Book of abstracts - British Neuroscience Association

6.03
Transcription factor expression analysis in adult human brain
stem/progenitor cells
Baer K*1, Curtis M A *2, Eriksson P S 2, Stapley H 1, Faull R L3, Mark
I. Rees1
* equal contribution; 1Molecular Neuroscience, School of Medicine,
Swansea University, Swansea SA2 8PP, UK; 2Centre for Brain Repair
and Rehabilitation, Göteborg University, Göteborg, Sweden;
3Department of Anatomy with Radiology, The University of Auckland,
Auckland, New Zealand.,
Transcription factors (TF) are responsible for the specification and fate
determination of cells as they develop from progenitor cells into
specific types of cells in the brain. Sox-2 and Pax-6 are TFs with key
functional roles in the developing brain, although less is known about
TFs in the rudimentary germinal zones in the adult human brain.
Endogenous stem/progenitor cells have been demonstrated in two
neurogenic regions in the adult human brain, the subependymal layer
(SEL) of the basal ganglia and the subgranular layer (SGZ) of the
hippocampus (Curtis et al., PNAS 2003; Eriksson et al., Nat Med
1998). Before potential therapeutic applications, we require a thorough
understanding of TF localization and the processes involved in
directing stem cells toward desired neuronal phenotypes.
Our hypothesis is that specific TF are responsible for the fate
decisions of stem/progenitor cells in the neurogenic regions of the
adult human brain. We aim to identify the key TF that are present in
the neurogenic regions of the adult human brain. In this study we have
investigated the distribution and characterization of Sox-2 and Pax-6
in the human subventricular zone (SVZ). Sox-2 immunoreactivity
showed a nuclear labelling pattern and colocalised on GFAP
immunoreactive cells, whereas Pax-6 immunoreactivity was
detectable in the nucleus and the cytoplasm of SVZ cells and
colocalised with PSA-NCAM positive progenitor cells. Thus, our data
surprisingly reveal that these TFs are differentially expressed in the
adult human SVZ where Sox-2 and Pax-6 specify a glial and neuronal
fate, respectively.
6.04
mGluR5 is involved in dendrite differentiation and excitatory synaptic
transmission in NTERA2 human embryonic carcinoma cell derived
neurons
Park H, Andrews P W, Molnar E, Cho K
1. Biomedical Science, University of Sheffield, Sheffield, 2. MRC Centre for
Synaptic Plasticity, University of Bristol, 3. Henry Wellcome Laboratories for
Integrative Neuroscience and Endocrinology, MRC Centre for Synaptic
Plasticity, University of Bristol, Bristol BS1 3NY, UK
The pluripotent human embryonic carcinoma cell line NTERA2 readily
differentiates into neurons when exposed to retinoic acid in vitro. These
neurons show characteristic morphology with long processes and they
express neuronal markers TUJ-1 and NeuN. NTERA2-derived neurons can
regulate Ca2+ signalling through ionotropic glutamate (iGluR) and
muscarinic receptors (mAChRs). Little is known, however, about the role of
metabotropic glutamate receptors (mGluRs) in these neurons. Here we
show that NTERA2-derived neurons express functional mGluR5, which is
involved in Ca2+ signalling. Blocking mGluR5 activity at early stages of
differentiation leads to fewer dendrites and a reduction in miniature
excitatory postsynaptic currents (mEPSCs). Furthermore, cells cultured in
the presence of the mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine
(MPEP) show reduced N-methyl-D-aspartate (NMDA) receptor-mediated
Ca2+ mobilisation but increased alpha-amino-3-hydroxy-5-methyl-4-
isoxazolepropionic acid (AMPA) receptor Ca2+ permeability. During normal
neuronal development the edited GluR2 renders AMPARs Ca2+
impermeable. The increased Ca2+ permeability of AMPARs in MPEPtreated
neurons is due to the reduced expression of GluR2 subunit protein.
Thus, mGluR5 activity at early stages of differentiation is likely to play a role
in the development of multipotent cell-derived neurons.
6.05
The effect of pro-inflammatory cytokines, IL-1 beta and TNF alpha
on embryonic rat hippocampal neuronal precursor cells
Keohane A, Sullivan A, Nolan Y
Anatomy Department, Biosciences Institute, University College, Cork,
EIRE.
6.06
Dopaminergic differentiation of human embryonal carcinoma cells
Bramwell T W 1, Lakics V 2, Przyborski S A 1
1School of Biological and Biomedical Sciences, University of Durham,
South Road, Durham, DH1 3LE; 2Eli Lilly & Co. Ltd. ,Erl Wood Manor,
Windlesham Surrey, GU20 6PH
Hippocampal neurogenesis occurs in the developing and adult brain
due to the presence of multipotent stem cells. These cells can be
cultured in vitro as spherical aggregates called “neurospheres” and
when given appropriate signals, can differentiate into neurons,
astrocytes and oligodendrocytes. Hippocampal neurogenesis is
impaired in Alzheimer’s disease. In post-mortem Alzheimer’s brains,
large numbers of activated microglia have a deleterious effect on
neurons through the action of the pro-inflammatory cytokines,
interleukin 1β (IL-1β) and tumour necrosis factor α (TNFα).
The aim of this study was to assess the effects of these two cytokines
on neuronal and astroglial differentiation in cultures of embryonic
hippocampal neurospheres. Hippocampus was isolated from
embryonic day (E)18 rat and cells were allowed to proliferate for 7
days in vitro (DIV) in the presence of appropriate growth factors. Cells
from the neurospheres were then differentiated for 7DIV in the
presence of either IL-1β or TNFα (10ng/ml - 100ng/ml) without growth
factors and stained immunocytochemically for βIII-tubulin (post-mitotic
neurons), doublecortin (newly-born neurons) and glial fibrillary acidic
protein (astrocytes).
Immunolabelling experiments demonstrated that the percentages of
post-mitotic and newly-born neurons were reduced significantly in the
presence of IL-1β or TNFα (p< 0.05; ANOVA, n=3). Conversely, the
percentage composition of astrocytes increased significantly after
incubation with IL-1β or TNFα (p< 0.01; ANOVA, n=3).
This study demonstrates that IL-1β and TNFα have a detrimental effect
on neuronal development by inhibiting the differentiation of precursor
cells to a neuronal phenotype, and by promoting their differentiation to
an astroglial phenotype.
Parkinson’s disease (PD) principally affects a discrete population of
mesencephalic dopamine producing neurons in the substantia nigra pars
compacta, representing an ideal candidate for potential cell replacement
therapies. Consequently, the development of methods to generate pure
populations of dopaminergic cells from various sources in vitro is of great
interest.
Human pluripotent embryonal carcinoma (EC) stem cells derived from
teratocarcinomas offer a robust model system to study neural differentiation.
Using the human EC cell line TERA2.cl.SP12 work is being undertaken to
elucidate the molecular mechanisms governing the production of
dopaminergic neurons. Currently our studies are focused on the effects of
the use of physiological oxygen culture conditions, previously shown to
increase the proportion of dopaminergic neurons derived from midbrain
precursor cells (Studer et al, J. Neurosci. 20, 2000) in combination with
retinoic acid-induced differentiation. To that end, real-time reverse
transcription PCR, flow cytometry and western blotting are being employed
to assess the expression of the key neuronal (e.g. Tuj1) and more
specifically, dopaminergic markers such as tyrosine-hydroxylase, Nurr1,
dopamine receptor 1 and 2 and dopamine transporter. Our preliminary gene
expression data show that these markers are present in differentiated EC
cells. We propose to further investigate the molecular mechanisms
controlling the development of dopaminergic neurons under a range of
alternative growth conditions, and assess markers of neuronal
differentiation, as outlined above. These studies will hopefully allow for
production of purer populations of this particular neuronal phenotype with a
range of potential applications in basic research and the development of
pharmaceuticals.
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