Development of an in vitro model for the study of HCN1 Developmental Epilepsy and Encephalopathy

Developmental and epileptic encephalopathies (DEEs) are a heterogeneous group of neurodevelopmental diseases, united by a dynamic interplay between epilepsy and abnormal brain maturation. The patients' genetic aetiologies are becoming more apparent thanks to screening advancement, as at least half of patients are diagnosed with pathogenic variants. The early onset and invalidating characteristics of DEEs urge us to find treatments tailored for each instance of this disease, with heterogeneous causes. A rare form of this disease (DEE24) is caused by mutations in the HCN1 gene, which encode the hyperpolarization-activated cyclic nucleotide-gated cation channel. This channel is expressed in many neuronal and non-neuronal cell types with a variety of functions, but mainly to regulate membrane potential and excitability properties. Even though HCN1 has been associated with epilepsy and neural development before, the mechanisms by which the pathology arises are still elusive. Therefore, we devised an in vitro model for studying the mutated channels in a context more fitting than a heterologous expression system. By electroporating mouse embryo cortices with expression vector inserted with mutated (M153I/A387S/G391D), wild-type human HCN1, or the empty vector at E16.5, we targeted the neural progenitor which would differentiate into upper layer pyramidal neurons. At DIV18.5 dissected and dissociated these to make primary cortical neuronal culture, then studied their activity with calcium imaging at DIV14. We found reduced synchronicity in the A387S cultures compared to every other condition and a slight increase in the frequency of population spikes in G391D and M153I compared to wild-type cultures, While A387S seems to trend downwards. These results show that, while all these mutations show cation leakage at resting membrane potential in heterologous expression systems, they influence the network dynamic differently, and may involve other pathological mechanisms specific to the single mutation. Furthermore, by taking advantage of qPCR and Calcein AM, PI, and Hoechst 33342 staining, we started to question whether this model would be fitting to study novel treatments for seizures in DEEs. In particular, we focus our attention on Soticlestat, an inhibitor of the degradation pathway of cholesterol in the brain, which has already shown good results in the ENDYMION phase 3 clinical study.