Dissecting the role of HCN1 in Developmental and Epileptic Encephalopathy (DEE) by exploiting patient-specific models of cerebral cortex development

Developmental and Epileptic Encephalopathies (DEE) are a heterogeneous group of devastating conditions characterized by very early onset, unprovoked and recurrent seizures, that often show poor response to pharmacological treatment. Recent advancements in genome sequencing have expanded our knowledge on DEE, and the number of associated mutations in voltage-gated ion channels has grown exponentially. Indeed, new evidence supports the surprising role of ion channels, classically studied exclusively in mature neurons, in developing neurons, and in prenatal human brain pathology. Among others, many mutations in Hyperpolarization-activated Cyclic Nucleotide-gated (HCN1) gene have been causative of the DEE24, whose affected patients show also impaired global development, intellectual disability, and behavioral abnormalities. Here, we aim to understand how brain developmental processes are altered by the HCN1 pathogenic variants associated with DEE24. We selected 3 de novo HCN1 variants (M153I, G391D, A387S) showing association with autistic/behavioral and morphological traits and evaluated their effect on electrophysiological properties in both heterologous cell lines and neurons. We observed that the HCN1^A387S mutant displays an abnormal electrophysiological profile, while HCN1^G391D shows a subcellular mislocalization. By exploiting a highly reproducible human cortical organoids (hCOs) system, we generated hCOs from HCN1-DEE24 patient-specific iPSC lines. Moreover, through in utero electroporation, we generated chimeric brains harboring these variants in a temporally and spatially regulated manner. We analyzed the activity and morphology of electroporated neurons in vitro, as well as their cell-intrinsic and non-cell autonomous effects on early cortical development in vivo.

Our preliminary results suggest that the generation of personalized models, bridging genetic data with molecular neurodevelopment, allows the identification of specific endophenotypes of distinct HCN1 variants and their effects on cortical assembly. The obtained results will open the way for future research and investigations, aiming at improving the clinical conditions of epileptic patients.