Projects on offer
1. The effect of Amyloid beta (Aβ) in
neurotransmitter release
Dr Pavlos Alifragis, School of Biological Sciences,
Royal Holloway University London, Egham,
Surrey, TW20 0EX. Pavlos.Alifragis@rhul.ac.uk
The aim of this project is to investigate the effects
of Aβ on neuronal and synaptic physiology.
Alzheimer’s disease is strongly associated with Aβ
deposits in the brain, and the soluble oligomeric
forms of Aβ which correlate with cognitive decline.
In certain brain regions, specifically the
hippocampus, Aβ potently disrupts synaptic
plasticity, by inhibiting the induction of LTP, and
altering synaptic morphology. The mechanisms
underlying this synaptic dysfunction remain largely
unclear. Furthermore, the site of Aβ action
remains elusive, with reports of toxic Aβ both
internally and extracellularly, and evidence
suggesting Aβ targets both the pre- and postsynapse. We have exciting data suggesting Aβ is
interacting with presynaptic proteins such as
Synaptophysin. Moreover we have shown that this
interaction is necessary and sufficient to disrupt
the interaction between Synaptophysin and
VAMP2. In addition to the formation of intact
synaptic vesicles, disrupting the interaction
between VAMP2 and synaptophysin at the
synapse affects the regulation of SNARE complex
formation, as synaptophysin bound to VAMP2,
prevents VAMP2 entering the SNARE complex
allowing SV fusion and exocytosis. Our data show
increased entry of VAMP2 into the SNARE
complex, as a result of Aβ induced disruptions to
the VAMP2-synaptophysin complex.
The student will employ a range of techniques
from culturing primary hippocampal neurons, to
confocal microscopy, immuno labelling, treatment
with Aβ etc. in order to investigate additional
effects induced by Aβ at the presynaptic contact.
The initial focus would be on Synapsin 1, a
presynaptic protein involved in regulating the
number of synaptic vesicles available for release
via exocytosis. The project will investigate whether
treatment of neurons with Aβ results in
dissociation of Synapsin clusters at the
presynaptic contact.
References:
1) Hall, A.C., et al., Mol Cell Neurosci, 2002.
20(2): p. 257-70.
2. Characterisation of Smart Contrast
Reagents for early Diagnosis of Alzheimer's
Disease by MRI
Prof Brian Austen, St. George’s University of
London, Cranmer Terrace, London SW17 0RE.
sghk200@sgul.ac.uk
Early diagnosis of Alzheimer's at the mild cognitive
impairment stage is essential if effective treatment
and prevention is to be implemented. We have
chemically synthesised several contrast agents,
peptides that bind aggregated beta-amyloid, and
contain ligated gadolinium(which enhances the T1
and suppresses the T2 modes of an MRI scan).
The project will involve screening further reagents
for binding to beta-amyloid dimers and fibrils using
ELISA assays, and for potential toxicity to
neuronal cells in culture, using biochemical tests.
An understanding of methods presently used for
imaging neurodegeneration will be obtained, along
with knowledge of the main pathological
mechanisms of Alzheimer's Disease. In
collaboration with others in the lab, the student will
examine the MRI changes resulting from the
contrast agents injected into transgenic mice
models of Alzheimer’s Disease.
References:
1) Journal of Peptide Science 14 (8) 44
2) Brian M. Austen,‡ Katerina E. Paleologou,§
Sumaya A. E. Ali,| Mohamed M. Qureshi,| David
Allsop,§ and Omar M. A. El-Agnaf, (2008)
Designing Peptide Inhibitors for Oligomerization
and Toxicity of Alzheimer’s /beta/-Amyloid
Peptide. Biochemistry, 47(7):1984-92
3. Investigating the effects of the anticonvulsant, valproic acid on NMDA receptor
function and pharmacology
Dr Philip Chen, School of Biological Sciences,
Royal Holloway University London, Egham,
Surrey, TW20 0EX. philip.chen@rhul.ac.uk
Fast neuronal communication at synaptic contacts
relies on neurotransmitter release from the
presynaptic terminal and the majority of excitatory
neurotransmission within the mammalian central
nervous system is mediated by the
neurotransmitter glutamate. Glutamate acts on a
number of ligand gated ion channels often referred
to as 'ionotropic' receptors which when opened
permit ion flux across the cell membrane and
result in a change in neuronal excitability. One
subtype of ionotropic glutamate receptor is the Nmethyl-D-aspartate receptor (NMDAR) and these
receptors play a number of roles in normal and
abnormal processes within the central nervous
system (CNS), ranging from learning and memory
to excitotoxic neuronal cell death following stroke
or brain trauma. The involvement of NMDARs in
such a broad range of CNS processes and
disorders have triggered a great deal of interest in
understanding the molecular determinants of
NMDAR function. NMDARs are tetrameric protein
complexes made up of different types of subunit
(two NR1s and two NR2s, of which there are four
subtypes; A, B, C and D) and the incorporation of
different subunits within the NMDAR complex
underlies the distinct functional properties seen
amongst NMDAR subtypes. The following project
will be offered in my laboratory.
Valproic acid (VPA) is the most common therapy
for epilepsy, however its influence on excitatory
neurotransmitter function is unclear. The project
will examine if valproic acid influences NMDA
receptor (NMDAR) function and pharmacology in
recombinant expression systems. We will examine
the pharmacology of valproic acid by recording
glutamate evoked inward currents from Xenopus
oocytes injected with different NMDAR subtypes
under two-electrode voltage clamp configuration.
Work on this project may also be extended to
examine the effects of valproic acid on native
NMDAR function using extracellular electrode
recordings from hippocampal slices and the effect
of VPA on NMDAR triggered neuronal cell death
in primary neuronal cultures.
Methods used: Two-electrode voltage clamp
electrophysiology, expression and functional
characterisation of recombinant receptors in
Xenopus oocytes, in vitro cRNA synthesis,
Xenopus oocyte injection and incubation and
primary neuronal cell culture.
References:
1) Chen PE et al. Structural features of the
glutamate binding site in recombinant NR1/NR2A
N-methyl-D-aspartate receptors determined by
site-directed mutagenesis and molecular
modeling. (2005) Molecular Pharmacology.
67:1470-1484.
2) Chen PE and Wyllie DJA. Pharmacological
insights obtained from structure-function studies of
ionotropic glutamate receptors. (2006) British
Journal of Pharmacology. 147: 839-853.
3) Terbach N and Williams RSB. Structurefunction studies for the panacea, valproic acid.
(2009) Biochemical Society Transactions 37:112632.
4. Generating and expressing an HA-tagged
Kinectin fusion protein
Dr Jennifer Murdoch, School of Biological
Sciences, Royal Holloway University London,
Egham, Surrey, TW20 0EX.
Jenny.murdoch@rhul.ac.uk
Tulp3 has been identified as a novel negative
regulator of the Sonic hedgehog signalling
pathway, but the precise molecular function of
Tulp3 is not known. Several putative interacting
proteins have been identified from a yeast 2hybrid screen. This project will aim to investigate
further the interaction with one of these proteins,
Kinectin. The project will aim to clone the Kinectin
cDNA into an appropriate expression vector, to
generate and express an HA-tagged fusion
protein.
This project will initially involve bacterial
transformation of an existing kinectin plasmid, to
allow preparation of larger quantities of plasmid
DNA. The kinectin cDNA will be subcloned from
this vector into an alternative expression vector,
using a PCR or restriction enzyme-based
subcloning approach. The student will be
expected to play a major role in designing an
appropriate subcloning strategy. Correct
subcloning will be confirmed by sequence
analysis, using the appropriate bioinformatics
tools. The HA-Kinectin construct will be
transfected into mammalian cells (293T or MDCK
cells) and the expression of tagged protein
assessed by Western blotting. If time allows, the
plasmid will be co-transfected with a construct for
V5-tagged Tulp3. Immunoprecipitation with antiHA and anti-V5 antibodies will be performed.
Evidence for Tulp3 and Kinectin coimmunoprecipitation will support the hypothesis
that these proteins interact.
References:
1) Patterson, V.L., Damrau, C., Paudyal, A.,
Reeve, B., Grimes, D.T., Stewart, M.E., Williams,
D.J., Siggers, P., Greenfield, A. and Murdoch, J.N.
(2009) Mouse hitchhiker mutants have spina
bifida, dorso-ventral patterning defects and
polydactyly: Identification of Tulp3 as a novel
negative regulator of the Sonic hedgehog
pathway. Human Molecular Genetics 18:17191739.
5. Molecular and pharmacological analysis of
the psychoactive plant, Khat
Dr. Jamal Nasir, St. George’s University of
London, Cranmer Terrace, London SW17 0RE.
jnasir@sgul.ac.uk
AIMS:


1. Using in vivo pharmacological assays to
identify the active components in Khat.
2. Using genomics technologies to generate
a molecular profile of Khat.
The psychoactive plant khat (Catha edulis), is
widely used as a stimulant in parts of Africa and
the Middle East. Although highly addictive and
potentially dangerous, it remains legal in Britain,
despite being banned across North America and
most of Europe. Here, it has historically been
widely used by the Somali community. However,
its more widespread use has recently caused
great concern and has been widely publicised in
the media. Known to induce euphoria and
improve mood, alertness, sexual potency and self
confidence, it can also induce more severe
symptoms, including sleeplessness and high
blood pressure leading to cardiac arrest. It can
also cause mental health problems involving
paranoia and delusions and other symptoms
resembling schizophrenia. These symptoms are
generally attributed to amphetamine like
compounds, but the biological properties of this
plant are poorly understood at the molecular
level. Further insights into the underlying
biochemical pathways could yield important clues
about the aetiology of schizophrenia. In
collaboration with Professor Bramley (RHUL), Dr.
James Moffatt and Hussein Mohamoud (SGUL),
we intend to isolate extracts of this plant and
undertake pharmacological and mass
spectroscopy studies to identify active
components of Khat.
6. The heparin/heparan sulphate binding
properties of the BMP antagonist protein
gremlin
Dr Chris Rider, School of Biological Sciences,
Royal Holloway University London, Egham,
Surrey, TW20 0EX. c.rider@rhul.ac.uk
The bone morphogenetic proteins have important
roles in the development of the nervous system,
as indeed in many other tissues. They are also
up-regulated in adult CNS in response to injury
and will undoubtedly influence cell fate at such
locations. A full understanding of their activities
will be important in developing new approaches to
the treatment of nervous system injury. BMP
activities are modulated by a number of selectivity
antagonist proteins which block receptor binding.
Some of these antagonists including noggin,
cerberus and chordin were first recognised
because of their pivotal roles in head and brain
development. An increasing number of the BMPs
are now known to bind to heparin and related
polysaccharides found on cell surfaces and the
extracellular matrix. This is also currently the case
for the two of the antagonists, but we do not yet
know how widespread this property is amongst
this class of proteins. Such interactions will have
the effect of restricting the diffusion of these
proteins, giving rise to localised concentration
gradients, which may be crucial in tissue
morphogenesis (3). This project seeks to study
the predicted heparin/heparin sulphate binding
properties of a further BMP antagonist, gremlin.
Using a heparin binding ELISA method (1) we
have clearly established that commercially
available recombinant human gremlin binds
strongly to heparin. However one caveat is that
this particular protein carries a poly-histidine tag,
and since this tag is polybasic it is possible that it
may generate an artefactual binding site for the
highly acidic heparin-like molecules. We therefore
need to confirm that gremlin without this tag still
binds strongly. To this end the student will
contribute to on-going efforts to express and purify
a recombinant gremlin tagged instead with the
peptide myc, using approaches we have
previously employed for other proteins (2). The
project will then progress to characterizing its
heparin/heparan sulphate binding properties and
cell culture studies aimed at elucidating the
physiological importance of gremlin interactions
with these polysaccharides.
References:
1) S.M. Rickard, R. S. Mummery, B. Mulloy and
C.C. Rider (2003) The Binding of Human Glial
Cell-Line Derived Neurotrophic Factor (GDNF) to
Heparin and Heparan Sulphate: Importance of 2O-Sulphate Groups and Effects on its Interaction
with its Receptor GFRa1. Glycobiology 13, 419426.
2) I. Alfano, P. Vora, R.S. Mummery, B. Mulloy
and C.C. Rider (2007) The Major Determinant of
the Heparin Binding of Glial Cell-Line Derived
Neurotrophic Factor is near the Aminoterminus
and is Dispensable for Receptor Binding.
Biochemical Journal 404, 131-140.
3) C. C. Rider and B. Mulloy (2010) The Bone
Morphogenetic Protein and Growth Differentiation
Cytokine Family, and their Protein Antagonists.
Biochemical Journal 429, 1-12.
7. The role of valproic acid in Ca2+ signalling
Dr Katalin Török, St. George’s University of
London, Cranmer Terrace, London SW17 0RE.
ktorok@sgul.ac.uk
Valproic acid is a major drug used in the treatment
of bipolar disorder and it has been established that
it affects the metabolism of the Ca2+ releasing
signalling molecule, IP3 (1). The downstream
effects of how it further affects cell function, e.g.
neuronal output, are not fully understood. The
Ca2+ signalling pathway is instrumental in
learning and responses of the limbic system to
stimulation (2). The aim of this project is to
determine how valproic acid, via IP3 modulates
neuronal Ca2+ signalling.
Plan of investigation:
A. Ca2+ imaging: Primary neuronal cultures will
be prepared from rat brain and its regions, e.g.
hippocampus and the Ca2+ signalling pathway will
be stimulated by glutamate and glutamate
receptor agonists. Intracellular Ca2+ transients will
be monitored by the fluorescent Ca2+ indicator
fluo-4 and imaged by confocal microscopy.
Valproic acid will be applied and the resulting
spatio-temporal patterns of Ca2+ will be compared
to the responses of untreated neurons.
B. Imaging of the response of the Ca2+-activated
regulatory protein calmodulin (CaM and the
‘memory molecule’ Ca2+.calmodulin-dependent
protein kinase II (CaMKII). Neuronal cultures will
be transfected with fluorescently tagged CaM and
CaMKII and their functional mutants and the
intracellular translocation of CaMKII will be
studied.
Outcome:
A. The mutants of Ca2+ downstream signalling
molecules CaM and CaMKII will provide novel
insight into neuronal Ca2+ signal transduction
mechanisms
B. A novel understanding of how valproic acid
affects Ca2+ and downstream Ca2+ signalling
providing new insights into its mechanisms of
action. This will be useful in considering novel
drug targets for bipolar disorder.
References:
1) R.S.B. Williams, L. Cheng, A. Mudge and A.J.
Harwood (2002) A common mechanism of action
for three mood-stabilizing drugs. Nature 417, 2925
2) P.A.A. Grant, S.L. Best, N. Sanmugalingam, R.
Alessio, A.M. Jama, and K. Török A two-state
model for Ca2+/CaM-dependent protein kinase II
(CaMKII) in response to persistent Ca2+
stimulation in hippocampal neurons Cell Calcium
(2008) 44, 465—478
8. Synapse to nucleus Ca2+ signalling and the
effects of valproic acid
Dr Katalin Török, St. George’s University of
London, Cranmer Terrace, London SW17 0RE.
ktorok@sgul.ac.uk
The metabolism of the Ca2+ releasing signalling
molecule, IP3 is affected in bipolar disorder and its
treatment by valproic acid (1). The longer term
manifestation of bipolar disorder and the effects of
valproic acid depend on synapse to nucleus
signalling resulting in changes in gene expression.
Our hypothesis is that Ca2+ signals are
transduced to the nucleus by the nuclear
translocation of the Ca2+ binding regulatory
protein, calmodulin (CaM). It has been established
that CaM nuclear transport is facilitated and our
hypothesis is that it is aided by Ca2+/CaMdependent protein kinases I (CaMKI) and
(CaMKIV) (2,3). The aim of this project is to test
this hypothesis and to determine how valproic acid
modulates neuronal Ca2+ signalling from the
synapse to the nucleus.
Plan of investigation:
A. CaM nuclear translocation. Primary neuronal
cultures will be prepared from rat brain and its
regions, e.g. hippocampus and the Ca2+
signalling pathway will be stimulated by glutamate
and glutamate receptor agonists. CaM nuclear
translocation monitored by already developed
fluorescently tagged CaM and its functional
muatants and imaged by confocal microscopy.
Valproic acid will be applied and CaM nuclear
translocation will be compared to the responses of
untreated neurons.
B. Imaging of the response and structure-function
relationship of CaMKI and CaMKIV in CaM
nuclear translocation and the effects of valproic
acid. Neuronal cultures will be transfected with
fluorescently tagged CaM and CaMKI and
CaMKIV mutants to determine the functionally
important portions of CaM and CaMKs in nuclear
translocation. The effects of valproic acid will be
tested.
Outcome:
A. Ca2+ signalling mechanism from the synapse
to the nucleus by CaM and CaMKs will be better
understood
B. A novel understanding of how valproic acid
affects Ca2 signalling to the nucleus will be
obtained. This will be useful in considering novel
drug targets for bipolar disorder.
References:
1) R.S.B. Williams, L. Cheng, A. Mudge and A.J.
Harwood (2002) A common mechanism of action
for three mood-sabilizing drugs. Nature 417, 292-5
2) K. Török PERSPECTIVE: The regulation of
nuclear membrane permeability by Ca2+
signaling: A tightly regulated pore or a floodgate?
Science STKE 386 (2007) pe24.
3) R. Thorogate and K. Török Ca2+-dependent
and independent mechanisms of calmodulin
nuclear translocation. J. Cell Science 117 (2004)
5923-5936.
9. Molecular analysis of the role of the
centrosome in neurogenesis
Dr Chris Wilkinson, School of Biological Sciences,
Royal Holloway University London, Egham,
Surrey, TW20 0EX.
Christopher.wilkinson@rhul.ac.uk
Genetic defects in centrosome proteins are linked
to a number of developmental diseases in which
the brain is affected. The disease microcephaly, in
which the size of the brain is considerably reduced
but its normal architecture retained, is caused in
several cases by mutations in genes that encode
centrosome proteins. This is the organelle that
organises the microtubule network and has
important roles in forming the mitotic spindle. The
mechanism by which these defects in this tiny
organelle have such a dramatic effect on brain
development is still not known. This project will
seek to investigate the role of the centrosome in
early embryonic neurogenesis by depleting these
proteins and observing the effect on brain
development. Early zebrafish embryos will be
used as a model as their rapid development and
transparency make them accessible to molecular
and microscopic analyses, including time-lapse
confocal microscopy of living embryos. This
project will seek to model microcephaly by making
zebrafish embryos with small brains. The
challenge will then be to work out the molecular
mechanisms underpinning the gross phenotype.
Techniques used: immunofluorescence
microscopy; confocal microscopy; RT-PCR;
Western blotting; molecular cloning; Zebrafish
embryo microinjection.
References:
1) Bond J, Woods CG. Cytoskeletal genes
regulating brain size. Curr Opin Cell Biol. (2006)
18(1):95-101.
2) Bond J, Roberts E, Springell K, Lizarraga SB,
Scott S, Higgins J, Hampshire DJ, Morrison EE,
Leal GF, Silva EO, Costa SM, Baralle D, Raponi
M, Karbani G, Rashid Y, Jafri H, Bennett C, Corry
P, Walsh CA, Woods CG. A centrosomal
mechanism involving CDK5RAP2 and CENPJ
controls brain size. Nat Genet. (2005) 37(4):353-5
3) Lucas EP, Raff JW. Maintaining the proper
connection between the centrioles and the
pericentriolar matrix requires Drosophila
centrosomin. J Cell Biol. (2007) 178(5):725-32.
4) Graser S, Stierhof YD, Nigg EA. Cep68 and
Cep215 (Cdk5rap2) are required for centrosome
cohesion. J Cell Sci. (2007) 120:4321-31.
10. Making better treatments for bipolar
disorder
Dr Robin Williams, School of Biological Sciences,
Royal Holloway University London, Egham,
Surrey, TW20 0EX. Robin.wiliams@rhul.ac.uk
Bipolar disorder (BD) is a devastating disorder
giving rise to cyclic changes of mood and often
leading to suicide. Understanding the aetiology of
the disorder, which is at least partially caused by
genetic factors, has so far proved impossible. One
of the most well-supported theory describing how
bipolar disorder drugs work is called ‘inositol
depletion’ (1). This mechanism was first proposed
by Berridge et al1 around 20 years ago, and
states that the over-activation of a cell signalling
pathway in bipolar disorder involves inositol, and
that bipolar disorder drugs thus function to deplete
cellular inositol. We have published widely on this
mechanism, based in a simple biomedical model,
Dictyostelium discoideum (2-4), and we
successfully applied these and subsequent results
to primary mammalian neurons.
This project will exploit a cellular response to
inositol depletion, whereby cells increase the
expression of inositol biosynthetic enzymes. The
project will therefore use a promoter for one of
these inositol biosynthetic enzymes to express
green fluorescent protein. We will then test
increased gene expression on exposure to inositol
depleting drugs.
The outcome from this project will be a
mechanism to identify novel compounds with
potential efficacy in bipolar disorder treatment, and
the isolation of potential new treatments based
around the structure of valproic acid.
Methods developed will include cell culture, PCR,
cloning, cell transformation, DNA isolation, colony
screening, fluorescent microscopy, and
pharmacology.
References:
1) Berridge MJ, Downes CP, Hanley MR. Cell.
1989;59:411-419.
2) Eickholt BJ, Towers G, Ryves WJ et al. Mol
Pharmacol. 2005;67:1-8.
3) Shimshoni JA, Dalton EC, Jenkins A et al. Mol
Pharmacol. 2007; 71, 884-892.
4) Williams RSB, Cheng L, Mudge AW, Harwood
AJ. Nature. 2002;417:292-295.