Organizer Neurotoxicity Society (NTS)

Organizing Committee

FINAL PROGRAM & ABSTRACTS

Juan Segura-Aguilar (Chile), Chair Richard M. Kostrzewa (USA), Vice-Chair Trevor Archer (Sweden), General Secretary Pablo Caviedes (Chile) David Sulzer (USA) Federico Dajas (Uruguay) Sergio T. Ferreira (Brasil) Marta Antonelli (Argentina) Fernando Díaz Grez (Chile) Wojciech Danysz (Germany) Anita Sidhu (USA) David Sulzer (USA) Ryszard Brus (Poland) Xin-Fu Zhou (Australia)

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Dear colleagues,

SPONSORS ISN ISN-CAEN IBRO IBRO-LARC IUBMB Merz Pharmaceuticals Boehringer Ingelheim

On behalf of the Organizing Committee, we would like to extend our warmest welcome to the III Meeting of the Neurotoxicity Society (NTS). Our previous meetings in Pucón in 2001, La Serena in 2003 and Viña del Mar in 2005 have been unique opportunities to discuss neurotoxicity mechanisms involved in a wide variety of pathological conditions, both from the basic and clinical points of view, and to identify possible therapeutical targets. The meetings also provide graduate students with the possibility of presenting and discussing their work with leading experts in the field, thus providing new insight to the research, and setting the pace for the development of future collaborations and postdoctoral training opportunities. We thanks all invited speakers who make this meeting possible and we wish you a most fruitful and pleasant stay in Pucón, and we look forward to seeing you again in future meetings. Juan Segura-Aguilar President, Organizing Committee

Program- NTS Meeting March 22, 2007 12.00-20.00 Registration 20.00 Welcome 20.30 Welcome Reception

Scientific Program

March 23, 2007

8.15-8.30 Welcome Symposium 1. Glia and neurodegeneration Chair: Italo Mochetti (USA) 8.30-9.00 Agustina Garcia (Spain) Regulation of the glial inflammatory response by cGMP-mediated pathways 9.00-9.30 Rommy von Bernhardi (Chile) Obtaining the most of microglial cells in neuroinflammation: their regulation by astrocytes and neurons 9.30-10.00 Wendy Macklin (USA) Signaling pathways regulating oligodendrocyte survival and myelination Symposium 2. Advances in Parkinson´s disease I. Chair: Rita Raisman-Vozari (France) 10.00-10.30 Claudio Fernandez (Argentina) Synucleinopathies: Structural features and molecular interactions 10.30-11.00 Coffee 11.00-11.30 David Sulzer (USA) Alpha-synuclein and autophagy: an early step in Parkinson's Disease? 11.30-12.00 Juan Segura-Aguilar (Chile) DT-diaphorase prevents neurotoxicity of aminochrome 12.00-12.30 Vladimir Buchman (UK) Mouse models for studying function of synuclein family members in the normal and degenerating brain

2007 nts meeting program AND ABSTRACTS 12.30-13.00 Richard Kostrzewa (USA) Serotonin and Parkinson's Disease 13.00-14.00 Lunch 14.00-14.30 E. Ch. Wolters (The Netherlands) Clinico-pathological correlations in Parkinson's Disease 14.30-15.15 Clinical Round Table: Present and Future in the treatment of Parkinson's disease: Chair: Fernando Diaz-Grez (Chile), Pablo Venegas (Chile); Federico Micheli (Argentina). 15.15-16.25 Student oral presentations: Gustavo da Costa Ferreira (Brazil) Gustavo da Costa Ferreira (Brazil) Possible additive effect between glutaric and 3hydroxyglutaric acids on rerebral energy metabolism. Bettina Bongiovanni (Argentina) 2,4-dichlorophenoxyacetic acid induced oxidative stress in cerebellar granule cells in cultures and amphetamine reduced effect Juan Tichauer (Chile) Effect of SR-A and SR-Marco ligands on glial NO production and the modulation of SR-A expression by TGF-β Patrícia Fernanda Schuck (Brazil) Quinilinic acid mildly impairs cerebral energy metabolism Lisette Blanco (Cuba) Impact of the cholinergic system manipouklations through system (-) Nicotine administration on the GLU and GABA release and Motor execution of hemiparkinsonian rats. Mandi Gandelman (Uruguay) Modulation of motor neuron survival by extracellular purines is mediated by astrocytes: implications for amyotrophic lateral sclerosis 16.25-16.55 Coffee Symposium 3: Neurodegeneration I Chair : Federico Dajas (Uruguay) 17.00-17.30 Arne Schousboe (Denmark) Energy substrates to support glutamatergic synaptic function: Role of glycogen, glucose and lactate 17.30-18.00 Marta Antonelli (Argentina) Glutamate and GABA /BDZ receptors in the adult prenatally stressed rat 19.00-21.00 Guided Poster sessions 21.00 Dinner

March 24, 2007

Symposium 4: Neurodegeneration II Chair : Marta Antonelli (Argentina) 8.30-9.00 Mario Herrera-Marschitz (Chile) Neuronal plasticity following metabolic insults at birth: effect of nicotinamide 9.00-9.30 Yousef Tizabi (USA) Nicotine effects in a neurotoxic model of sensorimotor gating impairment. neuroplasticity 9.30-10.00 Gilles Guillemin (Australia) Characterization of the kynurenine pathway in human neurons and neuroblastoma 10.00-10.30 Coffee Symposium 5: ALS & Neurodegeneration Chair: Loeffler Jean-Philippe (France) 10:30-11:00 Joe Beckman (USA) The role of oxidants and antioxidants in the progressive neurodegeneration of ALS 11.00-11.30 Luis Barbeito (Uruguay) Identification of NGF species that promote neuronal apoptosis

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11.30-12.00 Loeffler Jean-Philippe (France) Energy homeostasis and denervation process at the neuro-muscular junctions in ALS 12.00-12.30 Clinical Round Table: Present and Future in the treatment of Alzheimer disease. Chair: Patricio Fuentes (Chile); Maria Isabel Behrens (Chile) 12.30-13.30 Lunch Symposium 6: Alzheimer disease and Dementia. Chair: Pablo Caviedes (Chile) 13.30-14.00 Sergio Ferreira (Brazil) Molecular and cellular mechanisms of neuronal dysfunction induced by Abeta in Alzheimer's disease 14.00-14.30 Nibaldo Inestrosa (Chile) Amyloid-β oligomers alter the structure and function of the post-synaptic region. The role of Wnt signaling pathway 14.30-15.00 Coffee Symposium 7- Neurotrophic factors, signal transduction and neurodegeneration. Chair: Gilles Guillemin (Australia) 15.00-15.30 Italo Mochetti (USA) Experimental therapeutic approaches for HIV-1 associated neurotoxicity 15.30-16.00 Xin-Fu Zhou (Australia) Neuroxicity of the precursors for neurotrophins. 16.30-17.00 Rafael Linden (Brazil) Neurotrophic functions of the cellular prion protein: Possible implication for the lossof-function hypothesis of prion diseases 17.00-17.30 Cecilia Hidalgo (Chile) Generation of RyRmediated calcium signals in hippocampal neurons by NMDA, ROS and Iron 17.30-18.00 NTS Annual Meeting Plenary Lecture, Chair: Richard Kostrzewa 19.30-20.30 Peter Riederer (Germany) Modelling Alzheimer pathology by loss of insuline receptor function 21.00-Gala Dinner

March 25, 2007 Symposium 8: Neuroprotection and therapheutical approaches- New discoveries: Chair W. Danysz 8.30-9.00 Gary Wenk (USA) The role of glutamate in the inflammatory processes in Alzheimer's disease 9.00-9.30 Johan Luthman (Sweden) Therapeutic approaches for neuroprotection?. 9.30-10.00 Chris G. Parsons (Germany) Neuroprotective potential of group I metabotropic glutamate receptor antagonists 10.00-10.30 W. Danysz (Germany) Contribution of pre-clinical pharmacology to CNS drug discovery - highlights and limitations (translational medicine) 10.30-11.00 Coffee Symposium 9: Neurodegeneration and Metals Chair: Marco Tulio Nuñez (Chile) 11.00-11.30 Alan Hazell (UK) Manganese neurotoxicity in liver failure 11.30-12.00 Julie K Anderssen (USA) Glutathione depletion in Parkinson's disease impacts on both mitochondrial function and iron homeostasis 12.00-12.30 Tulio Nuñez (Chile) Iron dyshomeostasis in Parkinson's disease

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12.30-13.30 Lunch Symposium 10: Neurodegeneration II Chair: Richard Kostrzewa (USA) 13.30-14.00 Fernando Garcia de Melo (Brazil) Dopaminergic activity in the developing CNS: Its characteristics and functional significance 14.00-14.30 Michael A. Collins (USA) Protective mechanisms against neuroinflammatory proteins induced by preconditioning brain cultures with moderate ethanol concentrations 14.30-15.00 Pablo Caviedes (Chile) Knockdown of Down syndrome related proteins: Effect on cellular dysfunction 15.00-15.30 Coffee Symposium 11: Neurodegeneration III Chair: Mario Herrera-Marschitz, 15.30-16.00 Neels Van der Schyf (USA) A multiple disease etiology approach to drug discovery for neurodegeneration 16.00-16.30 Ryszard Brus (Poland) Revelation of dysplacement functions of monoaminergic nerves alter lesions 16.30-17.00 Federico Dajas (Uruguay) Agonists of central nicotinic acetylcholine receptors and neuroprotection 17.00-17.30 Rita Raisman-Vozari (France) Pleiotrophin is expressed in the rat adult striatum and mediates the differentiation of dopaminergic neurons in culture Conclusions 18.00- 19.00 Educational Lecture: How to write a successful grant application. Juan Segura-Aguilar (Chile) 19.00 - 20.00 Educational Lecture: How to write a scientific publication. Mario Herrera-Marschitz (Chile) 20.00 Dinner

PLENARY LECTURE: Prof. Dr. Peter Riederer (Germany) Modeling Alzheimer pathology by loss of insulin receptor function

2007 nts meeting program AND ABSTRACTS

PRESENTATIONS Symposium 1.

Glia and neurodegeneration Chair: Italo Mocchetti (USA) REGULATION OF THE GLIAL INFLAMMATORY RESPONSE BY CYCLIC GMP-MEDIATED PATHWAYS Agustina García Institute of Biotechnology and Biomedicine, Universidad Autónoma de Barcelona, Spain We previously showed that inflammatory compounds that induce NO synthase 2 (NOS2) expression in glial cells exert a biphasic effect on NO-dependent cGMP formation in astroglia as a result of an initial activation of NO-sensitive guanylyl cyclase (GCNO), followed by down-regulation of the enzyme. This observation led us to investigate the role of cGMP-mediated pathways in the glial inflammatory response. We have observed that activation of the NO-cGMP-protein kinase G (PKG) pathway is involved in LPS-induced alteration in astrocyte morphology and actin and GFAP cytoskeleton through inhibition of RhoA GTPase. Additionally, this cGMP effect is associated with enhanced astroglial migration in a scratch wound assay. In microglia, cells devoid of GCNO activity, stimulation of the cGMP-PKG pathway by atrial natriuretic peptide (ANP) induces a rapid change to an amoeboid morphology that is accompanied by enhanced phagocytic activity, but diminished LPS-induced NOS2 expression. These results indicate that cGMP-mediated pathways can regulate physiopathologically relevant inflammatory responses in glial cells. This work was supported by a SAF2004-01717 grant (Spain). MICROGLIAL CELLS IN NEUROINFLAMMATION: THEIR REGULATION BY ASTROCYTES. R von Bernhardi, B Godoy, R Herrera-Molina, G Ramírez Lab. Neuroscience, Dept. Neurology, Faculty of Medicine, Pontificia Universidad Católica de Chile, Chile. In Alzheimer, senile plaques-associated glia become activated, secreting cytokines, nitric oxide (NO) and O2._. Scavenger Receptors (SRs) are involved on the interaction of glial cells with Aβ. Here we show that various SR-type A ligands (Fucoidan, Poly I and Dextran Sulfate) increased 3-4-fold NO production by microglia,

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an effect potentiated by Aβ. In contrast, only Poly I (10-400 µg/ml) increased up to 5-fold the production of NO by astrocytes. NO-increase correlated with an increased expression of IL-1β. Aβ did not induce NO whereas it potentiated Poly I-induced production of IL-1β. We evaluated if IL-1β took part on the modulation of microglial activity under pro-inflammatory conditions. IL-1β reduced IFNγ-induced microglial NO-production by 70%. It also reduced phospho-ERK by 40%, without affecting phosphoSTAT1. In contrast, in microglial cultures exposed to astrocytes' conditioned media, IL-1β did not inhibit phospho-ERK whereas reduced activation of STAT1. Modulation by IL-1β was observed at 30 min, but not later, indicating that modulation was an early transient phenomenon. Our results suggest that SR-As, besides their participation on Aβ-uptake, also participate in the inflammatory activation of glial cells. Support: FONDECYT 1040831.

HYPERMYELINATION: TOO MUCH OF A GOOD THING. WB Macklin, S P Narayanan AI Flores, EN Morse Cleveland Clinic Foundation, Cleveland, OH 44195, USA. The current studies were undertaken to investigate the role of Akt in oligodendrocytes in vivo, with the expectation that Akt would protect oligodendrocytes from death during demyelination. Transgenic mice overexpressing constitutively active Akt (Akt-DD) driven by the myelin proteolipid protein promoter were generated (Plp-AktDD). They were analyzed from P10 through adulthood by immunohistochemistry, electron micros-copy and/or western blots for the number of oligodendrocytes, the amount of myelin, the amount of gliosis and the structure and organization of Nodes of Ranvier. Electron microscopic analysis indicated that myelin was thicker in PlpAkt-DD mice than in WT, but there were no more oligodendrocytes than in WT mice. Thus, more myelin was generated by each oligodendrocyte. As animals aged, they continued to myelinate, and all white matter areas continued to expand. The corpus callosum became enlarged, as did the optic nerve and brainstem. These animals appeared unable to stop myelination. There was increased gliosis in older adult mice, and these mice generally did not survive past 12-14 months of age. Understanding the pathways that Akt regulates in oligodendrocytes may provide exciting new targets for enhancing remyelination in multiple sclerosis, but clearly these pathways must be tightly regulated, since excess myelin appears to be pathologic. Studies supported by the Natl. Mult. Sclerosis Soc.

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Symposium 2.

Advances in Parkinson´s disease I. Chair: Rita Raisman-Vozari (France)

SYNUCLEINOPATHIES: STRUCTURAL FEATURES AND MOLECULAR INTERACTIONS CO Fernández Institute of Molecular and Cell Biology, Argentina and Max Planck Institute for Biophysical Chemistry, Germany. [email protected] Many proteins associated with neurodegenerative diseases (PrP, APP, Aβ peptide and SOD-1) have metal binding properties. In the documented examples metal binding relates to pathogenesis via an impact on aggregation or production of oxidative damage. Thus, defining binding sites and the molecular details of complex formation may provide insights into pathogenic processes and neuronal biology. Our objective is to establish the role of metal ions in synucleinopathies at the molecular resolution currently available for other amyloidoses. The interaction of Cu(II) and other divalent metal ions with α−Synuclein (AS) were studied using low and high-resolution spectroscopic techniques (Rasia et al., PNAS 102:4294, 2005). The metal-AS interactions were characterized at single-residue resolution by NMR. The influence of metals on inducing AS fibrillation was strongly linked to their binding properties. A comparative analysis reveals a hierarchy in metal binding to AS, dictated by structural factors involving different domains of the protein (Bertoncini et al., PNAS 102:1430, 2005; Rasia et al., 2005; Binolfi et al., JACS 128:9893, 2006). The new insights into the structural basis of metal ion interactions with AS support a tighter link with other amyloid-related disorders such as Alzheimer's disease and prion disease.

ALPHA-SYNUCLEIN AND AUTOPHAGY AS AN EARLY STEP IN PARKINSON'S DISEASE D Sulzer1, Z Talloczy1, EM Mosharov1, M Martinez-Vicente2, AM Cuervo2

1Depts.

Neurology, Psychiatry, Pharmacology, New York State Psychiatric Inst., Columbia Univ., 650 W. 168th Street, New York, NY; 2Dept. Anatomy and Structural Biology, Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NY. Altered degradation of α-synuclein (α-syn) has been

implicated in the pathogenesis of Parkinson's Disease (PD). We previously reported that α-syn can be turned over via chaperone-mediated autophagy (CMA), a selective lysosomal mechanism for degradation of cytosolic proteins, and that pathogenic mutants of α-syn block lysosomal translocation of CMA substrates into the lysosomal lumen, impairing their own degradation along with that of other CMA substrates. We now show that α-syn overexpression enhances cytosolic dopamine (DA). When cytosolic DA is enhanced (via L-DOPA), there is an inhibition of CMA in normal DA neurons, but much less so in α-syn knockout DA neurons. This toxic response might be due to a specific blockade of CMA by DA-modified α-syn. The resulting "multiple hit" mechanism could explain the selective cell death of substantia nigra DA neurons, the presence of Lewy bodies in PD, and the means by which α-syn mishandling causes the idiopathic disease.

DT-DIAPHORASE PROTECTION IN CATECHOLAMINERGIC CELL LINE AGAINST AMINOCHROME NEUROTOXIC EFFECTS Sergio Cardenas, Irmgard Paris, Patricio FuentesBravo, Rebecca Graumann, Alejandra Riveros, Jorge lozano, Melissa Calegaro, Pablo Caviedes, Juan Segura-Aguilar 1Programme of Molecular and Clinical Pharmacology and 2Morphology, ICBM, Faculty of Medicine, Casilla 70000, Santiago-7, Chile. Dopamine oxidation to the o-quinone aminochrome is a normal process, which results in the formation of neuromelanin. However, under certain conditions aminochrome can induce neurotoxicity in dopaminergic neurons when (i) aminochrome is reduced by one-electron to the endogenous neurotoxin leukoaminochrome o-semiquinone radical, which is very reactive with oxygen generating a redox cycling between this radical and aminochrome depleting NADH or NADPH and oxygen; or (ii) aminochrome form adducts with α-synuclein resulting in the stabilization of long-lived protofibrils intermediates. Several lines of evidence have shown that a prerequisite of α-synuclein pathology is its oligomerization into neurotoxic protofibrils. DT-diaphorase catalyzes the two-electron reduction of aminochrome to leukoaminochrome, preventing aminochrome neurotoxic pathways: (i) one-electron reduction of aminochrome; and (ii) the formation of α-synuclein adducts with aminochrome. These results suggest that DT-diaphorase plays a neuroprotective role in dopaminergic neurons. Supported by FONDECYT 1061083 and FIRCA TW007044-01.

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MOUSE MODELS FOR STUDYING FUNCTION OF SYNUCLEIN FAMILY MEMBERS IN THE NORMAL AND DEGENERATING BRAIN Vladimir L Buchman and Natalia Ninkina School of Biosciences, Cardiff University, Museum Avenue, Cardiff CF10 3US, United Kingdom

CLINICO-PATHOLOGICAL CORRELATIONS IN PARKINSON'S DISEASE E Ch Wolters Department of Neurology, Amsterdam VUMC Vrije Universiteit Medical Center, P.O. Box 7057, 1007 MB Amsterdam.

The role of α-synuclein in aetiology and pathogenesis of Parkinson's disease and other synucleinopathies is well documented however exact mechanism of its involvement in brain pathology is still unclear. Recent studies suggest that in healthy brain synucleins have neuroprotective function but mutations, overexpression or erroneous localisation could be harmful for certain types of neurons, primarily dopaminergic neurons of substantia nigra. To understand molecular and cellular processes associated with synucleinopaties we studied several strains of genetically altered mice with modified expression of synuclein genes. Results of behavioural, biochemical, toxicological and primary tissue culture studies will be discussed in the light of common and diverse functions of different members of synuclein family in the normal and degenerating nervous system. Supported by The Wellcome Trust.

In 1817, James Parkinson in his 'Essay of the Shaking Palsy' described for the first time the signs and symptoms of a motor syndrome later called Parkinson's disease. Initially it was thought that this disease was caused by a not well understood abiotrophy affecting the brainstem dopaminergic neurons. Although in this disease non-motor symptoms were recognized also, this idea was valid until recently Heiko Braak did put the cat among the pigeons by publishing a multitude of pathological data, leading to the conclusion that Parkinson's disease, at least the α-synucleinopathy held responsible for this disease, was a diffuse process, involving most if not all neurotransmitter systems in the central and peripheral nervous system. Indeed, from a clinical point of view his observations fitted beautifully in the explanation of the various clinical manifestations of this disease. Not only there was now a solid base to explain the early, pre-motor signs and symptoms of this disease, thanks to better insight in the pathophysiology of this disease also other disease entities such as 'REM sleep behavioural syndrome', 'Dementia with Lewy Bodies' and 'Pan Autonomic Failure' can now be understood as part of this α-synucleinopathic process. Although it will need probably years of semantic/scientific discussion to share all these clinical entities under the umbrella of Parkinson's Disease, clinicians are now equipped not only to better understand and to earlier diagnose this condition, but also to ameliorate the quality of life of the patients suffering this disease.

SEROTONIN IN A PARKINSONIAN RAT RM Kostrzewa1, JP Kostrzewa1, P Nowak2, R Brus2 1Depts. of Pharmacology, Quillen College of Medicine, East Tennessee State Univ., Johnson City, TN, USA; 2Medical Univ. Silesia, 41-808 Zabrze, Poland. [email protected] To determine the possible influence of serotonin (5-HT) nerves in Parkinson's disease (PD), a rat model of PD was produced by 6-hydroxydopamine (6-OHDA; 134 µg, icv) at 3d postbirth. In the PD rat, a neonatal 5-HT lesion with 5,7-dihydroxytryptamine (5,7-DHT) attenuated enhanced D1 agonist effects; the 5-HT2 antagonists ketanserin and mianserin likewise attenuated D1 agonistinduced motor and stereotyped effects; and the DA microdialysate level of DA was enhanced by p-chloroamphetamine in rats co-lesioned with 6-OHDA and 5,7-DHT. Hydroxyl radical (HO.) levels were elevated in striatal tissue but not in the microdialysate of 6-OHDAlesioned rats; and ketanserin did not alter the microdialysate HO. level in PD rats. Findings implicate 5-HT agents as potentially beneficial anti-dyskinetic agents that do not pose a risk of increasing reactive oxygen species in PD. Supported by NINDS NS39272 and Medical University of Silesia: NN-1-001/06.

CLINICAL ROUND TABLE Present and Future in the Treatment of Parkinson's Disease: Fernando Diaz-Grez (Chile), Pablo Venegas (Chile); Federico Micheli (Argentina).

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Symposium 3.

Neurodegeneration I. Chair : Federico Dajas (Uruguay) ENERGY SUBSTRATES TO SUPPORT GLUTAMATERGIC SYNAPTIC FUNCTION: ROLE OF GLYCOGEN, GLUCOSE AND LACTATE. Arne Schousboe1, Lasse Bak1, Helle Sickmann1, Ursula Sonnewald2, Helle Waagepetersen1 1Dept. Pharmacol. & Pharmacotherapy, Faculty of Pharm. Sci., Univ. Copenhagen, DK-2100 Copenhagen, Denmark, and 2Dept. Neurosci., Norwegian Univ. Sci. & Technol., N-7489 Trondheim, Norway. Homeostatic mechanisms operating to maintain normal glutamatergic activity, i.e., vesicular glutamate release and inactivation by plasma membrane transporters were investigated using cultured glutamatergic neurons (cerebellar granule cells) as well as astrocytes as model systems. The functional role of glycogen, glucose and lactate as energy substrates for the maintenance of these processes was studied using 13C-labeled substrates (glucose and lactate) and NMR spectroscopy or LC-MS to monitor metabolism. Moreover, pharmacological tools to specifically block enzymes involved in glycogen turnover (a phosphorylase inhibitor), glycolysis (iodoacetate) or TCA cycle activity (fluoroacetate) were included in the investigations. The main findings of these studies may be summarized as follows: Glycogen turnover in astrocytes appears to be important for biosynthesis of releasable glutamine which may serve as precursor for neurotransmitter glutamate (Sickmann et al., Neurochem. Res. 30:1295, 2005). Energy derived from glycolysis rather than TCA cycle activity appears to be the primary driving force for astrocytic glutamate transport which is important for inactivation of transmitter glutamate and for prevention of excitotoxic actions of glutamate (Schousboe et al., Neurotox. Res. 8:221, 2005). In cocultures of neurons and astrocytes, pyruvate metabolism was compartmentalized and a considerable pyruvate recycling was observed (Bak et al., Neurochem. Res. 32:671, 2007). The latter is interesting as this process is necessary for complete oxidation of glutamate. In glutamatergic neurons both glucose and lactate can serve as energy substrates but it was found that glucose was the superior substrate to support glutamatergic activity (Bak et al., J. Cereb. Blood Flow Metab. 26:1285, 2006). Altogether, these findings underline the concept of a complex interaction between neuronal and astrocytic metabolism in order to maintain optimal function of glutamatergic activity thereby preventing glutamate

induced neurodegeneration. GLUTAMATE AND GABA/BDZ RECEPTORS IN THE ADULT PRENATALLY STRESSED RAT. Marta C. Antonelli. IQUIFIB (UBA-CONICET), Facultad de Farmacia y Bioquímica. UBA. Buenos Aires. ARGENTINA. The ability of an organism to cope with a stressful event in adult life is greatly influenced by previous early experience with stress, specially during the perinatal period. Previous studies from our laboratory have shown that adult offspring of rats stressed during the last week of pregnancy exhibited higher levels of anxiety than control rats concomitantly with a long-lasting astroglial reaction and a reduced dendritic arborization with synaptic loss. Since metabolism of glutamate and GABA is intimately linked to a substrate cycle between neurons and sorrounding astroglia, our results prompted us to suggest that both glutamate and GABA/Benzodiazepine (BDZ) neurotransmitter pathways might be impaired in the brain of a prenatally stressed rat. Repeated restraint during the last week of pregnancy was used as a model of prenatal stress and quantitative autoradiography was employed to detect both ionotropic and metabotropic glutamate receptors as well as GABA/BDZ receptors. Adult offspring of rats stressed during pregnancy exhibited an increase in glutamate NMDA receptors in frontal cortex, hippocampal CA1, medial caudate-putamen nucleus accumbens. Group III metabotropic glutamate receptors increased only in frontal cortex of prenatally stressed rats, but remained unchanged in all other regions examined. The number of benzodiazepine receptor binding sites decreased in the central amygdaloid nucleus (Ce), CA1, CA3 and dentate gyrus regions of the hippocampus when compared to controls. Taken altogether these results show that both glutamate and GABA receptors are altered in prenatally stressed rats most probably as a consequence of an impairment on the metabolism of these neurotransmitters in the neuron-astrocytic cycle.

Symposium 4.

Neurodegeneration II. Chair : Marta Antonelli (Argentina) NEURONAL PLASTICITY FOLLOWING METABOLIC INSULTS AT BIRTH: EFFECT OF NICOTINAMIDE. M Herrera-Marschitz1, P Morales1, D Bustamante1, V Klawitter1, J Fiedler2, N Simola3, M Morelli3, D MacDonald4, A Tasker4, T Hökfelt5

2007 nts meeting program AND ABSTRACTS 1Programme

of Molecular & Clinical Pharmacology, ICBM, Medical Faculty; 2Dept. Neurochemistry, Chemical and Pharmaceutical Science Faculty, University of Chile, Santiago, Chile; 3Dept. Toxicology, Cagliari University, Cagliari, Italy; 4Dept. Biomedical Sciences, UPI, Canada; 5Dept. Neuroscience, Karolinska Institutet, Stockholm, Sweden. Interruption of oxygen availability and re-oxygenation at birth implies a severe metabolic insult, affecting the development of the central nervous system (CNS), increasing its vulnerability to challenges occurring at adult stages. It has been reported that perinatal asphyxia produces regionally specific neuronal decrease and neurite atrophy in basal ganglia, and hippocampus. In hippocampus, a concomitant increase of neurogenesis and neurite hypertrophy has also been observed. The potential neuroprotection of nicotinamide, a non-selective inhibitor of poly(ADP-ribose) polymerase (PARP-1), has been investigated, finding functional and morphological improvements when administered 24h after the insult (0.8 mmol/kg, i.p., 24, 48 and 72 h after birth.). The main effect of nicotinamide has been seen in neostriatum, preventing an asphyxia-induced decrease of the number of nNOS cells, and nNOS- and dopamine-like neurite atrophy. The present results support the idea that nicotinamide can prevent the effects elicited by a sustained energy-failure condition, as occurring during perinatal asphyxia, enlightening the enzyme PARP-1 as a novel target for neuronal protection. The support by FONDECYT and ICBM-Enlace grants is acknowledged. NICOTINE AND HYPOGLUTAMATERGIC HYPOTHESIS OF SCHIZOPHRENIA Yousef Tizabi, Ph.D. Department of Pharmacology, College of Medicine, Howard University, Washington, DC 20059. Schizophrenia is a complex neuropsychiatric disorder with devastating consequences. It is characterized by thought fragmentation, hallucination and delusion, collectively referred to as positive symptoms. In addition, mood changes or affective disorders, referred to as negative symptoms as well as cognitive impairments can be manifested along the positive symptoms. Arguably, modeling such a disorder in its entirety in animals might not be feasible. Despite this limitation, various models with significant construct, predictive and some face validity have been developed. One such model, based on hypoglutamatergic hypothesis of schizophrenia, makes use of administering NMDA receptor antagonists and evaluating behavioral paradigms such as sensorimotor gating. Because of very high incidence of smoking among

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schizophrenic patients, it has been postulated that some of these patients may actually be self medicating with tobacco's nicotine. Research on nicotinic-glutamatergic interactions using various animal models has yielded conflicting results. In this presentation, some of these models and possible confounding factors including pharmacokinetic and pharmacodynamic parameters will be discussed. Overall, a therapeutic potential for nicotinic agonists in some schizophrenic patients may be suggested. Supported by NIH/NIMH (2SO6 GM08016-36). CHARACTERIZATION OF THE KYNURENINE PATHWAY IN HUMAN PRIMARY NEURONS Gilles J Guillemin1, George A Smythe2, Vimal Kapoor3, Edwin Lim1 & Bruce J. Brew4 1Centre for Immunology, Neuroimmunology Dept.; 2UNSW, Biomedical Mass Spectrometry Facility; 3UNSW, Pharmacology Dept.; 4St Vincent Hospital, Neurology Dept., Sydney, Australia. The kynurenine pathway (KP) is a major route of L-tryptophan catabolism resulting in the production of neurotoxic, neuroprotective and immune tolerance-inducing intermediates. The KP has been shown to be involved in neurodegenerative diseases including Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis and Parkinson's disease with the balance of KP intermediates being neurotoxic and immune tolerance-inducing. The characterization of the KP in the normal human brain has been so far only partly delineated. It is important to know what KP metabolites are produced by each brain cell types to be able to understand their interactions and to appropriately design and test therapies. We have previously characterized the KP in human macrophages, microglial cells and astrocytes. In this study, we characterized the KP in human foetal neurons in comparison with the human neuroblastoma cell line SKNSH using RT-PCR, HPLC, mass spectrometry and immunocytochemistry. We found that neurons express all the KP enzymes but at different levels. Indoleamine 2,3 dioxygenase is strongly expressed by both primary neurons and SKNSH whereas kynurenine aminotransferase 2, kynureninase and kynurenine hydroxylase showed very low level expression. Picolinic carboxylase was found expressed only in primary neurons, not in SKNSH. Because of this "late switch" SKNSH were able to produce low amounts of the excitotoxin quinolinic acid whereas primary neurons preferentially produced the neuroprotective picolinic acid. Moreover, primary neurons were to catabolize quinolinic acid. The net result of neuronal KP induction is therefore towards neuroprotection and immune tolerance. This study represents the first comprehensive characterization of the KP in neuron.

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Symposium 5.

ALS & Neurodegeneration. Chair: Loeffler Jean-Philippe (France) THE LOSS OF ZINC FROM SOD AND ALS Joe Beckman Linus Pauling Institute at Oregon State University, USA Because superoxide reacts so rapidly to nitric oxide to form peroxynitrite, the balance between nitric oxide and copper, zinc superoxide dismutase (SOD) is critical in determining the biological actions of nitric oxide. In the neurodegenerative disease known as Lou Gehrig's disease or ALS, mutations to SOD cause about 2% of the cases. These mutations increase the probability that SOD will lose its structural zinc atom. Using mass spectrometry of native SOD directly from ALS-affected tissues, we can now measure zinc-deficient SOD to be almost equal in concentration as copper, zinc SOD specifically in the disease affected regions. Zinc-deficient SOD remains reduced in tissues by oxidizing ascorbate and reduced SOD reacts rapidly with peroxynitrite to form nitrogen dioxide. This tips the balance to driving nitric oxide from a benign signaling molecule towards the direction of tyrosine nitration and toxicity. The loss of ascorbate in motor neurons may also contribute to neurodegeneration. IDENTIFICATION OF NEW NGF SPECIES THAT PROMOTE NEURONAL APOPTOSIS Luis Barbeito Institut Pasteur de Montevideo, Uruguay. Nerve growth factor (NGF) overexpression and increased production of peroxynitrite occur in several neurodegenerative diseases. We investigated whether NGF could undergo post-translational oxidative or nitrative modification that would modulate its biological activity. Compared to native NGF, peroxynitrite-treated NGF showed the exceptional ability to induce p75NTR-dependent motor neuron apoptosis at physiologically relevant concentrations. While native NGF requires an external source of nitric oxide (NO) to induce motor neuron death, peroxynitrite-treated NGF induced motor neuron apoptosis in the absence of exogenous NO. Nevertheless, NO potentiated the apoptotic activity of peroxynitritemodified NGF. Blocking antibodies to p75NTR or downregulation of p75NTR expression by antisense treatment prevented motor neuron apoptosis induced by peroxynitrite-treated NGF. We investigated what oxidative modifications were inducing this toxic gain of function and

found peroxynitrite induced tyrosine nitration in a dose dependent manner. Moreover, peroxynitrite triggered the formation of stable high molecular weight oligomers of NGF. Preventing tyrosine nitration by urate abolished the effect of peroxynitrite on NGF apoptotic activity. These results indicate that the oxidation of NGF by peroxynitrite enhances NGF apoptotic activity through p75NTR 10,000 fold. To our knowledge, this is the first known post-translational modification that transforms a neurotrophin into an apoptotic agent. ENERGY HOMEOSTASIS AND DENERVATION PROCESSES AT THE NEURO-MUSCULAR JUNCTION IN ALS. Jean-Philippe Loeffler, PhD INSERM Res. Director, U692 Inserm/ULP,11, rue Humann, 67085 Strasbourg Cedex, France. loeffler@ neurochem.u-strasbg.fr. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by motor-neuron (MN) loss and muscle wasting. The nature of the selective degeneration of MN remains elusive, but several studies show a dying back process starting at the neuromuscular junction (NMJ). This might be indicative of early pathological processes in the muscle. Previous studies from our laboratory revealed that a mouse model of ALS (SOD G86R) presents with hypermetabolism (Dupuis et al., PNAS 101:11159, 2004) and that the muscular expression of a specific proteine : NOGO A, is sufficient to initiate the denervation process (Jokic et al., EMBO 11: 1162, 2006). The present study was aimed at elucidating whether muscular hypermetabolism can induce denervation at the NMJ. To this end we used a mouse model where hypermetabolism is experimentally produced by expressing the uncoupling protein 1 (UCP1) in skeletal muscle. Behavioural, electromyographic and biochemical studies revealed that increased energy demand in the muscle is sufficient to induce destruction of the NMJ and denervation.

CLINICAL ROUND TABLE Present and Future in the Treatment of Alzheimer Disease. Chair: Patricio Fuentes (Chile), Maria Isabel Behrens (Chile)

2007 nts meeting program AND ABSTRACTS

Symposium 6.

Alzheimer Disease and Dementia. Chair: Pablo Caviedes (Chile) MOLECULAR AND CELLULAR MECHANISMS OF NEURONAL DYSFUNCTION INDUCED BY Aβ OLIGOMERS IN ALZHEIMER'S DISEASE Sergio T. Ferreira1,2, Fernanda G. De Felice1,2, William L. Klein2 1Institute of Medical Biochemistry, Federal University of Rio de Janeiro, Rio de Janeiro, RJ 21944-590, Brazil; 2Dept. of Neurobiology and Physiology, Northwestern University, Evanston, IL 60208, USA. Alzheimer's disease (AD) is neuropathologically characterized by extracellular amyloid plaques, intracellular neurofibrillary tangles, accumulation of soluble oligomers of the β-amyloid peptide (Aβ), oxidative stress and synapse loss. Accumulating evidence indicates that soluble Aβ oligomers (also known as ADDLs, for "Abetaderived diffusible ligands") are the main neurotoxins that cause early synaptic dysfunction, memory loss and ultimately neuronal degeneration in AD. However, the molecular/cellular mechanisms by which Aβ oligomers cause synaptic dysfunction and neurodegeneration are only beginning to be unraveled. Here, we have investigated the relationship between oxidative stress, a major aspect of AD pathology, and neuronal binding of Aβ oligomers. Using hippocampal neuronal cultures, we found that ADDLs stimulated excessive formation of reactive oxygen species (ROS) through a mechanism requiring N-methyl-D-aspartate receptor (NMDA-R) activation. ADDL binding to neurons was reduced and ROS formation was completely blocked by antibodies to the extracellular domain of the NR1 subunit of NMDA-Rs. In harmony with a steric inhibition of ADDL binding by NR1 antibodies, ADDLs that were bound to detergentextracted synaptosomal membranes co-immunoprecipitated with NMDA-R subunits. The NR1 antibody did not affect ROS formation induced by NMDA, showing that NMDA-Rs themselves remained functional. Memantine, an open-channel NMDA-R antagonist prescribed as a cognition-enhancing drug for AD patients, completely protected against ADDL-induced ROS formation, as did other NMDA-R antagonists. Memantine and the antiNR1 antibody also attenuated a rapid ADDL-induced increase in intraneuronal calcium, which was essential for stimulated ROS formation. These results show that ADDLs bind to or in close proximity to NMDA-Rs, triggering neuronal damage through NMDA-R-dependent calcium flux. This response provides a pathologically-

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specific mechanism for the therapeutic action of memantine, indicates a role for ROS dysregulation in ADDLinduced cognitive impairment, and supports the unifying hypothesis that ADDLs play a central role in AD pathogenesis. Supported by Howard Hughes Medical Institute (USA) and the Brazilian agencies Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), Fundacao de Amparo `a Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Programa de Nucleos de Excelencia (Pronex). AMYLOID- β OLIGOMERS ALTER THE STRUCTURE AND FUNCTION OF THE POSTSYNAPTIC REGION. THE ROLE OF WNT SIGNALING PATHWAY. Inestrosa, N.C. Centro de Regulación Celular y Patología "Joaquin V. Luco" (CRCP), MIFAB, Facultad de Ciencias Biológicas, P. Universidad Católica de Chile, Santiago, Chile. Synaptic dysfunction is increasingly viewed as an early Alzheimer's Disease manifestation. The cellular mechanism by which Aβ affects synapses remains unclear. Aβ oligomers target synapses in cultured rat hippocampal neurons suggesting that they play a role in the regulation of synaptic structure and function. We have decided to study (1) the possible molecular players involved in concentrating Aβ at the synapses and (2) the synaptic effects of Aβ oligomers. Neuroligin is a post-synaptic cell adhesion protein. Its extracellular domain is mainly formed by an acetylcholinesterase-like domain. We propose that neuroligin could target of Aβ oligomers at the synapse. We have also studied the glutamatergic synaptic transmission of the Schaffer collateral excitatory synapses onto CA1 pyramidal cells in rat hippocampal slices. Aβ oligomers show a decrease in the evoked response over 50% in field recording and over 60% in intracellular recording. The analysis of the facilitation ratios suggest that Aβ oligomers effect depends on the postsynaptic site. This effect of Aβ oligomers correlates well with a reduction of PSD-95. A non-canonical Wnt ligand, Wnt-5a prevents from the decrease triggered by Aβ oligomers in the AMPA (GluR1), NMDA (NR2A) receptor and PSD-95. Interestingly, Aβ oligomers affect the maintenance of the cadherin catenin complex which has been shown to mediate synaptic plasticity. Altogether, our results suggest that Aβ oligomers decrease the amplitude of synaptic responses by affecting the postsynaptic region at different levels. The Wnt signaling activation opens a possibility in the prevention of these synaptic effects. Support by: FONDAP Nº13980001, MIFAB.

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Symposium 7.

Neurotrophic Factors, Signal Transduction and Neurodegeneration. Chair: Gilles Guillemin (Australia) BRIAN-DERIVED NEUROTROPHIC FACTOR PREVENTS THE NIGROSTRIATAL DEGENERATION INDUCED BY HUMAN IMMUNODEFICIENCY VIRUS-1 GLYCOPROTEIN 120. Italo Mocchetti1, Rachel L. Nosheny1, Farid Ahmed1, Edwin M. Meyer2, Ke Ren2 and Lino Tessarollo3. 1Dept. of Neuroscience, Georgetown Univ. Medical Center, Washington DC; 2Dept. of Pharmacology and Therapeutics, Univ. Florida, Gainesville, FL; 3Neural Development Group, National Cancer Institute, Frederick, MD. [email protected]. Human immunodeficiency virus type 1 (HIV-1) causes neuronal degeneration, and, at a late stage, creates dementia and other neurological abnormalities. Therefore, the need for neuroprotective agents is great. However, therapeutic agents that reduce HIV neurotoxicity are difficult to characterize and develop because rodents are not infected by HIV. This study was undertaken to develop an animal model of HIV neurotoxicity using the HIV-1 envelope glycoprotein 120 (gp120). Gp120 or vehicle was injected acutely in the striatum. Gp120 produced loss of nigro-striatal neurons, determined both by histochemical analysis of brain sections for apoptosis and biochemical determination of dopamine. The neurotrophin brain-derived neurotrophic factor delivered into the striatum by a recombinant adeno-associated viral vector prevented gp120 toxicity. The neuroprotective activity of BDNF was accompanied by a decreased expression of CXCR4, a chemokine receptor crucial for HIV infection. Conversely, BDNF heterozygous mice exhibited an increase in CXCR4 mRNA levels compared to wild type littermates. The results of the present study support the notion that gp120 produces a widespread neurotoxicity similar to that observed in HIV individuals and that BDNF may be a suitable neuroprotective agent for HAD. Supported by NIH grant NS 040670. ARE PRONEUROTROPHINS ANTAGONISTS OF MATURE NEUROTROPHINS? Xin-Fu Zhou Department of Human Physiology and Centre for Neuroscience, Flinders University, GPO Box 2100, Adelaide 5001, Australia

Neurotrophins are synthesized as precursors with around 240 amino acids which are enzymatically processed into mature molecules. Mature neurotrophins play important roles in the regulation of proliferation, differentiation, migration and survival of neurons during development. Neurotrophins are also important for regulation of synaptic plasticity and learning and memory. Recently, it was found that neurotrophins are not fully processed and in many cases proneurotrophins exist as the main form in pathological and physiological conditions. In addition, proNGF and proBDNF could induce apoptosis of sympathetic neurons via their receptors p75NTR and sortilin in neuronal cultures. However, whether these molecules cause neuron death in vivo conditions is not known. We have used several models to address this question. We have expressed and purified recombinant prodomain fragments of NGF and BDNF and raised antibodies specifically against the prodomains and tested their biological activities in vitro. Using a well characterized sciatic injury model in neonatal rats we found that proBDNF increased death of sensory neurons in the dorsal root ganglia (DRG), whereas its antibodies specifically against the prodomain of BDNF rescued the death of DRG neurons induced by axotomy. The death of DRG neurons can also be rescued by local application of sortilin-Fc, a recombinant soluble molecule which prevents binding of proneurotrophins to sortilin on cell membrane. Interestingly, antibodies to proBDNF increased the number of sensory neurons in the contralateral DRG. In contrast to proBDNF, proNGF in vivo may have neurotrophic functions. In neonatal sympathetic ganglia, antibodies to proNGF did not prevent the death of sympathetic neurons in vivo but caused a small reduction in the number of sympathetic neurons. ProNGF can induce trkA phosphorylation, suggesting that proNGF may signal via trkA to promote neurotrophic function in vivo. Our data suggest that proBDNF may act as an antagonist of mature BDNF whereas proNGF may have weak neurotrophic functions in vivo. NEUROTROPHIC FUNCTIONS OF THE CELLULAR PRION PROTEIN: POSSIBLE IMPLICATION FOR THE LOSS-OF-FUNCTION HYPOTHESIS OF PRION DISEASES. Rafael Linden Instituto de Biofísica da UFRJ, Rio de Janeiro, Brasil. Prion diseases are transmissible spongiform encephalopathies (TSEs), attributed to conformational conversion of the cellular prion protein (PrPC) into an abnormal conformer that accumulates in the brain. Understanding the pathogenesis and, particularly, loss-of-function components of TSEs requires the identification of functional

2007 nts meeting program AND ABSTRACTS

properties of PrPC. Both the expression and the engagement of PrPC with distinct ligands modulate a variety of functions of the nervous and immune systems, including memory and inflammatory reactions. Work in vitro using both tissue and dissociated neuron cultures showed that engagement of PrPC with one of its ligands, the cochaperone hop/STI-1, induces neurotrophic signaling. This may be relevant for the neurodegeneration associated with prion diseases. Nonetheless, many other functions are modulated by both expression and ligand binding of PrPC, and the mechanisms leading to signaling mediated by PrPC remain obscure. Data on structural changes, novel putative components of the PrPC-based multi-component cell surface complex, as well as the role of endocytosis upon PrPC-mediated signaling will be presented. These data suggest that the prion protein is a dynamic cell surface platform for the assembly of signaling modules, based on which selective interactions with many ligands and transmembrane signaling pathways translate into wide-range consequences upon both physiology and behavior. A ROLE FOR IRON, CALCIUM AND REACTIVE OXYGE SPECIES ON HIPPOCAMPAL SYNAPTIC PLASTICITY. Cecilia Hidalgo1,2, Pablo Muñoz1,3 M. Angélica Carrasco1,2 and Marco T. Núñez11,3. 1Centro FONDAP de Estudios Moleculares de la Célula; 2Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile; 3Department of Biology, Faculty of Sciences, and Cell Dynamics and Biotechnology Institute, Universidad de Chile, Santiago, Chile. Current evidence indicates that iron and calcium have central roles in neuronal function. Iron is needed for normal cognitive, motor, and social-emotional developments, while calcium signals are required for synaptic plasticity in the hippocampus. Likewise, increasing evidence supports a physiological role of reactive oxygen species (ROS) and nitrogen species (RNS) in synaptic plasticity. We will present some of our recent work in hippocampal neurons in culture showing how iron and calcium interact to stimulate signaling cascades known to participate in synaptic plasticity. Thus, ROS/RNS, including ROS generated by iron through the Fenton reaction, stimulate ryanodine receptor-mediated calcium release from internal stores. The resulting calcium signals activate the MAPK/ERK cascade that is known to promote the transcription of genes required for hippocampal long-term potentiation (LTP). Additionally, our results indicate a need for iron to induce LTP by high frequency stimulation of hippocampal CA1 neurons. We will discuss the possible contribution of calcium signals

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generated by ryanodine receptors jointly stimulated by calcium entry and iron-generated ROS to the stimulation of signaling pathways required for synaptic plasticity, and how oxidative stress may contribute to neurodegeneration by causing excessive calcium release. Supported by FONDECYT grants 1030988, 1060177 and 1040448, and by FONDAP CEMC 15010006.

Symposium 8.

Neuroprotection and Therapheutical Approaches - New Discoveries Chair: Wojciech Danysz (Germany) THE ROLE OF GLUTAMATE IN THE INFLAMMATORY PROCESSES IN ALZHEIMER'S DISEASE Gary L Wenk, Professor Departments of Psychology & Neuroscience, Ohio State University Degenerative diseases such as Alzheimer's disease show marked neuroinflammation that may contribute to cell death. Anti-inflammatory substances are being explored as therapeutic responses to these degenerative processes. This report will discuss anti-inflammatory approaches that focus directly or indirectly upon the action of glutamate. We have discovered that a diverse set of different pharmacological approaches can effectively reduce biomarkers of brain inflammation, including nitro releasing non-steroidal anti-inflammatory drugs, NMDA glutamate receptor antagonists, caffeine, curcumin and cannabinoid receptor stimulation. These findings provide insight into how neurons and glia communicate to regulate a complex immunosuppressive strategy that is able to limit the destructive consequences of brain inflammation. Adenosine receptor antagonism has been recently described as having antiinflammatory properties, although the mechanism by which this occurs is still undefined. We investigated the proposed anti-inflammatory role of adenosine antagonism through administration of caffeine using two different animal models of chronic neuroinflammation. The endocannabinoid system is the pharmacological target of cannabis. Cannabinoid receptors (CBr) are highly expressed in the brain and their stimulation induces numerous central and peripheral effects. A growing interest in the beneficial properties of the endocannabinoid system raised the possible implication of CBr in the control of inflammatory process in the brain and as a possible therapy in neurodegenerative diseases such as Alzheimer's disease. We have designed a novel animal model of the chronic neuroinflammation associated with AD in order to study the contribution of activated microglia to the mechanisms of

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2007 nts meeting program AND ABSTRACTS

degeneration within vulnerable brain regions. We have investigated whether the consequences of chronic inflammation within the temporal lobe can be reversed by treatment with memantine. Our findings suggest that memantine has a broad range neuroprotective effects and cognition enhancing properties that may involve its ability to attenuate the cumulative effects of neuroinflammation. Supported by NIH AG10546 and Merz. NEUROPROTECTIVE POTENTIAL OF GROUP I METABOTROPIC GLUTAMATE RECEPTOR ANTAGONISTS Chris G. Parsons and Wojciech Danysz Merz Pharmaceuticals GmbH, D-60318 Frankfurt am Main, Germany. Metabotropic glutamate receptors (mGluRs) consist of three major groups I-III which are involved in physiological processes such as synaptic transmission and neuronal plasticity as well as neuropathology. There are two group I mGluR subtypes, mGluR1 and mGluR5, which are mainly localized to postsynaptic membranes. Their activation usually evokes excitation in neurons via PLC conversion of PIP2 to DAG and IP3. DAG activates PKC whereas IP3 has numerous effects including stimulation of Ca2+ release from intracellular stores. The advent of potent and CNS penetrant subtypeselective allosteric modulators has led to an extensive investigation of the role of mGluRs in CNS diseases, e.g., pharmacological blockade of mGluR1/5 produces neuroprotection in a variety of in vitro and in vivo models suggesting a potential application of mGluR1/5 receptor antagonists in chronic neurodegenerative disorders, such as Alzheimer´s and Parkinson´s disease. The advantage of all these drugs with respect to ionotropic glutamate antagonists derives from the observation that mGluRs do not "mediate," but rather "modulate" excitatory synaptic transmission. It is predicted that mGluR ligands will have less undesirable effects resulting from the inhibition of physiological excitatory synaptic transmission. CONTRIBUTION OF PRE-CLINICAL PHARMACOLOGY TO CNS DRUG DISCOVERY HIGHLIGHTS AND LIMITATIONS (TRANSLATIONAL MEDICINE) Wojciech Danysz, Christopher G. Parsons Merz Pharmaceuticals, Eckenheimer Landstrasse 100, D-60318 Frankfurt, Germany The aim of the talk is on one hand to show the limitations of predictability of preclinical pharmacology for efficacy of substances to be used in man. On the other hand, it is stressed that being aware of such limitations may greatly

increase predictability value. The talk has been organised in 6 sub themes: 1) Human target vs. experimental target: The differences between human and experimental target could be both of pharmacokinetic and pharmacodynamic nature, e.g., faster metabolism/ elimination in animals, also qualitative differences in metabolism such as formation of species specific metabolites. Pharmacodynamic differences include differences in target molecule features resulting in changes in affinity and/or efficacy at the target. 2) Relation between pharmacokinetics and pharmacodynamics. We should be sure that at therapeutic doses (for animal experiment doses active in models for given indication) the drug is present at the target at concentration showing activity at the target in vivo (affinity). 3) Target vs. indication based drug discovery: This approach gives relative high chance of success (in particular in case of proven target) but is not very innovative. On the other hand indication based discovery involves research for new targets, often through support of basic science. It is bearing high risk potential, but in the long run it drives the real advancement. 4) Validity of animal models: construct validity, predictive validity and face validity. 5) Need for better animal models of CNS diseases. 6) Use of appropriate reference agents. In general the aim of R&D is to develop new treatments superior to existing therapies since without this, neither marketing force nor patients would benefit. This is however not equivalent to providing meaningful advantage. At present, most often superiority emerges from better pharmacokinetic or side-effects profile, rather than from real increases in efficacy.

Symposium 9.

Neurodegeneration and Metals Chair: Marco Tulio Nuñez (Chile) MANGANESE NEUROTOXICITY IN LIVER FAILURE Alan S. Hazell Department of Medicine, Hôpital Saint-Luc (CHUM), Univ. of Montreal, Montreal, Quebec, Canada H2X 3J4

Portal-systemic encephalopathy (PSE), the chronic form of hepatic encephalopathy (HE), is a major consequence of liver cirrhosis, in which the pathogenesis is unknown. It occurs as a result of shunting of portal blood into the systemic circulation. Recently, manganese has been linked to the pathophysiology of PSE in which the chronic liver failure and portal-systemic shunting together prevent its elimination from the blood, leading to uptake into brain. Clinical features of both PSE and manganese neurotoxicity (MN, manganism) include the

2007 nts meeting program AND ABSTRACTS

presence of signal hyperintensities in the globus pallidus (GP) using T1-weighted magnetic resonance imaging, reflecting an accumulation of the metal in this brain region in both maladies, along with the presence of extrapyramidal disturbances. Pathologically, both disorders show the presence of Alzheimer type II astrocytosis, suggesting an important role for manganese in this cellular transformation in PSE. Recent studies from our laboratory have established that manganese levels in the GP increase by over 5-fold in MN. These levels of manganese are also consistent with similar increases reported in cases of human PSE. In addition, we have identified 5-fold higher levels of the divalent metal transporter-1 (DMT-1) protein in the pallidum of normal rats, providing a possible explanation for the focal accumulation of manganese in this brain region in both MN and PSE. Furthermore, we have determined that rats with experimental PSE show increased deposition of the metal in the mitochondria of astrocytes in the GP. Current evidence suggests that astrocytes are the site of early dysfunction and damage in both PSE and MN, possessing a high-affinity transport mechanism for manganese and an impressive capacity to accumulate the metal. Our findings therefore indicate that PSE displays several characteristics of MN, suggesting that elements of MN are operating in this form of HE. Supported by the Canadian Institutes of Health Research.

IRON CHELATION IN PARKINSON'S DISEASE Julie K. Andersen, Ph.D. Buck Institute for Age Research, 8001 Redwood Blvd., Novato, CA 94945 Parkinson's disease (PD) is a neurodegenerative disorder that stems from the loss of dopaminergic (DAergic) neurons of the substantia nigra (SN). Several studies have demonstrated elevated levels of intracellular iron in the Parkinsonian brain but whether it is causatively involved in neuronal cell death has been controversial. We previously reported that iron chelation via expression of the major iron sequestering protein ferritin protects against PD-inducing MPTP toxicity in young animals yet following prolonged exposure on its own results in neurodegeneration by 12 months of age in these same animals (Kaur et al., 2003; 2006). We are currently exploring whether iron elevation in young animals is contributing to PD pathology via its activation of prolyl hydroxylase (PH). When activated, PH can act as an inhibitor of hypoxia-inducing factor 1 alpha (HIF-1α) which normally turns on various genes involved in iron metabolism including heme oxygenase-1 (HO-1) and the transferrin receptor (TfR). Recent data from our laboratory

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suggests that the PH inhibitor 3,4-dihydroxybenzoate (3,4-DHB) protect against SN DAergic cell loss in young MPTP-treated mice. We are currently assessing the hypothesis that iron chelation in young ferritin transgenics elicits its protective effects via inhibition of PH and increased HIF-1α levels suggesting that DHB-type PH inhibitors may be a novel treatment for the disorder. We hypothesize that prolonged iron chelation in older animals effects iron-sulfur center synthesis which can impact on both mitochondrial function and iron homeostasis suggesting that prolonged iron chelation may not be suitable for treatment of PD in older patients.

IRON DYSHOMEOSTASIS IN PARKINSON'S DISEASE. Julio Salazar1,2, Natalia Mena1, Daniel Bórquez1, Rita Raisman-V.2 and Marco T. Núñez1. 1Departamento de Biología, Facultad de Ciencias, Universidad de Chile & Instituto Milenio de Dinámica Celular y Biotecnología, Santiago, Chile and 2INSERM, UMR679, Neurology and Experimental Therapeutics Paris, France. Iron is perhaps the most necessary element for life on earth, participating in a myriad of cellular processes where one-electron transfer reactions are required. Because of its essentiality, and its scarcity in aqueous oxidative environments, live organisms developed mechanisms to ensure an adequate iron supply, with disregard to long-term deleterious effects derived from iron accumulation. Neurons from brain areas sensitive to degeneration, such as the substantia nigra pars compacta (SNpc), accumulate iron with age and thus are submitted to an always increasing oxidative stress and potential cellular damage. The mechanisms involved in normal neuronal iron homeostasis that are surpassed in Parkinson disease (PD) remain largely undetermined. How nigral neurons succumb or survive to iron-induced oxidative stress are relevant questions both to know about the etiology of the disease and to design neuroprotective strategies. In this work, we will review the components of SNpc iron homeostasis, and we present evidence from recent studies aimed to unravel the molecular basis of iron dyshomeostasis in PD, in which dysregulation of IRP1 and DMT1 seems to play a pivotal role. The work of the authors was supported by FONDECYT project 1040448 and by a Millennium Scientific Initiative (MIDEPLAN, Chile) grant to the Institute for Cellular Dynamics and Biotechnology and by a CONYCYTINSERM collaborative project on Parkinson's disease.

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Symposium 10.

Neurodegeneration II. Chair: Richard M. Kostrzewa (USA) DOPAMINERGIC ACTIVITY IN THE DEVELOPING CNS: ITS CHARACTERISTICS AND POSSIBLE SIGNIFICANCE. Fernando G. de Mello Instituto de Biofísica Carlos Chagas Filho, UFRJ, Rio de Janeir,o RJ, Brasil Glial cells have been shown to participate as supportive cells in the nervous system and to take part in cellular communication via the release of neuroactive substances. In this report we show that retina glial Müller cells are able to synthesize and release dopamine (DA). DA can be detected in cell extracts of cultured Müller cells exposed to the its precursor, L-DOPA. Moreover a large portion of DA synthesized by cultured Müller glia is recovered in the superfusing medium showing the tendency of the accumulated DA to be released to the extracelular space. Müller cells purified from developing chick and mouse retinas express both, L-DOPA decarboxylase and the dopamine transporter DAT. The synthesis of DA from exogenously supplied L-DOPA to avian Müller cultures is inhibited by m-hydroxybenzylhydrazine (HBH), a DDC inhibitor. DA release occurs, in part, via a transporter-mediated process and is coupled to the activation of dopaminergic D1 receptors expressed by glia itself or by neurons. The capacity of synthesis and release of DA is not restricted to the avian retina, since similar results were obtained in Müller cell cultures from mice. Finally it is shown that, in vivo, Müller cells concentrate its DDC-like immunoreactivity in the outer part of the retina, close to the retina pigmented epithelium, a potential endogenous source of L-DOPA. Together, these results indicate that Müller glial cells can modulate early dopaminergic activity in the retina, which is likely to influence retina development and eye growth. Supported by: CNPq, FAPERJ and Pronex. PROTECTIVE MECHANISMS AGAINST NEUROINFLAMMATORY PROTEINS INDUCED BY PRECONDITIONING BRAIN CULTURES WITH MODERATE ETHANOL CONCENTRATIONS Michael A. Collins, Ph.D. Department of Cell Biology & Neurobiology, Division of Biochemistry, Loyola University School of Medicine, Maywood IL 60153 USA.

It is widely appreciated that brain damage and neuronal dysfunction are caused by chronic abuse of alcohol (ethanol). However, in nearly 20 epidemiological studies in the past decade, light-to-moderate drinking has been associated with lower risks of aging-dependent cognitive decline and dementia, including Alzheimer's disease (AD), than either abstention or heavy chronic drinking. This J-shaped curve has been variously attributed to circulatory/cerebrovascular actions of moderate ethanol ingestion, but it could be related to ethanol "preconditioning" effects on brain glia and neurons as well. Indeed, with cultures of rat organotypic brain slices or mixed cerebellar cells, 6 days of moderate ethanol preconditioning (MEP, 20-30 mM) blocks neurodegeneration caused by amyloid-β, a key AD protein (Belmadani et al., NeuroRep., 2004), as well as by other neurotoxic proteins such as HIV-1 gp120, a player in AIDS dementia. The neuroprotection arises from suppression of neuroinflammatory elevations (by gp120) in superoxide, arachidonic acid, glutamate and nitrosylated cellular proteins (Belmadani et al., Neurosci., 2001), and correlates temporally with selective induction during "late" MEP of anti-inflammatory heat shock proteins (hsp70 and hsp27). Inhibiting hsp induction during MEP with quercetin or siRNA transfection largely abolishes the neuroprotection. Our further studies indicate that hsp elevations may be downstream of MEP-induced signaling events involving activation of NMDA receptors and certain kinases, e.g., protein kinase C isoforms and, interestingly, focal adhesion kinase (FAK) associated with the actin cytoskeleton. We suggest that moderate ethanol ingestion might counter cognitive decline by exerting preconditioning-like suppressive effects on ongoing neuroinflammation provoked by accumulation of amyloidogenic proteins within the aging human brain. Supported by USPHS NIAAA and the Loyola Alcohol Research Program. KNOCKDOWN OF DOWN SYNDROME RELATED PROTEINS: A STRATEGY TO RELATE SPECIFIC GENE OVEREXPRESSION TO NEURONAL DYSFUNCTION P. Opazo1, I. Díaz1, P. Fernández1, J. Mikkola1, A.M. Cárdenas2, J. Segura-Aguilar1, P. Lockman3, D.D. Allen4, T. Shimahara5, R. Caviedes1 and P. Caviedes1. 1Prog of Molec & Clinic Pharmacol, ICBM, Fac of Med, Univ of Chile, Santiago, CHILE; 2CNV, University of Valparaíso, Valparaíso, CHILE; 3Dept. of Pharmaceutical Sciences, Texas Tech Univ. Health Science Center, Amarillo, TX, USA; 4Dept. of Pharmaceutical Sciences, Northeastern Ohio Universities College of Pharmacy, Rootstown, OH., 5Laboratoire de Neurobiologie Cellulaire et Molèculaire, CNRS, Gif sur Yvette, France.

2007 nts meeting program AND ABSTRACTS

Human Down syndrome (DS) or trisomy of autosome 21, represents the hyperdiploidy that most frequently survives birth. The condition determines multisystemic anomalies, and central nervous system alterations that result in mental retardation. At the neuronal level, our group has shown that the condition causes altered action potential and ionic current kinetics, and severe cholinergic dysfunction. Such alterations are essentially similar in cultured mouse trisomy 16 (Ts16) neurons, an animal model of DS. Regrettably, Ts16 is unviable. Hence, we established immortal cell lines from cerebral cortex, hippocampus, spinal cord and dorsal root ganglia of normal and Ts16 animals, using a proprietary method. Cell lines from Ts16 cerebral cortex (named CTb) and hippocampus (named HTk) show altered intracellular Ca2+ signals in response to several neurotransmitters, and choline uptake and secretion deficits similar to those of primary Ts16 cultures. Several DS-related genes may underlie such alterations when overexpressed: App (Amyloid precursor protein), Sod1 (Cu/Zn superoxide dismutase), and Slc5a3 (Na+/Myoinositol cotransporter). We have now used RNA antisense sequences to knockdown the expression of these genes to normal levels in our CTb and HTk lines, and then studied cholinergic and glutamatergic function. Knockdown of App greatly normalizes the noted alterations, whereas Sod1 and Slc5a3 knockdown had lesser effects. The results indicate that our cell lines are adequate models to study DS-related neuronal dysfunction, and that gene knockdown is an adequate tool to determine specific contributions of DS-related genes, and hence in the identification of therapeutic targets.

Symposium 11.

Neurodegeneration III Chair: Mario Herrera Matschitz (Chile) A MULTIPLE DISEASE ETIOLOGY APPROACH TO DRUG DISCOVERY FOR NEURODEGENERATION. Cornelis J. Van der Schyf, Ph.D. Professor and Chair of Pharmaceutical Sciences, and Professor of Neurobiology, Northeastern Ohio Universities Colleges of Pharmacy and Medicine, State Route 44, Rootstown, OH 44272. [email protected] Most diseases have a complex etiological profile of cause and effect that result from insults originating from events that vary from, for example, exposure to environmental toxins, to homeostatic disregulation among many others. Drug discovery efforts have traditionally focused on targeting a potential drug molecule

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to one of these pathologies - an approach that became known as "targeted drug design", or a so-called "magic bullet" approach. Molecules with actions at a variety of pathologies were generally construed to be "dirty drugs". We and others have applied the "dirty drug" concept to design molecules that act in a multimodal way to target several etiologies of a particular disease in a concerted manner. Application of this approach holds particular promise for the treatment or prevention of neurodegenerative diseases. We will present data detailing molecules that act by targeting ion channel conduits both for the intracellular transport of extraneous calcium, as well as extraneous iron. In vitro data will be presented that indicate that these molecules protect neurons, astrocytes, and brain endothelial cells from calcium- and iron-induced toxicity.

REVELATION OF 'DYSPLACEMENT' FUNCTIONS OF MONOAMINERGIC NERVES AFTER LESIONS P. Nowak1, R. Szkilnik1, A. Bortel1, J. Niwinski1, G. Szczerbak1, M. Drosik1, L. Noras1, A. Kwiecinski1, R.M. Kostrzewa2, R. Brus1 1Dept. Pharmacology, Medical Univ Silesia, 41-808 Zabrze, Poland, 2Quillen College of Medicine, East Tennessee State Univ, Johnson City, TN. pharbrus@ slam.katowice.pl To study 'displacement' nerve function, neonatal rats were lesioned with DSP-4 and/or 6-OHDA, then observed as adults. Effectiveness of DSP-4 or 6-OHDA treatment was assessed by brain deficits in NA (after DSP-4) or DA (after 6-OHDA), or elevation in 5-HT levels (after 6-OHDA; 5-HT sprouting) [HPLC/EC method]. In rats co-lesioned with DSP-4 and 6-OHDA, stereotyped (i.e., oral activity, grooming, digging) and motor responses (i.e., locomotor and exploratory activities) to serotonin (5-HT) and dopamine (DA) -agonists or -antagonists were enhanced, vs unlesioned or singlelesion rats. Also, in co-lesioned rats DA- and 5-HT-agonists produced behaviors that were unexpressed in rats that were unlesioned or lesioned with a single neurotoxin. These findings demonstrate that both DA and 5-HT neurons assume new roles, or 'displacement' functions when coordinate neuronal phenotypes are destroyed or absent. Supported by Medical University of Silesia: NN-1-001/06.

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AGONISTS OF CENTRAL NICOTINIC ACETYLCHOLINE RECEPTORS AND NEUROPROTECTION. Dajas F1, A Abin-Carriquiry1, G Costa1, J Urbanavicius1, M Ferreira1 B Cassels2, S Wonnacott3. 1Dept. of Neurochemistry, Instituto de Investigaciones Biológicas Clemente Estable, Montevideo,Uruguay; 2Dept. of Chemistry, Faculty of Sciences, University of Chile, Santiago; 3Dept. of Biology and Biochemistry, University of Bath, UK. In the central nervous system nicotinic acetylcholine receptors (nAChR) modulate neurotransmitter release and are implicated in several brain functions in health and pathological conditions. Epidemiological evidence shows a negative correlation between smoking and the incidence of Parkinson´s Disease and several experimental models have reproduced this negative relationship. Nicotine prevents the decline of dopamine or the functional striatal disruptions in PD experimental models while other nAChR agonists like cytisine (cy) or epibatidine can protect PC12 cells or cortical cells in culture. Thus, subtype specific ligands of nAChR could be theoretically able to modulate specifically the release of dopamine in the nigrostriatal pathway and could both palliate extrapiramidal symptomatology in PD´s and delay the neurodegenerative process. In the present work we studied the effects of cy and the halogenated derivatives 3 Br-cytisine (3 Br-cy) and 5 Br-cytisine (5 Br-cy) on nAChR-evoked [3H] DA release from rat striatal slices in vitro and DA release in the rat striatum in vivo by microdyalisis as well as the protection given to dopaminergic pathways in a model of experimental parkinsonism with lesion of the substantia nigra with 6-OHDA and administration of the agonists. In the striatal slices, an assay governed mainly by α4β2 nAChRs, 3 Br-cy is more potent and efficacious while 5 Br-cy has diminished activity compared with cy. In contrast, in vivo, 5 Br-cy was much more efficacious, cy being in the same order than nicotine. Cy and 5 Br-cy prevented the decrease of the DA striatal levels while 3 Br-cy had no effects in a wide range of doses. Since cy and 3 Br-cy are more potent at receptors showing high sensitivity to acetylcholine while cy and 5 Br-cy have almost no efficacy at these receptors, it would be likely that protection in vivo is partially related to the presence of low affinity 4 2 nAChR on the striatal dopaminergic terminals. This research was supported by a Wellcome Trust Grant (CRIG 0739295/Z/03/Z), UK.

PLEIOTROPHIN IS EXPRESSED IN THE RAT ADULT STRIATUM AND MEDIATES THE DIFFERENTIATION OF DOPAMINERGIC NEURONS IN CULTURE Raisman-Vozari R1, I Taravini2, JE Ferrario1, S Mourlevat1, T Debeir1, J Delbe3, J Courty3, L Ginestet1, GM Murer2, M Ruberg1 & O Gershanik 2. 1INSERM U679, Paris, France; 2 ININFA-CONICET, Buenos Aires, Argentina; 3FRE CNRS 2412, Creteil, France Parkinson's disease is characterized by a selective loss of the dopaminergic nigrostriatal pathway. Surviving neurons undergo both spontaneous or levodopa induced plastic changes. We have recently reported the finding of an increased expression of Pleiotrophin (PTN) in the striatum of rats with an unilateral 6-OHDA lesion of the nigrostriatal tract chronically treated with levodopa. PTN has a strong influence on neuronal differentiation and proliferation during development, is a secreted protein produced by neurons, glial and neuroepithelial cells, and is highly expressed during early post-natal brain development. PTN enhances neurite outgrowth in cultured neurons and is involved in axonal guidance. We thus sought in this study to identify by immunohistochemistry the different cellular types that contain PTN at the striatal and nigral levels, and to detect the presence of the PTN receptor N-syndecan at the nigral level. Moreover, in vitro, we investigated whether PTN affects the differentiation of DA neurons in embryonic mesencephalic cultures. Our results indicate that PTN is selectively localized in two of the major classes of striatal interneurons (cholinergic interneurons and the GABAergic interneurons that express somatostatin/nitric oxide (cholinergic interneurons and the GABAergic interneurons that express somatostatin/nitric oxide synthase). The present work suggests new roles for striatal interneurons in the adult brain. It is tempting to speculate that striatal interneurons can supply a cocktail of neurotrophic and differentiationpromoting substances that can stimulate the remodeling of dopaminergic innervation in physiological and pathological conditions. At the nigral level we were able to show that both PTN and N-syndecan are present in a small number of tyrosine hydroxylase immunoreactive neurons. In vitro, our results showed that PTN can induce the differentiation of dopaminergic neurons. In conjunction, all of our results suggest that PTN would mediate some of the compensatory mechanisms that surviving dopaminergic nigrostriatal neurons undergo in the parkinsonian brain.

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POSTER PRESENTATIONS Session 1.

Neuroprotection Chairs: W Danysz & Y Tizabi 1. N-ACETYLCYSTEINE PROTECTS CULTURED HIPPOCAMPAL NEURONS FROM EXCITOTOXICITY ENHANCED BY L-TRASNPYRROLIDINE-2,4-DICARBOXYLIC ACID. Toshiyuki Himi, Maiko Kawaguchi Department of Pharmacology and Toxicology, Faculty of Pharmacy, Musashino University. 1-1-20 Nishi-Tokyo, Tokyo 202-8549, JAPAN We have shown that the inhibition of the glutamate transporters increased neuronal vulnerability against oxidative stress. Here we show that neuronal glutamate transporter EAAT3 plays an essential role in L-cysteine uptake in cultured neuronal cells. In mixed cell cultures, glutamate transporter inhibitors inhibited not only glutamate uptake but also L-cysteine uptake, though these inhibitors did not influence L-cysteine uptake in the pure astrocyte cultures. Increased level of extracellular cysteine enhanced the L-cysteine uptake and intracellular glutathione level, and inhibitors of glutamate transporters inhibited this effect of extracellular cysteine. Antisense oligonucleotide for EAAT3 decreased cysteine uptake and glutatione level in the neurons, and increased neuronal vulnerability against oxidative stress. These facts indicate that neuronal glutamate transporter EAAT3 plays an important role in neuronal cysteine uptake and glutathione synthesis. 2. KNOCK-DOWN OF IRP1 PROTECTS TYROSINE HYDROXYLASE-POSITIVE NEUROBLASTOMA FROM CELL DEATH Natalia Mena, Enrique Armijo, Daniel Bórquez and Marco T. Núñez. Departamento de Biología, Facultad de Ciencias, Universidad de Chile & Instituto Milenio de Dinámica Celular y Biotecnología, Santiago, Chile Iron is indispensable for function of many prosthetic groups, including heme and iron-sulfur clusters, and animals have accordingly developed sophisticated systems to maintain iron homeostasis. Thus, insufficient iron concentrations affect the functioning of numerous proteins, whereas its excess can oxidize and damage nucleic acids, proteins and lipids. The incorporation of cellular iron, its compartmentation and export must be regulated. To this end, cells have developed sophisticat-

ed systems to support the homeostasis of Fe. In vertebrates, the cellular levels of Fe are controlled postranscriptionally by the activity of regulatory proteins named IRP1 and IRP2, two cytosolic proteins that bind to iron response elements, named IREs, present in the nontranslated regions of the mRNA of proteins of iron homeostasis (Eisenstein, 2000). Investigations have demonstrated that the normal homeostasis of iron is sustained by IRP2. On the contrary, IRP1 does not have a significant role in the normal regulation of cell iron levels. IRP1, but not IRP2, is activated by oxidative stress generated by the accumulation of iron, which produce a vicious circle of more intracellular iron and more stress. This characteristic of IRP1 could be fundamental in the beginning of neurodegenerative diseases such as Parkinson. Due to these precedents we postulate that IRP1 is a "death protein", whose cellular function would be the induction of cellular death in states of disease or aging. Here, we present work with SHSY-5Y human neuroblastoma cells induced to differentiate into tyrosine hydroxylase-positive cells transfected with a plasmid carrying shRNA targeted to IRP1. We found that the knock-down of IRP1 induced decreased levels of the iron uptake transporter DMT1 and increased resistance to oxidative stress-mediated cell death induced by iron, 6-OHDA, LPS and rotenone. We conclude that IRP1 is an interesting target to induce the decrease of iron accumulation and cell death. Supported by FONDECYT project 1040448 and by a Millennium Scientific Initiative (MIDEPLAN, Chile) grant to the Institute for Cellular Dynamics and Biotechnology. 3. COMPARISON OF DIAZOXIDE AND PACAP AS NEUROPROTECTIVE COMPOUNDS ON DIFFERENT RETINADEGENERATION MODELS: HISTOLOGICAL AND IMMUNOCYTOCHEMICAL INVESTIGATIONS Babai N1,1 T Atlasz1, P Kiss2, R Gábriel1, F Bari3, F Domoki3 and D Reglodi2. Depts. of 1Exp. Zoology and Neurobiol., 2Anatomy, Univ. Pécs, 3Dept. of Physiol., Univ. Szeged, Hungary Several retinal degeneration models are described for different ophthalmic diseases. Pituitary adenylate cyclase activating polypeptide (PACAP) and diazoxide (DIAZ) are neuroprotective in animal models of different brain pathologies. We investigated the neuroprotective role of these compounds in two rodent model systems: monosodium-glutamate (MSG)- and chronic carotid occlusion (CCO)-induced retinal degeneration. Rats were subjected either to sc MSG treatment on P1,

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P5 and P9 days, or to CCO at 2 mths, followed by intravitreal DIAZ or PACAP treatment. Rats were sacrificed 21 days after. In MSG-treated retinas the inner nuclear and ganglion cell layers fused. Both compounds were protective, the order of neuroprotective efficacy was DIAZ > PACAP. CCO led to a severe degeneration of all retinal layers, and PACAP was more effective than DIAZ. Our results may have clinical implications in reducing glutamate-induced excitotoxicity or ischemic retinal degeneration in ophthalmic diseases. Immunocytochemical experiments are in progress to reveal the cell type-specific effects of the degeneration models and the neuroprotective treatments. Support: OTKAT046589/061766, ETT 439/2006. 4. THE NEUROPROTECTIVE EFFECT OF PACAP IN A 6-OHDA-INDUCED RAT MODEL OF PARKINSON`S DISEASE IS GENDERDEPENDENT Reglodi D, A Lubics, I Lengvari, G Toth, O Hegyi, P Kiss, B Gaszner and A Tamas. Dept. of Anatomy, Univ. Pecs, Hungary In the past few years we provided evidence that the neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) is protective in a rat model of Parkinson`s disease. PACAP decreases the loss of dopaminergic neurons in the substantia nigra after a unilateral 6-OHDAinduced lesion. This is accompanied by a significant amelioration of the behavioral symptoms: PACAPtreated animals show no hypokinesia and a better recovery in the asymmetrical signs. It is well known that the effects of the neurotoxin 6-OHDA and the human disease as well show gender differences. A few neuroprotective substances have been reported to be less effective in females. The aim of the present study was to compare the neuroprotective potential of PACAP in normal and gonadectomized male and female rats. A similar degree of neuroprotection was achieved in normal males and ovariectomized females. However, PACAP was not able to decrease cell loss in normal females and castrated males, where the effects of the 6-OHDA-treatment were not so severe. Our study shows that not only the effects of neurotoxins, but also the neuroprotective potential of drugs may be different in males and females in animal models of Parkinson`s disease. Support: OTKA T046589, T048848, ETT439/2006, 314/2006. 5. FRIZZLED RECEPTORS ARE LOCATED AT SYNAPTIC REGIONSAND FRIZZLED 1 MEDIATES NEUROPROTECTION AGAINST Aβ PEPTIDE. Lorena Varela-Nallar, Marcelo A. Chacon and

Nibaldo C. Inestrosa. Centro de Regulación Celular y Patología "Joaquín V. Luco" (CRCP), MIFAB, Facultad de Ciencias Biológicas, P. Universidad Católica de Chile. The Wnt signaling pathway is involved in neuroprotection against the toxicity of amyloid-β-peptide (Aβ). Binding of Wnt ligands to their receptors Frizzled (Fz) activates the Wnt signaling. We analyzed the distribution of different Fzs in cultured hippocampal neurons and determined that these receptors are located in synaptic regions. The markers used were synapsin-1 for the presynaptic region and PSD-95 for the postsynaptic region. We also evaluated the neuroprotective effect of Fzs expression. Results indicated that PC12 cells transfected with Fz1 are more resistant to Aβ toxicity when exposed to Wnt3a, as determined by MTT assay and caspase-3 activity. In agreement with these results, Fz1 knock down increased the susceptibility of PC12 cells and hippocampal neurons to Aβ neurotoxicity. These results, along with the synaptic localization of Fz receptors, suggest that the Wnt signaling pathway may indeed prevent the synaptic failure induced by Aβ peptide. Supported by FONDAP-Biomedicine (Nº13980001) and Millennium Institute for Fundamental and Applied Biology (MIFAB). 6. COMMON ANTIAPOPTOTIC MECHANISMS IN THE NEURO- AND CARDIOPROTECTIVE EFFECTS OF PACAP Racz B1, B Gasz1, F Gallyas2, P Kiss3, A Tamas3, R Jozsa3, A Lubics3, I Lengvari3, G Toth4, E Roth1 and D Reglodi3. Department of 1Surgical Research & Techniques, 3Biochemistry, 3Anatomy, Univ. Pecs, 4Dept. of Medical Chemistry, Univ. Szeged, Hungary. The neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) has well-known neuroprotective effects. This has been shown in neuronal cultures against various types of toxic agents and in vivo, in different models of neuronal injuries. One of the main mechanisms of the neuroprotective effects of PACAP is its antiapoptotic actions. PACAP acts on various, partially interacting, apoptotic signaling mechanisms. In the present study we compare the effects of PACAP on cell survival and apoptosis, and the underlying signal transduction pathways, in neuronal and cardiac cells. Cell survival MTT test and flow cytometric analysis show that PACAP significantly decreases both neuronal and cardiomyocyte apoptosis. We found that PACAP acts on similar pathways in both systems: the proapoptotic members of the MAPK family (JNK, p38) are inhibited,

2007 nts meeting program AND ABSTRACTS

while the antiapoptotic ERK is activated. The mitochondrial route of apoptosis is also inhibited in both systems. The effect of PACAP on these pathways leads to a significant decrease in the levels of caspase-3 in both cardiomyocytes and neuronal cells. Support: OTKA T046589/48848/F048908, ETT439/314/2006. 7. EFFECTS OF MINOCYCLINE ON DOPAMINERGIC NEURONAL DEATH INDUCED BY 6-HYDROXYDOPAMINE IN ORGANOTYPIC CULTURE OF THE RAT MESCENCEPHALON Carlos Juri MD, Gigliola Ramírez, Juan Tichauer and Rommy von Bernhardi MD PhD. Laboratory of Neurosciences. Neurology Department. School of Medicine. Pontificia Universidad Católica de Chile.Marcoleta 352. Santiago. Chile. [email protected] Minocycline is a member of the tetracycline antibiotics family with additional properties as inhibitor of microglial activation and apoptotis. Parkinson's disease is a neurodegenerative disorder characterized by preferential dopaminergic neuronal death. Microglial activation has been involved as one of its pathogenic mechanisms. The aim of this study is to evaluate the effect of minocycline on dopaminergic neuronal death induced by the neurotoxin 6 hydroxydopamine (6OHDA) in order to develop a new model of organotypic mescencephalic culture. Mescencephalic slices of 300 µm were obtained from neonatal rats, after proper selection, they were cultured for 5 days in a defined culture medium. At the third day neurotoxicity was induced by 6OHDA, one group of cultures was then treated with minocycline and the other with vehicle. After completing five days in culture, slices were analyzed by western blot to evaluate tyrosine hydroxylase and beta-tubulin III immunoreactivity. Minocycline reduced the dopaminergic cell death induced by 6OHDA in this new model of in vitro Parkinsonism neurodegeneration. The significance of our model for the evaluation of therapeutic actions will be discussed. 8. POSTTRAUMATIC ADMINISTRATION OF PACAP IN CENTRAL FLUID PERCUSSION INJURY IN RATS Kovesdi E1, A Tamas2, D Reglodi2, O Farkas3, J Pal1, P Bukovics1, T Doczi1, A Buki1. Depts. of 1Neurosurgery, 2Anatomy and 3Radiology, Medical Faculty, University of Pécs, Pécs, Hungary Pituitary adenylate cyclase activating polypeptide (PACAP) has neuroprotective effects in various in vitro and in vivo studies. The aim of our study was to analyze the effects of PACAP on diffuse axonal

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injury (DAI), an important contributor to morbidity and mortality associated with traumatic brain injury (TBI), in a central fluid percussion (CFP) model. Rats were subjected to moderate (2Atm) CFP injury. 30 min post-injury 100 µg PACAP was administered icv. DAI was assessed by immunohistochemical detection of β-amyloid precursor protein (β-APP), indicating impaired axoplasmatic transport, and RMO-14 antibody, representing foci of cytoskeletal alterations (neurofilament compaction), both considered classical markers of axonal damage. The mean density values of damaged axonal profiles revealed significant protection in the corticospinal tract of the PACAP-treated rats. In addition to our former observations in an impact acceleration model of diffuse TBI, the present study demonstrated that PACAP also inhibits DAI in the CFP injury model, a finding indicating that PACAP and derivates can be considered potential candidates for further experimental studies, or for clinical trials in the therapy of TBI. Support: OTKA T046589, T048724, ETT 439/2006. 9. ASSOCIATION OF GST M1 NULL POLYMORPHISM WITH PARKINSON´S DISEASE IN CHILEAN POPULATION WITH STRONG AMERINDIAN GENETIC COMPONENT Carolina Perez-Pastene1, Rebecca Graumann1, Fernando Díaz-Grez1, Marcelo Miranda3,5, Pablo Venegas3,4, Osvaldo Trujillo Godoy3, Roque Villagra3, Jose Manuel Matamala1, Luisa Herrera2 and Juan Segura-Aguilar1 1Molecular and Clinical Pharmacology and 2Human Genetic, ICBM, Faculty of Medicine, University of Chile , Casilla 70000, Santiago-7, Chile; 3Liga del Parkinson de Chile, Santiago, Chile; 4Clinic Hospital Universidad de Chile, Santiago, Chile; 5Clinica Las Condes, Santiago, Chile Aminochrome is the precursor of neuromelanin, but one electron reduction of aminochrome generates a potent neurotoxin or the formation of neurotoxic alphasynuclein protofibrils is enhanced and stabilized by aminochrome. GSTM1 or GSTM2 catalyze the conjugation of aminochrome to a GSH-conjugate, which is resistant to oxidation catalyzed by biological oxidizing agents such as oxygen superoxide radicals and hydrogen peroxide. In addition, GSTM2 catalyzes the conjugation of the precursor of aminochrome dopamine o-quinone to 5-glutathionyl dopamine, which is the precursor of 5-cysteinyl-dopamine encountered in rat, guinea pig and human brain. We have studied the association of null mutation Glutathione Transferase M1 (GST M1*0/0) with

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Parkinson's disease in a Chilean population with strong Amerindian genetic component. We determined the genotype in 349 patients with idiopathic Parkinson's disease and compared to 611 controls. A significant association of null mutation in GST M1 with the disease was found (p value 0.021). This association becomes more relevant in earlier range of age. The association of GSTM1*0/0 with Parkinson's disease support the idea that Glutathione Transferase M1 may play a role protecting the astrocytes against toxic dopamine oxidative metabolism since this enzyme prevent the toxic oneelectron reduction of aminochrome. Supported by FONDECYT 1061083 and FIRCA TW007044-01. 10. ENZYME GST M2-2 PREVENTS AMINOCHROME INDUCED CELL DEATH IN ASTROCYTES Rebecca Graumann and Juan Segura-Aguilar Programme of Molecular and Clinical Pharmacology and Morphology, ICBM, Faculty of Medicine, Casilla 70000, Santiago-7, Chile. Dopamine transporter facilitates the uptake of dopamine into dopaminergic neurons and astrocytes. At physiological pH products of oxidized dopamine such as aminochrome may act as a neurotoxin. Human glutathione transferase of the µ class, isoform 2-2 (GST M2-2) conjugates aminochrome and dopamine o-quinone, the products of which are resistant to oxidation. GST M2-2 is expressed and localized in astrocytes. The results of this study suggest that GST M2-2 functions in astrocytes as a protective enzyme against aminochrome. The importance of the protective function of GST M2-2 in astrocytes was analyzed using the human astrocyte cell line U373MG. Data from this study imply that U373MG cell line expresses GST M2-2 and takes up 3H-dopamine. Cells were incubated with dopamine in the presence of copper to facilitate the formation of aminochrome. U373MG cells appeared to have increased mortality during incubation with aminochrome compared with the cell line RCSN-3 derived from substantia nigra. Astrocytes transfected with the cDNA of GST M2-2 (NM_000850) expressed increased survival during incubation with aminochrome and copper. Electron spin resonance analysis indicated that there is a difference in copper incubation between native cells and transfected cells. Supported by FONDECYT 1061083 and FIRCA TW007044-01.

11. THE DT-DIAPHORASE PREVENT THE AGGREGATION OF α-SYNUCLEIN INDUCED FOR AMINOCHROME Cardenas SP, C Perez-Pastene, E Couve, J SeguraAguilar. Molecular and Clinical Pharmacology, ICBM, Faculty of Medicine, Casilla 70000, Santiago-7, Chile; α-Synuclein is a 140 aa protein of the neuron, that under pathological stimuli can aggregate to fibrils. The aggregation of α-synuclein has been related with the generation of Parkinson's Disease. The oxidation of the dopamine has been implicated in the aggregation of α-synuclein to prefibrillar structures called prothofibrils, very toxic structures that can induce damage to the neurons and kill it. We have found that a quinone derived of the oxidation of the dopamine, the aminochrome, is responsible of the agregation of α-synuclein, inducing the formation of globular polymeric structures and preventing the fibrils formation, but more important, we found that flavoenzymes that can reduce the aminochrome, like cytochrome C reductase and specially the DT-diaphorase can prevent this aggregation of α-synuclein. Supported by FONDECYT 1061083 and FIRCA TW007044-01. 12. EFFECTS OF PLANTS USED IN PERUVIAN FOLK MEDICINE ON MIXED CULTURES OF NEURONS AND GLÍA María S. Gonzales Champi Biochemistry and Nutrition Research Centre "Alberto Guzmán Barrón", State University of San Marcos, Lima, Perú. This research pretend to link ancient knowledge used by Peruvian population about plants used to treat illness related to nervous system with reliable information to improve their use. So that knows if the substances present in plants have an effect positive or negative in the function of neurons and glía, if they have beneficial influence on nervous cells to delay or arrest the damage that progress to death cell analyze variables as activation or inactivation or synthesis of molecules implicated, etc. First, make three types of cellular cultures of rats: neurons, glial cells and a mixed culture of both and will proceed to identify type of nervous cell, with control and experimental groups. In parallel I'll prepare extracts crude watery and alcoholic, after their fractions will obtain by Sephadex, the next step is a preliminary analyze of substances with HPLC-DAD. Subsequently a TLC assay to select the fractions to biological assays; depend of outcomes to continue HPLC-DAD and finally RMN.

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After add to the cultures: glutamate, LPS, staurosporine and privates of potassium to induce inflammation, excitotoxicity, degeneration and apoptosis. Later, apply test to determinate of cellular viability by FDA. Evaluation of the glial activation by: measurement of production of nitric oxide; ascertainment of stimulate of transcription factors NFκB and C/EBPs and; measurement of synthesis of TNF-α. Measurement of the proliferation of glial cells and; assessment of the microglial activity. Also will proceed to value the type of death cell using Hoechst´ dye and activation of Caspase-3. Finally will use immunofluorescence assays with lectin. And the results are analyzed with statistics test ANOVA and Student-t considered significant values of p <0.05.

survival of TH positive neurons compared to controls (P<0.0001). Additionally, 2 mM and 4 mM of inosine augmented survival to a greater extent than 0.5 mM and 1 mM. Treatment with 2 mM and 4 mM of insoine also protected TH positive neurons against 6-OHDA administration (P=0.01). Although preliminary, the current results combined with inosine's ability to transverse the BBB, indicate that inosine has the potential to be a noninvasive neuroprotective strategy in animal models of PD. Future experiments will analyze the mechanisms by which inosine protects DA neurons in vitro and the ability of inosine to protect the nigrostriatal system when administered peripherally to 6-OHDA lesioned rats. Supported by: University of Cincinnati Millennium Fund (CES).

Session 2.

14. NON-CANONICAL Wnt PATHWAY IS INVOLVED IN POSTSYNAPTIC DIFFERENTIATION AND SYNAPTIC PROTECTION AGAINST AMYLOID-β PEPTIDE (Aβ). Iván E. Alfaro, Ginny G. Farías, Margarita Dinamarca and Nibaldo C. Inestrosa. Centro de Regulación Celular y Patología "Joaquín V. Luco" (CRCP) and MIFAB, P. Universidad Católica de Chile.

Neurotrophic Factors, Signal Transduction Chairs: I Mocchetti & G Guillemin 13. THE PURINE NUCLEOSIDE INOSINE IS NEUROTROPHIC FOR MESENCEPHALIC DOPAMINE NEURONS IN VITRO BT Terpstra, KL Paumier, ND Levine, SL Wohlgenant and CE Sortwell Department of Neurology, University of Cincinnati, Cincinnati, OH 45267. Parkinson's Disease (PD) is characterized by a progressive loss of tyrosine hydroxylase (TH) positive neurons in the substantia nigra pars compacta, resulting in decreased levels of dopamine (DA) in the striatum. At present there is no clinically viable neuroprotective therapy for patients with PD and experimental neuroprotective strategies, such as direct infusion or gene transfer delivery of neurotrophic factors, are limited by their invasive nature. Inosine, a purine nucleoside that readily crosses the blood brain barrier (BBB), has been shown to have neuroprotective properties against ischemia in vivo (Shen et al., 2005). In the current study, we demonstrate that inosine is neurotrophic for DA neurons in vitro. Mesencephalic cell suspensions derived from E14 Sprague Dawley rats were plated at a concentration of 3000 cells/µl. Cultures were subjected to daily inosine treatment (0.5 mM-4 mM) and evaluated under two separate cell culture paradigms: at plating or twenty four hours prior to and following a 6-hydroxydopamine (6-OHDA) insult. After the cessation of inosine administration, immunocytochemistry for TH was performed. At all doses tested, inosine added at plating increased the

The synaptic hypothesis on Alzheimer´s disease has been supported by recent studies indicating that neurotoxic amyloid-β-peptide (Aβ oligomers specifically target central synapses, decreasing PSD-95 levels and reducing the surface expression of NMDA and AMPA glutamate receptors in both hippocampal neurons and APP transgenic animals. We have demonstrated that Wnt-5a is a synaptogenic non-canonical Wnt ligand that can promotes postsynaptic differentiation increasing the number of PSD-95 clusters and the density of dendritic spines in cultured hippocampal neurons. Therefore, we decided to examine whether the activation of the noncanonical Wnt-5a signaling was able to protect glutamatergic synapses from deleterious effects of Aβ oligomers and we would like to find out whether the Wnt5a can prevent the decrease in synaptic contacts, PSD-95 clusters and NMDA receptors clusters induced by the Aβ oligomers. Our results, suggest that the non-canonical Wnt signaling plays a pivotal role in the development and maintenance of the normal synaptic integrity, and its activation may be of therapeutic interest in patients with neurodegenerative diseases such as Alzheimer´s. Supported by FONDAP (Nº 13980001), Millennium Institute (MIFAB) and a pre-doctoral Fellowship from CONICYT to I.E.A.

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15. EFFECT OF SR-A AND SR-MARCO LIGANDS ON GLIAL NO PRODUCTION AND THE MODULATION OF SR-A EXPRESSION BY TGF-β Tichauer J, B Godoy, R von Bernhardi Neuroscience Laboratory, Neurology Department, Faculty of Medicine, Pontificia Universidad Católica de Chile. Alzheimer is a progressive neurodegenerative disease characterized by memory impairment and cognitive dysfunction. β-amyloid (Aβ) aggregates are a major pathological hallmark. Aβ and pro-inflammatory factors can activate astrocytes and microglial cells surrounding plaques, inducing various processes such as β-amyloid phagocytosis, release of short lived cytotoxic factors as nitric oxide (NO) and the induction of different transduction pathways. Previous reports have shown that astrocytes can modulate microglial cytotoxic reactivity through secretion of TGF-β and can also increase Aβ phagocytosis. SR-A and SR-MARCO are among the scavenger receptors expressed by glial cells involved in the uptake of Aβ, but its role in signaling cascades inducing an inflammatory response and TGF-β modulation are not clear. We evaluate the effect of SR-A ligands (Fucoidan and Poly I) and Aβ on NO secretion by astrocytes and microglial and evaluate the effect of TGF-β on scavenger expression in vitro. RESULTS: Poly I increased NO secretion by astrocytes and microglial cells of rat primary cultures whereas Fucoidan had little effect. TGF-β increased SR-A expression by the microglial cell line EOC20. We also found a decrease on the expression of Smad3 protein of the hippocampus that was age-dependent. CONCLUSION: Poly I (but not Fucoidan) increased NO production in a timeand concentration-dependent manner. Aging could diminish the modulatory effect of TGF-β on glial activation due to a decrease on Smad3 expression favoring neurodegenerative diseases. 16. Wnt SIGNALING INDUCES MOLECULAR MODIFICATIONS IN N-CADHERIN. Sebastián Belmar, Marcela Colombres and Nibaldo C. Inestrosa. Centro de Regulación Celular y Patología "Joaquín V. Luco" (CRCP) y MIFAB, Facultad de Ciencias Biológicas, P. Universidad Católica de Chile. The cadherin/catenin complex has been implicated in synaptic events such as synaptogenesis and synaptic plasticity. We are interested in the modulation of the biochemical properties of N-cadherin by the Wnt pathway, as well as the effects of the amyloid-β-peptide (Aβ) oligomers. We have found that the activation of the Wnt signaling increases the formation of N-cadherin dimers resistant to trypsin in rat hippocampal neurons. Our

results suggest that the activation of Wnt signaling induced a strong adhesive conformation of N-cadherin at synaptic sites. We have also observed an increase of N-cadherin puncta in hippocampal neurites, after exposure to the non-canonical Wnt-5a ligand. In addition, we prepared synaptosomes and post-synaptic densities (PSD) from rat brain, such preparation reveals an enrichment of N-cadherin and β-catenin in these fractions. Moreover, when we incubated rat hippocampal neurons with Wnt ligands, we found an increased in the β-actin association as observed by co-immunoprecipitation. Interestingly, the incubation of hippocampal neurons with Aβ oligomers disrupts such complex. We conclude, that the Wnt signaling modulation of the cadherin/ catenin complex, could has interesting implications on the neuronal plasticity required to prevent or reverse the synaptic failures observed in Alzheimer`s disease. Supported by FONDAP (Nº 13980001), Millennium Institute (MIFAB) and a pre-doctoral Fellowship from CONICYT to MC. 17. DOPAMINERGIC PLASTICITY IN THE ADULT DIFFERENTIATED AVIAN RETINA. MCF de Mello, I Henze, PF Gardino, R Fleming and FG de Mello. IBCCF-UFRJ, Brasil. [email protected] In the chick retina, differentiation of tyrosine hydroxylase phenotype (TH+) is observed after the 12th embryonic day (ED). The birth of committed dopaminergic amacrines occurs between the 5th and 7th ED. It was postulated that intrinsic retinal factors, in the interval between the 7th and 12th ED would signal maturation of the TH+ phenotype. Work from our group showed that TH+ differentiantion is dependent on signals that activate adenylyl cyclase. In the retina, PACAP, a neuroactive peptide, is capable of inducing TH+ phenotype in retina cell cultures. After ED 12, signaling via PACAP receptor (PAC 1) desensitizes, reaching low levels in the mature tissue, as observed by measuring cAMP accumulation. Cronic administration of the PAC 1 antagonist (PACAP 6-38) resensitizes the PAC-1/cyclase system in the mature retina. The presence of Nurr1 positive cells in the mature retina far exceeds the number of TH+ cells, suggesting that if one could resensitize PACAP signaling in the retina, one could activate TH+ plasticity in the differentiated tissue. When eyes from post-hatched chicken are exposed to PACAP 6-38 for 24 hours, the PACAP response of the tissue increases 2-3 fold, which resuls in 50% increase in the number of TH+ cells during this period. We suggest that, provided PACAP signaling can be reactivated in mature retinas, plastic changes in dopaminergic phenotype can be achieved. Supported by: CNPq, FAPERJ, Pronex.

2007 nts meeting program AND ABSTRACTS

18. REDOX MODULATION OF TRANSCRIPTION FACTORS AND HAT/HDAC ENZYMES IN NEURONS. Denise Riquelme, María Angélica Carrasco. Laboratorio de Bioquímica Celular. ICBM, Facultad de Medicina. Universidad de Chile. [email protected]. In neurons, low concentrations of ROS facilitate long term changes in the structure and neuronal functions necessary for synaptic plasticity. These changes require neuronal gene transcription; this is mediated by both, modulation of the transcriptional activators/repressors and changes in the structure of the chromatin. In neurons the study of the modulation of transcripcional regulators by redox state is substudied, but is possible that redox modulation of transcriptional factors (AP-1, NFκB, NFAT) and HDAC/HAT enzymes depends of the crosstalk between redox state and calcium. The aim of this work is studying the effect of neuronal redox state in both, activation of transcriptional factors and in the activity of HAT/HDAC enzymes. To develop this aim we will utilize electrical stimulated primary neurons in different intracellular redox conditions. We will evaluate the activation of transcriptional regulators by utilization of gene reporters and biochemical techniques. We expected that the electrical stimulation activates ROS production and these species will modulate, in calcium dependent way, both the activation of transcriptional factors and HAT/HDAC enzymes activity. FONDECYT 1060177. 19. EXPRESSION OF BDNF RECEPTORS, TRKB AND P75, AND OF SYNAPTIC VESICLE-RELATED PROTEIN SYNAPSIN I IN SUBSTANTIA NIGRA IN A RAT MODEL OF PRESYMPTOMATIC PARKINSON`S DISEASE. E. Riquelme, J. Abarca, C. Leon, and G. Bustos Lab. of Biochemical Pharmacology, Dept. of Cell and Molecular Biology, Pontifícia Universidad Católica de Chile, Santiago. By using a rat model of presymptomatic Parkinson`s disease (PD) we have reported early and transient increases in brain derived neurotrophic factor (BDNF) expression in substantia nigra (SN). These changes could modify, in turn, the expression of vesicle-related proteins in SN, in order to facilitate transmitter release from remaining undamaged cells, following partial dopamine cell population disappearance. We have now evaluated, by means of immunohistochemical (IHC) techniques, the expression in SN of BDNF receptors, trkB and p75, at earlier stages of our presymptomatic PD model. In parallel we have studied the expression in SN of the synaptic vesicle-related

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protein synapsin I. Ipsilateral intrastriatal 6-OH-DA injection was used to partially damage the nigro-striatal DA pathway. After one day of the neurotoxin injection, an increase in the number of phosphorylated synapsin I-immunoreactive (IR) cells was found in the ipsilateral SN. However, at this time period, no changes were detected in trkB, p75 and synapsin I IR. In contrast, one week after 6-OH-DA injection we observed a decrease in the expression of both trkB and synapsin I IR in the SN. At this last time-period we detected no IR changes for p75 in the ipsilateral SN of 6-OH-DA pretreated rats. These results indicate that during the first stages of this rat presymptomatic model of PD, transient increases of BDNF expression as well as phosphorylated synapsin IR in SN precede a decrease in the expression of the trkB receptor, whereas the expression of the p75 receptor in SN is unaffected. At later stages of this rat model, there is a decrease of synapsin I expression in SN which correlates with a "down-regulation" in the expression of both BDNF and trkB in this basal ganglia nucleus. It is possible that synaptic transmission may be modified in SN by biphasic changes in the expression of both BDNF and its trkB receptor during presymptomatic stages of PD. FONDECYT # 105-0981. 20. EXPRESSION OF DOPAMINERGIC TRANSCRIPTION FACTORS IN PRENATALLY STRESSED ADULT RATS. María Rosa Katunar1, Alicia Brusco2 and Marta C. Antonelli1. 1IQUIFIB (UBA-CONICET), Facultad de Farmacia y Bioquímica. UBA. 2IBCyN. Facultad de Medicina.UBA. Buenos Aires. Argentina. Stress applied to the gestant dam was shown to produce long-term effects on the adult offspring. We have previously shown that prenatal stress (PS) increases D(2)-type dopamine (DA) receptor levels with a concomitant reduction in DA release after amphetamine stimulation in prenatally stressed rats of 4 weeks old and a significant reduction of dendritic arborization, synaptic contacts and a pronounced astrocytic reaction. The precise anatomical localization and functional differentiation of dopaminergic neurons is achieved through the effect and gradient disposition of several induction signals. It has recently been identified two transcription factors Nurr1 and Pitx3 which are expressed at critical moments of DA neurons differentiation. Their genetic expression is activated immediately after these neurons determination and maintained through adult life. Employing an immunocitochemistry approach, we studied the expression levels of these factors in mesencephalon slices of prenatally stressed adult rats. Nurr1 expression was localized in

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periaqueductal ependymals cells, whereas Pitx3 expression was restricted to Sustantia Nigra, compact and reticular (SNc and SNr) and to ventral tegmental area (VTA). PS rats showed an increse in Pitx3 positive nucleus. Since Pitx3 modulates the expression of the key enzyme in DA synthesis, tyrosine hydroxylase, the increase of Pitx3 in the adult prenatally stressed rat might be a compensatory mechanism to counteract the decrease of DA levels observed at early stages.

21. GLUCOSE TRANSPORT STIMULATION IN ASTROCYTES REQUIRES NA+ AND CA2+ SIGNALLING. 1,2Loaiza A, 1OH Porras, 1I Ruminot, 1LF Barros 1Centro de Estudios Científicos CECS. 2Universidad Austral de Chile We have previously reported that glutamate activates glucose transport in astrocytes in culture. This effect is mediated by the Na+-glutamate co-transporter, which supports glutamate homeostasis in the central nervous system and particularly in the synapses. Besides the stimulation of glucose transport, glutamate increases cytosolic Na+ and Ca2+. These effects are mimicked by D-aspartate, a specific substrate of the Na+-glutamate co-transporter which does not stimulate glutamate receptors of astrocytes. Now we show that the inhibition of the intracellular Ca2+ increase, using either extracellular EGTA or intracellular BAPTA, prevented the stimulation of the uptake of the fluorescent glucose analogue 6-NBDG by D-aspartate, suggesting that Ca2+ is a mediator in the effect. However, the induction of an acute increase in intracellular Ca2+ by the agonists DHPG or endothelin-1 (ET-1) was not able to stimulate glucose uptake. Similarly, an acute Na+ increase induced with gramicidin or ouabain, also failed to stimulate glucose uptake. On the other hand, when Ca2+ and Na+ signals were induced simultaneously, with ET-1 and gramicidin respectively, hexose uptake was stimulated in a similar manner as with glutamate or D-aspartate. The activation of hexose transport can also be reproduced by sequential induction of Na+ and Ca2+ signals. These results suggest that acute regulation of glucose transport in astrocytes occurs by the coincident detection of Na+ and Ca2+ signals. The functional consequences of this novel mechanism will be discussed in the context of acute regulation of energy metabolism in the brain. FONDECYT 1051082.

22. ROLE OF THE EXPRESSION OF RYR AND TRKB IN MORPHOLOGICAL CHANGES INDUCED BY BDNF IN DENDRITIC SPINES OF HIPPOCAMPAL NEURONS Tatiana Adasme, Cecilia Hidalgo, MA Angélica Carrasco. Laboratorio de Señales Mediadas por Calcio; Laboratorio de Bioquímica Celular. ICBM, Facultad de Medicina. Universidad de Chile. [email protected] Synaptic plasticity involves synaptic efficiency changes induced by neuronal activity, which are related to learning and memory processes. Long-term changes like new synapse formation require mRNA and protein synthesis. Ca2+ release mediated by RyR has been linked to this process. RyR activation promotes dendritic spines growth, and its inhibition blocks transcription involved in synaptic plasticity. Additionally, BDNF/TrkB downstream signaling in hippocampus generates Ca2+ mobilization, activation of transcription factors, expression of genes like RyR2, and dendritic spines stretching. The aim of this work is to determine whether the morphological changes induced by BDNF are dependent on RyR2 and TrkB expression through the activation of CREB and NFAT, by a mechanism dependent on Ca2+ release and/or ROS production. We expect to find an increased RyR2 and TrkB expression related to Ca2+, ROS and RNS signaling, and to the morphological cell changes in response to BDNF. FONDAP 15010006 FONDECYT 1060177. 23. REDOX MODULATION OF THE RYANODINE RECEPTOR/CALCIUM RELEASE CHANNEL (RYR) ACTIVITY FROM RAT HYPOCAMPAL NEURONS" Humeres A1, C Hidalgo1,2, MT Nuñez3 1Centro FONDAP de Estudios Moleculares de la Célula, 2ICBM, Facultad de Medicina, Universidad de Chile, Santiago, 3Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Santiago. The ryanodine receptors/calcium release chanels (RyR chanels) mediate calcium (Ca2+) release from intracellular stores in several cellular types including neurons. These channels originate Ca2+-induce-Ca2+-release (CICR), a mechanism for the amplification and propagation of Ca2+ signals initially generated by Ca2+ entry into cells. Several intracellular components, such as Ca2+, Mg2+, ATP, protein kinases, phosphatases and redox species regulate RyR channels. In particular our lab has demonstrated that changes in the RyR redox state by endogenous redox compounds or by changes in cellular redox state show an important physiological relevance.

2007 nts meeting program AND ABSTRACTS

It has been reported in excitotoxicity by glutamate that NMDA receptors (NMDAR) have properties that permit them trigger Ca2+ overload and Ca2+-mediated neuronal damage more effectively than other Ca2+ sources. Together with Ca2+ dysregulation, excitotoxic stimulation of NMDAR produces reactive oxygen and nitrogen species (ROS and RNS) from intracellular sources. Our hypothesis is that ROS/RNS, produced by excitotoxic stimulation of the NMDAR, generate calcium signals sensitive to ryanodine that lead to death of hippocampal neurons in culture. Also the subcellular location of the NMDAR is important in this process. We will investigate the generation of intracellular calcium signals, the production of ROS and the cellular viability produced by stimulation of the NMDAR (synaptic and extrasynaptic) with different concentrations of NMDA. In addition to this, we will study the participation of RyR in the generation of these signals and cellular response, after the stimulation of NMDAR, to demonstrate the participation of RyR in the excitotoxicity by glutamate, process related with neuronal death in pathologies and neurodegenerative disease. Financiado por FONDAP CEMC 15010006 y Proyecto ICM P05-001F.

Session 3.

Neurodegeneration and Metals/Pollutants Chairs: J Andersen & M Collins 24. NEUROLOGICAL AND NEUROPSYCHOLOGICAL IMPAIRMENT BY MERCURY IN HANDMADE MINERS IN ANDACOLLO, FOURTH REGION CHILE. Floria Pancetti1, Gislaine Lam2, Patricia Lillo2, David Sáez2, Sebastián Corral1, Daniel Moraga1. 1Escuela de Medicina, Universidad Católica del Norte, Coquimbo, Chile and 2Universidad de Chile, Dpto. Neurología Campus Sur, Santiago, Chile. [email protected]; [email protected] Mercury produces damage to the nervous system, and this is proportional to the time of exposition. Handmade miners amalgam mercury with gold, then it is evaporated to take out the gold. The inhalation of this vapour is the main way of income to organism by this metal. We selected a population of handmade gold miners exposed to mercury by means of a survey poll. People were subjects to neurological examination , neuropsychological tests to measure executive functions, memory and attention. Also we measured the mercury level in blood and hair. We compared this population with other people with similar demograph-

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ic features, no exposures to mercury. The data actually obtained reveals a high prevalence of neuropsychological and neurological impairment in people exposed to mercury and high levels of this metal in their organism. Financial assistance: FONIS SA05I20032. 25. Al, Si, Fe, AND SILICA PRECIPITATION IN ELECTRIC ORGAN FROM ALIVE ELECTRIC FISH F Barrera1, Nora N. Cesaretti2, María Prado Figueroa1 1Instituto de Investigaciones Bioquímicas de Bahía Blanca (INIBIBB) CONICET, Universidad Nacional del Sur (UNS) 8000 Bahía Blanca, Argentina; 1Dept. de Geología, Universidad Nacional del Sur, Argentina. [email protected] Aluminum, iron and silicon were observed in electrocytes (family Rajidae) by SEM-EDS. Also, electric organ segments, cryostat sections and subcellular fractions were observed using mineralogical techniques. X-ray diffraction analysis showed low - quartz variety. When we observed through crossed polarized illumination, which allow for mineral identification, chalcedony was distributed in the electrocyte cytoplasm, nerves and all the fractions. It is rounded in shape, first order of birefringence colour and with undulatory extinction angle. Chalcedony (SiO2) is a typical mineral present in fossilization processes in different organic matter; its precipitation occurring at specific pH (7-8) and oxidation potential (Eh) (0.0 to -0.2) conditions. This observation supports the idea of an important role played by Eh - pH conditions for silica precipitation in electric organ as well as it was report in geological environments. This is the first mention that chalcedony was in relation with cholinergic nerve terminals, from a living animal. Acknowledgments: To SGCyT, UNS, Argentina. 26. CHALCEDONY (FOSSILIZATION PROCESS) IN HUMAN BRAIN FROM AGED PATIENTS María Prado Figueroa1, Luis Flores Valerio*, Juvenal Sanchez2, Nora N. Cesaretti3 1Instituto de Investigaciones Bioquímicas de Bahía Blanca (INIBIBB) CONICET, Universidad Nacional del Sur (UNS) 8000 Bahía Blanca, Argentina; 2Depto. Patología, Instituto de Enfermedades Neoplásicas, Lima, Perú; 3Depto. Geología, UNS, Argentina. [email protected] Aluminum and silicon were observed in human degenerated brain by X-ray microanalysis using EDS-SEM. Now, we explored the presence of silica by using a mineralogical microscope. Sections of human brain were

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collected after autopsy from aged patients (83 years old). Chalcedony (a silicon mineral) was detected in cerebral and cerebellar sections. In plane-polarized light it is translucent, rounded, 12 - 60 µm in size and in crossedpolarized light shows first order birefringence color and radial extinction. Chalcedony (SiO2) is a typical mineral of fossilization processes in organic matter and precipitated in certain conditions of pH and Eh in sedimentary environments but according our results also in biological systems. This is the first mention of chalcedony in human CNS and also the use of mineralogical techniques in biological study. SGCyT, UNS, Argentina. LFV was an IBRO fellow from UNMSM, Lima-Perú.

27. HYPOMYELINATION IN PUP RATS EXPOSED TO 2,4-DICHLOROPHENOXYACETIC ACID (2,4D) THROUGH MOTHER'S MILK Konjuh C1, B Bongiovanni1, LM López2, AM de Duffard1, A Brusco2, R Duffard1 1LATOEX - Facultad de Cs. Bioquímicas y Farmacéuticas, UNR. Suipacha 531. 2000.Rosario. 2LANAIS-MIE. Facultad de Medicina. UBA. Paraguay 2155. 1121. Bs As. Argentina. [email protected] 2,4-D is a phenoxyherbicide used for selective control of broadleaf weeds. Unfortunately, 2,4-D produces different harmful effects on mammals, including humans, which range from embryotoxicity and teratogenicity to neurotoxicity. Very little is known about the cellular mechanisms underlaying its neurotoxicity. Through this study we want to elucidate: which are the mechanisms by which 2,4-D produces hypomyelination in Central Nervous System, how 2,4-D affects the oligondendrocyte and finally, which of the myelin components are altered in these exposed pups. We analyzed myelin ultrastructure by electron microscopy; myelin proteins using western blot and immunohistochemical studies and myelin chemical composition, in control and exposed pups. All 2,4-D exposed pups showed deficits in several myelin components such as proteins (MBP, CNP, PLP), galactolipids, cholesterol and phospholipids. In agreement with myelin component deficits observed, the ultrastructural analysis showed a diminution in the number of sheets, increase in interperiod distance and presence of blebs in myelin sheath. Furthermore, the oligodendrocyte ultrastructure showed mitochondrial and reticular swelling. The results suggest that the alterations observed in the oligodendrocyte could lead to disturbances in myelin formation and compaction.

28. ONTOGENETIC NORADRENERGIC LESION ALTERS HISTAMINERGIC ACTIVITY IN ADULT RATS. P Nowak1, A Bortel1, J Jochem2, K Zwirska-Korczala2, J Niwinski1, M Drosik1, L Noras1, RM Kostrzewa3, R Brus1 1Depts. Pharmacology and 2Physiology, Medical Univ. Silesia, 41-808 Zabrze, Poland; 3Quillen College of Medicine, East Tennessee State Univ., Johnson City, TN, USA. [email protected] To determine if accentuated or new (revealed) locomotor or stereotyped activities would accrue for histaminergic neurons in the absence of noradrenergic neurons during postnatal ontogeny, neonatal rats were lesioned with DSP-4 (50 mg/kg sc) on the 1st and 3rd days after birth. Effectiveness of DSP-4 was assessed by a dramatic reduction in noradrenaline (NA) level in hippocampus, striatum and frontal cortex at 8 wk [HPLC/ED method]. By an immunoenzymatic method the histamine level of frontal cortex was also found to be reduced in DSP-4lesioned rats at 8 wk. Moreover, in DSP-4-lesioned rats, reactivity (stereotyped or locomotor activity; nociceptive response) to cimetidine (H2 antagonist) and thioperamide (H3 antagonist) was altered, while reactivity to the H1 antagonists diphergan and S(+)chlorpheniramine was virtually unaltered. These findings indicate that noradrenergic neurons normally exert a modulatory role on developing histaminergic neurons and associated receptor sensitivity level. Supported by Medical University of Silesia: NN-1-001/07. 29. EVIDENCE FOR OXIDATIVE STRESS IN TISSUES DERIVED FROM SUCCINATE SEMIALDEHYDE DEHYDROGENASE DEFICIENT MICE Alexandra Latini1, Karina Scussiato1, Guilhian Leipnitz1, K Michael Gibson2, Moacir Wajner1,2,3 1Departamento de Bioquímica, ICBS, UFRGS; 2Departments of Pediatrics, Children's Hospital of Pittsburgh, Rangos Research Center and the University of Pittsburgh School of Medicine; 4ULBRA In the present study we evaluated the tissue antioxidant defenses and lipid peroxidation in brain (cortex, cerebellum, thalamus and hippocampus) and in the liver of succinate semialdehyde dehydrogenase deficient mice. The total radical-trapping antioxidant potential (TRAP) and GSH levels, the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) and the levels of malondialdehyde (TBA) were investigated. The mutant animals showed reduced tissue non-enzymatic antioxidant defenses particularly in the

2007 nts meeting program AND ABSTRACTS

cerebral cortex (decreased GSH) and in the liver (decreased TRAP and GSH). Furthermore, SOD activity was significantly increased in the cerebellum and liver, whereas the activity of CAT was significantly increased in the thalamus. In contrast, GPx activity was significantly diminished in the hippocampus. Finally, we observed that lipid peroxidation (TBA levels) was stimulated in the cerebral cortex and liver, reflecting a high lipid oxidative damage in these tissues. Our data showing an imbalance between tissue antioxidant defenses and oxidative damage indicate that oxidative stress has a prominent role in the pathophysiology of this neurometabolic disorder. Financial support: CNPq, PRONEX, FAPERGS, PROPESQ-UFRGS, NIH NS40270 (KMG), Pediatric Neurotransmitter Disease Association. 30. 2,4-DICHLOROPHENOXYACETIC ACID INDUCED OXIDATIVE STRESS IN CEREBELLAR GRANULE CELLS IN CULTURES AND AMPHETAMINE REDUCED THIS EFFECT. B Bongiovanni1, A Brusco2, A Ferri1, M Rassetto1, M Lopez2, A Evangelista de Duffard1, R Duffard1 1LATOEX. Fac. Cs. Bioquímicas y Farmacéuticas. UNR. Suipacha 531. 2000. Rosario. 2LANAIS-MIE. Fac. Medicina. UBA. Paraguay 2155. 1121. Bs As. Argentina. [email protected] 2,4-Dichlorophenoxyacetic, a worldwide-used herbicide, produced from embryotoxicity and teratogenicity to neurotoxicity on different animal species. We have previously reported apoptosis in rat cerebellar granule cell (CGC) cultures by 2,4-D. Besides, amphetamine (AMPH) has consistently been reported to accelerate recovery of function in animals and humans with brain injury. In the present work neuronal morphology and biochemical events in rat CGC cultures have been analyzed in order to determine if the 2,4-D (1mM) toxicity mechanism involves an alteration in the oxidative cellular homeostasis and whether d-AMPH reduced this effect. A drastic decrease in growing cerebellar granule cells - detection of apoptotic and necrotic cells - together with an increased ROS generation, a decreased GSH level, CAT activity, as well as an increase in the GPx activity with respect to the control group by the 2,4-D were observed. However, the effect of 2,4-D was partly reverted and apoptotic-like cells were and ROS generation detected not found in cultures exposed to 2,4-D plus 10 µM d-AMPH. In this way, d-AMPH could act as a neuroprotector of the 2,4-D toxic effects

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Session 4.

Neurodegeneration Chairs: R Kostrzewa, CJ Van der Schyf 31. QUINOLINIC ACID MILDLY IMPAIRS CEREBRAL ENERGY METABOLISM Patrícia F. Schuck, Gustavo C. Ferreira1, Anelise Tonin1, Paula C. Ceolato1, Moacir Wajner1 Dept. Bioquímica, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil. [email protected] Quinolinic acid (QA) is found at increased concentrations in brain of patients affected by various neurodegenerative disorders, including Huntington's and Alzheimer´s diseases. Considering that the neuropathology of these disorders has been recently attributed at least in part to energy deficit, we investigated the in vitro effect of QA on various parameters of cerebral energy metabolism. We observed that QA increased glucose uptake, whereas 14CO2 generation from glucose, acetate and citrate was inhibited. Furthermore, QA-induced increase of glucose uptake was prevented by the NMDA receptor antagonist MK-801. Complex II activity was also inhibited by QA, and this inhibition was prevented by pre-incubating homogenates with catalase plus superoxide dismutase, indicating that this effect was probably mediated by reactive oxygen species. In addition, the activity of lactate dehydrogenase was decreased by QA. The other activities of the respiratory chain complexes, Krebs' cycle enzymes, creatine kinase and Na +,K +-ATPase and lactate production were not affected by the acid. QA did not change the oxygen consumption in the presence of glutamate/malate or succinate as well, suggesting that its effect on cellular respiration was rather weak. The data provide evidence that QA provokes a mild impairment of cerebral energy metabolism and does not support the view that the brain energy deficiency associated to neurodegenerative disorders could be solely endorsed to QA accumulation. 32. EFFECTS OF NICOTINAMIDE ON HIPPOCAMPAL PLASTICITY FOLLOWING PERINATAL ASPHYXIA EVALUATED WITH ORGANOTYPIC CULTURES Morales P1, P Huaiquín1, D Bustamante1, J Fiedler2, M Herrera-Marschitz1 1Programme of Molecular & Clinical Pharmacology, ICBM, Medical Faculty; 2Dept. of Neurochemistry, Chemical and Pharmaceutical Science Faculty, University of Chile, Santiago, Chile.

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The potential neuroprotection of nicotinamide on the consequences of perinatal asphyxia was investigated with hippocampal organotypic cultures. Perinatal asphyxia was induced in vivo by immersing foetusescontaining uterine horns removed from ready-to-deliver rats into a water bath for 20 min. Sibling caesareandelivered pups were used as controls. Nicotinamide, a non-selective inhibitor of poly(ADP-ribose) polymerase (PARP-1), (0.8 mmol/kg, i.p.) or saline was administered to asphyxia-exposed and caesarean-delivered control pups 24, 48 and 72 h after birth. At seven postnatal day, tissue from hippocampus was dissected and placed on a coverslip. After a month, the cultures were processed for immunocytochemistry with neuronal marker against MAP-2 and cell proliferation marker against BrdU. Perinatal asphyxia produced a significant decrease in the number of MAP-2-positive cells/mm3 in CA1 and DG region of hippocampus. Perinatal asphyxia also induced an increase in the number of BrdU-positive cells/mm3 and in number of BrdU/MAP-2 double labelled cells in DG. Nicotinamide treatment prevented the asphyxia-induced decrease of the number of MAPpositive cells in CA1. Moreover, nicotinamide induced a decrease in the number of BrdU positive cells but the number of BrdU/MAP-2 positive cells was preserved. The present results support the idea that nicotinamide can prevents long-term neuronal deficits induced by a sustained energy-failure condition, as occur during perinatal asphyxia. The support by DID (I2-02/8-2), FONDECYT, and ICBM-Enlace grants is acknowledged. 33. EFFECT OF ALTERED LIGHTING REGIMENS AND CHRONIC MILD STRESS ON THE CIRCADIAN RHYTHMS IN THE RAT Olah A2, V Csernus1, J Sandor2, A Muller2, R Jozsa1 1Dept. of Anatomy, University of Pecs, 2Faculty of Health Sciences, University of Pecs Recent data confirm that the chronic mild stress (CMS) model of depression has high validity, however, CMS may have opposite effects, termed as 'anomalous' behavioural profile. Dyregulation of the hypothalamus-pituitary-adrenal (HPA) axis is related to endogenous depression. In our study, decreases in reactivity to rewards in rats following exposure to CMS were observed. The aim was to investigate the changes of the daily hormonal levels following CMS or various lighting schedules and to analyse rhythm parameters. 102 adult rats of both genders were randomly distributed in the following groups: 1) control, 2) using CMS and 3) rats kept under inverted lighting regimens. Sucrose consumption was measured weekly. Corticosterone and serotonin levels were measured in blood samples obtained every 4 h over

24 h. The circadian rhythm parameters were compared in the different groups. Daily mean levels of total corticosterone and serotonin did not show significant differences. However, the daily mesor and amplitude showed clear group- and gender differences. In conclusion, CMS and also disturbing lighting schedule itself result in the impairment of certain hormonal rhythms and these disturbances appear in an early stage of anhedonic behaviour. Support: ETT314/2006 and 439/2006. 34. IMPACT OF THE CHOLINERGIC SYSTEM MANIPULATIONS THROUGH SYSTEMIC (-) NICOTINE ADMINISTRATION ON THE GLU AND GABA RELEASE AND MOTOR EXECUTION OF HEMIPARKINSONIAN RATS. Lisette Blanco Lezcano, Lourdes Lorigados Pedre, Ma. Elena González Fraguela, Lisis Martínez Martí, Nancy Pavón Fuentes, Teresa Serrano Sánchez, Yovany Bauza Calderín, Yovany Coro. International Center of Neurological Restoration, Havana City, Cuba. The pedunculopontine nucleus (PPN) projects the only cholinergic pathways that reach the substantia nigra pars compacta (SNc) and additionally it send a glutamatergic projection to this structure. For this reason the study of the impact of the cholinergic management through systemic administration of nicotine solution is a very important topic taking into considerations the data available about the neuroprotective actions of this drug on the dopaminergic cells. Methods. The (-) nicotine (1mk/kg of weight) schedule administration was 3 dose s.c in 3 steps. The motor execution, the motor asymmetry, GLU and GABA release in PPN and the survival of dopaminergic cells were the variables studied. The quantification of the aminoacids (GLU and GABA) was carried out employing the high performance liquid chromatography (HPLC) coupled flourimetric detector. Results: The (-) nicotine systemic administration induced a significant decrease (p<0.01) of the rotatory activity and also the animals exhibited a significant decrease in the platform latency with the highest bridges: square and circular. The rats tumbled down more lately in the lesser bridges in comparison to the control groups. Nevertheless the motor execution of the treated rats didn't reach the normal patterns in this test. The nicotine systemic treatment induced also a significant decrease (p<0.01) of the GLU and GABA extracellular level in PPN and also it improved the dopaminergic cell bodies in SNc in comparison to the hemiparkinsonian rats without pharmacologic treatment. Conclusions. These results could reflect the changes in the cholinergic system and the nicotine receptors in the 6-OHDA models and the direct role of the PPN in the modulation of the cholinergic activity in the Parkinsonism status.

2007 nts meeting program AND ABSTRACTS

35. AGING AND SPATIAL MEMORY IN THE RODENT OCTODON DEGUS Alejandra Ponce1, Waldo Cerpa2, Pablo Muñoz1, Nibaldo Inestrosa2, Adrian Palacios1 1Centro de Neurociencia de Valparaíso, Fac de Ciencias, Universidad de Valparaíso. 2Centro de Regulacion Celular y Patologia "Joaquin V. Luco" eInstituto Milenio MIFAB, Universidad Católica de Chile The Octodon degus is an endemic rodent of our country. Aging brains of these animals show important similarities with Alzheimer landmark diseases (AD) in human. The spatial memory performance during aging was evaluated with a behavioral alternance T-maze task, and with histochemistry methods to visualize markers for gliosis, β-amyloid peptide, and body neurons, the presence of neurodegenerative landmarks found in AD. Four experimental groups differing in age: 3m (G1), 12m (G2), 24m (G3) and 36m (G4) months subjects were used. The results show that G1 needs 8.8; G2 6.5 and G3 7.3 sessions on average to reach an 80% criteria performance, during two consecutives sessions. In G4 only 2 of 8 subjects reached the learning performance criteria and there was an increment of oligomer β-amyloid accumulation in individuals that didn't reach the learning criteria. Other results indicate that a difference doesn't exist in gliosis and number of body neurons in the hippocampus in animals at 12 and 36 months. Suggesting to the 36 months of O. degus we are even speaking of Alzheimer's presenile, and in this stage apparently not yet there is a noticeable neuronal death. We suggest that our Octodon degus model shows excellent similarities with neurodegeneratives process and memory decay failures affecting AD patients. 36 CIS-4-DECENOIC ACID ALTERS RESPIRATORY PARAMETERS IN RAT BRAIN Gustavo C. Ferreira, Patrícia F. Schuck, Paula C. Ceolato, Anelise Tonin, Carolina M. Viegas, Moacir Wajner Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil. Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most frequent disorder of fatty acid oxidation. Biochemically, MCAD-deficient patients present tissue accumulation of the medium-chain fatty acids octanoic, decanoic and cis-4-decenoic (cDA) acids. Clinical presentation of MCAD deficiency is related to fasting and increased metabolic stress, which precipitate acute symptoms such as drowsiness or lethargy that may develop into coma or even death. In the present work, we investigated the in vitro effect

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of cDA on respiratory parameters in mitochondrial preparations from rat brain, namely states III and IV, as well as the respiratory control ratio (RCR), using glutamate/malate and succinate as substrates. It was observed that cDA diminished state II and RCR, while increased state IV in the presence of glutamate/malate and succinate as substrate. Taken together, these data strongly suggest that cDA may lead to impairment of ATP production acting as an uncoupler of oxidative phosphorylation, which could explain, at least in part, the characteristic brain dysfunction observed in MCAD deficient patients. Financial Support: CNPq, FAPERGS, PROPESQ/ UFRGS. 37. GENETIC DIAGNOSIS OF CADASIL: REPORT OF THREE CASES Gonzalo Farías Gontupil1,3, Patricia Iturra Constant2, Osvaldo Trujillo Godoy1, David Sáez Méndez1 1Neurology Department. Barros Luco-Trudeau Hospital, Santiago, Chile. 2Human Genetics Department, ICBM, Universidad de Chile. 3Medical Sciences doctoral program, Universidad de Chile. [email protected]. Cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL) is an inherited disease characterized by subcortical cerebral ischemic events, migraine, dementia and mood disorders. It is produced by mutations in the transmembrane receptor NOTCH 3 gen, which are commonly found in exons 3 and 4. We review the clinical and imagenologic data of two patients of our hospital with the diagnosis of CADASIL. We purified genomic DNA of the two patients and also analyzed a sample obtained from the sister of one of the patients. PCR amplification and direct sequentiation of both strands of DNA for NOTCH 3 exons 3 and 4 was performed. The C346T mutation in exon 3 was found in the first patient, while in the second patient and his sister, who has history of migraine, C475T mutation in exon 4 was present. Our results are consistent with previous reports which indicate that exons 3 and 4 are frequent sites of CADASIL causing mutations. We also discuss the diagnostic utility of genetic testing for CADASIL. 38. ENDOSOMAL-LYSOSOMAL PATHWAY ABNORMALITIES IN CEREBRAL CORTEX CELL LINE DERIVED FROM TRISOMY 16 MOUSE, AN ANIMAL MODEL OF DOWN SYNDROME Christian Arriagada1, Eduardo Rojas2, Illani Atwater2, Raúl Caviedes3, Pablo Caviedes3 1Anatomy and Developmental Biology Program, 2Human Genetics Program, 3Clinical and Molecular Pharmacology Program, ICBM, Faculty of Medicine, University of Chile.

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Endosomal-lysosomal pathway is used in neurons to degrade proteins that have been internalized from the cell surface. Between these proteins is found the amyloid precursor protein and its processing derivates peptides, involved in Alzheimer's disease (AD). This fact has been observed in neurons derived from Down Syndrome (DS) and AD individuals and could be the starting point of the neuropathology observed in both conditions. In the other hand, cholesterol stimulates amyloidogenic pathway of processing of the amyloid precursor protein and enhances the intracellular accumulation of amyloid peptides. In the present work, we observed a decrease in the time course of this proteolytic pathway and enlargement of the involved compartments in a cell line derived from cerebral cortex of trisomy 16 mouse, an animal model of DS compared to the normal condition, in cultures supplemented with cholesterol associated to lipoproteins, reflecting a putative dysfunction in this pathway or associated pathways such as the recycling of proteins to the secretory pathway. These results reflect the use of these cell lines as an in vitro model to the study the early neurobiological mechanisms involved in DS. 39. MODULATION OF MOTOR NEURON SURVIVAL BY EXTRACELLULAR PURINES IS MEDIATED BY ASTROCYTES: IMPLICATIONS FOR AMYOTROPHIC LATERAL SCLEROSIS. MM Gandelman1, JM Garré2, MP Cassina3, V Abudara2, L Barbeito1 1Programa en Neurodegeneración, Institut Pasteur Montevideo, Montevideo, Uruguay. 2Departamento de Fisiología y 3Departamento de Histología y Embriología, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay. Extracellular purines, ATP, ADP, AMP and adenosine (EP's) are important signaling molecules in the nervous system. Acting through the purinergic P1 and P2 receptors, they can control differentiation, proliferation, survival and cell death. EP's are released in perisynaptic areas where they act as neurotransmitters and neuromodulators and also from damaged cells. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the progressive death of motor neurons and strong astrogliosis. Transgenic rats expressing the G93A mutation of Cu-Zn superoxide dismutase 1 (SOD-1) constitute a model for the disease. The possible role of EP's in ALS, acting either on astrocytes or motor neurons is currently unknown. Our results show that primary cultures of astrocytes expressing SOD-1G93A accumulated 4 times more ATP than non transgenic ones.

EP's affected the astrocytic redox state and reduced the transmembrane mitochondrial potential by 10%. Motor neuron survival in vitro was not affected by EP's, however it decreased to 45% when plated over EP-treated astrocyte monolayers. These results suggest that increased production of EP's in SOD-1G93A astrocytes may contribute to motor neuron death and play a role in ALS pathogenesis. This study was funded by PEDECIBA, Uruguay. 40. COMPARISON OF THE NEUROBEHAVIORAL DEVELOPMENT FOLLOWING TOXIC, HYPOXIC INJURIES, AND MATERNAL DEPRIVATION IN NEONATAL RATS Kiss P, D Reglodi, J Farkas, M Szalai, A Tamas, A Lubics, I Lengvari, B Gaszner, D Szogyi, D Hauser, M Koppan Department of Anatomy, Univ. Pecs, Hungary The present study investigated the effects of various perinatal events on the neurobehavioral development of newborn rats. One group of pups was subjected to monosodium glutamate (MSG) treatment on postnatal days 1,3,5,7 and 9. Another group underwent unilateral carotid occlusion followed by hypoxia exposure on postnatal day 7. Perinatal asphyxia was induced by caesarian delivery of pups 15 min after decapitation of the mother. Finally, maternal deprivation was induced by separating the pups for 3 hrs daily for 2 weeks. Somatic growth, appearance of physical characteristics and neurological reflexes as well as performance in certain reflexes and the development of motor coordination were tested for 5 weeks. MSG treatment and hypoxic/ischemic injuries caused significant delay in the neurobehavioral development and motor coordination. Pups performed worse in negative geotaxis, righting and gait reflexes. In contrast, maternal deprivation enhanced neurobehavioral development compared to control rats. Our study gives a summary of our findings on the neurobehavioral development in each model. Support: OTKA T046589, ETT439/2006. 41. POSSIBLE ADDITIVE EFFECT BETWEEN GLUTARIC AND 3-HYDROXYGLUTARIC ACIDS ON CEREBRAL ENERGY METABOLISM Gustavo C. Ferreira1, Patrícia F. Schuck, Anelise Tonin, Paula C. Ceolato, Carolina M. Viegas, Moacir Wajner Dept. Bioquímica, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil. [email protected]

2007 nts meeting program AND ABSTRACTS

Increased concentrations of glutaric acid (GA) and 3-hydroxyglutaric acid (3HG) are found in the body fluids and brain tissue of patients affected by glutaric acidemia type I (GAI), which present a markedly striatal and cortical degeneration. In addition, a toxic role was recently postulated for quinolinic acid (QA) in the neuropathology of GAI. Therefore, we investigated whether the combination of QA with GA or 3HG or the mixture GA plus 3HG could alter energy metabolism in cerebral cortex from young. The parameters evaluated were glucose uptake, lactate formation and 14CO 2 production from labeled glucose and acetate, as well as the activities of pyruvate dehydrogenase (PDH) and creatine kinase (CK). We first observed that 5 mM GA, 1 mM 3HG and 0.1 µM QA per se did not alter these parameters. Similarly, no change occurred when combining GA with QA or 3HG with QA. In contrast, coincubation of GA plus 3HG increased glucose uptake, decreased 14 CO generation from glucose, inhibited PDH activ2 ity as well as total and mitochondrial CK activities. Furthermore, GA plus 3HG-induced inhibitory effects on CK were prevented by the antioxidants glutathione and catalase plus superoxide dismutase, indicating the participation of reactive oxygen species. Our data indicate a synergistic action of GA and 3HG disturbing energy metabolism in cerebral cortex of young rats. Financial support: CNPq, PROPESQ-UFRGS, FAPERGS. 42. SYNTHESIS AND PURIFICATION OF AMINOCHROME SP Cárdenas, P Iturriaga, J Segura-Aguilar Molecular & Clinical Pharmacology, ICBM, Faculty of Medicine, University of Chile. Aminochrome is a derivative molecule of the oxidation of dopamine, that is a precursor of neuromelanin. It has been shown that many flavoenzyme reductases can reduce aminochrome with one electron, generating many reactive oxygen species and free radicals that can be very toxic for the neuron. Other authors have suggested that the aminochrome can form adduct with α-synuclein, inducing its aggregation and stabilizing the prothofibrillar structure, that is very toxic for the neuron. Aminochrome is a molecule involved in toxic mechanisms in dopaminergic neurons, is very important in the study of it, but is a very unstable molecule that quickly polimerizes to melanin, and up to now the synthesis or purification has been imposible. We have designed a very simple and quick system to obtain purified aminochrome that is very stable for many hours. This system can be used in future studies about toxic mechanism of this quinone and even in the study of melanin synthesis. Supported by FONDECYT 1061083 and FIRCA TW007044-01.

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43. STUDIES ON THE EFFECT OF THE AMINOCHROME TREATMENT IN THE AXONAL TRANSPORT OF ALPHA SYNUCLEIN Alejandra Riveros, Inmaculada Cuchillo, Diane Hanger, Juan Segura-Aguilar, John Stephenson. Department of Neurocience, Institute of Psychiatry, King´s College London.; Molecular & Clinical Pharmacology, ICBM, Faculty of Medicine, University of Chile. Several genes have been linked to Parkinson's disease, but alpha-synuclein has attracted most attention since alpha-synuclein is a major constituent of Lewy bodies. Given that alpha-synuclein assumes a fibrillar betapleated sheet structure in Lewy bodies in PD and related alpha-synucleinopathies, the leading hypothesis for its pathogenicity is the formation of toxic aggregates. Several lines of evidence have shown that a prerequisite of alpha-synuclein pathology is its oligomerization into cytotoxic protofibrils. Alpha-synuclein has been shown to bind synaptic vesicles, and protofibrils can form pores that could lead to permeabilization of the vesicle membranes, thereby releasing dopamine into the cytosol. Formation of protofibrils is enhanced and stabilized by aminochrome derived from the oxidation of dopamine, and this could account for the selective toxicity of alphasynuclein protofibrils in the substantia nigra. We tested the hypothesis that the formation of alpha-synucleinaminochrome adducts, which accelerate and stabilize the formation of protofibrils, affect the axonal transport of alpha-synuclein since the failure of axonal transport has been implicated in the development of several neurodegenerative disorders. We used time-lapse video microscopy to obtain images of live neurons that had been transfected with plasmids expressing proteins tagged with enhanced green fluorescent protein to investigate the effect of aminochrome on the axonal transport of alpha-synuclein. We found a significant difference in the average velocities, between alpha-synuclein in the absence and presence of aminochrome. These results support the neurotoxic role of the formation of alphasynuclein adducts with aminochrome. Supported with grants from London, King's College London, Alfa Neuralnet, EuroAid Program, EU and FONDECYT 1061083.

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44. PARKINSON'S DISEASE POPULATION WITH STRONG AMERINDIAN INFLUENCE FROM SANTIAGO Fernando Díaz-Grez, Carolina Perez-Pastene, Jose M. Matamala, Juan Segura-Aguilar Molecular & Clinical Pharmacology, ICBM, Faculty of Medicine, University of Chile. In Chile Amerindian genetic component correlates with the socioeconomic level: it is small (typically less than 10%) in the upper strata, and greater in lower strata (about 40 %). Santiago is a city of about 5-6 millions inhabitants with a heterogeneous population with areas of marked European influence with high economical and educational level. There are also areas with a majority of native genetic influence with low economical and educational level. The data was obtained from Melipilla and Maipu from Santiago, which are areas with low economical level and recruited by announcements published in seniors' organizations or local radios from the same geographic areas. In Melipilla, an experimental neurologist examined 269 persons (206 female and 73 male) with an age 67 ± 8 years old. We found 9 persons (3.3%) with Parkinson's disease (6 female and 3 male) with an age 69 ± 6 years old. In Maipu, 107 persons were examined by the same neurologist (88 female and 9 male) with an age of 56 ± 14 years old, and we found 3 persons with Parkinson's disease (2.8%) with an age of 79 ± 12 years old. Parkinson's disease patients included in this study were diagnosed by the neurologists with idiopathic Parkinson's disease and presented at least two of four cardinal signs: bradykinesia, rest tremor, rigidity, and postural reflex impairment group. Exclusion criteria for a diagnosis of idiopathic disease were the use of medications (e.g., phenothiazines) during the 12 months preceding symptom onset; MRI, or CT evidence of multiple cerebrovascular events prior to symptom onset; evidence of another known cause of parkinsonism (e.g., history of brain tumor or encephalitis), atypical Parkinson's disease presentation, dementia or essential tremors. Supported by FONDECYT 1061083.

45. EVALUATION OF THE NEUROTOXIC ACTIVITY OF THE ACETOGENINS OF Annona muricata IN INDUCTION OF PARKINSON'S DISEASE USING SLICE CULTURE BIOASSAY Gladys Buitrón, Silvia Suarez Instituto de Bioquímica y Nutrición "Alberto Guzman Barrón". Universidad Nacional Mayor de San Marcos, Lima, Perú. The present study consists of the evaluation of the neurotoxic activity of the acetogenins isolated of the leaves of Annona muricata. The acetogenins have verified cytotoxic activity but in one acetogenin (annonacin) was found neurotoxic activity. The hypothesis is that the concentration of the acetogenins of the Annona muricata has relation with the neurotoxic activity on the induction of the Parkinson`s disease. The method in vitro is a model of induction of Parkinson's disease by neurotoxins using slice culture bioassay. In this model will be used 6-hydroxydopamine (6-OHDA) and it will be compared with neurotoxin (acetogenin). The aim of the present work consists of evaluating the neurotoxic activity of the acetogenins of Annona muricata from Peru, where population consumes it. The experimental method includes isolation of different acetogenins, a model of induction of Parkinson's disease by neurotoxins, such as 6-OHDA and using slice culture bioassay. To verify the neurotoxic activity we visualized in real time the decrease of the tyrosine hydroxylase positive cell bodies and fibers, and it is observed also the effect on the proteins of the extracellular matrix.

2007 nts meeting program AND ABSTRACTS

Patagonia Satellite Meeting

Neurotoxins, Neurodegeneration and Neuroprotection: Past, Present and Future March 26, 2007 20.00-21.00 Registration 21.00-22.00 NEUROTOXINS: WHERE WE'VE BEEN, WHERE WE ARE, WHERE WE'RE GOING. RM Kostrzewa1, J Segura-Aguilar2 Depts. Pharmacology, 1Quillen College of Medicine, East Tennessee State Univ., Johnson City, TN, USA; 2ICBM Med. Faculty, Univ. Chile, Santiago, Chile. [email protected] Although classically viewed as substances that overtly destroy nerves, neurotoxins are now seen as tools that disrupt neural function, even temporarily. Selectivity is imparted by affinity to proteins or other cellular elements. Endogenous neurotoxins are known, and synuclein, Fe, plaques, neuroinflammatory cytokines, and like neurotoxic species fall under the definition of a neurotoxin. Chemicals that disrupt astrocytes and other glial cells, ultimately inducing neural dysfunction, might be termed "indirectly acting" neurotoxins. Adaptations in psychiatric disorders and in substance abuse could reasonably be considered to be the product of neurotoxic events. The naturally occurring programmed neuronal cell death during ontogeny, might invoke the mediators of this process as neurotoxins. In summary, the concept of a selective neurotoxin has evolved to incorporate endogenous cellular mediators of glial and neuronal cell death, and the role of neurotoxins is thus central in the pathophysiology of neurodegenerative, psychiatric, and substance abuse disorders. March 27, 2007 21.00-22.00 ARE THE EXPERIMENTAL MODELS USED TO STUDY NEURODEGENERATION SUITABLE TO COLLECT INFORMATION TO DEVELOP NEW THERAPY ACTIONS IN NEURODEGENERATIVE DISEASES? Gilles Guillemin Centre for Immunology, Neuroimmunology Dept. Sydney, Australia. Over the last two decades, understanding of the molecular mechanisms involved in neurodegenerative diseases has significantly increased. This is particularly true for Alzheimer's disease (AD) and motor neuron diseases such as amyotrophic lateral sclerosis (ALS). We aim to

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discuss here the relevance of studies performed on small animal models with emphasis on genetically engineered rodent models for such diseases. These animal models have been extensively used as precious tools to try to understand some of the mechanistic aspects of AD or ALS and to validate potential therapeutic targets. What we have learned from them? How have they improved with time? How valid are these models? What were the "bad surprises" arising from therapeutic translation to human? March 28, 2007 21.00-22.00 IS NEUROPROTECTION THE FUTURE OF THE TREATMENT OF NEURODEGENERATIVE DISEASES Wojciech Danysz, Christopher G Parsons Merz Pharmaceuticals, Eckenheimer Landstrasse 100, D-60318 Frankfurt, Germany The aim of the workshop is to tackle aspects seldom being topics of the discussion among scientists. These aspects are, in our opinion necessary for creating long term strategies in seeking for treatments that are supposed to affect the progression of the neurodegeneration. Below such topics for discussion are listed with the hope to create open, fretful discussion resulting in conclusion that should be made, then published in Neurotoxicity Research. 1. Given high costs of health care should we for any price try to prolong life of patients? In our opinion always the following factors should be considered. a. Stage (disability status) at the time of treatment initiation (role of early diagnosis) b. Type of disability - care costs c. Patients consciousness d. Duration of the disease e. Mode of action: delay in progression, stopping the progression, reversing (restoration) f. What are accepted side-effects costs ? 2. How to prove in clinical trials, neuroprotective efficacy, given the following problems: a. necessity of using add-on to symptomatological treatment b. regulatory requirements to show functional improvement - NMR, PET, or other biomarkers not accepted (this requires wash-out period) c. requirement of long duration of studies 3. How preclinical studies should be adjusted to realities of clinical studies i.e., #2a and #2b?

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List of Students who received a fellowship with funds from ISN, IBRO, IUBMB, ISN-CAEN, IBRO-LARC 1. Luis A. Flores Valerio (Peru) 2. Lisette Blanco (Cuba) 3. Mandi Gandelman (Uruguay) 4. Paola De Lorenzi (Argentina) 5. Anitsi Loaiza Díaz (Chile) 6. Pablo Guzman (Chile) 7. Alejandra Riveros (Chile) 8. Alexies Humeres (Chile) 9. Rebecca Graumann (Chile) 10. Patrícia Fernanda Schuck (Brazil) 11. Gustavo da Costa Ferreira (Brazil) 12. Alejandra Ponce Cid (Chile) 13. Eduardo Riquelme Silva (Chile) 14. Katunar Maria Rosa (Argentina) 15. Maria Gonzalez (Peru) 16. Juan Tichauer (Peru) 17. Carlos Juri (Chile) 18. Natalia Mena (Chile) 19. Denise Riquelme P. (Chile)

20. Gonzalo Andrés Farías Gontupil (Chile) 21. Facundo Barrera (Argentina) 22. Cintia Nadina Konjuh (Argentina) 23. Mauricio Perez (Chile) 24. Gonzalo Farias G (Chile) 25. Javier Cisternas Recaro (Chile) 26. Gislaine Lam Esquenazi (Chile) 27. Javier Gaete (Chile) 28. Jose Manuel Matamala Capón (Chile) 29. Rene Meza Flores (Chile) 30. Tatiana Adasme (Chile) 31. Bettina Bongiovanni (Chile) 32. Cristian Arriagada (Chile) 33. Brian T. Terpstra (USA) 34. Patricio Zavala (Chile) 35. Gladyz Buitron (Peru) 36. Sergio Cardenas (Chile) 37. Katherine Saud (Chile) 38. Enrique Armijo (Chile)