Profile of N6-methyladenosine of Pb-exposed neurons presents epitranscriptomic alterations in Pi3K-AKT pathway-associated genes
Lead (Pb) is a pervasive heavy metal with multi-organ toxicity. However, the molecular mechanisms of Pb-induced neurotoxicity are not fully understood. The dynamics of N6-methylademine (m6A) is an emerging regulatory mechanism for gene expression, which is closely related to nervous system diseases. To elucidate the association between m6A modification and Pb-mediated neurotoxicity, primary hippocampal neurons exposed to 5 µM Pb for 48 h were used as the paradigm neurotoxic model in this study. According to the results, Pb exposure reprogrammed the transcription spectrum. Simultaneously, Pb exposure remodeled the transcriptome-wide distribution of m6A while disrupting the overall level of m6A in cellular transcripts. United analysis of MeRIP-Seq and RNA-Seq was applied to further identify the core genes whose expression levels are regulated by m6A in the process of lead-induced nerve injury. GO and KEGG analysis unveiled that the modified transcripts were overrepresented by the PI3K-AKT pathway. Mechanically, we elucidated the regulatory role of the methyltransferase like3 (METTL3) in the process of lead-induced neurotoxicity and the downregulation of the PI3K-AKT pathway. In conclusion, our novel findings shed new light on the functional roles of m6A modification in the expressional alternations of downstream transcripts caused by lead, providing an innovative molecular basis to explain Pb neurotoxicity. Overall design: P0 Sprague-Dawley (SD) rats were obtained from the Experimental Animal Center of Anhui Medical University (Auhui, China). Berif, the hippocampal tissue was detached stereoscopically from whole brain of P0 rats and digested by trypsinization (0.03%). The digested tissue were dissociated by trituration through sterile, fire-polished Pasteur pipettes. The dissociated neurons were plated in poly-L-lysine-precoated (0.5 mg/mL) dishes (100 cells per mm2) with serum-free neurobasal medium containing B27 and GlutaMAX. At DIV7, half the volume of media were removed and replaced with fresh neurobasal feeding media supplemented with Ara-C (2?µL/mL). Neurons were fed with PbAc (5?µM) at DIV12. The Pb dosage used in the cell experiment is chosen on the basis of its demonstration in the previous reports (Ding et al., 2018; Gu et al., 2019; Prins, Park, & Lurie, 2010) that it does not cause excessive neuronal death and leads to neuronal damage, as a way of establishing a replicable model for the study of lead-induced neurotoxicity (Data not shown). After culturing at 37 ? for 48 h in a 5% CO2 incubator, neurons were hrvested.