Demethylase FTO in the cerebrospinal fluid-contacting nucleus of mice contributes to neuropathic pain via mediating m6A demethylation of P2rx4 mRNA.

N6-methyladenosine (m6A) modification plays a crucial role in pain regulation by modulating pain-related gene expression. The cerebrospinal fluid-contacting nucleus (CSF-contacting nucleus) is closely associated with pain, and downregulation of P2X4 receptor (P2X4R) expression in this region alleviates hyperalgesia. However, the relationship between m6A modification and P2X4R in CSF-contacting nucleus remains unclear. This study aims to investigate the role and potential mechanisms of the m6A demethylase fat mass and obesity-associated protein (FTO) and P2X4R in neuropathic pain (NP) induced by spared nerve injury (SNI) in male mice. We observed decreased m6A levels and upregulated FTO expression in the CSF-contacting nucleus of SNI mice. FTO was primarily expressed in neurons of the CSF-contacting nucleus, with symmetrical distribution across its bilateral regions. In CSF-contacting nucleus, FTO overexpression reduced m6A methylation and promoted pain, while FTO inhibition increased m6A levels and alleviated pain hypersensitivity. The administration of the FTO inhibitor meclofenamic acid (MA) into CSF-contacting nucleus alleviated pain. FTO regulated the expression of P2rx4 mRNA and protein in CSF-contacting nucleus. Furthermore, P2rx4 mRNA is a downstream target of FTO-mediated m6A demethylation. In summary, the m6A demethylase FTO contributes to NP by upregulating the expression of P2rx4 mRNA and protein through mediating m6A demethylation of P2rx4 mRNA. Therefore, the m6A demethylase FTO in CSF-contacting nucleus may represent a novel therapeutic target for NP.