TRIM8 Promotes Epileptiform Activity by Destabilizing the Glucocorticoid Receptor NR3C1 and Enhancing AMPA Receptor Phosphorylation

Background: The glucocorticoid receptor NR3C1 exhibits antiepileptic properties, but the mechanisms governing its stability during epileptogenesis remain elusive. This study investigated whether the E3 ubiquitin ligase TRIM8 regulates neuronal hyperexcitability and epileptic activity by modulating NR3C1. Methods: We established an in vivo epilepsy model via intrahippocampal kainic acid (KA) injection and an in vitro epileptiform model using Mg2+-free artificial cerebrospinal fluid in primary hippocampal neurons. The roles of TRIM8 and NR3C1 were assessed using in vivo and in vitro gain- and loss-of-function approaches, alongside co-immunoprecipitation, Western blotting, immunofluorescence and whole-cell patch-clamp recording. Results: TRIM8 is significantly upregulated in hippocampal and temporal lobe neurons in epileptic mice. TRIM8 was markedly upregulated in the hippocampal neurons of epileptic mice, inversely correlating with NR3C1 levels. Mechanistically, TRIM8 interacted with NR3C1, promoting its polyubiquitination and proteasomal degradation. This TRIM8-mediated NR3C1 reduction enhanced the phosphorylation of AMPA receptor (AMPAR) subunits GluR1 (Ser831) and GluR2 (Ser880) without affecting total receptor expression. In vitro, TRIM8 overexpression exacerbated calcium dysregulation, neuronal injury, and AMPAR phosphorylation; crucially, concurrent NR3C1 overexpression rescued these effects. In vivo, knockdown of TRIM8 significantly reduced seizure frequency, prolonged the latency to the first Stage III seizure, shortened average seizure duration, and decreased total seizure burden in KA-induced epileptic mice. Electrophysiologically, TRIM8 overexpression significantly increased the frequency of spontaneous action potentials and amplitudes of spontaneous excitatory postsynaptic currents under Mg2+-free conditions. Furthermore, in vivo knockdown of TRIM8 attenuated KA-induced seizure severity, restored NR3C1 protein stability, and suppressed aberrant AMPAR phosphorylation in the hippocampus. Triple immunofluorescence staining showed that KA-induced epilepsy increased TRIM8 but decreased NR3C1 immunoreactivity in NeuN+ hippocampal neurons, and TRIM8 knockdown reversed these changes. Conclusions: TRIM8 acts as a critical driver of epileptiform activity by targeting NR3C1 for degradation, thereby disinhibiting AMPAR phosphorylation and enhancing network hyperexcitability. The TRIM8-NR3C1-AMPAR axis emerges as a previously unrecognized molecular pathway in epileptogenesis, highlighting its potential as a promising therapeutic target for epilepsy.