N-Homocysteinylation of Cofilin-1 Aggravates Epileptic Pathophysiology via Disruption of Actin Dynamics.

Elevated homocysteine (Hcy) levels contribute to the onset and progression of childhood epilepsy. However, Hcy-lowering therapies provide limited benefits, suggesting additional pathogenic mechanisms. Here, we identified N-homocysteinylated Cofilin-1 (N-Hcy-CFL1) as a molecular link between elevated Hcy levels and childhood epilepsy. We detected N-Hcy-CFL1 in patients with epilepsy and in a mouse model of hyperhomocysteinemia. Mass spectrometry identified Hcy-dependent lysine modifications in CFL1, and functional assays, including actin depolymerization, the F/G-actin ratio, and axonal elongation, demonstrated impaired CFL1 activity and consequent cytoskeletal rigidity. These changes manifested as increased F-actin levels, increased F/G-actin ratio, and abnormal axonal growth. In vivo, hyperhomocysteinemic mice exhibited prolonged seizures and axonal remodeling, both of which were attenuated by CFL1-directed interventions. Clinically, blood Hcy levels correlated with N-Hcy-CFL1 levels in patients' brain tissues, supporting its potential as a biomarker. Together, these findings identify N-Hcy-CFL1 as a pathogenic mediator and suggest that N-homocysteinylation is a therapeutic target beyond conventional Hcy-lowering strategies.