Clinical trials using fetal tissue have provided the necessary proof-of-principle evidence that transplanted dopamine neurons can appropriately integrate into the brain and alleviate motor symptoms in Parkinson’s disease patients for > 20 years. Pluripotent stem cells (PSCs), are now being pursued as an alternative cell source, circumventing ethical and availability issues, given their competent self-renewal and differentiation capabilities. Despite rapid progress in the field over the past decade, and recent advancement of these cells into clinical trials (Japan October 2018), we recognise a number of short-comings that require further attention. Despite the existence of efficient protocols for the directed differentiation of human PSC into ventral midbrain (VM) progenitors (i.e. cells capable of maturing into dopamine neurons and amenable to transplantation), a small proportion (10%) of cells in culture remain incorrectly specified. These cells are capable of significant expansion after transplantation, such that grafts commonly contain <5% dopamine neurons when examined after many months. These highly proliferative cells not only present an evident risk of neural overgrowth, but there also remains little knowledge of the identity these cells become and the impact they may have on the graft and patient. For these reasons, strategies have been pursued to improve the purity of donor tissue for grafting. However, to date, cell sorting approaches, to select for correctly patterned human PSC-derived cells prior to implantation have been suboptimal. In chapter 3 of the thesis we employ two human PSC reporter lines to demonstrate that the isolation of vm progenitors, but not vm precursors, results in viable cell grafts that are more predictable in their composition, retain integration and functional capacity to restore motor deficits in Parkinsonian rats, and importantly eliminate highly proliferative cells and populations known to contribute to graft-induced dyskinesias. An alternative safeguarding approach for transplantation studies is to employ suicide gene therapy – targeted at eliminating unwanted cells after transplantation. In Chapter 4, we employ a human PSC line carrying a suicide gene (thymidine kinase) that can be activated by administration of a prodrug (ganciclovir), to enable remote killing of proliferating cells within the transplant. We demonstrate that the timely activation of this ‘suicide switch’ can prevent excessive expansion of undesirable cells in the graft whilst preserving DA integrity - yet surprisingly, a small proportion of persistent dividing cells remained. We speculate that this was a result of suboptimal delivery of ganciclovir caused by insufficient vascularisation of dopamine grafts. In a third approach to improve graft outcomes, we address the impact of human PSC-derived VM progenitor donor age (Chapter 5). Despite historical efforts to understand the impact of different aged VM fetal tissue on graft survival, dopamine contribution and functional integration, there remains no comparable assessment of human PSC-derived vm progenitor age. While the current clinical trial in Japan has elected to use late stage human PSC-derived VM progenitors, in Chapter 5 we surprisingly show that VM-specified early-stage progenitors generated the most homogeneous DA grafts, containing a considerably lower component of unwanted, off-target cells than grafts derived from older progenitors. Importantly we demonstrated that while this effect was extremely consistent within human PSC lines, considerable inter-line variability was evident, highlighting the need for rigorous assessment on a line to line basis, prior to translation. In summary, the work provided within this thesis provides significant new insight into strategies to standardize human PSC-derived transplantation for Parkinson’s disease – importantly addressing the need to consider graft survival, proportion of dopamine neurons and their function, as well as the critical requirement to eliminate unwanted cell types to ensure maximal safety.

The peptide hormone relaxin is involved in reproductive processes but has also been investigated for several decades as a treatment for a range of disease states such as scleroderma, acute heart failure, and fibrotic conditions. The receptor for relaxin, RXFP1, is an integral membrane protein belonging to the G protein coupled receptor (GPCR) family. RXFP1 is therefore a therapeutically tractable target for which a thorough understanding of its mechanism of binding and activation is required to develop better relaxin-like drugs. The aims of these studies are to investigate the mechanism by which relaxin binds and activates RXFP1 using a variety of molecular pharmacology approaches in a HEK293T cell model system recombinantly expressing RXFP1 in various forms. Specifically, a hypothesis was tested that a homodimer of RXFP1 might be the minimal functional unit required for receptor activation. GPCR dimers are postulated to interact via their transmembrane helices, so initial investigations aimed to disrupt RXFP1 homodimerisation by incorporation of peptides representing single transmembrane segments of RXFP1 as well as recombinant expression of RXFP1 transmembrane domains. There was no evidence that RXFP1 homodimerisation is required for receptor activation. Following this, the evidence for RXFP1 homodimerisation was re-evaluated in the development of two methods which utilise principles of Bioluminescence Resonance Energy Transfer (BRET). Firstly, split Nanoluciferase was used to tag cell surface localised RXFP1 receptors in combination with mCitrine-tagged RXFP1 and BRET was measured to assess relative receptor proximity. This indicated that RXFP1 is unlikely to be a stable homodimer, intracellularly localised receptors predominate, and there is no change in receptor:receptor proximity upon relaxin stimulation. Secondly, Nanoluciferase-tagged RXFP1 receptors were used in combination with fluorescently labelled relaxin and BRET was measured to track relaxin:RXFP1 binding interactions. This allowed sensitive, real time measurements of the relaxin:RXFP1 binding interactions, demonstrating a multi-step mechanism of relaxin binding in which the linker domain of RXFP1 is critical for high-affinity interactions. Furthermore, there was no evidence of negative co-operativity of relaxin binding, contrary to previous reports which were used as evidence of RXFP1 homodimerisation. Overall, these studies indicate that relaxin does not activate RXFP1 via a mechanism involving a receptor homodimer. Several molecular tools were developed which will be useful for future investigations into RXFP1 pharmacology. This work adds incremental detail to the understanding of how relaxin activates RXFP1, hopefully leading to the development of novel therapeutically useful relaxin-like molecules in future.

Although amyotrophic lateral sclerosis (ALS) typically presents in mid to late life, there is increasing evidence in patients and mouse models for a protracted preclinical period of motor neuron vulnerability and damage before clinical onset. We hypothesized that the seeds for the development of ALS may be sown shortly after conception and motor neuron vulnerability is induced in the perinatal period of life, leading to subsequent neuronal dysfunction and degeneration. The goal of this study was to identify the earliest gene expression patterns in the vulnerable lower motor neurons at very early and key developmental ages in the superoxide dismutase 1 (SOD1)G93A mouse model of ALS. We have implemented and fully characterised HB9:GFP reporter mouse to unambiguously identify and isolate the spinal alpha motor neurons for transcriptomic profiling using RNA sequencing, pathway analysis and target validation. Isolation of HB9:GFP+ spinal motor neurons using FACS was employed at embryonic day 12.5 (E12.5), E17.5, postnatal day 3 (P3) and P8 in SOD1G93A mice and control littermates. Purification of enriched motor neurons was only successful from E12.5 mice. At E17.5, P3 and P8 samples were enriched in glial cells. Gene expression profile of HB9:GFP+ spinal motor neurons from E12.5 SOD1G93A mice revealed significant dysregulation of RNA processing genes, consistent with key pathological pathways in ALS. Dysregulation of Gria2, an AMPA receptor subunit gene, implicated in Ca2+ mediated excitotoxicity was confirmed by real-time qPCR and immunohistochemical analyses in motor neurons from embryonic SOD1G93A mice and induced pluripotent stem cell lines of ALS patient carrying SOD1 mutations. We propose that dysregulation of the AMPA receptor may lead to mitochondrial pathology induced by increased Ca2+ influx and the intracellular increase of free radicals which confers an early susceptibility of spinal motor neurons to excitotoxicity in ALS. In summary, this study provides the first insights into gene expression profiles of motor neurons from SOD1G93A mice in utero. Importantly, our data identified dysregulation of RNA processing and the AMPA-mediated excitotoxicity receptor subunit as early as E12.5 which may account for an early, and likely a causal event in triggering selective motor neuron vulnerability in ALS, arguing for administration of early interventions using anti-excitotoxic therapeutic approaches in ALS.

Clinical trials have provided evidence that the transplantation of new dopamine neurons (DAn) into the striatum of Parkinson’s disease (PD) patients can survive, integrate and restore motor function. Demonstrated first using fetal donor tissue isolated from the developing ventral midbrain (VM), the procedure was hindered by challenges of poor tissue standardisation, limited availability and ethical considerations. Recently, studies have seen a rapid advancement in the use of human pluripotent stem cells (hPSC), differentiated into dopamine progenitors, as a viable alternative and standardized donor source. Despite recently advancing to clinical trials, several questions remain regarding the safety, predictability and efficacy of these cells. While fetal tissue has an estimated 20% cell survival and just 10% of cells within the graft are DA neurons, hPSC-derived neural transplants are notably poorer – remaining highly heterogeneous with a high proportion of non-dopaminergic cells and display inferior reinnervation of target tissues compared to their fetal counterpart. Such observations suggest that a more conducive environment, reminiscent of the developing brain, to support newly integrate fetal and hPSC-DA progenitors could improve the differentiation and functional capacity of neural transplants for PD. The present thesis had focused on improving the host environment into which cells are delivered by using a tissue-specific hydrogel/biomaterial to mimic the extracellular matrix (ECM). The employed biomaterial was capable of signalling to the cells by presenting a high density of a laminin epitope (the main brain ECM component) capable of influencing not only cell adhesion but also survival, differentiation and plasticity. In addition, the biomaterial was utilised to sustain the delivery of trophic proteins, absent in adult brain. This thesis demonstrated the synergistic capacity of the tissue-specific hydrogel, sustaining the delivery of stromal derived factor1 (SDF1) or glial cell-derived neurotrophic factor (GDNF) to enhance grafting outcome for rodent fetal tissue grafts and human pluripotent stem cell derived grafts, respectively, in rodent models of PD. Finding demonstrated these functionalised scaffolds promoted graft survival, improved the specification of A9 dopaminergic neurons (the subpopulation of DA neurons responsible influencing motor function) and enhance plasticity – resulting in restoration of motor deficits. Recognising the heterogeneity of hPSC-derived VM grafts to date, yet with little knowledge of the cellular composition, the work within this thesis also developed a new method to rapidly and efficiently transcriptionally profile xenografts. The method employs bulk tissue dissection, inclusive of the graft and surrounding host tissue, and utilises differences in the RNA sequences between the species to discriminate the xenograft from host gene expression -using either real-time qPCR (qPCR) or whole RNA sequencing (RNAseq). The technique is validated and demonstrated by assessing and comparing the composition of undifferentiated hPSC grafts/teratomas to hPSC-derived VM progenitor grafts in the rodent brain. The approaches enable identification of known and novel genes and provides the first complete characterisation of human VM grafts to date. Collectively, this thesis provides new insight into improving the functional outcomes of VM progenitors’ grafts, as well as a means to screen grafts for both safety and predictable efficacy. Such knowledge will be of critical important as these cells continue to move into clinical trials.

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.

The integration and interpretation of sensorial information is a fundamental requirement for cognition. The thalamus is the sensory gate to the cortex. With its widespread cortical and subcortical connectivity, the thalamus has the network capabilities to function as an integrator of sensory relevant information necessary to drive behavior. Higher order regions of the thalamus send extensive projections to the cortex which exert a powerful influence on cortical encoding of sensory information. However, despite both anatomical and functional evidence, this aspect of thalamic functions has remained largely overlooked likely due to the complexity of investigating thalamocortical circuits in behaving animals. The posteromedial nucleus of the thalamus (POm) is the higher order thalamic nucleus mediating somatosensation. Studies have shown that the POm can modulate sensory processing of cortical pyramidal neurons in the primary somatosensory cortex and drive the cortical plasticity thought to underlie perceptual learning. Despite these reports however, the extent to which POm can influence sensory processing remains unclear and little is known about the role of the POm during sensory-based behavior, let alone goal- directed behavior. In this thesis, I aimed to investigate the effect of POm modulatory input on the primary somatosensory cortex during sensory encoding and goal-directed behavior and determine how this thalamic influence contributes to correct behavioral performance. To achieve this, I used a combination of patch clamp electrophysiology, optogenetics and two-photon Ca2+ imaging of POm axonal projections in the forepaw area of the primary somatosensory cortex (forepaw S1) in anaesthetized and behaving mice trained to perform a sensory based goal-directed task. The work presented in this thesis demonstrates that the activity of POm axonal projections within forepaw S1 encodes correct goal-directed active behavior and dynamically shifts according to task requirements and brain state. In addition, by activating cortical pyramidal neurons either directly or indirectly (through GABAergic interneurons), we show that POm input can both enhance or suppress sensory responses, exerting a powerful modulatory influence over cortical sensory processing. These findings expand the known roles of the higher-order-somatosensory thalamus, from sensory encoding to action selection and flexible switching. Overall, the thalamus is not a simple relay system but mediates complex cognitive functions which are crucial to guide behavior.

Schizophrenia is a severe neuropsychiatric disorder with unknown aetiology, however, environmental stressors that operate early in life are proposed to increase the vulnerability to schizophrenia, but how they act is unknown. One mechanism may be that the diverse environmental risk factors converge on stress-immune pathways and the resultant immune activation perturbs growth factor systems, with consequential neurodevelopmental changes that increase vulnerability to schizophrenia. This research examined the epidermal growth factor (EGF) system, a key growth factor system, the immune system and potential interactions between them in schizophrenia to determine a plausible pathological mechanism. In a targeted candidate gene and pathway approach, expression of >200 selected EGF and immune system genes and the relevant signalling pathways were studied in microarray data derived from BA9 and BA46 of dorsolateral prefrontal cortex from schizophrenia, mood disorder and healthy control subjects. In BA46 and BA11 (orbitofrontal cortex), mRNA of >100 relevant genes, and protein levels of eight molecules were quantified. To determine EGF and immune system changes in association with environmental insult, six markers were protein quantified in brain of spiny mice offspring exposed to maternal immune activation (MIA). In BA9, IFNA1 emerged as the only differentially expressed gene, which was upregulated in both schizophrenia and mood disorder groups. Microarray gene expression profiles from BA46 revealed more transcriptomic changes in schizophrenia than in bipolar affective disorder and major depressive disorder patients across the whole genome and in the cohort of selected EGF and immune genes. Pathway analysis showed dampened EGF system signalling as indicated by the downregulation of the MAPK-ERK and PI3K-AKT-mTOR pathways, in association with upregulated immune pathways TLR-NFkB, TNF, JAK-STAT and the complement cascade in schizophrenia. In BA46, 68 genes showed differential mRNA expression, predominantly in schizophrenia, where decreased EGF system signalling, as indicated by attenuated expression of the MAPK-ERK, NRG1-PI3K-AKT and mTOR cascades, and similarly diminished immune molecular expression, notably in TLR, TNF and complement pathways, along with low NF-kB1 and elevated IL12RB2 protein levels were noted. Additionally, nominal evidence for altered convergence between ErbB-PI3K and TLR pathways was found in BA46 in schizophrenia. Comparatively minimal changes were noted in BA11. Spiny mouse MIA offspring had elevated NF-kB1 protein in nucleus accumbens but only trend EGF system changes in the brain. This research revealed that the EGF and immune systems play a significant role in the pathology of schizophrenia, potentially aberrantly converging, and that the PI3K-AKT-mTOR, TLR and complement pathways may be more closely implicated in the illness pathology. Distinct pathway changes between schizophrenia and mood disorder may reflect variant pathological processes between them. The DLPFC, specifically BA46, appeared a critical regional substrate in schizophrenia in relation to EGF and immune system dysregulation. Further, exposure to environmental insult during gestation can lead to immune dysregulation in the postnatal brain not necessarily associated with marked EGF system perturbations. These findings could inform on a plausible pathological mechanism in schizophrenia linked with environmental risk factors, and lay a foundation for the development of a biological risk profile to allow early recognition and intervention.

Synapses are the basic computational units in the brain which higher level information processing depends on. Neuroligins are a family of postsynaptic cell adhesion molecules which bind to presynaptic neurexins to form a trans-synaptic complex. Neuroligin-1 (Nlgn1) is a member of the neuroligin family and has been extensively studied for its role in regulating postsynaptic function. However, whether Nlgn1 also regulates presynaptic function via the neuroligin-neurexin complex and how does its function at the synapse impact behaviour remain underexplored. This thesis aims to shed more light on these two outstanding questions. In Chapter 1, we assessed the impact of constitutive Nlgn1 deletion (Nlgn1-/-) on the size of different populations of synaptic vesicles (synaptic vesicle pools) in the presynaptic terminal and the rate at which synaptic vesicles are mobilised to release neurotransmitters, a process called presynaptic exocytosis. We found that the loss of Nlgn1 moderately reduced the size of the total recycling pool of synaptic vesicles only when two sub-populations called the readily releasable pool and reserve pool were sequentially released. However, the rate at which the total recycling pool undergoes exocytosis was identical in Nlgn1-/- and WT synaptic terminals. In Chapter 3, we performed detailed dissections of the behavioural phenotype of Nlgn1-/- mice. Despite having robust and well-characterised impairment in postsynaptic mechanisms thought to be required for learning, Nlgn1-/- mice showed normal ability to learn complex associations and flexibility when the learnt associations changed in several instrumental conditioning tasks. However, we found that Nlgn1-/- mice were consistently less willing to overcome effort cost to earn rewards but were more willing to exert effort to escape from an aversive situation. To infer the underlying cognitive processes that generate these behavioural observations, we demonstrated in the form of a simple computational model and behavioural simulations that an increased subjective weighting on negative utilities could, at least in principle, capture the Nlgn1-/- behavioural phenotype across different tasks. Our findings provide new insights into the trans-synaptic function of Nlgn1 and identify a novel role of Nlgn1 in regulating utility trade-off in decision-making. Taken together, this thesis contributes towards a greater goal of neuroscience: understanding the generation of behavioural phenomena across different levels of the brain.

The cardiovascular system is under the constant control of a specialised neural network spanning a number of brain regions. This includes a distinct population of neurons found within the paraventricular nucleus (PVN) of the hypothalamus. Activation of these neurons increases sympathetic nerve activity to cardiovascular organs and raises blood pressure. The mechanisms driving increased activity in the network has not been fully elucidated. Recent evidence suggests that invasion of peripheral immune cells into key regions of the brain might play a role in this regard. The chemokine known as C-C- Ligand 2 (CCL2) is a key activator of peripheral immune cells in hypertension. Whether CCL2 facilitates the entry of immune cells into brain regions that are critical for blood pressure regulation is one of the fundamental questions that has not been addressed. Therefore, I hypothesised that CCL2 causes immune cells to invade specific brain regions to increase nerve activity and blood pressure. In the first study, I found that central injections of CCL2 activated and recruited peripheral circulating macrophages that infiltrated into the PVN. This invasion of macrophages into the brain increased sympathetic nerve activity to the kidney and increased blood pressure. In the second study, CCL2 levels were increased in the plasma and the cerebrospinal fluid at the pre-hypertensive stage in the renovascular model of hypertension. Increased CCL2 caused the recruitment of macrophages into the PVN during the pre-hypertensive stage. This critical finding suggests the involvement of macrophage invasion into the PVN during the early stages of hypertension, possibly leading to blood pressure increases and the development of hypertension. These data are supported by the key finding of the third study, that macrophages are prevented from entering the PVN by antagonising CCL2 signalling mechanisms in the brain; leading to a decrease in blood pressure in renovascular hypertensive rats. In conclusion, I found that the chemokine, CCL2, activates and recruits peripherally circulating immune cells to infiltrate the PVN, leading to increases in sympathetic nerve activity and blood pressure. This action, by CCL2, in the early pre-hypertensive stage of hypertension, might be a mechanism by which hypertension becomes established, viz by activating and recruiting immune cells that infiltrate into the brain to activate key areas and neural networks critical for the moment-to-moment control of the cardiovascular system. Blocking the receptor via which CCL2 acts, decreases blood pressure. The findings reported here highlights the importance of this immune-brain pathway in the development of hypertension.

Abnormal iron elevation and the associated cell death pathway ferroptosis are implicated in the pathogenesis of several neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease. Ferritin, the major iron binding protein of the body, is present in cerebrospinal fluid (CSF) where it has been shown to be associated with Alzheimer’s disease progression, however the function of extracellular ferritin is not known. Ferroptosis involves the degradation of cytosolic ferritin, but whether extracellular ferritin impacts on ferroptosis is unknown. To better understand the role of extracellular ferritin in neurodegenerative diseases, this project aims 1) to characterize extracellular ferritin in response to iron and 2) to investigate the effect of extracellular ferritin on neuronal ferroptotic cell death in vitro. This thesis demonstrated that the expressions of intracellular and extracellular ferritin are associated with intracellular iron levels in both glia and neuronal cell lines. Extracellular vesicles from cell culture and from brain tissue were found to contain ferritin. Though the levels of soluble ferritin secreted by neurons increased with an increase in intracellular iron levels, the levels of vesicular ferritin remained unchanged. The levels of iron in neuronal extracellular vesicles increased with cellular iron burden. Endocytosis of soluble apo-H ferritin protected cultured neurons against ferroptosis. Taken together, changes in biofluid ferritin levels may represent a response to ferroptosis in neurodegenerative diseases.

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.

Background: Dravet Syndrome (DS) is classified by severe seizures and progressive developmental delay. In 80% of cases, DS is caused by mutations in the SCN1A gene. Despite a growing understanding of the underlying pathomechanisms of DS thanks to animal and heterologous expression models, clinical outcomes for DS remain poor. This has led to the investigation of new high throughput models for diagnosis and drug discovery as an avenue for precision medicine. Multielectrode arrays (MEAs) allow dissociated neurons to form networks on a surface embedded with microelectrodes that record their extracellular activity. This model is promising in the field of precision medicine due to a greater complexity than heterologous expression systems and potential for higher throughput drug screening than can be achieved using animal model. Following this premise, this thesis addresses the following aims. Aim 1: Establish a workflow for culturing primary cortical neurons on MEAs and determine the ideal culture conditions to rapidly generate stable networks for further high-throughput disease modelling and drug screening. Aim 2: Evaluate the potential of the MEA system in modelling DS. Sensitivity of the system will be determined by comparing activity from networks sourced from animal models that exhibit a severe or mild phenotype as a result of SCN1A haploinsufficiency due to their genetic background. Aim 3: Determine the predictive validity of MEAs for screening therapeutic compounds by applying known anti-epileptic drugs to the DS model networks. Method: Primary cortical mouse neurons were cultured on multiwell MEA plates. Various seeding densities and lengths of maturation were tested to determine the optimal parameters for generating stable and reproducible networks. Stability was assessed by comparing a variety of features describing spiking, bursting, and network connectivity. Genetic epilepsy was assessed by culturing neurons derived from mouse models with SCN1A haploinsufficiency. These mice are of different strains and have genetic backgrounds that influence the severity of the SCN1A+/- phenotype (severe or mild expression). Once again spiking, bursting and network connectivity parameters were compared. Finally, stiripentol, lamotrigine and cannabidiol were applied to DS networks and the discriminatory effects between the DS and normal networks were compared. Results: A stable baseline was generated at DIV21 using seeding density 11,646 cells/mm2. SCN1A haploinsufficiency in the C57BL/6 and SV129 backgrounds mimicked what is observed in vivo, with a significant epilepsy phenotype observed in the C57BL/6 background that was not seen in the SV129 background. While stiripentol, lamotrigine and cannabidiol all had significant effects on the overall activity profile of the networks, the response to the drugs was largely indiscriminate between normal and SCN1A+/- networks. Conclusion: A workflow to generate stable networks was achieved and was successfully implemented when characterizing the genetic DS network models. The MEAs captured the DS phenotype and discriminated between severe and mild phenotypic expression. While there were occasional significant differences between normal and SCN1A+/- network responses to known AEDs, these small differences alone were not enough to confirm that this assay showed predictive validity for drug screening. Further studies examining different drugs and concentrations on the networks, as well as more sensitive analysis methods should be performed to further understand the potential of this assay.