No abstract

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.

No abstract

The σ receptors (S1Rs) are implicated in a variety of diseases including Alzheimer disease and cancer. Previous PET S1R radiotracers are characterized by slow kinetics or off-target binding that impedes their use in humans. Here, we report the first PET imaging evaluation in rhesus monkeys of 4 F-labeled spirocyclic piperidine-based PET radiotracers (F- to F-). Baseline scans for the 4 radiotracers were obtained on an adult male rhesus monkey. Blocking scans were obtained with the S1R-selective agonist SA4503 to assess binding specificity ofF- and F- Arterial input functions were measured, and binding parameters were determined with kinetic modeling analysis. In the rhesus brain, all 4 radiotracers showed high and fast uptake. Tissue activity washout was rapid forF- and F-, and much slower for F- and F-, in line with their respective in vitro S1R-binding affinities. Both the 1-tissue-compartment and multilinear analysis-1 kinetic models provided good fits of time-activity curves and reliable estimates of distribution volume. Regional distribution volume values were highest in the cingulate cortex and lowest in the thalamus for all radiotracers. F- showed greater differential uptake across brain regions and 3-fold-higher binding potential than F- SA4503 at the dose of 0.5 mg/kg blocked approximately 85% (F-) and 95% (F-) of radiotracer binding. TracersF- and F- displayed high brain uptake and fast tissue kinetics, with F- having higher specific binding signals than F- in the same monkey. Taken together, these data indicate that both F- and F- possess the requisite kinetic and imaging properties as viable PET tracers for imaging S1R in the human brain.

Purpose The adenosine A2A receptor has emerged as a therapeutic target for multiple diseases, and thus the non-invasive imaging of the expression or occupancy of the A2A receptor has potential to contribute to diagnosis and drug development. We aimed at the development of a metabolically stable A2A receptor radiotracer and report herein the preclinical evaluation of [18F]FLUDA, a deuterated isotopologue of [18F]FESCH. Methods [18F]FLUDA was synthesized by a two-step one-pot approach and evaluated in vitro by autoradiographic studies as well as in vivo by metabolism and dynamic PET/MRI studies in mice and piglets under baseline and blocking conditions. A single-dose toxicity study was performed in rats. Results [18F]FLUDA was obtained with a radiochemical yield of 19% and molar activities of 72–180 GBq/μmol. Autoradiography proved A2A receptor–specific accumulation of [18F]FLUDA in the striatum of a mouse and pig brain. In vivo evaluation in mice revealed improved stability of [18F]FLUDA compared to that of [18F]FESCH, resulting in the absence of brain-penetrant radiometabolites. Furthermore, the radiometabolites detected in piglets are expected to have a low tendency for brain penetration. PET/MRI studies confirmed high specific binding of [18F]FLUDA towards striatal A2A receptor with a maximum specific-to-non-specific binding ratio in mice of 8.3. The toxicity study revealed no adverse effects of FLUDA up to 30 μg/kg, ~ 4000-fold the dose applied in human PET studies using [18F]FLUDA. Conclusions The new radiotracer [18F]FLUDA is suitable to detect the availability of the A2A receptor in the brain with high target specificity. It is regarded ready for human application.

No abstract