Mettl3/Nrf2 Axis Suppresses Parkinson's Disease Progression via Inhibiting NLRP3-Induced Pyroptosis in Serotonin Neurons

Summary

Here, we present a protocol to investigate how Mettl3 regulates Nrf2 via m6A modification, thereby suppressing microglial pyroptosis and safeguarding serotonin neurons in Parkinson's disease models, with applications in epitranscriptomic neuroinflammation research.

Abstract

The precise mechanisms underlying Parkinson's disease (PD) pathogenesis remain incompletely understood, particularly regarding the role of microglial inflammation and serotonin neuron survival. This protocol delineates a comprehensive framework for elucidating how methyltransferase-like 3 (Mettl3) modulates nuclear factor erythroid 2-related factor 2 (Nrf2) through N6-methyladenosine (m6A) modification, thereby attenuating microglial pyroptosis and preserving serotonin neurons in both in vitro and in vivo PD models. The primary goal is to furnish researchers with reproducible methodologies for dissecting epitranscriptomic regulation of neuroinflammatory pathways, commencing with lipopolysaccharide (LPS)-induced microglial activation in BV2 cells to simulate inflammatory cascades, followed by methylated RNA immunoprecipitation quantitative PCR (MeRIP-qPCR) for m6A analysis. In vivo, we detail the establishment of an MPTP-induced PD mouse model, complemented by stereotactic delivery of adeno-associated virus serotype 9 (AAV9) vectors for targeted Nrf2 modulation in the striatum. Behavioral evaluations encompass forelimb placement, accelerating rotarod, and open field tests to quantify motor deficits, while molecular assays include Western blotting for pyroptosis markers (e.g., NLRP3, cleaved-caspase-1), enzyme-linked immunosorbent assay (ELISA) for cytokines, and dihydroethidium (DHE) staining for reactive oxygen species (ROS) detection in serotonin neurons. Advanced microscopy techniques, such as immunohistochemistry for Iba1 and TPH2, enable visualization of microglial dynamics and serotonergic integrity. Results substantiate that Mettl3 deficiency exacerbates Nrf2 downregulation, NLRP3 inflammasome hyperactivation, pyroptotic cell death, and consequent serotonin neuron degeneration. This method not only provides a robust experimental scaffold for probing m6A-mediated neuroprotection but also highlights potential therapeutic avenues for mitigating PD progression through targeted modulation of the Mettl3/Nrf2 axis in neurodegenerative contexts.

Introduction

Parkinson's disease (PD) ranks as the second most prevalent neurodegenerative disorder among the elderly, affecting around 2-3% of individuals aged 65 and older, which poses a significant burden on both families and society¹. Although the precise mechanisms behind PD remain partially understood, accumulating evidence indicates a connection between PD development and challenges in neuronal transmission, along with neuroinflammation driven by microglial cells, which is a common trait seen in aging brains and various neurodegenerative diseases, including PD^2,3,4,5. Microglia serve as the innate immune cells within the central nervous system (CNS) and are vital for sustaining brain homeostasis⁶. However, persistent overactivation of these microglia can trigger chronic neuroinflammatory responses, ultimately contributing to neurodegenerative disease progression.

Both central and peripheral inflammatory processes significantly influence the pathology of PD^7,8. The activation of microglial cells prompts inflammatory responses that impact neuronal survival⁹, with emerging evidence highlighting its priming by ubiquitin ligases¹⁰. Among various inflammatory pathways, NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome activation serves as a primary contributor to microglial inflammatory regulation¹¹. Activation leads to NLRP3 expression and subsequent assembly of the inflammasome complex composed of the caspase activation and recruitment domain (CARD) adapter protein and pro-caspase-1, culminating in protein cleavage and cytokine release¹². Elevated NLRP3 activation has been observed in PD patients and various animal models of the disease, resulting in neuronal death. Notably, inhibiting NLRP3 has demonstrated protective effects against PD pathology in mouse models, underscoring the NLRP3 inflammasome's crucial role in PD onset^13,14.

Serotonin (5-HT) signaling is a significant mechanism of neural regulation, influencing numerous behaviors and physiological functions through interactions with at least 14 postsynaptic receptor subtypes^15,16, including roles in CNS pathology as detailed in comprehensive overviews¹⁷. The extensive neuromodulatory influence of the 5-HT system is governed by approximately 26,000 neurons in the rodent brain¹⁸. While substantial literature associates PD predominantly with dopaminergic neuron loss, the relationship between 5-HT neurons and PD is less thoroughly examined.

N6-methyladenosine (m6A) modification, the most prevalent mRNA modification in eukaryotic cells, is key to regulating mRNA splicing, stability, and export, thereby influencing various cellular activities¹⁹. m6A levels are modulated by methyltransferases and demethylases. Elevated m6A modifications in the brain have been linked to neurodevelopment, with its dysregulation being closely tied to neurodegenerative conditions^20,21, including altered METTL3 expression in Alzheimer's models²². For example, the accumulation of methyltransferase-like 3 (METTL3) in the insoluble fraction of post-mortem brain tissues from Alzheimer's patients has been positively correlated with levels of insoluble Tau protein²³. Furthermore, a significant decrease in m6A levels in the striatum can lead to a substantial decline in dopamine neurotransmitter levels²⁴. Notably, twelve m6A-related single-nucleotide polymorphisms have shown significant associations with PD susceptibility²⁵. This protocol establishes comprehensive methodologies for investigating how METTL3-mediated m6A modifications regulate microglial pyroptosis through the Nrf2/NLRP3 axis, ultimately affecting serotonin neuron survival in PD models. The acute MPTP in vivo model and LPS in vitro model were selected for their robust induction of neuroinflammatory responses, though chronic paradigms could complement future studies as discussed below.

Protocol

All animal experiments were conducted under the approval of the Institutional Animal Care and Use Committee of the Feicheng People's Hospital (approval number IACUC-2024-118) and performed in strict accordance with institutional guidelines and established ethical principles for laboratory animal research. The study protocols ensured humane care and treatment of all animals, with particular emphasis on minimizing suffering and distress, while adhering to both institutional standards and ARRIVE guidelines for responsible animal research practices.

1. Cell culture and microglial activation model

1. BV2 cell preparation and maintenance
   1. Rapidly thaw frozen BV2 microglial cell stocks in a 37 °C water bath for 2 min, ensuring gentle swirling to prevent thermal shock.
   2. Centrifuge the thawed cell suspension at 300 × g for 5 min at room temperature (RT) to remove cryoprotectant.
   3. Discard the supernatant and resuspend the cell pellet in 10 mL of complete high-glucose DMEM medium supplemented with 10% FBS, 1% penicillin-streptomycin, and 2 mM L-glutamine.
   4. Perform cell viability assessment using the trypan blue exclusion method (mix 10 µL of cell suspension with 10 µL of 0.4% trypan blue, count using hemocytometer), ensuring >95% viability before proceeding.
   5. Plate cells in T75 culture flasks at a density of 1 × 10⁶ cells per flask and maintain in a humidified cell culture incubator at 37°C with 5% CO₂.
   6. Passage cells every 48 h when reaching 80-85% confluency using standard trypsinization procedures.
      NOTE Maintain consistent passage numbers (passages 3-8) to ensure reproducible inflammatory responses. All cell culture experiments were independently repeated at least three times, with three technical replicates per condition to minimize variability.
2. LPS-induced microglial activation
   1. Seed BV2 cells in 6-well plates at 2 × 10⁵ cells per well in 2 mL of complete medium 24 h before treatment.
   2. Prepare LPS stock solution at 1 mg/mL in sterile PBS, filter-sterilize through a 0.22 µm filter, and store in single-use aliquots at -20 °C.
   3. Validate LPS activity using Limulus Amebocyte Lysate (LAL) assay to confirm endotoxin concentration before each experiment²⁶.
   4. Dilute LPS stock to a working concentration of 1 µg/mL in serum-free DMEM immediately before use.
   5. Remove culture medium from wells and wash cells twice with sterile PBS.
   6. Add 2 mL of LPS-containing medium (1 µg/mL final concentration) to treatment wells and serum-free DMEM to control wells.
   7. Incubate cells for 24 h at 37 °C with 5% CO₂ for inflammatory activation.
   8. Include positive control wells treated with 100 ng/mL tumor necrosis factor (TNF)-α and negative control wells with vehicle only (PBS) to validate the inflammatory response.
      NOTE: Prepare fresh LPS solution for each experiment to maintain consistent bioactivity. If the inflammatory response is weak (e.g., <2-fold cytokine increase), check reagent stability or extend incubation to 48 h.
3. Mettl3 overexpression and knockdown
   1. Design and synthesize small interfering RNA (siRNA) sequences targeting Mettl3 and scrambled control sequences using standard oligonucleotide synthesis protocols. Select target sequences from the coding region of Mettl3 mRNA (GenBank accession: NM_019721) with 19-21 nucleotide length, ensuring GC content of 40-60%²⁷.
   2. Validate siRNA sequences using BLAST analysis to confirm specificity and avoid off-target effects (ensure <70% homology with non-target genes).
   3. Prepare overexpression vectors containing full-length Mettl3 cDNA cloned into the pcDNA3.1 vector using restriction sites EcoRI and XhoI.
   4. Transfect cells at 70-80% confluency using a cationic lipid transfection reagent:
      1. Mix 2 µL of cationic lipid transfection reagent with 50 pmol siRNA or 2 µg of plasmid DNA in 100 µL of Opti-MEM medium.
      2. Incubate mixture for 15 min at RT.
      3. Add the transfection complex dropwise to cells and incubate for 4-6 h before changing to fresh complete medium.
   5. Incubate transfected cells for 48 h before LPS treatment to allow adequate protein expression changes.
   6. Confirm transfection efficiency using fluorescent reporter co-transfection (100 ng of pEGFP-C1 per well), targeting >70% efficiency by fluorescence microscopy before proceeding.

2. Animal model development and experimental design

1. Animal preparation and randomization
   1. Obtain C57BL/6J mice from Vital River Laboratory, ensuring equal male-to-female ratios, aged 8 weeks, weighing 19-26 g.
   2. House animals in specific pathogen-free (SPF) conditions with 12:12 h light-dark cycle, controlled temperature (22 ± 2°C), and humidity (50-60%).
   3. Allow a 7-day acclimatization period with ad libitum access to standard chow and water.
   4. Perform baseline behavioral assessments during acclimatization: Conduct comprehensive behavioral evaluations, including forelimb placement test (10 trials per side), accelerating rotarod assessment (4-40 rpm over 5 min), and open field locomotor activity analysis (10-min sessions in a 50 cm × 50 cm × 50 cm apparatus)^28,29,30.
   5. Randomly assign animals to experimental groups (n = 6 per group) using computerized randomization to minimize bias: sham, Model, Model+Nrf2-KD, Model+oe-Nrf2.
   6. Implement a blinded experimental design where investigators performing behavioral assessments are unaware of group assignments.
2. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease model
   1. Prepare MPTP solution at 30 mg/kg in sterile saline immediately before injection by dissolving 15 mg of MPTP hydrochloride in 5 mL of sterile saline and adjusting volume based on animal weight.
      ​CAUTION: MPTP is highly neurotoxic. Handle in a chemical fume hood with appropriate personal protective equipment, including double gloves and a lab coat.
   2. Administer MPTP via intraperitoneal injection at 30 mg/kg body weight (injection volume 10 mL/kg) for three consecutive days at the same time each day (09:00 ± 30 min).
   3. Inject sham group animals with equivalent volumes of sterile saline following an identical injection schedule.
   4. Monitor animals for signs of distress, weight loss (>15% considered endpoint), or adverse reactions daily during the MPTP administration period using standardized scoring sheets.
   5. Maintain animals for 8 days post-final injection to allow neurodegeneration development.
      NOTE: Animals can be housed normally during this period with continued monitoring.
3. Stereotactic viral injection
   1. Prepare AAV9 viral vectors by triple transfection of HEK293T cells using polyethylenimine (PEI; 1:3 DNA:PEI ratio), harvest at 72 h post-transfection, purify using iodixanol gradient ultracentrifugation (177,000 × g for 2 h), and concentrate using centrifugal filter devices to achieve 1 × 10¹² genome copies/mL.
   2. Validate viral titer via quantitative polymerase chain reaction (PCR) using primers targeting the ITR regions, ensuring titers reach 1 × 10¹² genome copies/mL with standard curve analysis.
   3. Validate viral titer using SYBR Green-based quantitative PCR with ITR-specific primers, performing serial dilutions of known standards and calculating genome copies using standard curve analysis. Confirm absence of replication-competent virus contamination using p24 ELISA.
   4. Anesthetize mice with isoflurane (3% induction, 1.5-2% maintenance) delivered at 0.8-1.0 L/min oxygen flow rate through a nose cone, monitoring respiratory rate (60-100 breaths/min) and toe pinch reflex throughout the procedure. Secure the mouse in the stereotactic frame and apply ophthalmic ointment to prevent corneal drying.
   5. Secure the mouse in the stereotactic frame and apply ophthalmic ointment to prevent corneal drying.
   6. Make a 1.5 cm midline scalp incision using a sterile scalpel blade and identify the bregma landmark using stereotactic coordinates with 0.1 mm precision.
   7. Calculate injection coordinates for striatum: anterior-posterior -0.5 mm, medial-lateral ±2.0 mm, dorsal-ventral -3.0 mm from bregma.
   8. Perform pilot injections with fluorescent tracer (e.g., green fluorescent protein [GFP]) to validate targeting accuracy, confirming >80% co-localization with microglial markers (Iba1) via immunohistochemistry before experimental injections.
   9. Lower injection needle (33-G) to target depth at 1 mm/min rate and inject 2 µL of viral suspension at 0.2 µL/min using a micro syringe pump with an automated controller. Lower the injection needle to the target depth and inject 2 µL of viral suspension at a rate of 0.2 µL/min using a microsyringe pump.
   10. Maintain needle position for 5 min post-injection to prevent backflow.
   11. Slowly withdraw the needle at a 0.5 mm/min rate, close the incision with 4-0 silk sutures using an interrupted pattern, and allow recovery from anesthesia on a heated recovery pad.
   12. Administer post-operative analgesia (carprofen 5 mg/kg subcutaneously once daily for 3 days) and monitor recovery for 24 h with detailed observation sheets.

3. Molecular biology techniques and validation

1. RNA extraction and quality control
   1. Harvest cells or striatal brain tissue samples immediately after experimental endpoints by euthanizing animals humanely via CO₂ inhalation followed by cervical dislocation; dissect tissue on ice, mince finely, and enzymatically dissociate with 0.25% trypsin for 10 min at 37 °C if harvesting cells.
   2. Extract total RNA using TRIzol reagent (1 mL per 1 10⁶ cells or 100 mg of tissue) with mechanical homogenization (20-25 strokes in a dounce homogenizer) and chloroform phase separation (0.2 mL of chloroform per 1 mL of TRIzol).
   3. Assess RNA quality using spectrophotometry (A260/A280 ratio 1.8-2.0) and gel electrophoresis (1% agarose) to confirm 28S and 18S ribosomal bands.
   4. Quantify RNA concentration using a spectrophotometer.
   5. Store RNA samples at -80 °C in nuclease-free water at -80 °C with the addition of RNase inhibitor (40 units/µL) until analysis.
2. Nuclear-cytoplasmic fractionation
   1. Harvest cells (2 × 10⁶ minimum) and wash twice with ice-cold PBS (5 mL per wash, 5 min centrifugation at 300 × g).
   2. Utilize a commercial cell fractionation kit with the following protocol.
      1. Resuspend the cell pellet in 200 µL of cytoplasmic extraction buffer with protease inhibitors.
      2. Incubate on ice for 10 min with vortexing every 3 min.
      3. Centrifuge at 16,000 × g for 5 min at 4 °C and collect supernatant (cytoplasmic fraction).
      4. Wash the pellet twice with cytoplasmic extraction buffer.
      5. Resuspend the pellet in 100 µL of nuclear extraction buffer.
      6. Incubate on ice for 40 min with vortexing every 10 min.
      7. Centrifuge at 16,000 × g for 10 min at 4 °C and collect the supernatant (nuclear fraction)
   3. Validate fractionation efficiency by analyzing nuclear marker (U6) and cytoplasmic marker (GAPDH) distribution using Western blot.
   4. Extract RNA from nuclear and cytoplasmic fractions separately.
   5. Analyze Nrf2 mRNA levels in each fraction using RT-qPCR with fraction-specific reference genes.
3. Quantitative real-time PCR (RT-qPCR)
   1. Synthesize cDNA from 1 µg of total RNA using a reverse transcription reagent kit with random primers.
   2. Perform qPCR using Premix Ex Taq II kit with gene-specific primers on a real-time PCR system.
   3. Validate primer specificity using melt curve analysis and confirm a single amplicon by gel electrophoresis.
   4. Use the following thermal cycling conditions: initial denaturation at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s.
   5. Calculate relative mRNA expression levels using the 2^-ΔΔCt method with β-actin as an internal reference.
   6. Include no-template controls and validate reference gene stability across experimental conditions using geNorm analysis (stability value M < 0.5).
      NOTE: Reproducibility scores: intra-assay coefficient of variation (CV) <10% across three independent runs.
4. MeRIP-qPCR for m6A modification analysis
   1. Extract total RNA (minimum 20 µg) and fragment to 100-200 nucleotides using RNA fragmentation reagent (10 mM ZnCl₂, 10 mM Tris-HCl, pH 7.0) at 70 °C for 5 min.
   2. Perform immunoprecipitation using m6A-specific antibody (2 µg per 20 µg of RNA) overnight at 4 °C with rotation (10 rpm) in immunoprecipitation buffer (50 mM Tris-HCl, pH 7.4, 750 mM NaCl, 0.5% NP-40).
   3. Validate antibody specificity using known m6A-modified and unmodified RNA controls.
   4. Wash immunoprecipitated complexes five times with washing buffer.
   5. Elute bound RNA and reverse transcribe using random primers.
   6. Reverse transcribes eluted RNA using random primers and perform qPCR analysis using Nrf2-specific primers covering potential m6A sites.
   7. Perform qPCR analysis using Nrf2-specific primers and calculate enrichment relative to input RNA.
      NOTE: If detection is inconsistent, optimize fragmentation time (4-6 min) to improve reproducibility; quantitative benchmark: signal-to-noise ratio >15:1 compared to IgG controls.

4. Protein analysis and validation

1. Protein extraction and quantification
   1. Lyse cells (1 × 10⁶ cells) or homogenize tissue samples (100 mg of striatal tissue) in 200 µL of radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride (PMSF), 1 µg/mL leupeptin, 1 µg/mL pepstatin A) and phosphatase inhibitors (1 mM sodium orthovanadate, 5 mM sodium fluoride).
   2. Incubate lysates on ice for 30 min with periodic vortexing (every 10 min for 10 s).
   3. Centrifuge at 12,000 × g for 15 min at 4 °C to remove cellular debris and collect supernatant.
   4. Quantify protein concentration using bicinchoninic acid (BCA) assay with bovine serum albumin standards (0, 25, 125, 250, 500, 750, 1000, 1500, 2000 µg/mL) in triplicate measurements.
   5. Validate protein integrity by analyzing housekeeping protein levels (GAPDH, expected MW: 36 kDa) across all samples using Western blot.
2. Western blot analysis
   1. Prepare protein samples with equal loading (30-50 µg) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer (final concentrations: 62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol, 0.003% bromophenol blue) and denature at 95 °C for 5 min.
   2. Separate proteins by SDS-PAGE using 10% polyacrylamide gels, running at 80 V for 30 min followed by 120 V for 90 min in Tris-glycine-SDS buffer.
   3. Transfer proteins to polyvinylidene fluoride (PVDF) membranes using a semi-dry transfer system (400 mA for 90 min) in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol).
   4. Confirm transfer efficiency using reversible protein staining (Ponceau S: 0.1% in 5% acetic acid for 5 min) before proceeding to antibody incubation.
   5. Block membranes with 5% non-fat milk in TBST (TBS + 0.1% Tween-20) for 1 h at RT with gentle agitation.
   6. Incubate with primary antibodies diluted 1:1000 in blocking buffer overnight at 4 °C with gentle agitation.
   7. Wash membranes three times with TBST (10 min each) and incubate with HRP-conjugated secondary antibodies (1:5000 dilution) for 1 h at RT.
   8. Detect protein bands using enhanced chemiluminescence and quantify using densitometry.
   9. Normalize target protein expression to loading control (GAPDH) and validate antibody specificity using appropriate controls (peptide blocking for primary antibodies, secondary-only controls).

5. Behavioral assessment and functional analysis

1. Forelimb placement test
   1. Allow the mouse to acclimate to the testing environment (quiet room, 22 °C, dim lighting) for 30 min before assessment.
   2. Grasp the mouse gently by the dorsal skin, suspending all four limbs approximately 0.5 cm above the table edge while ensuring the mouse cannot see the table surface.
   3. Brush vibrissae on each side against the table edge to trigger the placing reflex and record successful forelimb placement within 2 s.
   4. Perform 10 trials per side with 30-s intervals between trials, alternating left and right sides.
   5. Perform testing in consistent lighting conditions (50-100 lux) and time of day (13:00-17:00) to minimize circadian influences.
   6. Calculate success rate as a percentage of successful placements: (successful placements/total trials) × 100.
2. Accelerating the rotarod test
   1. Set the rotarod apparatus to an initial speed of 4 rpm with linear acceleration to 40 rpm over 5 min duration.
   2. Train mice on the rotarod for 3 consecutive days (3 trials per day, 15-min intervals) before testing to minimize learning effects.
   3. Standardize testing conditions, including RT (22 ± 2 °C), lighting (100 lux), and background noise levels (<50 dB).
   4. Place the mouse on the rotating rod facing forward and start the acceleration protocol immediately.
   5. Record latency to fall (time from start until mouse falls or completes 5-min trial) for each trial, testing 5 times per animal with 15-min rest intervals.
   6. Calculate average latency from the three longest trials to minimize variability due to motivational factors.
3. Open field test
   1. Use an open field apparatus (50 cm × 50 cm × 50 cm clear acrylic box) with a video tracking system positioned 1 m above the arena.
   2. Clean apparatus with 70% ethanol between animals (spray and wipe, 5-min air dry) to eliminate odor cues.
   3. Place the mouse in the center of the apparatus and record activity for 10 min under consistent lighting (200 lux) with video capture at 30 fps.
   4. Analyze multiple parameters using automated tracking software:
      Total distance traveled (cm)
      Center zone time (defined as 10 cm from walls, reported in s)
      Movement velocity (cm/s)
      Time spent in periphery vs center zones
4. Define the central zone as 10 cm from the walls and quantify time spent and distance traveled in the center versus the periphery zones.

6. Advanced microscopy and imaging

1. DHE staining for ROS detection
   1. Prepare fresh dihydroethidium (DHE) solution at 10 µM in PBS immediately before use (protect from light).
   2. Perform co-immunofluorescence staining combining DHE with tryptophan hydroxylase 2 (TPH2) antibody (1:500 dilution) to specifically identify serotonin neurons.
      ​NOTE: This dual-labeling approach enables discrimination of ROS production within TPH2-positive neuronal cell bodies from oxidative stress in surrounding glial cells, based on the principle that DHE, upon oxidation by superoxide, forms membrane-impermeant fluorescent products that remain localized within the cell of origin.
   3. Incubate tissue sections (20 µm thickness) with DHE solution for 30 min at 37 °C in dark conditions in a humidified chamber.
   4. Include negative controls (no DHE) and positive controls (100 µM H₂O₂ treatment for 30 min) to validate ROS detection specificity.
   5. Wash sections three times with PBS and mount with anti-fade mounting medium.
   6. Image using fluorescence microscopy (DHE: 518 nm excitation/605 nm emission, TPH2: 488 nm excitation/525 nm emission) with consistent exposure settings (200 ms) across samples; imaging systems were calibrated weekly using standard fluorescence beads (signal-to-noise ratio > 20:1).
   7. Quantify fluorescence intensity in TPH2-positive cells only using ImageJ software with standardized analysis protocols (threshold: 50-255, particle size: 10-∞ pixels²). Establish ROI boundaries based on TPH2 immunoreactivity to ensure measurement of oxidative stress specifically within serotonergic neurons while excluding signal from adjacent microglia, astrocytes, or other cell types. For each animal, analyze a minimum of 50 TPH2-positive neurons across multiple tissue sections.
2. Immunohistochemistry
   1. Fix tissue sections in 4% paraformaldehyde in PBS for 24 h at 4 °C with gentle agitation.
   2. Dehydrate through a graded ethanol series (70%, 80%, 90%, 95%, 100% × 2, 1 h each) and embed in paraffin using an automated processor.
   3. Cut 5 µm sections using a microtome and mount on charged glass slides, ensuring 3-4 sections per slide.
   4. Deparaffinize sections using xylene (2 × 10 min) and rehydrate through graded ethanol to water.
   5. Perform antigen retrieval using citrate buffer (10 mM sodium citrate, pH 6.0) in a microwave (750 W for 10 min with 2-min cooling intervals).
   6. Validate antibody specificity using appropriate negative controls (no primary antibody) and positive control tissues (brain sections known to express target proteins).
   7. Block endogenous peroxidase activity with 3% hydrogen peroxide in methanol for 10 min at RT.
   8. Apply primary antibodies overnight at 4 °C in a humidified chamber.
   9. Detect using HRP-conjugated secondary antibodies (1:500 dilution, 1 h at RT) and DAB chromogen (incubate until brown color develops, typically 2-5 min).
   10. Counterstain with hematoxylin (30 s), dehydrate, clear, and mount with permanent mounting medium.
   11. Quantify positive cells using stereological methods with systematic random sampling (every 5th section, 3 counting frames per section, 40× magnification).

7. Statistical analysis and data validation

1. Statistical design and analysis
   1. Perform power analysis to determine adequate sample sizes for detecting biologically meaningful differences (effect size ≥ 0.8, power ≥ 0.80, α = 0.05) using G*Power 3.1.9.7 software.
   2. Validate data normality using the Shapiro-Wilk test and assess homogeneity of variance using Levene's test before parametric analysis .
   3. Analyze data using a statistical software with appropriate statistical tests based on experimental design and data distribution.
   4. Apply one-way analysis of variance (ANOVA) for single-factor comparisons and two-way ANOVA for factorial designs (e.g., treatment × time interactions).
   5. Use Tukey's post hoc test for multiple comparisons when ANOVA shows significance (p < 0.05), applying Bonferroni correction when appropriate.
   6. Set statistical significance threshold at p < 0.05 for all analyses with additional notation for p < 0.01 and p < 0.001.
   7. Report effect sizes (Cohen's d or η²) and 95% confidence intervals alongside p-values to provide a comprehensive statistical interpretation.
   8. Present data as mean ± SEM from a minimum of three independent experiments, with individual data points shown when n ≤ 10.
      NOTE: All cell culture experiments should be independently repeated at least three times, with each experiment including appropriate controls and multiple technical replicates, and observed variability (e.g., CV <15% for behavioral data) was minimized through blinding and standardized timing.

Results

The protocol successfully demonstrates that Mettl3 expression decreases in LPS-treated microglial cells (Figure 1), as evidenced by both mRNA and protein level reductions compared to control groups (Figure 1B,C). ELISA analysis confirms successful LPS-mediated inflammatory activation through elevated IL-6 and TNF-α levels in culture supernatants (Figure 1A).

MeRIP-qPCR analysis reveals that Mettl3 overexp...

Discussion

There are several critical steps that require careful attention to ensure reproducible results. The LPS activation timing is crucial - inflammatory responses peak at 24 h post-treatment, and we strongly recommend preparing fresh LPS solution for each experiment, as stored solutions can lose bioactivity^19,20. For the MeRIP-qPCR analysis, antibody quality is paramount. We have found that validating antibodies using known positive and negative controls before experi...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
AAV9 viral vectors	Vector Core Facility	Custom	Containing Nrf2 constructs
Accelerating rotarod	Ugo Basile	47600	For behavioral testing
Anti-GAPDH antibody	Cell Signaling Technology	5174	Primary antibody, 1:5000
Anti-GSDMD antibody	Abcam	ab219800	Primary antibody, 1:1000
Anti-Iba1 antibody	Wako	019-19741	Primary antibody, 1:500
Anti-NLRP3 antibody	AdipoGen	AG-20B-0014	Primary antibody, 1:1000
Anti-Nrf2 antibody	Abcam	ab62352	Primary antibody, 1:1000
Anti-SLC6A4 antibody	Novus Biologicals	NBP1-85726	Primary antibody, 1:500
Anti-TPH2 antibody	Millipore	MAB847	Primary antibody, 1:500
BCA Protein Assay Kit	Pierce	23225	For protein quantification
BV2 microglial cells	Shengen Biology	SG-BV2	Mouse microglial cell line
C57BL/6J mice	Vital River Laboratory	213	8-week-old, 19-26 g
Cell Fractionation Kit	Cell Signaling Technology	9038	Nuclear-cytoplasmic separation
Complete high-glucose DMEM	Gibco	11965092	Cell culture medium
DAB Chromogen	Vector Laboratories	SK-4100	For immunohistochemistry
Dental drill	Fine Science Tools	18000-17	For burr hole drilling
DHE (Dihydroethidium)	Molecular Probes	D11347	ROS detection
ELISA Kit (IL-1β)	R&D Systems	MLB00C	Mouse IL-1β detection
ELISA Kit (IL-18)	R&D Systems	7625	Mouse IL-18 detection
ELISA Kit (IL-6)	R&D Systems	M6000B	Mouse IL-6 detection
ELISA Kit (TNF-α)	R&D Systems	MTA00B	Mouse TNF-α detection
Fetal Bovine Serum	Gibco	16000044	Cell culture supplement
HRP-conjugated secondary antibody	Jackson ImmunoResearch	111-035-003	Anti-rabbit, 1:10000
Isoflurane	RWD Life Science	R510-22	Anesthetic agent
LAL Assay Kit	Lonza	50-647U	LPS activity validation
Lipofectamine transfection reagent	Invitrogen	11668019	For cell transfection
LPS (Lipopolysaccharide)	Sigma-Aldrich	L2630	From E. coli, 1 mg/mL stock
m6A antibody	Synaptic Systems	202003	For MeRIP, 1:200
Microsyringe pump	Harvard Apparatus	70-3007	For stereotactic injection
MPTP	Sigma-Aldrich	M0896	Neurotoxin, 30 mg/kg
Open field apparatus	ANY-maze	Custom	50 cm ´ 50 cm ´ 50 cm
Paraformaldehyde	Sigma-Aldrich	P6148	4% in PBS
pcDNA3.1 vector	Invitrogen	V79020	Expression vector
Penicillin-Streptomycin	Gibco	15140122	Antibiotic solution
Premix Ex Taq II Kit	Takara	RR820A	For qPCR
PrimeScript RT Kit	Takara	RR037A	Reverse transcription
PVDF membrane	Millipore	IPVH00010	For Western blot
RIPA buffer	Cell Signaling Technology	9806	Protein extraction
siRNA (Mettl3)	RiboBio	Custom	Target sequence validation
Stereotactic frame	RWD Life Science	68001	For brain surgery
TRIzol reagent	Invitrogen	15596026	RNA extraction
Trypan blue	Sigma-Aldrich	T8154	Cell viability staining