King's College London - Current PhD Projects

This project aims to identify susceptibility and phenotype modifier genes for sporadic amyotrophic lateral sclerosis by searching for linkage disequilibrium in a genome wide screen utilising the novel method of DNA pooling and gene-targeted, densely spaced markers. Statistical analysis of the data produced will generate information on areas requiring individual genotyping. In areas where association is confirmed, candidate genes will be mutation screened.
[back to top]

A yeast cell model of SOD1 protein aggregation in amyotrophic lateral sclerosis: a novel tool for identifying proteins and compounds that inhibit or accentuate the pathogenic process

SOD1 mutants are detected in 20% of familial and 3% of sporadic ALS cases. Transgenic and knockout mice studies have confirmed that mutations cause motor neurone degeneration by a toxic gain of function. The finding that copper-free SOD1 retains its toxicity abrogates a role for aberrant catalysis and evidence increasingly points to micro-aggregates of SOD1 being mechanistic in pathogenesis. The aim of this project is to establish a yeast based assay to assess the aggregation of SOD1. To achieve this cDNA of SOD1 ALS mutants G37R, G85R and G93A was cloned into a yeast expression vector, and the construct transformed into S.cerevisiae. Protein expression was confirmed using western blots. Aggregation was confirmed by probing supernatant and pellet fractions (generated by centrifugation) with anti SOD1 antibodies. Future work will involve filter trap dot blot assays and non-denaturing gel electrophoresis. Initial data has shown that mutant SOD1 aggregates in yeast to form high molecular weight species, similar to those observed in transgenic mouse spinal cord and some mammalian cell lines. Wild type SOD1 does not aggregate and is observed as monomers. High molecular weight species are observed in yeast transformed with G37R, G85R and G93A mutants. They are detergent insoluble (resistant to 2% SDS and 8M urea), and able to form in many different strains of S.cerevisiae.
[back to top]

Axonal transport in motor neurone disease

Mutations in the superoxide dismutase 1 (SOD1) gene account for 20% of familial cases of amyotrophic lateral sclerosis (ALS). Several theories have been put forward as to why and how these mutations, found in many different sites of the SOD1 protein, cause disease. One theory is that mutant SOD1 disrupts axonal transport, and mutations in components of the axonal transport system in ALS patients support such a notion. The aims of this research were firstly to characterise axonal transport of wild-type and ALS mutant SOD1, and secondly, to gain insight by which SOD1 is transported through axons. SOD1 movement was visualised by transfection of green fluorescent protein (GFP) tagged SOD1 cDNAs into neurons. GFP tagging of SOD1 was shown to not affect the activity or binding properties of SOD1. Analyses of the movement characteristics of wild-type and four ALS mutants (A4V, G37R, G85R, and G93A) revealed that SOD1 is transported at fast speeds (~1µm/sec) in a predominantly retrograde direction and ALS mutant SOD1 transport is perturbed. Immunoprecipitation studies reveal that SOD1 binds directly or indirectly to the molecular motor dynein. SOD1 obtains copper via a chaperone, the copper chaperone for SOD1 (CCS). CCS also binds to the neuronal adaptor protein X11a, and X11a has been implicated in molecular motor receptor function. Using different fluorescent protein tags, SOD1, CCS and X11a have been shown to be transported as a complex through axons. This thesis reports novel data about SOD1 movement characteristics and transport partners in neurons and demonstrates defective transport of ALS mutant SOD1s.
[back to top]

Biochemical and histological staging of neuropathology in progressive supranuclear palsy

Progressive supranuclear palsy (PSP) is a progressive neurodegenerative disorder that presents clinically as a movement disorder with parkinsonian features. The aetiology of PSP remains unknown, but a definitive diagnosis is achieved only after neuropathological investigation. PSP brain commonly exhibits abnormal neuronal and glial inclusions of the microtubule-associated protein, tau, along with neuronal loss and gliosis that affect primarily subcortical regions. Due to the presence of tau pathology, PSP is classified as a tauopathy. Although abnormal tau pathology is a central feature in the neuropathological diagnosis of PSP, this may not be the best indicator of disease progression since it is not yet known if the deposited tau persists after degeneration of affected neurons and glia. Other changes are also apparent in PSP brain and recent studies in Dr Dickson’s laboratory have shown that gliosis and inflammation are also prominent pathological features of PSP. The purpose of this study is to perform a full neuropathological analysis of a range of PSP autopsy brains selected to encompass a wide range of ages of onset and disease durations with the aim of formulating staging criteria to represent the neuropathological progression of the disease. A similar process, termed Braak staging, is used to describe Alzheimer brains and this has been extremely useful in determining the regional and temporal pathogenesis of this related tauopathy. We propose to quantitatively study tau burden, gliosis and inflammatory markers using standard biochemical techniques including gel electrophoresis, western blotting and ELISA of extracted proteins, coupled with immunohistochemistry. PSP tissue will be analysed in parallel compared with appropriate age-matched controls. Tissue is available, with full ethical permission for its use, from the Brain Bank at the Mayo Clinic, Jacksonville, USA. The results of this study will inform the neuropathological diagnosis of PSP which is sometimes complicated by the lack of a clear distinction between PSP and other tauopathies. Quantification of the relative contributions of tau, gliosis and inflammatory processes in staged PSP brain will provide insight into the pathological origin(s) and pathogenesis of this disease and the possibility of contributing to the development of a therapeutic strategy for PSP.
[back to top]

Bioinformatics for intelligent candidate gene selection in neurodegenerative disorders

Neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and Amyotrophic Lateral Sclerosis are well known for their multifactorial and complex genetic nature. For the past few years, genetics research in neurodegenerative disorders has identified many putative loci for susceptibility genes. However the traditional approaches (e.g. polymorphism detection and genotyping) to find genes responsible for these diseases from such broad linked regions are rather labour intensive and not completely systematic. This project aims to improve the gene analysis and gene search processes by making use of all available biological information and bioinformatics resources. With the availability of complete human genome sequences and extensive public databases (e.g. the human transcriptome map, the human protein reference database, the gene knockout database, etc.), it is feasible to build a knowledge base of neurodegenerative disorders by integrating this data with the linkage data. Knowledge mining technique and many bioinformatics technologies can be explored to develop an intelligent information system to interrogate the knowledge base. The target information system needs to be flexible so that newly available information can be added for enhancement. This project is aimed to build the knowledge base and develop the information system. It will involve extensive analysis work in many aspects of neurodegenerative disorders, biological data from multiple heterogeneous data sources, bioinformatics tools and computing techniques. Analyses will be followed by database modeling, identification and defining system components. Once the database is built, raw data needs to be transformed and mapped to the database before database population. The system development will involve programming and use of different software packages.
[back to top]

Cellular and molecular studies of Alsin/ALS2

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease involving selective degeneration of lower motor neurons in the spinal cord and brainstem, and upper motor neurons in the motor cortex. Some forms of ALS are familial and a few causative genes have now been identified. Mutations in Alsin/ALS2 have been identified that cause a rare recessive form of juvenile ALS (ALS2), juvenile Primary Lateral Sclerosis (jPLS) and infantile onset ascending hereditary spastic paraplegia (IAHSP). Alsin contains three functional domains that show sequence homology to guanine nucleotide exchange factors (GEFs) for the Ras superfamily of GTPases. The Ras superfamily comprises five subfamilies: Ras, Rho, Rab, Arf and Ran, all of which are monomeric proteins that bind and hydrolyse guanosine nucleotides and possess a common structurally-conserved GTP-binding region known as the ‘G domain’. By cycling between GTP-bound (active) and GDP-bound (inactive) conformations, Ras GTPases act as molecular switches, controlling a wide range of critical cellular processes such as signal transduction pathways, regulation of the cytoskeleton and intracellular trafficking. GEFs act as positive regulators of Ras GTPase activity by promoting GDP release resulting in activation through GTP binding. The first putative GEF domain of Alsin shows most similarity to a Ran GEF (RCC1-like domain), the second to Rho GEFs (DH/PH domain) and the third to a Rab GEF (VPS9 domain). In these studies, a polyclonal antibody to Alsin was generated and used to investigate Alsin localisation in the mammalian central nervous system. Alsin was found to be present in a number of neuronal populations in the brain including neurons of the cortex, hippocampus and cerebellum, and in motor neurons of the spinal cord. Since the DH/PH domain of Alsin shows close homology to Rho GEFs, assays were performed to investigate the in vivo effect of Alsin on the activity of the Rho subfamily members RhoA, Rac1 and Cdc42. Alsin was shown to activate Rac1 and to a lesser extent RhoA. Rac1 is known to regulate neurite outgrowth during development. Experiments were therefore performed to investigate the role of Alsin in neurite outgrowth. Immunofluorescence showed that Alsin is localised to the growth cones of rat embryonic cortical and hippocampal neurons (as well as the cell body and processes), which further implicates a role for the protein in neurite outgrowth. Finally, the phosphorylation state of Alsin was investigated, as the activity of Rho GEFs is often regulated by means of phosphorylation. Alsin was found to be a phosphoprotein in vivo and several serine/threonine phosphoylation sites were identified.
[back to top]

Genetic influences on the non-cognitive symptomatology of Alzheimer's disease

The aim of this project is to study the genetic influences on behavioural and psychiatric disturbances in Alzheimer’s disease (AD). The project is divided into two stages. At first, Principal Component Analysis followed by Cluster analysis will be applied to a sample of 967 patients with probable AD in order to a) identify clusters of symptoms which represent unique behavioural sub-syndromes and b) assign patients into these clusters. During the second stage, I will be trying to identify genes responsible for the observed behavioural phenotypes. This will be achieved by performing an association study initially between polymorphisms in 8 candidate genes and behavioural sub-syndromes in a 300 sample of patients with probable AD. In addition, I will utilize the results from a recent genome wide linkage study which suggested that there is a candidate locus on chromosome 6 which modifies the risk of developing psychotic symptoms in AD. An association study will be performed between polymorphisms located in potential candidate genes in this region and psychosis in AD patients.
[back to top]

In-situ characterisation of the recessive motor neurone protein ALS2/Alsin

Several mutations in the ALS2 gene are responsible for a number of recessive motor neuron disorders, which are characterised by the specific loss of upper motor neurons. Most pathogenic mutations of the ALS2 gene result in premature termination of translation, leading to partial or complete loss of ALS2 protein. To gain further insight into the normal function of ALS2 and how the partial or total loss of its function leads to motor neuron degeneration, a polyclonal antibody (Ab5817) was raised against a peptide derived from the ALS2 protein. This antibody defines an antigen referred to as ALS2 RLD-derived antigen (ARDA). Double immunostaining of cultured embryonic neurons and mouse spinal cord sections for ARDA and MAP2, Tau, or phosphorylated neurofilaments, markers of dendrites and axons, respectively, demonstrated that the ARDA antigen was specifically localised in the somatodendritic compartment and excluded from axons. ARDA was also selectively localised to the somatodendritic compartment of motor neurons in human spinal cord and the same type of staining was observed, but with a slight decrease in the intensity, on brain and spinal cord sections from ALS2 knockout mice as on sections from control animals. These data show that Ab 5817 detects the ALS2 protein in situ and are consistent with the existence of at least one ALS2 homologous gene, as recently reported. In order to analyse expression patterns of the different known ALS2 isoforms as well as its homologous gene in neuronal and non-neuronal tissues, an RT-PCR assay was developed. ALS2 long and short forms as well as the homologous gene are expressed ubiquitously in brain, liver and heart both in adult and embryonic mice. ALS2 long form has been suggested to protect neuronal cells from toxicity by SOD1 mutants. Therefore, we looked at expression patterns in brain and spinal cord of SOD1 transgenic mice and their littermates. No significant differences were found in transgenic mice compared to controls. This project has made significant progress towards the characterisation of the ALS2 protein in situ by showing that at least one of the ALS2 protein displays a preferential somatodendritic localisation and that ALS2 is part of a family of complex proteins.
[back to top]

Insulin signalling: coupling APP processing to tau phosphorylation in Alzheimer's disease

Alzheimer’s disease (AD) is pathologically characterised by the presence of extracellular amyloid-ß (Aß) plaques and intraneuronal neurofibrillary tangles (NFTs) composed of highly phosphorylated tau. The contribution of each of these pathologies towards neurodegeneration is unclear. Recent evidence suggests a causal link between Aß deposition and tau phosphorylation, but the mechanisms mediating these connections remain to be established. The insulin-signalling pathway is associated with cognitive function and prevents amyloidogenic APP processing and tau phosphorylation by inhibiting glycogen synthase kinase-3 (GSK-3) activity. We hypothesise that defects in insulin signalling may mediate the connection between plaque and tangle pathologies in AD. We aim to further investigate the role of the insulin signalling pathway in connecting Aß and tau pathologies in vivo using a combination of genetic and pharmacological techniques. Tau phosphorylation and APP processing will be examined in transgenic murine models of insulin resistance, generated by eliminating specific proteins in the insulin signalling cascade. An in vivo model of Aß-induced tau phosphorylation will be generated and, using specific inhibitors of insulin signalling, the mechanisms by which insulin disrupts the amyloid cascade established. Preliminary studies revealed that tau is hyperphosphorylated in IRS2 and PTEN knockout mice compared with their littermate controls. This elevated tau phosphorylation contradicts the observed reduction in GSK3ß activity in these animals and the mechanisms mediating such effects require further investigation. The effects of Aß42 injection on tau phosphorylation in P301L transgenic mice were unclear. GSK3ß activity, however, was inhibited in wild type (wt) mice following lithium treatment (3g/L) in drinking water for 1 week. Preliminary studies suggested that tau phosphorylation is inhibited by lithium treatment in P301L mice. Investigating the role of insulin signalling in AD may elucidate a common mechanism between Aß and tau pathologies and provide novel therapeutic targets: increasing insulin sensitivity and reducing GSK3ß activity should delay Aß deposition and subsequent tau phosphorylation and neurodegeneration.
[back to top]

Investigating the link between tau phosphorylation and progressive supranuclear palsy

Tau is a micortubule associated protein which an important pathogenic factor in a number of diseases (tauopathies) including Alzheimers disease and Progressive Supranuclear palsy (PSP). More is known about the phosphorylation and subsequent aggregation of tau in Alzheimers disease, but it is thought that hyperphosphorylation of tau also leads to its aggregation, tangle formation and then cell death in PSP. It is known that the phosphorylation of tau differs between PSP and Alzheimers disease, but it is not known which phosphorylation sites are PSP specific. Tau phosphorylation differs not only between Alzheimers and PSP, but also between PSP cases themselves. A range of confirmed PSP cases will be screened in order to determine some that are suitable for further analysis. Insoluble tau will be purified from the suitable cases using methods previously employed in the purification of tau from Alzheimers disease brain. Once purified, the tau will be subjected to a biochemical analysis using technquesd including mass spectrometry to determine phosphorylation sites, which can be compared with ones already known for Alzheimer tau. Phosphorylation sites that are unique to PSP tau can then be used to determine the protein kinases involved. Ultimately, assuming the kinases involved in phosphorylating PSP tau are distinct from those involved in the phosphorylation of Alzheimer tau, this study will provide potential drug targets for the treatment of PSP.
[back to top]

Is cortical excitability abnormal in motor neuron disease (MND)? Studies using transcranial magnetic stimulation (TMS) and functional magnetic resonance imaging (fMRI)

Motor Neuron Disease (MND), also known as Amyotrophic Lateral Sclerosis (ALS), is progressive and ultimately fatal disease characterised by degeneration of corticospinal tract and spinal motoneurons. The precise pathogenic mechanisms underlying the disease process are unknown, but excitotoxic mechanisms have been strongly implicated. In vivo evidence in support of this has come from studies using transcranial magnetic stimulation (TMS) studies which have shown that ALS subjects show reduced intra-cortical inhibition.In this study we will test the hypothesis that abnormal fMRI activation is related to reduced intra-cortical inhibition. As a first step we will develop a motor fMRI paradigm where the precise grip force and EMG activity from the muscles can be recorded in the MRI scanner. This will define the relationship between muscle output and fMRI signals from various cortical fields. By rigorously standardising the motor task to a percentage of each individual’s maximum force, the motor effort between the different subject groups will be comparable, therefore controlling for artefacts that might interfere with interpretation of blood oxygen level depend (BOLD) signal changes.We will then group ALS patients on the basis of normal or abnormal intra-cortical excitability using paired TMS stimuli. We hypothesise that ALS subjects with impaired intra-cortical inhibition will show abnormal (i.e. increased) fMRI activation in the primary motor cortex, whereas those in whom intra-cortical inhibition is normal will not show significantly increased motor cortex fMRI activationWe will investigate excitatory and inhibitory processes in the motor cortex of patients at a synaptic level by developing pharmacological challenge studies for fMRI. Using a GABAergic agent (midazolam) we predict that patients in whom there exists abnormal motor cortex excitability will show an abnormal (decreased) inhibitory response compared to controls and patients with weakness due to pure LMN syndromes. Conversely, using the GABAA antagonist flumazenil, we hypothesise that MND subjects will show an increase of motor cortex activation compared to controls. This aspect of the work is novel and exploratory but has the potential to provide major new insights into the physiological basis of brain activation if a sound methodological basis can be developed.In addition to studying subjects with MND, we have the opportunity to study patients with “pure” Hereditary Spastic Paraperesis (HSP) with spastin gene mutations. It is likely that the cellular pathology differs from that of MND. These subjects therefore provide a different pathophysiological model in which to test our hypothesis.
[back to top]

Language function in older adults with mild cognitive impairment and Alzheimer's disease

Language dysfunction is a less consitent feature of early Alzheimer's disease than memory disorder. However, there is evidence that problems such as word-finding abaility may be an early feature of dementia in some patients and therefore a possible biomarker. To study this issue, multiple aspects of language function and word-finding ability are to be assessed in a group of patients with mild AD, in a group of inividuals with MCI and a group of age matched healthy controls. Follow-up of the MCI and control groups will reveal the degree to which language function changes over time and predicts more general cognitive deterioration.
[back to top]

Mapping signalling pathways in the rat brain using monoamime oxidase inhibitors

Depression is a complex, heterogeneous disorder. Several neurotransmitter pathways have been implicated in its pathogenesis and treatment; namely the monoamines, serotonin, noradrenaline and dopamine. This system has therefore been the focus of drug development for decades. The selective serotonin reuptake inhibitors (SSRIs) are highly effective in treating depression, producing a reduction in unwanted side effects relative to previous therapies available. However, they do exhibit a clinical lag phase of several weeks and up to a third of patients exhibit treatment resistance. Therefore, it is pertinent to develop more efficacious treatments. The main aim of this project is to characterise patterns of cerebral activation in the rat following the administration of SSRIs, selective noradrenaline re-uptake inhibitors (SNRIs) and a combination of these classes. In previous studies, various neuroimaging techniques have been used to further our insight into the neuroanatomical sites of antidepressant action. This study will employ c-fos and 2-DG, to examine the temporal effects of antidepressants on neural activity combined with pharmacological MRI to map whole brain in vivo activity. Through both acute and chronic administration of the antidepressants, temporal changes in their neural effects may be observed. Therefore, by using invasive and non-invasive technique in animals, the pharmacodynamic mechanisms underlying the therapeutic effects of antidepressants may be further understood. This may facilitate the development of more clinically effectual treatments for depression. Further studies will attempt to recapitulate work conducted on the social deficits observed in the Mongolian gerbil following pair bond disruption (Hendrie et al., 2003). Following exposure to a stressful environment, this species has been shown to exhibit similar hippocampal atrophy as observed in depressed patients. It is predicted that by using structural MRI studies to quantify hippocampal atrophy, combined with behavioural tests (e.g. tail suspension) during chronic SSRI administration a greater understanding of SSRI therapeutic effects will be achieved.
[back to top]

Mapping the neural correlates of sensory stimulation using functional magnetic resonance imaging (fMRI)

The aim of this investigation is to provide a preclinical model aligned to a human volunteer trial model (Aziz et al., 2000). This human study exploited the fact that the oesophagus develops as both a somatic and visceral structure. The proximal (somatic) and distal (visceral) parts of the oesophagus were each stimulated by distension of a balloon. Functional MRI showed differing areas of activation in the brain when the two different areas were stimulated. This model is of relevance to a group of disorders known as functional gastrointestinal disorders (FGD). The pathophysiology of FGD is incompletely understood, but gut nerve hypersensitivity, spinal nerve hypersensitivity and/or aberrant brain processing of sensory information are thought to play a role in the generation of symptoms. Phase 1 in the present investigation will be a study on the bench using anaesthetised rats to assess physiological responses to somatic and visceral oesophageal stimulation of varying intensity using a balloon inserted into the oesophagus per-orally. Phase 2 will be a dose-response-type experiment carried out in the MRI scanner. The balloon will be inflated to varying degrees, from non-noxious to noxious, in both the proximal and distal positions. The BOLD response in the brain will be measured in an attempt to show a dose-response relationship between the level of stimulation and the BOLD response obtained. If the first two phases of the investigation yield good results, further work that could potentially be carried out includes sensitisation of the oesophagus followed by imaging of the response to balloon stimulation. Also, once this model has been established as a preclinical model, the effects of therapeutic agents could also be assessed. Aziz, Q., Thompson, D.G. et al. (2000) Cortical processing of human somatic and visceral sensation. J Neurosci. 20: 2657-63.
[back to top]

Measuring neurodegeneration in Parkinsonian syndromes using multi-modal MRI

Although Idiopathic Parkinson’s Disease (IPD), Multiple System Atrophy (MSA) and Progressive Supranuclear Palsy (PSP) are distinct conditions, each with a characteristic molecular pathology, they may be clinically indistinguishable at presentation (1-4). Identification of the different distribution of molecular pathology at an early stage of the disease using multi-modal magnetic resonance imaging (MRI) may prove to be important for differential diagnosis and subsequent management. At a more mechanistic level, such studies will provide the basis for a more precise understanding of the functional anatomy of evolving motor and cognitive changes in these neurodegenerative disorders, and ultimately of genotype/phenotype interactions.
[back to top]

Molecular genetics of amyotrophic lateral sclerosis (ALS)

Amyotrophic Lateral Sclerosis (ALS) is a lethal neurodegenerative condition in which selective death of the motor neurones leads to muscle wasting and atrophy and death after 3 to 5 years [1]. Most cases of the disease are sporadic but 5-10% of cases are familial and commonly inherited as an autosomal dominant trait [2]. Mutations in the gene SOD1 [3] and VAPB [4] have been shown to cause the familial form of the disease. Two families that do not have mutations in any genes currently associated with ALS have been studied. Family 1 shows strong linkage to chromosome 16 [5] with a 2-point LOD score above 3 but this is a large region containing approximately 150 known genes of which 20 have been sequenced in part or full with no polymorphisms that so far. Family 2 has had linkage to chromosome 16 excluded by genotyping of polymorphic markers. In order to determine linkage in this second family, Affymetrix SNP chips are to be used [6]. In addition haplotype studies across loci thought to be important in the pathogenesis of the disease such as VEGF [7] [8] and Angiogenin [9] are to be carried out to determine whether these influence the sporadic form of the disease in our population. These will study the groupings of different polymorphisms in 250 affected individuals compared to 250 controls. 1. Talbot, K., Motor neurone disease. Postgrad Med J, 2002. 78(923): p. 513-9. 2. Camu W, K.J., Moulard B, Salachas F, Briolotti V, Rouleau GA, Meininger V, Genetics of familial ALS and consequences for diagnosis. French ALS Research Group. J Neurol Sci., 1999. 165(S1): p. S21-S26. 3. Rosen, D.R., et al., Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis [published erratum appears in Nature 1993 Jul 22;364(6435):362] [see comments]. Nature, 1993. 362(6415): p. 59-62. 4. Nishimura, A.L., et al., A Mutation in the Vesicle-Trafficking Protein VAPB Causes Late-Onset Spinal Muscular Atrophy and Amyotrophic Lateral Sclerosis. Am J Hum Genet, 2004. 75(5). 5. Ruddy, D.M., et al., Two families with familial amyotrophic lateral sclerosis are linked to a novel locus on chromosome 16q. Am J Hum Genet, 2003. 73(2): p. 390-6. 6. Sellick GS, L.C., Tolmie J, Newbury-Ecob R, Geenhalgh L, Hughes S, Whiteford M, Garrett C, Houlston RS., Genomewide linkage searches for Mendelian disease loci can be efficiently conducted using high-density SNP genotyping arrays. Nucleic Acids Res, 2004. 32(20): p. 164. 7. Oosthuyse, B., et al., Deletion of the hypoxia-response element in the vascular endothelial growth factor promoter causes motor neuron degeneration. Nat Genet, 2001. 28(2): p. 131-8. 8. Lambrechts, D., et al., VEGF is a modifier of amyotrophic lateral sclerosis in mice and humans and protects motoneurons against ischemic death. Nat Genet, 2003. 9. Greenway MJ, A.M., Ennis S, Traynor BJ, Corr B, Frost E, Green A, Hardiman O, A novel candidate region for ALS on chromosome 14q11.2. Neurology, 2004. 63(10): p. 1936-1938.
[back to top]

NSAIDs, insulin signalling and Alzheimer's disease: molecular mechanisms

A number of epidemiological studies have demonstrated that non-steroidal anti-inflammatory drugs (NSAIDs) reduce the risk of developing Alzheimer’s disease (AD). However, the molecular mechanisms underlying this effect are unclear. Identifying the molecular target(s) of these compounds relevant to the pathogenesis of AD would be an important step in the development of more potent and selective drugs One possible mechanism is through an effect on insulin signalling. NSAIDs are insulin sensitizers, and epidemiological, genetic and cellular studies suggests that dysfunctional insulin signalling may be involved in the pathogenesis of AD. The current project will seek to identify an effect on insulin signalling by NSAIDs that prevents the abnormal phosphorylation of tau induced by Aß1-42. In addition certain NSAIDs have also been shown to reduce the ratio of Aß1-42 /Aß1-38. It has been reported that this effect is mediated by a reduction in ROCK activity via the direct inhibitory action of this particular sub-set of NSAIDs on the small Gprotein RhoA. The current study will therefore also examine the effects of NSAIDs in models of RhoA induced tau hyperphosphorylation, neurite retraction and Aß1-42 production to obtain further insights into the protective effects of NSAIDs in AD.
[back to top]

Pathophysiological mechanisms in motor neuron disease: a neuroimaging and neurophysiological approach

abstract currently unavailable
[back to top]

Phosphoproteomics and long-term plasticity in mice

The aim of this research is to use mass spectrometry (MS) to study the phosphorylation of proteins that are involved in synaptic plasticity during long-term potentiation (LTP) and long-term depression (LTD) at the Schaffer collateral-CA1 synapse in C57BL/6 and DBA/2 mice. Methanol and deuterated methanol will be used to convert proteins from experimental and control tissue, respectively, to methyl esters differing by a mass of 3. This will enable the phosphorylation differences between the two states to be determined precisely, an obvious advantage over using Western blots. Memories are formed by experience-based learning, giving rise to the hypothesis that experience-dependent changes in synaptic plasticity form the basis of information storage in the brain. The best mechanisms by which synaptic strength is altered as a consequence of experience are long-term potentiation (LTP) and long-term depression (LTD), increases and decreases, respectively, in synaptic gain. The mechanisms by which LTP and LTD change synaptic strength are not fully understood but probably involve post-translational modifications to signal transduction pathways, the most important of which is protein phosphorylation. In recent years mass spectrometry (MS) techniques have been used to identify the protein complexes and signal transduction pathways that occur at activated synapses. Many proteins suspected of involvement in LTP/LTD have been identified and studied using MS and biochemical methods, e.g., protein kinases and phosphatases, cytoskeletal proteins, scaffold and receptor-clustering proteins, etc. This project will quantify precisely, the effects of long-term plasticity changes on their phosphorylation status and on that of other proteins shown to be involved.
[back to top]

Positional candidates in Alzheimer's disease

abstract currently unavailable
[back to top]

Protein kinase N and neurodegenerative diseases

Protein kinase N (PKN) is a 120 kDa serine/threonine kinase with a catalytic domain towards the C-terminus of the protein and an N-terminus with a regulatory domain. Cleavage of PKN has been shown in response to toxic insults and the cleaved product is constitutively active due to loss of the regulatory N-terminus. One protease that cleaves PKN under these conditions is caspase-3, which has been implicated in motor neurone disease (MND). Tau and neurofilaments are both phosphorylated by PKN, which is significant because aberrant phosphorylation and accumulation of these proteins are found in Alzheimer’s disease (AD) and MND respectively. Neurotoxic stimuli that are associated with AD and MND that may cause caspase-3 activation, and therefore PKN activation, include glutamate and amyloid-ß (Aß). This study aims to use a combination of confocal and live microscopy to examine the subcellular localisation of full length and cleaved PKN forms in neurones. The effects of neurotoxic insults on PKN subcellular distribution will also be investigated.
[back to top]

Proteomic analysis of disease-associated mitochondrial protein changes in amyotrophic lateral sclerosis (ALS)

abstract currently unavailable
[back to top]

Proteomic analysis of mutant SOD1 transgenic mouse tissue to identify disease-specific changes relevant to motor neurone disease

abstract currently unavailable
[back to top]

Regulation of gene expression in Alzheimer's disease

The two hallmarks of Alzheimer’s disease are extracellular amyloid beta plaques and intracellular neurofibrillary tangles comprised primarily of tau protein. Abeta is acknowledged to play a major role in the disease progression of Alzheimer’s disease but the mechanisms underlying its neurotoxic effect have not yet been fully explained. Studies have shown that Abeta affects gene transcription and recent work using DNA microarrays has established a gene expression profile of Alzheimer’s disease brains and shown pronounced differences between diseased and control brains. This study set out to determine the differential expression of genes in primary mouse cortical neuronal cell cultures using Affymetrix DNA microarrays after Abeta stimulation. Treatments were carried out with fibrillar Abeta 1-42, fibrillar Abeta 25-35 (active fragment of Abeta 1-42) and A? 1-42 oligomers (ADDL’s) in order to determine genes that may be involved in A? neurotoxicity. Abeta toxicity was demonstrated by the LDH cytotoxicity assay. Furthermore, Caspa Tag fluorescent staining of caspase enzymes and immunofluorescent staining of active caspase 3 revealed activation of caspases and induction of apoptotic cell death by Abeta peptides. Fibrillar Abeta 25-35, fibrillar Abeta 1-42 and ADDL’s were shown to differentially regulate gene expression using Affymetrix microarrays. Microarray gene expression data was corroborated using PCR and protein expression was also examined using western blot. An immediate early gene, early growth response 1 (egr-1), was shown to be induced by Abeta 25-35. Several downstream targets of egr-1 (presenilin 2, p53, p35 activator of Cdk5) are involved in Alzheimer’s disease therefore the expression of these was also examined using PCR and western blot. In summary, a differential gene expression profile of primary mouse neuronal cultures was established after stimulation by fibrillar and oligomeric species of Abeta peptide. Changes in gene expression may help elucidate the mechanism of Abeta toxicity.
[back to top]

Regulation of the transcription factor Oct-6 in CNS neurones

Oct6 is a class III POU domain transcription factor that shows restricted expression during central nervous system development in the cerebral cortex. Post-mitotic neurons leaving the ventricular zone express Oct6 as they migrate to the developing cerebral wall. Normal adult expression is more restricted, becoming confined to the neurons of cortical layers 2/3 and 5, and the CA1 region of the hippocampus. Our laboratory has demonstrated increased OCT6 immunoreactivity relative to age-matched controls in the brains of individuals suffering from various neurological disorders. Up-regulation has also been observed in the brains of rodents exposed to neurotoxins, mechanical lesions or in various neurodegenerative models. Moreover, if lesioned rats are transplanted with conditionally immortalised neural stem cells (NSC), a procedure shown to induce repair, OCT6 immunoreactivity is lost. In this study it has been observed that the NSC’s in question are positive for Oct6 mRNA but express at most only basal levels of protein, implying that a post-transcriptional mechanism of Oct6 regulation operates in these cells. Attempts to further characterise the type of regulation tentatively suggest that mRNA stability may be a significant factor. To establish whether there was a similar disparity between protein and mRNA localisation in vivo, mouse brain tissue sections were doubled labelled for OCT6 protein and mRNA, and at the ages examined this was found not to be the case. The role of Oct6 and its regulation has been extensively studied in the peripheral nervous system, where its involvement in Schwann cell differentiation has been well characterised. Conversely, its presence in the developing and adult CNS is less clear, and in this study cultured mouse cortical cells were used as a model system for investigating how neuronal Oct6 regulation compares with that of glia. Preliminary data suggest that the factors that regulate glial Oct6 expression may well differ from neuronal ones.
[back to top]

Role of lipid rafts in neuronal amyloidogenesis

Aß originates from the amyloid precursor protein (APP) by regulated proteolysis, to generate Aß. APP internalization from the cell surface is required for the efficient secretion of Aß, and the isolation of APP as well as of the enzymatic machinery involved in Aß generation, in specialized domains of the cell surface termed lipid rafts, suggests that internalization through these domains may be one major route of Aß generation. We propose to explore the potential contribution of APP internalization via lipid rafts to the generation and secretion of Aß. Our working hypothesis states that: Internalization of APP through lipid rafts is central to amyloidogenesis. We will test our working hypothesis by accomplishing the following specific aim: To understand the role of lipid rafts on Aß generation. We will disrupt lipid raft integrity by manipulating the levels of their main lipid components, cholesterol and sphingolipids, in primary neurons expressing wildtype or endocytosis-deficient APP, and will measure the relative partitioning of APP metabolites, as well as Aß secretion rates, before and after lipid manipulation. Our work will involve the generation and analysis of lipid rafts using primary neurons from transgenic mice of different APP genotypes.
[back to top]

Stem cell transplantation in models of neurodegeneration: an integrated analysis by neuroimaging, behaviour and histology

This work will investigate how a conditionally immortalised neural stem cell line (MHP36) survives, migrates, integrates and modifies diseased brains of rodent models of neurodegeneration. This will be done by serial in vivo magnetic resonance imaging, which will determine how stem cell transplantation affects the structure of the diseased brain, by allowing direct comparison between grafted and lesioned animals. The imaging will be paralleled by behavioural assessment that will determine the progression of behavioural impairment over time and any recovery following stem cell transplantation. Such correlations may reveal anatomical changes in neurodegenerative models, which could serve as surrogate markers for the assessment of stem cell transplant efficacy. Post-mortem histological analysis will be used to validate the in vivo neuroimaging, providing a detailed measure of the effects of grafting on neurodegeneration. Survival, migration, integration, differentiation and host immune response will be determined using appropriate histological markers. This work will provide detailed in vivo validation of anatomical and functional changes resulting from disease onset and assess the efficacy of neural stem cell grafts in inducing recovery.
[back to top]

The effects of phosphorylation by identified protein kinases on the axonal transport and aggregation of tau

Tau is a neuronal microtubule associated protein, the most well characterised function of which is its binding to and stabilisation of microtubules, which serve as tracks for axonal transport in neurons. Abnormalities in tau have been linked to a number of neurodegenerative disorders including Alzheimer’s disease (AD). In AD brain tau is the main constituent of neurofibrillary tangles, which are composed of paired helical filaments of insoluble tau. Tau in these inclusions has been shown to be abnormally phosphorylated, although the mechanisms connecting this and the formation of aggregates remain unclear. Phosphorylation may induce aggregation by changing the physical properties of tau, or may mediate tangle formation through a change in another property of tau such as its microtubule binding or axonal transport Several groups of protein kinases are seen as potential mediators of the abnormal phosphorylation of tau. One such group are the stress-activated protein kinases, which have been shown to phosphorylate tau in vitro and in cultured cells. Additionally activated SAPKs have been found localised with deposits of phosphorylated tau in samples from human AD brain and mouse models. In this project I will examine the effects of phosphorylation by stress-activated protein kinases on the axonal transport and aggregation of tau, using EGFP-tagged tau constructs and modulators of SAPK activity in cultured neurons
[back to top]

The mode of action of neural stem cells

Oct-6 (also known as SCIP and Tst-1) is a member of the POU-III sub-family of the POU-domain transcription factors. Members of this sub-family have been shown to have important functions in neurodevelopment. Recent experiments in our laboratory have revealed Oct-6 expression in the neurons of postmortem human schizophrenic brain whereas it was undetectable in matched controls, this puts Oct-6 forward as a possibly being the first definitive marker for schizophrenia. Observations from our laboratory reveal that Oct-6 immunoreactivity can either be nuclear, cytoplasmic or both in different populations of neurons. As Oct-6 is a transcriptional regulator it contains the common nuclear localization signal so it’s presence in the cytoplasm raises the possibility of Oct-6 sequestration by another protein into the cytoplasm. The discovery of binding partners for Oct-6 is, therefore, important to understanding its mechanism of action. Little is known of specific Oct-6 interactions within either the CNS or PNS with the exception of a well-documented interaction with Sox 10 in glial cells. Here Sox 10 acts synergistically with Oct-6 to regulate downstream genes. We would therefore like to identify similar factors functioning in the Schizophrenic neuron. The aims of this project, therefore, are to determine the mechanisms that regulate the expression of Oct-6 (notably those mechanisms relating to the synergistic/antagonistic effects of binding partners) and to elucidate the genes that Oct-6 regulates downstream and the function of these genes in relation to schizophrenia.
[back to top]

The relevance of positional specification to the differentiation of neural stem cells

The mammalian telencephalon is a complex yet widely studied region in developmental neurobiology. The telencephalon is patterned by gradients of signalling molecules such as BMPs, Wnts, FGFs and Shh, that in turn lead to region-specific transcription factor expression. As a result neural progenitor cells (NPCs) in the dorsal telencephalon generate glutamatergic neurons and astrocytes and in the ventral telencephalon generate GABAergic neurons and oligodendrocytes in vivo. This patterning information is retained in cultured E14 rat neural progenitor cells. The cardinal features of neural stem cells (NSCs) are self-renewal, multipotentiality and the ability to functionally repopulate damaged tissue. However for these cells to be used therapeutically a more in-depth knowledge of stem cell biology is required. To generate sufficient numbers of NSCs/NPCs to be used in potential transplantation therapy two methods have been explored. Cells have been grown as neurospheres in the presence of mitogens such as fibroblast growth factor 2 (FGF2) and/or epidermal growth factor (EGF). Alternatively cells have been immortalised by an oncogene and again cultured with FGF2 and/or EGF. Research has suggested that FGF2 may influence the positional information retained by NPCs and thus expanded cells seem to possess a broader developmental potential than in vivo cells. The proposed research will develop understanding of the effects of expansion and immortalisation on NPC gene expression and cell fate. The project will explore the possibility of manipulating cell fate using exogenous signalling molecules in order to generate specific neural cell types since these would be useful in treating neurodegenerative diseases.
[back to top]

The role of inhibition in cortical map reorganisation

Sensory deprivation alters the short-term synaptic dynamics of connected pairs of neurons in rodent primary sensory cortex. Less is known about modifications in inhibitory circuitry. I will trim rats’ whiskers for a period of ten days and then make whole cell patch clamp recordings of monosynaptically connected pyramidal and interneuron pairs in vitro. Presynaptic pyramidal cells will be stimulated to fire action potentials and responses from the postsynaptic interneuron will be recorded. I will assess the effect of whisker trimming on intrinsic excitation and short-term dynamics of unitary synaptic connectivity. Addition of a flurophore to the internal solution will enable me to image the recorded neurons using confocal microscopy. Subsequent 3D reconstruction of the cells will identify potential synaptic connections between the neurons.
[back to top]

The role of plasticity of excitatory connections between pyramidal neurons in cortical map reorganisation

Rearrangement of cortical maps, which represent information obtained from different sensory areas, occurs not only during development but, also, in response to alterations in sensory input. However, the mechanisms underlying experience-dependent plasticity in adults remain unclear. The organisation of the rodent whisker representation in the primary somatosensory cortex, which can be visualised as solid ‘barrels’ in Layer 4, make it a good model in which to study the mechanisms underlying cortical map reorganisation. We induce brief periods of non-traumatic sensory deprivation by whisker trimming, to generate adjacent areas of barrel cortex which are either deprived of, or receive normal, sensory input. Multiple changes in short-term synaptic dynamics in excitatory inputs to Layer 2 pyramidal neurons in deprived rat barrel cortex have been described. I will make whole cell recordings of pairs of synaptically-connected pyramidal neurons in Layer 2/3 at the junction of deprived and spared cortex, where cortical plasticity is greatest. Recorded neurons will be filled with fluorophores and reconstructed in three dimensions using confocal microscopy. My aim is to determine the cellular mechanisms to the observed changes in synaptic efficacy. I will focus on the relative contributions of long-term potentiation and long-term depression of the amplitude of excitatory postsynaptic potentials, versus changes in synaptic number, to synaptic plasticity in this system.
[back to top]

The role of the Oct-6 transcription factor in development and the repair of brain damage

My project focuses on the role of the POU-domain transcription factor Oct-6, examining its expression in embryonic development and affective disorders. I plan to look at the expression of Oct-6 mRNA and protein at different stages of neurodevelopment in the mouse, using both in situ hybridization and immunohistochemistry. In addition to looking at murine expression of Oct-6, I also hope to investigate its human developmental expression. I plan to examine expression patterns of genes such as Emx-1, -2 and Pax-6 proposed to function upstream of Oct-6 and genes such as Mash-1 and the Neurogenins proposed to function in conjunction with or downstream of Oct-6, with a view to trying to elucidate a pathway by which oct-6 expression could shape developmental events. I also intend to look at the expression of Oct-6 in the brains of patients diagnosed with bipolar disorder and depression. Work in our lab has previously shown Oct-6 to be expressed in the brains of schizophrenics, by investigating Oct-6 expression in affective disorders it can be ascertained whether Oct-6 is specific to schizophrenia or whether it more a general marker. Work in our lab has identified two genes, 8R1B and 3R2C, proposed to act downstream of Oct-6, I also will look at the expression of these genes in schizophrenia, bipolar disorder and depression.
[back to top]

The use of pharmacological MRI to characterise the ketamine model of schizophrenia in rodents

The administration of ketamine results in an NMDA hypofunction state potentially analogous to the aetiology of schizophrenia. The current project aims to investigate neural activity in this model of psychosis, using dynamic magnetic resonance imaging (MRI) in rats. By using this non-invasive technique, temporal and spatial changes in brain activity can be detected by changes in blood oxygenation level dependant (BOLD) signal. The prefrontal and retrosplenial cortices, thalamus and hippocampus have been noted to be of relevance in earlier autoradiography and immunocytochemistry studies (Duncan, Moy et al. 1998) and hence particular interest will be paid to these areas. Corroborative behavioural studies have been performed, following the administration of subanaesthetic doses of ketamine, and correlated with temporal changes in neural activity visualised to delineate pharmacodynamics with MRI. Most of this work is now completed, and currently only increases in the number of animals in each experimental group is being undertaken. Administration of each enantiomer of ketamine hydrochloride is also intended to be used to further characterise the ketamine effects on the brain. Following these preliminary investigations, I intend to use the imaging data I have collected to date, to identify brain areas of interest for subsequent microdialysis investigations. These measures should allow us to distinguish neurotransmitter systems which are modulated in the psychotic state for temporal correlation with the BOLD effect. Various antagonists and agonists of these endogenous compounds will then be administered in the pharmacological MRI paradigm and the resultant changes in neural activity analysed. It is envisaged that existing antipsychotic and antiepileptic treatment shall also be administered to rats which are under the influence of ketamine to elucidate the drugs’ mode of action. Initially, the work will focus on the amelioration of the psychotic state with Clozapine pre-dosing followed by agonists of the metabotropic mGluII/III receptors. As ketamine is also safe to administer to humans, neuroimaging studies of the animal model could be directly translated into human subjects, thus identifying brain areas and neurotransmitter systems that are of significant interest in the psychotic state of the disease. Duncan, G. E., S. S. Moy, et al. (1998). "Metabolic mapping of the rat brain after subanesthetic doses of ketamine: potential relevance to schizophrenia." Brain Res 787(2): 181-90.
[back to top]

Transcriptional and epigenetic changes during neural stem cell differentiation

abstract currently unavailable
[back to top]

X11-mediated reduction of Aß in the APPswe (Tg2576) mouse model of Alzheimer’s disease: in vivo analysis of neuronal function

It has recently been reported that the neuronal X11a adaptor protein reduces cerebral amyloid-ß (Aß) levels and Aß deposition in the APPswe (Tg2576) transgenic mouse model of the amyloid pathology of Alzheimer’s disease. APPswe mice overexpress the “Swedish” mutant form of the amyloid precursor protein (APPswe) and have been reported to have electrophysiological and cognitive deficits attributable to Aß pathology. The hypothesis that forms the basis of this proposal is that X11a -mediated reduction in cerebral Aß will reduce, or delay the onset of, these electrophysiological deficits and result in improved neuronal function. To a certain extent, it should also restore cognitive deficits observed in APPswe mice. The aims of this study are: 1. To use in vivo electrophysiology methods to prospectively characterise synaptic function in double transgenic APPswe /X11a mice compared to APPswe littermates. 2. To characterise prospectively the effects of APPswe/X11a? in cognitive test compared to APPswe littermates 3. To correlate cerebral Aß levels and amyloid deposition with changes over time in cognition and synaptic function in double transgenic APPswe/ X11a mice compared to APPswe littermates.
[back to top]

A genome-wide association study in amyotrophic lateral sclerosis by candidate gene targeting and DNA pooling

The aim of this project is to search for susceptibility genes responsible for sporadic amyotrophic lateral sclerosis (ALS). The list of candidate genes is very large and derived from pathological observations, known pathogenic mechanisms, and areas of linkage demonstrated in families. Because of the limitations of present genetic technology, it is not practical to individually genotype large numbers of individuals. I will therefore use DNA pooling to perform a targeted candidate gene based scan, on pools of 300 DNA samples divided phenotypically into four distinct pools, each age and sex matched to four corresponding control pools. A set of 3000 primer pairs specific for highly polymorphic microsatellite markers will be used to genotype each pool. I expect to find regions showing a significant difference in allelic frequencies in comparison control samples. Further statistical analysis of these areas may lead to the identification of the gene(s) that, upon mutation, increase the susceptibility to ALS.
[back to top]

An investigation into the influence of premorbid factors of personality, social networks and depression on the manifestation of Alzheimer’s disease

There is considerable heterogeneity in the manifestation of Alzheimer’s Disease (AD) in terms of the presence of behavioural and psychological symptoms of dementia (BPSD), age of onset and speed of progression. Although a large body of work has considered factors affecting AD risk, much less attention has been paid to factors influencing disease presentation. The current study assesses the influence of premorbid factors of personality, social support, depression and anxiety on the phenotype of AD. I hypothesise that these factors will be associated with age of onset and symptomatic profile of disease, as well as show interaction effects with each other and with known genetic modifiers of disease. Premorbid factors may also modify the nature and timing of help-seeking in dementia. 200 cases of probable AD will undergo a thorough assessment in order to enable the research diagnosis to be confirmed, and to collect detailed information from a family member on the manifestation of disease and the way in which help was sought for the condition. Cases will also receive a cognitive assessment and a blood sample will be obtained for genetic analysis. For each case, either one, or ideally two informants, will complete a questionnaire and telephone interview to retrospectively assess their premorbid personality, social support and lifestyle when the case was aged in his/her 40’s, as well as retrospective assessment of premorbid depression and anxiety. The main analyses will be performed using a range of regression techniques. Where two informants exist for assessment of premorbid factors, mean scores will be taken.
[back to top]

Applied proteomics in the Neurosciences

Analysis of proteins and peptides by mass spectrometry became widely applicable in the mid 80s following the advent of ionisation processes such as Matrix-assisted Laser Desorption Ionisation (MALDI) and electrospray ionisation (ESI). Nowadays, mass spectrometry has come to underpin the majority of proteomics research initiatives, being the analytical method of choice, largely because of the high sensitivity afforded by modern technology platforms. Although it is now possible to detect low abundant proteins, the ability to separate cellular proteins into highly resolved subsets is of equal importance. Techniques such as ion exchange chromatography or immobilised metal affinity chromatography (IMAC) can be used in series with reversed phase chromatography to provide exquisite two-dimensional separations, which can also be directly interfaced to the mass spectrometer. The aim of this project is to investigate cellular models of Alzheimer's disease (AD) pathogenesis and bipolar disorder therapy using several mass spectrometry-based strategies for differential display of peptides and phosphopeptides. It is envisaged that four different peptide tagging techniques will be applied to investigate biologically relevant systems. Isotope coded affinity tag (ICAT) reagents have been designed to specifically label cysteine residues and subsequent purification of the labelled peptides is achieved via a Streptavidin-Biotin affinity step. Peptide sequence tag (PST) and Tandem mass tag (TMT) reagents facilitate the purification of N-terminal peptides and differential isotopic labelling by O-methylation (DILM) will be applied specifically for the analysis of phosphopeptides. The use of this combination of tagging approaches will increase protein coverage, improve the detection of low abundance proteins and allow new insights into neurological and psychiatric disorders.
[back to top]

Biologically-inspired robots

abstract currently unavailable
[back to top]

Candidate gene screening in the motor neurone disorders

abstract currently unavailable
[back to top]

Developing a standardised psychometric test battery for functional neuroimaging

abstract currently unavailable
[back to top]

Development of novel perfusion based magnetic resonance imaging techniques

abstract currently unavailable
[back to top]

Fate commitment and differentiation of conditionally immortalised neural stem cells

not available
[back to top]

Functional magnetic resonance imaging in animal models of pain

abstract currently unavailable
[back to top]

Functional neuroimaging of the depressed brain

abstract currently unavailable
[back to top]

Functional significance of altered splicing of the microtubule-associated protein, tau, modeled by rna reprogramming

Tau protein is important for the establishment and maintenance of neuronal morphology and it is involved in the stabilisation of microtubules. Tau has an important role in organising the cytoskeleton, which serves as tracks for organelle transport. Tau protein is encoded by a single gene that generates, by alternative splicing, six brain isoforms. These isoforms contain either three (3R) or four (4R) microtubule binding repeats. Certain pathologies, such as Alzheimer’s disease, referred to as tauopathies, are characterised by tau inclusions. Some inherited tauopathies are caused by mutations in the tau gene that lead to an excess of 4R tau. The aim of this project is to study the effects of changes in the tau isoform ratio in microtubules stabilisation, tau protein aggregation and changes in organelle transport. Tau transcripts will be reprogrammed using the SMaRT (spliceosome-mediated RNA trans-splicing) technology. The SMaRT technology promotes the splicing between an endogenous transcript and an exogenously delivered RNA or pre-trans-splicing molecule (PTM). We have shown that Tau is a suitable target for RNA reprogramming using SMaRT. We have designed and characterised different tau-targeted SMaRT constructs to reprogram Tau in neuroblastoma SHSY5Y cells and post-mitotic neurons. In co-transfection experiments using a tau minigene as a splicing model we have confirmed that we are able to interfere with exon 10 splicing and that the product from the trans-splicing reaction is accurate. Our objective will be to reprogram endogenous Tau and check the efficiency of trans-splicing at the RNA and protein level. Stable cell lines expressing 4R tau isoform will be generated and characterised. We will analyse changes in the microtubule network, organelle transport and processing of tau (aggregation and degradation). With this model we will also study if some tau mutations related to frontotemporal dementia (FTDP-17) affect the normal function and processing of the tau protein, especially the degradation pathway.
[back to top]

In vitro differentiation of conditionally immortalised neuroepithelial stem cells

abstract currently unavailable
[back to top]

Investigation of behaviour, neurochemistry, and in vivo imaging in the MPTP mouse model of nigro-striatal degeneration

abstract currently unavailable
[back to top]

Mapping the progressive changes in pathology and behaviour following stroke

abstract currently unavailable
[back to top]

MND as a multisystem disorder: Quantitative studies on selective vulnerability and cellular and molecular pathology

Although motor neurone disease (MND) has often been regarded as a purely motor syndrome, evidence from neuroimaging and neuropsychological studies imply that it is a multi-system disorder. Involvement of the “extra-motor” area is evident in patients in whom MND is associated with frontal lobe type dementia (MND-D). In this study, neuronal quantitative analysis of post-mortem tissue showed some degeneration of pigmented neurones in the substantia nigra in MND-D brains compared to controls but not in MND inclusion body (MND-IB) dementia or MND cases. In addition to the extra-motor involvement, magnetic brain stimulation and positron emission tomography studies suggest that abnormal GABAergic neurotransmission might contribute to excitotoxicity in MND. This excitotoxicity hypothesis was investigated at the cellular and molecular level using immunohistochemistry and Real Time PCR. Glutamatergic neurones were labelled with antibody against non-phosphorylated neurofilament (SMI-32) and different populations of GABAergic interneurones with antibody against calcium binding proteins (calbindin-D28K, CB; parvalbumin, PV; calretinin, CR). The density of CB-immunoreactive (-IR) and SMI-32-IR neurones in the primary motor, prefrontal, and anterior cingulate cortex in MND subjects were found to be significantly decreased compared to controls. Reduction in the densities of PV-IR and CR-IR neurones in MND subjects were not significant however. In addition, quantitative RT-PCR studies showed that mRNA levels of CB, CR, glutamic acid decarboxylase 65 and 67, and 5HT-1A receptor in the precentral gyrus and anterior frontal gyrus was slightly decreased in MND cases compared to those of controls. This investigation suggests that involvement of the glutamatergic and GABAergic neurotransmitter systems is more widespread than previously thought. Disturbances in glutamatergic neurotransmission, possibly mediated or exacerbated by alterations in GABAergic functioning, may be the cause of excitotoxicity. These findings also support the notion that MND should be considered a multi-system disorder rather than a motor syndrome alone.
[back to top]

Molecular genetic studies of Alzheimer's disease

abstract currently unavailable
[back to top]

Neurocognitive and behavioural aspects of negative symptoms in schizophrenia

abstract currently unavailable
[back to top]

Neuroimaging of cognitive control in major depresssion

abstract currently unavailable
[back to top]

Neurophysiological predictors of respone to repetitive transcranial magnetic stimulation (rTMS)

abstract currently unavailable
[back to top]

Proteomics in the early diagnosis of Alzheimer’s disease

Aims: A biological marker would improve the accuracy of the early diagnosis of Alzheimer’s disease (AD). An ideal biomarker should reflect the neuropathology of AD, and thus be available in monitoring the progression of the disease and in evaluating the efficacy of therapy AD. Therefore our aim is to identify a plasma/serum marker, which will be able to distinguish AD from normal aged people. Background: At present diagnosis of Alzheimer’s disease is identified by a combination of clinical tests, neuropsychological tests, brain imaging and neuropathlogical diagnosis. Currently there exists no simple test or biological marker that could detect early AD cases or to monitor progression of the condition. Various proteins have been investigate as candidate markers the major Alzheimer’s markers, such as Ab and tau. Other promising protein markers include the iron binding protein p97 the angiotensin-converting enzyme, plasma a1 antichymotrypsin and apolipoprtein E. However no study yet examined a large complement of proteins without a priori decisions regarding likely candidates.Method: We have used proteomics to screen for protein changes in plasma from AD (n=50) and normal control subjects (n=50). Samples were resolved by two-dimensional electrophoresis (2DGE) and analysed by using Melanie 3 software (Genebio, SA. Protein differences between cases and controls will be identified by image analysis and mass spectrometry. Further analyses will use both 2DGE and candidate protein approaches to correlate changes with progression and AD genotype and phenotype.
[back to top]

Repetitive transcranial magnetic stimulation (rTMS) studies in depression

abstract currently unavailable
[back to top]

Semi-automated analysis of brain volumetry in CNS disorders

abstract currently unavailable
[back to top]

Serial neuroimaging assessment of the progressive changes in ALS

not available
[back to top]

Testing cognitive models of persecutory delusions across diagnises

abstract currently unavailable
[back to top]

Time-use diablement and outcome in patient's with Parkinson's disease and their carers

This is a cross-sectional study that is exploring the impact of chronic neurological disease (Parkinson's disease) on patterns of time-use in patients and their carers. The study assesses time-use within the WHO model of disablement and examines the relationship to more usual subjective indicators of quality of life.
[back to top]

Current phd opportunities

King's College London - Homepage

Sitemap Site help Terms and conditions Accessibility Recruitment News Centre Contact us