Incomplete Caspase 3 Activation and Mitigation of Apoptosis in Hibernating Ground Squirrels, Spermophilus lateralis.

AbstractHibernating golden-mantled ground squirrels, Spermophilus [Callospermophilus] lateralis, tolerate proapoptotic conditions, such as low body temperature, anorexia, acidosis, and ischemia/reperfusion. Avoiding widespread apoptosis is critical for hibernator survival. Caspase 3, the key executioner of apoptosis, cleaves a majority of apoptotic targets. Under proapoptotic conditions, inactive procaspase 3 (32 kDa) is activated when cleaved into 17- and 12-kDa fragments (p32, p17, and p12, respectively). Caspase 3 activation results in extreme enzymatic activation. Activity increases >10,000-fold followed by apoptotic execution. Is widespread apoptosis occurring during the proapoptotic hibernation season? Western blots showed p17 increased ∼2-fold during hibernation, indicating caspase 3 activation. However, in vitro caspase 3 activity assays found no extreme increases in activity. Downstream caspase 3 targets ICAD (inhibitor of caspase-activated deoxyribonuclease) and PARP (poly (ADP-ribose) polymerase) did not experience elevated cleavage during hibernation, which is inconsistent with caspase 3 activation. TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling) assays from multiple tissues found only 0.001%-0.009% of cells were TUNEL positive during winter, indicating negligible apoptosis during hibernation. Typically, caspase 3 activation generates a strong commitment toward apoptosis. We found that despite a ∼2-fold increase in active caspase 3, hibernators experience no downstream caspase 3 activity or widespread apoptosis. A systems-level approach suggests an incomplete signaling cascade wherein some caspase 3 activation during hibernation does not necessarily lead to bona fide apoptosis.

Mitigating Apoptotic and Inflammatory Signaling via Global Caspase Inhibition in Hibernating Ground Squirrels, Spermophilus lateralis.

AbstractIn most systems, the caspase cascade is activated during cellular stress and results in inflammation and apoptosis. Hibernators experience stressors such as extremely low body temperatures, bradycardia, possible ischemia and reperfusion, and acidosis. However, widespread inflammation and apoptosis would represent an energetic expense that is incompatible with hibernation. To better understand global caspase regulation during hibernation, we employed a systems-level approach and analyzed 11 caspases in ground squirrel liver that are involved in inflammatory (caspases 1, 4, 5, 11, and 12) and apoptotic (caspases 2, 6, 7, 8, 9, and 10) pathways. Western blots revealed liberation of active forms for two inflammatory (caspases 11 and 12) and two apoptotic (caspases 6 and 9) caspases during hibernation (e.g., p15, the most active fragment of caspase 6, increased 8.26±0.70-fold in interbout-aroused animals). We used specific peptide substrates to interrogate the four seemingly activated caspases and demonstrated no expected increases in proteolytic activity. Specific targets of these four caspases were similarly not cleaved, demonstrating that initiation of caspase activation may occur without concomitant downstream effects. Similarly, we found no evidence for upstream activation for caspase 9 signaling based on permeabilization of the outer mitochondrial membrane. We contend that these caspases are suppressed after seeming activation during hibernation. Incomplete caspase signaling is effectively mitigating the induction of widespread inflammation and apoptosis during hibernation.

An Experimental Evolution Test of the Relationship between Melanism and Desiccation Survival in Insects.

We used experimental evolution to test the 'melanism-desiccation' hypothesis, which proposes that dark cuticle in several Drosophila species is an adaptation for increased desiccation tolerance. We selected for dark and light body pigmentation in replicated populations of D. melanogaster and assayed several traits related to water balance. We also scored pigmentation and desiccation tolerance in populations selected for desiccation survival. Populations in both selection regimes showed large differences in the traits directly under selection. However, after over 40 generations of pigmentation selection, dark-selected populations were not more desiccation-tolerant than light-selected and control populations, nor did we find significant changes in mass or carbohydrate amounts that could affect desiccation resistance. Body pigmentation of desiccation-selected populations did not differ from control populations after over 140 generations of selection, although selected populations lost water less rapidly. Our results do not support an important role for melanization in Drosophila water balance.

Selection for desiccation resistance in adult Drosophila melanogaster affects larval development and metabolite accumulation.

We studied larval development and acquisition of metabolic resources in Drosophila melanogaster selected for adult desiccation resistance. Desiccation-selected (D) flies had longer developmental times in comparison with control populations, resulting in significantly higher body mass. No differences were found in larval growth rates, suggesting that increased body mass results from the extended larval feeding period. Larvae from both D and control lines molted from second to third instar 81-84 h after egg laying, whereas D larvae pupated 5-6 h later than controls. This indicates that selecting adults for desiccation resistance results in longer third larval instar. Newly eclosed D flies had higher carbohydrate and water contents in comparison with control populations. No differences were found in body mass, water or metabolite contents of newly eclosed selected and control flies after larvae were prevented from further feeding from 96 h after egg laying onwards. This shows that differences in accumulated carbohydrate and water stores are a result of evolved differences in the duration of third larval instar. The contribution of third instar feeding to desiccation resistance of adult selected flies is higher than that of controls. Thus, selection for adult stress resistance has resulted in correlated changes in larval physiology.