Technical Reference #3239

3239.

Inhibitors of Catalase-Amyloid Interactions Protect Cells from -Amyloid-Induced Oxidative Stress and Toxicity Lila K. Habib; Michelle T. C. Lee; and Jerry Yang, University of California San Diego, Journal of Biological Chemistry, 285(3239), (2010)
Link To Paper

Abstract:
Compelling evidence shows a strong correlation between accumulation of neurotoxic -amyloid (A ) peptides and oxidative stress in the brains of patients afflicted with Alzheimer disease (AD). One hypothesis for this correlation involves the direct and harmful interaction of aggregated A peptides with enzymes responsible for maintaining normal cellular levels of reactive oxygen species (ROS). Identification of specific destructive interactions of A peptides with cellular anti-oxidant enzymes would represent an important step toward understanding the pathogenicity of A peptides in AD. This report demonstrates that exposure of human neuroblastoma cells to cytotoxic preparations of aggregated A peptides results in significant intracellular co-localization of A with catalase an anti-oxidant enzyme responsible for catalyzing the degradation of the ROS intermediate hydrogen peroxide (H2O2). These catalase-A interactions deactivate catalase resulting in increased cellular levels of H2O2. Furthermore small molecule inhibitors of catalase-amyloid interactions protect the hydrogen peroxide-degrading activity of catalase in A -rich environments leading to reduction of the co-localization of catalase and A in cells inhibition of A -induced increases in cellular levels of H2O2 and reduction of the toxicity of A peptides. These studies thus provide evidence for the important role of intracellular catalase-amyloid interactions in A - induced oxidative stress and propose a novel molecular strategy to inhibit such harmful interactions in AD.

Materials & Methods:
Assessment of the Cellular Internalization of A and BTAEGx Molecules by Fluorescence Deconvolution Microscopy— SH-SY5Y cells were plated in DMEM without phenol red (supplemented with 10% FBS and 4 mM L-glutamine) on 35 mm glass bottom dishes (MatTek Co. Ashland MA) and incubated overnight. The growth medium was removed and solutions of pre-aggregated fluorescently labeled A (5 M dissolved in medium) with or without BTA-EGx (40 M dissolved in medium) were added to the cells and allowed to incubate for 12 h before imaging. The cells were washed with DMEM (without phenol red or FBS) immediately prior to imaging with a Delta Vision Deconvolution Microscope System (Applied Precision Issaquah WA) equipped with a Nikon TE-200 inverted light microscope with infinity corrected lenses and with a mercury arc lamp as the illumination source. The co-localization was determined using softWoRx image analysis software (Applied Precision Issaquah WA). The intrinsic fluorescence of the BTA-EGx molecules (Abs/Em: 360/ 440 nm (60)) was observed by excitation with bandpass (bp) filtered light (Ex/bp: 360/40 nm) and the emission monitored at Em/bp: 457/50 nm. The fluorescence of the HiLyte Fluor 488- labeled A (Abs/Em: 503/528 nm) was detected using an Ex/bp: 490/20 nm excitation filter and an Em/bp: 528/38 nm emission filter. The images shown in Fig. 5 are fluorescence micrographs of representative z-slices within cells.

Microscopic Technique
Fluorescence Deconvolution Microscopy

Cell Type(s)
SH-SY5Y cells