The SIK1-NR4A2 axis: a core driver of epilepsy identified by integrated machine learning and experimental validation

Epilepsy remains a major clinical challenge, suggesting that mechanisms beyond the classical excitation-inhibition imbalance contribute to epilepsy. The Liver kinase B1 (LKB1) signaling pathway coordinates energy homeostasis and cell survival, but its key downstream effectors in epilepsy remain largely unknown.

We integrated machine learning screening of LKB1 pathway genes using public Gene Expression Omnibus datasets (GSE143272) with in vivo and in vitro experimental validation. A PTZ-induced status epilepticus rat model, AAV-mediated gene knockdown/overexpression, lentiviral transfection of primary hippocampal neurons, Electroencephalography recording, whole-cell patch-clamp recording, and Western blot analysis were employed to investigate the functional role and molecular mechanisms of candidate genes.

Machine learning identified SIK1 (salt-inducible kinase 1) as the top-ranked epilepsy risk gene within the LKB1 pathway. SIK1 expression was significantly upregulated in the hippocampus of SE rats. AAV-mediated SIK1 knockdown prolonged seizure latency and reduced seizure duration, while SIK1 overexpression exacerbated seizure severity. Mechanistically, through PPI network analysis and mediation analysis, we identified NR4A2 as a downstream effector of SIK1. SIK1 negatively regulated NR4A2 expression: SIK1 knockdown increased NR4A2, whereas SIK1 overexpression decreased NR4A2. Rescue experiments showed that co-overexpression of SIK1 and NR4A2 reversed the pro-epileptic effect of SIK1 alone. Whole-cell patch-clamp recordings demonstrated that SIK1 knockdown or NR4A2 overexpression restored action potential amplitude to near-normal levels in magnesium-free-treated neurons.

SIK1 negatively regulates NR4A2, and restoring NR4A2 expression counteracts SIK1-induced neuronal hyperexcitability. These findings suggest that targeting the SIK1-NR4A2 axis may represent a potential therapeutic strategy for epilepsy.