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

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.