Vacancies and studentships

Development of in vivo patch clamp methods and their application to investigations of neurophysiological deficits in transgenic models of Alzheimer’s disease pathology.
Supervisors: Andy Randall and Matt Jones

Our own work and that of many other groups has uncovered a range of neurophysiological deficits in mouse models of Alzheimer’s disease associated pathology (Randall et al. 2010). These have largely been studies performed either in vitro, or using extracellular recording methods in vivo. We wish to understand if and how these existing data translate to the cellular level in vivo. Consequently, the key front-end objective of this project is a technology development goal. Namely, to successfully establish in vivo patch clamp recording from cortical and hippocampal neurones of adult mice in our laboratories.

Once established we will use the additional analytical power provided by these cellular level electrophysiological techniques to investigate how neurophysiological function is disturbed in the intact CNS of transgenic mouse lines that are commonly used to model discrete aspects of AD pathology. In particular, we will examine PS1M146LAPPSwe mice ((Holcomb et al., 1998) which overproduce Abeta peptides and Tg4510 mice (Santacruz et al., 2005) which over-express human P301L tau and generate an age-dependent forebrain tauopathy.

Having identified and characterised functional deficits with these in vivo methods we will establish if these can be reversed by interventional measures. In the PS1M146LAPPSwe mice we will employ gamma secretase inhibitors. In the Tg4510 mice we will use the ability to suppress expression of P301L tau using in-life dosing with doxycycline.

Randall, A.D., Witton, J., Booth, C., Hynes-Allen, A. & Brown, J.T. (2010) The functional neurophysiology of the amyloid precursor protein (APP) processing pathway. Neuropharmacology, 243-267.

Understanding synaptic and cognitive deficits in mental retardation and Autism
Supervisors: Prof Zafar Bashir and Dr Clea Warburton

Fragile X syndrome (FXS) is the most common form of heritable mental retardation and the leading identified cause of autism. FXS is caused by transcriptional silencing of the FMR1 gene that encodes the fragile X mental retardation protein (FMRP), but the pathogenesis of the disease is unknown.

According to one proposal, many psychiatric and neurological symptoms of FXS result from unchecked activation of mGluR5, a metabotropic glutamate receptor. Much of what is known so far about FXS is based on work in the hippocampus of FMR1 knockout mice. However, many of the behavioural and cognitive effects of the human disease are much more likely to be associated with deficits in brain regions other than hippocampus. One such brain region is the prefrontal cortex. We have shown that the prefrontal cortex is crucial for associative forms of memory and higher cognitive function and have described forms of synaptic plasticity that very likely underlie these higher functions.

This project will assess in FMR1 KO mice synaptic transmission and synaptic plasticity in prefrontal cortex and assess associative cognitive learning tasks that rely on prefrontal cortex. In addition, whether inhibition of mGluR5 can reverse synaptic and cognitive deficits associated with FMR1 KO will be examined.

In this project the student will utilise extracellular and whole cell recording from slices of prefrontal cortex from FMR1 KO and WT mice to examine synaptic transmission and synaptic plasticity and will utilise behavioural experiments to examine effects on cognition. This combination of techniques will provide multidisciplinary and multilevel analyses of the synaptic bases of mental retardation and autism.

Barker GR, Warburton EC. (2008) Critical role of the cholinergic system for object-in-place associative recognition memory. Learn Mem. 16: 8-11

Griffiths S, Scott H, Glover C, Bienemann A, Ghorbel MT, Uney J, Brown MW, Warburton EC, Bashir ZI. (2008) Expression of long-term depression underlies visual recognition memory. Neuron 58:186-94.

Genetic susceptibility of preterm infants to white matter brain injury
Supervisors: Prof. Elek Molnar1, Dr. Karen Luyt2, Prof. Aniko Varadi3

1MRC Centre for Synaptic Plasticity, School of Physiology and Pharmacology, University of Bristol
2School of Clinical Sciences, Neonatology, St Michael’s Hospital, University of Bristol
3Genomics Research Institute, Faculty of Health and Life Sciences, University of the West of England

Brain injury in preterm infants typically affects white matter, causing cognitive impairment and cerebral palsy. 20% of preterm infants (<32 weeks gestation) suffer white matter injury (WMI). Glutamate is the major excitatory neurotransmitter in the CNS [1]. Following its release, glutamate is removed from the synaptic cleft by glutamate transporters. Hypoxic-ischaemic insult to the brain causes excessive release of glutamate, which ultimately leads to neural cell death (excitotoxicity). The aetiology of WMI in the preterm brain is multifactorial, however the final common mechanism causing injury/death to white matter cells (oligodendrocytes) is thought to be ionotropic glutamate receptor-mediated excitotoxicity [2]. Glutamate transport is the only mechanism for maintaining extracellular glutamate concentrations below excitotoxic levels. Of the five identified transporters, EAAT2 is responsible for 90% of glutamate uptake. A common gene mutation (single nucleotide polymorphism, SNP) in EAAT2 leads to impaired uptake, with resultant higher levels of glutamate and increased excitotoxicity. This SNP is associated with more severe neurological impairment in stroke patients [3]. This project aims to establish genetic susceptibility to WMI due to defective glutamate transport in preterm infants using our recently established DNA bank and database of preterm infants. The results of the genetic screening will be correlated to clinical data obtained by neuroimaging and functional neurodevelopmental assessment. This study will directly contribute to the development of a rapid genetic screening assay that can provide an early and accurate prediction of white matter injury that is essential for more effective targeted treatment strategies.

[1] Molnar E (2008) Molecular organization and regulation of glutamate receptors in developing and adult mammalian central nervous systems. In: Handbook of Neurochemistry and Molecular Neurobiology:  Neurotransmitter Systems (eds: Lajtha A, Vizi ES), Third edition, Springer Reference, pp415-441.

[2] Luyt K, Varadi A, Durant CF, Molnar E (2006) Oligodendroglial metabotropic glutamate receptors are developmentally regulated and involved in the prevention of apoptosis. J Neurochem 99:641-656.

[3] Mallolas J, Hurtado O, Castellanos M, Blanco M, Sobrino T, Serena J, Vivancos J, Castillo J, Lizasoain I, Moro MA, Davalos A (2006) A polymorphism in the EAAT2 promoter is associated with higher glutamate concentrations and higher frequency of progressing stroke. J Exp Med 203:711-717.

Characterising the function of genes implicated in the aetiology of Alzheimer’s using human induced pluripotent stem (iPS) cells and transgenic techniques
Supervisors: Prof. James Uney, Dr Clea Warburton and Prof. Malcolm Brown

It is now possible to reprogram human adult somatic cells by ectopically expressing nuclear reprogramming genes such that they are induced to become pluripotent stem cells. Induced pluripotent stem (iPS) cell technology clearly has tremendous potential for disease modelling in human cells and derivation of pluripotent cells for regenerative medicine. In collaboration with Dr. Nick Allen in Cardiff University we have been using iPS cells derived from human patients to study the neuronal function of genes recently implicated in AD (by a Genome Wide association study) and have found that these ‘AD genes’ regulate vesicle recycling and dendritic spine size. Dr. Allen has also generated transgenic knockout mice to allow the behavioural effects of these genes to be studies. This project will investigate the role genes recently implicated in AD (e.g. PICALM) have on glutamate receptor expression using: iPS neurons derived from patients; electron microscopy; lentiviral overexpression and knockdown strategies. Under the supervision of Dr. Clea Warburton behavioural tests to investigate memory function in AD gene knockout mice will also be carried out.

Neuronal excitability, spike-timing synaptic plasticity and ageing
Supervisors: Dr. Jon Brown, Prof. Richard Apps and Prof. Neil V. Marrion

The electrical properties of the brain are thought to change as we get older, resulting in cognitive decline in the elderly.  However, the nature and functional consequences of these changes to neuronal excitability are poorly understood.  We have recently identified an important and novel reduction in the excitability of hippocampal neurones which is likely have profound consequences as to how these cells interact within neuronal circuits.  This change in excitability results from a change in sodium current, while other data suggests that the slow afterhyperpolarization is also changed in ageing.  The student will work as part of a larger collaborative effort focusing ageing research (including Drs. Nina Balthasar and Matt Jones), and use a combination of in vitro and in vivo electrophysiological and molecular approaches to understand how these changes modify spike-timing dependent synaptic plasticity and neural circuits.

Can stress induce Alzheimer’s disease?
Supervisors: Profs Kei Cho and Elek Molnar

Alzheimer’s disease (AD) is the epitome of a devastating type of illness confronting healthcare systems worldwide – it has a prolonged course, is increasing in prevalence and is very debilitating both for the patient and the patient’s family. Recent studies have shown that environmental factors may be involved in AD and cognitive deficits. Stress is emerging as one of the key risk factors of Alzheimer’s disease and dementia. Using integrated electrophysiological, molecular, biochemical and imaging approaches, we will investigate the molecular and cellular mechanisms by which ‘stress’ causes synaptic dysfunction and triggers neurodegeneration. We will use the well established model of ‘stress’-induced regulation of synaptic transmission in the perirhinal cortex which we have extensively validated. We will record neuronal activity using extracellular field potentials to study neuronal populations and whole-cell patch-clamp recording to investigate changes within individual neurons [1, 2]. Our initial work will build upon our discoveries of the important role of ‘stress’ in glutamate and acetylcholine receptors in the perirhinal cortex and long-term synaptic plasticity that likely to underly various types of memory. Functional changes will be correlated to ‘stress’-induced changes in the molecular composition, distribution and signalling of glutamate and acetylcholine receptors.

Electrophysiological studies will be supervised by Prof Kei Cho (School of Clinical Sciences). Cellular, molecular and imaging components of the study will be carried out in Prof. Elek Molnar’s laboratory (School of Physiology and Pharmacology).

[1] Jo J, Ball SM, Seok H, Oh S-B, Massey PV, Molnar E, Bashir ZI, Cho K (2006) Experience-dependent modification of mechanisms of long-term depression. Nature Neurosci 9:170-172.

[2] Jo J, Son GH, Winters BL, Kim MJ, Whitcomb DJ, Dickinson BA, Lee YB, Futai K, Amici M, Sheng M, Collingridge GL, Cho K (2010) Muscarinic receptors induce LTD of NMDAR EPSCs via a mechanism involving hippocalcin, AP2 and PSD-95. Nat Neurosci 13:1216-1224.

Endosomal sorting in synaptic plasticity and Alzheimer’s disease
Peter J. Cullen and Jeremy M. Henley

Endosomal sorting of internalised neurotransmitter receptors to recycling or degradative pathways is a key decision step for physiological and pathophysiological neuronal function. For example, AMPA receptor (AMPAR) sorting underlies synaptic plasticity including long term potentiation (LTP) and long term depression (LTD), and defective endosomal sorting of APP (and possibly AMPARs) has been proposed as a factor in Alzheimer’s disease (AD). Cullen’s lab focuses on molecular aspects of the retromer complex, which regulates sorting from early endosomes into recycling pathways. Henley’s lab studies aspects of the regulation of synaptic AMPA and kainate receptor trafficking and recycling. It has been noted by many groups that NMDA receptors and Aβ-invoked AMPAR trafficking share many properties, suggesting common pathways. This project will exploit the synergistic expertise of both labs to study the role of endosomal sorting and especially the retromer complex on AMPAR trafficking in normal and diseased neurones. 

i) Preliminary data from the Cullen lab suggests that sorting nexin-27 (SNX27), a brain-enriched retromer-binding protein, binds the AMPAR subunit, GluA1 via a PDZ interaction. In dendrites SNX27 partially co-localizes with GluA1 and we hypothesize that the retromer-SNX27 complex may regulate endosomal sorting of GluA1-containing AMPAR during the initial phase of LTP. We already have a library of tools to build on these initial observations, including custom antibodies to retromer components and SNX27 as well as validated shRNAs and RNAi-resistant mutants. The aim of this project is to use these tools in primary cultured neurons to determine the role of the retromer and SNX27 in AMPAR trafficking under basal and potentiated conditions. All of the necessary experimental protocols are in routine use in the host labs.

ii) Cullen’s lab has also shown that another class of proteins sorted by the retromer are the Sortilin receptors, including SorLA, which regulates the endocytic sorting of APP and is closely linked to late-onset AD. Retromer defects lead to aberrant APP processing and retromer-deficient mice develop hippocampal memory and synaptic dysfunction associated with elevated endogenous Aβ peptide. A current model is that under normal conditions, APP is recycled through a retromer-dependent pathway, but in late-onset AD retromer dysfunction causes APP retention in endosomes and elevated Aβ formation. Thus, the second part of the project will investigate the role of the retromer in APP processing initially in cultured neurones, again using available tools and techniques well-established in our labs.

This PhD provides an exciting opportunity for training in modern techniques in membrane biology, protein biochemistry and cell biology, and the application of these approaches to a fundamental area of neurobiology. The student will be based primarily in the Cullen lab but will generate cultures and tools in the Henley lab. S/he will participate fully in group meetings and actively engage in day-to-day collaboration and information exchange that will benefit both the student and the host labs. 

Cullen, P.J. Endosomal sorting and signalling: an emerging role for sorting nexins. Nat Rev Mol Cell Biol 9, 574-582 (2008).

Cullen, P.J. Emerging roles for retromer in development and disease. Nat Cell Biol invited review (2011).

Small, S.A. Retromer sorting: a pathogenic pathway in late-onset Alzheimer disease. Arch Neurol 65, 323-328 (2008).

Henley, J.M., Barker, E. and Glebov, O.O. Routes, destinations and delays; recent advances in AMPAR trafficking. Trends Neurosci. (2011) - invited review in submission.

The interaction of dietary and pharmacological agents on BDNF production and related pathways
Professor David Jane and Dr Shelley J Allen

The neurotrophin brain derived neurotrophin (BDNF) is implicated in long term potentiation (LTP) and proBDNF in long term depression (LTD), via the TrkB and p75 receptors respectively. Diseases such as Alzheimer’s and Huntingdon’s are associated with a marked reduction in BDNF levels, whilst impaired BDNF/TrkB signalling is linked to schizophrenia. BDNF may thus be a key protein in the underlying pathogenesis of these disorders and is therefore a likely therapeutic target. Augmentation of the protein BDNF has successfully ameliorated symptoms and pathology in rodent and primate models of Alzheimer’s. However this approach is intrusive because of the requirement to cross the blood-brain-barrier for efficacy. Ultimately a small molecule approach or a BDNF modulator may be required. In the last few years a number of pharmacological interventions and dietary or lifestyle influences have been investigated with regard to treatment in Alzheimer’s disease or related dementias. On further investigation, interestingly, we find that many of these appear to be involved in the regulation of BDNF. This studentship would encompass the investigation of both negative and positive modulating effects of drugs commonly used in the management of Alzheimer’s disease, such as the cholinesterase inhibitors, NMDA antagonists, antidepressants and antiepileptics, as well as probing the molecular mechanisms for potential protective effects of certain dietary supplements. Obtaining additional novel compounds by in silico searches of virtual compound databases would also be explored. Further to this it has been noticed that preclinical studies carried out in rodents often produce conflicting results to that seen in human clinical trials or in epidemiological studies. Recently, our work has revealed the importance of investigating the human/rodent receptor differences with regard to drug efficacy and we feel this might reflect a wider problem. We have expertise in the production of various wild-type and modified protein constructs including neurotrophins and their receptors and relevant NMR constructs; also in silico drug discovery, compound design, structure-activity relationships, bioassays and cell signalling. We also have a number of appropriate cell models. The student would explore differences between human and rodent receptor/pathway interactions as seen in vivo and in vitro in order to establish where divergence may result in potentially misleading conclusions being drawn from animal models of Alzheimer’s disease. The ultimate aim would be to gain an understanding as to how BDNF may be modulated towards a potential therapeutic in Alzheimer’s disease.

Control of a neuromodulatory system by light
Supervisors: Dr Jack Mellor and Dr Nina Balthasar (University of Bristol) and Dr John Isaac (Eli Lilly and Company)

Neuromodulatory systems such as acetylcholine, dopamine or serotonin control vital brain functions and are major therapeutic drug targets for the treatment of diseases such as Alzheimer’s and schizophrenia. However, we know surprisingly little about the way in which these neuromodulatory systems work. Recently, it has become possible to selectively control neuronal activity within the brain by genetically targeting light sensitive proteins to specific neuronal subtypes (Zhang et al., 2007). These light sensitive proteins can either trigger the neurons to fire action potentials or inhibit their firing depending on the wavelength of light used. These tools can then be used to investigate the functions of identified neurons. The aim of this project is to specifically target light activated proteins to neurons that release the neuromodulator acetylcholine. We will then use this tool to investigate the role of acetylcholine in synaptic plasticity, network activity, and learning and memory. By combining state-of-the-art techniques, such as the use of these virally delivered light-activated systems with neuronal sub-population-specific genetically modified mice, 2-photon live cell imaging and electrophysiology the student will develop a rare, highly desirable skill set and use it to address a question of fundamental importance to how the brain works.

Reference:

Zhang F, Aravanis AM, Adamantidis A, de Lecea L, Deisseroth K. (2007). Circuit-breakers: optical technologies for probing neural signals and systems. Nat Rev Neurosci. 8: 577-81.