ABSTRACT
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.
Subject(s)
Alzheimer Disease/drug therapy , Amyloid beta-Peptides/antagonists & inhibitors , Amyloid beta-Protein Precursor/antagonists & inhibitors , Dimercaprol/analogs & derivatives , Neuroprotective Agents/pharmacology , Protein Biosynthesis/drug effects , Protein Synthesis Inhibitors/pharmacology , 5' Untranslated Regions/drug effects , 5' Untranslated Regions/genetics , Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/genetics , Amyloid beta-Protein Precursor/metabolism , Animals , Cell Line, Tumor , Chelating Agents/pharmacology , Dimercaprol/pharmacology , Dimercaprol/therapeutic use , Dose-Response Relationship, Drug , Down-Regulation/drug effects , Down-Regulation/genetics , Drug Evaluation, Preclinical , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Humans , Iron/metabolism , Neuroprotective Agents/therapeutic use , Organometallic Compounds/pharmacology , Organometallic Compounds/therapeutic use , Paroxetine/pharmacology , Paroxetine/therapeutic use , Protein Biosynthesis/genetics , Protein Processing, Post-Translational/drug effects , Protein Synthesis Inhibitors/therapeutic use , RNA, Messenger/drug effects , RNA, Messenger/metabolism , Receptors, Transferrin/geneticsABSTRACT
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.
Subject(s)
Amyloid beta-Peptides/chemistry , Isoenzymes/metabolism , Prostaglandin-Endoperoxide Synthases/metabolism , Animals , Blotting, Western , Brain/metabolism , Chromatography, High Pressure Liquid , Cross-Linking Reagents/chemistry , Cyclooxygenase 2 , Dimerization , Humans , Hydrogen Peroxide/chemistry , Hydrogen Peroxide/pharmacology , Immunoblotting , Membrane Proteins , Models, Chemical , Oxidative Stress , Peptides/chemistry , Precipitin Tests , Protein Binding , Rats , Tyrosine/chemistryABSTRACT
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.
