Florey Department of Neuroscience and Mental Health - Theses
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ItemEpileptic Encephalopathies: Identification and Characterization of Disease Mechanisms( 2022)Pathogenic mutations in the KCNQ2 gene, encoding the KV7.2 voltage - gated potassium channel, are known to cause neonatal seizure disorders, including severe Epileptic Encephalopathies. Epileptic Encephalopathies are characterised by pharmacoresistant seizures, developmental delay, and behavioural and cognitive deficits. Current therapies show limited efficacy in the treatment of seizures and fail to address the devastating comorbidities 1–5. KCNQ2 de novo variant K556E was identified in a patient with Epileptic Encephalopathies 6. In this research project we aimed to characterize the KCNQ2 K556E variant to better understand the underlying mechanisms of the disease and to set the stage for therapeutic screens that will help finding better treatments for this patient and for other patients like her. In order to assess the disease mechanisms of this variant, three disease models were used. Initially, the biophysical properties of the variant were investigated using in vitro expression in Xenopus oocytes followed by two - electrode Voltage - clamp Recordings. The data suggest a loss-of-function with no dominant negative effect caused by the variant. A loss of KV7 channel function indicate a significant reduction in the production of the potassium M - current. The M - current main biophysical role is setting the neuronal resting membrane potential and protecting against uncontrolled repetitive action potential firing. The loss-of-function might be the underlying cause of an excitable phenotype. We hypnotised that application of KV7 channels opener might rescue the significant reduction current seen in KCNQ2 K556E variant. However, retigabine, a KV7 opener, had no significant effect on the variant’s biophysical properties when the mutant channel was expressed as a homotetramer in Xenopus laevis oocytes. The second disease model we have generated and characterized is an in vivo disease model. A knock - in mouse model carrying the corresponding amino acid exchange in its KCNQ2 gene. Our observational studies revealed that both homozygous and heterozygous mice develop spontaneous seizures and present with increased mortality rates and premature death compared to wild-type littermate controls. The heterozygous mice are more susceptible to both heat induced seizures and chemically induced seizures. The heterozygous mice also expressed some behavioural changes when compared with wild-type littermate controls. The heterozygous mice bury less marbles in the marble burying test when compared with wild-type littermate controls. This might suggest a less anxious like behaviour and reduced cognitive abilities. Furthermore, the significant difference found between genotypes in this specific behavioural test could be used as an efficacy marker in a drug screening set of experiments in later stages. Lastly, we have generated and characterized patient specific in vitro iPSC-derived neuronal cultures. To this end we exploited a differentiation method based on the overexpression of Neurogenin 2 (NGN2) transcription factor to generate excitatory cortical neurons and investigated the electrophysiological characteristics of the neuronal cultures using whole cell patch clamping technique. Our findings suggest a more excitable phenotype of the patient-derived neurons in comparison with a control cell line from a healthy subject. In conclusion, the disease models indicate that a loss-of-function caused by the K556E variant likely leads to an increase in neuronal excitability which may be responsible for the increased susceptibility to epileptic seizures.
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ItemInvestigating SCN2A dysfunction in later-onset epileptic encephalopathy and autism( 2022)Voltage-gated sodium channels are protein complexes that underlie action potential electrogenesis in excitable cells. Genetic variation in channel genes is a major cause of neurodevelopmental disorders including epilepsy, autism spectrum disorder (ASD), and intellectual disability. SCN2A, encoding voltage-gated sodium channel 1.2 (NaV1.2), is one of the most significant single-gene contributors in all neurodevelopmental disorders, with genetic variants reported in several conditions ranging in severity from benign temporary seizure syndromes to phenotypically devastating developmental and epileptic encephalopathies. Genetic variants in SCN2A are typically described as either gain- or loss-of-function (LOF), with evidence to suggest that there is strong correlation between genotype and phenotype. SCN2A ASD and later-onset developmental and epileptic encephalopathy (LOEE) are severe life-long disorders with no targeted pharmacological interventions currently available. Anti-epileptic pharmaceuticals have variable efficacy in treating the seizures associated with LOEE, and they do not target the associated features of the disorder, some of which are common with ASD, including intellectual disability, developmental delay, movement disorders, and behavioural issues. This thesis is the culmination of four years of study of voltage-gated sodium channel patients, gene regulation, and neuron models, and includes the first phenotypic analysis of patient-derived induced-pluripotent stem cell (iPSC) models of SCN2A in ASD and LOEE, two developmental disorders with enigmatic mouse models and an unmet clinical need. This project is designed to elucidate the mechanisms behind LOF SCN2A in these two disorders using electrophysiological and molecular techniques on selected candidate variants in each disorder. The hypothesis of this PhD project is that patient-derived iPSC models of SCN2A LOF will reveal disorder-specific phenotypes in neurons, informing the pathomechanisms of disease and useful for future drug screening projects. This thesis is divided into four chapters, each chapter exploring a different aspect of SCN2A in development and disease. Chapter 1 serves as an introduction to sodium channel subtypes, structure, function, and contains a review of the SCN2A literature describing patient genetic variants, associated phenotypes, published laboratory models and therapeutic strategies. Chapter 2 is a published study on the developmental expression and transcript regulation of voltage-gated sodium channel genes relevant to neurodevelopmental disorders. Chapters 3 and 4 explore the pathomechanisms of disease in SCN2A LOEE and ASD, respectively, and include the first functional characterisation of SCN2A patient-derived iPSC models of these disorders.
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ItemMechanisms Underlying Excitability in an HCN1 Developmental and Epileptic Encephalopathy( 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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ItemComputational modelling of pathologic mechanisms in genetic epilepsies: ion channels, single neurons and neural networks( 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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ItemTargeting HCN4 channels in epilepsy( 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.
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ItemA platform for analysis of in vitro neuronal networks for the development of precision therapeutics in SCN2A disease( 2019)Background and Purpose Developmental and epileptic encephalopathies are a group of devastating neurological disorders in which the patients have developmental impairment as well as refractory seizures. Comorbid states are common and include cognitive and movement disorders. SCN2A, which encodes the brain sodium channel Nav1.2, has emerged as one of the most prominent developmental and epileptic encephalopathy genes. Based on the onset of disease, patients with SCN2A epilepsy variants can be divided into two major groups. In the early onset group, seizures start within the first three months of life, whereas in the second group, the onset is after three months of age. Sodium channel blockers such as phenytoin are effective in some of the early onset patients. In contrast phenytoin is ineffective and may in fact worsen seizure outcomes in late onset disease. This suggests different molecular pathomechanisms. The lack of efficacious therapies underscores an urgent need for novel treatment strategies. Experimental Approach Two knock-in mouse lines were generated carrying Scn2a p.R1883Q and p.R854Q variants corresponding to human SCN2A p.R1882Q and p.R853Q variants. These are the most common recurrent variants found in the early and the late onset group of SCN2A developmental and epileptic encephalopathies, respectively. In vitro signatures of neuronal network behaviour were assessed using multi-electrode array analysis of the primary cortical cultures obtained from postnatal day 0-1 animals carrying the respective variants up to 28 days in vitro. Acute pharmacological effects were evaluated around 22 days in vitro. Key Results After 2-4 weeks network analysis in culture showed increased activity for neurons harbouring the heterozygous p.R1882Q variant associated with early onset disease. Conversely, a decreased firing rate was observed in cultures in which neurons carried the heterozygous p.R853Q variant associated with late-onset disease. The excitability in both cultures was reduced by phenytoin, which resulted in shifting the p.R1882Q in vitro phenotype towards the wild types and p.R853Q away from the wild types, consistent with clinical observations. Interestingly, one of the tested antiepileptic drugs changed the activity of both cultures was towards the wild type phenotype indicating potential benefits of this drug for both early and late onset SCN2A developmental and epileptic encephalopathies. Conclusion and Implications The assumption that early onset SCN2A variants are more likely to cause gain-of-function and the late onset SCN2A variants to a loss-of-function of the Nav1.2 channel was confirmed for the two studied variants using in vitro neuronal cultures. Moreover, the clinical observations regarding the effectiveness of the sodium channel blocker phenytoin in patients with early and late onset seizures was corroborated by our in vitro models. Lastly, an antiepileptic drug was identified as a potential treatment for both early and late onset SCN2A developmental and epileptic encephalopathies.
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ItemAntisense oligonucleotide precision therapy in KCNT1 - severe epilepsy( 2019)In recent years, the advancement of sequencing technology used in conjunction with thorough clinical evaluation has enabled clinicians to attribute genetic factors to the etiology of epilepsy. A significant number of mutations in genes encoding ion channels have been identified as the cause of the developmental and epileptic encephalopathies (DEE), a group of severe epilepsy syndromes of childhood and infancy characterized by the presence of abundant epileptiform activity, refractory seizures, intellectual disability, developmental regression, movement disorders, and increased mortality. The gene KCNT1 encodes the sodium activated potassium channel subunit KNa1.1. De novo mutations in this gene have been identified in patients with both severe and milder forms of epilepsy. The most commonly associated phenotypes are epilepsy of infancy with migrating focal seizures (EIMFS) and autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). EIMFS is situated on the severe end of the spectrum and manifests during the first six months of life with frequent multifocal seizures in addition to developmental plateau or regression. Patients with KCNT1 associated epilepsy typically respond poorly to conventional anti-seizure medications, resulting in an unfavorable prognosis and compromising their quality of life. Biophysical studies in heterologous expression systems have shown that KCNT1 pathogenic variants found in epilepsy result in strongly increased potassium currents. This overall gain of function (GoF) is currently accepted as the primary mechanism of disease in KCNT1 associated epilepsy. To directly address this pathologic mechanism, antisense oligonucleotide (ASO) mediated knockdown was used to test the idea that reducing Kcnt1 expression would be therapeutic in a mouse model of KCNT1 associated epilepsy. This approach was supported by the tolerance for KCNT1 loss of function (LoF) observed in the general population and the mild phenotype found in Kcnt1 knockout mice. Using the CRISPR Cas9 system, an orthologous pathogenic variant found in KCNT1 EIMFS was inserted in Kcnt1 to develop a rodent model. Mice, homozygous for the mutation display frequent spontaneous seizures, abundant interictal activity in the electrocorticogram (ECoG), behavioral abnormalities and early death. Thus, recapitulating key aspects of KCNT1 severe epilepsy and offering a valuable tool for therapeutic screening. Then, an ASO was designed to specifically hybridize with Kcnt1. An additional nontargeting sequence was used as a control. After a single intracerebroventricular bolus injection, homozygous mice showed a marked knockdown of Kcnt1 mRNA and in comparison to control treated animals, displayed almost complete abolition of seizures, prolonged survival, and improved cognition. Exaggerated pharmacology was explored in wild type mice treated with a dose of ASO that produced more than 90 percent knockdown. Here, behavioral measures revealed some anxiety like traits in ASO treated mice, but knockdown was otherwise tolerated. Additional seizure susceptibility in LoF was tested in Kcnt1 knock out mice, which indicated that LoF was not related to a reduction in seizure threshold. To conclude, the preclinical evidence presented in this thesis supports ASO based gene silencing as a therapeutic approach in KCNT1 GoF epilepsies. This body of work provides proof of concept for such approach and encourages the translation of ASO based therapies for genetic epilepsies, in particular for those with an underlying GoF pathomechanism.