Hypoxic Preconditioning Requires the Apoptosis Protein CED-4 in C. elegans

Abstract

Hypoxic preconditioning (HP) is a rapid and reversible proadaptive response to mild hypoxic exposure with such a response protecting cells from subsequent hypoxic or ischemic insult. HP mechanisms are of great interest because of their therapeutic potential and insight into metabolic adaptation and cell death. HP has been widely demonstrated in the vertebrate subphylum but not in invertebrates. Here, we report that the nematode Caenorhabditis elegans has a potent HP mechanism that protects the organism as well as its neurons and myocytes from hypoxic injury. The time course of C. elegans HP was consistent with vertebrate-delayed HP, appearing 16 hr after preconditioning and lasting at least 36 hr. The apoptosis pathway has been proposed as either a trigger or target of HP. Testing of mutations in the canonical C. elegans apoptosis pathway showed that in general, genes in this pathway are not required for HP. However, loss-of-function mutations in ced-4, which encodes an Apaf-1 homolog, completely blocked HP. RNAi silencing of ced-4 in adult animals immediately preceding preconditioning blocked HP, indicating that CED-4 is required in adults during or after preconditioning. CED-4/Apaf-1 is essential for HP in C. elegans and acts through a mechanism independent of the classical apoptosis pathway.

Systematic Identification of Gene Activities Promoting Hypoxic Death

“…We have previously shown that high-level resistance to hypoxic death of the whole organism is possible in C. elegans by mutation or knockdown of single genes 1 (Scott et al 2002;Dasgupta et al 2007). Organismal death is preceded by permanent behavioral deficits, neuronal, and myocyte loss, and necrotic-like cell death (Scott et al 2002).…”

“…Organismal death is preceded by permanent behavioral deficits, neuronal, and myocyte loss, and necrotic-like cell death (Scott et al 2002). Mutations in the canonical programmed cell death pathway result in moderate hypoxia resistance of the adult animal, indicating that apoptotic cell death contributes to the demise of the animal after hypoxia (Dasgupta et al 2007). We made use of the reliable C. elegans death phenotype following hypoxic incubation and an available whole-genome RNAi library to perform a systematic screen for determinants of hypoxic sensitivity in an intact organism.…”

An Anoxia-starvation Model for Ischemia/Reperfusion in <em>C. elegans</em>

“…Furthermore, temperature increase is an additional stress, so it is important to monitor closely. Strictly speaking, starvation does not contribute significantly to the degree of mortality observed 7 , per se, but it appears to reduce variability among experimental replicates. Given that there can be significant variability from day-to-day, it is extremely important to compare samples run directly in parallel, and to repeat experiments over multiple days.…”

“…Although this model is technically an anoxia-starvation (AS) condition, cell death occurs through mechanisms that are conserved in mammals, including damage induced by oxidants during reperfusion 5 . Furthermore, similar to mammalian IR, damage induced by AS in C. elegans can be prevented by ischemic preconditioning 6,7 or anesthetic preconditioning 8,9 .…”

Non‐apoptotic cell death in Caenorhabditis elegans

“…This response is adaptive and reversible. Hypoxic preconditioning also confers resistance to hypoxia in neurons and myocytes of C. elegans (Dasgupta et al, 2007). Hypoxic preconditioning is dependent on CED-4, while other molecules of the core apoptotic machinery (EGL-1, CED-3, and CED-9) are not required.…”