Inhibition of TNF-alpha protein synthesis restores neuronal function and reverses cognitive deficits induced by chronic neuroinflammation

Poster Presentations P3 the peptides are internalised and bind PSD95, disrupting its interaction with PMCA2B. The peptides protect SHSY-5Y neuronal cell lines against cell death induced by glutamic acid (10mM); however, the peptides are not protective against toxicity induced by aggregated beta-amyloid or camptothecin, suggesting that toxicty of these compounds is mediated by other mechanisms. Conclusions: Cyclic PDZ-binding peptides have potential in disrupting interactions of functional proteins in post-synaptic complexes; hexa-D Arg offers an cost-effective method of internalising peptide ligands without the use of the full penetratin sequence on which it is based. The potential of these peptides against neuronal destruction in diseases such as status elipticus or Alzheimer’s disease warrants further investigation. P3-417

STIMULATION OF ENDOCANNABINOID RECEPTORS IS REQUIRED FOR REDUCTION OF NEUROINFLAMMATION AND RESTORATION OF NEUROGENESIS IN THE AGED BRAIN

Isabelle Bardou1, Holly Brothers2, Yannick Marchalant3, Gary Wenk2, 1 Ohio State University Department of Psychology, Columbs, Ohio, United States; 2The Ohio State University, Columbus, Ohio, United States; 3NICN UMR 6184 CNRS, Marseille Cedex 20, France. Background: Chronic neuroinflammation is associated with many neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases inducing an elevation of pro-inflammatory cytokines, an impairment of spatial memory and, a dysregulation of glutamatergic neurotransmission via a loss of NMDA receptors. Cannabinoids constitute a class of compounds which includes natural (e.g. endocannabinoid) or synthetic molecules. Two types of endocannabinoid receptors have been characterized (CB1 and CB2). The endogenous ligands of these receptors can prevent neuroinflammation and decrease the glutamate release. Due to their psychoactive effects, the use of some cannabinoids in human therapy is limited despite their therapeutic potential. In order to avoid undesirable psychoactive effects, we investigate the effects of two non-psychoactive cannabinoids (dexanibinol, HU211 and cannabidiol) on neuroinflammation and cognitive processes in aged rats and compare their actions with an NMDA receptors antagonist (memantine) and endocannabinoid receptor agonist (WIN-55212-2) which we have already demonstrated can reduce microglia activation in young rats given experimental neuroinflammation. Methods: Old rats (24 mo) were treated for 3 weeks by daily subcutaneousl injections with vehicle, WIN-55212-2, memantine, HU211 or cannabidiol. During the third week of the treatment, spatial memory was assessed using the Morris water maze task. Afterwards, we examined by immunohistochemistry the number of activated microglia (OX-6) and neurogenesis (BrdU & DCX) in different brain regions (e.g. hippocampus, entorhinal cortex, cingulate gyrus and frontal cortex). Results: WIN-552122 decreased the number of activated microglia and increased neurogenesis in the hippocampal dentate gyrus that correlated with an attenuation of the spatial memory impairment, as compared to vehicle-treated rats. The reduction of activated microglia was brain region dependent and differed according to the drug treatment group. Cannabidiol produced a slight, but non-significant increase in the number of activated microglia within the hippocampus. Conclusions: These data highlight a critical role of the endocannabinoid system in the control of neuroinflammation and suggest a possible beneficial effect of the administration of these compounds during aging. In contrast to what we’ve observed in young rats, NMDA antagonism cannot affectively reduce neuroinflammation in the aged brain. Within the aged brain, the only way to reduce neuroinflammation and restore neurogenesis is to stimulate the psychoactive endocannabinoid system. P3-418

INHIBITION OF TNF-ALPHA PROTEIN SYNTHESIS RESTORES NEURONAL FUNCTION AND REVERSES COGNITIVE DEFICITS INDUCED BY CHRONIC NEUROINFLAMMATION

Karim Belarbi1, David Tweedie2, Carla Arellano1, Timothy Jopson1, Nigel Greig2, Susanna Rosi1, 1Brain and Spinal Injury Center, University of

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California San Francisco, San Francisco, California, United States; National Institute of Aging, National Institutes of Health, Baltimore, Maryland, United States. 2

Background: Alzheimer’s disease (AD) is characterized by progressive memory loss, due, in part, to impairment in synaptic plasticity preceding the neurodegenerative changes. The early memory loss associated with AD is coincident with increased neuroinflammation in brain regions involved in learning and memory. Neuroinflammation is reliably detected by the presence of activated microglia and proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha). Given the role of TNF-alpha on the early progression of AD there is a compelling rationale to investigate new therapeutic approaches that selectively target TNF-alpha. The main goal of our study was to investigate the effect of a novel blood-brain barrier permeable TNF-alpha protein synthesis inhibitor, 3,6’-dithiothalidomide (DT), on neuronal dysfunctions induced by chronic neuroinflammation. Methods: Neuroinflammation can be induced by slow chronic infusion of lipopolysaccharide (LPS) into the fourth ventricle of the rat. LPS-induced neuroinflammation results in microglia activation and over expression of TNF-alpha and interleukin-1beta (IL-1beta) in the hippocampus, impaired hippocampus-dependent cognitive functions and altered expression of the plasticity-related immediate early gene Arc (activity regulated cytoskeletal protein) essential for memory formation. LPS or artificial cerebrospinal fluid (aCSF) was infused into the 4th ventricle for 28 days via an osmotic minipump (0.25 mg/h). Starting on day 29 LPS and aCSF-infused animals received daily intraperitoneal injections of DT (56mg/kg) or vehicle for 14 days. Novel object, novel place recognition and Morris water maze testing were used to assess hippocampal dependent and independent cognitive functions. All animals were euthanized 30 min after the water maze probe trial. Results: Treatment of animals with DT returned hippocampal TNF-alpha to control levels and rescued LPS-induced hippocampal-dependent cognitive impairments. Changes in hippocampus-dependent cognitive functions were associated with a normalization of the fraction of pyramidal and granular neurons expressing the plasticity-related Arc protein in the hippocampus. Conclusions: Inhibition of TNF-alpha synthesis reversed cognitive deficits induced by chronic neuroinflammation and restored neuronal functioning. Our study indicates that TNF-alpha is a critical mediator of the effects of neuroinflammation in cognitive dysfunctions; therefore, targeted inhibition of TNF-alpha synthesis may provide a novel therapeutic approach to modify the disease progression during the early stages of AD. P3-419

NEURONAL NICOTINIC ACETYLCHOLINE A4B2 RECEPTOR OCCUPANCY ASSAY IN RATS USING UNLABELLED ZW-104 AS TRACER

Gopinadh Bhyrapuneni, Vishwottam Kandikere, Nageswararao Muddana, Ramanatha Saralaya, Murugesan Arumugam, Suraj Tubuchi, Devender Reddy Ajjala, Ramakrishna Nirogi, Suven Life Science Ltd, Hyderabad, India. Background: The neuronal nicotinic acetylcholine receptors (nAChRs) belongs to the family of cholinergic receptors which are coupled with ligand gated ion channels. Due to the diverse mechanism and therapeutic potential of a4ß2 receptor this is a current attractive target for pharmaceutical discovery research. The radio labeled 5-iodo, 2-fluro, 6-fluro and 5-methyl substituted A-85380 was used as a PET tracer in preclinical and clinical studies. However the rate of elimination was very slow and the ratio between signal and noise also found to be less as compared to novel ZW-104. Due to less non specific binding and fast elimination kinetics than 2-fluro-A85380 the ZW-104 could be suitable candidate for in vivo receptor occupancy studies. Methods: Male Wistar rats fasted for overnight and treated per orally vehicle or TC-1734 (0.3, 1, 3 and 10 mg/kg) or ABT-089 (1, 3, 10 and 30 mg/kg) or ABT-0594 (0.1, 0.3, 1 and 3 mg/kg) or A-366833 (0.03, 0.1, 0.3, 1, 3 and 6 mg/kg) or cytisine (0.03, 0.1, 0.3, 1 and 3 mg/kg). After 30 minutes of test compounds or vehicle animals were treated with ZW-104 by later tail vein. All animals were sacrificed by cervical decapitation at tracer elimination phase and brain regions (cerebellum thalamus) were rapidly dissected out and frozen on dry ice until analysis. From all test