Screening the activation state of multiple phosphoproteins reveals the in vivo impact of stressors on brain signaling pathways controlling cellular structure.
Toxicologist 2008 Mar; 102(1):372
Stress results in brain plasticity; these changes likely involve transduction of signals in a complex signaling network. We exposed C57Bl6/J male mice to various stressors and examined the changes in the phosphorylation state of multiple proteins to determine how stress affected signaling. Phosphorylation, the dominant mode of post-translation modification involved in cellular signaling, is controlled through the enzymatic action of both kinases and phosphatases. As these enzymes remain active under a variety of post-mortem conditions all analyses were conducted on tissue obtained after microwave euthanasia, a procedure that ensures death within no more than 50 msec. Moreover, because brain temperature of approximately 90 degrees C is achieved all enzymatic activity is destroyed, preserving in vivo phosphorylation state. Mice were exposed to one of the following: (1) a single 2-hr restraint, (2) a single 5-min swim in 4o C water, (3) 3 consecutive 10 min swims at room temper-ature separated by 5-min periods of non-swim, (4) 7 days of continuous exposure to corticosterone through the implant of a 100 mg pellet or (5) 21 consecutive days of stressors 1, 2, or 3. Hippocampus, cortex and striatum were collected following a stress session, at 24 hrs or 1 week later. A large-scale analysis of signaling networks was performed using Kinetworks Phospho Site Screens; this technique involves the use of phosphospecific antibodies to proteins from many of the currently known signaling pathways & allows the simultaneous determination of the phosphorylation status of approximately 80 phosphoproteins. Cell structure alterations characterize brain plasticity; we observed activation changes in multiple phosphoproteins linked to cell structure including the cytoskeletal proteins alpha- and gamma-adducin, GSK3 alpha, & tau. The direction and intensity of the change depended on the stressor and the brain area examined. Our data suggest an examination of the signaling pathways involved in the control of the cytoskeleton may be warranted to increase our understanding of stress-induced plasticity.
Biological-effects; Neuropathology; Quantitative-analysis; Laboratory-animals; Skeletal-system; Stress; Neurophysiological-effects; Neurophysiology; Neurotransmitters; Brain-function; Brain-electrical-activity; Signaling-systems; Cell-function; Cell-metabolism; Cellular-transport-mechanism
The Toxicologist. Society of Toxicology 47th Annual Meeting and ToxExpo, March 16-20, 2008, Seattle, Washington