Combined lead exposure and high-copper intake exacerbates synaptic loss via METTL3-mediated m6A methylation in mice

Lead (Pb) exposure is a well-established environmental risk factor for cognitive impairment. Emerging evidence indicates that the combined effects of co-occurring metals may modify their neurotoxicity, but the underlying mechanisms remain elusive. The present study investigated how high copper (Cu) intake exacerbates Pb-induced synaptic loss, with a specific focus on the critical mediating role of N6-methyladenosine (m6A) RNA methylation in this process. We established a mouse model divided into four groups: control, Pb exposure, high Cu, and lead-copper co-exposure (Pb+Cu, PC). Cognitive function was assessed using the Morris water maze test and novel object recognition test. Golgi-Cox and immunofluorescence staining for postsynaptic density protein 95 (PSD95) were utilized to quantify dendritic spine density and synaptic number. Additionally, quantitative real-time PCR (RT-qPCR), Western blot, and dot blot assays were performed to analyze global m6A methylation levels and METTL3 expression, the core catalytic subunit of the m6A methyltransferase complex. Results showed that lead-copper co-exposure significantly exacerbated spatial learning and memory impairments in mice, accompanied by more severe loss of hippocampal dendritic spines and synapses. Mechanistically, heavy metal co-exposure induced an aberrant elevation of PSD95 mean methylation in the hippocampus, a dysregulation of epitranscriptomic modification that serves as a core driver of synaptic impairment. This elevated m⁶A level was due to the specific upregulation and enhanced activity of METTL3, the core catalytic subunit of m⁶A methyltransferase. Specific knockdown of METTL3 in vitro in primary hippocampal neurons and HT22 cells attenuated co-exposure-induced m6A hyper-methylation and partially restored synaptic structure and function, supporting the mediating role of aberrant m6A modification in synergistic heavy metal neurotoxicity. This study first identifies that high Cu intake synergistically exacerbates Pb neurotoxicity by activating the METTL3-driven m6A epitranscriptomic pathway, offering a potential target for intervening in cognitive impairment related to combined heavy metal exposure.