MODULATION OF CHOLESTEROL METABOLISM AMELIORATES NEURONAL HYPEREXCITABILITY AND NEUROINFLAMMATION IN A <EM>GRIN2D</EM>-RELATED DEVELOPMENTAL AND EPILEPTIC ENCEPHALOPATHY MODEL

Developmental and Epileptic Encephalopathies (DEEs) caused by gain-of-function variants in N-methyl D-aspartate receptor subunits are associated with severe epilepsy, cognitive impairment, motor dysfunction, and premature death, yet effective treatments addressing the full neurological phenotype are lacking. The GRIN2D c.1999G>A (p.Val667Ile) variant enhances GluN2D receptor activity, leading to neuronal hyperexcitability and neurotoxicity. Here, we investigated the therapeutic potential of Compound A, a selective cholesterol 24-hydroxylase inhibitor, in a Grin2d Val667Ile mouse model that recapitulates key features of human DEE. Mice were assigned to two different treatment windows corresponding to distinct developmental ages and assessed using a battery of motor, cognitive, and behavioural assays, alongside electroencephalogram recordings, survival analysis, and immunohistochemical analyses. Treatment with Compound A significantly extended lifespan and reduced premature mortality across both treatment windows. Electrophysiological analyses revealed normalization of abnormal brain activity, including restoration of background oscillatory patterns and improvement of epileptic phenotypes. Behavioural testing demonstrated robust rescue of motor coordination, balance, and hyperactivity, along with improvements in cognitive performance. In addition, Grin2d mice exhibited marked astrocytic activation in the hippocampus, as indicated by elevated glial fibrillary acidic protein expression, reflecting an underlying neuroinflammatory state. Treatment normalized astrocytic activation in hippocampal subregions, reducing glial fibrillary acidic protein levels to those observed in wild-type mice. These findings indicate that modulation of cholesterol metabolism ameliorates both neuronal hyperexcitability and neuroinflammation, improving epileptic and non-epileptic manifestations of GRIN2D-related DEE. This work supports Compound A as a disease-modifying therapeutic strategy with potential to improve neurological outcomes and quality of life in affected patients.