This Doctoral Training Centre offers research PhDs in childhood-onset epilepsies.
Projects with a September 2023 start date are listed below. If you would like to know more about a project, please feel free to contact the supervisors of the project.
Primary supervisor: Prof Cathy Abbott (Centre for Genomic & Experimental Medicine)
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The ease of use of gene editing tools developed over the last 5 years has resulted in many new models of genetic epilepsies that recapitulate clinical mutations precisely . However, in spite of the severity of the conditions being modelled, these new mouse lines frequently display phenotypic abnormalities but stop short of showing face validity in the form of spontaneous seizures, at least at a high enough frequency to be readily observable. Clearly any therapeutic strategy (whether genetic or drug-based) needs to be tested on suitable animal models, but the translational potential of such studies is limited if there is no ability to test the impact of the intervention on seizures.
In this project the student will explore several different approaches to solving the problem of low frequencies/absence of spontaneous seizures in the context of one to two specific genes in which de novo mutations cause childhood-onset epilepsy (such as EEF1A2 and CDKL5), and for which well characterised mouse models are available. The use of multiple lines will allow the student to make direct comparisons and judge how widely applicable any specific approach would be. They will explore a range of different methods to enhance face validity including continuous EEG monitoring, in-cage behavioural monitoring coupled with video recording, altering the genetic background of the parent strain, and measuring the threshold for seizure induction.
The student will, in parallel, also investigate molecular mechanisms underlying at least one of these genetic disorders; this could range from the use of proteomics to look at changes in binding partners resulting from specific mutations, to detailed characterisation of expression patterns in specific neuronal populations and/or mapping expression throughout the brain using lightsheet microscopy. This combination of sub-projects will allow the student to be trained in a wide variety of lab disciplines from basic to preclinical research. This approach will also allow the student to generate their own hypotheses to be tested in the later stages of the PhD. They will thus combine important research into practical solutions for the development of face valid preclinical models to maximise their translational potential, with more fundamental research into mechanisms underlying childhood-onset epilepsies.
1. Fallah MS, Eubanks JH. Seizures in mouse models of rare neurodevelopmental disorders. Neuroscience. 2020 Feb 12;
2. Davies FCJ, Hope JE, McLachlan F, Marshall GF, Kaminioti-Dumont L, Qarkaxhija V, et al. Recapitulation of the EEF1A2 D252H neurodevelopmental disorder-causing missense mutation in mice reveals a toxic gain of function. Hum Mol Genet. 2020 Jun 27;29(10):1592–1606.
3. Amendola E, Zhan Y, Mattucci C, Castroflorio E, Calcagno E, Fuchs C, et al. Mapping pathological phenotypes in a mouse model of CDKL5 disorder. PLoS One. 2014 May 16;9(5):e91613.
4. Marshall G, Gonsalez-Sulser A, Abbott CM. Modelling epilepsy in the mouse: challenges and solutions. Dis Model Mech 2021. 14(3):dmm047449. doi: 10.1242/dmm.047449
Primary supervisor: Prof Emily Osterweil (Centre for Discovery Brain Sciences)
Additional supervisors: Prof Matt Nolan (Centre for Discovery Brain Sciences)
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Childhood-onset epilepsy is a feature of several neurodevelopmental disorders, including Fragile X Syndrome and SYNGAP1 haploinsufficiency, two monogenic causes of autism and intellectual disability1,2. The aetiology of seizures in these disorders is poorly understood. However, treatments that normalize protein synthesis have been shown to prevent the emergence of pathological changes in both Fmr1-/y and Syngap+/- mouse models, including epileptiform activity and susceptibility to audiogenic seizures (AGS) 3-6. This suggests that dysregulation of protein turnover causes the generation of epilepsy (epileptogenesis) in both disorders.
Identifying the functional consequences of dysregulated protein turnover that contribute to epileptogenesis could provide unique insights and potentially identify new therapeutic targets. The student will use cutting edge techniques in molecular biology, electrophysiology and imaging to assess the functional consequences of changes in protein synthesis and degradation that occur within seizure-generating neurons.
The specific aims are to: (1) elucidate the critical neuron populations contributing to AGS in Fmr1-/y and Syngap+/- rat models, and (2) to identify the functional changes that occur as a results of dysregulated protein synthesis and degradation in these neurons and that contribute to seizure generation
Although AGS is a highly reproducible phenotype in the Fmr1-/y and Syngap+/- models, the underlying circuitry has not been identified. To identify activated neurons over a time-course post-AGS induction, the student will use a combination of immunostaining for cFos (which reports recently active neurons) and whole-brain imaging. This approach will enable the student to focus on detailed analysis of brainstem nuclei implicated in audiogenic seizures in other models and unbiased brain-wide analyses to capture a global picture of candidate neural populations. The student will then use animals treated with lovastatin, a therapeutic that prevent AGS in the Fmr1-/y model, to more discretely identify the circuits that generate seizure activity 4.
To understand how contributes to epileptogenesis, brain slice patch-clamp electrophysiology approaches will be used to identify ion channel mechanisms active in the candidate neuronal populations that lead to seizure generation. This strategy will take advantage of TRAP-seq profiling and proteomic data from the Osterweil lab, which has reveal novel targets for therapeutic intervention in developmental disorders and childhood-onset epilepsies7. The initial focus will be on excitatory neurons in the Inferior Colliculus that have been implicated in AGS 8. The ion channel mechanisms that promote epileptogenesis in mutant neurons will then be targeted for knock-down using viral injection of shRNAs or using relevant pharmacological strategies.
1. Berry-Kravis, E. Epilepsy in fragile X syndrome. Dev Med Child Neurol 44, 724-728 (2002).
2. Mignot, C. et al. Genetic and neurodevelopmental spectrum of SYNGAP1-associated intellectual disability and epilepsy. Journal of medical genetics 53, 511-522, doi:10.1136/jmedgenet-2015-103451 (2016).
3. Yan, Q. J., Rammal, M., Tranfaglia, M. & Bauchwitz, R. P. Suppression of two major Fragile X Syndrome mouse model phenotypes by the mGluR5 antagonist MPEP. Neuropharmacology 49, 1053-1066, doi:10.1016/j.neuropharm.2005.06.004 (2005).
4. Osterweil, E. K. et al. Lovastatin corrects excess protein synthesis and prevents epileptogenesis in a mouse model of fragile X syndrome. Neuron 77, 243-250 (2013).
5. Barnes, S. A. et al. Convergence of Hippocampal Pathophysiology in Syngap+/- and Fmr1-/y Mice. J Neurosci 35, 15073-15081, doi:10.1523/JNEUROSCI.1087-15.2015 (2015).
6. Wang, C. C., Held, R. G. & Hall, B. J. SynGAP regulates protein synthesis and homeostatic synaptic plasticity in developing cortical networks. PLoS One 8, e83941, doi:10.1371/journal.pone.0083941 (2013).
7. Thomson, S. R. et al. Cell-Type-Specific Translation Profiling Reveals a Novel Strategy for Treating Fragile X Syndrome. Neuron 95, 550-563 e555, doi:10.1016/j.neuron.2017.07.013 (2017).
8. Gonzalez, D. et al. Audiogenic Seizures in the Fmr1 Knock-Out Mouse Are Induced by Fmr1 Deletion in Subcortical, VGlut2-Expressing Excitatory Neurons and Require Deletion in the Inferior Colliculus. J Neurosci 39, 9852-9863, doi:10.1523/JNEUROSCI.0886-19.2019 (2019).
Primary supervisor:Prof David Wyllie (Centre for Discovery Brain Sciences)
Additional supervisors: Dr. Katie Marwick (Centre for Clinical Brain Sciences)
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N-methyl-D-aspartate (NMDA) receptors are a class of ligand-gated ion channel that are activated by L-glutamate, the major excitatory neurotransmitter in the mammalian central nervous system (CNS). Physiologically NMDA receptors play critical roles in fast excitatory neurotransmission and signalling, in neurodevelopment, synaptic plasticity while dysfunctional NMDA receptor function leads failure certain forms of learning and memory, cell death via excitotoxicity leading to neurodegenerative disease, and where there is imbalance in the excitation:inhibition ratios this is thought to be a significant factor that leads to several psychiatric disorders. Importantly for this project impaired NMDA receptor function can give rise to epilepsy.
Fundamental to the normal physiological functioning of NMDA receptors is the voltage-dependent block that is mediated by Mg2+ ions – at resting membrane potentials the permeation pathway (i.e. the ion channel pore) of NMDA receptors is blocked by Mg2+ and only when the membrane potential depolarizes as a result of increased electrical activity does the pore become permeable to cations, most notably Na+, K+ and Ca2+ ions. Without voltage-dependent Mg2+ block, NMDA receptors would be active at rest and this would lead to excessive excitatory drive and uncontrolled neuronal activity and massive seizure activity. Hence, voltage-dependent Mg2+ block is critical for normal glutamatergic synaptic function. In recent years many mutations have been identified in GRIN genes (the genes that encode NMDA receptor subunits) which lead to expression of NMDA receptors with either gain- or loss- of function (1).
In this PhD project the student will study the effects of a mutation that in humans causes a severe childhood-onset epilepsy (2) and results from the asparagine residue in the GluN2A subunit being replaced by a lysine residue – GluN2AN615K. Our lab has previously characterized the physiological and pharmacological properties of GluN2A-containing NMDA receptors harbouring this mutation in heterologous expression systems (3, 4) but in this project we will use a transgenic rat model in which half of the GluN2A NMDA receptor subunits express the mutation – thus the heterozygous nature of the expression seen in humans is reflected in the model.
The student will use multidisciplinary approaches to gain mechanistic understanding of how the reduced voltage-dependent Mg2+ block exhibited by NMDA receptors expressing GluN2A NMDA receptor subunits leads to pathophysiological signalling in the CNS. The N615K mutation can be considered to be both a loss-of-function mutation (reduced Mg2+ block and reduced Ca2+ permeability) while at the same time it can be thought of as being a gain-of function mutation as the activity of NMDA receptors will not be inhibited at hyperpolarized membrane potentials. To assess the extent to which these two sides of altered function contribute to dysregulated signalling the student will combine electrophysiological recording from ex vivo brain slices to assess synaptic function and Ca2+-imaging to monitor network activity. Moreover, using a variety of pharmacological seizure-inducing paradigms the student will determine whether this pre-clinical model of childhood-onset epilepsy shows either spontaneous seizure-like activity or increased susceptibility to seizure initiation. Finally, using in vivo recording the student will assess whether the extent to which this mutation leads to epileptic-like activity and how such activity can be ameliorated by drug interventions.
1. XiangWei W, Jiang Y, Yuan H: De Novo Mutations and Rare Variants Occurring in NMDA Receptors. Curr Opin Physiol 2018, 2:27-35.
2. Endele S, Rosenberger G, Geider K, Popp B, Tamer C, Stefanova I, Milh M, Kortum F, Fritsch A, Pientka FK et al: Mutations in GRIN2A and GRIN2B encoding regulatory subunits of NMDA receptors cause variable neurodevelopmental phenotypes. Nat Genet 2010, 42(11):1021-1026.
3. Marwick KFM, Hansen KB, Skehel PA, Hardingham GE, Wyllie DJA: Functional assessment of triheteromeric NMDA receptors containing a human variant associated with epilepsy. J Physiol 2019, 597(6):1691-1704.
4. Marwick KFM, Skehel PA, Hardingham GE, Wyllie DJA: The human NMDA receptor GluN2A(N615K) variant influences channel blocker potency. Pharmacol Res Perspect 2019, 7(4):e00495.