Book of abstracts - British Neuroscience Association

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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. Page 16/101 - 10/05/2013 - 11:11:03
10.02 The effect of PAT (thymulin related peptide) on pain related behavior and cytokine production in the brain in a rat model of neuroinflammation. Safieh-Garabedian B, Poole S, Mazih B, Saade N E Neuroscience Program, American University of Beirut, Bliss Street, Beirut, Lebanon. Parenterals Section, NIBSC,, Blanche lane, South Mimms, Potters Bar, Herts EN6 3QG, UK, , Despite the relative protection of the brain, intracerebral inflammation can be detrimental for neurons. Our aim in this study, was to investigate the possible protective role of PAT against inflammation. Brain inflammation was induced in Sprague-Dawley rats by intracerebroventricular (ICV) injection of endotoxin (1µg/5µl). Separate groups of rats (n= 5-6 each) were pretreated with different doses of PAT (ICV; 0.2µg, 1µg and 5µg) 30 minutes before ET. One group of rats was treated with PAT only and sham operated animals served as controls. The effects of pretreatment with PAT were assessed on pain related behavior and on the increased intracerebral levels of cytokines induced by ICV injection of ET. Under deep anesthesia, tissue samples were isolated from different brain areas (hippocampus, diencephalon and brainstem) and were processed for the determination of interleukins (IL)-6, IL-8, IL-10 and NGF concentrations by ELISA. ET injection produced sustained thermal and cold hyperalgesia and significant upregulation of the levels of cytokines and NGF. Pretreatment with PAT prevented in a dose-dependent manner the ETinduced hyperalgesia and altered the cytokine levels in different areas of the brain. The results suggest that PAT might have an important protective role of the brain against inflammation. (Supported by grants from URB and Lebanese National Scientific Research Council) 10.03 Systemic inflammation alters expression of the kynurenine pathway enzymes indolamine 2,3-dioxygenase and kynurenine 3- monooxygenase in rat brain Connor T J, Starr N, Harkin A Trinity College Institute of Neuroscience, University of Dublin, Trinity College, Dublin 2, Ireland Indoleamine 2,3-dioxygenase (IDO) is the rate limiting enzyme in the kynurenine pathway; the main pathway for tryptophan metabolism in the brain. Here we demonstrate that systemic administration of the inflammagen bacterial lipopolysaccharide (LPS) to rats induces IDO mRNA expression in cortex and hippocampus 4hr post administration. Induction of IDO was associated with a large increase in expression of the proinflammatory cytokines TNF-alpha and IL-6, with a modest increase in IFNgamma mRNA expression. Following its synthesis by IDO, kynurenine can be further metabolised down one of two pathways. Specifically, the enzyme kynurenine-3-monooxygenase (KMO) metabolises kynurenine into the free radical generator 3-hydroxykynurenine; a compound the can be further metabolized to form the excitotoxic N-methyl-D-aspartate receptor agonist quinolinic acid. Alternatively, the enzyme kynurenine aminotransferase (KAT II) metabolises kynurenine into kynurenic acid; a neuroprotective compound with NMDA receptor antagonist properties. Consequently, maintaining an appropriate balance between these two arms of the kynurenine pathway is critical for a healthy brain. Here we demonstrate that LPS suppressed KMO mRNA expression 4hr post administration, with a rebound increase observed at 24hr post LPS. In contrast, LPS failed to alter KAT II expression. This is the first demonstration that an inflammatory stimulus impacts upon expression of KMO; an enzyme that drives kynurenine metabolism in a neurotoxic direction. We speculate that the reduction in KMO expression observed 4hr post LPS may represent a physiological mechanism to limit production of neurotoxic products following IDO induction in response to an inflammatory insult. 10.04 Noradrenaline reuptake inhibitors inhibit neuroinflammation induced by a systemic inflammatory challenge O`Sullivan J B, Harkin A, Connor T J Trinity College Institute of Neuroscience, University of Dublin, Trinity College, Dublin 2, Ireland. Evidence suggests that the monoamine neurotransmitter noradrenaline elicits anti-inflammatory actions in the central nervous system (CNS), and consequently may play an endogenous neuroprotective role in CNS disorders where inflammatory events contribute to pathology. In line with this hypothesis, we demonstrate that noradrenaline suppresses expression of the pro-inflammatory cytokines IL-1beta and TNF-alpha and induction of iNOS/nitric oxide production from mixed glial cultures prepared from rat cortex, in response to the inflammagen bacterial lipopolysaccharide (LPS). As previous studies indicate that the noradrenaline reuptake inhibitor (NRI) desipramine has anti-inflammatory properties, we examined the ability of desipramine and more selective NRI’s to alter glial proinflammatory cytokine production. However, treatment of mixed glial cells with NRI’s largely failed to alter inflammatory events induced by LPS. In contrast to the in vitro situation, acute in vivo treatment of rats with NRI’s elicited an anti-inflammatory effect in the CNS characterised by reduced mRNA expression of the pro-inflammatory cytokines IL-1beta and TNF-alpha and iNOS in cortex in response to systemic LPS administration. The data also suggest that in vivo treatment with NRI’s inhibited microglial activation in the cortex indicated by reduced expression of the microglial activation makers CD40 and CD11b. These data indicate that NRI’s do not have a direct modulatory effect on the inflammatory response in glial cells, however when administered in vivo can limit inflammatory events in the brain. Overall, this study has yielded significant insights into the ability of noradrenaline augmentation strategies to limit neuroinflammation. 10.05 The PPARã agonist, rosiglitazone, attenuates LPS-induced changes in vitro in an IL-4-dependent manner. Griffin R J, Loane D J, Deighan B F, Lynch M A Trinity College Institute of Neuroscience, Physiology Department, Trinity College, Dublin 2, Ireland. Inflammatory changes in the brain contribute to neuronal deficits associated with age and neurodegenerative conditions. These changes include activation of microglia and the associated release of proinflammatory cytokines. Evidence from this and other laboratories suggest that rosiglitazone, a selective peroxisome proliferator-activated receptor gamma (PPARγ) agonist, exerts an anti-inflammatory action in brain. We investigated the modulatory effect of rosiglitazone on LPS-induced changes in cultured rat cortical glia and report that it significantly reduced the LPS-triggered increases in interleukin-1β (IL-1β) and tumour necrosis factor-α (TNFα) (p < 0.001). The mechanisms underlying the antiinflammatory effects of rosiglitazone are not thoroughly understood but our data show that treatment with rosiglitazone increases hippocampal concentration of the anti-inflammatory cytokine IL-4. To address this further, we investigated the effect of rosiglitazone on LPS-induced changes in primary glial cells prepared from 1-day old wild-type and IL-4-/- mice. The data show that rosiglitazone attenuates the LPS-induced increase in IL-1β concentration (p < 0.05) and the expression of major histocomaptabilty complex class (MHC) II mRNA (p < 0.05) in cells prepared from in wild-type but not IL-4-/- mice. In contrast, rosiglitazone attenuated the LPS-induced increase to a similar extent in both preparations and had no effect on the LPS-induced increase on IL-6. Our findings confirm an anti-inflammatory role for rosiglitazone and suggest that the action may be mediated by IL-4. Acknowledgements: Science Foundation Ireland, The Higher Education Authority Ireland (PRTLI), G03007 8AA. Page 17/101 - 10/05/2013 - 11:11:03
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Page 3 and 4: 3.07 Congenital hypothyroidism alte
Page 5 and 6: 4.01 The effect of hippocampal slic
Page 7 and 8: 4.09 ß-adrenergic modulation of in
Page 9 and 10: 5.03 Constitutively active Ras and
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Page 31 and 32: 18.03 Finding cells for replacement
Page 33 and 34: 20.01 Cannabinoid neuropharmacology
Page 35 and 36: 21.05 Spatial arrangement of cortic
Page 37 and 38: 23.04 Adenosine, astrogliosis and e
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Page 41 and 42: 28.05 The effects of the FAAH inhib
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43.10 Biochemical estimates of SNAR
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45.01 Mutant SOD1-PUMA knockout dou
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49.04 Regulation of synaptic functi
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51.04 The use of MRI for tracking d
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53.04 The role of the pedunculopont
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56.06 Dissecting exploratory behavi
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57.01 Opioid Receptor Agonists’-
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57.09 Cognitive rigidity and altere
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57.17 An analysis of DJ-1 (PARK7) a
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58.05 Differential effects of Inter
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60.07 2-Dimensional gel electrophor
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63.02 Subpyrogenic systemic inflamm
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64.08 Modulation of ketamine-induce
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65.08 The anti-inflammatory role of
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68.01 A comparison of carbachol and
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