CPEB4 mediated EZH2 dependent beclin1 methylation affecting neuronal ferroptosis in epilepsy

Cytoplasmic polyadenylation element-binding protein 4 (CPEB4) regulates protein expression by modulating mRNA translation and stability. Previous investigations have reported adaptive upregulation of CPEB4 in epilepsy, and knockout of the CPEB4 gene aggravates status epilepticus and neuronal degeneration in mice. However, the mechanisms through which CPEB4 influences epileptic seizures remain poorly defined. This investigation sought to elucidate the role of CPEB4 in epilepsy by examining its regulation of downstream targets involved in neuronal ferroptosis. Lentivirus was stereotactically injected into the hippocampus of pilocarpine-induced status epilepticus (SE) mice to achieve CPEB4 overexpression or knockdown, and its effects on seizure activity were assessed. Mouse behavioral performance was evaluated using the open field and wire hang tests. The neuroprotective effects of CPEB4 overexpression were further examined in a glutamate (Glu)-induced neuronal HT22 cell injury model. The RBPsuite database identified enhancer of zeste homolog 2 (EZH2) as a downstream target of CPEB4, which was validated through RNA immunoprecipitation (RIP). Cistrome database analysis indicated enrichment of trimethylation of histone H3 lysine 27 (H3K27me3) in the Beclin1 promoter region, which was confirmed by chromatin immunoprecipitation (ChIP) assays demonstrating EZH2-mediated H3K27me3 modification. Lipid peroxidation markers, including reactive oxygen species, malondialdehyde, and glutathione, as well as ferroptosis-associated indicators such as SLC7A11, GPX4, and FTH expression, were quantitatively analyzed. CPEB4 expression was compensatorily upregulated following status epilepticus in mice. CPEB4 overexpression mitigated seizure severity and cognitive impairments in post-SE mice, while CPEB4 knockdown exacerbated lipid peroxidation and ferroptosis. Conversely, CPEB4 overexpression inhibited both processes. Treatment with the ferroptosis inhibitor ferrostatin-1 attenuated CPEB4 knockdown-induced lipid peroxidation and ferroptosis, accompanied by increased EZH2 expression and decreased Beclin1 expression. In the Glu-induced HT22 cell injury model, RIP analysis confirmed the binding of CPEB4 to the 3’-untranslated region of EZH2 mRNA. ChIP assays further verified that EZH2 mediated H3K27me3 modification of the Beclin1 promoter. In vitro, EZH2 knockdown and Beclin1 overexpression diminished the protective effects of CPEB4 overexpression on neuronal viability, lipid peroxidation inhibition, and ferroptosis suppression. CPEB4 expression increases compensatorily following status epilepticus. By regulating EZH2, CPEB4 mediates H3K27me3 modification of the Beclin1 gene, downregulates Beclin1 expression, and suppresses lipid peroxidation and ferroptosis. These effects confer neuroprotection to hippocampal neurons, mitigate epileptogenesis, and alleviate epilepsy-induced neuronal injury.