Book of abstracts - British Neuroscience Association

9.04
Inducing neural differentiation of embryonal carcinoma stem
cells using natural and synthetic retinoids: screening,
characterisation and mode of action
Bridgens C E, Barnard J H, Collings J C, Cartmell E J, Goss H M,
Whiting A, Marder T B, Przyborski S A
School of Biological and Biomedical Sciences; Department of
Chemistry, Durham University, South Road, Durham, DH1 3LE;
ReInnervate Limited, Old Shire Hall, Durham, DH1 3HP.
Retinoids are a group of natural and synthetic molecules that are
structurally and/or functionally analogous to all-trans-retinoic acid
(ATRA), the major active metabolite of vitamin A. ATRA regulates a
broad range of essential processes during mammalian embryogenesis
and adult homeostasis, including vision and cellular differentiation,
proliferation and apoptosis. Consequently, retinoids have the potential
to be used in numerous clinical indications including treatment of
neurological tumours.
We have commenced a study directed at the synthesis of stable
alternatives to ATRA and investigation of the molecular pathways that
regulate cell development in response to natural and synthetic
retinoids. A small library of synthetic retinoids have been designed and
prepared, some of which are efficacious at inducing the differentiation
of human embryonal carcinoma (EC) stem cells into both neural and
non-neural cell types. The most potent compounds can up-regulate
antigens associated with neural phenotypes at comparable rates and
in similar patterns to that which is observed in response to ATRA.
These compounds are more stable than ATRA, which is susceptible to
photo- and thermal-isomerisation under laboratory conditions, and
thus provide a more convenient reagent to modulate reproducible
differentiation in cultured stem cells.
9.05
Investigating the role played by microtubule-associated protein 1–LC2
(MAP1-LC2) in AMPA receptor trafficking and targeting.
Hann V, Ives J H, Fung S, Payne H L, Thompson C L
School of Biological and Biomedical Sciences, Durham University, South
Road, Durham, DH1 3LE.
Stargazin is a member of the Transmembrane AMPA Receptor Regulatory
Protein (TARP) family. Stargazin has been shown to be involved in both cell
surface trafficking (extrasynaptic) and synaptic targeting of AMPA receptors.
The last six amino acids of stargazin, –RRTTPV interacts with PDZ domains
of the membrane-associated guanylate kinase family of proteins, in
particular PSD-95. This association is pivotal to stargazin’s ability to target
AMPA receptors to the synapse, disruption of this interaction by deletion of
the TTPV sequence eliminates synaptic targeting. However, cell surface
trafficking (extrasynaptic) of AMPA receptors is not affected thus this aspect
of stargazin-mediated processing is independent of interactions through the
TTPV domain and must therefore be governed by sequences up-stream of
the extreme C-terminus.
Using the entire intracellular C-terminus of stargazin as ‘bait’ in a yeast-2
hybrid screen of a mouse brain cDNA library we identified MAP1-LC2 as a
‘RRTTPV’ sequence-independent interactor. Anti-stargazin antibody
immunoaffinity purifications of Triton X-100 soluble cerebellar membranes
followed by MAP1A-LC2 and GluR2 pull-downs confirmed that these
proteins exist in a tripartite complex in vivo.
By yeast-mating assays using stargazin and MAP1A-LC2 deletion clones
we are narrowing down the interaction domains in order to use dominantnegative
approaches to interfere with this interaction in neurones to
evaluate its functional significance.
Funded by the BBSRC.
9.06
Evaluation of RNA preservation in the human post-mortem brain
tissue and its suitability for the application of molecular biological
techniques
Fernando S, Dexter D, Reynolds R
BrainNet Europe Consortium, Dept of Cellular & Molecular
Neuroscience, Imperial College Faculty of Medicine, Charing Cross
Hospital, London
Brain Net Europe is an FP6 Network of Excellence incorporating 19
established brain banks in Europe, whose main objective is to collect
and distribute well-characterised high quality post-mortem brain tissue
for basic research in neuroscience.
In this collaborative study we have evaluated the quality of RNA in
archived tissue samples of 155 human post-mortem brains obtained
from 8 different brain banks within the network. Brain tissue from
several neurological disorders and controls was included in the study
with post-mortem delays of up to 100 hrs and freezer intervals up to 7
years. The quality of isolated total RNA was analysed against age,
gender, post-mortem delay, freezer interval and CSF pH to establish
the effects of post-mortem variables. We have also examined regional
variation in RNA quality using tissue obtained from anatomically
defined areas of the brain. Additionally some of the samples were
analysed on different microarray platforms and a selected set of genes
studied by quantitative RT-PCR.
All samples yielded good quantities of RNA with varying quality and
>70% of samples were found to be of suitable quality for molecular
biological purposes. None of the above mentioned factors analysed
seemed to have a significant effect on the quality of RNA. We conclude
that intact RNA for molecular biology can be obtained from human
post-mortem brain tissue, even with long post-mortem delays and
freezer intervals. However, the successful expression of certain genes
from post-mortem brain tissue may require enhanced procurement
efforts to maximize RNA integrity.
9.07
Disrupted-In-Schizophrenia 1 (DISC1) and protein kinase A signalling
Nick Bradshaw, Shaun Mackie, Christie S, Porteous D, Millar K
Molecular Genetics, University of Edinburgh, Molecular Medicine Centre,
Western General Hospital, Crewe Road South, Edinburgh, EH4 2XU
Disrupted-In-Schizophrenia 1 (DISC1) is a widely accepted risk factor for
schizophrenia and related psychiatric disorders. We have previously
demonstrated that DISC1 interacts with phosphodiesterase 4B (PDE4B),
an independently identified risk factor for psychiatric illness. Type 4
phosphodiesterases are of interest because they are homologous to the
Drosophila learning and memory mutant Dunce, consistent with the
cognitive deficits that characterise schizophrenia. Moreover PDE4s are
specifically inhibited by the prototypic antidepressant rolipram, and PDE4-
deficient mice behave as if on antidepressants. PDE4B hydrolyses cAMP, a
key signalling molecule in the Protein Kinase A (PKA) pathway. PDE4B
cAMP hydrolysing activity is regulated by PKA phosphorylation, forming a
negative feedback loop. Intriguingly, PDE4B binding to DISC1 is dynamic
and cAMP-dependent, suggesting that DISC1 sequesters PDE4B in a low
activity state until cAMP hydrolysing activity is required to switch off cAMP
signalling, at which time PDE4B is released. We now demonstrate that
DISC1 is also phosphorylated by PKA in vivo, suggesting that DISC1
function may be modulated indirectly by PDE4B. This interaction and
potential for regulation of two independently identified genetic risk factors
for psychiatric illness implies that they are key to psychosis-related
molecular pathways.
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