Minocycline-mediated protection from Aβ is mediated primarily by suppression of astrocytic inflammatory responses

S264 P1-312

Poster Presentations P1 BLOCKING INTERLEUKIN-1 FUNCTION ATTENUATES ALZHEIMER’S PATHOLOGY VIA REDUCING ANTI-INFLAMMATORY RESPONSES IN A TRANSGENIC MOUSE MODEL

Masashi Kitazawa, David Cheng, Michelle Tsukamoto, Vitaly Vasilevko, David C. Cribbs, Frank M. LaFerla, University of California, Irvine, Irvine, CA, USA. Contact e-mail: [email protected] Background: Inflammation plays an important role in the progression of Alzheimer disease (AD) pathologies. Its effects, however, are dichotomous, and the exact mechanisms of action during the disease progression remain to be elucidated. Epidemiological studies reveal a suppression of inflammation reduces a risk of AD, and clinical data from AD patients show aberrant increases in pro-inflammatory cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) while marked decreases in anti-inflammatory cytokines in the plasma as well as brain. To support these findings, a large body of in vitro and in vivo studies demonstrated that upregulation of IL-1beta or TNF-alpha exacerbates AD-like pathologies. We previously demonstrated that aberrant upregulation of IL-1beta may mediate AD-like pathologies in 3xTg-AD mice. Methods: Nine-month old 3xTg-AD mice received anti-IL-1 receptor blocking antibody (antiIL-1Rab; 200 micro-g, every 9-10 days) intraperitoneally for 6 months. Sham-treated mice received isotype-matched IgG injection in the same manner, and control mice were untreated throughout the treatment period. Results: Cognitive functions were assessed at the end of the treatment period, and we found that mice with anti-IL-1Rab treatment showed a significantly better cognitive functions than control or sham-treated mice in Morris water maze, novel object recognition and contextual fear conditioning tests. Pathologically, anti-IL-1Rab-treated mice have markedly reduced Abeta plauque burden and hyperphosphorylated tau accumulations in CA1 hippocampus region although quantitative Abeta ELISA analysis revealed a significant increase in soluble Abeta42 species in the brain. Interestingly, levels of oligomeric Abeta species detected by antibodies A11 or OC were significantly reduced by the anti-IL-1Rab treatment. Pro-inflammatory responses in the brain were altered as detected by significant reductions of IL-1beta and TNF-alpha, while increased number of microglia containing 6E10-positive APP fragments was observed in the anti-IL-1Rab-treated mice. Changes in tau phosphorylation was mediated by an inactivation of glycogen synthase kinase-3beta, and cellular catenin signaling was also altered. Conclusions: These data suggest that an inhibition of a specific pro-inflammatory cytokine may modulate the overall brain inflammatory responses as well as the progression of AD-like pathologies. Therefore, targeting specific inflammatory molecules could be beneficial and may help to develop potential therapeutic strategies for AD. P1-313

EVALUATION OF THE EXPRESSION OF ADHESION MOLECULES ON ENDOTHELIAL CELLS TREATED WITH AMYLOID-BETA AND OXIDIZATED LDL

Emma Rodriguez1, Nancy Gomez1, Claudia Huesca1, Felipe Masso1, Luis Felipe Montan˜o2, 1Instituto Nacional de Cardiologia, Mexico, Mexico; 2 Universidad Nacional Autonoma de Mexico, Mexico, Mexico. Contact e-mail: [email protected] Background: Accumulation of amyloid beta (Ab) is the hallmark in Alzheimer disease (AD), which is increased by injury to or inflammation of the brain. An augment of blood LDLox has been associated with a risk for inflammatory diseases like atherosclerosis. Inflammatory process begins with adhesion of leucocytes on the endothelium, regulated by adhesion molecules, but it is very-little known the regulatory role of Ab on the expression of those molecules and if LDLox could be relationed. Our aim was to determinate the effect of LDLox and/or Ab on adhesion molecules expression on endothelial cells (HMEC-1). Methods: Expression of adhesion molecules was made through flow cytometry using specific FITC labeled antibodies and adhesion assays were made also, with thymidine-labeled monocytes U937. Results: Our results show that Ab in different concentrations (0, 5, 10 and 20 mM) increases the expression of E-selectin (2.28, 4.2, 4.1 and 6.61 X 1000 FU (flouorescence units), respectively); ICAM-1 (61.2, 65.5,

75.9 and 66.1 X 1000 FU) and VCAM-1 (11.5, 14.1, 17.9 and 17.4 X 1000 FU). Adhesion Assays were made with Ab and LDL ox (100 mg/ml) and an augment on percentage adhesion was seen proportionally with Ab concentrations (13.5, 25.8 and 39.1%, respectively), meanwhile LDLox alone showed an increment of 77.85%, while it was better with 5 mM (105. 5%) and 10 mM (88.9%) but no with 20 mM (69.7%) Ab. Conclusions: These results show that the Ab can induce the expression of adhesion molecules on endothelial cells and the LDLox may increase that effect. It is possible that synergic action of Ab and LDLox could be a major risk for cardiovascular events. P1-314

SIGNIFICANCE OF 18-KDA TRANSLOCATOR PROTEIN IN MONITORING AND REGULATING MICROGLIAL RESPONSE TO Ab AMYLOIDOSIS: AN IN VIVO PET STUDY OF MICROGLIAIMPLANTED MOUSE MODELS

Bin Ji1, Makoto Sawada2, Satoko Hattori1, Masahiro Maruyama1, Jun Maeda1, Maiko Ono1, Takashi Okauchi1, Hiromi Suzuki2, MingRong Zhang1, Makoto Higuchi1, Tetsuya Suhara1, 1National Institute of Radiological Sciences, Chiba, Japan; 2Nagoya University, Nagoya, Japan. Contact e-mail: [email protected] Background: Removal of amyloid b peptide (Ab) deposits and neurotoxic inflammation mediated by microglia may have contradictory impacts on the pathogenesis and treatment of Alzheimer’s disease (AD). We previously reported the role of 18-kDa translocator protein (TSPO, also known as peripheral benzodiazepine receptor) as a marker for the neurotoxic activation of microglia. On this basis, the present study is aimed at clarifying the significance of TSPO in therapeutic approaches to AD Ab pathologies by in vivo monitoring of amyloid precursor protein transgenic (APP Tg) mice, which underwent implantation of microglial clones expressing TSPO at different levels. Methods: Two microglial cell lines dubbed Ra2 and HS1 were implanted into the hippocampi of APP Tg mice, and amyloid loads and TSPO levels during the course of treatments were non-invasively assayed by high-resolution positron emission tomography (PET) with specific radioligands. Ab burden, microglial activation, TSPO upregulation and cytokine/ chemokine releases were also investigated by immunohistochemical, biochemical and autoradiographic techniques. Results: Implantation of Ra2 resulted in local elimination of Ab lesions without overt changes in TSPO levels. By contrast, HS1 induced no Ab removal but notable TSPO rises around the injection site. Microscopic examinations illustrated activation of TSPO-negative, endogenous microglia in the vicinity of Ra2 implants, which was in turn suppressed in the case of HS1 cells. In cell cultures, numerous inflammatory chemokines were abundantly produced by HS1 as compared with Ra2, and a subset of these chemokines were shown to increase in brains of HS1-implanted mice and AD patients. The chemokine release was also provoked in cultured Ra2 cells treated with lipopolysaccharides, and this upregulation was attenuated by a ligand for TSPO, PK-11195. Conclusions: Our data imply that low TSPO expressor microglia are likely prone to the Ab elimination and can stimulate the involvement of neighboring microglia in this process, which may be disrupted by upregulation of TSPO and consequent overproduction of inflammatory chemokines. TSPO is a PETmeasurable biomarker potentially useful for directing microglial responses to the Ab removal. Pharmacological regulation of TSPO alone or in combination with other immunotherapeutic interventions would also be efficacious against AD pathologies. P1-315

MINOCYCLINE-MEDIATED PROTECTION FROM Ab IS MEDIATED PRIMARILY BY SUPPRESSION OF ASTROCYTIC INFLAMMATORY RESPONSES

Claire J. Garwood, Diane P. Hanger, Wendy Noble, MRC Centre for Neurodegeneration Research, Institute of Psychiatry, Kings College London, London, United Kingdom. Contact e-mail: [email protected] Background: Minocycline is a second generation tetracycline derivative with potent, neuroprotective actions in various models of neurodegenerative disease including Alzheimer’s disease that are likely associated with its antiapoptotic and anti-inflammatory properties. Objectives: Firstly, to

Poster Presentations P1 determine if the effectiveness of minocycline in reducing b-amyloid-induced abnormal tau processing and neuronal death is mediated by glia. Secondly to investigate the role of glia in Ab-mediated degenerative pathways. Methods: We have investigated the effect of minocycline treatment on Ab-induced tau processing and neuronal death in primary neuronal, mixed and glial cultures. Mixed cortical cultures were prepared from E18 rat embryos, and neuronal cultures were derived following the suppression of glial proliferation with cytosine arabinoside. Analysis of mixed cultures by immunocytochemistry determined that these cultures contain 4.3 6 0.4% astrocytes and negligible numbers of microglia (<0.0001%). Glial cultures, containing astrocytes, microglia and small numbers of oligodendrocytes were prepared from P2 rat pups. Cultures were pre-treated with 20mM minocycline for 24h prior to treatment with 10mM soluble oligomeric Ab. Neuronal cell death, caspase activation, pathological changes in tau proteins and the secretion of inflammatory mediators were assessed by a variety of methods. Results: We show that the presence of astrocytes accelerates the onset of neuronal death in response to Ab, and that the neuroprotective influence of minocycline is only observed when astrocytes are present. Furthermore, we observe that astrocytes are required for Ab-induced tau phosphorylation, and that astrocytes mediate caspase-3-mediated cleavage of tau in response to Ab. In glial cultures, we find increased secretion of several inflammatory factors upon Ab treatment, including IL-1a, IL6 and IL10. Pre-treatment of glial cultures with minocycline suppresses the secretion of these pro-inflammatory mediators. The influence of specific inflammatory factors on Ab-induced neurotoxicity is currently under investigation. Conclusions: These results suggest that glia, and more specifically astrocytes, play a major role in Ab-induced abnormal tau processing and neuronal death. These findings also confirm that glia are a primary target of minocycline. It is possible that suppression of specific neuroinflammatory responses, either alone or in combination with other treatments, may represent an effective therapeutic strategy for the treatment of Alzheimer’s disease. P1-316

INVOLVEMENT OF PHOSPHOINOSITIDE 3-KINASE GAMMA IN THE NEUROINFLAMMATORY RESPONSE AND COGNITIVE IMPAIRMENTS INDUCED BY BETA-AMYLOID 1-40 PEPTIDE IN MICE

Giselle F. Passos1, Cla´udia P. Figueiredo2, Rui D. S. Prediger2, Kathryn A. B. S. Silva2, Jarbas M. Siqueira2, Filipe S. Duarte2, Paulo C. Leal2, Rodrigo Medeiros1, David H. Cribbs1, Joa˜o B. Calixto2, 1 University of California, Irvine, CA, USA; 2Universidade Federal de Santa Catarina, Florianopolis, Brazil. Contact e-mail: [email protected] Background: In addition to neurofibrillary tangles (NFT) and deposition of beta-amyloid (Abeta) in extracellular plaques, chronic inflammation due to recruitment of activated glial cells to amyloid plaques are an invariant component in Alzheimer’s disease (AD), and several studies have reported that the use of non-steroidal anti-inflammatory drugs (NSAIDs) may provide a measure of protection against AD. The objective of this study was to investigate whether phosphoinositide 3-kinase gamma (PI3Kgamma), which is important in inflammatory cell migration, plays a role in the neuroinflammation, synaptic dysfunction, and cognitive deficits induced by Abeta. Methods: Male C57Bl/6 mice (20-30 g) were injected intracerebroventricularly (icv) with Abeta(1-40) (400 pmol/mouse) and received the selective inhibitor of PI3Kgamma, AS605240 (30 mg/kg, p.o., twice daily). After 7 days, animals were tested for cognitive deficits using the Morris water maze. The expression of CD68, glial fibrilary acid protein (GFAP), p-Akt, cyclooxygenase-2 (COX-2), and synaptophysin was examined by immunohistochemistry. To further assess the role of PI3Kgamma in Abeta(1-40)-induced cell responses, chemotaxis of macrophages towards the supernatant from Abeta(1-40)-stimulated macrophages (Abeta(1-40)-treated media) and cytokine production were assessed in the presence of AS605240. Results: We found that the selective inhibitor of PI3Kgamma, AS605240, was able to attenuate the Abeta(1-40)-induced activated phenotypes of astrocytes and microglia in the hippocampus, and decrease immunostaining for p-Akt and COX-2. Importantly, AS605240 administration to mice prevented

S265

Abeta(1-40)-induced cognitive deficits and synaptic dysfunction. Moreover, macrophages treated with AS605240 displayed impaired chemotaxis in vitro towards Abeta(1-40)-treated media, but their expression of tumour necrosis factor-a (TNF-a) and interleukin-1b (IL-1b) was unaffected. Conclusions: Our data suggest that selective PI3Kgamma inhibitors may represent interesting targets for AD drug development. P1-317

ROLE OF GAP-43 IN AXONAL DAMAGE AND REGENERATION IN NEUROINFLAMMATORY SITUATIONS

Kameshwar R. Ayasolla1, Aileen Lynch2, 1Bar-Ilan University, Ramat Gan, Israel; 2Trinity College, University of Dublin, Dublin 2, Ireland. Contact e-mail: [email protected] Background: It has been lately established that inflammation plays a significant role in the pathogenesis of brain injury. Both Ab generation inside the brain or its uptake from systemic circulation into the brain via the blood brain barrier is known to cause brain inflammation and brain injury. Released cytokines are neuro-toxic to the brain and may prevent neural stem cell differentiation, prevent proper gaseous exchange, and increase risk of exposure to hypoxic conditions. Stem cell therapy seem to be a good way to replace the depleted stem cell population, although the altered redox conditions as well the inflammatory cytokines may not permit infused stem cells to thrive well. Here in the following study our objectives were to understand the possible mechanisms involved in axonal damage and regeneration under neuroinflammatory conditions. Several reports suggest possible axonal loss, neuronal stress, leading to neuronal apoptosis resulting in memory loss and associated cognitive defecits. Our earlier studies demonstrated elevation in inflammatory cytokines in reponse to LPS/Ab peptide in glial cells, is mediated by sphingolipid ceramide together with ROS generation and activation of various stress signaling cascades. Methods: a) Here in the following studies we studied PC-12 cells in response to NGF induced neurite outgrowth and in its inhibition either to glial cell conditioned medium (glia previously stimulated with LPS + Ab) or to cytokines (IL-1b/Ab).b) In addition we studied GAP43 phosphorylation under inflammatory conditions to either cytokine or Ab stress. Results: Investigation of the possible mechanisms showed that these stressors induced elevation in ceramide, ROS generation as well a alteration in GAP-43 phosphorylation, stress kinase cascades (MAP kinases) which were all normalized by blocking ceramide generation using neutral sphingomyleinase inhibitor 3-o-methyl Sphingomyelin or with ceramide synthase inhibitor fumonisin B1. Conclusions: GAP-43 a major phosphoprotein in the CNS plays a major role in microtubular protein stability promoting axonal extensions. Here in these studies we show evidence of possible phosphorylation of GAP-43 by PKC-delta that may facilitate axonal growth and discuss possible alterations in GAP-43 phosphorylation/PKC co-localization in the detergent resistant membranes (DRMs) in inflammatory conditions, that all these deleterious effects might be possibly reversed by blocking ceramide generation. P1-318

ABLATED TNF RECEPTOR EXPRESSION DIFFERENTIALLY AFFECTS MICROGLIAL ACTIVATION AND AMYLOID DEPOSITION IN THE 3XTG-AD MOUSE MODEL

William J. Bowers, Sara L. Montogomery, Michael A. Mastrangelo, Wade C. Narrow, University of Rochester Medical Center, Rochester, NY, USA. Contact e-mail: [email protected] Background: Progressive memory and cognitive deterioration represent primary symptoms of Alzheimer’s disease (AD), which is a neurodegenerative disorder that evolves in a temporal and spatial manner within the afflicted brain. AD is pathologically characterized by extracellular Ab peptide deposition, intracellular neurofibrillary tangle formation, decreased synaptic integrity, and neuronal loss. Neuroinflammatory processes are highly active in AD, and efforts have focused on determining the functions particular inflammatory mediators play in the disease. Our laboratory previously reported that enhanced expression of the potent pro-inflammatory cytokine, tumor necrosis factor-alpha (TNF-a) and concomitant increase in microglial activation are specifically detected within the entorhinal cortex of young 3xTg-AD