all participants (IK-2018-061-01). All participants underwent a comprehensive general and neurologic examination. Patients with PD were referred by senior neurologists, and disease severity was assessed via the United Parkinson disease rating scale (UPDRS). Healthy controls without any signs of neurological disease were recruited from routine physical examinations.

RNA interference and plasmid transfection

To knockdown the Ulk1 gene, BV2 cells were transfected with Ulk1 siRNA (sense: 5′-GGGUAGUAAUGACACCACCUCGGAA-3′; antisense: 5′-UUCCGAGGUGGUGUCAUUACUACCC-3′) or scrambled siRNA (GenePharma, Shanghai, China) as a control via Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA). The plasmids used for Stat1 OE (Flag-Stat1) and knockdown (sh-Stat1) were a gift from Professor Hui Zheng at Soochow University (Suzhou, China). Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) was used for plasmid transfection. The genetic editing efficiency was verified via western blotting.

Open field test

Locomotor activity was evaluated via an open field test. The apparatus consisted of a white chamber (40 × 40 × 40 cm). The mice were allowed to adapt to the apparatus for one hour before the test. Each mouse was placed in the center and allowed to explore the arena for 10 min. The locomotor trajectories were automatically recorded with a video camera and analyzed with an ANY-Maze system (Stoelting, USA). The total distance traveled was measured to evaluate locomotor activity. The arena was cleaned with 70% ethanol after each trial.

Rotarod test

The rotarod test was used to assess motor coordination. Before testing, the mice were trained at a constant speed of 20 rpm for three sessions on three consecutive days. Each session included two separate trials. During the testing, the rotarod speed was increased from 0 to 40 rpm within 5 min. The latency to fall off the rod was recorded automatically for each mouse, and the average latency of three trials, with an interval of 15 min, was calculated for further analyses.

Balance beam test

The balance beam test was used to assess motor function. The apparatus consisted of a beam (1 cm wide and 100 cm long), which was placed 60 cm above the floor with a black shelter at one end. The mice were trained to cross a thicker beam (3 cm wide) three days before formal testing. On the testing day, each mouse was placed on one end. The time to cross the beam was recorded. Three consecutive trials with 30-min intervals were performed for each mouse to minimize individual variance. The mean time of three trials was calculated for analyses.

Generation of Stat1-KO MEFs

The plasmids for Streptococcus pyogenes Cas9 and sgRNA expression were a gift from Guanghui Wang (Soochow University, Suzhou, China). MEFs were cotransfected with the Cas9 expression plasmid and Stat1 sgRNA (5’-TACTACAGAGCAAACGCTGG-3’; 5’-GGAGAGTGTCGACCTGCTGG-3’) plasmid via Lipofectamine 2000. After transfection, MEFs were cultured under blasticidin (2 µg/ml) and puromycin (2 µg/ml) selection for two weeks. Single clones were picked for culture, and the KO efficiency was verified by western blotting.

Luciferase reporter gene assay

To assess Ulk1 promoter activity, we constructed a plasmid carrying a reporter (pGL3-Ulk1-luc) by cloning the Ulk1 promoter cDNA into a pGL3 enhancer vector with a luciferase reporter. The pGL3-Ulk1 mutant was obtained via site-directed mutagenesis via the wild-type pGL3-Ulk1 plasmid as a template with the following primer sequences: 5’-ccccgcagggaaacccaagatccgcggcac-3’; 5’-gtgccgcggatcttgggtttccctgcgggg-3’. The constructs were verified via DNA sequencing.

To evaluate the impact of STAT1 on Ulk1 promoter activity, HEK293T cells were cotransfected with pGL3-Ulk1-luc and a Flag-Stat1 or vector, along with a Renilla plasmid, to normalize the transfection efficiency. Twenty-four hours after transfection, dual-luciferase reporter assays were performed via a Vazyme kit (DL101-01; Nanjing, China). The Ulk1 promoter activity was expressed as the relative firefly luciferase activity over the Renilla luciferase activity.

Chromatin immunoprecipitation (ChIP)

The ChIP assay was carried out according to the protocol of the SimpleChIP® Plus Sonication Chromatin IP Kit (56383, CST, USA). In brief, BV2 cells were fixed with 1% formaldehyde solution for 10 min to crosslink proteins to DNA. Glycine was added to quench excess formaldehyde. After nuclear isolation, the chromatin was sonicated with a Bioruptor Plus sonication device (Diagenode, Belgium). ChIP was performed using 2 µg of either anti-STAT1 (9172, CST) or rabbit IgG at 4°C overnight. The protein‒DNA complexes were precipitated with protein G magnetic beads, followed by elution. Next, the crosslinking was reversed, and the DNA was purified. The enrichment of the ULK1 promoter fragments was assessed via qPCR with the following primers: forward, 5’- CATCAAGTGCTCCAACATGC-3’ and reverse, 5’- GTTAGAGGGGAAGTTTGAGG-3’. The results are presented as the percentage of input.

Statistical analyses

All the results are presented as the means ± SEM of at least three independent experiments unless otherwise specified. The statistical significance was evaluated via GraphPad Prism 8 (San Diego, CA, USA). Student’s t tests were used to compare the differences between two groups. For multiple group comparisons, statistical significance was determined by one-way analysis of variance (ANOVA) followed by Tukey’s test, Dunnett’s post hoc test, or two-way ANOVA followed by Tukey’s post hoc analysis, as indicated in the figure legends. Differences were considered significant at P < 0.05.