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Deposition of cross-linked insoluble protein aggregates such as amyloid plaques is characteristic for Alzheimer's disease. Microglial activation by these extracullar deposits has been proposed to play a crucial role in functional degeneration as well as cell death of neurones. A sugar-derived post-translational modification of long-lived proteins, advanced glycation endproducts (AGEs), activate specific signal transduction pathways, resulting in the up-regulation of various proinflammatory signals such as cytokines [interleukin-6 (IL-6), tumour necrosis factor-alpha (TNF-a)] and inducible nitric oxide synthase (iNOS). Our goal was to study AGE-activated signal transduction pathways involved in the induction of proinflammatory effectors in the murine microglial cell line N-11. Chicken egg albumin-AGE (CEA-AGE), used as model AGE, induces nitric oxide (NO), TNF-a and IL-6 production. The AGE receptor, RAGE, and the transcription factor, nuclear factor kappa B (NF-jB), appear to be involved in all pathways, since a neutralizing RAGE antibody and a peptide inhibiting NF-jB translocation down-regulated NO, TNF-a and IL-6 production. NO and TNF-a, but not IL-6 production appear to be regulated independently, since NOS inhibitors did not decrease TNF-a secretion and a neutralizing TNF-a antibody did not reduce NO production, while employment of NOS inhibitors reduced significantly the secretion of IL-6. Inhibition of the MAP-kinase-kinase (MEK) and phosphatidylinositol 3-kinase (PI 3 K) pathway, but not that of mitogen-activated protein kinase-p38 (MAPK-p38), reduced NO, TNF-a and IL-6 significantly, suggesting that simultaneous activation of the first two pathways is necessary for the AGE-induced induction of these pro-inflammatory stimuli.

Advanced glycation endproducts (AGEs) accumulate on long-lived protein deposits including beta-amyloid plaques in Alzheimer's disease (AD). AGE-modified amyloid deposits contain oxidized and nitrated proteins as markers of a chronic neuroinflammatory condition and are surrounded by activated microglial and astroglial cells. We show in this study that AGEs increase nitric oxide production by induction of the inducible nitric oxide synthase (iNOS) on the mRNA and protein level in the murine microglial cell line N-11. Membrane permeable antioxidants including oestrogen derivatives (e.g. 17beta-oestradiol) thiol antioxidants (e.g. (R+)-alpha-lipoic acid) and Gingko biloba extract EGb 761, but not phosphodiesterase inhibitors such as propentophylline, prevent the up-regulation of AGE-induced iNOS expression and NO production. These results indicate that oxygen free radicals serve as second messengers in AGE-induced pro-inflammatory signal transduction pathways. As this pharmacological mechanism is not only relevant for Alzheimer's disease, but also for many chronic inflammatory conditions, such membrane-permeable antioxidants could be regarded not only as antioxidant, but also as potent therapeutic anti-inflammatory drugs.

Activation of glial cells has been proposed to contribute to neuronal dysfunction and neuronal cell death in Alzheimer's disease. In this study, we attempt to determine some of the effects of secreted factors from activated murine N-11 microglia on viability and morphology of neurons using the differentiated neuroblastoma cell line Neuro2a. Microglia were activated either by lipopolysaccharide (LPS), bacterial cell wall proteoglycans, or advanced glycation endproducts (AGEs), protein-bound sugar oxidation products. At high LPS or AGE concentrations, conditioned medium from microglia caused neuronal cell death in a dose-dependent manner. At sublethal LPS or AGE concentrations, conditioned media inhibited retinoic acid-induced neurite outgrowth and stimulated retraction of already extended neurites. Among the many possible secreted factors, the contribution of NO or NO metabolites in the cytotoxicity of conditioned medium was investigated. Cell death and changes in neurite morphology were partly reduced when NO production was inhibited by nitric oxide synthase inhibitors. The results suggest that even in the absence of significant cell death, inflammatory processes, which are partly transmitted via NO metabolites, may affect intrinsic functions of neurons such as neurite extension that are essential components of neuronal morphology and thus may contribute to degenerative changes in Alzheimer's disease.

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Summary β‐Amyloid (Aβ) plaques are characteristic hallmarks of Alzheimer's disease (AD). In AD, it has been suggested that activation of microglial cells might be the link between Aβ deposition and neuronal degeneration. Activated microglia are associated with senile plaques and produce free radicals and inflammatory cytokines. However, it is still not clear whether Aβ needs a prestimulated environment to exert its proinflammatory potential. Advanced glycation endproducts (AGEs), protein‐bound oxidation products of sugars, have been shown to accumulate in senile plaques and could induce a silent but chronic inflammation in the AD brain. We tested whether Aβ acts as an amplifier of a submaximal proinflammatory response initiated by exposure to chicken egg albumin‐AGE, lipopolysaccharide or interferon‐γ. Synthetic Aβ was used to produce three different samples (Aβ‐fibrilar; Aβ‐aggregated; Aβ‐AGE), which were characterized for β‐sheeted fibrils by the thioflavin‐T test and electron microscopy. As markers of microglial activation, nitric oxide, interleukin‐6, macrophage‐colony stimulation factor and tumour necrosis factor‐α production was measured. All three Aβ samples alone could not induce a detectable microglial response. The combination of Aβ preparations, however, with the coinducers provoked a strong microglial response, whereby Aβ‐AGE and fibrilar Aβ were more potent inflammatory signals than aggregated Aβ. Thus, Aβ in senile plaques can amplify microglial activation by a coexisting submaximal inflammatory stimulus. Hence, anti‐inflammatory therapeutics could either target the primary proinflammatory signal (e.g. by limiting AGE‐formation by AGE inhibitors or cross‐link breakers) or the amplifyer Aβ (e.g. by limiting Aβ production by β‐ or γ‐secretase inhibitors).

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