Abstracts

This is a Late Breaking abstract

Rationale: De novo mutations in ion channel genes have been associated with an increasing number of cases of developmental and epileptic encephalopathy (DEE). Here, we examine two newly generated knock-in mouse models that replicate de novo sequence variations in the HCN1 voltage-gated ion channel, p.G391D and p.M153I (Hcn1^G380D/+ and Hcn1^M142I/+ in mouse), associated with severe drug-resistant neonatal- and childhood-onset epilepsy, respectively. Our study aims to establish a causal relation between HCN1 mutations and epilepsy; characterize the mice seizure phenotypes and associated comorbidities to verify their appropriateness in modeling the human disease; investigate basic cellular mechanisms, and assess the animals’ response to pharmacological intervention, both to replicate clinical findings in HCN1 patients and explore new approaches to the treatment of DEE linked to HCN1 sequence variations.

Methods: Mice were generated using CRISPR/Cas9 genome editing, and seizure activity assessed via electrocorticogram (ECoG) recordings. Behavioral paradigms included open field and gait analysis (Catwalk XT); pole climbing; and spontaneous alternation and novel object recognition for testing short- and long-term memory, respectively. Immunohistochemical analysis was performed on fixed mouse brains using fluorescent antibody labeling and confocal microscopy. Whole-cell patch-clamp recordings were performed using ex vivo hippocampal slices to probe for effects of HCN1 mutations on CA1 pyramidal neuron physiology, and heterologous expression in HEK293T cells to test for effects of select drugs on wildtype and mutant HCN1 channels.

Results: Both mouse lines showed spontaneous, generalized tonic-clonic seizures and alterations in hippocampal morphology as well as expression of epilepsy markers. Locomotor hyperactivity was observed in both mouse lines, while defects in motor coordination and cognitive function were limited to the more severe p.G391D (Hcn1^G380D/+ in mouse) mutation. HCN1 protein expression was decreased in both lines, with mutant HCN1 subunits replicating p.G391D markedly absent from the axonal terminals of parvalbumin-positive basket cell interneurons, a normal site of high expression for HCN1 channels. In line with clinical reports from patients with pathogenic HCN1 variants, administration of the antiepileptic Na⁺ channel antagonists lamotrigine and phenytoin resulted in the paradoxical induction of seizures in both mouse lines, consistent with an impairment in inhibitory neuron function.

Conclusions: Our findings demonstrate the role of HCN1 mutations in causing seizures, and suggest a potential contribution of interneuronal dysfunction in HCN1-linked epilepsy, with consequent alterations in the antiseizure drug response profile. The availability of strong preclinical models, such as the Hcn1^G380D/+ and Hcn1^M142I/+ mouse lines presented here, overall provides an ideal platform for investigating the mechanisms underlying epileptic encephalopathies as well as testing new therapeutic approaches.

Funding: National Institutes of Health NS106983, NS109366, NS123648; Deutsche Forschungsgemeinschaft FOR 2715 (IS63/10-1/2), CRC 1451 (project ID 431549029 - B01); Fondazione Telethon, GGP20021