The Caspase Cascade during Hibernation in the Golden-Mantled Ground Squirrel, Spermophilus lateralis

In several human pathologies like heart attack, stroke, neurodegenerative diseases, and autoimmune disorders, widespread cell death, or apoptosis, is a major cause of organ dysfunction and death. Hibernating golden-mantled ground squirrels, Spermophilus lateralis, experience numerous conditions during the winter that are known to be pro-apoptotic in other mammal systems (e.g. extreme hypothermia, ischemia and reperfusion, acidosis, increased reactive oxygen species, bone and muscle disuse). However, studies suggest that hibernators may invoke a protective phenotype to limit widespread cell damage and loss during the hibernation season. Could regulating apoptosis provide protection against the harmful conditions experienced during the hibernation season? Could the lessons learned from studying the mechanisms of hibernation provide insights into new therapies for human pathologies? To address potential apoptotic regulation, I systematically examined a class of crucial apoptotic regulators, the caspase cascade (caspases 1-12), for evidence of apoptotic signaling and regulation during hibernation. Caspases comprise a family of cysteine-aspartate proteases that, upon proteolytic processing and activation, participate in a complex signaling cascade involved in apoptosis and inflammation. Using ground squirrel liver, I determined the availability and activation status of caspases with western blots, performed caspase-specific enzymatic activity assays, and analyzed multiple caspase-mediated cellular events for indications of downstream caspase signaling during hibernation. Surprisingly, I found the canonical apoptotic executioner caspases 3 and 6, as well as inflammatory caspases 11 and 12, appeared activated during hibernation. Caspase activation typically has dramatic effects on enzymatic activity. For instance, in other systems, when caspase 3, the key executioner of apoptosis, is processed into the active 17 kDa (p17) fragment, caspase 3 enzymatic activity can increase \u3e10,000X compared to the procaspase form. Therefore, caspase 3 activation is thought to commit a cell to apoptosis. I found caspase 3 p17 increased ~2X during hibernation which may indicate significant apoptotic commitment. Did these seemingly winter-activated caspases display increased activity? Using in vitro enzymatic assays, I found no indications of dramatically increased caspase activity. To better understand the implications of seeming caspase activation during hibernation, I used a systems-level approach to analyze several events downstream of caspase activation. I looked for indications of caspase 3 activity via degradation of the inhibitor of caspase-activated DNAse (ICAD), inactivation of DNA repair enzyme poly (ADP-ribose) polymerase (PARP), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) activity. Caspase 6 activity was determined via nuclear lamin A cleavage and inflammatory caspase activity was analyzed through IL-1 and IL-18 cleavage as well as serum transaminase levels. Despite the pro-apoptotic conditions of hibernation and the seeming caspase activations, I found no evidence of increased downstream caspase activity or evidence of widespread apoptosis and inflammation during hibernation. My data demonstrate that regulation of apoptosis during hibernation does not involve the prevention of caspase activation. Instead, partial activation of the caspase cascade does not result in predictable downstream processing, thus demonstrating regulation of apoptosis during hibernation occurs at an unexpected locus. These data demonstrate the importance and utility of the systems-level approach in studying complex cellular signaling pathways like apoptosis during hibernation