Post-mortem Interval Effects on the Phosphorylation of Signaling Proteins

Abstract

Post-mortem brain tissue provides a unique opportunity to uncover the genes or proteins involved in the pathophysiology of neuropsychiatric disorders. Protein phosphorylation is a common protein modification within intracellular signaling pathways that affects the distribution and function of protein, and has been hypothesized to be of major importance in both the pathophysiology and treatment of major neuropsychiatric disorders. Thus, we were interested in ascertaining the stability of the phosphorylated forms of proteins that are involved in cellular signaling. Antibodies against phospho-tyrosine, phospho-threonine, and phospho-PKA substrates were used to examine the PMI effects on the general amounts of proteins in their phosphorylated form. Phospho-specific antibodies for ERK, JNK, RSK, CREB, and ATF-2 were used to test the effects of PMI on specific proteins whose functioning are known to be regulated markedly by phosphorylation. We found that PMI rapidly decreased the levels of proteins in their phosphorylated states and also decreased the total levels of certain proteins. The PMI effects were observed in the samples stored at both 41C and room temperature, in both frontal cortex and hippocampus. Thus, it appears that measurements (such as two-dimensional gel electrophoresis and functional assays) that rely on the phosphorylation state of proteins would be extremely sensitive to PMI.

Postmortem Brain: An Underutilized Substrate for Studying Severe Mental Illness

“…Postmortem variables such as PMI may also impact phosphorylation (Funk et al, 2012;Li et al, 1996Li et al, , 2003Li et al, , 2005O'Callaghan and Sriram, 2004;Oka et al, 2011). The often transient nature of phosphorylation is well characterized, and in part led to the development of strategies (such as microwave devices) for rapid 'fixation' of brain tissues particularly for proteins with a rapid turnover cycle for phosphorylation/dephosphorylation (O'Callaghan and Sriram, 2004).…”

ATF2, A Member of the CREB/ATF Family of Transcription Factors, in Chronic Stress and Consequent to Antidepressant Treatment: Animal Models and Human Post-Mortem Brains

“…We did not, however, observe a significant increase in pATF2 in nonmedicated depressed patients compared to normal controls. Li et al (2003) have recently shown that postmortem interval (PMI) produces profound and relatively rapid effects on the phosphorylation of proteins involved in intracellular signaling, including specifically the phosphorylation of ATF2. The inclusion of PMI as a covariate in our model did not result in a significant effect, indicating that differences in PMI between groups could not account for the lack of effect observed.…”

Impact of Temperature Dependent Sampling Procedures in Proteomics and Peptidomics – A Characterization of the Liver and Pancreas Post Mortem Degradome

“…The properties of the usual 2-DE based approach therefore clearly influence the degradome data, making it tempting to define these results as "2-DEgradomes." It should also be noticed that the PMI PTM knowledge is mainly based on protein phosphorylation, with different proteins displaying varying levels of PMI sensitivity within the first 10h (1,3,9,17) but there are also reports on PMI sensitivity among acetylated lysines (9). The more proteolytic nature of the pancreas is probably responsible for the fact that ϳ99% of the liver proteome is similar between D and FD whereas this is only the case for ϳ89% in pancreas.…”

“…Post mortem degradation in during PMI is a well known compromising problem when studying endogenous peptides (2,3) and has also been proven to affect the results of polypeptide (here defined as proteins larger than 10 kDa) studies (3)(4)(5)(6)(7)(8). PMI degradation has mainly been studied on human or mouse brain tissue, using two-dimensional electrophoresis (2-DE), SDS-PAGE, and immunoblotting (1,(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). There are also a few proteomic studies on muscle tissue degradation in livestock (13)(14)(15)(16).…”