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UCD School of Biomolecular & Biomedical Science

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Neuroscience Day

University College Dublin

School of Biomolecular andBiomedical Science

9th October 2009

Images from UCD Images of Research competitionsSee www.ucd.ie/sbbs for details


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Programme

Venue: UCD Conway InstituteTalks and seminars will take place in the UCD Conway lecture theatre

9.20 am Welcome

9.30 am Jessica McDonald: SDS-stable Ab dimers extracted in the water- and triton-X100-solublefractions of brain are specific for Alzheimer-type dementia

9.50 am Charles Metais: Chronic and acute effects of simvastatin on synaptictransmission in wild type and APPswe/PSN1dE9 mice

10.10 am Oktay Kaplan: Cooperation of AP1 clathrin adaptin complexes and RAB-8 in protein transportto cilia and cilia morphology determination

10.30 am Orla Watters: The effects of Tumour Necrosis Factor-alpha & Glutamate preconditioning; an invitro model of stroke

10.50 am Coffee/Tea and Poster viewingUCD Conway Foyer

11.30 pm Laura Batti: A role for the Prolyl Hydroxylase inhibitor, DMOG, on synaptic transmission andneuroprotection in an ex-vivo model of cerebral ischemia.

11.50 am Sebiha Cevik: Joubert syndrome-associated ARL13B/ARL-13 functions at the ciliarymembrane and is required for ciliary protein transport in C. Elegans

12.10 pm Bartek Lukasz: Immune contributions to behavioural deficits in rodent models of Schizophrenia

12.30 pm Poster Viewing/Lunch breakSandwich Lunch will be served at 1.30pm

2 pm CLASS Seminar: Dr. John F. Cryan, College of Medicine and Health, University College Cork

KILLING THE BLUES: NOVEL NEUROPHARMACOLOGICAL STRATEGIES TO DEVELOPANTIDEPRESSANT THERAPIES

3 pm Reception/Prize-givingUCD Conway Foyer


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Contents

4 Welcome from Dr. Jana Haase, SBBS

5 Invited Speaker Profile – Dr. John F. Cryan

6-11 Oral Presentation Abstracts

6 Jessica McDonaldSDS-stable A dimers extracted in the water- and triton-X100-soluble fractions of brain arespecific for Alzheimer-type dementia

7 Charles MetaisChronic and acute effects of simvastatin on synaptic transmission in wild type andAPPswe/PSN1dE9 mice

8 Oktay KaplanCooperation of AP1 clathrin adaptin complexes and RAB-8 in protein transport to cilia and ciliamorphology determination

9 Orla WattersThe effects of Tumour Necrosis Factor-alpha & Glutamate preconditioning; an in vitro model ofstroke

10 Laura BattiA role for the Prolyl Hydroxylase inhibitor, DMOG, on synaptic transmission and neuroprotectionin an ex-vivo model of cerebral ischemia.

10 Sebiha CevikJoubert syndrome-associated ARL13B/ARL-13 functions at the ciliary membrane and is requiredfor ciliary protein transport in C. Elegans

11 Bartek LukaszImmune contributions to behavioural deficits in rodent models of Schizophrenia

12-15 Poster Abstracts:

12 J. O’ Sullivan: The prion protein as a stress regulator

12 R. Ring: Identification of Captodiamine as a Putative Antidepressant

13 D. Corbett: Characterisation of SLITRK2-Deficient Mice

13 A.J. Mably: Assessment of the effects of human Aβ on spatial reference memory

14 C. Foley-Fisher: The C. elegans PQR neuron: a model for ciliogenesis14 K. McEvoy: Sulphated cyclodextrins acting as heparin mimetics in prion disorders15 S. Kandil: Upregulation of the Transsulfuration Pathway during Gliotoxin Mediated

Inhibition of GSH in C6 Glioma Cells

15 C. Downey: The effects of MDMA and caffeine co-administration on rat blood pressure,heart rate and temperature

16 Sponsors in Attendance

17 Notes


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WELCOME

DR. JANA HAASEUCD SCHOOL OF BIOMOLECULAR AND BIOMEDICAL SCIENCE

I would like to welcome you all to the SBBS Neuroscience Day 2009. As in previous years, thisevent showcases the achievements of SBBS graduate students in the area of Neuroscience,which is one of the key research areas in the School.

This year’s event also marks the launch of the SBBS Thematic PhD Programme inNeuroscience. From this year onwards, all SBBS postgraduate students associated withPrincipal Investigators in the area of Neuroscience will be enrolled into the programme. Inaddition to their individual research projects, Thematic PhD students will complete taughtmodules, aiming to equip them with state-of-the-art knowledge in Neuroscience, as well as withimportant transferable skills enhancing career prospects upon graduation.

I am very grateful to PerkinElmer Ireland, who is sponsoring the Best Oral Presentation Prizetoday. I am sure the representative, Colm Berry, who is here today will be happy to discussPerkinElmer’s latest products and offers with us.

I sincerely hope you enjoy this scientific programme and I would like to thank everyone whohelped with the organisation of this event; in particular Mary Gallagher, Jacqueline Jago,Margaret Doherty, and John Murray.

With best wishes,

Dr. Jana Haase


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Invited SpeakerSpeaker Profile

Dr. John F. Cryan

School of Pharmacology, University College Cork

Presentation title:KILLING THE BLUES: NOVEL NEUROPHARMACOLOGICAL STRATEGIES TO DEVELOPANTIDEPRESSANT THERAPIES

Biography

Dr. John F. Cryan is a Senior Lecturer in Pharmacology in the School of Pharmacy and in theDept. Pharmacology Therapeutics UCC. He received a B.Sc. (Hons) in Biochemistry and a PhDin Pharmacology from the National University of Ireland, Galway, Ireland. He was a visitingfellow at the Dept Psychiatry, University of Melbourne, Australia, which was followed bypostdoctoral stints at the University of Pennsylvania, Philadelphia, USA and The ScrippsResearch Institute, La Jolla, California. He spent four years at the Novartis Institutes forBioMedical Research in Basel Switzerland, as a LabHead, Behavioural Pharmacology wherehis research group were responsible for the preclinical characterisation of novel therapeutictargets and ligands for psychiatric disorders prior to joining UCC in 2005. Currently he is also aPrincipal Investigator in the Alimentary Pharmabiotic Centre(http://www.ucc.ie/research/apc/content) and Food Health Ireland (www.fhi.ie ). Dr. Cryan is amember of the International Union on Pharmacology (IUPHAR) Committee on GABAB

receptors. He is member of the Full Committee of the European Behavioural PharmacologySociety and served as its Meeting Secretary and Executive Committee Member from 2007-2009. Dr Cryan has been honoured with the European College of Neuropsychopharmacology(ECNP) Fellowship Award, the Wyeth Psychopharmacology Award from British Association ofPsychopharmacology and the Young Scientist Award from the European BehaviouralPharmacology Society.

Dr. Cryan is an Editor of both British Journal of Pharmacology and Neuropharmacology. He isAdvisory Editor of Psychopharmacology; on the Board of Reviewing Editors of Brain Research;an Associate Editor of Frontiers in Behavioural Neuroscience; an Editorial Board Member ofBehavioural Pharmacology and has acted as Guest Editor of both Neuroscience andBiobehavioral Reviews and International Journal of Neuropsychopharmacology . He haspublished over 80 articles and has an H-Index of 30. Dr. Cryan’s current research interestsinclude the neuropharmacology of stress-related neuropsychiatric disorders includingdepression, anxiety and drug dependence. Moreover, his group is also focused onunderstanding the interaction between the brain and gut and how it applies to stress andimmune-related disorders, including irritable bowel syndrome, obesity and sepsis. He is alsointerested in applying novel approaches to facilitate drug/siRNA delivery to the brain in vivo.


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Oral Presentation abstracts

AβMONOMER AND SDS-STABLE AβDIMERS EXTRACTED IN THE WATER- AND TRITON-X100-SOLUBLE FRACTIONS OF BRAIN ARE SPECIFIC FOR ALZHEIMER-TYPE DEMENTIA

Jessica M. Mc Donald1, George M. Savva2, Carol Brayne2, Fiona E. Matthews3,Alfred T. Welzel1, Ganesh M. Shankar4, Dennis J. Selkoe4, Paul G. Ince5and Dominic M. Walsh1 .

1Laboratory for Neurodegenerative Research, School of Biomolecular and BiomedicalScience, Conway Institute, University College Dublin, Dublin 4, Republic of Ireland;2Department of Public Health & Primary Care, Institute of Public Health, CambridgeUniversity, Cambridge, CB2 0SR, UK; 3MRC Biostatistics Unit, Institute of PublicHealth, Cambridge University, Cambridge, CB2 0SR, UK; 4Center for NeurologicDiseases, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA02115; 5Department of Neuroscience, Sheffield University, Sheffield, S10 2JF, UK.

By current criteria a positive diagnosis of Alzheimer’s disease (AD) requires both the clinical confirmationof dementia and post-mortem detection of amyloid plaques and neurofibrillary tangles in the neocortex ofthe brain. The amyloid protein (Aβ) is a small hydrophobic protein (~4 kDa) believed to play a causalrole in AD and can exist in multiple different assembly forms including fibrils found in amyloid plaques,which are present in AD brain. However, the quantity of A-containing plaques does not correlate wellwith clinical status suggesting that non-plaque Aβ may be pathogenic. Using 45 brains from the Newcastle cohort of the Cognitive Function and Ageing Study we initiated an unbiased analysis toexamine the relationship between biochemically distinct forms of A and the presence of Alzheimer-typedementia. Cortical samples were serially extracted with Tris-buffered saline (TBS), TBS containing 1%TX-100 (TBS-TX) and 88% formic acid (FA) and extracts analyzed for A byimmunoprecipitation/Western blotting. The cohort was divisible into those with dementia at death with(14) or without (10) significant Alzheimer-type pathology (ATP), and those who were not demented with(2) or without significant ATP (17). TBS and especially TBS-TX A monomer was highly specific for dementia with ATP and SDS-stable A dimer was detected specifically and sensitively in TBS, TBS-TXand formic acid (FA) extracts of Alzheimer brain. As expected, A monomer in the FA fraction closely correlated with diffuse and neuritic plaque burden and was not specific for AD. These findings supportthe hypothesis that soluble A monomer and SDS- stable dimeris a major correlate of dementia associated with ATP and is likely to be intimately involved in the pathogenesis of cognitive failure.


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ACUTE AND CHRONIC EFFECTS OF SIMVASTASTIN TREATMENT ON SYNAPTIC TRANSMISSIONAND LONG TERM POTENTIATION (LTP)

Charles Metais and Caroline HerronSchool of Biomolecular and Biomedical Sciences,Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland.

Alzheimer’s disease (AD) is the most common neurodegenerative disease in elderly people. Thisdementia is characterised by impairments in learning and memory, and by deposition of amyloid plaquesand neurofibrillary tangles. Animal models have been successfully developed to mimic the disease(Jankowsky et al., 2004, Savonenko et al., 2005). In these models, several studies have demonstrated inthe hippocampus, a structure involved in learning and memory, that LTP, a stimulus dependent increasein synaptic transmission, considered to be a cellular form of learning and memory, is impaired (Trincheseet al., 2004). Recently, chronic treatment with simvastatin, a cholesterol-lowering agent, improved thescore of both control and AD mice in learning and memory tasks (Li et al., 2006). However little has beenreported on the acute and chronic effects of simvastatin on hippocampal synaptic transmission andplasticity. In our study we have investigated, first the acute effects of simvastatin in the CA1 region ofhippocampal slices from young mice (8weeks old). We found that simvastatin (35M) significantly increased the excitatory postynaptic potential (EPSP) slope, indicating an increase in excitability.Moreover the paired pulse facilitation (PPF) ratio decreased significantly suggesting thatneurotransmitter release increased after drug application. We then focused on the acute effect ofsimvastatin on compound action potential (cAP). We also found that simvastatin (35M) increased significantly the amplitude of the cAP. This result could explain the increase of excitability we haveobserved previously. We also investigated the effects of simvastatin on LTP. LTP was induced usingeither 100 or 200 Hz. 100 Hz protocol: Two trains of stimuli at 100Hz for one second applied 30s apart.200 Hz protocol: Two sets of ten trains of ten stimuli at 200Hz; with 2s inter-train interval; 30s betweensets.

We found that acute application of simvastatin significantly decreased the magnitude of LTP in slicesfrom young animals with either induction protocol. On another aspect of the study we assessed thepossible positive effect of chronic simvastatin treatment (50mg/kg/diet Li et al for 180 days) on LTP inslices from 16-month-old animals slices from APPswe/PS1dE9 double transgenic mice and their age-matched controls were used. We found that chronic simvastatin treatment restored LTP in our transgenicmodel. No significant difference of LTP magnitude was observed with aged matched controls an hourafter 100Hz HFS. All extracellular field recordings were performed in the stratum radiatum of the CA1region of transverse hippocampal slices. Stimuli were applied every 30s. Stable baseline for 20 minuteswas necessary prior to any drug application or LTP induction.

ReferencesJankowsky J. et al., “Mutant presenilins specifically elevate the levels of the 42 residue b-amyloidpeptide in vivo: evidence for augmentation of a 42-specific c secretase” 2004 Human MolecularGenetics, 2004, Vol. 13, No. 2 159–170.Ling Li et al., “Simvastatin Enhances Learning and Memory Independent of amyloid load in Mice.” AnnNeurol 2006;60:729-739.Savonenko A. et al., “Episodic-like memory deficit in the APPswe/PS1dE9 mouse model of Alzheimer`sdisease: Relationships to β-amyloid deposition and neurotransmitter abnormalities.” Neurobiology ofdisease 18 2005 602-617.rinchese F. et al., “Progressive Age-Related Development of Alzheimer-like Pathology in APP/PS1 Mice”Ann Neurol 2004;55:801-814.


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COOPERATION OF AP1 CLATHRIN ADAPTIN COMPLEXES AND RAB-8 IN PROTEIN TRANSPORTTO CILIA AND CILIA MORPHOLOGY DETERMINATION

Oktay I. Kaplan1, Katarzyna Kida1, Sebiha Cevik1, & Oliver E. Blacque1*

1School of Biomolecular and Biomedical Science, UCD Conway Institute, University College Dublin,Belfield, Dublin 4, Ireland.

Clathrin and clathrin adaptor (AP) complexes mediate protein sorting and vesicular trafficking betweenintracellular membrane compartments and the plasma membrane. One such compartment is the cilium,which extends from the surface of most vertebrate cells. Here, using C. elegans and mammalian cellculture models, we investigate the role of AP-1 in metazoan cilium formation/function and in proteintargeting to cilia. First, we show that the clathrin heavy chain (CHC) and AP-1 complex components(UNC-101, APS-1) are required for determining cilium morphology, positioning and ultrastructure, and formediating vesicle transport of transmembrane proteins (ODR-10) to cilia. AP-1 functions independent ofintraflagellar transport (IFT), since IFT proteins display normal ciliary localizations and motilities in unc-101 mutants, and the ciliary localisation and dendritic motilities of ODR-10 are unaffected in IFT mutants.In contrast, unc-101 and rab-8(Q67L) (GTP-locked RAB8) mutations both prevent the formation of ODR-10 transport vesicles, cause ODR-10 to become trapped at the plasma membrane, and suppress AWBcilia membranous fan formation in grk-2 mutants. Consistent with related roles for RAB-8 and AP-1 inmembrane trafficking to cilia, GFP-tagged RAB-8, UNC-101 and ODR-10 possess overlappingsubcellular localizations and dendritic trafficking behaviours. Together, our results suggest that AP-1 andRAB-8 cooperate in a CHC-dependent manner to coordinate membrane trafficking to cilia.


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THE EFFECTS OF TUMOUR NECROSIS FACTOR-ALPHA & GLUTAMATE PRECONDITIONING;AN IN VITRO MODEL OF STROKE

Orla Watters and John O’ConnorUCD School of Biomolecular and Biomedical Science,UCD Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland.

Glutamate-induced excitotoxicity is a well established contributor to neuronal damage during a cerebralischemic event such as stroke. Physiological levels of glutamate and proinflammatory cytokines such asTNFα play a role in the regulation of synaptic plasticity within the hippocampus, the brain region involvedin memory processing and consolidation. At pathophysiological levels which arise during stroke,dysregulation of these processes may occur, giving rise to enhanced vulnerability of these cells to theischemic insult. However, previous studies suggest that a mild transient ischemic attack (TIA) within 72 hof a stroke may result in attenuation of its clinical severity (Castillo et al. 2003). In this study wedeveloped an in vitro model of TIA using organotypic hippocampal cultures. These cultures werepretreated with a mild acute dose of glutamate (30 µM) or TNFα (5 ng/ml) for 30 min, then allowed arecovery period of 24 h before application of a second glutamate insult. As NMDA receptors arepermeable to calcium ions, and calcium is a well-established mediator of glutamate-inducedexcitotoxicity, we investigated whether preconditioning with or glutamate altered glutamate-inducedcalcium influx during the second insult. We also investigated whether the pretreatments themselves hadan effect on cell viability and on basal calcium levels.

We found that pre-treatment with glutamate or TNFα for 30min 24 h before the second insult resulted ina significant reduction of glutamate-induced calcium influx in both groups compared to controls. It ispossible that the pre-treatments conditioned the cultures to become less responsive to the second insult,preventing a large influx of calcium into the cells which could lead to initiation of apoptotic pathways andexcitotoxicity. The NMDA receptor antagonist, D-AP5 (100 µM) and the p38 MAPkinase inhibitor SB203580 (10 µM) did not significantly alter the preconditioning effect of glutamate when co-applied, asthere no significant difference in the glutamate-induced calcium influx between these groups was found.However the mGluR5 antagonist MPEP (10uM), when added alongside TNFα resulted in significantlyamplifying the dampening effect of TNFα pre-treatment on glutamate-induced calcium influx 24 h postrecovery. This suggests that mGluR5 receptor activation may modulating the effect of TNFα at itsreceptors. Inhibition of the MAP kinase pathway during TNFα preconditioning using SB 203580 (10 uM),successfully reversed the preconditioning effect of TNFα, suggesting that this downstream signallingpathway has a role to play in the TNFα-mediated preconditioning effect. Extracellular field recording wascarried out to investigate the immediate effects of these pre-treatments on CA1 pyramidal neurons inacute cut hippocampal slices. We found that neither 30 uM glutamate nor 5 ng/ml TNFα significantlyaltered synaptic activity in this region and no effect on the level of LTP generated by high frequencystimulation was found.

Reference: Castillo J, Moro MA, Blanco M, Leira R, Serena J, Lizasoain I, Dávalos A. (2003) Therelease of tumor necrosis factor-alpha is associated with ischemic tolerance in human stroke. AnnNeurol. 54(6): 811-9.

This work was funded by Science Foundation Ireland.


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A ROLE FOR THE PROLYL HYDROXILASE INHIBITOR DMOG, ON SYNAPTIC TRANSMISSIONAND NEUROPROTECTION IN AN EX VIVO MODEL OF CEREBRAL ISCHEMIA

Laura Batti1, C. Taylor,2 J.J. O’Connor1

1UCD School of Biomolecular and Biomedical Science & 2School of Medicine and Medical Science,Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland.

A reduction in oxygen availability in neurons triggers a broad spectrum of transcriptional and non-transcriptional responses inducing adaptation to insufficient oxygen supply. One of the mainmechanisms leading to these adaptations is the activation of hypoxia-inducible transcription factor-1α(HIF-1α). Hypoxic regulation of gene expression can be mimicked by dimethyloxalylglycine (DMOG), acell permeable competitive inhibitor of HIF1-α prolyl hydroxylase, which leads to the stabilization of HIF.Although DMOG application appears as a highly attractive target for therapeutic intervention inischaemic disease, its effect on synaptic activity is unknown. We have investigated the action of DMOGon transcriptional and non-transcriptional changes in rat hippocampal slices and compared the changesin synaptic transmission to those occurring in brain slices exposed to low oxygen.

We also investigated putative therapeutic effects of DMOG pre-conditioning on neuronal viability in an exvivo model of cerebral ischaemia. External field recording and staining experiments were carried out inthe rat CA1 hippocampus a region found to be very vulnerable to hypoxic injury. Both treatmentsresulted in reversible depression of synaptic transmission (at 0.1 and 1 mM DMOG) (p<0.01), increasedpaired pulse facilitation and reduced NMDA receptor activation (p<0.05). In addition, pre-conditioningwith DMOG in organotypic hippocampal slices showed a neuroprotective effect in an ex vivo model ofcerebral ischaemia. These results indicate a possible for the first time a putative role for PHD inhibition insynaptic transmission, which may have some similarities with the hypoxia-induced depression andsuggest a potential use of PFD inhibitors as pharmacological neuroprotection in the hippocampus.

JOUBERT SYNDROME-ASSOCIATED ARL13B/ARL-13 FUNCTIONS AT THE CILIARY MEMBRANEAND IS REQUIRED FOR CILIARY PROTEIN TRANSPORT IN C. elegans

Sebiha Cevik

School of Biomolecular and Biomedical Science, UCD Conway Institute, University College Dublin,Belfield, Dublin 4, Ireland.

The small ciliary G-protein ARL13B is required for cilium biogenesis, sonic hedgehog signallingand is mutated in patients with Joubert syndrome (JS). Here, using C. elegans and mammalian cellculture systems, we investigated the poorly understood ciliary and molecular basis of ARL13B function.First we show that the localisation of ARL13B/ARL-13 is frequently restricted to a proximal ciliarycompartment, where it associates with the ciliary membrane via palmitoylation modification motifs. Nextwe find that loss-of-function C. elegans arl-13 mutants possess defects in cilium morphology andultrastructure, as well as defects in ciliary protein localisation and transport; ciliary transmembraneproteins abnormally accumulate, PKD-2 ciliary abundance is elevated and anterograde intraflagellartransport (IFT) is destabilized. Finally, we show that arl-13 interacts synthetically with other ciliogenic andciliary transport-associated genes (e.g., bbs-8, dyf-5) in maintaining cilium integrity and anterograde IFTstability. Together, these data implicate a role for JS-associated ARL13B at the ciliary membrane, whereit regulates the transport and/or stability of ciliary transmembrane proteins and IFT assemblies.


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ABERRANT IMMUNE SIGNALLING IN RODENT MODELS OF SCHIZOPHRENIA

Bartek Lukasz, N.C. O’Sullivan, J.S. Loscher, K.J. Murphy.Applied Neurotherapeutics Research Group, UCD School of Biomolecular and Biomedical Science, UCDConway Institute, University College Dublin, Belfield, Dublin 4.

Schizophrenia is unusual among neurodevelopmental disorders in that the positive, negative andcognitive symptoms that characterise the disorder are not evident in the early years of life but ratheremerge in the late teenage period. Studies of the aetiology of schizophrenia suggest genetic riskinteracts with environmental insults, such as second trimester influenza infection or traumatic childhoodevents, to impinge on cortical development in particular in the prefrontal cortex and hippocampus.

Here, we have investigated the consequences of combining two environmental models ofschizophrenia-like behavioural deficits, isolation rearing from postnatal day 25 and immune challengewith viral dsRNA analogue, poly(I:C), in male Wistar rats across the critical postnatal day 36 to 44 periodof prefrontal cortical maturation.

Our results confirmed isolated animals to have a deficit in sensorimotor gating as revealed bydecreased pre-pulse inhibition (PPI). Interestingly, animals treated with polyI:C appeared normal atpostnatal day 60 with a PPI deficit only emerging at postnatal day 80. Combination of the insults did notexacerbate the deficits seen with either insult alone suggestive of a convergence on a commonmechanism of disruption. To further investigate this possibility, using qPCR, we assayed thehippocampal dentate gyrus of isolated animals for alterations viral response genes, namely, Irf7, Mx2,Pkr and Ifit2. These analyses revealed a profound decrement (6.5 – 46.9%) in expression of thesegenes across the postnatal day 30-40 period. Moreover, western blot analysis showed an associateddecrease (18.7%) of Irf7 protein in P40 isolated animals. In summary, our findings suggest thatdisruption of specific immune response systems may be an important common component of severalenvironmental insults known to increase risk of developing schizophrenia.

This work was funded by EU Marie Curie Programme & Science Foundation Ireland.


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Poster abstracts

THE PRION PROTEIN AS A STRESS REGULATOR

Jack O’ Sullivan, Emma J. Comerford and Hilary E.M McMahon

Prion Research Group, UCD School of Biomolecular and Biomedical Science, Conway Institute for Biomolecularand Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland

Prion diseases or Transmissible Spongiform Encephalopathies (TSEs) are a group of neurodegenerative disordersthat affect both animals and humans. Gerstmann-Straussler Scheinker syndrome (GSS), Fatal Familial Insomnia(FFI) and Creutzfeldt-Jakob Disease (CJD) are amongst those affecting humans. The spectrum of human TSEsmay now be greater with the discovery of proteinase-sensitive prionopathy (PSPr). It is now reported that the prionprotein is also associated with Alzheimer’s disease (AD), and cancer development. In general TSEs are linkedwith the accumulation within the brain of an abnormal proteinase resistant isoform (PrPSc) of the normal hostencoded prion protein (PrPC).

The function of PrPC has remained an enigma for many years since its discovery and linkage with Prion disorders.As the disease process in prion disorders is linked with oxidative damage and impaired anti-oxidant defencesystems, it is proposed that PrPC plays a role in the protection of the cell against oxidative stress. In fact inneuronal cells both the absence of PrPC and the presence of PrPSc results in increased cell sensitivity to oxidativestress and a reduction in the level of Superoxide Dismutase (SOD) activity. Here the effect of scrapie infection onthe antioxidants SOD1 and SOD2 are examined.

IDENTIFICATION OF CAPTODIAMINE AS A PUTATIVE ANTIDEPRESSANT

Rebecca Ring, Shane Gannon, Mark Pickering, Adrian Coyle, Lisa Conboy, Noel O’Boyle, Darren Scully andCiaran M Regan.School of Biomolecular and Biomedical Sciences, UCD Conway Institute, Dublin 4, IRELAND.

Using a behavioural tier we re-evaluated a series of clinically-safe drugs which were removed by the FDA duringthe Drug Efficacy Study Implementation programme in the period 1968-1974. Chronic treatment (8 days) ofC57Bl6 mice with drug doses (3-5 mg/kg) previously used in the clinic revealed captodiamine to exhibit ananxiolytic action in the open-field and elevated X-maze paradigm. Captodiamine was also found to reduce latencyto despair in the forced swim test but was without effect on sensorimotor processing (prepulse inhibition). Theeffects of captodiamine on behavioural despair suggested this drug to be a putative antidepressant. Furthermore,receptor displacement studies demonstrated captodiamine to have significant affinities for the 1, D3 and 5HT2C

receptors which have been previously implicated as drug targets in the treatment of depression. Captodiamine wasdemonstrated to be an agonist at the 1 receptor, as the rimcazole 1 receptor-specific antagonist blocked itsaction in the forced swim test, an agonist at the D3 receptor, as demonstrated by its blockade of electrical field-induced relaxation of the rat pylorus, and a 5HT2C receptor antagonist, as judged by its ability to block 5HT-inducedcalcium release in 5HT2C receptor-transfected HEK cells. Enantiomeric analysis revealed the antidepressant actionof captodiamine to reside in the R-enantiomer which had the highest affinity for the 5HT2C receptor (5HT2C receptor:R=1.6 ηM; S=44.9 ηM; 1 receptor: R=0.094 μM; S=0.036 μM; D3 receptor: R=0.96 μM; S=0.31 μM). The abilityof captodiamine to modulate BDNF expression in a brain region-specific manner is currently being used as asurrogate marker to understand the unique polyvalent pharmacology of this putative antidepressant.

The support of Enterprise Ireland is gratefully acknowledged. RR is supported by the HEA PRTLI cycle 4(Bio)pharmaceutical and Pharmacological Sciences initiative.


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CHARACTERISATION OF SLITRK2-DEFICIENT MICE

Danielle Corbett1, Kei-ichi Katayama2, Yoshifumi Matsumoto2, Naoko Morimura2, Kazuyuki Yamada2, Maya Ota2,Veravej G. Ornthanalai4, Chihiro Honma3, Niall P. Murphy4 and Jun Aruga2

1School of Biomolecular and Biomedical Science, Applied Neurotherapeutics Research Group, UCD ConwayInstitute, University College Dublin, Ireland, 2Laboratory for Behavioral and Developmental Disorders, 3Support Unitfor Animal Experiments, 4Neuronal Circuit Mechanisms Research Group, RIKEN Brain Science Institute (BSI),Wako-shi, Saitama 351-0198, Japan

The Slitrk family comprises type I neuronal transmembrane proteins that control neurite outgrowth (Aruga andMikoshiba, 2003) and share structural similarities with the axonal guidance molecule Slit and with the Ntrkneurotrophin receptors (Brose and Tessier-Lavigne, 2000; Huang and Reichardt, 2003). Slitrk proteins shareleucine-rich domains, known to be involved in many protein-protein interactions (Kobe and Kajava, 2001). Recently,mutations in SLITRK1 were found to be related to neuropsychiatric disorders, including Tourette's syndrome andtrichotillomania (Abelson et al., 2005; Züchner et al., 2006), and Slitrk1-deficient mice were shown to displayincreased anxiety and depression-like behaviour and noradrenergic abnormalities (Katayama et al., 2008).Nonetheless, the function of other proteins in this family remain largely unknown. In order to investigate the role ofSlitrk2 in vivo, we developed Slitrk2-knockout mice and analysed their behavioural and neurochemical phenotypes.Slitrk2-deficient mice exhibit hyperactivity in several behaviour tests (e.g. open field, elevated plus maze, marbleburying test, rotarod test) and neurochemical alterations. These results suggest that Slitrk2 may be involved inneuropsychiatric diseases with a hyperactivity component and neurochemical abnormalities. Further studies arecurrently being conducted to obtain a detailed characterisation of Slitrk2-deficient mice neurochemical profile andbehavioural phenotype.

ASSESSMENT OF THE EFFECTS OF HUMAN Aβ ON SPATIAL REFERENCE MEMORY

Alexandra J. Mably1, Stephanie Daumas 2, Jessica M. McDonald1, Richard G.M. Morris2 and Dominic M. Walsh1

1Laboratory for Neurodegenerative Research, School of Biomolecular and Biomedical Science, University CollegeDublin, Dublin 4, Republic of Ireland; 2Laboratory for Cognitive Neuroscience, CCNS, University of Edinburgh, UK.

Alzheimer’s disease (AD) is the most common cause of late-life dementia and in its early phase is characterized byimpairment of various facets of episodic memory. Although the cause of AD remains uncertain extensive geneticevidence indicates that the amyloid β-protein (Aβ) plays a central role in pathogenesis. Aβ is prone to self-association and aggregation and can exist in a multitude of different assembly states, but as yet the form or formsof Aβ that mediate cognitive impairment are ill-defined. Prior studies have shown that water-soluble forms of Aβare elevated in AD brains and that the levels of water-soluble Aβ strongly correlate with synaptic compromise. Thecurrent study seeks to determine if water-soluble non-fibrillar AD brain-derived Aβ can alter episodic memory wheninjected into the lateral ventricle of a normal adult rat.For these experiments a cohort of 30 samples of human temporal cortex were homogenized in Tris-buffered saline,ultra-centrifuged and A detected in the supernatant by immunoprecipitation (IP) and western blotting (WB). Usingthis procedure 21 out of 30 brains were found to contain Aβ monomer and SDS-stable Aβ dimer, with total Aβvalues in the range of 0.95 – 407.9ng/g. A sample containing abundant Aβ monomer (51.6ng/g) and SDS-stabledimer (23.36ng/g) was selected for injection into rats for assessment of spatial reference memory and was de-salted into sterile ammonium acetate buffer to remove bacteria and small molecule drugs. Using Lister Hoodedand Wistar rat strains pilot studies were carried out to determine the effectiveness of a water-maze training regimefor 48 hr post-training spatial memory recall; and to select the strain of rat most suitable for the proposedbehavioural study. Rats underwent two days of cue-task training in the water-maze, followed by one day of spatialreference memory training (SRM) consisting of 8 trials. Following the 8th trial the rats underwent a 10 minute probetest (PT) that acted as a reinforcement trial and allowed assessment of the level of spatial memory prior to the 24hrand 48hr PTs. The rats then underwent PTs at 24hrs and 48hrs post-training. The Atlantis Platform was used toprevent memory extinction. Both rat strains showed rapid learning during the SRM training and robust memory atboth the 24hr and 48 hr PT. However, the Lister-Hooded rats performed in a more consistent manner andappeared to be more confident in the maze, having a much lower percentage thigmotaxis (time spent within 5cm ofthe walls of the water-maze), for this reason they were selected as the strain to go forward into the behaviouralstudy. Experiments testing the effects of water-soluble Aβ containing brain extracts are ongoing.

This work was supported by the European Community’s Seventh Framework Programme (FP7/2007-2013) underGrant Agreement No. 200611 (DMW).


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THE C. ELEGANS PQR NEURON: A MODEL FOR CILIOGENESIS

Christian Foley-Fisher

Cilia are evolutionarily conserved microtubule-based cell motile or non-motile appendages foundthroughout the animal kingdom. Non-motile or primary cilia possess important sensory roles (e.g., olfaction,chemoreception, mechanosensation, and photoreception) and defects in these functions can lead to humanailments such as cystic kidney disease, retinal dystrophy, bone abnormalities, organ laterality defects andphenotypically complex disorders such as Bardet-Biedl syndrome (BBS). In the nematode, C. elegans, all ciliaextend from the dendritic tips of sensory neuronal cells. Whilst significant strides are being made in many differentresearch models to discover the protein machinery (eg. intraflagellar transport (IFT) proteins) that builds andmaintains cilium structures, surprisingly little is known about the early stages of metazoan cilium development. Inthe current model for ciliogenesis a centriole migrates to membrane surface where it forms a basal body from whicha ciliary axoneme is extended by IFT. The PQR neuron develops after the worm has hatched from the egg and thecilium tipped dendrite is exposed to the pseudocoelomic cavity.

We have used protein-tagging and time-specific bleaching to track hourly the early stages of PQRdevelopment. This has shown that the PQR dendrite outgrowth begins almost immediately after hatching and thatrecruitment of the cilia protein ARL-13 begins between 6 and 7 hours post-hatch, indicating that the PQR cilium isformed during or very shortly after PQR dendrite extension. To assess if cilium formation or function is required forPQR dendritogenesis, we have been examining dendrite morphology/length in ciliary gene mutants. In arl-13mutants, which possess subtle defects in cilium morphology, the PQR dendrite of 25% of adult worms abnormallyextend past the cilium. This data indicates that the ciliary function of ARL-13 is required for determining dendritelength/morphology and refutes a previous suggestion that PQR dendrite outgrowth is terminated by ciliumformation. Presently we are examining the role of other ciliogenic genes in PQR dendrite outgrowth, as well asinvestigating the very earliest steps of cilium formation, using centriolar protein markers to assess centriolemigration to distal dendrite tips and subsequent basal body formation.

SULPHATED CYCLODEXTRINS ACTING AS HEPARIN MIMETICS IN PRION DISORDERS

Karl McEvoy, Hilary E.M McMahon

Prion Research Group, UCD School of Biomolecular and Biomedical Science, Conway Institute for Biomolecularand Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland

Prion disorders are characterised by the accumulation of a misfolded isoform (PrPSc) of the host encoded prionprotein (PrPC). We have identified the antiprion potential of cyclodextrin (CD) analogues and have identifiedsulphated-β-cyclodextrin, with a half-maximal inhibitory concentration (IC50) of 2.4µM, as having 31 fold greaterantiprion activity than that previously reported for β-Cyclodextrin (β-CD).Scrapie infected cells were treated with a range of βCD analogues. This enabled a CD structure to antiprion activityanalysis to be carried out. The metachromatic activity of each cyclodextrin was determined; this test is employed tomimic complexation of glycosylaminoglycans to a cell membrane. Sulphated-βCD had an IC50 of 2.4µM and it wasthe only CD found to have a metachromatic activity. Its activity was equivalent to that of heparin and heparinsulphate, this may account for sulphated-βCD’s superior antiprion action.

In solution heparin can form a helical structure with a hydrophobic interior, the hydrophobic interior of cyclic CD’s isvital for CD molecule encapsulation. The controlled CD structure, however, restricts degradation by humanenzymes; consequently sulphated-CD’s could be ideal candidates in the search for prion therapeutics. Sulphated-CD’s may open up avenues for the treatment of TSEs.


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UPREGULATION OF THE TRANSSULFURATION PATHWAY DURING GLIOTOXIN MEDIATEDINHIBITION OF GSH IN C6 GLIOMA CELLS

Sarah Kandil1, Lorraine Brennan2, Gethin McBean1

School of 1Biomolecular and Biomedical Science, UCD Conway Institute and School of 2Agriculture, Food Scienceand Veterinary Medicine, UCD Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland

L-aminoadipate (L-AA) and L-β-N-oxalyl-amino-L-alanine (L-BOAA) belong to a group of L-glutamate analoguesthat are toxic to astrocytes and are known collectively as gliotoxins. Both compounds target the xc

- cystine-glutamate exchanger, leading to a reduced intracellular concentration of cysteine and, consequently, aconsiderable reduction in the major antioxidant, glutathione (GSH). Recently, a functional transsulfuration pathwayhas been identified in astrocytes, in which cysteine can be supplied from methionine and thus contribute to the poolof cysteine required for GSH synthesis.

In this study, we have used gliotoxin-mediated inhibition of the xc- exchanger to evaluate the relative contribution

of the exchanger and the transsulfuration pathway to the supply of cysteine. Incubation of C6 glioma cells witheither gliotoxin significantly inhibited the xc

- exchanger and reduced GSH content of the cells to 44% and 46% ofcontrol, respectively, after 24 h. The cysteine content was 34% and 26% of control, respectively. Blockade of thetranssulfuration pathway with propargylglycine (PPG) caused a 22% reduction in GSH. A further reduction in GSHwas observed when the cells were co-incubated with L-BOAA (to 63% of control), but not when the cells were co-incubated with PPG and L-AA. Neither L-AA nor L-BOAA inhibits cystathionine--lyase; on the contrary,expression of this enzyme following incubation of C6 glioma cells with either gliotoxin was enhanced, leading to anincreased contribution of the transsulfuration pathway in GSH synthesis. It is concluded that an increased fluxthrough the transsulfuration pathway promotes de novo synthesis of GSH when the supply of cysteine via the xc

-

exchanger is limited.

THE EFFECTS OF MDMA AND CAFFEINE CO-ADMINISTRATION ON RAT BLOOD PRESSURE,HEART RATE AND TEMPERATURE

C. Downey1, J.J. Callanan2, K. Murphy1 K.M. O’Boyle1

1UCD School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Belfield, Dublin4, Ireland.2Department of Veterinary Pathology, University College Dublin, Belfield, Dublin 4, Ireland.

3,4-methylenedioxymethamphetamine (MDMA, ecstasy) is a common recreational drug of abuse. MDMA inducedtoxicity can be exacerbated by poly drug use. Ecstasy users commonly co-ingest psychoactive drugs to increase orprolong their “high”. Drugs such as ketamine, amphetamines and selective serotonin reuptake inhibitors (SSRI’s)are often used for this purpose. Along with knowingly ingesting various drugs ecstasy tablets are often containother ingredients such as caffeine, amphetamines, opioids and hallucinogens. Along with increasing evidence ofMDMA induced toxicity in humans, several drug interactions have been shown to occur which potentiate thedetrimental effects of MDMA. Caffeine a drug which is often consumed in the form of high energy drinks has beenshown to increase the levels of hyperthermia and serotonergic loss in rats when administered prior to MDMA. Ourown studies have shown that single doses of caffeine (20 mg/kg) and MDMA (20 mg/kg) which are well toleratedlead to increased mortality rates when combined. As caffeine is regularly consumed in the form of tea, coffee, highenergy drinks and can also often be found as an added ingredient in ecstasy tablets, this drug interaction couldhave serious health implications for human ecstasy users. Radiotelemetry devices capable of measuring ECG,blood pressure, body temperature and activity were used to monitor Wistar rats treated with either saline, caffeine(20 mg/kg), MDMA (20 mg/kg) or a combination of both caffeine (20 mg/kg) and MDMA (20 mg/kg). Caffeine alonehad little effect on blood pressure or temperature. MDMA administration resulted in increased locomotion, heartrate, blood pressure and body temperature.

The co-administration of MDMA and caffeine increased the MDMA induced hyperthermia and mean arterialpressure. Furthermore blood serum from co-treated rats contained higher levels of urea, creatinine and alkalinephosphatase than MDMA treated animals. This altered blood biochemistry indicates an impaired renal functionwhich may be attributable to increased dehydration and heart failure. These results would indicate that thecombination of MDMA and caffeine can have serious implications on cardiovascular function and body temperatureregulation which may prove to have a role in unexpected human fatalities resulting from ecstasy use.


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Sponsors in Attendance

PerkinElmer Ireland has kindly sponsored today’s Best Oral Presentation Prize


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Notes

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