Epilepsy & Neuropharmacology
See also The Ioin Channels and Disease Laboratory
Neuropharmacological strategies for disease modification and prevention of the development of epilepsy - also offered as MBiomedSc
Supervisors: Dr Pablo Casillas-Espinosa, Dr Kim Powell, Prof Terence O’Brien, Dr. Sandy Shultz, A/Prof. Nigel Jones.
Project Site: The Department of Medicine, The Royal Melbourne Hospital, and The Melbourne Brain Centre, Parkville.
Contact: Pablo Casillas-Espinosa E:pablo.casillas@unimelb.edu.au; Kim Powell E:kpowell@unimelb.edu.au
Project description: Current therapies for epilepsy are symptomatic, only suppressing the symptoms (seizures), but do not impact the development or progression of disease. Many groups around the world, including ours, are testing novel therapies to impact epileptogenesis, intervening very early in epilepsy development to limit the severity of disease, with some preclinical success. But most patients present at the clinic already experiencing seizures, so a more practical strategy would be to attempt to modify epilepsy disease progression.
For this project, we will investigate whether our novel treatments can reverse epilepsy severity in a rat model of acquired epilepsy in cases of established epilepsy. We then evaluate if the animals are having less seizures, behavioural comorbidities and neuroimaging changes after the completion of treatment. If the results are positive, they would have major clinical implications in patients with already established acquired epilepsy. Moreover, the experimental drugs that we will be tested have a favorable safety profile in early phase clinical trials facilitating the translation of the results of this preclinical study into a clinical trial.
Skills: The skills expected to be learnt from this projects include: Small animal handling and neurosurgery (electrode implantations), animal models of temporal lobe epilepsy, behavioral neuroscience, magnetic resonance imagining interpretation and analysis.
Projects available
- Anti-epileptogenic effects of novel T-type calcium channel blocker.
- Behavioural changes and Imaging the during the epileptogenic proces
Biomarkers of epileptogenesis and epilepsy disease progression - also offered as MBiomedSc
Supervisors: Dr Pablo Casillas-Espinosa, Dr Kim Powell, Dr. Sandy Shultz, A/Prof. Nigel Jones, Prof Terence O’Brien
Project Site: The Department of Medicine, The Royal Melbourne Hospital, and The Melbourne Brain Centre, Parkville.
Contact: Pablo Casillas-Espinosa E:pablo.casillas@unimelb.edu.au; Kim Powell E:kpowell@unimelb.edu.au
Project description: A biomarker is an objectively measured characteristic of a normal or pathologic biological process. The development of novel interventions to treat, cure, and prevent epilepsy would benefit greatly from the identification and validation of such biomarkers. In addition, identification of biomarkers may facilitate the development of novel interventions to prevent epilepsy; to prevent the occurrence of epileptic seizures, reverse progression of epilepsy, and potentially even cure epilepsy after it is established. This project will investigate blood- and brain-derived biomarkers of epileptogenesis (the development of epilepsy) and of disease progression of epilepsy using small animal models.
Skills: The skills expected to be learnt from this project include: Small animal handling and neurosurgery (electrode implantations), models of acquired epilepsy, blood and cerebrospinal fluid (CSF) collection, EEG recordings and analysis, and biochemical and molecular analysis (subcellular fractionation, western blotting), magnetic resonance imagining interpretation and analysis.
Reducing Epilepsy Deaths - Learning from the NCIS (National Coronial Information System)
Supervisors: Dr Rosemary Panelli
Project Site: The Melbourne Brain Centre, The Department of Medicine (RMH)
Contact: Dr Rosemary Panelli E: rpanelli@unimelb.edu.au
Project Description: The most common epilepsy-related cause of death remains a mystery. Epilepsy carries a risk of premature death that is 2-3 times higher than for the general population and a risk of sudden death 20 times higher. The mean age of death is low and the number of Years of Life Lost is high. Sudden Unexpected Death in Epilepsy (SUDEP) is the term now used to describe these unexplained deaths but recognition and appropriate reporting is inconsistent internationally and the incidence is difficult to assess.
The Australian National Coronial Information System is unique internet-based data storage and retrieval system and research access to such a comprehensive database is rare in the international context.The objective of this study is to identify and analyse all information held in the NCIS database concerning epilepsy-related deaths. The NCIS data is extensive and valuable due to the large number of these deaths which occur in the community setting. A systematic examination of the NCIS documents (police reports, post-mortem results, toxicology, and coroners’ findings), will allow the researchers to clarify the frequency of SUDEP and to identify any patterns or common factors associated with the deaths, thus enabling a more informed characterisation of epilepsy-related death and risk in this country.
The project will include extensive assessment and interpretation of forensic and police reports, database development, critical analysis of the data, and preparation of information for publication.
Keeping the Brain and the Heart in Sync - HERG channels in the CNS - also offered as MBSc
Supervisors: Dr Chris French,
Project Site: Department of Medicine RMH/MBC Neurosciences Building, Parkville
Contact: Chris French frenchc@unimelb.edu.au
Project description: (H)ERG (“human ether a go-go”) ion channels are important in for pacing the heart. Genetic disorders of this channel or drug inhibition lead to serious cardiac arrhythmias. It is known that (H)ERG channels are also in the mammalian CNS, but there is almost no data on their effects on neural function. Recent studies in this lab have disclosed evidence of electrical activity of these channels in rat hippocampus, and that they are exquisitely sensitive to antipsychotic drugs. Additionally, computer simulations show activity of this channel may modulate brain rhythms known to be important in epilepsy and schizophrenia. The project will involve further characterization of these channels in single neurons, as well as looking at how brain rhythms and epileptic activity in brain slices are affected by these channels, especially their modulation by antipsychotic drugs.
Additionally, we will have the unique opportunity of studying these channels in human brain tissue obtained from neurosurgical procedures.
Modelling Epilepsy and Epilepsy Drug Effects-Computational Neuroscience Project
Supervisors: Dr Chris French,
Project Site: Department of Medicine RMH/MBC Neurosciences Building, Parkville
Contact: Chris French frenchc@unimelb.edu.au
It is unclear how large scale electrical oscillations in the CNS are produced with epileptic seizures. Simple hyper-excitability of individual ion channel types and abnormalities of synaptic transmission are undoubtedly important. However, at the network level, recurrent excitation and inhibition from interneurons must be crucial, and may explain why some anti epileptic drugs (AED's) produce paradoxical exacerbation of seizures. This project involves modelling small networks (initially just 2 neurons) to examine the dynamics of seizure production, as well as how certain anti-epileptic drugs suppress or occasionally exacerbate network oscillations. This modelling involves incorporating novel experimental data from this laboratory on normal and drug affected ion channel mechanisms, as well as the effect of glial (supporting cells) cell interactions. The program "Neuron" will be mainly used for the simulations. Some programming experience is necessary, but the modelling language is relatively simple. This project provides an opportunity to gain an in-depth understanding of ion channel kinetics and non-linear behaviour of individual neurons and networks, with a strong clinical relevance.
Sodium Channels in Epilepsy - also offered as MBiomedSc
Supervisors: Dr Chris French, Prof Terence O’Brien
Project Site: Department of Medicine (RMH), MBC Neurosciences Building, Parkville
Contact: Dr Chris French T: 9035 6376 E: frenchc@unimelb.edu.au
Laboratory Overview. The O’Brien Laboratory in the Department of Medicine, University of Melbourne, has a wide range of research activities related to the neurological disorder epilepsy. Projects include molecular biological studies, in vivo and in vitro electrophysiology, advanced imaging techniques, animal behaviour models, pharmacogenomics as well as comprehensive clinical
Project Overview. The project will be to study voltage-gated sodium channels, membrane proteins that are the basis of almost all electrical signaling in the nervous system, and so of the greatest significance in normal function, as well as disease states including epilepsy. Properties of normal channels in rat brain cells and cloned channels in tissue culture will be studied, as well as the effects of common anti-epileptic drugs (AED’s). We are particularly interested in examining how minor genetic variations impact on AED action. Opportunities for mathematical modeling and computational simulations of nerve cell activity are also available.
The project thus offers a very wide range of possibilities for advanced skill acquisition, including molecular biological techniques, patch-clamping and computational neuroscience. Several publications are anticipated. Additionally, a very high priority is placed on basic research skill acquisition, including experimental design and analysis, statistical techniques, familiarity with common molecular biological methods, as well as public presentation of research findings.
Long-term outcome of newly diagnosed epilepsy - also offered as MBiomedSc
Supervisors: Prof. Patrick Kwan
Projects site: Department of Medicine (RMH), University of Melbourne
Contact: Professor Patrick Kwan, E: patrick.kwan@unimelb.edu.au
Project description: Seventy million people have epilepsy with 34–76 per 100,000 developing the condition every year. To formulate rational treatment plans, it is important to understand the different clinical courses and patterns of response to antiepileptic drugs, ideally by following outcomes from the point of treatment initiation.
This project will perform analysis focusing on response to the initial therapies and their relationship with long-term treatment outcomes and development of pharmacoresistance in newly treated epilepsy patients. The student will be involved in recruiting and following up eligible patients. Basic knowledge and skills in biostatistics is preferred
Does epilepsy cause a secondary cardiac channelopathy?
Supervisors: Dr. Kim Powell, Prof Terence O’Brien, Dr. Marian Todaro
Project Site: The Department of Medicine, The Royal Melbourne Hospital, The University of Melbourne.
Contact: Dr KimPowell E: kpowell@unimelb.edu.au ; Prof Terence O’Brien E: obrientj@unimelb.edu.au
Project description: People with epilepsy are at a higher risk of death than the general population. People with epilepsy may die suddenly without an obvious pathologic cause for death. Such deaths are termed Sudden Unexpected Death in EPilepsy (SUDEP), and this is the major clinical problem facing epilepsy patients, accounting for 17-38% of all epilepsy related deaths. Basic research investigating the causal mechanisms underlying SUDEP is lacking. Alterations in function or expression of ion channels expressed in both cerebral and cardiac tissue represent strong candidate mechanisms for SUDEP - defects in membrane excitability could predispose an individual to a dual phenotype of epilepsy and cardiac arrhythmia. In both a genetic and an acquired animal model of epilepsy we have identified altered cardiac electrophysiological function with an associated down-regulation of the cardiac pacemaker HCN2 channel. Based on this data We have hypothesised that the development of epilepsy itself can results in secondary changes in cardiac ion channel expression and function that could contribute to an increased risk of cardiac arrhythmias and therefore SUDEP.
Aims: To investigate whether patients with chronic epilepsy have alterations in cardiac electrophysiology and ion channel expression compared to matched non-epileptic control subjects.
Methods: This will be investigated by examining cardiac tissue from patients with chronic epilepsy collected during open heart surgery at the Royal Melbourne Hospital and Melbourne Private. This tissue collected will be atrial muscle, which is routinely excised, and discarded as part of the routine cannulation of patients that are being placed on cardiopulmonary bypass for cardiac surgery. These patients would be identified by using a screening questionnaire given to all patients during the pre-admission clinic assessment. Identified patients will then be given a more detailed interview collecting data about their epilepsy syndrome, aetiology, duration, seizure frequency, and medication history. Control subjects will be patients without a history of epilepsy matched to the epilepsy patients for age, sex, cardiac disease status in a ratio of 1:3 (i.e. three controls for each patient with epilepsy). The mRNA and protein levels for the ion channels, HCN2 and 4 channels, which are expressed both in the hearth and the brain will be measured, and compared between the epilepsy and control patients. The patients’ ECG recordings will also be compared for significant electrophysiological difference. Any significant molecular or electrophysiological changes identified will be correlated with the epilepsy syndrome (i.e. genetic vs. acquired), the duration of epilepsy and the seizure frequency. Parrellel studies are being undertaken in animal models of chronic epilepsy to enable the mechanisms causing the epilepsy-associated cardiac changes to be better elucidated.
Outcome: This study has the potential to identify the mechanism responsible for epilepsy-associated cardiac dysfunction and thereby provide an opportunity to target interventions that can prevent the cardiac dysfunction, and mitigate the risk of SUDEP.
Investigating molecular and physiological determinants of Suddent Unexplained Death in Epilepsy in acquired and genetic animal models of epilepsy - also offered as MBiomedSc
Supervisors: Dr Kim Powell, Dr Pablo Casillas-Espinosa and Prof Terry O’Brien
Project Site: Department of Medicine (RMH), MBC Neurosciences Building, Parkville
Contact: Dr. Kim Powell T: 9035 6394 E: kpowell@unimelb.edu.au;
Project Description: Epilepsy is associated with an increased risk of sudden unexplained death (SUDEP), possibly due to cardiac arrhythmias, although the precise mechanism remains unknown. SUDEP is considered the most important direct epilepsy-related mode of death and accounts for up to 30% of all deaths in the epilepsy population, being particularly prevalent amongst young patients with uncontrolled or drug-resistant, frequent and severe generalized tonic-clonic seizures.
Ion channels that coexist in the brain and heart would make ideal candidates for SUDEP because defects in intrinsic membrane excitability could predispose an individual to a dual phenotype of epilepsy and cardiac arrhythmias culminating in sudden death. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and T-type calcium channels play an important role in the generation of pacemaker activity in the brain and heart. Furthermore, its functional role becomes more marked in the process of pathological cardiac hypertrophy and heart failure. Thus HCN and T-type calcium channels are attractive candidates for investigating molecular mechanisms of SUDEP. Our research has identified a cardiac transcriptional channelopathy of HCN2 and Cav3.1 and Cav3.2 T-type calcium channels, with associated detrimental cardiac electrophysiological changes, in rat models of both genetic generalised epilepsy (GAERS) and acquired temporal lobe epilepsy (kainic acid (KA) induced post-status epilepticus (SE)).
Several projects will be offered to investigate different aspect of SUDEP and cardiac dysfunction in animal models of genetic and acquired epilepsy
Project 1: To investigate the molecular mechanisms contributing to the cardiac dysfunction on genetic and acquired animal models of epilepsy.
Project 2: To investigate if decreased HCN2 expression translates to a decrease in HCN channel current (If) in cardiomyocytes in animal models of genetic and acquired epilepsy.
Project 3: To investigate if by pharmacologically suppressing seizures we can alleviate the altered cardiac electrophysiological function and HCN2 and T-type calcium channel transcriptional repression
Skills: The skills expected to be learnt from this project include: Small animal handling and surgery, Drug testing in animal models of epilepsy, electrophysiology recordings and analysis, biochemical and molecular analysis (real time PCR, western blotting).
Stargazin and AMPA receptor expression at cortical synapses in epileptic rats - also offered as MBiomedSc
Supervisors: Dr Kim Powell, Professor Terence O’Brien
Project Site:: Department of Medicine (RMH), MBC Neurosciences Building, Parkville
Contacts: Dr. Kim Powell T: 9035 6394 E: kpowell@unimelb.edu.au
Project Description: Absence seizures, one of the most common seizure types in humans with idiopathic generalised epilepsy (IGE), are generalised non-convulsive events characterised by recurrent episodes of staring with unresponsiveness. Absence seizures most commonly affect children and adolescents who can experience hundreds of seizures per day and if left untreated can lead to disruptions in learning. Despite the important recent identification of genetic mutations in some rare families with IGEs showing a monogenic inheritance, in the common situation (>95% of sufferers) with complex inheritance patterns the genetic determinants of the absence seizures are still unknown. These epilepsies are presumed to be polygenic, with more than one genetic variation contributing to the phenotype, but the nature of these variations and how they interact to result in epilepsy remains to be determined. GAERS are a strain of rats which spontaneously develop generalized absence seizures.
AMPA receptors are ionotropic transmembrane receptors for the excitatory neurotransmitter glutamate, which mediates fast synaptic transmission in the central nervous system. Stargazin is the archetypal member of a family of proteins called Transmembrane AMPA Receptor regulatory Proteins (TARPs), and is critical for the trafficking and anchoring of AMPA receptors to synaptic membranes. Stargazin also influences electrophysiological properties of AMPA receptors including the slowing of deactivation and reducing desensitization rates. This newly identified TARP role for stargazin may have major functional implications on the homeostatic balance of neuronal excitation, and potentially for the pathophysiology of epilepsy. Recent work from our lab has shown increased expression of stargazin at neuronal membranes in the somatosensory cortex of epileptic GAERS animals, a brain region thought to be involved in the generation of absence seizures. These animals also show increased membrane AMPA receptor expression, which may be driven by elevated stargazin levels. Stargazin is known to interact with other synaptic proteins to localise AMPA receptors to the post-synaptic density (PSD), the region of the postsynapse opposite sites of neurotransmitter release.
The specific aims of this project are
- To biochemically isolate the PSD from the somatosensory cortex of epileptic GAERS and non-epileptic control (NEC) rats
- To compare PSD localization of stargazin, AMPA receptor subunits and other synaptic proteins in GAERS and NECs
- To correlate membrane and synaptic expression of stargazin and AMPA receptors with seizure parameters
Skills: The skills expected to be learnt from this project include: Small animal handling and neurosurgery (electrode implantations), EEG recordings and analysis, and biochemical and molecular analysis (subcellular fractionation, western blotting)
Investigating the role of a Cav3.2 calcum channel mutation in contributing to the epileptic phenotype using congenic rat strains and a knock in mouse model - also offered as MBiomedSc
Supervisors: Dr Kim Powell, ProfessorTerry O’Brien
Project Site: Department of Medicine (RMH), MBC Neurosciences Building, Parkville
Contact: Dr. Kim Powell T: 9035 6394 E: kpowell@unimelb.edu.au;
Prof. Terry O’Brien E: obrientj@unimelb.edu.au
Project Overview: Absence seizures, one of the most common seizure types in humans with genetic generalised epilepsy (GGE), are generalised non-convulsive events characterised by recurrent episodes of staring with unresponsiveness. Absence seizures most commonly affect children and adolescents who can experience hundreds of seizures per day and if left untreated can lead to disruptions in learning. Despite the important recent identification of genetic mutations in some rare families with IGEs showing a monogenic inheritance, in the common situation (>95% of sufferers) with complex inheritance patterns, the genetic determinants of the absence seizures is still unknown. These epilepsies are presumed to be polygenic, with more than one genetic variation contributing to the phenotype, but the nature of these variations and the mechanisms by which they act to result in epilepsy remains to be determined. In an important, well characterised model of GGE with absence seizures, the Genetic Absence Epilepsy Rats from Strasbourg (GAERS), our research group has discovered a homozygous, missense, single nucleotide (G to C) mutation in the Cav3.2 T-type calcium (Ca2+) channel gene (Cacna1h) resulting in an amino acid from arginine to proline (R1584P). The R1584P mutation correlates with the epileptic phenotype in GAERS doubled crossed with Non-Epileptic Control (NEC) rats. Additionally, the R1584P mutation increases the rate of recovery from channel inactivation in a splice variant specific manner, producing a predicted gain-of-function phenotype.
We have a knock-in mouse model of the R1584P Cav3.2 mutation as well as two congenic rat strains; a NEC strain expressing the R1584P mutation and a GAERS strain without the R1584P mutation which we will use as tools to investigate the neurobiological mechanisms by which the R1584P mutation results in is pro-absence effects. These experiments will explore further the specific role played by the R1584P mutation in the absence phenotype of GAERS and the effect of genetic background.
Project 1: To examine the expression of spike-wave-discharges (SWD) in two different congenic rat strains, an NEC congenic strain expressing the R1584P mutation and a GAERS congenic rat strain without the R1584P mutation.
Project 2: To characterise the epileptic phenotype of a knock-in mouse expressing the R1584P mutation and to investigate the effect of genetic background.
Skills: The skills expected to be learnt from this project include: Small animal handling and surgery, EEG recording and analysis.
Investigations into the role of neuropeptide y in a genetic rat model of absence epilepsy - also offered as MBiomedSc
Supervisor: Prof Margaret Morris, Prof Terence O’Brien, Dr Kim Powell
Project Site: Department of Medicine (RMH), MBC Neurosciences Building, Parkville.
Contact : Dr Kim Powell T : 9035 6394 E : kpowell@unimelb.edu.au
Project Description: The Genetic Generalised Epilepsies (GGE) account for approximately 30% of all epilepsy patients, and patients may manifest a variety of different seizure types. Absence seizures are one of the most common seizure type in GGE and are characterised by a classical 3Hz generalised spike-and-wave discharge (SWD) on EEG resulting in abrupt episodes of interrupted consciousness. SWDs develop from natural neural oscillations within the thalamocortical circuitry, which is comprised of three structures; somatosensory cortex (SCx), ventrobasal nucleus and reticular nucleus (nRT) of the thalamus. The underlying aetiology for this disorder is still unknown. However, there is rapidly accumulating evidence for an anti-seizure role of Neuropeptide Y (NPY) in animal models of both focal and of generalised epilepsies. NPY is a 36-amino acid neuropeptide that acts as a neurotransmitter in the brain and has several different functions including increasing food intake and storage of energy as fat, reducing anxiety and stress, reducing pain perception, affecting the circadian rhythm, lowering blood pressure, and controlling epileptic seizures.
This study will investigate the efficacy of enhancing NPY expression focally in selected thalamocortical brain regions using a recombinant adenovirus viral vector in suppressing seizures in Genetic Absence Epilepsy Rats of Strasbourg (GAERS) model of GGE.
Skills: Small animal handling and neurosurgery (electrode implantations, microinjection catheter implantations), rat electroencephalography recordings, brain perfusion, fixation and histological preparation, immunohistochemistry.
Serotonin in epilepsy
Supervisorr: A/Prof. Nigel Jones ncjones@unimelb.edu.au
Project Site: Department of Medicine RMH, MBC Neurosciences Building Parkville
Contact: A/Prof Nigel Jones E: ncjones@unimelb.edu.au
Project description: Any type of brain injury can result in epilepsy, a chronic neurological condition associated with seizures or ‘fits’. The pathological processes occurring in the brain which drive the development of epilepsy following brain injury are not clear, but certain drugs acting at serotonin receptors, including SSRI antidepressants, accelerate these processes. Using animal models, this project will investigate serotonin signalling in epilepsy, and attempt to understand why SSRIs accelerate the development of disease following injury. We will utilise a variety of techniques, including assessment of serotonin levels, molecular consequences of serotonin activity, immunocytochemical identification of serotonin receptors, and pharmacological manipulation of the serotonin system, all in the context of epilepsy. Available as Honours, Masters or PhD projects
Skills: Small animal handling; animal models of epilepsy; small animal surgery and EEG recording; pharmacology; microdialysis; fast-scan cyclic voltammetry; molecular biology techniques, such as real-time qPCR, Western blotting; histology, including immunocytochemistry.
THE ION CHANNELS AND DISEASE LABORATORY
Our laboratory is located on the first floor in the Melbourne Brain Centre, Kenneth Myer Building, and is fully equipped with state-of-the art neurophysiological and imaging capabilities. We are a 20 person multidisciplinary team working on individual and joint projects in the neurosciences. Our primary interest is in diseases and therapies that involve ion channels with a particular focus on epilepsy. In epilepsy our work begins with clinical and genetics collaborators who identify gene mutations. Many of these are in ion channels and we seek to understand how these mutated genes lead to behavioural seizures. We use a range of methods, appropriate to the scale of investigation and combine, genetic, molecular, biophysical, computational, neurophysiological and behavioural approaches. In addition, our laboratory houses the Australian Optogenetics Repository and we are well positioned to exploit this exciting new method. The projects below give a sample of the work being undertaken and available for suitable candidates.
Elucidating the pharmacology and mechanism of action of phrixotoxin on voltage-gated sodium channels – also offered as MBiomedSc
Supervisors: Dr Geza Berecki and Prof Steven Petrou
Project Site: Epilepsy and Ion Channels Group, The Florey Institute of Neuroscience and Mental Health, Kenneth Myer Bldg
Contact: Dr Geza Berecki E : geza.berecki@florey.edu.au
Project description: Voltage-gated Nav1.2 sodium channel mutations are associated with a number of neurological disorders such as epileptic encephalopathies. Clinically used drugs and experimental compounds can target Nav1.2 channels and modulate neuronal excitability. Among these, phrixotoxin-3 (PTx3) from the venom of the tarantula Grammostola rosea blocks the inward Nav1.2 channel current (INa) by altering Nav1.2 channel gating. Remarkably, PTx3 is one of the most potent and selective peptide modulators of Nav1.2 channels, with a half-maximum inhibitory concentration of 0.6 nM and ~100 fold selectivity for Nav1.2 over other neuronal voltage-gated Nav channels. Therefore PTx3 could emerge as a valuable research tool capable of selectively targeting Nav1.2 channels.
The goal of this project is to elucidate the effect of PTx3 on neuronal human Nav1.1, Nav1.2, and Nav1.6 channels stably expressed in mammalian cell lines using the conventional voltage-clamp (VC) technique, and to study the effect of PTx3 on neuronal firing using the novel dynamic-clamp (DC) technique. The biophysical properties of these Nav channels, including current-voltage characteristics, voltage-dependence of (in)activation, and recovery from inactivation will be determined in the absence and presence of PTx3. In DC configuration, Nav1.2, Nav1.2, or Nav1.6 currents will be implemented as external current input to a realistic cortical pyramidal neuron model cell. This model cell incorporates all major neuronal channel currents; however its Nav channel current is replaced with external Nav1.2, Nav1.2, or Nav1.6 current. The model cell’s membrane potential is continuously computed in real time and used to clamp the membrane voltage of the mammalian cell expressing the Nav channel under investigation. This unique DC recording configuration provides a dynamic voltage environment capable of mimicking the physiology of the cell and provides a direct readout PTx3 modulation on neuronal model cell excitability.
Electrophysiological properties of patient derived stem cell neurons harbouring SCN2A mutations – also offered as MBiomedSc
Supervisors: Dr Geza Berecki, Ben Rollo and Prof Steven Petrou
Project Site: Epilepsy and Ion Channels Group, The Florey Institute of Neuroscience and Mental Health, Kenneth Myer Bldg
Contact: Dr Geza Berecki E : geza.berecki@florey.edu.au
Project description: Epileptic encephalopathies (EE) are a group of devastating disorders with poor prognosis and complex etiology presenting in childhood. De novo mutations in the SCN2A gene encoding for the voltage-gated Nav1.2 sodium channel represent a relatively common cause of EE. Recent landmark technological advances enable patient-specific cells reprogramming to pluripotency by creating induced pluripotent stem cells (iPSC). iPSCs can then be differentiated into neurons, resulting in stem cell (SC) neurons. These patient-derived SC neurons can serve as models of EEs and help clarify the contribution of Nav1.2 channel mutations to these conditions.
In this project, the candidate will study the electrophysiological properties of SC neurons derived from patients with R1882Q or R853Q mutations in SCN2A, whereas SC neurons resulting from CRISPR/Cas9 gene correction of the R853Q mutation to wild-type (wt) and SC neurons generated from healthy subjects will serve as controls. It has been suggested that the R1882Q mutation may result in Nav1.2 channel gain in function, whereas the R853Q mutation may be associated with loss of function and presence of aberrant so-called omega currents, facilitated by the movement of the mutated voltage-sensor S4 segments. Nevertheless, the effects of such altered Nav1.2 functions on the overall neuronal activity are unknown.
Conventional whole-cell current-clamp and voltage-clamp techniques will be used to characterize the activity and action potential (AP) characteristics of various SC neurons, including firing frequency, rheobase, and threshold for AP initiation, AP amplitude, and AP waveform. These experiments will deliver a direct readout of the impacts of Nav1.2 dysfunction on SC neuron excitability and will provide the basis for a detailed understanding of disease mechanisms and allow the development of effective therapies.
Wetware in a loop: voltage-clamp and dynamic-clamp studies of SCN2A sodium channel mutations underlying childhood epilepsy – also offered as MBiomedSc
Supervisors: Dr Geza Berecki and Prof Steven Petrou
Project Site: Epilepsy and Ion Channels Group, The Florey Institute of Neuroscience and Mental Health, Kenneth Myer Bldg
Contact: Dr Geza Berecki E : geza.berecki@florey.edu.au
Project description: Epileptic encephalopathies (EE) are a group of devastating disorders with poor prognosis and complex etiology presenting in childhood. De novo mutations in the SCN2A gene encoding for the voltage-gated sodium (Nav) channel, type II α subunit (Nav1.2), represent a major cause of EE. Prior to understanding and treatment of a particular EE, the contribution of Nav1.2 channel mutations to individual neuronal excitability need to be determined.
In this project the candidate will use both conventional voltage-clamp (VC) and novel dynamic clamp (DC) techniques to investigate the pathogenicity of selected SCN2A mutations affecting Nav1.2 channel function. Transiently expressed human Nav1.2 channels carrying K905N and D1598G mutations (associated with severe EE), R937C and R1902C mutations (associated with autism and milder epileptic syndromes), and wild-type (wt) Nav1.2 channels (control) will be studied in transfected mammalian cells. Sodium currents through wt or mutant Nav1.2 channels will be recorded in VC mode and applied as external current input to a realistic cortical pyramidal neuron model cell in real time. This model cell incorporates all major neuronal channel currents; however its Nav channel current is replaced with external wild-type or mutant Nav1.2 current. The model cell’s membrane potential is continuously computed in real time and it is used as a voltage clamp command for the HEK cell expressing wt or mutant Nav1.2 channels. This unique DC recording configuration provides a dynamic voltage environment capable of mimicking the physiology of the cell and provides a direct readout of the impacts of Nav1.2 dysfunction on neuronal excitability. This is a significant advance over conventional electrophysiological and computational modelling approaches that are the only option currently available, and DC should improve the throughput of mutation analysis and quality of predictions.
Multielectrode array analysis of neuronal networks derived from an epilepsy mouse-model – also offered as MBiomedSc
Supervisors: Dr. Snezana Maljevic, Prof Steven Petrou
Project Site: Florey Institute of Neuroscience & Mental Health, Division of Epilepsy, Kenneth Myer Bldg
Contact: Snezana Maljevic, Steve Petrou E-mail: snezana.maljevic@florey.edu.au, steven.petrou@florey.edu.au
Project description: A recurrent mutation, Arg320His, in the KCNC1 gene, encoding voltage-gated potassium channel Kv3.1, has been recently identified as one of the main causes of progressive myoclonus epilepsy (PME), a rare, inherited disorder manifesting with myoclonus, tonic-clonic seizures, and ataxia. In vitro analysis in Xenopus laevis oocytes revealed that the mutation causes a loss of channel function (Muona et al., Nat Genet 2015).
To provide in-depth analysis of disease mechanisms, we generated knock-in mouse model carrying Arg320His mutation. The project aims at examining properties of neuronal networks derived from this mouse model using multielectrode array (MEA) analysis. To this end, primary neuronal cultures will be plated on MEA dishes and their activity analysed at different time points. We will also examine the effects of elevated temperature on the network activity, as clinical data suggest improvement of symptoms in patients with fever. This platform will be further used to test the efficiency of different drugs, including specific Kv3.1 channel openers, by assessing their impact on the signatures of network activity altered by the mutation.
Apart from cell culture methods and MEA analysis, the project will include Ca imaging of neuronal network activity, as well as immunostaining to assess the localization of mutant channels in neurons. We expect that the obtained results will lead to the clinical translation and precision medicine approaches in the treatment of the affected individuals.
Epilepsy in a Dish – Disease Modelling and Treatment of Severe Epileptic Encephalopathies – also offered as MBiomedSc
Supervisors: Dr Ben Rollo, Dr Snezana Maljevic, Prof Steve Petrou
Project Site: Epilepsy and Ion Channels Group, The Florey Institute of Neuroscience and Mental Health, Kenneth Myer Bldg
Contact: Ben Rollo ben.rollo@unimelb.edu.au
Project description: The Ion Channel and Disease Laboratory are investigating new treatments for a class of severe genetic epilepsy known as epileptic encephalopathies (EE). In most cases EE are caused by mutations in genes coding for membrane-bound ion channels, such as the sodium and potassium channel proteins SCN2A and KCNT1, respectively. Since EE patients do not respond to mainstream anti-epileptic treatments our laboratory has created patient-derived neurons for the purpose of creating a disease model to test novel anti-epileptic therapeutics.
Specifically, we have generated induced pluripotent stem (iPS) cells from EE patients who carry point mutations in the sodium channel gene Nav1.2 (which codes for the SCN2A protein). Cortical neurons have been generated from these iPS cells by neural differentiation protocols using either small molecules or forced expression of neural genes. These EE patient-derived cortical neurons are capable of firing action potentials and forming functional synapses in culture. In this project we will investigate the action of a novel anti-epileptic therapy which will utilize gene-targeting anti-sense oligonucleotides (ASOs). ASOs designed to bind Nav1.2 will be studied for their ability modulate the behavior of SCN2A and restore normative behavior to EE patient-derived neurons. The functional outcomes of ASO treatment will be assessed by single cell patch clamping, and for network analysis by multi electrode arrays (MEAs). In addition, this project will require the techniques of in vitro cell culture, immunohistochemistry (including western blot and single cell immunofluorescence) and gene expression analysis using real time PCR.
Evaluating the impact of dietary C10 and C8 fatty acids on spontaneous seizures and behavior in mouse models of epilepsy – also offered as MBiomedSc
Supervisors: Nikola Jancovski-PhD and Professor Steve Petrou
Project Site: Florey Institute of Neuroscience & Mental Health, Division of Epilepsy,
Kenneth Myer Building
Contact: Nikola Jancovski: nikola.jancovski@florey.edu.au/jancovski.n@unimelb.edu.au
Project description: Increasing evidence suggests that medium-chain triglyceride (MCT) diet is one of the most effective therapeutic approaches in patients with drug-resistant epilepsy. Octanoic acid (C8) and decanoic acid (C10) are major constituents in the diet and research has shown increased quantities of these compounds in plasma of children with intractable epilepsy treated with the diet. The antiepileptic properties of MCT diet might be attributed to C10 and C8, and recent studies reported acute anticonvulsant effects of C10 and C8 in several seizure tests in mice. Although it is suggested that C10 acid exerts direct inhibition of excitatory neurotransmission and therefore decreases seizure activity, the exact mechanisms of action remain elusive. Further studies with C10 and C8 acids using animal models are needed to evaluate the role of these acids in seizures control. It is very important to complete important pre-clinical work in rodent models of epilepsy before these diets are used in clinical trials.
In this project the student will have the possibility of using a range of experimental techniques; from behavioural studies using different mouse models of epilepsy to recording electrical activity of the brain. The results might lead to developing of new therapeutic approaches for patients with drug-resistant epilepsy.
“CLARITY” based glass brain mapping in health and disease – also offered as MBiomedSc
Supervisors: Dr Tim Karle, Dr Kay Richards, Prof Steve Petrou,
Project Site: Florey Institute
Contact: Prof Steven Petrou E: spetrou@unimelb.edu.au;
Project description: Histochemical optically clearing of whole tissue samples and the development of new microscopes that can image deep within the tissue have created unprecedented insight into the wiring of neural networks. Changes in the wiring of cortical neurons, in particular, have been implicated in a number of disorders such as epilepsy, schizophrenia, autism and depression. In this project the candidate will clear whole brains; allowing imaging of neurons, which have been labelled with fluorescent tags. Multi-photon excitation and custom laser light-sheet based microscopy will allow acquisition and reconstruction of exquisite 3D images in key regions of the mouse cortex. The workflow will include chemical clearing, optical microscopy and software deconvolution of the big data sets which will be generated. By comparing normal and epilepsy models this work will begin to unravel the changes that occur prior to and after the occurrence of seizures. This will shed important light on the scale on which structural changes occur in epilepsy and will guide future experimental and clinical work.
Multiphoton imaging of induced pluripotent-stem cell derived brainoids – also offered as MBiomedSc
Supervisors: Dr. Tim Karle, Dr. Snezana Maljevic, Prof Steven Petrou
Project Site: Florey Institute of Neuroscience & Mental Health, Division of Epilepsy, Kenneth Myer Bldg
Contact: Dr Tim Karle, Dr Snezana Maljevic E: tkarle@florey.edu.au, snezana.maljevic@florey.edu.au
Project description: Development of induced pluripotent stem cell (iPSC) approaches has enabled studies of epilepsy mechanisms in patient –based models. This can be achieved by generating 3-D iPSC-derived neuronal cultures, so called brainoids, which present self-organised neuronal assemblies. Video rate imaging of neuronal activity in these millimetre sized assemblies is made possible using short intense pulses of infrared light to image inside the living tissue. Scanning the light rapidly through the tissue causes fluorescence of genetically tagged populations of neurons, expressing Calcium sensitive fluorescent indicators. This allows multiphoton optical mapping of electrical activity in the neural networks. This project combines stem cell biology with novel imaging techniques to aid the understanding of genetic epilepsy mechanisms.
Pharmacological modulation of KCNT1 by quinidine in a genetic mouse of epilepsy – also offered as MBiomedSc
Supervisors: Melody Li, Nikola Jancovski, Kay Richards, Steven Petrou
Project Site: Florey Institute of Neuroscience & Mental Health
Contact: Dr Melody Li E: melody.li@florey.edu.au, Prof Steven Petrou E: steven.petrou@florey.edu.au
Project description: Pathological mutations in the gene, KCNT1, are the primary cause of a spectrum of severe epilepsies characterized by early disease onset, often with cognitive and developmental impairments. Recently, we identified a clinically used anti-arrhythmic, quinidine, could reverse the pathological effects of KCNT1 mutations in vitro. While clinical trials resulting from our in vitro work showed clear benefits of quinidine, the effects were not as dramatic as expected, presumably as a consequence of poor blood brain barrier penetration and the late age of treatment initiation.
This project aims to address the above factors of quinidine administration in a KCNT1 epileptic mouse model. Techniques will involve: [1] administering quinidine directly to brain using intra-cerebroventricular injection, [2] examining the potential correlation between seizure and age of treatment initiation using pro-convulsant assay and/or electroencephalogram (EEG). Outcomes from this study are expected to inform future clinical studies on the quinidine dosing regimen in patients.
Evaluating the efficacy of antisense oligonucleotides in a mouse model of Epilepsy of Infancy with Migrating Focal Seizures (EIMFS) – also offered as MBiomedSc
Supervisors: Melody Li, Nikola Jancovski, Kay Richards, Snezana Maljevic, Steven Petrou
Project Site: Florey Institute of Neuroscience & Mental Health
Contact: Dr Melody Li E: melody.li@florey.edu.au,
Prof Steven Petrou E: steven.petrou@florey.edu.au
Project description: Antisense oligonucleotide (ASO) has recently emerged as a novel therapeutic strategy and several ASOs have entered Phase I clinical trials for neurological disorders and cancer. ASOs are chemically modified short strand of nucleotides that are designed to “switch off” a gene of interest. Using cell-based assay, we identified increased KCNT1 gene function as the key disease mechanism in several types of epileptic encephalopathies. Thus, ASOs that are designed to downregulate KCNT1 is a promising therapeutic strategy.
This study aims to evaluate the effect of ASOs designed by a pharmaceutical company in the KCNT1 EIMFS mouse model. Specific techniques will involve [1] intra-cerebroventricular administration [2] electroencephalogram (EEG) [3] pro-convulsant assay.
This is an important pre-clinical test that aims to motivate precision medicine application of ASO technology in genetic epilepsy
Functional characterization of KCNA2 epilepsy – causing mutations – also offered as MBiomedSc
Supervisors: Dr. Snezana Maljevic, Prof. Steven Petrou
Project Site: Ion Channels and Human Diseases Group, The Florey Institute of Neuroscience and Mental Health
Contact: E: snezana.maljevic@florey.edu.au,
Project description: Increasing number of genetic variants affecting ion channel genes and associated with different forms of epilepsy has been identified in the recent years. One of the important steps in understanding if and how these variants contribute to the disease phenotype is their functional characterization using different in vitro and in vivo approaches. The initial screen of detected variants is often performed in Xenopus laevis oocytes or HEK cells and involves site-directed mutagenesis, RNA production and injection, cell culture methods and two-microelectrode or patch clamp technique. In addition, biochemical methods and immunocytochemistry are applied to examine the expression and localization of affected channels. Positions are currently available for examining several novel mutations detected in the KCNA2 and KCNC1 gene, encoding voltage-gated potassium channel Kv1.2 and Kv3.1, respectively. Both genes have recently been associated with different forms of epilepsy, and we aim to examine the common disease mechanisms and select variants for further in depth analysis using mouse and stem cell models
Problematic pumps: the mechanistic basis of Na K-ATPase mutations – also offered as MBiomedSc
Supervisors: Ian C Forster, Melody Li, Steve Petrou
Project Site: Florey Institute of Neuroscience & Mental Health
Contact: E: ian.forster@florey.edu.au
Project description: Mutations in the ubiquitous Na-K-ATPase (sodium potassium pump) have been implicated in several neurological disorders such as rapid-onset parkinsonism and alternating hemiplegia. Understanding the molecular basis for these clinical disorders is key to developing appropriate treatments as well as gaining deeper insights into the physiological role and molecular mechanism of the wild-type protein. We will use real-time biophysical assays, combining conventional electrophysiology and fluorometry, to elucidate the mechanistic dysfunction. Interested applicants will gain first-hand experience with molecular biology, electrophysiology, fluorometry and computational biology. Some basic knowledge of molecular biology techniques and basic laboratory practices would be desired.
Structure function studies on phosphate transporters – also offered as MBiomedSc
Supervisors: Ian C Forster, Melody Li, Steve Petrou
Project Site: Florey Institute of Neuroscience & Mental Health
Contact: E: ian.forster@florey.edu.au
Project description: Sodium-coupled phosphate transporters provide the main means by which dietary phosphate is absorbed in the gut and reabsorbed in the kidney, to achieve phosphate homeostasis. Understanding the molecular basis of the transport mechanism at the molecular level is essential for developing clinically effective drugs to target phosphate transporters in clinically prevalent conditions such as end-stage kidney disease. We will use biophysical assays, combining conventional electrophysiology and fluorometry, to study the transport dynamics in real time and elucidate potential drug interaction sites. Interested applicants will gain first-hand experience with molecular biology, electrophysiology, fluorometry and computational biology. Some basic knowledge of molecular biology techniques and standard laboratory practices would be desired.
Zinc and Seizures
Supervisors: A/Prof Chris Reid, A/Professor Steve Petrou, Dr Paul Adlard - also offered as MBiomedSc
Project Site: Florey Institute of Neuroscience & Mental Health, Division of Epilepsy, Kenneth Myer Bldg
Contact: Chris Reid E : careid@unimelb.edu.au
Project description: Zn2+ is an essential element having a multitude of biological functions throughout the body. Our research has demonstrated that low brain Zn2+ can increase seizure susceptibility (Hildebrand et al 2015 Sci Rep). This highlights Zn2+ supplementation as a potentially good therapeutic strategy for seizure conditions. Before clinical trials can begin we need to complete important pre-clinical work in rodent models of epilepsy. We also need to better understand the mechanisms through which Zn2+ modulates neuronal excitability. In this project the student will learn a range of experimental techniques aimed at understanding the role Zn2+ plays in changing neuronal excitability. This will include using established rodent models to test diet and drug manipulations of brain Zn2+ levels on seizure susceptibility and electrophysiological investigations looking at how neuron excitability is changed by Zn2+. The results have particularly relevance for developing countries, where epilepsy rates are high and nutritional supplementation is a potential practical therapy
Novel antiepileptic drug targets based on HCN channel antagonists - also offered as MBiomedSc
Supervisors: A/Prof Chris Reid, A/Professor Steve Petrou
Project Site: Florey Institute of Neuroscience & Mental Health, Division of Epilepsy, Kenneth Myer Bldg
Contact: Chris Reid E : careid@unimelb.edu.au
Project description: About 30% of epilepsy patients are not controlled on currently available antiepileptic drugs. Our laboratory has discovered a novel anti-epileptic drug target. HCN channels are an ion channel in the brain that regulates rhythmic behaviour which is a hallmark of a seizure. In collaboration with Italian scientists we have demonstrated that a compound that blocks a certain subtype of this channel reduces seizures. Based on this the NIH Anticonvulsant Screening Program in USA will test this compound on a range of seizure models. In this project we want to begin to understand how blockers of this channel reduce seizures. We have assembled a range of state-of-the-art tools to answer this question. This includes a viral-based knock-down strategy, a conditional knock-out mouse model and pharmacological tools. In this project the student will have the possibility of using a range of experimental techniques; from behaviour to recording single neuron activity. These channels are also thought to be important to the generation of pain and drugs based on this target may be useful in this condition as well.
How does pH change brain excitability? - also offered as MBiomedSc
Supervisors: Christopher A. Reid, Nikola Jancovski, Steven Petrou
Project Site: Florey Institute of Neuroscience & Mental Health, Division of Epilepsy, Kenneth Myer Bldg
Contact: Chris Reid E: Christopher.reid@florey.edu; careid@unimelb.edu.au
Project description: Brain pH levels have long been known to modify seizure susceptibility. Breathing too quickly results in brain alkalosis and can trigger seizures. In contrast, acidic shifts in brain pH induced by respiration of increased CO2 concentrations can reduce seizure susceptibility. In fact, a gas found in emergency departments called carbogen (5% CO2 – 95% O2), is a rapid and effective anti-seizure therapy that could be used clinically. Our laboratory is working on how pH causes change in neuronal excitability. We have already discovered that pH impacts excitatory and inhibitory neurons differently. This project will investigate the impact of pH in a mouse model that is missing an acid sensitive channel. It will involve testing the behaviour of the mouse and looking at neuron and network excitability. By understanding these mechanisms we will be better able to develop more targeted therapeutic strategies for stopping seizures.
Functional characterization of the temperature sensitive component of the KCNCI epilepsy causing mutation R320H
Supervisors: Dr Carol Milligan & A/Professor Steve Petrou
Project Site: Florey Institute of Neuroscience & Mental Health, Division of Epilepsy, Kenneth Myer Bldg
Contact: Steven Petrou T : 9035 3628 E : spetrou@unimelb.edu.au; Carol Milligan E : carol.milligan@florey.edu.au
Project Description: A KCNC1 gene mutation (p.Arg320His) has recently been identified in 20 cases of progressive myoclonus epilepsy (PME), a distinctive epilepsy syndrome characterised by myoclonus, generalised tonic-clonic seizures and progressive neurological deterioration. KCNC1, not previously associated with human disease, encodes the voltage-gated potassium channel KV3.1 which is expressed predominantly in inhibitory interneurons. This recurrent, loss of function missense mutation is pathogenic in the heterozygous state (Muona et al., Nat Genet 2015). Unexpectedly, transient improvement in gait and myoclonus with fever was noted in six cases, typically lasting just hours or a few days while the subject was ill.
This project aims to explore the mechanism of action of this fever effect on both homomeric and heteromeric channels, using in vitro high throughput automated electrophysiology and temperature. The candidate will first have to produce mutant cDNAs then transiently transfect into HEK293 cells prior to analysis on the automated platforms. Candidates will be trained in the necessary molecular biological methods and then in ion channel electrophysiology and will work closely with a senior member of the team to ensure success