$540 Poster Session P4: Molecular Mechanisms of Neurodegeneration - Oxidative Stress

by A[3) in comparison to the CC promoter (1.3× stimulation by LPS vs. 1.7× stimulation by A[3). Conclusions: Our results suggest that the C/T conversion increases the activity of the promoter, especially with low dosages of inflammatory stimuli, which may result in an upregulation of the IL-lc~ protein and an amplification of the "cytokine cycle" stimulating IL-11~ as well as other cytokines. Increased cytokines may play an important role in promoting AD development. Acknowledgements: We sincerely thank the support of a grant from the National Institutes of Health (AG20248).

~ - ~ LIPOPOLYSACCHARIDE AS A NEUROINFLAMMATORY AGENT: EVALUATION OF THE TIME COURSE OF INFLAMMATORY MARKERS USING QUANTITATIVE REAL TIME PCR, BEHAVIORAL OUTCOMES, AND IMMUNOHISTOCHEMISTRY

Donna L. Herber*, Lisa M. Roth, Melissa J. Freeman, Dave Morgan, Marcia N. Gordon. University of South Florida, Tampa, FL, USA. Contact e-mail: dherber@hsc.usfedu

Background: Inflammation has been argued to play a primary role in the pathogenesis of Alzheimer's disease. Lipopolysaccharide (LPS) activates the innate immune response and triggers gliosis when injected into the central nervous system. Previous work by our lab has demonstrated the ability of the innate immune system to remove A[~ in APP transgenic mice when stimulated by intrahippocampal injections of LPS. Objective(s): In studies described here, we evaluated the expression of scavenger receptors, cytokines, the LPS signaling cascade through TLR4, and other acute phase proteins in response to LPS. Methods: Nontransgenic mice were injected bilaterally into the hippocampus with 4 Ixg of LPS. Behavioral outcomes were assessed using a y-maze at 1, 3, 7, 14, or 28 days post injection. Quantitative PCR analysis of gene expression was conducted at 1, 6, and 24 hours, as well as 3, 7, 14, and 28 days post injection. Immunohistochemistry was also performed for reactive gfial markers. Results: No effect of LPS was seen in the Y-maze percent alternations made by the mice compared to either uninjected or saline injected control mice. A significant decrease in the number of ann entries was demonstrated at 24 hours post-injection, indicating sickness behavior. At 6 hours post treatment, GFAP and the CD36 scavenger receptor mRNA were significantly elevated. Markers of inflammation including GFAP, CD45 and TLR4 mRNA were elevated after 24 hours and peaked at 3 days. At 7 and 14 days, the levels were decreasing from peak values, but remained significantly elevated at 28 days relative to controls. At three days post injection, mRNA for the cytokines ILI~ and TNFc~, LPS-signaling cascade related NF~:B and CD14, the class A scav- enger receptor, and the acquired immunity Fcy receptor were also increased. Synaptotagmin was unchanged throughout the time course, suggesting a lack of general synaptic toxicity. Immunohistochemical data corroborated gene expression data. Conclusions: These findings indicate that the innate response occurred within 6 hours of LPS injection, triggered cytokine production, increased transcripts involved with the TLR4 signaling cascade, and caused upregulation of scavenger receptors that may be responsible for phagocytosing A[3 in APP mouse models. Supported by AG15490.

~ I L - 8 POTENTIATION OF AMYLOID-BETA (AOl.42)-INDUCED FUNCTIONAL RESPONSES IN CULTURED HUMAN MICROGLIA

Sonia Franciosi*, Hyun B. Choi, Jae K. Ryu, Seung U. Kim, James G. McLarnon. University of British Columbia, Vancouver, BC, Canada. Contact e-mail: soniaf@ interchange, ubc.ca

Amyloid beta (A[3) stimulated microglia mediate inflammatory processes which likely contribute to pathogenesis of Alzheimer's Disease (AD). IL-8 is a chemokine produced from activated microglia and is reported to be upregulated in AD brains. We have investigated the influence of 1L-8 on full length (A131_42)-induced responses in cultured human microglia using RT-PCR, ELISA and immunocytochemistry assays. Unstimulated microgfia show constitutive expression of IL-I~ and the anti-inflammatory cytokines

IL-10 and TGF-[31. Application of either A~142 (5 IxM) or IL-8 (100 ng/mL) for 8 hrs induced the expressions of the pro-inflammatory cytokines IL-6 and TNF-c~ and the enzyme COX-2; in addition IL-113 was increased from consti- tutive levels. In the presence of A~1-42 and IL-8 together, expressions of the pro-inflammatory factors IL-I[~, IL-6, TNF-c~ and COX-2 were significantly increased compared to either A131-42 or IL-8 stimulation alone. However, expressions of the anti-inflammatory cytokines IL-10 and TGF-[31 remained unchanged from basal levels despite stimulation with either A~1-42, IL-8 or A~1-42 mad IL-8 combined. ELISA assays showed unstimulated human microglia produced low levels of IL-113, IL-6, TNF-ct and COX-2. Stimu- lation with A~1-42 or IL-8 separately for 24 hrs led to a significant increase in production of IL-I[3 and IL-6. A13142 also increased TNF-~ and COX-2 production whereas IL-8 was ineffective. IL-8 in combination with A[31.42 significantly enhanced IL-113, IL-6, TNF-c~ and COX-2 levels compared with either stimulus alone. These results suggest that IL-8 potentiates A~1-42 induced expression and production of pro-inflammatory factors in human microglia which may have relevance to inflammation in AD.

Poster Session P4: Molecular Mechan i sms of N e u r o d e g e n e r a t i o n - Oxidat ive Stress

• HIGHLY REACTIVE y-KETOALDEHYDES IN ALZHEIMER'S DISEASE

Scan S. Davies* 1, Nathalie Bemoud-Hubac 2, Sandra Olsen 1, Thomas J. Montine 3, L. Jackson Roberts II 1. tVanderbilt University, Nashville, TN, USA," 2 INSERM/1NSA Lyon, Lyon, France; 3 University of Washington, Seattle, WA, USA. Contact e-mail: sean.davies@vanderbilt.edu

Background: Free radical mediated oxidation of polyunsaturated fatty acids forms a number of lipid aldehydes capable of adducting to and altering protein function. We recently discovered novel series of highly reactive y-ketoaldehydes (yKAs), termed isoketals and neuroketals, that are formed during oxidation of arachidonic acid and docosahexanoic acid, respectively. These yKAs rapidly adduct to lysine residues at a rate that far exceeds that of 4-hydroxynonenal. Moreover, these yKAs exhibit a remarkable proclivity to crosslink proteins. Proteins adducted by these yKAs exhibit a markedly reduced rate of degradation by the proteasome and nM concentrations of yKAs are cytotoxic to neuronal cell lines. Therefore, formation of yKAs in vivo would be expected to have profound deleterious effects. Objectives: Determine the relative levels and location of yKAs adducted to proteins in AD brain versus aged-matched controls. Methods: We used an anti-yKA adduct single chain antibody (Dl l ScFv) to examine to the distribution of yKA protein adducts in post-mortem brain sections from patients with AD and aged-matched controls by immunohistochemistry. We also measured the levels of yKA adducts in brain homogenates by tandem mass spectrometry. Sandwich ELISAs were performed using anti-tan or anti-tubulin antibodies to capture the respective proteins and D11 ScFv antibody to measure yKA adducts. We also determine the effect of adducting recombinant human tan with a synthetic yKA on immunoreactivity to the anti-PHF antibody Alz50. Results: A striking finding was intense staining in almost all of the neuronal bodies and neuropil in AD hippocampus, which was absent in the cerebellum of these same subjects and the hippocampus from aged-matched controls. Significantly increased levels of neuroketal adducts were also found in the hippocampus from patients with AD compared to aged-matched control subjects when measured by tandem mass spectrometry. Sandwich ELISA suggested increased levels of yKA adducts on tan and tubulin in AD brains. Adduction of synthetic yKA to tan substantially increased immunoreactivity with Alz50. Conclusions: These results suggest that these yKAs may contribute importantly to the neurotoxicity and pathogenesis of AD.