Age-Related Diseases (Wolfson Centre for)
|MPhil/PhD, MD (Res)
|Part Time, Full Time
My work covers many areas of bioinformatics with particular emphasis on structural biology. Current projects are microarray search engines, protein dynamical simulation and interaction site prediction.
Our group is interested in the development of orally available retinoids for the treatment of CNS disorders. We are developing a number of novel retinoid receptor specific agonists and tool compounds to look at the mechanisms of retinoid signalling pathways in Alzheimer’s disease, spinal cord injury and neurogenesis.
Nuclear receptor signalling
Cellular effects of retinoic acid (RA) are mediated by binding to nuclear receptors - the retinoic acid receptors (RARs) and retinoid X receptors (RXRs). There are three subtypes of each receptor, alpha, beta and gamma, and multiple isoforms of each subtype due to alternative splicing and differential promoter usage. RARs mediate gene expression by forming heterodimers with RXRs, whereas RXRs can mediate gene expression either as homodimers or by forming heterodimers with orphan receptors, which are also members of the nuclear receptor. The RAR/RXR heterodimers regulate transcription by binding to retinoic acid response elements (RAREs) in the upstream regions of target genes. Because the RAR genes contain RAREs, one notable effect of RA is its ability to induce the expression of the RARs themselves, thus stimulating various RA signalling pathways.
RARbeta2 signalling and neurite regeneration
We have shown that the RARbeta2 receptor induces neurite regeneration in vitro in both embryonic and adult neurons. In the adult spinal cord little or no expression of RARbeta2 can be detected, however when this tissue is transduced with RARbeta2 using lentivirus vectors neurite outgrowth occurs. By microarray analysis we have identified a number of genes involved in neurite regeneration, which are regulated by RARbeta2.
RARalpha signalling and neuronal survival
By generating retinoid deficient rats we have shown that RARalpha as opposed to other RAR receptors is required for the survival of motoneurons, Purkinje neurons and cerebral cortex neurons the same receptor deficit is found in human pathology samples of spontaneous cases of motoneuron disease and Alzheimer’s disease (AD). By using both in vitro and in vivo assays a number of target genes known to be involved in AD have been identified which are regulated by RARalpha signalling. Current work involves manipulating the retinoid pathway in mouse models of neurodegeneration and assaying for target genes and behavioural analysis including open field, novel object recognition and T maze.
RAR signalling and stem/progenitor cell differentiation
We have identified specific roles of RARbeta and alpha signalling in neural progenitor cell (NPC) differentiation. This will allow the transplantation of stem cells with a defined lineage into the injured nervous system or the stimulation of endogenous progenitor cells in the injured CNS both of which may lead to functional repair.
Screening for novel retinoids
Retinoids are small molecules which have been shown to cross the blood brain barrier, and therefore have therapeutic potential for the treatment of CNS disorders. However, to date very few retinoids with drug like properties have been developed. We have set up screening assays for both binding (IC50) and potency (EC50) of retinoids at the RARs from which we can identify specific receptor agonists. These will be used in models of CNS injury described above.
Understanding neurodegeneration is key to developing and taking forward new treatments for Alzheimer’s disease, Parkinson’s disease and stroke. Central to our approach is translating basic science into improved clinical treatment. Key elements of our work include experimental studies to understand basic disease mechanisms, the Brains for Dementia Research initiative, enabling us to apply this understanding to the human brain, and a substantial focus on biomarkers and clinical trials.
Major clinical trials have address psychiatric and behavioural symptoms associated with dementia and the use of antipsychotic and sedative drugs. The Neurodegeneration group is led by Professor Clive Ballard.
Nuclear receptor signalling
Cellular effects of retinoic acid (RA) are mediated by binding to nuclear receptors - the retinoic acid receptors (RARs) and retinoid X receptors (RXRs). There are three subtypes of each receptor, alpha, beta and gamma, and multiple isoforms of each subtype due to alternative splicing and differential promoter usage. RARs mediate gene expression by forming heterodimers with RXRs, whereas RXRs can mediate gene expression either as homodimers or by forming heterodimers with orphan receptors, which are also members of the nuclear receptor. The RAR/RXR heterodimers regulate transcription by binding to retinoic acid response elements (RAREs) in the upstream regions of target genes. Because the RAR genes contain RAREs, one notable effect of RA is its ability to induce the expression of the RARs themselves, thus stimulating various RA signalling pathways.
RARbeta2 signalling and neurite regeneration
We have shown that the RARbeta2 receptor induces neurite regeneration in vitro in both embryonic and adult neurons. In the adult spinal cord little or no expression of RARbeta2 can be detected, however when this tissue is transduced with RARbeta2 using lentivirus vectors neurite outgrowth occurs.
By microarray analysis we have identified a number of genes involved in neurite regeneration, which are regulated by RARbeta2.
RARalpha signalling and neuronal survival
By generating retinoid deficient rats we have shown that RARalpha as opposed to other RAR receptors is required for the survival of motoneurons, Purkinje neurons and cerebral cortex neurons the same receptor deficit is found in human pathology samples of spontaneous cases of motoneuron disease and Alzheimer’s disease (AD). By using both in vitro and in vivo assays a number of target genes known to be involved in AD have been identified which are regulated by RARalpha signalling. Current work involves manipulating the retinoid pathway in mouse models of neurodegeneration and assaying for target genes and behavioural analysis including open field, novel object recognition and T maze.
RAR signalling and stem/progenitor cell differentiation
We have identified specific roles of RARbeta and alpha signalling in neural progenitor cell (NPC) differentiation. This will allow the transplantation of stem cells with a defined lineage into the injured nervous system or the stimulation of endogenous progenitor cells in the injured CNS both of which may lead to functional repair.
Screening for novel retinoids
Retinoids are small molecules which have been shown to cross the blood brain barrier, and therefore have therapeutic potential for the treatment of CNS disorders. However, to date very few retinoids with drug like properties have been developed. We have set up screening assays for both binding (IC50) and potency (EC50) of retinoids at the RARs from which we can identify specific receptor agonists. These will be used in models of CNS injury described above.
The focus of work in my research group is on finding new approaches for the improved treatment of Parkinson's disease. Current strategies under investigation including targeting metabotropic glutamate receptors and replenishing depleted levelos of neurotrophic factors. It is hoped such strategies will help combat the progressive neurodegenertion that underlies this disease.
Parkinson Disease
Parkinson’s disease is a debilitating movement disorder that results from degeneration of dopamine-containing neurones in the nigrostriatal pathway. The resultant loss of striatal dopamine sets up downstream changes in firing within the basal ganglia motor loop that are manifest clinically as deficits in movement. Current treatments like L-DOPA, which replenish lost dopaminergic transmission, are initially effective at reversing the motor symptoms. However, these drugs do little to halt the relentless degeneration of dopaminergic neurones and the increasing doses of drug needed to counter ever-worsening symptoms bring about disabling side-effects such as dyskinesia. For this reason, alternative treatments that offer protection against the degeneration or do not evoke dyskinesia are eagerly awaited.
Metabotropic Glutamate Receptors
Excess glutamate transmission from an overactive subthalamic nucleus which innervates the substantia nigra controbutes not only to symptom generation, but is also one of many factors thought to contribute to the progressive nigral cell death. G-protein coupled group III metabotropic glutamate (mGlu) receptors that signal through Gi/Go are implicated in the negative regulation of glutamate release. Using in situ hybridisation and immunohistochemistry we have demonstrated expression of these receptors in the substantia nigra; electron microscopy studies of others have shown this localisation to be upon presynaptic glutamatergic terminals. If functional, activation of these receptors should reduce glutamate release in the substantia nigra and thereby provide a means not only of correcting the motor symptoms, but also of potentially offering much-needed neuroprotection. Our in vitro brain slice work has confirmed that at least two of the group III mGlu receptors (mGlu4 and mGlu7) have the capacity to reduce glutamate release in the substantia nigra, an effect we have since demonstrated in vivo for this class of receptor, using microdialysis. Activation of mGlu4 and mGlu7 receptors also reversed symptoms in rodent models of Parkinson’s disease. Of great promise, these targets also show considerable neuroprotective potential, being able not only to protect the neurones from degenerating, but also preserving motor function in rodent models of the disease. We are currently identifying which of the group III mGlu receptors holds the greatest neuroprotective potential and hope to follow the targeting of this receptor over a longer time frame to check on potential dyskinetic side-effects, for example. We also wish to explore in more detail the cellular and molecular mechanisms behind these beneficial effects.
Growth Factors and Neuro-repair
We have also started to examine the potential of biologicals, specifically fibroblast growth factor-20 (FGF-20). A number of growth factors appear to malfunction in Parkinson’s disease and FGF-20 in particular has genetic links with the disease which may result in a hostile environment for cells to survive in. Work carried out in our group so far has shown that replacement of this endogenous protective factor is an effective way of providing protection against toxin-mediated cell death both in dopaminergic cells in culture and in rodent models of Parkinson’s disease. It is not yet known how these beneficial effects are brought about – whether by direct actions on the dopainergic neurones themselves, which do express the relevant FGFR1 receptors (see Figure 1) or, as is the case with related factors (e,g. FGF2) via increasing the synthesis and release of other growth factors from glial cells. We hope our future studies will shed light on these mechanisms and inform us how best to effect protection with these agents.
Around 60% neurones have already degenerated when Parkinson’s disease signs first appear. In the adult brain, residual neurones have limited capacity for optimal rewiring and compensation because of certain molecules within the extracellular matrix of damaged sites that inhibit collateral sprouting and plasticity. In future studies we hope to find ways of countering these inhibitory molecules to encourage plasticity and rewiring of the brain in Parkinson’s disease.
The development of an undifferentiated cell into a neuron is a process that is fundamental to the formation of sensory tissues. In many tissues neurogenesis is preceded by a period of proliferation before cells exit the cell cycle and differentiate. Our goal is to understand the mechanisms by which developing tissues coordinate proliferation and neuronal differentiation.
We use the Drosophila eye to study the signals which control neurogenesis. Prior to photoreceptor differentiation an extensive proliferative phase generates a large pool of undifferentiated cells, which are then specified sequentially through reiterative use of the Notch and EGF receptor pathways. Neurogenesis in the Drosophila eye occurs in a spatio-temporal manner making it particularly well suited for studying temporal controls during differentiation.
Insulin receptor signalling and neurogenesis
We have shown that the conserved insulin receptor (InR)/Tor pathway plays a key role in controlling the timing of neuronal differentiation in Drosophila (Bateman and McNeill 2004, Cell 119, p.87-96). By using mutants in various components of the InR/Tor pathway, we showed that activation of this pathway causes precocious differentiation of neurons. Conversely, inhibition of InR/Tor signalling significantly delays neurogenesis. Correct timing of neuronal differentiation is essential for tissue pattern formation and consequently mutations in components of InR/Tor signalling cause pattern defects in the adult. One of the aims of our research is to determine the molecular mechanism by which InR/Tor signalling regulates the timing of neuronal differentiation in Drosophila.
Neurogenesis and disease
We are also interested in diseases in which Inr/Tor signalling plays a role. One such disease is Tuberous Sclerosis Complex (TSC). TSC affects 1 in 6000 live births and is caused by mutations in one of two genes (TSC1 or TSC2). The pathology of TSC is typified by the formation of benign tumours in the brain, kidneys and other organs, beginning in early childhood. One of the most debilitating manifestations among patients with TSC is the high prevalence and severity of epilepsy, with patients suffering from up to hundreds of seizures per day. TSC1/2 are core components of the InR/Tor pathway and we have shown that loss of TSC1 causes precocious neuronal differentiation in Drosophila. The demonstration that TSC1 controls the timing of neuronal differentiation and hence neuronal patterning in Drosophila is intriguing, since abnormal neuronal development and migration are a major part of the neuropathology of TSC.
The eukaryotic cell harbours a diverse repertoire of endocytic mechanisms that regulate manifold aspects of homeostasis, signalling and function. In the neuron, this complexity is well documented within the framework of membrane recycling at the presynaptic terminal. Our recent findings suggest that a similar arrangement may exist at the postsynaptic compartment, where a novel pathway for receptor trafficking does not require clathrin and dynamin – the defining proteins of the classical endocytic pathway (Fig. 1). I now plan to use imaging and biochemical approaches in cultured neurons to gain a better understanding of the role this pathway plays in the physiological and pathological context of neuronal (and possibly glial) function.
Imaging synaptic geometry
Existing evidence suggests that synapses are remarkably dynamic structures that persistently undergo structural modification. So far, however, direct visualization of nanoscale synaptic dynamics has been unfeasible due to the lack of suitable methodology. I have developed an assay that utilizes the principle of trans-synaptic fluorescence resonance energy transfer (FRET) to assess the synaptic geometry (Fig. 2). I will apply this approach to established cellular paradigms of synaptic maturation, plasticity and pathology in order to delineate the mechanisms underlying synaptic dynamics, with specific aim at the multiple cell adhesion molecules resident at the synapse.
Regulation of neuronal membrane trafficking by activity and pathology – a systemic approach
In order to fulfil their function, neurons must constantly readjust their functional properties in response to the activity of the system. A crucial element of this readjustment is dynamic regulation of surface expression of neurotransmitter receptors, ion channels and other membrane proteins. Through regulation of trafficking at (or near) the synapse, neurons achieve tight control over their most fundamental properties – synaptic transmission and intrinsic excitability. While much research has been focusing on activity-regulated surface expression of particular proteins, the comprehensive picture of activity-dependent regulation of neuronal membrane trafficking is lacking and the mechanisms underlying it are poorly understood. I propose to undertake a systemic characterization of the activity-dependent regulation of neuronal surfaceome (i.e. proteins expressed at the surface of the neuron) in the context of Alzheimer’s disease, using affinity purification, proteomics and subsequent characterization by imaging and biochemistry
Nuclear receptor signalling
Cellular effects of retinoic acid (RA) are mediated by binding to nuclear receptors - the retinoic acid receptors (RARs) and retinoid X receptors (RXRs). There are three subtypes of each receptor, alpha, beta and gamma, and multiple isoforms of each subtype due to alternative splicing and differential promoter usage. RARs mediate gene expression by forming heterodimers with RXRs, whereas RXRs can mediate gene expression either as homodimers or by forming heterodimers with orphan receptors, which are also members of the nuclear receptor. The RAR/RXR heterodimers regulate transcription by binding to retinoic acid response elements (RAREs) in the upstream regions of target genes. Because the RAR genes contain RAREs, one notable effect of RA is its ability to induce the expression of the RARs themselves, thus stimulating various RA signalling pathways.
RARbeta2 signalling and neurite regeneration
We have shown that the RARbeta2 receptor induces neurite regeneration in vitro in both embryonic and adult neurons. In the adult spinal cord little or no expression of RARbeta2 can be detected, however when this tissue is transduced with RARbeta2 using lentivirus vectors neurite outgrowth occurs.
By microarray analysis we have identified a number of genes involved in neurite regeneration, which are regulated by RARbeta2.
RARalpha signalling and neuronal survival
By generating retinoid deficient rats we have shown that RARalpha as opposed to other RAR receptors is required for the survival of motoneurons, Purkinje neurons and cerebral cortex neurons the same receptor deficit is found in human pathology samples of spontaneous cases of motoneuron disease and Alzheimer’s disease (AD). By using both in vitro and in vivo assays a number of target genes known to be involved in AD have been identified which are regulated by RARalpha signalling. Current work involves manipulating the retinoid pathway in mouse models of neurodegeneration and assaying for target genes and behavioural analysis including open field, novel object recognition and T maze.
RAR signalling and stem/progenitor cell differentiation
We have identified specific roles of RARbeta and alpha signalling in neural progenitor cell (NPC) differentiation. This will allow the transplantation of stem cells with a defined lineage into the injured nervous system or the stimulation of endogenous progenitor cells in the injured CNS both of which may lead to functional repair.
Screening for novel retinoids
Retinoids are small molecules which have been shown to cross the blood brain barrier, and therefore have therapeutic potential for the treatment of CNS disorders. However, to date very few retinoids with drug like properties have been developed. We have set up screening assays for both binding (IC50) and potency (EC50) of retinoids at the RARs from which we can identify specific receptor agonists. These will be used in models of CNS injury described above.
We use the Drosophila eye to study the signals which control neurogenesis. Prior to photoreceptor differentiation an extensive proliferative phase generates a large pool of undifferentiated cells, which are then specified sequentially through reiterative use of the Notch and EGF receptor pathways. Neurogenesis in the Drosophila eye occurs in a spatio-temporal manner making it particularly well suited for studying temporal controls during differentiation.
Insulin receptor signalling and neurogenesis
We have shown that the conserved insulin receptor (InR)/Tor pathway plays a key role in controlling the timing of neuronal differentiation in Drosophila (Bateman and McNeill 2004, Cell 119, p.87-96). By using mutants in various components of the InR/Tor pathway, we showed that activation of this pathway causes precocious differentiation of neurons. Conversely, inhibition of InR/Tor signalling significantly delays neurogenesis. Correct timing of neuronal differentiation is essential for tissue pattern formation and consequently mutations in components of InR/Tor signalling cause pattern defects in the adult. One of the aims of our research is to determine the molecular mechanism by which InR/Tor signalling regulates the timing of neuronal differentiation in Drosophila.
Neurogenesis and disease
We are also interested in diseases in which Inr/Tor signalling plays a role. One such disease is Tuberous Sclerosis Complex (TSC). TSC affects 1 in 6000 live births and is caused by mutations in one of two genes (TSC1 or TSC2). The pathology of TSC is typified by the formation of benign tumours in the brain, kidneys and other organs, beginning in early childhood. One of the most debilitating manifestations among patients with TSC is the high prevalence and severity of epilepsy, with patients suffering from up to hundreds of seizures per day. TSC1/2 are core components of the InR/Tor pathway and we have shown that loss of TSC1 causes precocious neuronal differentiation in Drosophila. The demonstration that TSC1 controls the timing of neuronal differentiation and hence neuronal patterning in Drosophila is intriguing, since abnormal neuronal development and migration are a major part of the neuropathology of TSC.
Chronic pain is a debilitating disorder that affects millions of people world-wide and has a considerable detrimental impact on quality of life. There are multiple events which can lead to chronic pain including trauma, diabetes, surgical procedures, cancer and HIV. Effective analgesic therapies are inadequate in the majority of chronic pain patients and are often associated with unpleasant side-effects. Consequently at present there is a substantial, unmet, clinical need for more effective analgesics for chronic pain patients.
Nuclear receptor signalling
Cellular effects of retinoic acid (RA) are mediated by binding to nuclear receptors - the retinoic acid receptors (RARs) and retinoid X receptors (RXRs). There are three subtypes of each receptor, alpha, beta and gamma, and multiple isoforms of each subtype due to alternative splicing and differential promoter usage. RARs mediate gene expression by forming heterodimers with RXRs, whereas RXRs can mediate gene expression either as homodimers or by forming heterodimers with orphan receptors, which are also members of the nuclear receptor. The RAR/RXR heterodimers regulate transcription by binding to retinoic acid response elements (RAREs) in the upstream regions of target genes. Because the RAR genes contain RAREs, one notable effect of RA is its ability to induce the expression of the RARs themselves, thus stimulating various RA signalling pathways.
RARbeta2 signalling and neurite regeneration
We have shown that the RARbeta2 receptor induces neurite regeneration in vitro in both embryonic and adult neurons. In the adult spinal cord little or no expression of RARbeta2 can be detected, however when this tissue is transduced with RARbeta2 using lentivirus vectors neurite outgrowth occurs.
By microarray analysis we have identified a number of genes involved in neurite regeneration, which are regulated by RARbeta2.
RARalpha signalling and neuronal survival
By generating retinoid deficient rats we have shown that RARalpha as opposed to other RAR receptors is required for the survival of motoneurons, Purkinje neurons and cerebral cortex neurons the same receptor deficit is found in human pathology samples of spontaneous cases of motoneuron disease and Alzheimer’s disease (AD). By using both in vitro and in vivo assays a number of target genes known to be involved in AD have been identified which are regulated by RARalpha signalling. Current work involves manipulating the retinoid pathway in mouse models of neurodegeneration and assaying for target genes and behavioural analysis including open field, novel object recognition and T maze.
RAR signalling and stem/progenitor cell differentiation
We have identified specific roles of RARbeta and alpha signalling in neural progenitor cell (NPC) differentiation. This will allow the transplantation of stem cells with a defined lineage into the injured nervous system or the stimulation of endogenous progenitor cells in the injured CNS both of which may lead to functional repair.
Screening for novel retinoids
Retinoids are small molecules which have been shown to cross the blood brain barrier, and therefore have therapeutic potential for the treatment of CNS disorders. However, to date very few retinoids with drug like properties have been developed. We have set up screening assays for both binding (IC50) and potency (EC50) of retinoids at the RARs from which we can identify specific receptor agonists. These will be used in models of CNS injury described above.
In animals, the family of TRP (transient receptor potential) ion channels fulfil important roles as transduction molecules, converting chemical and physical information into electrical signals. TRP channels are particularly prominent for transduction mechanisms in specialised sensory cells, where TRP channels are essential for the ability of animals to detect physical and chemical changes within the body, as well as in the external environment. In this way, animals exploit TRP channels to detect light, touch, temperature (hot and cold), pH and a wide range of chemicals. The functions of some of these ion channels are familiar to all of us. TRPV1 responds to stimulation with hot temperatures and the pungent ingredient capsaicin from chili peppers, which is why we perceive chilies as hot. Menthol from peppermint (and other chemicals that evoke a cooling sensation) shares a molecular target with cold temperatures in the ion channel TRPM8, whereas mustard, raw onion, tear gas and many other irritants evoke tearing, cough or pain by stimulating TRPA1. Experiments with transgenic mice have confirmed that without these ion channels, animals are unable to sense heat, cold and irritants, respectively. TRP channels in sensory neurons have attracted particular interest as potential drug targets for the development of novel analgesic drugs, since it has become clear that the expression of these ion channels determines which modalities different populations of sensory neurons respond to (e.g., heat, cold and irritants), since they act as the primary transducers of the painful stimuli.
Much is still unknown about the roles different members of this large family of ion channels fulfil in physiological and pathophysiological situations. This includes fundamental questions such as how many of these proteins are activated and regulated by endogenous molecules and how TRP channels in sensory neurons contribute to chronic pain conditions. This is the main focus of my lab. I use electrophysiological, microfluorometric, behavioural and molecular methods to elucidate sensory transduction mechanisms and to identify molecules or physical stimuli that act as agonists or modulators of sensory neuron TRP channels. Together with Prof. Stuart Bevan, these studies have previously led to the identification of the first endogenous agonists for TRPM8 and TRPA1 and identification of TRPA1 as the molecular target for paracetamol
EphB receptors and chronic pain
My laboratory recently identified an entirely novel role for the ephrinB/EphB system as modulators of synaptic efficacy in the spinal cord, contributing to sensory abnormalities in persistent pain states. Chronic pain syndromes are a clinical problem of considerable importance. Despite the rapid growth of the area of pain research over the past two decades, the molecular and cellular mechanisms that underlie chronic pain states are still incompletely understood, and this incompleteness limits the range of therapeutic strategies that can be adopted.
It is known that some pain states are due to a ‘sensitisation’ of the central nervous system, as a consequence, for example, of prolonged activation of pain receptors due to inflammation or nerve injury. We have found that EphB receptors and ephrins are crucially involved in the processing of painful stimuli at the level of the spinal cord, and could thus be potential targets for the development of new analgesic drugs. Activation of the EphB receptors in the spinal cord induces a reduction in the threshold of response to painful thermal stimuli. Conversely, blocking in the spinal cord the interactions between EphB receptors and their cognate ligands, ephrin Bs, produces analgesia in a number of experimental models.
We are currently using a combination of biochemical, anatomical, behavioural and electrophysiological techniques on rats and transgenic mice to understand in more detail the role of the Eph/ephrin system in chronic pain, using several well characterised laboratory models of inflammatory and neuropathic pain. This work is funded by the Wellcome Trust.
Eph receptors, ephrinBs and spinal cord injury
Our understanding of the factors that are responsible for the failure of CNS regeneration, whilst improving, remains incomplete. One of the best characterised functions of Eph receptors and ligands during development is to act as negative or repellent guidance cues. It is thus reasonable to hypothesise that alterations in the expression of these molecules may be one of the events that follow injury to the CNS, and render an injury site inhibitory to axonal regeneration.
We have identified a number of ephrin-Bs and their Eph receptors that exhibit a pattern of expression and regulation after injury which correlate with areas of regenerative failure. Utilising a number of animal models of acute and chronic spinal cord injury we aim to characterise the dynamic expression and interaction of these receptors and ligands with a view to interfering with their activation and signalling in vivo, thus hopefully promoting CNS repair. We have financial support from the International Spinal Research Trust to carry out this work.
Nuclear receptor signalling
Cellular effects of retinoic acid (RA) are mediated by binding to nuclear receptors - the retinoic acid receptors (RARs) and retinoid X receptors (RXRs). There are three subtypes of each receptor, alpha, beta and gamma, and multiple isoforms of each subtype due to alternative splicing and differential promoter usage. RARs mediate gene expression by forming heterodimers with RXRs, whereas RXRs can mediate gene expression either as homodimers or by forming heterodimers with orphan receptors, which are also members of the nuclear receptor. The RAR/RXR heterodimers regulate transcription by binding to retinoic acid response elements (RAREs) in the upstream regions of target genes. Because the RAR genes contain RAREs, one notable effect of RA is its ability to induce the expression of the RARs themselves, thus stimulating various RA signalling pathways.
RARbeta2 signalling and neurite regeneration
We have shown that the RARbeta2 receptor induces neurite regeneration in vitro in both embryonic and adult neurons. In the adult spinal cord little or no expression of RARbeta2 can be detected, however when this tissue is transduced with RARbeta2 using lentivirus vectors neurite outgrowth occurs.
By microarray analysis we have identified a number of genes involved in neurite regeneration, which are regulated by RARbeta2.
RARalpha signalling and neuronal survival
By generating retinoid deficient rats we have shown that RARalpha as opposed to other RAR receptors is required for the survival of motoneurons, Purkinje neurons and cerebral cortex neurons the same receptor deficit is found in human pathology samples of spontaneous cases of motoneuron disease and Alzheimer’s disease (AD). By using both in vitro and in vivo assays a number of target genes known to be involved in AD have been identified which are regulated by RARalpha signalling. Current work involves manipulating the retinoid pathway in mouse models of neurodegeneration and assaying for target genes and behavioural analysis including open field, novel object recognition and T maze.
RAR signalling and stem/progenitor cell differentiation
We have identified specific roles of RARbeta and alpha signalling in neural progenitor cell (NPC) differentiation. This will allow the transplantation of stem cells with a defined lineage into the injured nervous system or the stimulation of endogenous progenitor cells in the injured CNS both of which may lead to functional repair.
Screening for novel retinoids
Retinoids are small molecules which have been shown to cross the blood brain barrier, and therefore have therapeutic potential for the treatment of CNS disorders. However, to date very few retinoids with drug like properties have been developed. We have set up screening assays for both binding (IC50) and potency (EC50) of retinoids at the RARs from which we can identify specific receptor agonists. These will be used in models of CNS injury described above.
In animals, the family of TRP (transient receptor potential) ion channels fulfil important roles as transduction molecules, converting chemical and physical information into electrical signals. TRP channels are particularly prominent for transduction mechanisms in specialised sensory cells, where TRP channels are essential for the ability of animals to detect physical and chemical changes within the body, as well as in the external environment. In this way, animals exploit TRP channels to detect light, touch, temperature (hot and cold), pH and a wide range of chemicals. The functions of some of these ion channels are familiar to all of us. TRPV1 responds to stimulation with hot temperatures and the pungent ingredient capsaicin from chili peppers, which is why we perceive chilies as hot. Menthol from peppermint (and other chemicals that evoke a cooling sensation) shares a molecular target with cold temperatures in the ion channel TRPM8, whereas mustard, raw onion, tear gas and many other irritants evoke tearing, cough or pain by stimulating TRPA1. Experiments with transgenic mice have confirmed that without these ion channels, animals are unable to sense heat, cold and irritants, respectively. TRP channels in sensory neurons have attracted particular interest as potential drug targets for the development of novel analgesic drugs, since it has become clear that the expression of these ion channels determines which modalities different populations of sensory neurons respond to (e.g., heat, cold and irritants), since they act as the primary transducers of the painful stimuli.
Much is still unknown about the roles different members of this large family of ion channels fulfil in physiological and pathophysiological situations. This includes fundamental questions such as how many of these proteins are activated and regulated by endogenous molecules and how TRP channels in sensory neurons contribute to chronic pain conditions. This is the main focus of my lab. I use electrophysiological, microfluorometric, behavioural and molecular methods to elucidate sensory transduction mechanisms and to identify molecules or physical stimuli that act as agonists or modulators of sensory neuron TRP channels. Together with Prof. Stuart Bevan, these studies have previously led to the identification of the first endogenous agonists for TRPM8 and TRPA1 and identification of TRPA1 as the molecular target for paracetamol
We use the Drosophila eye to study the signals which control neurogenesis. Prior to photoreceptor differentiation an extensive proliferative phase generates a large pool of undifferentiated cells, which are then specified sequentially through reiterative use of the Notch and EGF receptor pathways. Neurogenesis in the Drosophila eye occurs in a spatio-temporal manner making it particularly well suited for studying temporal controls during differentiation.
Insulin receptor signalling and neurogenesis
We have shown that the conserved insulin receptor (InR)/Tor pathway plays a key role in controlling the timing of neuronal differentiation in Drosophila (Bateman and McNeill 2004, Cell 119, p.87-96). By using mutants in various components of the InR/Tor pathway, we showed that activation of this pathway causes precocious differentiation of neurons. Conversely, inhibition of InR/Tor signalling significantly delays neurogenesis. Correct timing of neuronal differentiation is essential for tissue pattern formation and consequently mutations in components of InR/Tor signalling cause pattern defects in the adult. One of the aims of our research is to determine the molecular mechanism by which InR/Tor signalling regulates the timing of neuronal differentiation in Drosophila.
Neurogenesis and disease
We are also interested in diseases in which Inr/Tor signalling plays a role. One such disease is Tuberous Sclerosis Complex (TSC). TSC affects 1 in 6000 live births and is caused by mutations in one of two genes (TSC1 or TSC2). The pathology of TSC is typified by the formation of benign tumours in the brain, kidneys and other organs, beginning in early childhood. One of the most debilitating manifestations among patients with TSC is the high prevalence and severity of epilepsy, with patients suffering from up to hundreds of seizures per day. TSC1/2 are core components of the InR/Tor pathway and we have shown that loss of TSC1 causes precocious neuronal differentiation in Drosophila. The demonstration that TSC1 controls the timing of neuronal differentiation and hence neuronal patterning in Drosophila is intriguing, since abnormal neuronal development and migration are a major part of the neuropathology of TSC.
The eukaryotic cell harbours a diverse repertoire of endocytic mechanisms that regulate manifold aspects of homeostasis, signalling and function. In the neuron, this complexity is well documented within the framework of membrane recycling at the presynaptic terminal. Our recent findings suggest that a similar arrangement may exist at the postsynaptic compartment, where a novel pathway for receptor trafficking does not require clathrin and dynamin – the defining proteins of the classical endocytic pathway (Fig. 1). I now plan to use imaging and biochemical approaches in cultured neurons to gain a better understanding of the role this pathway plays in the physiological and pathological context of neuronal (and possibly glial) function.
Imaging synaptic geometry
Existing evidence suggests that synapses are remarkably dynamic structures that persistently undergo structural modification. So far, however, direct visualization of nanoscale synaptic dynamics has been unfeasible due to the lack of suitable methodology. I have developed an assay that utilizes the principle of trans-synaptic fluorescence resonance energy transfer (FRET) to assess the synaptic geometry (Fig. 2). I will apply this approach to established cellular paradigms of synaptic maturation, plasticity and pathology in order to delineate the mechanisms underlying synaptic dynamics, with specific aim at the multiple cell adhesion molecules resident at the synapse.
Regulation of neuronal membrane trafficking by activity and pathology – a systemic approach
In order to fulfil their function, neurons must constantly readjust their functional properties in response to the activity of the system. A crucial element of this readjustment is dynamic regulation of surface expression of neurotransmitter receptors, ion channels and other membrane proteins. Through regulation of trafficking at (or near) the synapse, neurons achieve tight control over their most fundamental properties – synaptic transmission and intrinsic excitability. While much research has been focusing on activity-regulated surface expression of particular proteins, the comprehensive picture of activity-dependent regulation of neuronal membrane trafficking is lacking and the mechanisms underlying it are poorly understood. I propose to undertake a systemic characterization of the activity-dependent regulation of neuronal surfaceome (i.e. proteins expressed at the surface of the neuron) in the context of Alzheimer’s disease, using affinity purification, proteomics and subsequent characterization by imaging and biochemistry
In animals, the family of TRP (transient receptor potential) ion channels fulfil important roles as transduction molecules, converting chemical and physical information into electrical signals. TRP channels are particularly prominent for transduction mechanisms in specialised sensory cells, where TRP channels are essential for the ability of animals to detect physical and chemical changes within the body, as well as in the external environment. In this way, animals exploit TRP channels to detect light, touch, temperature (hot and cold), pH and a wide range of chemicals. The functions of some of these ion channels are familiar to all of us. TRPV1 responds to stimulation with hot temperatures and the pungent ingredient capsaicin from chili peppers, which is why we perceive chilies as hot. Menthol from peppermint (and other chemicals that evoke a cooling sensation) shares a molecular target with cold temperatures in the ion channel TRPM8, whereas mustard, raw onion, tear gas and many other irritants evoke tearing, cough or pain by stimulating TRPA1. Experiments with transgenic mice have confirmed that without these ion channels, animals are unable to sense heat, cold and irritants, respectively. TRP channels in sensory neurons have attracted particular interest as potential drug targets for the development of novel analgesic drugs, since it has become clear that the expression of these ion channels determines which modalities different populations of sensory neurons respond to (e.g., heat, cold and irritants), since they act as the primary transducers of the painful stimuli.
Much is still unknown about the roles different members of this large family of ion channels fulfil in physiological and pathophysiological situations. This includes fundamental questions such as how many of these proteins are activated and regulated by endogenous molecules and how TRP channels in sensory neurons contribute to chronic pain conditions. This is the main focus of my lab. I use electrophysiological, microfluorometric, behavioural and molecular methods to elucidate sensory transduction mechanisms and to identify molecules or physical stimuli that act as agonists or modulators of sensory neuron TRP channels. Together with Prof. Stuart Bevan, these studies have previously led to the identification of the first endogenous agonists for TRPM8 and TRPA1 and identification of TRPA1 as the molecular target for paracetamol.
The development of an undifferentiated cell into a neuron is a process that is fundamental to the formation of sensory tissues. In many tissues neurogenesis is preceded by a period of proliferation before cells exit the cell cycle and differentiate. Our goal is to understand the mechanisms by which developing tissues coordinate proliferation and neuronal differentiation.
Insulin receptor signalling and neurogenesis
We have shown that the conserved insulin receptor (InR)/Tor pathway plays a key role in controlling the timing of neuronal differentiation in Drosophila (Bateman and McNeill 2004, Cell 119, p.87-96). By using mutants in various components of the InR/Tor pathway, we showed that activation of this pathway causes precocious differentiation of neurons. Conversely, inhibition of InR/Tor signalling significantly delays neurogenesis. Correct timing of neuronal differentiation is essential for tissue pattern formation and consequently mutations in components of InR/Tor signalling cause pattern defects in the adult. One of the aims of our research is to determine the molecular mechanism by which InR/Tor signalling regulates the timing of neuronal differentiation in Drosophila.
Neurogenesis and disease
We are also interested in diseases in which Inr/Tor signalling plays a role. One such disease is Tuberous Sclerosis Complex (TSC). TSC affects 1 in 6000 live births and is caused by mutations in one of two genes (TSC1 or TSC2). The pathology of TSC is typified by the formation of benign tumours in the brain, kidneys and other organs, beginning in early childhood. One of the most debilitating manifestations among patients with TSC is the high prevalence and severity of epilepsy, with patients suffering from up to hundreds of seizures per day. TSC1/2 are core components of the InR/Tor pathway and we have shown that loss of TSC1 causes precocious neuronal differentiation in Drosophila. The demonstration that TSC1 controls the timing of neuronal differentiation and hence neuronal patterning in Drosophila is intriguing, since abnormal neuronal development and migration are a major part of the neuropathology of TSC.
A number of neurodegenerative diseases, including Alzheimer’s disease and the transmissible spongiform encephalopathies, are caused by errors in the way membrane proteins ‘traffic’ within neurons (errors in the way they are assembled, moved to and removed from the cell surface). We study particularly how prion protein misfolds into a pathogenic form that kills neurons, causing Creutzfeld-Jacob disease in man.
Dynamin-independent receptor trafficking in neurons
The eukaryotic cell harbours a diverse repertoire of endocytic mechanisms that regulate manifold aspects of homeostasis, signalling and function. In the neuron, this complexity is well documented within the framework of membrane recycling at the presynaptic terminal. Our recent findings suggest that a similar arrangement may exist at the postsynaptic compartment, where a novel pathway for receptor trafficking does not require clathrin and dynamin – the defining proteins of the classical endocytic pathway (Fig. 1). I now plan to use imaging and biochemical approaches in cultured neurons to gain a better understanding of the role this pathway plays in the physiological and pathological context of neuronal (and possibly glial) function.
Imaging synaptic geometry
Existing evidence suggests that synapses are remarkably dynamic structures that persistently undergo structural modification. So far, however, direct visualization of nanoscale synaptic dynamics has been unfeasible due to the lack of suitable methodology. I have developed an assay that utilizes the principle of trans-synaptic fluorescence resonance energy transfer (FRET) to assess the synaptic geometry (Fig. 2). I will apply this approach to established cellular paradigms of synaptic maturation, plasticity and pathology in order to delineate the mechanisms underlying synaptic dynamics, with specific aim at the multiple cell adhesion molecules resident at the synapse.
Regulation of neuronal membrane trafficking by activity and pathology – a systemic approach
In order to fulfil their function, neurons must constantly readjust their functional properties in response to the activity of the system. A crucial element of this readjustment is dynamic regulation of surface expression of neurotransmitter receptors, ion channels and other membrane proteins. Through regulation of trafficking at (or near) the synapse, neurons achieve tight control over their most fundamental properties – synaptic transmission and intrinsic excitability. While much research has been focusing on activity-regulated surface expression of particular proteins, the comprehensive picture of activity-dependent regulation of neuronal membrane trafficking is lacking and the mechanisms underlying it are poorly understood. I propose to undertake a systemic characterization of the activity-dependent regulation of neuronal surfaceome (i.e. proteins expressed at the surface of the neuron) in the context of Alzheimer’s disease, using affinity purification, proteomics and subsequent characterization by imaging and biochemistry
The main interest of my lab is endocannabinoid (eCB) signalling in the nervous system, and this stems from our interest in regeneration, and in particular the role that adhesion molecules play in promoting axonal growth. Relevant contributions include the first report that polysialic acid is required for NCAM stimulated axonal growth, the observation that threshold levels and alternative splicing regulates NCAM function, the observation that signalling rather than adhesion accounts for the neurite outgrowth response stimulated by at least three adhesion molecules (CAMs, NCAM, L1 and N-cadherin); the consequent development of the CAM/FGF receptor hypothesis which identified a completely novel signalling axis in neurons. More recent work has identified cross-talk between the FGFR and eCB signalling in axonal growth and guidance.
Our current focus is on the role of endocannabinoid (eCB) signalling in adult neurogenesis. eCB signalling, driven by activation of the CB1 and/or CB2 cannabinoid receptors, has widespread functions during development and in the adult. Conditions that might benefit from intervention include pain, obesity, liver disease, spastic disorders, epilepsy, various psychiatric conditions as well as Parkinson’s and Alzheimer’s. As some drugs have been approved for use in man, with many others in clinical trials, it is important to fully understand the role of eCB signalling in adult neurogenesis. Our major contributions to this field to date have been: - (1) To use a bioinformatics approach to identify and then clone the diacyglycerol lipases (DAGL alpha and DAGL Beta) that synthesis 2-AG, one of the two well characterised eCBs (2) To generate the first knockout animals for each enzyme and provided conclusive evidence that these enzymes are responsible for the synthesis of 2-AG, and that DAGL beta function is required the best studied eCB function in the adult brain, namely retrograde synaptic signalling. (3) To provide both pharmacological, and more recently genetic, evidence that DAGL alpha and DAGL beta play a substantial role in the generation of new neurons in both the hippocampus and olfactory bulb in adult animals.
One fascinating aspect of neurogenesis is the dramatic decline that is seen during aging. We have shown that this can be circumvented by (a) preventing the degradation of eCBs and/or (b) direct activation of CB2 receptors. This suggests that the machinery to make eCBs and respond to them is intact, and leads us to hypothesis that the reduced neurogenesis seen in older animals most probably stems from a loss of “drive” upstream from the DAGLs. To be able to fully test this hypothesis we need to understand the factors that normally activate DAGLs in NSCs, as well as the actual mechanism that results in enzyme activation. The study of the structure and function of the DAGLs, and the pathways that regulate their activity, will open up new avenues for developing agents that can modulate enzyme activity in either a positive or negative manner – and perhaps lead to novel therapeutic tools.
A negative feature and unwelcome prospect of aging is the increased incidence of symptoms of sensory dysfunction that occur in old people e.g. chronic pain syndromes. Sensory dysfunction can be associated with nerve damage or tissue damage. Sensory neurones provide a pathway for information transfer between a site of dysfunction and the central nervous system where conscious perception occurs. So the sensory neurones are a physiological bottleneck for this information transfer. That makes them an attractive target for drugs and other therapies that aim to control sensation e.g. analgesic drugs for the control of nociception. Understanding how sensory neurones work is a necessary and fundamental step in defining and evaluating mechanisms that are suitable targets for therapeutic intervention.
Ion channels in sensory neurones
Research over the last 10 - 20 years has shown us that many of the specialized functions of sensory neurones, including nociception, are derived from tissue selective expression of proteins. Some of these proteins are ion channels. We are interested in ion channel expression and function in sensory neurones in general but focus on two ion channels in particular. These are NaV1.8 and TRPV1. NaV1.8 is responsible for most of the tetrodotoxin-resistant voltage-gated sodium current in sensory neurones. A few years ago we demonstrated the importance of this ion channel in the mechanism by which prostaglandins 'sensitize' sensory neurones making them more excitable - probably the cellular basis for a significant component of inflammatory hyperalgesia. Factors that control the activity of NaV1.8 and other voltage-gated sodium channels in sensory neurones are of interest. In particular, plasticity of the expression and pharmacology of the channels associated with disease states is of interest. Our attention (Clare Farmer) is currently focused on peripheral demyelinating disease in collaboration with Professor Ken Smith's laboratory in the Wolfson CARD.TRPV1, formerly known as the capsaicin receptor, is an ion channel that is found in the plasma membrane of nociceptive sensory neurones. Chilli peppers contain capsaicin which binds to and opens the channel, exciting the neurones and giving rise to the perception of hot, burning pain. A few years ago we put forward the idea, now widely substantiated, that capsaicin receptors are dephosphorylated by the calcium-dependent enzyme calcineurin and this is associated with desensitization of the channel. As well as contributing to our understanding of sensory mechanisms this idea has special importance because it gives us a basis for explaining the paradoxical phenomenon of capsaicin-induced analgesia whereby capsaicin - a pain provoking chemical - can produce profound and broad spectrum analgesic effects. We have proposed that the mechanism of capsaicin induced analgesia is a calcineurin-mediated cross-desensitization that occurs between TRPV1 and other receptors and ion channels in sensory neurones. We are currently investigating this idea in collaboration with Dr Derek Knight.
Non-neuronal nociceptors?
Sensory neuronal somata are ensheathed by a layer of satellite cells. If nociceptive sensory neurones are 'princes' amongst drug targets for novel analgesics then the satellite cells may be their cinderellas. It has recently been shown that the satellite cells interact with sensory neurones and participate in complex responses to tissue injury
Neurones release NO gas that provokes changes in the behaviour of satellite cells but the precise role of the cells and the mechanisms by which they communicate with their host neurones are not yet known and are just beginning to be studied. We have shown that the cells play a critical role in provoking the response of sensory neurones to bradykinin - an important inflammatory mediator - so the satellite cells may act as nociceptors in their own right. Further, bradykinin provides us with a chemical probe that we can use to investigate the satellite-cell neurone signalling mechanism. Satellite cells may prove to be critical for neurone-neurone cross talk in the sensory ganglia with all that this implies including specification of the size of sensory receptive fields and even modality switching.
Sensory function and dysfunction in relation to ageing or disease have been our major focus for several years but recently the opportunity to collaborate with Dr Stephen Minger in the Wolfson CARD has inspired a project (Siobhan Connor) to study the regulation of ion channels in neuronal stem cells. Using differentiated neural stem cells produced in Dr Minger's lab we can demonstrate the expression of several species of voltage-gated and ligand-gated ion channels and measure synaptic activity in stem cell cultures. We are currently investigating the mechanism of cell-cell interactions that specify the electrical phenotype of the stem cells and induce synaptogenesis.
