Animals and Ethics
Male C57BL/6J mice (7–8 weeks old, 22–25 g) were procured from Nanjing Gempharmatech Co. Ltd. A53T transgenic (A53T) mice were obtained from Beijing HuAFukang Biotechnology Co., Ltd. The mice were housed in a specific pathogen-free animal facility under standard conditions, maintained at 21–25° C with a 12-h light/dark cycle, and free access to food and water. A53T mice began to exhibit behavioral abnormalities and arched back at 8–9 months, and most mice had obvious movement disorders at 14–15 months. In this study, male A53T mice aged 10–12 months with onset disease were used for follow-up experiments, and C57BL/6J mice matched by age and sex were used as WT controls. All animal procedures were approved by the institutional animal care and use committee of Tongji Hospital.
Establishment of the PD mouse model and drug treatment
A subacute PD mouse model was established using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Twenty-five mice were divided randomly into three groups: control group (PBS), model group (MPTP), and positive drug group (2-DG+MPTP). Mice in the MPTP group and 2-DG+MPTP group were intraperitoneally injected with MPTP (MPTP, 30 mg/kg, Sigma) once daily for five consecutive days, while mice in the PBS group received a similar volume of PBS injected intraperitoneally. For 2-DG treatment, mice in the 2-DG+MPTP group were intraperitoneally injected with 2-DG (250 mg/kg) 3 days before receiving the MPTP injection; 2-DG was administered 2 h before the MPTP injection. Mice in the PBS group and MPTP group received a similar volume of PBS injected intraperitoneally.
An inflammatory PD mouse model was established by injecting LPS into the SN. Briefly, 30 mice were divided randomly into three groups: control group (PBS), model group (LPS), and positive drug group (2-DG+LPS). For the LPS group and 2-DG+LPS group, 2.5 μg of LPS was injected into the right SN using a stereotaxic apparatus, while mice in the PBS group received a similar volume of PBS. For 2-DG treatment, mice in the 2-DG+LPS group were intraperitoneally injected with 2-DG (250 mg/kg) 3 days before the LPS injection and continued for 5 days after the LPS injection. Mice in the PBS group and MPTP group received a similar volume of PBS by intraperitoneal injection.
To explore the effect of the SLC7A11 inhibitor sulfasalazine (SAS) on microglial activation, 32 mice were divided randomly into four groups: control group (PBS), model group (LPS), drug group (SAS), and drug+ model group (SAS+LPS). We first established an inflammatory PD mouse model. For SAS treatment, 150 mg/kg of SAS was administered intraperitoneally twice daily before LPS stereotaxic injection and continued for 7 days after LPS injection. Mice in the PBS group and LPS group received a similar volume of PBS injected intraperitoneally.
Behavioral test
Behavioral tests were performed on day 7 after the last MPTP administration and LPS injection.
Rotarod test: The Rotarod Treadmill (IITC) was used after a three-day training period. In the formal test, mice were placed on the rotarod instrument, set to uniformly accelerate from 5 to 45 rpm over 5 min. Each mouse underwent three trials with at least 30 min between tests, and the results were averaged. The time that mice stayed on the rotating axis before falling was recorded.
Pole test: A vertical wooden pole 50 cm long and 1 cm in diameter, with a cork at the top was employed. Before the formal test, the mice were trained for two consecutive days; each training session comprised three test trials. In the formal test, mice were placed on the cork, and the time it took to climb from the cork to the bottom of the pole was recorded.
Open-field test (OFT): Mice were placed and allowed to explore freely in an open-field reaction box (30 cm in length, 30 cm in width, and 35 cm in height) for 5 min. The ANY-MAZE video tracking system (Version 7.16, Stoelting, USA) was used to record their movement distance and immobility time.
Preparation of brain samples
After behavioral testing, mice were euthanized using 5% isoflurane to induce deep anesthesia. For immunofluorescence analysis, mice underwent cardiac perfusion with 30 mL of pre-cooled 1% phosphate-buffered saline (PBS) followed by 30 mL of pre-cooled 4% paraformaldehyde (PFA). After perfusion, the brain was removed, post-fixed in 4% PFA at 4 °C for 12 h, and then completely dehydrated in 30% sucrose. Serial 20 μm coronal sections were cut by a constant temperature (-23 °C) frozen slicer. For qPCR and western blot analysis, mice were perfused with 30 mL of pre-cooled 1% PBS. Their brains were extracted, rapidly frozen in liquid nitrogen-cooled isopentane, and stored at -80 °C.
Measurement of lactate concentration
Lactate concentrations in the cell culture medium were determined using a Lactate Assay Kit (A019-2-1, Jiancheng Bioengineering Institute, Nanjing, China) following the manufacturer's recommendations. Lactate concentrations in the SN and striatum were measured using the L-Lactate Assay Kit (Colorimetric/Fluorometric, ab65330, Abcam) according to the manufacturer's protocol.
Immunofluorescence staining
Brain slides were permeabilized with 0.25% Triton X-100 and blocked with QuickBlock™ Blocking Buffer (Beyotime, Nanjing, China) for 15 min. Brain sections were then incubated with primary antibodies for 12 h at 4 °C: rabbit anti-tyrosine hydroxylase (TH) (1:500; Abcam), sheep anti-TH (1:500; Novus), goat anti-IBA1 (1:500; Abcam), rat anti-CD68 (1:500; Bio-Rad), rat anti-CD86 (1:500; Abcam), rabbit-Mannose Receptor 1 (MRC1/CD206), rabbit anti-Lactate dehydrogenase A (LDHA) antibody (1:200, Cell Signaling Technology, CST), mouse anti-GFAP (1:200, CST), mouse anti-NeuN antibody (1:100, Proteintech), rabbit anti-pan histone lysine lactylation (pan-Kla) (1:100; PTM BIO), rabbit anti-H3K9la (1:100; PTM BIO), and rabbit anti-SLC7A11 antibody (1:100, Proteintech). Subsequently, slides were incubated with Alexa Fluor 488 or Alexa Fluor 594 (Yeasen Biotechnology) in the dark at room temperature for 1 h, and then stained with 4,6-diamidino-2-phenylindole. Images were captured using fluorescence and confocal microscopy (Olympus FV1200, Japan) and analyzed using ImageJ software (Java 1.8.0, National Institutes of Health, USA).
Culture and stimulation of primary microglia
Primary microglia were isolated from neonatal (P0–P3) C57BL/6J mice. Briefly, the brain tissue was digested with 0.125% trypsin for 15 min at 37 °C. The cells were resuspended in DMEM/F12 medium containing 20% fetal bovine serum and cultured in a 5% CO2 incubator at 37 °C. After 36 h, the medium was replaced with high-glucose DMEM containing 20% fetal bovine serum. After 10–12 days of culture, microglia were isolated from the mixed glial cultures.
Primary microglia were stimulated with 0.2 µg/mL LPS (0111: B4, Sigma-Aldrich, St. Louis, MO, USA) for 24 h. In some experiments, microglia were pretreated with 2-DG (0.5, 1, and 2 μM) to inhibit glycolysis, sodium oxalate (10 μM) to reduce the production of lactate, C646 (5 μM) to inhibit p300/CBP, or erastin (20 μM) to block SLC7A11 before addition of LPS. siRNA was used to knock down p300, Cbp, Ldha, and Slc7a11, and transfection was performed using Lipofectamine 3000. The cells were collected for subsequent experiments.
Primary microglia-conditioned medium (MCM) treatment SH-SY5Y cells
Briefly, primary microglia were treated with 20 μM erastin for 30 min and then with 0.2 µg/mL LPS for 4 h, followed by medium replacement. After 24 h, the medium was collected and mixed with fresh medium in a 1:1 ratio to obtain an MCM (Figure S5F). The MCM was used to stimulate human neuroblastoma SH-SY5Y cells, and the activity of cells was evaluated by CCK8 assay.
Cell viability assay
Cell viability was assessed using the CCK-8 kit (Yeasen, Shanghai, China). Briefly, 100 µL of medium containing 10 µL of CCK-8 reagent was added to each well and incubated at 37 °C for 4 h. Absorbance was measured at 450 nm using a microplate reader (Thermo Fisher Scientific).
Western blot analysis
Primary microglia and brain tissue were lysed in RIPA buffer with PMSF, phosphatase inhibitor, and a protease inhibitor cocktail (Servicebio, Wuhan, China). Protein concentrations were measured using a bicinchoninic acid assay kit (Beyotime, Shanghai, China). Equal amounts of protein extracts (20–40 μg) were separated by 8–15% SDS-PAGE and transferred onto 0.22 μm nitrocellulose membranes. The membranes were blocked with Quick Block western blocking buffer and incubated with primary antibodies for 12 h at 4 °C and subsequently with secondary antibodies (Servicebio, Wuhan, China) for 1 h at room temperature. Images were acquired using an imaging system (GelView 6000 Pro, Guangdong, China). The signals' integrated optical density (OD) was semi-quantified with ImageJ, using β-actin, GAPDH, tubulin, or Histone 3 as internal references. The primary antibodies used as follows: anti-TH (1:1000; Abcam), anti-LDHA (1:1000, CST), anti-Hexokinase 2 (HK2; 1:1000, Abcam), anti-Interleukin-1β (IL-1β) antibody (1:1000; Abcam), anti-IL-6 (1:1000, CST), anti-inducible nitric oxide synthase (iNOS; 1:1000; Abcam), anti-tumor necrosis factor-α (TNF-α; 1:1000, CST), anti-Arginase 1 (ARG1; 1:1000, CST), anti-NOD-like receptor thermal protein domain associated protein 3 (NLRP3; 1:1000, abclonal), anti-CREB-binding protein C (CREB-BP, CBP; 1:1000, Affinity), anti-p300 (1:1000, Affbiotech), anti-SLC7A11 (1:100, Proteintech), anti-pan-Kla, H3K9la, H3K18la, H3K56la, H4K12la, H4K8la, H4K5la, and H4K16la (1:1000; PTM BIO). Secondary antibody reactions were anti-mouse or anti-rabbit IgG-HRP antibodies (1:5000; Cat#SA00001-2; Proteintech).
Quantitative real-time PCR (qPCR)
Total RNA was isolated from brain tissue or primary microglia using RNAiso Plus reagent (Beijing Tsingke Biology Co., Ltd.). cDNA was synthesized using a PrimeScript RT Reagent Kit (Vazyme). Quantitative real-time PCR (qPCR) was performed on a CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories, Inc, Hercules, USA) or LightCycler 480II (Roche Diagnostics GmbH, Mannheim, Germany) using the SYBR Green Quantitative RT-PCR Kit (Yeasen Biotechnology Co., Ltd. Shanghai). Relative transcription levels were determined using the 2−ΔΔCT method with Actb as the reference gene. Primer sequences used in this study are listed in Table. S1.
Table S1. Primer sequences.
Gene name
|
Species
|
Primer direction
|
Primer sequence
|
Actb
|
Mouse
|
Forward primer
|
GTACTCTGTGTGGATCGGTGG
|
|
|
Reverse primer
|
AAAACGCAGCTCAGTAACAGTC
|
Nos2
|
Mouse
|
Forward primer
|
GTTCTCAGCCCAACAATACAAGA
|
|
|
Reverse primer
|
GTGGACGGGTCGATGTCAC
|
Glut1
|
Mouse
|
Forward primer
|
CCCCGTCCTGCTGCTATTG
|
|
|
Reverse primer
|
GCACCGTGAAGATGATGAAGAC
|
Hk2
|
Mouse
|
Forward primer
|
ATGATCGCCTGCTTATTCACG
|
|
|
Reverse primer
|
CGCCTAGAAATCTCCAGAAGGG
|
Ldha
|
Mouse
|
Forward primer
|
TGTCTCCAGCAAAGACTACTGT
|
|
|
Reverse primer
|
GACTGTACTTGACAATGTTGGGA
|
Nlrp3
|
Mouse
|
Forward primer
|
TGGATGGGTTTGCTGGGAT
|
|
|
Reverse primer
|
CTGCGTGTAGCGACTGTTGAG
|
Il1b
|
Mouse
|
Forward primer
|
TCTTTGAAGTTGACGGACCC
|
|
|
Reverse primer
|
TGAGTGATACTGCCTGCCTG
|
Tnf
|
Mouse
|
Forward primer
|
AGACCCTCACACTCACAAACCAC
|
|
|
Reverse primer
|
GCACGTAGTCGGGGCAGC
|
Il6
|
Mouse
|
Forward primer
|
TAGTCCTTCCTACCCCAATTTCC
|
|
|
Reverse primer
|
TTGGTCCTTAGCCACTCCTTC
|
Mrc1
|
Mouse
|
Forward primer
|
CTCTGTTCAGCTATTGGACGC
|
|
|
Reverse primer
|
TGGCACTCCCAAACATAATTTGA
|
Arg1
|
Mouse
|
Forward primer
|
TTGGGTGGATGCTCACACTG
|
|
|
Reverse primer
|
GTACACGATGTCTTTGGCAGA
|
Ym1
|
Mouse
|
Forward primer
|
CAGGGTAATGAGTGGGTTGG
|
|
|
Reverse primer
|
CACGGCACCTCCTAAATTGT
|
p300
|
Mouse
|
Forward primer
|
GAACAGGAAGAGGAAGAGAGGAAAC
|
|
|
Reverse primer
|
TGAGAAAGGTCATTAGACACATTGG
|
Crebbp
|
Mouse
|
Forward primer
|
GGCCAGGACCGCTTTGTTTATA
|
|
|
Reverse primer
|
ATCTTATGGGTGTGGCTCTTTGT
|
Slc7a11
|
Mouse
|
Forward primer
|
CTTTGTTGCCCTCTCCTGCTTC
|
|
|
Reverse primer
|
CAGAGGAGTGTGCTTGTGGACA
|
Cd80
|
Mouse
|
Forward primer
|
ACCCCCAACATAACTGAGTCT
|
|
|
Reverse primer
|
TTCCAACCAAGAGAAGCGAGG
|
Cd86
|
Mouse
|
Forward primer
|
TGTTTCCGTGGAGACGCAAG
|
|
|
Reverse primer
|
TTGAGCCTTTGTAAATGGGCA
|
Nlrp3
|
Mouse
|
Forward primer
|
TGGATGGGTTTGCTGGGAT
|
|
|
Reverse primer
|
CTGCGTGTAGCGACTGTTGAG
|
RNA-sequencing
RNA was extracted using TRIzol, and its quality was assessed using the NanoDrop and Agilent systems. The mRNA was purified, fragmented, and used for cDNA synthesis. The library was generated using Hieff NGS® DNA Selection Beads and quantified using a Qubit. The library was then sequenced using DNBSEQ-T7 (Wuhan Bioyi Biotechnology Co., Ltd.). Differential gene expression analysis was performed using DESeq2 (v1.30.1) with the following filtering criteria: fold change |log2FoldChange| > 1 and P < 0.05.
Cleavage under targets and tagmentation (CUT&Tag)
The CUT&Tag experiment was performed using the Hyperactive In-Situ ChIP Library Prep Kit for Illumina (pG-Tn5) (TD901, Vazyme, Nanjing, China). Briefly, the primary microglia were collected and bound to Concanavalin A-coated beads. The cells were then resuspended in an antibody buffer and sequentially incubated with primary antibodies against H3K9la and secondary antibodies. The samples were treated with pA/pG-Tn5 transposase. After transposon activation and tagmentation, DNA was isolated, amplified, and purified to create a library. VAHTS DNA Clean Beads (N411, Vazyme, Nanjing, China) were used during the purification steps of the library construction process. The library was quantified using the VAHTS Library Quantification Kit for Illumina (Vazyme Biotech) and sequenced on an Illumina NovaSeq 150PE.
Chromatin immunoprecipitation assay (ChIP)-qPCR
ChIP analysis was carried out with an anti-H3K9la antibody (PTM Bio, Hangzhou, China) according to the manufacturer's instructions of the ChIP Assay Kit (CST, #9005). Fold enrichment was determined by qPCR and expressed as a percentage of input chromatin (percentage of input). The primer sequences for the Slc7a11 promoter were 5′-CACTGTGGCAAGCCCTACA TA-3′ (forward) and 5′-CAGTGTAGGCAGGTCCCA TC-3′ (reverse).
Statistical analysis
Statistical analyses were performed using GraphPad Prism version 8.0.1. Data are presented as mean ± standard error of the mean (SEM). Two-tailed Student's t-tests or one-way analysis of variance (ANOVA) followed by Tukey's multiple test was performed to compare data among groups. P < 0.05 was considered statistically significant.