Mechanisms Underlying Excitability in an HCN1 Developmental and Epileptic Encephalopathy

© 2021 Lauren Elizabeth BleakleyEpilepsy is a neurological disorder characterized by seizures, which occur due to excessive and hypersynchronous neuronal activity. The Hyperpolarisation-activated Cyclic Nucleotide-gated channels (HCN channels) are a family of ion channels encoded by the genes HCN1, HCN2, HCN3 and HCN4, which are widely expressed throughout the brain and play key roles in regulating neuronal excitability and synchrony. Dysfunction and dysregulation of HCN channels has been closely linked to epilepsy. In particular, an increasing number of pathogenic variants in HCN1, which encodes the HCN1 channel isoform, have been identified and shown to give rise to epilepsy. Many of these variants cause Developmental and Epileptic Encephalopathy (DEE), a severe condition characterised by early-onset, pharmacoresistant seizures as well as developmental delays. The work described in this thesis aimed to identify the mechanisms underlying how HCN1 channel dysfunction can cause hyperexcitability and subsequent epilepsy, and to explore the best ways of treating this condition. To do so, we generated the first mouse model of HCN1 epilepsy, the Hcn1M294L heterozygous knock-in mouse. This mouse carries the murine homologue of the human HCN1 M305L variant, which has been identified in two unrelated patients with HCN1 DEE. The Hcn1M294L mouse accurately recapitulates several of the major phenotypic features of human HCN1 DEE, including having spontaneous seizures, epileptiform activity on electroencephalography (EEG), susceptibility to heat-induced seizures, and a learning deficit. Electrophysiological studies in Xenopus laevis oocytes and layer V somatosensory cortical pyramidal neurons in ex vivo tissue from Hcn1M294L mice revealed that the disease variant causes a loss of voltage dependence, resulting in a constitutively open HCN1 channel that allows cation ‘leak’ at depolarised membrane potentials. Consequently, Hcn1M294L layer V somatosensory cortical pyramidal neurons were significantly depolarised at rest and fired action potentials more readily, contributing to the hyperexcitability underlying the epilepsy. Pharmacological studies revealed the Hcn1M294L mouse to have similar pharmacoresponsiveness to the anti-epileptic drugs (AEDs) sodium valproate and lamotrigine as a human HCN1 DEE patient. These results positioned this mouse as a strong preclinical model with good face validity, on which potential treatments for HCN1 epilepsy could be trialled. A broad screen of ten currently available AEDs tested in Hcn1M294L mice revealed four drugs which significantly improved and three which significantly worsened neuronal epileptiform activity, providing a potential framework for the clinical treatment of HCN1 epilepsy. Finally, experiments exploring potential precision medicine treatments for HCN1 epilepsy demonstrated that the blood-brain barrier penetrant, broad-spectrum HCN channel blocking drug PTX-002 significantly reduced neuronal epileptiform activity in Hcn1M294L mice, providing an initial proof-of-concept that HCN channel block may be an effective treatment for HCN1 epilepsies caused by cation ‘leak’. Together, these results provide novel insights into the mechanisms underlying hyperexcitability in HCN1 epilepsies, and offer promising directions for future research and for the development of improved treatments for patients who live with these conditions