Book of abstracts - British Neuroscience Association

9.08
Regulation of potassium channel genes in response to neuronal
activity
Mucha M, Mordaka P M, Dalle C, Wood I C
Institute of Membrane and Systems Biology, University of Leeds,
Leeds LS2 9JT, UK
Epilepsy is the second most common neurological disease and 1 in 20
individuals will suffer an epileptic event sometime during their lifetime.
Once a seizure has occurred an individual becomes more susceptible
to further seizures. Although it is known that this effect requires
changes in gene expression, the exact nature of those changes is not
understood. It is likely that changes in expression of genes that
regulate neuronal excitability would be important for this response to
seizure activity. To investigate how such genes are regulated we have
isolated the genes, KCNQ2 and KCNQ3, which encode two potassium
channel subunits of the M-channel and function to regulate neuronal
excitability. Mutations in either KCNQ2 or KCNQ3, resulting in
diminished function in humans are responsible for a form of epilepsy
indicating the important role of these subunits. We have used
bioinformatic analysis, DNase I sensitivity, gel retardation assays and
reporter gene assays to identify functional promoter elements within
the human KCNQ2 and KCNQ3 genes. We show that KCNQ2 and
KCNQ3 are regulated by at least some shared transcription factors
that respond to seizure activity and that expression levels of these
genes in cultured neurons are responsive to neuronal activity.
9.09
The phosphorylation status of the p65 NF-kB subunit determines
whether NF-kB promotes or inhibits neurite growth
Gutierrez H, O`Keefe G, Davies A
Humberto Gutierrez PhD , Research Associate., Cardiff School of
Biosciences,, Biomedical Sciences Building 3,, Museum Avenue,, PO Box
911,, Cardiff CF10 3US,, Wales, UK, e-mail:gutierrezh@cf.ac.uk, Tel:
+(44)29 20 87 51 76
NF-kB is a ubiquitously expressed transcription factor that has recently
been shown to promote the growth of neural processes in the developing
peripheral and central nervous systems. The NF-kB dimer is held in an
inactive form in the cytoplasm by a member of the IkB family of inhibitory
proteins, and NF-kB is normally activated by IkB kinase
(IKK)-mediated phosphorylation of IkB on serine residues 32 and 36,
resulting in ubiquitination and proteosome-mediated degradation of IkB and
nuclear translocation of the liberated NF-kB. Overexpressing the
common p65/p50 NF-kB dimer in nodose ganglion sensory neurons
cultured from newborn mice enhanced NF-kB transcriptional activity and
increased neurite growth. Paradoxically, overexpressing IKK2 (but not
IKK1) also
enhanced NF-kB transcriptional activity but strongly inhibited neurite
growth. Furthermore, both p65/p50 growth stimulation and IKK2 growth
inhibition were reversed by expressing an IkBa mutant that cannot be
phosphorylated on serine residues. In addition to phosphorylating IkBa,
IKK2 (but not IKK1) phosphorylated p65 on serine 536 in these neurons.
Overexpression of a p65 mutant (S536A) that cannot be phosphorylated at
this site restored growth in IKK2 overexpressing neurons. Overexpression
of a phosphomimetic p65 mutant (S536D) led to a strong inhibition of
neurite growth. Taken together these results suggest that NF-kB can both
stimulate and inhibit neurite growth depending on the phosphorylation
status of p65. The implications for other neural populations and
extracellular signals involving NFkB activation are discussed
9.10
A novel TNF-alpha autocrine loop activates the canonical NF-kB
pathway to limit NGF-promoted axonal growth in developing
neurons.
O’Keeffe G W, Gutierrez H, Gavalda N, Davies A M
Cardiff School of Biosciences, Museum Avenue, Cardiff CF10 3US,
Wales.
Multiple signals from various cells control the growth, guidance and
terminal sprouting of axons in the developing nervous system as they
grow towards and within their innervation targets. Here we report a
novel autocrine signalling loop involving TNF-alpha produced by the
neurons themselves. This autcrine signalling loop functions to
regulate NGF-dependant axonal growth during the postnatal
development of mouse sympathetic neurons.
We found that TNF-alpha, signalling through TNFR1 significantly
reduced NGF-dependant neurite growth in sympathetic neurons
during a "developmental window". In these neurons, TNF-alpha
activated NF-kB as measured by degradation and Ser32/36
phosphorylation of IkB-alpha and an increase in NF-kB dependant
gene transcription using a GFP-reporter NF-kB construct. Using
selective mutants of proteins involved in the canonical and noncanonical
NF-kB signalling pathways, we found that that selectively
inhibiting NF-kB activation through the canonical pathway prevented
the reduction in growth triggered by TNF-alpha in sympathetic
neurons.
These results describe a novel function and mechanism of action for
TNF-alpha during neuronal development and they reveal a
unsuspected autocrine signalling loop in developing neurons that acts
to limit the extent of NGF-dependent axonal growth.
10.01
Interleukin-1 exacerbates excitotoxic brain injury via a neutrophil
dependant mechanism
LM McCluskey, SM Allan
Faculty of Life Sciences, University of Manchester, UK
Interleukin-1 (IL-1) is a pro-inflammatory cytokine that exacerbates
neurodegeneration, including alpha-amino-3-hydroxy-5-methyl-4-
isoxazolepropionate (AMPA) induced excitotoxic neuronal death. The aim of
this study was to test the hypothesis that neutrophils contribute to IL-1
mediated exacerbation of injury.
Male Sprague-Dawley rats were injected in the striatum with vehicle, AMPA,
IL-1 or AMPA plus IL-1 (A+IL-1), sacrificed 4 or 8h post-injection and serum,
brain and liver removed. Immunohistochemistry was performed to
investigate neutrophil recruitment and c-fos expression. Serum, brain and
liver cytokine-induced neutrophil chemo-attractant-1 (CINC-1) levels were
determined by ELISA. A separate cohort was pre-treated with neutrophil
depletion serum or normal rabbit serum (control) before intra-striatal
injection of A+IL-1. The animals were killed 24h later and whole blood and
brain removed. Brain lesion volume was calculated from cresyl-violet
stained sections. Immunohistochemistry was used to detect neutrophils
within the brain and circulating neutrophils were counted in the blood.
Injection of AMPA+IL-1 caused extensive neutrophil recruitment and c-fos
expression at 4 and 8h compared to injection of vehicle, AMPA or IL-1.
CINC-1 levels in the serum, brain and liver were induced to a similar extent
at 4h by IL-1 or A+IL-1, but not by vehicle or AMPA. Serum and brain CINC-
1 levels declined between 4 and 8h after IL-1 treatment, but remained
elevated at 8h after A+IL-1 treatment. Neutrophil depletion of animals prior
to injection of A+IL-1 reduced the resulting total lesion volume by 60%
compared to control.
These data suggest that IL-1 exacerbates excitotoxic brain injury by a
neutrophil dependant mechanism, possibly via CINC-1.
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