P3-299: Protein degradation related with post-mortem delay in the study of human neurodegenerative diseases. A proteomics approach

Abstract

Research on the human brain is basic to increase understanding about human neurological disorders such as Alzheimer disease (AD). Yet post–mortem delay is an unavoidable factor in the studies based on human post–mortem brain tissue. Brain proteins, when exposed to long post–mortem interval, may be altered by endogenous proteases and phosphatases, thus resulting in pitfalls and confusing results. The present study stresses the use of proteomics in the identification of vulnerable proteins to post–mortem delay, and illustrates the necessity for careful control of protein degradation when using post–mortem brain tissues. In the present work, brains of AD and age–matched control cases were analysed by two–dimensional electrophoresis at different artificial post–mortem intervals (0, 3, 6, 12, 18, 24 and 48 hours) subjected to variable temperature conditions. Silver–stained gels of the same case with increased artificial post–mortem delay disclosed differential spot expression which was the result of target protein degradation as revealed with mass spectrometry. We also studied by Western blot the post–mortem–dependent degradation of tau protein in paired helical filament (PHF)–enriched fractions from AD brains. Several proteins were identified and categorized as differentially vulnerable to post–mortem delay. Following standard paradigms (post–mortem delay at 4°C, beginning two h after death) phospho–tau degradation, manifested as a reduction in the number and intensity of the bands, occurred between 6 and 24 h of post–mortem and was universal in samples with post–mortem delays of 48 h. Again, these studies indicate the importance in considering post–mortem delay artefacts related with post–mortem delay in the study of phospho–tau patterns in AD and other tauopathies.