University of Chicago1, Neurobiology, Pharmacology, and Physiology, Chicago, IL 60637 Butenandt-Institute, University of Munich2, Munich, Germany, 80336
AMIODARONE INHIBITS APP PROCESSING BY INCREASING ENDOSOMAL PH, J.A. Hopson1, L.V. Deriy1, S.F. Lichtenthaler2, D.J. Nelson*1, University of Chicago1, Department of Neurobiology, Pharmacology, and Physiology, Chicago, IL 60637, University of Munich2, Butenandt-Institute, Munich, Germany, nelson@uchicago.edu. Alzheimer’s Disease (AD) is a late-onset neurodegenerative disease affecting 5 million Americans. Primary symptoms include memory loss followed by decreased motor function and death. On the cellular level, the neuronal death caused by AD is believed to be primarily due to two misprocessed, toxic proteins: extracellular amyloid-β (Aβ) plaques and intracellular τ tangles. Though virtually all cells in the human body make Aβ monomers, it appears that in AD, the plaque polymers are caused either by decreased clearance of Aβ from the extracellular space or overactive Aβ production. Aβ monomers are formed by the cleaving of amyloid precursor protein (APP), a transmembrane protein. Once APP is internalized for recycling via the endocytic pathway, it is cleaved by two proteases, β- and γ-secretase, and Aβ monomers are formed. These monomers are exocytosed, and due to their hydrophobic nature, form aggregates and create the toxic plaques characteristic of AD. Thus, decreasing Aβ production is a possible avenue for decreasing Aβ plaque formation and treatment for AD. Amiodarone is an FDA-approved anti-arrhythmic drug which blocks Na+, K+, and Ca2+ channels as well as β-adrenergic receptors. It is highly lipophilic and readily intercalates into cell membranes, including endosomal membranes. Based on the observation that amiodarone reduces β-secretase cleavage, we hypothesize that the nitrogen moiety present on the end of amiodarone faces the endosomal lumen, available to buffer protons, thereby alkalinizing the lumen. β-secretase requires an acidic environment to optimally cleave APP; if the environment is alkalinized, β-secretase cleavage is reduced or completely inhibited. Thus amiodarone, via its luminal-buffering capability, would prevent β-secretase cleavage and Aβ formation. To test this hypothesis, HEK cells stably transfected with APP were cultured and used for live-cell confocal microscopy. Endosomes were loaded with a doubly-conjugated, pH-sensitive dextran dye. We have demonstrated that amiodarone at concentrations of 30 µM or greater increases the endosomal pH to 5.82, compared to the control at pH 5.17, consistent with our hypothesis. This pH response is dose-dependent with an IC50 = 16.27 µM. Our data are consistent with previous data which show that increased endosomal pH causes decreased Aβ production 1. Therefore, amiodarone may serve as a lead compound in potential therapies targeted at reducing and preventing Aβ plaque formation. J.A. Hopson was supported by NIH R01 GM36823 and a grant from the Brain Research Foundation.
1. Vingtdeux, V., et al. Intracellular pH regulates amyloid precursor protein intracellular domain accumulation. Neurobiology of disease 25, 686-696 (2007).
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