Florey Department of Neuroscience and Mental Health - Theses
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ItemMass Spectrometry As A Tool For Drug Development In SCN2A Developmental and Epileptic Encephalopathies( 2023-11)Mutations within the SCN2A are recognized as a prominent cause of autism spectrum disorder and a spectrum of developmental and epileptic encephalopathies (DEEs). As more patients are affected by mutations in SCN2A, it drives the need for precision medicines and to better understand the biology and pathogenesis of the disorder. The SCN2A gene encodes the voltage-gated sodium channel, Nav1.2. Antisense oligonucleotides (ASOs) are a class of drugs being developed to treat SCN2A disorders by knocking down SCN2A mRNA and therefore protein levels. In this study, targeted mass spectrometry methods are utilised to measure Nav1.2 protein levels directly and untargeted, or “discovery”, proteomic methods are used to measure the entire proteome in brain tissue collected from various SCN2A mouse models and mice treated with an experimental ASO therapy. Three SCN2A knock-in missense mutation mouse models are included in the study, each representing a phenotypic group within the SCN2A disease population. These results all support that the ASO has strong target engagement on the protein expression similar to mRNA level. The mutant mouse models are R1882Q representing the early seizure onset phenotype, R853Q representing the late seizure onset phenotype, and S1758R representing the autism with no seizure phenotype. When measuring Nav1.2 in R1882Q mouse whole-brain treated with an ED80 dose (80% knockdown Scn2a mRNA) of an Scn2a-targeting ASO, Nav1.2 was reduced 72% compared to R1882Q mice treated with a scrambled-control ASO. WT mice treated with an ED50 dose of SCN2A-targeting ASO at P30 (post-natal day 30) with brain tissue collected over a 5-week period showed consistent knockdown of Nav1.2 protein of approximately 50% 2- and 3-weeks post-injection in cerebellum, hippocampus, and cerebellum. In the mutant models of Scn2a encephalopathy, Nav1.2 expression remained unchanged in R1882Q and R853Q mutant mice compared to WT littermates while Nav1.2 expression was reduced ~50% in S1758R mutant mice compared to WT littermates, suggesting haploinsufficiency may be a major driver of the autism phenotype. Global proteomic analysis revealed several potential off-target and/or toxicity biomarkers of ASO treatment. These biomarkers were primarily associated with neuroinflammation, including neurofilament heavy (Nefh) and programmed cell death 5 (Pdcd5). Global proteomic analysis in the 3 mutant models showed unique proteomic profiles in each, with minimal overlap, suggesting the very different phenotypes also lead to differences in protein expression and dysregulation. However, dysregulated proteins across the 3 models were involved in several shared pathways, including those responsible for regulation of synaptic signalling and mitochondrial function and metabolism. The exploration of novel epileptic mouse models and mice treated with experimental antisense oligonucleotides through proteomic analysis has unveiled promising prospects for potential new biomarkers. This integrated approach has provided invaluable insights. It is anticipated that certain biomarkers identified may undergo further validation and potentially be employed in clinical trials for emerging SCN2A drugs. These biomarkers could serve to monitor disease progression and assess the effectiveness of innovative treatments, building upon prior research efforts.
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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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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.