Book of abstracts - British Neuroscience Association

3.03
TNF-related protein LIGHT reduces BDNF-dependant neuronal
growth in sensory neurons
Nuria Gavalda, Alun M Davies
Cardiff School of Biosciences,, Biomedical Sciences Building 3,,
Museum Avenue,, Cardiff CF10 3US,, Wales, UK
LIGHT is a protein belonging to the TNF superfamily that was originally
identified as a weak inducer of apoptosis. LIGHT has been shown to
be involved in the costimulation and homeostasis of T-cells, however,
no data exist that describe a function for this ligand during the
development of the nervous system. Our present work reveals a novel
role for LIGHT in the regulation of neurite growth during the
development of mouse sensory neurons. LIGHT is capable of binding
to both the lymphotoxin-beta (LTβR) and HVEM receptors, both of
which were expressed by nodose neurons. Nodose neurons that were
transfected with a plasmid encoding LIGHT had significantly reduced
neurite growth, during a specific period of embryonic through to
postnatal development.
The activation of several signalling pathways have been shown to
mediate the effects of LIGHT in T-cells. These include NF-κB, JNK and
ERK/MAPK. In our cellular model, only ERK/MAPK is activated by
LIGHT, but not NF-κB nor JNK. Specifically blocking the LIGHT
induced ERK activation with the selective inhibitors, U0126 and
PD98059, inhibits the ability of LIGHT to affect neurite growth in
nodose neurons. In contast to this, inhibiting the NF-κB pathway using
specific NF-κB mutant proteins or the JNK pathway, using specific JNK
inhibitor, had no effect on the ability of LIGHT to affect neurite growth
in nodose neurons. All together, these results show a novel role of
LIGHT in sensory neuron development.
3.04
Competitive, intrinsic and stochastic determinants of neuromuscular
synapse elimination in transgenic YFP expressing mice
Teriakidis A, Jenkins N, Willshaw D J, Ribchester R R
Centres for Neuroscience Research and Neuroinformatics DTC, University
of Edinburgh
Competition at motor endplates strongly influences elimination of
polyneuronal innervation in neonatal muscles but it is still not known
whether intrinsic properties of motor neurones also determine ultimate
motor unit size. We are using transgenic mice expressing fluorescent
protein in motor neurones to measure and model the changes in motor unit
size as neuromuscular synapses form or become eliminated. First we
designed experiments to test whether motor unit size in adult lumbrical
muscles becomes reduced even when all other competing motor units are
removed, by partial denervation at birth (neonates anaesthetised by
chilling). We found that motor unit size was not reduced when these mice
reached adulthood, suggesting that competition is sufficient to account for
synapse elimination during development. Next we examined whether the
initial motor innervation pattern is random. Analysis of YFP-expressing
motor units in lumbrical muscles of thy1.2-YFPH mice indicated that all
connections made by a motor neurone are restricted to a single lumbrical
muscle with no divergence between muscles. Stochastic modeling
suggested that a key indicator of randomness is initial converge of
branches of the same axon on single motor endplates. To test this we
examined adult muscles in the earliest stages of reinnervation (12-14 days)
after nerve crush under halothane/N20 anaesthesia. We found several
compelling instances of within-unit convergence on motor endplates. Thus,
stochastic properties play an important role in establishing the divergent
and convergent innervation pattern within muscles but competitive
interactions at polyinnervated junctions are more important than intrinsic
properties of neurones during synapse elimination.
3.05
Investigation into the differentiation of motor neurons derived
from human pluripotent stem cells and assessment of their
function in vitro
Pan C, Przyborski S
School of Biological and Biomedical Science, Durham University,
South Road, Durham DH1 3LE
It is understood that retinoic acid (RA), sonic hedgehog (Shh) and
bone morphogenic proteins (BMPs) play an important role in cell fate
determination and the specification of inter-neurons and motor
neurons along the dorsal-ventral axis in the neural tube. In this study,
we propose to evaluate the function of these signalling molecules to
instruct the differentiation of human pluripotent stem cells.
TERA2.cl.SP12 embryonal carcinoma (EC) cells are a robust
caricature of human embryogenesis and an accepted model of neural
differentiation. Gene and protein expression analyses indicate that
human EC cells respond to RA, BMPs and Shh in a conserved
manner and regulate neural transcription factors and structural
proteins in a predicted way as cells commit toward the motor neuron
phenotype. To assess the function of these differentiated neurons, we
tested their ability to innervate skeletal muscle myotubes and induce
contraction. We showed that muscle contraction could be manipulated
pharmacologically: curare and atropine blocked myotube contraction,
whereas acetylcholine and carbachol increased the number of
contractile events. In other experiments, we have also shown that cells
exposed to RA and Shh in conjunction with other growth factors over
different time periods, preferentially form oligodendrocytes and/or
interneurons. These results indicate it is feasible to control and direct
the differentiation of human stem cells and produce specific neuron
subtypes in vitro as a model to investigate the molecular mechanisms
and signalling pathways that control these processes in man.
3.06
Clonally derived adult stem cell-derived soluble factors and their
instructive effects on neural stem and progenitor cells
Emmerson R, Croft A, Przyborski S A
School of Biological and Biomedical Sciences, University of Durham, South
Road, Durham, DH1 3LE; ReInnervate Ltd, Durham, DH1 3HP
Transplantation of both mesenchymal stem cells (MSCs) and dermal cells
has been shown to result in functional improvement in animal models of
neurological disease, however, the mechanism by which this occurs
remains unclear. We have shown that MSCs produce factors that instruct
neural stem cells (NSCs) to adopt neuronal and glial lineages, and this is
dependent on the developmental status of the MSC. MSCs induced to form
cellular aggregates, express neural antigens such as nestin and GFAP, and
in co-culture studies instruct NSCs to adopt a predominantly neuronal fate.
We have clonally derived MSC cell lines that express neural markers such
as Tuj-1 and GFAP without induction of cell aggregates. Therefore clonal
MSCs expressing neural antigens could have a similar instructive effect on
NSCs as their parent MSC population. Induction of neural antigen
expression such as nestin and Tuj-1 has also been demonstrated in dermal
populations of stem cells. The dermal papilla (DP) region is thought to be a
niche of dermal cells able to express neural proteins. Clonal cell lines
derived from the DP and the dermal sheath (DS) form aggregates that are
similar to both those derived from dermal stem cells and induced MSCs.
We are investigating whether dermal aggregates could have an instructive
effect on NSCs that is similar to MSCs. The study of clonally derived hair
follicle and MSC populations will enable a better understanding of the
effects of soluble factors produced by adult stem cell populations on
neurogenesis.
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