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To identify genetic determinants of hypoxic cell death, we screened for hypoxia-- resistant (Hyp) mutants in Caenorhabditis elegans and found that specific reduction-of-function (rf) mutants of daf-2, an insulin/insulinlike growth factor (IGF) receptor (INR) homolog gene, were profoundly Hyp. The hypoxia resistance was acutely inducible just before hypoxic exposure and was mediated through an AKT-1/PDK-1/forkhead transcription factor pathway overlapping with but distinct from signaling pathways regulating life-span and stress resistance. Selective neuronal and muscle expression of daf-2(+) restored hypoxic death, and daf-2(rf) prevented hypoxia-induced muscle and neuronal cell death, which demonstrates a potential for INR modulation in prophylaxis against hypoxic injury of neurons and myocytes.
Although genetically tractable model organisms have made longstanding contributions to our understanding of programmed cell death (1) and recently to identification of molecular mechanisms of hypoxic adaptation and sensing (2, 3), direct genetic screens for hypoxia-resistant mutants have been relatively unexplored. To identify genes that regulate hypoxic cell death, we screened new and existing mutant strains for animals that survived exposure to either hypoxia or sodium azide (4), an electron-- transport chain inhibitor used as a chemical surrogate for hypoxia. High-level resistance to hypoxia or azide was an uncommon phenotype. We identified only two new mutants and a few existing ones that had significantly improved survival. We found the strongest Hyp strains among existing mutants with reduced activity of the insulin/IGF receptor (INR) signaling pathway. daf-2(e1370), which carries a rf mutation in the homolog of the human insulin/IGF receptor (5), was markedly azide resistant compared with wild-type strain N2 (13.2 +/- 1.8% dead versus 80.8 +/- 5.90%; P < 0.0001). Subsequent hypoxic incubation demonstrated that daf-2(e13 70) was indeed Hyp (Fig. 1, Table 1). Genetic mapping confirmed the e1370 mutation was responsible for the Hyp phenotype (4).
daf-2(e]370) not only survived but fully recovered normal locomotion behavior after as long as 20 hours of hypoxic incubation (Fig. 1A, movies S1 and S2). N2 displayed significant locomotion defects after recovery from a 6.5-hour incubation. Hypoxic sensitivity was not stage or age specific with the exception of N2 dauers (a long-lived alternative larval stage), which were Hyp (Fig. IC). The Hyp phenotype of daf-2(e1370) was markedly sensitive to temperature; e1370 animals were less Hyp when raised at 150C than at 20...