Cell Culture and Treatment

In this study, we utilized two cellular models to investigate neuroinflammatory processes and dopaminergic cell death: the murine microglial cell line BV2 for neuroinflammation studies, and the human neuroblastoma cell line SH-SY5Y for examining dopaminergic neuronal apoptosis. Both cell lines were obtained from the Central Laboratory of the First Affiliated Hospital of Nanchang University (Nanchang, China). BV2 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM; 11,965,084; Gibco), while SH-SY5Y cells were cultured in DMEM/F12 medium (11,330,032, Gibco). Both media were supplemented with 10% heat-inactivated fetal bovine serum (FBS; 10,099,141; Gibco) and 1% penicillin–streptomycin (100 U/mL penicillin and 100 μg/mL streptomycin; 15,140,122; Gibco). Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2 and passaged every 2–3 days when reaching 80–90% confluence. For experimental treatments, BV2 cells were seeded at a density of 1 × 105 cells/mL and allowed to adhere for 24 h prior to treatment. Neuroinflammation was induced by exposing cells to recombinant human α-synuclein protein (10 μg/mL; S7820; Sigma-Aldrich). LRRK2 kinase activity was inhibited using PF-06447475 (2 μg/mL; PZ0185; Sigma-Aldrich). To investigate ferroptotic mechanisms, cells were treated with either the ferroptosis inducer Erastin (10 μM; E7781, Sigma-Aldrich) or the ferroptosis inhibitor Ferrostatin-1 (0.5 μM; SML0583; Sigma-Aldrich). All pharmacological agents were reconstituted and stored according to the manufacturer’s specifications.

Experimental Animals and Treatments

Male C57BL/6 mice (8 weeks old, 22–25 g) were purchased from the Laboratory Animal Center of Nanchang University and maintained under controlled environmental conditions (22 ± 2 °C temperature, 55 ± 5% humidity, and a 12-h light/dark cycle) with ad libitum access to standard chow and water. Following a one-week acclimatization period, a total of 84 mice were included in the final analyses. All procedures involving animals were reviewed and approved by the Nanchang University Ethics Committee [Ethics Approval No. (2023)CDYFYYLK(05–017)].

To establish the Parkinson’s disease model, mice received intraperitoneal injections of MPTP-HCl (30 mg/kg body weight; Sigma-Aldrich) once daily for five consecutive days, whereas control animals received equivalent volumes of sterile saline (0.9% NaCl). PF-06447475 (20 mg/kg, twice daily) was administered via oral gavage, beginning two days prior to MPTP treatment and continuing throughout the 14-day experimental period. Oral gavage was performed using a 20 G blunt-ended stainless steel feeding needle (approximately 5 cm in length; sterilized before each use), ensuring accurate and safe drug delivery. This dosing protocol was previously optimized based on pilot studies to achieve maximal LRRK2 inhibition [33].

The study comprised two separate experiments. In Experiment 1, mice were randomly allocated to four groups: Saline control, 0 days post-MPTP, 7 days post-MPTP, and 21 days post-MPTP. In Experiment 2, mice were allocated to three groups: Saline control, MPTP, and MPTP + PF-06447475. Each group contained an equal number of animals, resulting in a total of 84 mice across both experiments. The cohort was systematically divided to facilitate multiple analytical procedures, including behavioral assessments and immunohistochemical evaluations (42 mice), Western blot analyses (21 mice), and RNA extraction followed by reverse-transcription quantitative real-time PCR (RT-qPCR) (21 mice) (Figures S1, Supporting Information).

At the conclusion of the experimental period, all mice were euthanized, and the substantia nigra pars compacta (SNpc) was rapidly dissected. Tissues were either processed immediately or flash-frozen in liquid nitrogen and stored at − 80 °C for subsequent analyses. For behavioral and immunohistochemical assays, 6 mice per group were used. For molecular and biochemical analyses, tissues were obtained from three independent animals per group, with all measurements performed in technical triplicates (see Supplementary Table S1).

Primary Microglia Extraction and Culture

Murine primary microglial cells were isolated from 1–3-day-old C57BL/6 mice (Laboratory Animal Center of Nanchang University, China) in accordance with the guidelines approved by the Nanchang University Ethics Committee. Briefly, whole brains were carefully dissected under aseptic conditions and transferred to ice-cold phosphate-buffered saline (PBS; pH 7.4). Meninges and visible blood vessels were meticulously removed, and the remaining cortical tissue was mechanically minced into small fragments before enzymatic digestion with 0.25% trypsin (Servicebio; G4012) at 37 °C for 15 min to liberate individual cells. The digestion was halted by adding an equal volume of Dulbecco’s Modified Eagle Medium (DMEM; 11,965,084, Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS; 10,099,141, Gibco) and 1% penicillin–streptomycin (15,140,122, Gibco). The cell suspension was passed through a 70-μm cell strainer to remove debris, centrifuged at 200 × g for 5 min, and resuspended in fresh DMEM containing 10% FBS and 1% penicillin–streptomycin. Subsequently, the cells were plated into T75 flasks and maintained at 37 °C in a humidified 5% CO2 incubator, with medium changes every 3 days. After approximately 10–12 days, when the mixed glial cultures reached confluence, the flasks were gently shaken at 200 rpm for 4 h at 37 °C to dislodge the loosely adherent microglia, which were collected from the supernatant. The harvested cells were centrifuged (200 × g, 5 min), resuspended in fresh DMEM supplemented with 10% FBS and 1% penicillin–streptomycin, and reseeded in appropriate culture plates or dishes for subsequent experiments. The isolated microglial population was assessed using immunostaining with the microglial marker Iba1, demonstrating that microglia consistently accounted for more than 95% of the cultured cells (Figures S2, Supporting Information).

RNA Extraction and Quantitative RT–PCR Analysis

Total RNA was isolated from BV2 cells utilizing the EZ-PRESS RNA Purification Kit (EZ-Bioscience) in accordance with the manufacturer’s instructions. The mRNA expression levels of TNF-α, IL-1β, IL-6, LRRK2, p62, Keap1, and Nrf2 were subsequently determined. The quantity and quality of extracted RNA were assessed using a NanoDrop™ spectrophotometer (Thermo Fisher Scientific), with samples exhibiting A260/A280 ratios between 1.8 and 2.0 being selected for subsequent analysis. Complementary DNA (cDNA) was synthesized from 1 μg of total RNA using the PrimeScript™ RT Reagent Kit (Takara Bio) under the following conditions: 37 °C for 15 min followed by 85 °C for 5 s. RT-qPCR was performed using SYBR® Premix Ex Taq™ II (Takara Bio) on a CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories). The relative expression levels of target genes were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal reference gene. The ΔΔCt method was utilized to compute and normalize the relative expression of each gene.

Western Blot

Western blotting was employed to determine the expression levels of proteins LRRK2, p62, Keap1, Nrf2, GPX4, p-p65, p65, p-rab10 and β-actin, in BV2 cells and mice. Cells were harvested and lysed in RIPA buffer (P0013B; Beyotime) supplemented with protease and phosphatase inhibitors (B14001 and B15001; Biotools). Protein concentrations were quantified using the Bicinchoninic Acid (BCA) Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of protein (20 μg) from each sample were separated by electrophoresis on 10% SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes (Solarbio). The membranes were blocked in 5% non-fat dry milk dissolved in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature and then incubated overnight at 4 °C with specific primary antibodies: anti-LRRK2 (ab133474; Abcam; 1:10,000), anti-p62 (D6M5X; Cell Signaling Technology; 1:1000), anti-Keap1 (ab227828; Abcam; 1:2000), anti-Nrf2 (D1Z9C; Cell Signaling Technology; 1:1000), anti-GPX4 (ab125066; Abcam; 1:1000), anti-p-p65 (Ser536; Cell Signaling Technology; 1:1000), anti-p65 (D14E12; Cell Signaling Technology; 1:1000), anti-p-rab10 (phospho T73; ab230261; Abcam; 1:1000) and anti-β-actin (ab8227; Abcam; 1:1000) as an internal loading control. Following primary antibody incubation, membranes were washed with TBST and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000; Cell Signaling Technology) for 1 h at room temperature. The immunoreactive bands were detected using an enhanced chemiluminescence (ECL) kit (Thermo Fisher Scientific) and densitometric analysis of band intensities was performed using ImageJ software, and the protein expression levels were normalized to those of β-actin.

Cell Viability Assessment

Cell viability was determined using the Cell Counting Kit-8 (CCK-8) assay (96,992; Sigma-Aldrich) according to the manufacturer’s instructions. BV2 microglial cells were seeded into 96-well plates at a density of 5 × 103 cells per well and allowed to adhere overnight. Following treatment with α-synuclein (10 μg/mL), PF-06447475 (2 μg/mL), Erastin (10 μM), or Ferrostatin-1 (0.5 μM) for 24 h, 10 μL of CCK-8 solution was added to each well containing 100 μL of culture medium. After 2 h incubation at 37 °C in a humidified atmosphere containing 5% CO2, the absorbance was measured at 450 nm using a microplate reader (SpectraMax M5, Molecular Devices). Cell viability was expressed as a percentage relative to the control group. Each experiment was performed in triplicate.

Transfection

To overexpress LRRK2 and p62 in BV2 microglial cells, we utilized plasmid constructs encoding the mouse LRRK2 (NM_025730) and p62 (NM_011018) genes, which were obtained from OriGene Technologies (Rockville). BV2 cells were seeded in 6-well plates at a density of 1 × 105 cells per well and allowed to adhere overnight under standard culture conditions (37 °C, 5% CO2) in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin. On the day of transfection, the culture medium was replaced with fresh DMEM, and the cells were transfected with 4 μg of the respective plasmid DNA (LRRK2 or p62) using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. After 6 h of incubation, the transfection medium was replaced with complete growth medium, and the cells were cultured for an additional 48 h before being harvested for further analyses. The efficiency of LRRK2 and p62 overexpression was confirmed by Western blot analysis.

CD40 Expression Analysis

BV2 microglial cells were seeded in 12-well plates at a density of 2 × 105 cells per well and cultured until reaching 70–80% confluency in complete growth medium under standard conditions (37 °C, 5% CO2). Cells were then subjected to various treatments including α-synuclein (10 μg/mL), PF-06447475 (2 μg/mL), Erastin (10 μM), and Ferrostatin-1 (0.5 μM) according to the experimental design parameters. Following the treatment period, cells were harvested using non-enzymatic cell dissociation solution to preserve surface protein integrity, and subsequently washed with ice-cold flow cytometry staining buffer (phosphate-buffered saline containing 2% fetal bovine serum). Surface staining was performed by incubating cells with FITC-conjugated anti-mouse CD40 monoclonal antibody (124,608; BioLegend) for 15 min at 4 °C in the dark. After incubation, cells were washed twice with staining buffer to remove unbound antibodies and resuspended in 500 μL of flow cytometry buffer supplemented. Cell fluorescence analysis was performed using an LSRII flow cytometer (BD Biosciences). Data acquisition and analysis were conducted using FlowJo software (TreeStar).

Enzyme-Linked Immunosorbent Assay (ELISA)

The concentrations of pro-inflammatory cytokines in cell culture supernatants were quantified using commercial ELISA kits. Briefly, BV2 microglial cells were seeded in 24-well plates at a density of 1 × 105 cells per well and allowed to adhere overnight. Following treatment with α-synuclein, PF-06447475, Erastin, or Ferrostatin-1 for 24 h, culture supernatants were collected and centrifuged at 1,000 × g for 10 min at 4 °C to remove cellular debris. The levels of pro-inflammatory cytokines, including IL-6 (PI326; Beyotime), tumor necrosis factor-alpha (TNF‐⍺) (PT512; Beyotime), and IL-1β (PI301; Beyotime), were measured according to the manufacturer’s protocols. The absorbance was measured at 450 nm with wavelength correction at 570 nm using a microplate reader (SpectraMax M5, Molecular Devices). All samples were analyzed in triplicate, and results were expressed as pg/mL.

Quantification of Intracellular Iron

Mouse BV2 microglial cells were cultured in 12-well plates at a density of 2 × 105 cells per well and allowed to reach 70–80% confluency. Cells were then subjected to treatment with α-synuclein (10 μg/mL), PF-06447475 (2 μg/mL), Erastin (10 μM), and Ferrostatin-1 (0.5 μM) according to the experimental protocol. Following treatment, cells were lysed, and cell lysates were collected by centrifugation. Intracellular iron levels were quantified using an Iron Assay Kit (Sigma‐Aldrich; MAK025) following the manufacturer’s instructions. Absorbance was measured at 595 nm using a microplate reader.

Measurement of ROS Production

Mouse BV2 microglial cells were seeded in 12-well plates at a density of 2 × 105 cells per well and cultured until 70–80% confluency was reached. Cells were then treated with α-synuclein (10 μg/mL), PF-06447475 (2 μg/mL), Erastin (10 μM), and Ferrostatin-1 (0.5 μM) based on experimental design. ROS production was assessed using DCFDA-DA (Sigma‐Aldrich). Cells were incubated with 10 μM DCFDA for 30 min at 37 °C. Fluorescence intensity was then measured using an LSRII cytometer (BD Biosciences). Data were analyzed using the FlowJo software program.

Measurement of Malondialdehyde (MDA) Levels

Mouse BV2 microglial cells were seeded in 12-well plates at a density of 2 × 105 cells per well and cultured until reaching 70–80% confluency. Cells were then treated with α-synuclein (10 μg/mL), PF-06447475 (2 μg/mL), Erastin (10 μM), and Ferrostatin-1 (0.5 μM) as per experimental requirements. Post-treatment, cells were harvested, and MDA levels were measured to assess lipid peroxidation. Cell lysates were prepared, and MDA levels were determined using a Lipid Peroxidation (MDA) Assay Kit (Sigma-Aldrich) following the manufacturer’s instructions. The assay involves the reaction of MDA with thiobarbituric acid (TBA) to form a colored complex, which was measured at 532 nm using a BioTek microplate reader.

Determination of Glutathione (GSH) Levels

Mouse BV2 microglial cells were cultured in 12-well plates at a density of 2 × 105 cells per well and allowed to reach 70–80% confluency. Cells were then treated with α-synuclein (10 μg/mL), PF-06447475 (2 μg/mL), Erastin (10 μM), and Ferrostatin-1 (0.5 μM) as needed for the specific experimental setup. Following treatment, cells were lysed and deproteinated, and the supernatants were analyzed using a Glutathione Assay Kit (S0053; Beyotime) according to the manufacturer’s instructions. Absorbance at 405 nm was measured using a microplate reader, and GSH concentrations were extrapolated from a standard curve and normalized to the protein concentration of the samples.

Immunofluorescence Staining

In the in vitro experiments, the cells used for immunofluorescence staining were primary microglia. Primary microglial cells were cultured on poly-D-lysine-coated glass coverslips in 12-well plates until reaching 70–80% confluence. Following experimental treatments, cells were fixed with 4% paraformaldehyde for 30 min at room temperature and washed thoroughly with phosphate-buffered saline (PBS, pH 7.4). Cell membranes were permeabilized using PBS containing 0.03% Triton X-100, and non-specific binding was blocked with 5% goat serum albumin (C0265; Beyotime). The cells were then incubated with primary antibodies diluted in blocking solution overnight at 4 °C. After extensive washing with PBS, cells were incubated with fluorophore-conjugated secondary antibodies for 1 h at room temperature in darkness. Nuclei were counterstained with DAPI (S2110; Solarbio). Immunofluorescence images were acquired using a Leica confocal laser scanning microscope.For in vivo studies, mice were deeply anesthetized and perfused transcardially with PBS followed by ice-cold 4% paraformaldehyde. Brains were carefully dissected and post-fixed in 4% paraformaldehyde for 48 h at 4 °C, followed by cryoprotection in 30% sucrose solution for an additional 48 h at 4 °C. Mesencephalic tissues were sectioned coronally (12 μm thickness) using a Leica freezing microtome. Sections were mounted on poly-D-lysine-coated slides and processed for immunofluorescence staining. The sections were then subjected to incubation with the specified primary antibodies. Subsequently, the slices were subjected to incubation with secondary antibodies.

The following primary antibodies were used: anti-LRRK2 (1:100), anti-p62 (1:400), anti-p-p65 (1:1000), anti-Iba1 (1:500), Cy3-conjugated goat anti-rabbit IgG (GB21303; 1:200; Servicebio) and Alexa Fluor 488-conjugated goat anti-mouse IgG (GB25301; 1:200; Servicebio). Immunofluorescence was visualized using a Zeiss LSM 880 laser scanning confocal microscope. Image acquisition and analysis were performed using ZEN light software (Zeiss). Quantitative analysis of immunofluorescence intensity was conducted using ImageJ software.

SNpc Sample Collection and Homogenization

At the conclusion of the experimental period, each mouse was euthanized, and the SNpc was carefully dissected from each animal individually. For Western blot, tissue from the SNpc of each mouse was immediately homogenized in ice-cold RIPA buffer supplemented with protease and phosphatase inhibitors. We used either a motorized homogenizer, ensuring thorough dissociation of the tissue. The homogenate was then centrifuged to collect the supernatant, and protein concentration was determined using the BCA assay.For RT-qPCR, the fresh SNpc was homogenized directly in the provided lysis buffer from the EZ-PRESS RNA Purification Kit (EZ-Bioscience). This approach allowed efficient tissue disruption and preserved RNA integrity for subsequent extraction and cDNA synthesis.

Immunohistochemistry

Immunohistochemistry staining was performed to analyze dopaminergic neurons in the substantia nigra. Briefly, mouse brain tissues were fixed in 4% paraformaldehyde for 24 h, dehydrated through a graded ethanol series, and embedded in paraffin. Coronal brain Sects. (5 μm thickness) encompassing the SNpc region were prepared using a rotary microtome. The sections were deparaffinized with xylene, rehydrated through decreasing concentrations of ethanol, and subjected to heat-mediated antigen retrieval in citrate buffer (10 mM, pH 6.0) for 20 min. Endogenous peroxidase activity was quenched with 3% H2O2 for 10 min, followed by blocking with 5% bovine serum albumin (BSA) in PBS for 1 h at room temperature. Dopaminergic neurons were identified by overnight incubation at 4 °C with anti-tyrosine hydroxylase (TH) primary antibody (25,859–1-AP; 1:5000; Proteintech). After washing with PBS, sections were incubated with an HRP-conjugated goat anti-rabbit IgG secondary antibody (SA00001-2; Proteintech) for 1 h at room temperature. Streptavidin-HRP ABC solution (PK-4001; Vector Laboratories) was then applied according to the manufacturer’s instructions. The immunoreactive signals were visualized using 3,3′-diaminobenzidine (DAB) as a chromogen. Images were captured using a Leica confocal microscope and analyzed with ImageJ software.

Molecular Docking

Molecular docking analysis was performed using the HDOCK server (http://h-dockphys.hust.edu.cn/) to evaluate the potential interaction between LRRK2 and p62. The three-dimensional structure of LRRK2 was retrieved from the Protein Data Bank (PDB ID: 7li4), while the p62 structure was obtained from the AlphaFold protein database. Prior to docking simulation, protein structures were processed using PyMOL software (version 2.3.0, https://pymol.org) to remove original ligands, water molecules, and other organic compounds. The structures were further optimized using the "prepare" module in Discovery Studio software for hydrogenation and protonation states adjustment. The protein–protein interactions were analyzed using LigPlus software to evaluate the two-dimensional binding forces, while the protein–protein interaction interface was examined using the "analysis interface" module in Discovery Studio. The visualization of interacting amino acid residues between LRRK2 and p62 was accomplished using PyMOL software (version 2.3.0).

Microglial Supernatant Transfer Model

BV2 microglial cells were seeded in 6-well plates at a density of 2 × 105 cells/well and cultured in DMEM supplemented with 10% FBS until reaching 70–80% confluence. Cells were then exposed to various treatments: α-synuclein (10 μg/mL), PF-06447475 (2 μg/mL), Erastin (10 μM), or Ferrostatin-1 (1 μM) for 24 h under standard culture conditions (37 °C, 5% CO2). Following the treatment period, conditioned media were collected and centrifuged at 1000 × g for 10 min at 4 °C to remove cellular debris. The supernatants were then filtered through 0.22 μm sterile filters.SH-SY5Y neuroblastoma cells were pre-seeded in 6-well plates at a density of 1.5 × 105 cells/well and allowed to adhere for 24 h. The filtered BV2-conditioned media were mixed with fresh complete medium at a 1:1 ratio and applied to SH-SY5Y cells. Following 24 h of exposure to the conditioned media, SH-SY5Y cells were harvested and analyzed for apoptosis using Annexin V-FITC/PI double staining followed by flow cytometric analysis.

Apoptosis Analysis

After 24 h of treatment with BV2 microglial supernatants, SH-SY5Y cells were collected for apoptosis analysis using the Annexin V-FITC/PI Apoptosis Detection Kit (LiankeBio). SH-SY5Y cells were stained with Annexin V-FITC and Propidium Iodide (PI) according to the manufacturer’s protocol provided by LiankeBio.Stained cells were then analyzed using a flow cytometer (BD Biosciences) to determine the percentage of apoptotic cells based on Annexin V-FITC and PI staining patterns.

Mouse Behavioral Scoring System

A comprehensive scoring system was employed to assess the severity of motor and non-motor symptoms in the mouse model. The scoring criteria were as follows:

1 point: Mice exhibited reduced escape behavior, piloerection, yellowing and soiling of fur, hunched posture, and decreased voluntary activity.

2 points: In addition to the symptoms described for 1 point, mice displayed a marked reduction in voluntary activity, lethargy, and potential tremors or unstable gait.

4 points: Mice presented with symptoms as described for 2 points, along with an unstable gait, inability to walk in a straight line, or rotational walking.

6 points: Mice demonstrated lateral recumbency, paralysis of one side’s forelimb and/or hindlimb, difficulty in walking and feeding.

8 points: Mice exhibited complete paralysis of one side’s forelimb and/or hindlimb, spasticity of limbs, significant weight loss, and inability to feed independently.

10 points: Mice were near death or deceased. Scores ranging from 2 to 6 were considered indicative of successful modeling of the disease phenotype.

Mouse Behavioral Assessments

Rotarod Test: To evaluate motor coordination and balance, mice were subjected to the rotarod test. Prior to testing, mice underwent a training session on the apparatus, maintaining a constant speed of 20 rotations per minute (rpm) for a duration of 5 min. During the test, mice were placed on an accelerating rod, with the speed gradually increasing from 4 to 40 rpm over a period of 300 s. The latency to fall, defined as the time the mice were able to remain on the accelerating rod, was recorded for each animal. Pole Test: The pole test was employed to assess motor function and coordination. A sturdy wooden pole, measuring 50 cm in length and 1 cm in diameter, was vertically affixed to a base. At the top of the pole, a wooden ball with a diameter of 2 cm was attached. Mice were carefully placed on the ball and allowed to descend the pole. The time required for the mouse’s front paws to reach the base of the pole from the moment of release was measured. Each mouse underwent three consecutive trials, with a 30-min interval between each trial. The average time across the three trials was calculated and recorded for data analysis.

Statistical Analysis

All experiments were performed in triplicate unless otherwise specified. Data are presented as the mean ± standard deviation (SD). Statistical analyses were conducted using GraphPad Prism 9.0 software. The normality of the data distribution was assessed using the Shapiro–Wilk test. For comparisons between two groups, an unpaired two-tailed Student’s t-test was employed. For comparisons among multiple groups, one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was utilized. A p-value less than 0.05 was considered statistically significant.