Florey Department of Neuroscience and Mental Health - Theses

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    Mechanisms Underlying Excitability in an HCN1 Developmental and Epileptic Encephalopathy
    Bleakley, Lauren Elizabeth ( 2021)
    Epilepsy 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.
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    Computational modelling of pathologic mechanisms in genetic epilepsies: ion channels, single neurons and neural networks
    Bryson, Alexander Samuel ( 2021)
    Epilepsy is a common and chronic neurological condition characterised by the emergence of excessive or hypersynchronous electrical activity within the brain. A significant proportion of epilepsy is caused by gene mutations, many of which disrupt the function of subcellular protein structures known as ion channels that regulate the excitability of nerve cells (neurons). Despite the prevalence of epilepsy and its societal and economic impact, the mechanisms relating ion channel dysfunction to abnormal electrical activity within neuronal networks remain unclear. This is a matter of importance as approximately one-third of patients with epilepsy suffer intractable seizures despite treatment with modern anti-seizure pharmacotherapy. A more comprehensive understanding of epilepsy pathophysiology that that links ion channel pathology to network dysfunction may reveal new avenues for treatment. In this thesis, the biophysical consequences of two ion channel mutations associated with genetic forms of human epilepsy are explored using computational modelling and experimental electrophysiology. The first is a mutation of the NaV1.1 channel: a voltage-gated sodium channel that serves as an important regulator of neuronal excitability. In this work, we find that a NaV1.1 mutation associated with a severe form of epilepsy leads to impaired cortical inhibition through depolarisation block of inhibitory interneurons. Our results also suggest that NaV1.1 plays a central physiological role for sustaining high firing rates within cortical inhibitory interneurons. The second is a mutation of the GABAA (gamma-aminobutyric acid) receptor: a ligand-gated ion channel that mediates a powerful inhibitory influence within the brain known as tonic inhibition. Using computational modelling we predict that tonic inhibition can selectively modulate the excitability of subtypes of cortical interneurons according to their intrinsic electrophysiological properties. Our models suggest that differential modulation of neuronal excitability occurs via a novel electrophysiological mechanism that is mediated through the dendritic tree. These predictions are supported by in-vitro experiments, and further analysis suggests that modulation of neuronal excitability is dependent upon the expression of certain subtypes of voltage-gated potassium channels, such as the KV3.1 channel. A theme arising from this work is the relevance of distinct subtypes of inhibitory interneurons for regulating excitability in the brain. Therefore, this idea is explored in further detail using a cortical network model that incorporates different interneuron subtypes. Our model suggests that interneurons with properties typical of Parvalbumin-positive subtypes – a prevalent interneuron class within the cortex – are crucial for regulating the extent of internally-driven excitatory activity within a neuronal network. Reductions of excitability in Parvalbumin-positive interneurons promote a network state characterised by strong coupling between excitatory neurons. Known as an inhibition-stabilised network, this network regime is associated with certain cortical computational abilities and the potential to generate epileptic seizures.
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    Targeting HCN4 channels in epilepsy
    Kharouf, Qays ( 2020)
    Epilepsy is a prevalent neurological disorder that affects around 65 million people worldwide. Despite optimal treatment with modern antiepileptic drugs, about one third of patients will continue to have seizures, along with undesirable side effects. Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels are encoded by four genes (HCN1-4). HCN channels have four isoforms (HCN1-4) and produce HCN-mediated currents (Ih) that exhibit pacemaker properties critical for regulating the hyperexcitable neuronal activity seen during seizures. This thesis explores the impact of both pharmacological and molecular HCN channel block on seizure susceptibility and neuronal excitability. Broad-spectrum HCN channel block using ivabradine significantly reduced the seizure susceptibility of wildtype and Scn1a Dravet mice in two proconvulsant assays. Testing isoform-selective HCN channel blockers, the HCN2/1-preferring channel blocker, MEL55A, increased seizure susceptibility. Whereas, the HCN1-preferring channel blocker, MEL57A, had no effect on seizure susceptibility. The HCN4-preferring channel blocker, EC18, significantly reduced seizure susceptibility in two proconvulsant assays in vivo while displaying a safe drug profile. Furthermore, the conditional knockout of HCN4 channels in adult mice was also sufficient to significantly reduce seizure susceptibility in proconvulsant tests with minimal behavioural effects. Interestingly, EC18 showed no effect on seizure susceptibility when administered intraperitoneally to the conditional HCN4 knockout mouse model indicating seizure protection is HCN4-dependant. Moreover, electrophysiological as well as multi-electrode array (MEA) recordings indicated a significant reduction in parameters relating to neuronal excitability after treatment with the HCN4-preferring channel blocker, EC18. Together these results indicate that HCN4 channels are important mediators of neuronal network excitability suggesting they are promising anti-seizure drug targets with minimal adverse effects.