Rapid Microwave Fixation of the Brain Reveals Seasonal Changes in the Phosphoproteome of Hibernating Thirteen-Lined Ground Squirrels

Hibernating mammals such as the thirteen-lined ground squirrel (Ictidomys tridecemlineatus) experience significant reductions in oxidative metabolism and body temperature when entering a state known as torpor. Animals entering or exiting torpor do not experience permanent loss of brain function or other injuries, and the processes that enable such neuroprotection are not well understood. To gain insight into changes in protein function that occur in the dramatically different physiological states of hibernation, we performed quantitative phosphoproteomics experiments on thirteen-lined ground squirrels that are summer-active, winter-torpid, and spring-active. An important aspect of our approach was the use of focused microwave irradiation of the brain to sacrifice the animals and rapidly inactivate phosphatases and kinases to preserve the native phosphoproteome. Overall, our results showed pronounced changes in phosphorylated proteins for the transitions into and out of torpor, including proteins involved in gene expression, DNA maintenance and repair, cellular plasticity, and human disease. In contrast, the transition between the active states showed minimal changes. This study offers valuable insight into the global changes in brain phosphorylation in hibernating mammals, the results of which may be relevant to future therapeutic strategies for brain injury.