De novo mutations in voltage- and ligand-gated channels have been associated with an increasing number of cases of developmental and epileptic encephalopathies, which often fail to respond to classic antiseizure medications. Here, we examine two knock-in mouse models replicating de novo sequence variations in the HCN1 voltage-gated channel gene, p.G391D and p.M153I (Hcn1G380D/+ and Hcn1M142I/+ in mouse), associated with severe drug-resistant neonatal- and childhood-onset epilepsy, respectively. Heterozygous mice from both lines displayed spontaneous generalized tonic-clonic seizures. Animals replicating the p.G391D variant had an overall more severe phenotype, with pronounced alterations in the levels and distribution of HCN1 protein, including disrupted targeting to the axon terminals of basket cell interneurons. In line with clinical reports from patients with pathogenic HCN1 sequence variations, administration of the antiepileptic Na+ channel antagonists lamotrigine and phenytoin resulted in the paradoxical induction of seizures in both mouse lines, consistent with an effect to further impair inhibitory neuron function. We also show that these variants can render HCN1 channels unresponsive to classic antagonists, indicating the need to screen mutated channels to identify novel compounds with diverse mechanism of action. Our results underscore the necessity of tailoring effective therapies for specific channel gene variants, and how strongly validated animal models may provide an invaluable tool towards reaching this objective.

De novo mutations in voltage- and ligand-gated channels have been associated with an increasing number of cases of developmental and epileptic encephalopathies, which often fail to respond to classic antiseizure medications. Here, we examine two knock-in mouse models replicating de novo mutations in the HCN1 voltage-gated channel gene, p.G391D and p.M153I (Hcn1G380D/+ and Hcn1M142I/+ in mouse), associated with severe drugresistant neonatal- and childhood-onset epilepsy, respectively. Heterozygous mice from both lines displayed spontaneous generalized tonic-clonic seizures. Hcn1G380D/+ animals had an overall more severe phenotype, with pronounced alterations in the levels and distribution of HCN1 protein, including disrupted targeting to the axon terminals of basket cell interneurons. In line with clinical reports from HCN1 patients, administration of the antiepileptic Na+ channel antagonists lamotrigine and phenytoin resulted in the paradoxical induction of seizures in both lines, consistent with an effect to further impair inhibitory neuron function. We also show that these variants can render HCN1 channels unresponsive to classic antagonists, indicating the need to screen mutated channels to identify novel compounds with diverse mechanism of action. Our results underscore the need to tailor effective therapies for specific channel gene variants, and how strongly validated animal models may provide an invaluable tool towards reaching this objective.

A significant proportion of temporal lobe epilepsy (TLE) patients experience drug-resistant seizures associated with mesial temporal sclerosis, in which there is extensive cell loss in the hippocampal CA1 and CA3 subfields, with a relative sparing of dentate gyrus granule cells and the CA2 pyramidal neurons. A role for CA2 in seizure generation was suggested based on findings of a reduction in synaptic inhibition (Williamson & Spencer, 1994) and the presence of interictal-like spike activity in resected hippocampal tissue from TLE patients (Wittner et al., 2009). We recently found that in the pilocarpine-induced status epilepticus mouse model of TLE there was an increase in CA2 intrinsic excitability associated with a loss of CA2 synaptic inhibition. Furthermore, chemogenetic silencing of CA2 significantly reduced seizure frequency, consistent with a role of CA2 in promoting seizure generation and/or propagation (Whitebirch et al., 2022). In the present study we explored the basis of this inhibitory deficit using immunohistochemical and electrophysiological approaches. We report a widespread decrease in the density of pro-cholecystokinin-immunopositive interneurons and a functional impairment of cholecystokinin-expressing interneuron-mediated inhibition of CA2 pyramidal neurons. We also found a decrease in the density of CA2 parvalbumin-immunopositive interneurons and disruption to the pyramidal neuron-associated perisomatic perineuronal net in the CA2 subfield. These data reveal a set of pathological alterations that may disrupt inhibition of CA2 pyramidal neurons and their downstream targets in epileptic mice.

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