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,

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 prepar....

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 ov.......

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