29.05 - 01.06.2013 Gdynia, Poland
PROGRAM
ABSTRACT BOOK
2
Neuropeptides 2013 Conference
10
thJoint Meeting of the European Neuropeptide Club and the Summer
Neuropeptide Conference
29.05-01.06.13 Gdynia, Poland
PROGRAM
ABSTRACT BOOK
Index
General information ... 5
Program ... 9
Abstracts – Oral presentations ... 17
Abstracts – Poster presentations ... 45
List of authors ... 108
Notes ... 113
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Dear Colleagues,
We have a great pleasure to welcome you in Gdynia, at the 10th Meeting of the European Neuropeptide Club and the Summer Neuropeptide Conference.
The meeting is organized jointly by Department of Animal Anatomy, Department of Pathophysiology, Forensic Veterinary and Administration of the University of Warmia and Mazury in Olsztyn, and Olsztyn Division of the Polish Society of Veterinary Sciences.
The meeting is organized, according to the will of the participants of the 9th ENC meeting in London, in Gdynia, a city being a part of the 3City agglomeration, one of the most beautiful cities in Poland.
The venue, the Nadmorski Hotel, is located in a cosy and quiet neighbourhood, yet located virtually in the center of Gdynia. It offers exquisite conditions to have a successful meeting, as well as to enjoy the stay on seaside.
Organizers have done their best to attract speakers presenting talks covering hot topics in the field of neuropeptide research and related fields. We hope you will enjoy the stay in Gdynia both for scientific and personal reasons.
We also hope that this meeting will be a good occasion to make scientific and personal ties that will last for many years and bring many profits.
Have a great time in Gdynia!
Krzysztof Wąsowicz
Chair of the 2013 ENC Conference
General information
Venue
Nadmorski Hotel, 2 Ejsmond Street, Gdynia, Poland
Website
http://www.uwm.edu.pl/enc2013
Organizers
Organizing Committee
Miroslaw Lakomy, Honorary Chair Krzysztof Wasowicz, Chair
Zenon Pidsudko, Vice Chair
Michał Załecki, Administrative Secretary Piotr Podlasz, Scientific Secretary
Magdalena Klimczuk
Amelia Franke-Radowiecka Małgorzata Chmielewska Katarzyna Łosiewicz Oliwia Tomaszewska
European Neuropeptide Club
Erika Pinter, President
Susan Brain, Vice President
Eberhard Weihe, Past President, Vice President Illana Gozes, Secretary General
Barbara Kofler, Treasurer
Manfred Zimmerman, Honorary President
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Summer Neuropeptide Conference
Illana Gozes, Chair
Douglas E. Brenneman, Co-Chair Y. PengLoh, Co-Chair
Don Gehlert, Secretariat
Ildikó Antal, Treasurer, Finances
Scientific advisory board Ildikó Antal
Sándor Benyhe Anna Borsodi Susan D. Brain
Douglas E. Brenneman Don Gehlert
Pierangelo Geppetti Illana Gozes
Daniel Hoyer Jerzy Kaleczyc Andrzej Koncicki Y. PengLoh
Jason McDougall Inga Neumann
Riccardo Patacchini John Quinn
Registration
Registration desk is located at the main hall of the Nadmorski Hotel.
Registration fee includes admission to all sessions including the welcome reception, the conference materials, daily lunch and coffee (from Thursday to Friday), participation in Gala diner and 3City Sightseeing.
Slide and oral presentations
For computer presentations a CD-ROM, or USB device should be left to the audiovisual staff at the registration desk at least half an hour before start of the session. The presentation should be identified by the shortened title and the name of the first author. The presentation
should be saved in Microsoft Windows Power Point format (PC format).
All speakers are kindly requested to stay strictly within determined time limits.
Poster presentations
The size of poster should be not more than 80 cm (width) by 120 cm (height). Materials for mounting posters will be supplied.
Poster presentation is arranged in two sessions. Posters should be mounted in the morning on the day of the session and removed after the session to free space for next presentation. Posters not removed by owners will be removed by organizers.
Poster sessions will be held in the conference room.
Publication
Abstracts will be published in the Meeting Proceedings and full papers can be published in the Journal of Molecular Neuroscience For full paper preparation please refer
to http://www.editorialmanager.com/jomn/
Food and drinks
Coffee, tea and small refreshments will be served during coffee breaks and poster sessions. Lunches will be served at the hotel restaurant on Thursday and Friday. The badge serves as a lunch ticket.
Social programme
Wednesday, May 29, 2013
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Welcome reception will be held at the Nadmorski Hotel in the restaurant.
Thursday, May 30, 2013
Gala diner will be held at the Nadmorski Hotel in the restaurant.
Friday, May 31, 2013
3City sightseeing: Kosciuszko Square (the most representative and touristic place in Gdynia), Gdansk Oliwa - quick sightseeing of the Cathedral, Gdansk Center (Shipyard), Gdansk Old Town. Dinner.
Hotel plan
1. Conference room
2. Venezia Club ( entrance ) 3. Strefa restaurant
4. Breakfast lounge
5. Morskie Oko Banquet hall 6. Lobby bar
7. Reception Desk 8. Cloakroom 9. Toilets 10. Lifts
11. Genesis SPA 12. Business center 13. Kids playground
Program
WEDNESDAY (May 29th, 2013)
14.30-15.45 Registration
15.45-16.00 Opening
16.00-18.00 Symposium I: Neuroprotection Chair: Illana Gozes
16.00-16.30 Novel peptides related to activity-dependent neuroprotective protein: link to autophagy.
Illana Gozes (The Lily and Avraham Gildor Chair for the Investigation of Growth Factors
Director, Adams Super Center for Brain Studies, Sackler Faculty of Medicine, Tel Aviv University, Israel)
16.30-17.00 Examination of Ca2+ binding protein expression in the inner ear of wild type, PACAP-heterozygous and PACAP-deficient mice after kanamycin treatment Andrea Tamás (Department of Anatomy, University of Pécs, Hungary)
17.00-17.30 New advances in the neuroprotective effects of PACAP
Dóra Reglődi (School University of Pécs Medical School, Institute of Biochemistry and Medical Chemistry, Pécs, Hungary)
17.30-18.00 Orexins (hypocretins): pro- or antisurvival neuropeptides?
Jolanta B. Zawilska, (Department of Pharmacodynamics Medical University of Lodz, Poland)
18.00-19.00 Free program 19.00- Welcome drink
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THURSDAY (May 30th 2013)
8.00-9.00 Registration
9.00-10.30 Symposium II: TRPA1 and TRPV1 Chair: Julie Keeble
9.00-9.30 TRPA1-mediated vasodilatation in the peripheral vasculature
Aisah Aubdool, King's College London
9.30-10.00 Vascular effects of hyperthermic TRPV1 antagonists
Khadija Alawi, King's College London
10.00-10.30 The role of CGRP in TRPA1 receptor-mediated effects in skin
Erika Pintér (University of Pecs, Hungary)
10.30-11.00 Coffee break
11.00-12.30 Symposium III: CGRP Chair: Sue Brain
11.00-11.30 CGRP and cardiovascular biology
Susan Brain (King’s College London, UK)
11.30-12.00 The trigeminal nocisensor complex and CGRP in headaches
Mária Dux (University of Szeged, Hungary) 12.00-12.30 CGRP - the initial or final key mediator in
meningeal nociception and headache?
Karl Messlinger, (University of Erlangen, Germany)
13.00-14.30 Lunch break
14.30-16.00 Poster Session I (coffee).
16.00-17.30 Symposium IV: Galanin and other neuropeptides in various states
Chair: Mirosław Łakomy
16.00-16.30 Localization and immunocytochemical
characterization of the dorsal vagal nucleus (dmx) neurons projecting to the porcine stomach
prepyloric region in physiological state, following stomach partial resection and after prolonged acetylsalicylic acid supplementation
Jarosław Calka (University of Warmia and Mazury, Poland)
16.30-17.00 Galanin as anticonvulsant neuropeptide. studies in zebrafish (Danio rerio) model.
Piotr Podlasz (University of Warmia and Mazury, Poland)
17.00-17.30 Changes in the expression of neuropeptides and their receptors in enteritis in the pig
Krzysztof Wąsowicz (University of Warmia and Mazury, Poland)
17.30-20.00 Free program
20.00- Gala diner
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FRIDAY (May 31st 2013)
10.00-11.30 Symposium V: "Non-coding genomic influences on Neuropeptide activity and function”
Chair: Alasdair MacKenzie
10.00-10.30 Glucocorticoid receptor modulation of the TAC1 promoter in amygdala; polymorphisms, decoys, stress and anxiety.
Alasdair MacKenzie (University of Aberdeen, UK)
10.30-11.00 Genetic and epigenetic contribution to mood
disorders and obesity; the BDNF locus and beyond Benjamin Hing (University of Iowa, USA)
11.00-11.30 Use of online tool for in-depth SNP analysis Andrew Starkey (University of Aberdeen)
11.30-13.00 Poster Session II (coffee).
13.30-15.00 Lunch break
15.00-16.30 Concluding remarks and Young Investigator Awards
17.00- 3City sightseeng
Poster list
THURSDAY (May 30th 2013), 14.30-16.00 - Poster Session I.
Nr. Author Title
T1 Scheich B. INHIBITORY FUNCTION OF THE SOMATOSTATIN RECEPTOR SUBTYPE 4 (SST4) IN ANXIETY AND DEPRESSION-LIKE BEHAVIOURS IN THE MOUSE T2 Majewski M. THE COMPARISON OF THE EFFECTS OF
GUANETHIDINE (GUA), RESINIFERATOXIN (RTX) AND TETRODOTOXIN (TTX) ON THE CHEMICAL CODING OF INFERIOR MESENTERIC GANGLION (IMG)
NEURONS SUPPLYING PORCINE URINARY BLADDER.
T3 Balasko M. AGE-RELATED PATTERN OF ACUTE EFFECTS OF CORTICOTROPIN-RELEASING FACTOR ON ENERGY BALANCE
T4 Dudek A. CHEMICAL CODING AND DISTRIBUTION OF THE
TRAPEZIUS MUSCLE-PROJECTING MOTONEURONS IN THE PIG.
T5 Petervari E. EFFECTS OF ANOREXIGENIC AND OREXIGENIC NEUROPEPTIDES IN SPONTANEOUSLY
HYPERTENSIVE RATS
T6 Łosiewicz K. CHANGES IN THE EXPRESSION OF GAL AND THEIR RECEPTORS GALR1, GALR2 AND GALR3 IN THE COLITIS CAUSED BY BRACHYSPIRA
HYODYSENTERIAE INFECTION
T7 Kemény Á. CHANGES OF CYTOKINE PROFILE IN MONOSODIUM- IODOACETATE-INDUCED JOINT INFLAMMATION IN TRPA1 WT AND KO MICE
T8 Jakimiuk A. THE EXPRESSION OF RECEPTORS FOR SUBSTANCE P (NK1, NK2, NK3) IN TISSUES IN PIG (Sus scrofa)
T9 Cowie P. EXPLORING MECHANISMS OF DISEASE SUSCEPTIBILITY AND DRUG RESPONSE
STRATIFICATION AT THE CANNABINOID RECEPTOR 1 (CNR1) GENE LOCUS.
T10 Kun J. TRANSIENT RECEPTOR POTENTIAL ANKYRIN 1
(TRPA1) AND VANILLOID 1 (TRPV1) RECEPTOR mRNA EXPRESSION IN INFLAMMATORY BOWEL DISEASE (IBD) AND DEXTRAN SULFATE-INDUCED COLITIS IN MICE
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T11 Baryła M. THE EXPRESSION OF CALCITONIN GENE-RELATED PEPTIDE (CGRP) IN ZEBRAFISH (D. RERIO)
T12 Koziatek S. ANALYSIS OF GALANIN RECEPTORS GENES AND THEIR EXPRESSION IN ZEBRAFISH (DANIO RERIO) T13 Sandor B. TOOTH DEVELOPMENTAL DIFFERENCES BETWEEN
WILD TYPE AND PACAP-DEFICIENT MICE
T14 Pidsudko Z. IMMUNOHISTOCHEMICAL CHARACTERISTICS AND DISTRIBUTION OF SENSORY DORSAL ROOT GANGLIA NEURONS SUPPLYING THE URINARY BLADDER IN THE MALE PIG.
T15 Borbély É. ROLE OF THE CAPSAICIN-SENSITIVE SENSORY
NERVES IN AUTOANTIBODY-INDUCED ARTHRITIS OF THE MOUSE
T16 Zalecki M. THE INTRINSIC INNERVATION OF THE CARDIAC GLAND AND PYLORUS IN THE EUROPEAN BEAVER (CASTOR FIBER).
FRIDAY (May 31st 2013), 11.30-13.00 - Poster Session II
Nr. Author Title
F1 Biernacka B. THE ROLE OF GALANIN IN MODIFYING GI MORPHOLOGY IN A RAT MODEL OF POST- TRAUMATIC STRESS DISORDER
F2 Tékus V. ROLE OF THE TRANSIENT RECEPTOR POTENTIAL ANKYRIN 1 (TRPA1) ION CHANNEL IN NOCICEPTIVE PROCESSES
F3 Bossowska A. THE COMPARISON OF CONANTOKIN G (CTG),
RESINIFERATOXIN (RTX) AND BOTULINUM TOXIN A (BTX) EFFECTS ON THE CHEMICAL CODING OF
DORSAL ROOT GANGLIA (DRG) NEURONS SUPPLYING PORCINE URINARY BLADDER . F4 Botz B. PRO-INFLAMMATORY AND PRO-NOCICEPTIVE
ROLES OF PITUITARY ADENYLATE-CYCLASE ACTIVATING POLYPEPTIDE (PACAP) IN A SERUM TRANSFER ARTHRITIS MOUSE MODEL
F5 Szitter I. ROLE OF NEUROKININ 1 (NK1) RECEPTORS IN DEXTRAN SULFATE-INDUCED COLITIS: STUDIES WITH GENE-DELETED MICE AND A SELECTIVE RECEPTOR ANTAGONIST
F6 Dudek A. CHEMICAL CODING AND DISTRIBUTION OF THE TRAPEZIUS MUSCLE-PROJECTING SENSORY NEURONS IN THE PIG.
F7 Szőke É. EFFECT OF RESOLVIN D1 AND RESOLVIN D2 ON TRP ION CHANNEL ACTIVATION
F8 Atlasz T. PITUITARY ADENYLATE CYCLASE-ACTIVATING POLYPEPTIDE (PACAP) PREVENTS MONOSODIUM GLUTAMATE (MSG) INDUCED FUNCTIONAL
DISTURBANCES IN THE MOUSE RETINA IN VITRO.
F9 Franke-
Radowiecka A.
INNERVATION OF THE MAMMARY GLAND IN THE BEAVER (CASTOR FIBER). PRELIMINARY STUDY.
F10 Sághy É. RECEPTOR ACTIVATION BY PITUITARY
ADENYLATE-CYCLASE ACTIVATING POLYPEPTIDE ANALOGUES ON TRANSFECTED CELL LINES AND CELL BODIES OF PRIMARY SENSORY NEURONS F11 Klimczuk M. IMMUNOHISTOCHEMICAL CHARACTERISATION OF
NERVE FIBRES SUPPLYING THE MANDIBULAR,
SUBLINGUAL AND PAROTID GLAND IN THE BEAVER (CASTOR FIBER)
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F12 Borbély É. ROLE OF HEMOKININ-1 AND NK1 RECEPTORS IN ANXIETY, STRESS AND DEPRESSION-LIKE
BEHAVIOUR IN MICE
F13 Pidsudko Z. IMMUNOHISTOCHEMICAL CHARACTERISTICS AND DISTRIBUTION OF NEURONS IN THE
PARAVERTEBRAL, PREVERTEBRAL AND PELVIC GANGLIA SUPPLYING THE URINARY BLADDER IN THE MALE PIG.
F14 Hajna Zs. TRANSIENT RECEPTOR POTENTIAL ANKYRIN 1 (TRPA1) RECEPTOR PLAYS A PROTECTIVE ROLE IN ENDOTOXIN-INDUCED ACUTE PNEUMONIA, BUT NOT IN SMOKING-RELATED CHRONIC BRONCHITIS OF THE MOUSE
F15 Zalecki M. EXTRINSIC AFFERENTS PROJECTING TO THE PYLORUS IN THE DOMESTIC PIG - LOCALIZATION AND NEUROCHEMICAL CHARACTERISTIC
F16 Perkecz A. HEMOKININ-1 IS A KEY MEDIATOR OF CHRONIC ARTHRITIS-ASSOCIATED AND ACUTE ENDOTOXIN- INDUCED LUNG INFLAMMATION OF THE MOUSE
Abstracts - Oral presentations
EXAMINATION OF CA2+ BINDING PROTEIN EXPRESSION IN THE INNER EAR OF WILD TYPE, PACAP-
HETEROZYGOUS AND PACAP-DEFICIENT MICE AFTER KANAMYCIN TREATMENT
Tamas A.1, Fulop B.1, Szabadfi K.2, Kiss P.1, Nemeth A.3, Hashimoto H.4, Baba A.4,. Gabriel R.2, ReglodiD.1
1Departments of Anatomy, PTE-MTA „Lendület” PACAP Research Team,
2Experimental Zoology and Neurobiology, 3 Otorhinolaryngology, University of Pecs, Hungary; 4Graduate School of Pharmacological Science, Osaka
University, Osaka, Japan
Pituitary adenylate cyclase activating polypeptide (PACAP) is a multifunctional neuropeptide with well known neuroprotective and neurotrophic effects. Recently, we have shown that PACAP protects cochlear cells against oxidative stress-induced apoptosis in vitro, but there are no data about its effects in different ototoxic insults such as aminoglycoside-induced toxicity.
In this study we examined the effect of a single dose of ototoxic kanamycin treatment (1g/kg) on Ca2+ binding protein expression in hair cells of wild type, PACAP-heterozygous and PACAP-deficient mice. We treated 5-day-old mice with kanamycin subcutaneously and 2 days later we examined the Ca2+ binding protein (parvalbumin, calretinin) expression of the hair cells with immunohistochemistry.
Contol animals received physiological saline.
We found that the inner and outer hair cells of control PACAP- deficient mice and outer hair cells of PACAP-heterozygous mice showed more pronounced parvalbumin and calretinin immunopositivity compared to control wild-type mice. Elevated endolymphatic Ca2+ is deleterious for the cochlear function, against which the high concentration of Ca2+ buffers in hair cells may protect. Meanwhile, the increased immunoreactivity of Ca2+ binding proteins in the absence of PACAP provide further evidence for the important protective role of PACAP in hair cells against pathological
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conditions. Kanamycin induced a significant elevation in Ca2+
binding protein expression in hair cells of wild-type and PACAP- heterozygous mice, but the baseline higher expression in PACAP- deficient mice did not change significantly after the treatment. Our results showed significant differences in the inner ear of wild type, PACAP-heterozygous and PACAP-deficient mice after kanamycin treatment, which indicate the important role of PACAP in ototoxicity, but further investigations are necessary to examine the exact role of endogenous PACAP in ototoxic insults.
This work was supported by PTE-MTA “Lendület” Program, Richter Foundation, Arimura Foundation, OTKA K104984, TAMOP 4.2.1.B- 10/2/KONV-2010-002, 4.2.2.B-10/1-2010-0029, 4.2.2.A-
11/1/KONV-2012-0024, 4.2.4.A/2-11-1-2012-0001.
NEW ADVANCES IN THE NEUROPROTECTIVE EFFECTS OF PACAP
Reglodi D.1, Kiss P.1, Jungling A.1, Rivnyak A.1, Szabadfi K.2, Gabriel R.2, Atlasz T.3, Opper B.1, Horvath G.1, Tizabi Y.4, Brown
D.4, Szabo A.5, Kovacs K.5, Welke L.6, Tamas A.1
1Department of Anatomy, PTE-MTA “Lendület” PACAP Research Team,
2Department of Experimental Zoology and Neurobiology , 3Department of Sportbiology, 5Department of Biochemistry and Medical Chemistry, University
of Pecs, Hungary; 4Department of Pharmacology, Howard University College of Medicine Washington DC, USA; 6Department of Anatomy, Ross University
School of Medicine, Dominica
Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide originally isolated from the hypothalamus based on its cAMP-elevating effect in the pituitary. During the last 20 years a vast amount of evidence has emerged showing that PACAP acts on different receptors and has diverse biological effects. One of the well- studied effects of this neuropeptide is the strong neuroprotective effect. Its neuroprotective action was first shown in global and focal cerebral ischemia, and subsequently in numerous different in vitro and in vivo models of nervous system pathologies. In vitro, the peptide has been shown to protect neurons and glial cells against oxidative stress, hypoxia, different toxic agents, ceramide and ethanol. In vivo, PACAP has protective effects in animal models of Parkinson`s disease, Huntington chorea, retinal degeneration, multiple sclerosis, traumatic brain injury and spinal cord injury. The aim of the present study is to show recent progress in the neuroprotective effects of PACAP. In Parkinson`s disease, our laboratory and others have demonstrated that PACAP-deficient mice show higher susceptibility to toxic agents causing degeneration of the substantia nigra dopaminergic neurons. In vitro, we have shown that PACAP is protective against salsolinol-induced neuronal death (an in vitro model of Parkinson`s disease), involving decreases in caspase-3 levels and increases in brain-derived neurotrophic factor and CREB phosphorylation. In the retina, our resent results show that PACAP has protective effects in diabetic retinopathy and increased level of
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degeneration can be observed in PACAP knockout mice. In diabetic retinopathy, increases in anti-apoptotic factors and decreases in pro- apoptotic signaling molecules have been detected. In summary, recent results support the neuroprotective effects of both endogenous and exogenous PACAP and provide further insight into its antiapoptotic effects in neuronal cells.
Support: MTA Lendulet Program, TAMOP (4.2.1.B-10/2/KONV- 2010-002, 4.2.2.A-11/1/KONV-2012-0024), NIH/NIGMS 2 SO6 GM08016-39, Arimura Foundation, Ross University Research Fund, PTE AOK Research Fund, Richter Foundation, Bolyai Scholarship and OTKA K104984
NOVEL PEPTIDES RELATED TO ACTIVITY-DEPENDENT NEUROPROTECTIVE PROTEIN: LINK TO AUTOPHAGY
Gozes I., Malishkevich A., Wagner A.M.
The Adams Super Center for Brain Studies; The Lily and Avraham Gildor Chair for the Investigation of Growth Factors; The Elton Laboratory for
Neuroendocrinology; Department of Human Molecular Genetics and Biochemistry, School of Neuroscience, Sackler Faculty of Medicine, Tel Aviv
University, Tel Aviv 69978, Israel
Using protein structure activity scanning, we discovered the novel peptide drug candidate NAP (davunetide) in activity-dependent neuroprotective protein (ADNP). The ADNP-derived peptide NAP provides protection to the microtubule system, an essential component of the nerve cell skeleton (1), defending against the microtubule- associated protein tau pathology (2), which results as a consequence of ADNP deficiency (3, 4). Published data in a tauopathy model showed reduced neurodeneration by increased autophagy (5) at the level of activation of microtubule-associated protein 1 light chain 3 (LC3) cleavage. Autophagy is a highly regulated process, having a crucial impact on cellular homeostasis. One of the main autophagy markers is the family of the LC3 proteins (LC3, LC3A and LC3B);
(6). LC3 is cleaved at the C-terminal to LC3-I and transformed by phosphatidylethanolamine (PE) to LC3-II. LC3II connects to the autophagosome membrane (7). Published two-hybrid system analyses showed a potential direct interaction between LC3B and ADNP (8).
Our immunoprecipitation studies further suggested an interaction between ADNP and NAP and LC3B. Thus, NAP and related pipeline peptides may provide protection through regulation of autophagy (9).
Selected reading:
1. S. Oz, Y. Ivashko-Pachima, I. Gozes, PLoS One 7, e51458 (2012).
2. A. Idan-Feldman, R. Ostritsky, I. Gozes, Int J Alzheimers Dis 2012, 493670 (2012).
3. I. Vulih-Shultzman et al., J Pharmacol Exp Ther 323, 438 (Nov, 2007).
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4. N. Shiryaev, R. Pikman, E. Giladi, I. Gozes, Curr Pharm Des 17, 2603 (2011).
5. V. Schaeffer et al., Brain 135, 2169 (Jul, 2012).
6. J. Wu et al., Biochem Biophys Res Commun 339, 437 (Jan 6, 2006).
7. N. Mizushima, Genes Dev 21, 2861 (Nov 15, 2007).
8. A. Vinayagam et al., Sci Signal 4, rs8 (Sep 6, 2011).
9. A. Merenlender-Wagner, Z. Shemer, E. Giladi, A. Andrieux, I.
Gozes, (Sumbitted for publication).
OREXINS (HYPOCRETINS): PRO- OR ANTISURVIVAL NEUROPEPTIDES?
Zawilska J. B.
Institute of Medical Biology Polish Academy of Sciences, PL93-232 Lodz, 106 Lodowa Str, Department of Pharmacodynamics, Medical University of Lodz, PL
90-151 Lodz, Poland
Orexin A and orexin B (also known as hypocretin 1 and hypocretin 2) are closely related neuropeptides that were concurrently discovered in 1998 by two research groups. The most striking feature of orexins is their predominant expression in a few thousand neurons in the lateral and dorsomedial hypothalamus that project throughout the central nervous system. The peptides exert numerous biological functions by binding to two subtypes of G protein-coupled receptors, OX1R and OX2R. OX1R has higher affinity for orexin A than for orexin B, while OX2R binds orexin A and orexin B with a similar potency. Accumulating experimental evidence demonstrate an important role of orexins in the regulation of vigilance and the sleep/wake cycle, feeding, appetite and metabolic processes. In addition to that, the peptides are involved in the control the hypothalamo-pituitary-adrenal axis and functions of miscellaneous peripheral organs. Stimulation of orexin receptors can induce cell death in recombinant CHO cells, native colon carcinoma and neuroblastoma cells, and a potent growth reduction of human colon tumors in mice xenografts.
In our studies we investigated effects of orexins on survival of cultured astrocytes and neurons from rat cerebral cortex and compared them with those exerted on rat C6 glioma cells, an experimental model for studies on glioblastoma multiforme. Quantitative real-time PCR revealed the presence of orexin receptors in all studied cell types, with the expression level in neurons being several-fold higher than in astrocytes and C6 cells. Orexins and [Ala11-D-Leu15]orexin B (a selective agonist of OX2), used at 0.001-1 microM concentrations, markedly increased neuronal cells viability. The pro-survival properties of orexins were associated with an attenuation of caspase-3 activity. Comparable potency of the three peptides suggests a
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predominant role of OX2R in the studied phenomenon. Orexin A also increased neuronal cells survival under conditions of chemical hypoxia. This effect was blocked by U0126 and 10-DEBC, inhibitors of MEKK and Akt, respectively. Orexins did not affect viability of cultured cortical astrocytes. Incubation of C6 cells with orexin A (0.001-1 microM), but not with orexin B, resulted in a marked decrease of cell viability and a parallel increase in caspase-3 activity.
The inhibitory effect of orexin A on survival of rat C6 glioma cells was blocked by SB 202190 (a specific p38 MAPK inhibitor) and by Z-VAD-fmk (a pan-caspase inhibitor). Direct assessment of DNA synthesis by [3H]thymidine incorporation into DNA of rat C6 glioma cells revealed that orexins did not alter cell proliferation. Our results provide further support for the hypothesis that cell neoplastic transformation might be associated with an activation of OX1R- mediated apoptosis. On the other hand, orexins acting at OX2R could exert neuroprotective activity.
Supported by the grant from the Ministry of Science and Higher Education, Warsaw, Poland (NN 301 425438).
TRPA1-MEDIATED VASODILATATION IN THE PERIPHERAL VASCULATURE
Aubdool A.1, Graepel R.1, Kodji X.1, Fernandes E. S.1, Bevan S.2, Brain S. D1
1Cardiovascular Division BHF-Centre of Cardiovascular Excellence and Centre of Integrative Biomedicine, King's College London, 2Wolfson CARD Centre,
King's College London, London, SE1 9NH, UK
Transient receptor potential ankyrin-1 (TRPA1) is a non-selective thermosensitive cation channel which is widely expressed in a subset of sensory neurons. We have previously shown that intraplantar injection of the TRPA1 agonist cinnamaldehyde mediates vasodilatation in the mouse hindpaw via a TRPA1-dependent manner (Pozsgai et al., 2010). However, the relevance of this to cardiovascular regulation is not known. Here, we have investigated the ability of TRPA1 to influence cold responses, in addition to responses induced by cinnamaldehyde in the peripheral vasculature.
Initial studies used the mouse ear model where cinnamaldehyde was applied topically in the anaesthetised mice (ketamine-75mg/kg and medetomidine-25mg/kg, i.p.) and blood flow was measured in vivo using laser Doppler flowmetry. Results are shown as area under the response flux curve and expressed as mean + S.E.M. Subsequent studies investigated the effects of local cold exposure to the mice plantar skin. Blood flow was measured before (5-10 min for baseline readings) and after local cold exposure of the mouse hindpaw (30 min). Both cold exposure and cinnamaldehyde mediates vasodilatation in the hindpaw and ear, respectively. Cold-induced vasodilatation was substantially reduced in TRPA1 KO (92.1 + 10.8 x 1000 flux units), as compared to WT mice (226.8 + 21.4 x 1000 flux units, n=12, p<0.01) and significantly inhibited by the selective TRPA1 antagonist HC030031 (100mg/kg, i.p., n=10-12, p<0.001). Both cinnamaldehyde- and cold-induced vascular responses were shown to be significantly reduced in WT mice pre-treated with the selective calcitonin gene related peptide (CGRP) receptor antagonist, CGRP8-37, the substance P neurokinin-1 receptor antagonist SR140333 and a non-selective nitric oxide synthase inhibitor L-NAME, suggesting a
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prominent role of neuropeptides and nitric oxide in this vasoactive response. We provide novel evidence of a major involvement of TRPA1-containing sensory nerves in local cold-induced vascular responses in vivo.
Pozsgai et al. (2010) Cardiovasc. Res., 87(4), 760-8.
This study was supported by a BBSRC-led IMB capacity building award.
INVESTIGATING TRPV1 –DEPENDENT EFFECTS ON THE VASCULATURE IN VIVO
Alawi K., Keeble J.
Institute of Pharmaceutical Science, School of Biomedical Sciences, King’s College London
The transient potential receptor vanilloid 1 (TRPV1) receptor is a non- selective cation channel that is activated by capsaicin, (the pungent component of hot peppers), temperatures within the noxious range (>43 degrees C) and low pH (<pH 6.0). TRPV1 receptors are expressed in primary afferent fibres, such as A-delta and C-fibres, and non-neuronal cells such as vascular smooth muscle. Capsaicin- induced TRPV1 activation on sensory nerves leads to vasodilation via neuropeptide release, whereas TRPV1 activation on vascular smooth muscle causes vasoconstriction (Kark et al., 2008). It has been hypothesized that the distinct effects of TRPV1 activation on vascular beds is due to different subsets of TRPV1 receptors; neuronal TRPV1 activations results in vasodilation whilst non-neuronal TRPV1 activation results in vasoconstriction. We have studied the paradoxical effect of TRPV1 activation on the vasculature utilizing pharmacological tools, genetic ablation of the receptor and laser Doppler flowmetry/imagery.
Male & female, age matched TRPV1 Knockout (KO) and Wild Type (WT) mice and CD1 strain mice (Charles River UK) were used in all experiments, in accordance with the UK Scientific Procedures Act 1986. Animals were pre-treated with the selective TRPV1 antagonist, AMG9810 (50mg/kg) or vehicle, and capsaicin-induced blood flow changes were investigated in the pinnae of the ears (20 µl, 10mg/ml in ethanol) and the exposed synovial membrane of the knee joints (10 µl, 10 nmol in 100% ethanol) by laser Doppler imagery under ketamine (75mg/kg) & medetomidine (25mg/kg i.p.) anaesthesia, The contralateral ear/knee joint was treated with vehicle.
In separate studies, acute changes in blood flow in the pinnae of the ears of anaesthetised mice (ketamine & medetomidine) was investigated utilising the moorVMS-HEAT Skin Heater Module
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(VHP2) with an integrated P3 needle probe connected to the moorVMS-LDF Laser Doppler blood flow and temperature monitor (Moor Instruments, UK). Baseline blood flow was obtained for 5 min and then the ears were heated to 45°C for a duration of 5 min, in 0.1°C/sec increments; blood flow was monitored following the cessation of heating for 30 min.
Capsaicin induced a significant increase in blood flow of the ears, which was significantly inhibited by the TRPV1 antagonist AMG9810. However, capsaicin induced a rapid and significant reduction in blood flow in the exposed synovial membrane, which was not attenuated with AMG9810 treatment. All the vascular effects of capsaicin were abolished in TRPV1 KO mice. Heating of the ears resulted in a profound increase in cutaneous blood flow, which was significantly attenuated in TRPV1 KO mice vs. TRPV1 WT mice.
Interestingly, AMG9810 did not result in any attenuation of the significant increase in blood flow in the ears of CD1 mice in comparison to vehicle-treated mice. The heat-induced vasodilation provides a novel tool in investigating heat-induced TRPV1 activation.
Further experiments are necessary to identify the mediators involved downstream of heat-induced vasodilation.
THE ROLE OF CGRP IN THE TRPA1 RECEPTOR- MEDIATED EFFECTS
Pintér E., Hajna Z., Pozsgai G., Bagoly T., Helyes Zs., Szolcsányi J.
Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, University of Pécs, Hungary
Background: Capsaicin-sensitive sensory neurons express several ion channels of the TRP family, such as transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) receptors. TRPA1 can be activated by several chemical stimuli, such as allylisothiocyanate (AITC) or cinnamaldehyde resulting in the release of inflammatory neuropeptides, e.g. calcitonin gene-related peptide (CGRP). Recently, the gaseous mediator hydrogen sulphide (H2S) was suggested to have effect on capsaicin-sensitive sensory neurons acting via TRPA1 receptors. TRPA1 activation-induced CGRP release from sensory neurons of isolated rat trachea and microcirculatory changes in the mouse ear have been investigated in this study.
Methods: Male Wistar rats (180–250 g) were deeply anaesthetised with sodium thiopentone (100 mg/kg, i.p.) and exsanguinated. Isolated tracheas were stimulated by allyl-isothiocyanate (AITC) or H2S donor sodium hydrogen sulphide (NaHS). Two tracheas were placed in the same organ bath (1.8 mL) and perfused with oxygenated (95%
oxygen, 5% CO2) Krebs solution (37°C, 1 mL/min). CGRP release was measured by radioimmunoassay. Blood flow experiments were carried out using Balb/c, C57BL/6, TRPA1 KO and TRPV1 KO mice (20-30 g). In the first part of the experiment Balb/c mice were treated with the selective TRPA1 antagonist HC-030031 (i.p., 30 or 100 mg/kg) 30 minutes before application of AITC or NaHS. In the second phase of the investigation TRPA1 and TRPV1 gene-deleted mice and their wild-type littermates were used. In both phases, right ears of the animals were treated with 2% AITC or 5% NaHS, while left ears received respective vehicles. Skin blood flow of the ears was detected by laser Doppler imaging (Perimed) for 30 minutes in case of AITC-, and for 50 minutes in case of NaHS-application.
Results: AITC induced CGRP releasing from isolated rat trachea (EC50: 61.42 μmol/L). NaHS evoked a concentration-dependent
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increase of CGRP releasing (EC50: 0.96 mmol/L) which was inhibited by HC-030031 but was not affected by TRPV1 receptor blocker BCTC. AITC treatment of mouse ears led to 29.8±2.8%
increase in blood flow. NaHS elevated cutaneous blood flow by 61±4.5%. Effect of NaHS on microcirculation was ameliorated by HC-030031. Blood flow of TRPA1 KO mice showed significantly smaller increase in response to NaHS compared to the wild-type counterparts. Vasodilatation in TRPV1 KO animals did not differ from control. CGRP-8-37 peptide antagonist of CGRP only partly inhibited the response.
Conclusions: We conclude that activation of TRPA1 receptors causing CGRP release from sensory nerves of rat trachea, as well as inducing cutaneous vasodilatation in the mouse ear. TRPV1 receptors are not involved in these processes.
This work supported by research grants OTKA K-81984 and Baross Gábor Program.
CGRP A POTENT VASODILATOR, BUT WHAT IS ITS FUNCTIONAL RELEVANCE IN THE VASCULATURE?
Smillie S-J., King R., Brain S. D.
BHF- Centre of Cardiovascular Excellence and Centre of Integrative Biomedicine, King’s College London, London SE1 9NH
The 37 amino acid peptide calcitonin gene-related peptide (CGRP) primarily localised to sensory nerve fibres that innervate throughout the body, with an extensive perivascular localisation and a dual role in sensory and efferent function. The major cardiovascular activity of CGRP is its potent vasodilator activity that is obvious when exogenous CGRP is administered at fentomolar doses to the skin of human and animal species. However, CGRP is not generally thought to have a role in the regulation of baseline blood pressure. Extensive studies in animal models indicate that deletion or blockade of CGRP worsens cardiovascular disease primarily through effects on the heart and kidney.
We have been investigating the role of CGRP in the regulation of blood pressure using wildtype (WT) and CGRP knock out (KO) mice.
The WT and CGRPKO mice show similar blood pressure. On the other hand the chronic administration of Ang II (1.1 mg/kg/day for 14 days), to model a hypertensive state was associated with a raised blood pressure that was significantly higher in CGRPKO mice. The KO mice also exhibited enhanced vascular inflammation, in terms of enhanced aortic fibrosis. In addition increased gene and protein expression of VCAM-1 and decreased expression of endothelial- derived nitric oxide synthase was observed in CGRPKO mice, in keeping with the concept that CGRP protects against vascular inflammation.
The results support the concept that CGRP may have therapeutic potential for protecting against the onset of hypertension and vascular disease. However, progress has been limited in understanding the relevance of CGRP in human disease.
This study is supported by the British Heart Foundation and the BBSRC via a Mammalian Biology Capacity Building Grant.
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THE CONCEPT OF THE TRIGEMINAL NOCISENSOR COMPLEX OFFERS A NEW APPROACH TO THE
UNDERSTANDING OF THE PATHOPHYSIOLOGICAL ROLE OF CGRP IN HEADACHES
Dux M., Sántha P., Jancsó G.
Department of Physiology, University of Szeged, Dóm tér 10, Szeged, Hungary
The involvement of the trigeminovascular system in the pathomechanisms of primary headaches is well established. Our studies focused on the role of trigeminal chemosensitive primary afferents, which express the nociceptor ion channels TRPV1 and TRPA1 and mediate both the central transmission of nociceptive signals and the peripheral release of the vasodilator neuropeptide CGRP in the dura mater. Morphological studies using electron microscopy and immunohistochemistry disclosed the existence and distribution of C-fiber chemosensitive TRPV1- and CGRP-positive sensory nerves in the rat dura mater. Functional studies suggest that exogenous and endogenous activators of TRP channels, such as inhaled environmental irritants, ethanol or membrane lipid metabolites may provoke headaches through the activation of chemosensitive trigeminal afferents and induce changes in local blood flow of the meninges. Activation by mast cell tryptase of presynaptic PAR-2 receptors localized on chemosensitive trigeminal afferents sensitizes the nociceptive TRP channels and, consequently, the trigeminal nocisensor complex. Crosstalk between TRPV1 and TRPA1 channels might bear of special importance under conditions of receptor sensitization. Pharmacological manipulations which interfere with the activation of these TRP channels, and reduce neuropeptide release from both the peripheral and the central terminals of chemosensitive trigeminal afferents, may offer new approaches for the management of primary headaches. Supported by OTKA K-101873, TÁMOP 4.2.2.A- 11/1/KONV 2012-0052 and TÁMOP 4.2.2./B-10/1-2010-0012.
CGRP - THE INITIAL OR FINAL KEY MEDIATOR IN MENINGEAL NOCICEPTION AND HEADACHE?
Messlinger K., Albrecht S., Denner A.-C., De Col R., Nixdorf- Bergweiler B.
Institute of Physiology and Pathophysiology, University of Erlangen-Nuernberg, Germany
Calcitonin gene-related peptide (CGRP) is regarded as a key mediator in migraine and other primary headaches. Elevated concentrations of CGRP have been found during migraine attacks in the jugular venous plasma. Migraine pain is alleviated by triptans inhibiting CGRP release and by blockade of CGRP receptors with
“gepants”, suggesting the involvement of CGRP receptors in trigeminal nociceptive processes. However, CGRP is not activating nociceptive afferents, rather CGRP release requires the activation of peptidergic afferents. This discrepancy shows that the role of CGRP and the site of its action in the cascade of nociceptive events leading to headaches are unclear. Several lines of evidence from recent animal experiments indicate an exclusive central effect of CGRP acting as neuromodulator in the spinal trigeminal nucleus and possibly at higher centers of the headache producing pathway. In addition, CGRP release is linked to metabolic processes that include nitroxyl (NO-), a congener of nitric oxide (NO), and the activation of transient receptor potential ankyrin channels (TRPA1). In the hemisected rat and mouse cranial peparation in vitro, Angeli’s salt, a donor of nitroxyl, caused transient activation of meningeal afferents, whereas NONOate, an NO donor, was ineffective. Angeli’s salt and TRPA1 receptor agonists like acrolein also caused increased CGRP release from trigeminal tissues, which was abolished by blocking TRPA1 receptors. However, TRPA1 activation did not cause propagated meningeal afferent activity in vitro and did not increase neuronal activity in the rat spinal trigeminal nucleus in vivo. In mouse trigeminal brainstem slices superfusion of CGRP and Angeli’s salt caused depolarization of neurons and facilitated spiking activity. Taken together, we conclude that the neuronal activity in the trigeminal brainstem, which is critical for
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headache generation, may be controlled by local nitroxyl generation and CGRP release possibly involving TRPA1 receptor channels.
LOCALIZATION AND IMMUNOCYTOCHEMICAL
CHARACTERIZATION OF THE DORSAL VAGAL NUCLEUS (DMX) NEURONS PROJECTING TO THE PORCINE STOMACH PREPYLORIC REGION IN PHYSIOLOGICAL STATE, FOLLOWING STOMACH PARTIAL RESECTION
AND AFTER PROLONGED ACETYLSALICYLIC ACID SUPPLEMENTATION.
Gańko M., Całka J.
Dept. of Clinical Physiology, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Oczapowskiego 13, 10-718 Olsztyn, Poland
To investigate localization and chemical coding of the parasympathetic DMX neurons supplying the porcine stomach prepyloric area Fast Blue was injected into the studied region of control, resection (RES) and acetylsalicylic acid (ASA) group.
Following paraformaldehyde perfusion fixation the DMX sections were stained immunocytochemically to ChAT, PACAP,VIP, NOS, GAL, CART, SP and LENK.
Fluorescence microscopy revealed 485,2 ± 42,7, 575,2 ± 76,22 and 705,8 ± 61,04 of the FB+ perikarya in DMX of control, RES and ASA group, respectively. All FB+ cells were ChAT+. In the control DMX 30,08 ± 1,97% of the FB+ neurons expressed PACAP, while no other peptides were found in the FB-labeled perikarya. In the RES DMX PACAP was found in 45,58 ± 2,2%, VIP in 28,83 ± 3,63%, NOS in 21,22 ± 3,32% and GAL in 5,67 ± 1,49% of the FB+ perikarya. In the ASA DMX PACAP was revealed in 49,53 ± 5,73%, VIP in 40,32±
7,84%, NOS in 25,02 ± 6,08% and GAL in 3,37 ± 0,85% of the labeled neurons.
Our research for the first time revealed:
I. Expression of PACAP in the porcine vagal parasympathetic neurons projecting to the stomach prepyloric region.
II. Numerical increase of the FB+/PACAP+ somata in the DMX of the RES and ASA group.
III. De novo synthesis of the VIP, NOS and GAL in the retrogradely traced neurons following resection of the FB-injected area of the prepyloric stomach region as well as a result of the long term
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acetylsalicylic acid administration. Acquired data indicate possible participation of PACAP, VIP, NOS and GAL in neural response to studied pathological states.
GALANIN AS ANTICONVULSANT NEUROPEPTIDE.
STUDIES IN ZEBRAFISH (DANIO RERIO) MODEL
Podlasz P.1, Chmielewska M.1, 2, Łosiewicz K.2, Panula P.3, Wąsowicz K.2
1Department of Animal Anatomy and 2Department of Pathophysiology, Forensic Veterinary and Administration, Faculty of Veterinary Medicine, University of
Warmia and Mazury in Olsztyn, Poland.
3Neuroscience Center, Institute of Biomedicine/Anatomy, University of Helsinki, Finland.
Galanin is a 29-30 amino acids-long neuropeptide widely expressed in the central and peripheral nervous system. In the brain galanin may function as an inhibitory neurotransmitter or as a hypophysiotropic messenger in the anterior pituitary.
Neuroanatomical localization and physiological properties of galanin suggest that the peptide may be involved in the regulation of seizures.
Indeed, during the last years galanin emerged as a powerful endogenous inhibitor of epileptic activity. The zebrafish (Danio rerio) is a potential model for animal and human disease and drug screening.
The aim of this study was to develop a zebrafish model to investigate the antiepileptic properties of galanin. Immunohistochemical stainings were performed in embryos, larvae and adult zebrafish to localize galanin-IR fibers in the brain regions essential for the initiation of seizures. Automated analysis of behavior in 5dpf zebrafish larvae was also conducted with pentylenotetrazole that cause seizures. In this experiment, the effect of a systemically-active galanin analogs NAX 5055 and galnon was investigated. Used software allows to track zebrafish larval movement for measurement of kinetic parameters and determination of behavioural patterns. Preliminary results suggested that both analogs show anticonvulsant activities in the studied system.
We also demonstrated using qPCR that galanin analogs used in the studies significantly inhibit increase of expression of c-fos gene after exposure to the pentylenotetrazole. The results confirm that zebrafish can be an excellent model for the study function of neuropeptides including galanin and their possible use in the treatment of epilepsy.
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CHANGES IN THE EXPRESSION OF NEUROPEPTIDES AND THEIR RECEPTORS IN ENTERITIS IN THE PIG
Wąsowicz K.1, Podlasz P.2, Chmielewska M.1, 2,Łosiewicz K.1, Załęcki M.2, Pidsudko Z.2, Kaleczyc J.2, Łakomy M.2
1Department of Pathophysiology, Forensic Veterinary and Administration, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn,
Poland.
2Department of Animal Anatomy, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Poland.
The anatomy and physiology of the gastrointestinal tract of the domestic pig reselmbles that of humans so much that this species makes an ideal model animal for studies of the physiology and pathology of the alimentary tract. Ulcerative colitis and inflammatory bowel disease are debiliating diseases affecting the growing share pf the human population, especially in developed countries. Combining these two facts we decided many years ago to adopt the porcine model to study the influence of enteritis on many aspects of physiology of porcine gut, mainly on the autonomic innervation and function with a special stress put on the role of neuropeptides. We used two main models, one associated with proliferative inflammation occurring in Lawsonia intracellularis infection and in ulcerative inflammation caused by Brachyspira hyodysenteriae.
We studied changes in neuropeptide concentration, as well as changes in the expression of neuropeptides and neuropeptide receptors. The studied aspects varied with time according to changing research abilities.
In proliferative enteritis we found dramatic changes in the concentration of galanin (Gal), vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY) in the colon, with concentrations of GAL and NPY raised by the factor of ten. In the ileum the concentration of Gal and VIP rose two-fold and the concentration of NPY rose three- fold.
In swine dysenteria the concentrations of Gal in the ileum rose two-fold and that of Substance P (SP) rose by 60%. In the colon we studied the changes in the expression of Gal and its three receptors, as
well as the expression of SP and three tachykinin receptors in the mucosa, mucosal lymphocytes and muscular membrane. We detected downregulation of the expression of Gal, as well as GalR1, GalR2 and GalR3 receptors, with the most dramatic changes found in mucosal lymphocytes. Similar changes regarded the expression of SP and NK1, NK2 and NK3 receptors, except for NK2 receptor whose expression rose in colonic muscular membrane.
Our data indicate the clear relation between the inflammatory process and the neuropeptides and their receptors in the gut.
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GLUCOCORTICOID RECEPTOR MODULATION OF THE TAC1 PROMOTER IN AMYGDALA; POLYMORPHISMS,
DECOYS, STRESS AND ANXIETY.
MacKenzie A.,. Hay C. W., Shanley L., Davidson S., Cowie P.
School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Aberdeen, Scotland, UK, AB39 3UW.Phone 01224 437380. E-mail;
alasdair.mackenzie @abdn.ac.uk.
Numerous studies have established a strong link between stress hormone release and the development of anxiety related disorders.
Intriguingly, over-expression of substance P (SP; encoded by the TAC1 gene) in the amygdala increases anxiety related behaviour. In the current study we show that the TAC1 gene is up-regulated in amygdala neurones in response to activation of the glucocorticoid receptor (GR). Using a combination of bioinformatics, electrophoretic mobility shift assays (EMSA) and reporter plasmid magnetofection into primary amygdala neurones we identified a highly conserved dexamethasone (Dex) and forskolin responsive sequence (2GR) that binds the GR and is positioned 448 base pairs 5’ (-448 bp) of the TAC1 transcriptional start site (TSS) in humans. We also identified a second GR binding site that was polymorphic (SNPGR, rs17169049- G/T, -650bp) and whose T-allele is found in 20% and 12% of Japanese and Chinese populations respectively. We present evidence that the T-allele of SNPGR increases the activity of the TAC1 promoter through de-sequestration or de-repression of 2GR. The identification of a Dex/forskolin response element in the TAC1 promoter is an important component for understanding the complex links between stress and anxiety. In addition, the discovery of a SNP which can alter this response may have important implications for our understanding of the role of regulatory variation in increasing susceptibility to stress related disorders in Asian populations.
GENETIC AND EPIGENETIC CONTRIBUTION TO MOOD DISORDERS AND OBESITY; THE BDNF LOCUS AND
BEYOND
Hing B.1, Davidson S.2, Tamashiro K.3, Lutter M.1, Potash J.1, MacKenzie A.4
1 University of Iowa, Department of Psychiatry, Medical Laboratories, Iowa City, Iowa, USA.
2 University of Toronto, Department of Cell & Systems Biology, Toronto, Ontario, Canada
3 Johns Hopkins University School of Medicine, Department of Psychiatry &
Behavioral Sciences, Baltimore, Maryland, USA.
4 Institute of Medical Sciences, University of Aberdeen, Foresterhill, Scotland, United Kingdom
Major depression and obesity are medical conditions that have a significant negative impact on health and quality of life. Studies have identified genetic and environmental contribution to the development of these complex disorders. As appropriate level of gene expression plays an important role in behaviour and cognition, we hypothesise that altered gene expression by polymorphic variation and environmentally induced epigenetic modification such as DNA methylation can contribute to these complex disorders. To test this hypothesis, we first investigated how a single nucleotide polymorphism (SNP) that is associated with obesity in the brain derived neurotrophic factor (BDNF) gene affects its expression.
BDNF is an important regulator of neuroplasticity which is a cellular brain adaptation mechanism that regulates behaviour and cognition.
Altered BDNF levels can impair this process. Transgenic mouse analysis of BDNF promoter IV (BP4), which is a promoter that mediates activity mediated neuroplasticity, showed its spatial activity in hippocampus, amygdala and cortex consistent with its role in the limbic system. A highly conserved region (BE5.1), identified by comparative genomics, selectively inhibited BP4 response to the protein kinase A agonist, forskolin, in primary cortical cultures but not hippocampal cultures. The minor allele of the obesity associated SNP however did not alleviate this inhibition. In contrast, the minor allele of a second SNP that is in linkage disequilibrium, abolished BE5.1
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inhibition to forskolin. Both SNPs also showed combinatorial regulation of BP4 activity. This study highlights that disease associated regulatory polymorphisms can function in combination with other polymorphisms to regulate gene expression. In a second study, we investigated the effect of stress on DNA methylation in a mouse model of anxiety and depression using a novel genome-wide assay called Methyl-Seq. This study provides a unique glimpse into the epigenome where regions of differentially methylated DNA could alter expression of genes in the brain contributing to mood disorders.
Together, these studies underscore the effect of genetic and epigenetic risk factors in the development of complex phenotypes like obesity and major depression.
USE OF ONLINE TOOL FOR IN-DEPTH SNP ANALYSIS.
Starkey A.1, Royer B.2, Davidson S.3, MacKenzie A.2
1School of Engineering, College of Physical Sciences, University of Aberdeen, Aberdeen, UK, AB24 3UE. Phone: +44 (0) 1224 282701, email:
a.starkey@abdn.ac.uk
Human genetic variation has been the main determinant that has produced the enormous diversity of human forms obvious around the world that has allowed Homo sapiens to meet the challenges associated with colonising hostile new environments. However, genetic diversity is a double edged sword and it is also believed to be responsible for promoting individual susceptibilities to genetic disease. The most common form of genome diversity comes in the form of single nucleotide polymorphisms (SNPs) (3), which are simple base pair changes in the genome that occur within at least 1%
of the human population and can confer susceptibility to heritable disease by compromising aspects of genome function (4) (5).
Evolution depends upon random mutation within genomes, filtered by the process of natural selection.
Much data exists in a variety of online resources for the analysis of SNPs and related data. This paper presents a free to use online tool that combines publically available data from evolutionary conserved elements from various species, frequency of SNP alleles and SNP attributes, the underlying human genome data and genome data from other species including mouse, rat, chicken and opossum, transcription factor data and position weight matrices from the Transfac Public data set and Jaspar, and linkage disequilibrium data from the HapMap project for various populations. In addition, GWAS study information has also been included in the analysis phase of the results, and the website also links with PubMed to further filter the results that are found.
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This paper presents results from previous work from a published paper which presents the culmination of intensive study of a particular SNP and shows how the use of the online tool can replicate this work in a matter of minutes, thereby speeding up the process of SNP analysis and greatly simplifying the task of understanding what effect individual SNPs can have.
Abstracts – Poster presentations
INHIBITORY FUNCTION OF THE SOMATOSTATIN RECEPTOR SUBTYPE 4 (SST4) IN ANXIETY AND DEPRESSION-LIKE BEHAVIOURS IN THE MOUSE
Scheich B.1, Kormos V.1, Tékus V.1, Hajna Z.1, Gaszner B.2, Pintér E.1,3,4, Szolcsányi J.1,3,4, Helyes Zs.1,3,4
1Department of Pharmacology and Pharmacotherapy, 2Department of Anatomy, Faculty of Medicine, University of Pécs, Hungary, 3János Szentágothai Research Center, University of Pécs, Hungary, 4PharmInVivo Ltd., Pécs,
Hungary
Although the involvement of somatostatin has been suggested in psychiatric disorders, there is little known about the roles of its Gi- protein-coupled receptors (sst1-sst5). Sst2, sst3 and sst5 are responsible for the endocrine actions, and we earlier showed that sst4 mediates anti-inflammatory and analgesic actions in the periphery. Since sst4 is also localized in stress-related brain regions, we aimed at analyzing its role in anxiety and depression using behavioral and immunohistochemical techniques in mouse models.
Anxiety was examined in the open field test (OFT) by the duration of spontaneous movements and the number of fields crossed during 5 minutes. Time spent in the dark part of the light-dark box (LDB) was also measured. Stress-coping behavior, as an indicator of depression, was assessed in the forced swim (FST) and tail suspension tests (TST) by measuring the duration of immobility within 4-minute-periods. 2 hours after the TST mice were perfused with paraformaldehyde under deep anesthesia and c-fos immunohistochemistry was performed in a variety of stress-related brain regions to detect early neuronal activation. Genetic deletion of sst4 (sst4-/-) and its pharmacological activation with a selective synthetic non-peptide agonist, J-2156, were used as experimental tools.
The spontaneous locomotor activity of sst4-/- mice was significantly lower than that of their C57Bl/6 wildtype counterparts in the OFT, but no difference was observed in the LDB. Sst4 deficient mice showed
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significantly longer immobility in both the TST and FST. Injection of J-2156 (100 µg/kg; i.p.) significantly decreased immobility in the TST 15 minutes after administration compared to saline treatment. It did not influence immobility in the FST, but according to the literature antidepressants are also ineffective in this test. J-2156 remarkably increased the number of c-fos positive neurones in the periaqueductal grey matter, nucleus interstitialis striae terminalis, nucleus raphe dorsalis and ventralis septi lateralis in the TST test.
It is concluded that sst4 activation decreases anxiety and depression- like behaviours in mice, therefore, it might be a potential drug target in stress-related disorders. These effects might be due to increasing neuronal activation in certain brain regions, but the precise neurophysiological mechanisms and its human relevance needs further investigations.
Support: SROP-4.2.2.A-11/1/KONV-2012-0024, SROP-4.2.2.B- 10/1/2010-0029
