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Oxidative stress is associated with the neuropathology of Alzheimer's disease. We have previously shown that human Abeta has the ability to reduce Fe(III) and Cu(II) and produce hydrogen peroxide coupled with these metals, which is correlated with toxicity against primary neuronal cells. Cyclooxygenase (COX)-2 expression is linked to the progression and severity of pathology in AD. COX is a heme-containing enzyme that produces prostaglandins, and the enzyme also possesses peroxidase activity. Here we investigated the possibility of direct interaction between human Abeta and COX-2 being mediated by the peroxidase activity. Human Abeta formed dimers when it was reacted with COX-2 and hydrogen peroxide. Moreover, the peptide formed a cross-linked complex directly with COX-2. Such cross-linking was not observed with rat Abeta, and the sole tyrosine residue specific for human Abeta might therefore be the site of cross-linking. Similar complexes of Abeta and COX-2 were detected in post-mortem brain samples in greater amounts in AD tissue than in age-matched controls. COX-2-mediated cross-linking may inhibit Abeta catabolism and possibly generate toxic intracellular forms of oligomeric Abeta.

We screened for drugs that specifically interact with the 5'-untranslated region of the mRNA encoding the Alzheimer's amyloid precursor protein (APP). Our goal was to use newly discovered APP 5' UTR directed compounds to limit amyloid-beta (Abeta)-peptide output in cell culture systems. The APP 5' UTR folds into a stable RNA secondary structure (Gibbs free energy: DeltaG = -54.9 kcal/mol) and is an important regulator of the amount of APP translated in response to IL-1 (Nilsson et al., 1998; Rogers et al., 1999) and iron (Rogers et al., 2002). Seventeen drug "hits" were identified from a library of 1,200 FDA preapproved drugs (Rogers et al., 2002). Six of the original 17 compounds were validated for their capacity to suppress reporter gene expression in stable neuroblastoma transfectants expressing the dicistronic reporter construct shown in Fig. 2. These six leads suppressed APP 5' UTR driven luciferase translation while causing no effect on the translation of dicistronic GFP gene translated from a viral IRES (negative control to ensure specificity during drug screens). In this report, we show that paroxetine (serotonin reuptake blocker) and dimercaptopropanol (Hg chelator) exerted significant effects on APP expression (steady-state levels of APP), whereas Azithromycin altered APP processing. None of these three compounds altered APLP-1 expression. In the future, we will identify further novel compounds that influence Abeta levels, either via translation inhibition or by changing the activity of proteins coupled between APP translation and APP processing.

The 5' untranslated region (5'UTR) of the transcript encoding the Alzheimer's amyloid precursor protein (APP) is a key regulatory sequence that determines the amount of intracellular APP holoprotein present in brain derived cells. Using neuroblastoma cells (SY5Y) we developed a transfection based screen of a library of FDA drugs to identify compounds that limited APP luciferase reporter expression translated from the APP 5'UTR. Paroxetine (Paxil trade mark ), dimercaptopropanol, phenserine, desferrioxamine, tetrathiolmobdylate, and azithromycin were six leads that were subsequently found to also suppress APP holoprotein levels or to alter APP cleavage (azithromycin). Since APP holoprotein levels are proportionate to Abeta peptide output in many systems we tested the efficacy of paroxetine and dimercaptopropanol to limit Abeta secretion as measured by ELISA assays. Paroxetine and dimercaptopropanol limited Abeta peptide secretion from lens epithelial cells (B3 cells). Interestingly, paroxetine changed the steady-state levels of transferrin receptor mRNAs. These data suggested that this serotonin reuptake inhibitor (SSRI) provided extra pharmacological action to chelate interacellular iron or change the intracellular iron distribution. An altered iron distribution would be predicted to indirectly limit APP holoprotein expression and Abeta peptide secretion.

A growing body of evidence indicates that dysregulation of cerebral biometals (Fe, Cu, Zn) and their interactions with APP and Abeta amyloid may contribute to the Alzheimer's amyloid pathology, and thus metal chelation could be a rational therapeutic approach for interdicting AD pathogenesis. However, poor target specificity and consequential clinical safety of current metal-complexing agents have limited their widespread clinical use. To develop the next generation of metal chelators, we have designed and synthesized a new bifunctional molecule-XH1, based on a novel 'pharmacophore conjugation' concept. This lipophilic molecule has both amyloid-binding and metal-chelating moieties covalently connected by amide bonds. It achieved a putative binding geometry with Abeta1-40 peptide by the computational chemistry modeling and reduced Zn(II)-induced Abeta1-40 aggregation in vitro as determined by turbidometry. Moreover, our pilot data indicated that XH1 has no significant neurotoxicity at low micromolar concentrations and acute animal toxicity. XH1 specifically reduced APP protein expression in human SH-SY5Y neuroblastoma cells and attenuated cerebral Abeta amyloid pathology in PS1/APP transgenic mice without inducing apparent toxicity and behavior disturbances. Collectively, these preliminary findings carry implication for XH1 being a BBB-permeable lead compound for AD therapeutics targeting Alzheimer's amyloidogenesis, although further studies are needed.

We performed a screen for drugs that specifically interact with the 5' untranslated region of the mRNA coding for the Alzheimer's Amyloid Precursor Protein (APP). Using a transfection based assay, in which APP 5'UTR sequences drive the translation of a downstream luciferase reporter gene, we have been screening for new therapeutic compounds that already have FDA approval and are pharmacologically and clinically well-characterized. Several classes of FDA-pre-approved drugs (16 hits) reduced APP 5'UTR-directed luciferase expression (> 95% inhibition of translation). The classes of drugs include known blockers of receptor ligand interactions, bacterial antibiotics, drugs involved in lipid metabolism, and metal chelators. These APP 5'UTR directed drugs exemplify a new strategy to identify RNA-directed agents to lower APP translation and A beta peptide output for Alzheimer's disease therapeutics.