MAPK3 as a ferroptosis driver in excitatory neurons in Alzheimer’s disease: from genetic causal inference to in vivo functional validation

Introduction Alzheimer’s disease (AD) is a progressive neurodegenerative disorder, and ferroptosis-an iron-dependent form of regulated cell death-has been increasingly implicated in AD-related neuronal loss. However, the causal relationship between specific ferroptosis-related genes and AD, particularly within defined neuronal subtypes, remains incompletely understood. Methods We integrated two-sample Mendelian Randomization (MR) with single-cell expression quantitative trait locus (eQTL) analysis, followed by in vivo validation using AAV9-c-fos-mediated MAPK3 knockdown in APP/PS1 transgenic mice and in vitro validation using siRNA-mediated MAPK3 silencing in Aβ-treated HT22 neurons. Results MR analysis of large-scale genetic datasets identified seven ferroptosis-related genes with causal associations with AD. Single-cell eQTL MR prioritized MAPK3 as a significant risk factor specifically within excitatory neurons. AAV9-c-fos-mediated MAPK3 knockdown in APP/PS1 mice significantly ameliorated cognitive deficits in the Morris water maze. Mechanistically, MAPK3 knockdown inhibited aberrant CaMKII phosphorylation, activated the Nrf2-GPX4 antioxidant axis (upregulation of GPX4, HO-1, and FTH1; downregulation of ACSL4 and KEAP1), and reestablished the Bax/Bcl-2 equilibrium, thereby reducing both ferroptosis and apoptosis. These effects were reproduced in Aβ-treated HT22 neurons following siRNA-mediated MAPK3 silencing. Discussion Our findings identify MAPK3 in excitatory neurons as a potential causal link between genetic risk and AD pathogenesis, bridging population-level genetic risk and cellular vulnerability. Targeting the MAPK3-CaMKII-Nrf2 axis may represent a promising, cell-type-specific therapeutic strategy for AD.