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Inhibition of NGF deprivation-induced death by low oxygen involves suppression of BIMEL and activation of HIF-1.

Xie L, Johnson RS, Freeman RS - J. Cell Biol. (2005)

Bottom Line: Forced BIM(EL) expression removed the block to cytochrome c release but did not prevent protection by low O(2).Exposing neurons to low O(2) also activated hypoxia-inducible factor (HIF) and expression of a stabilized form of HIF-1alpha (HIF-1alpha(PP-->AG)) inhibited cell death in normoxic, NGF-deprived cells.Targeted deletion of HIF-1alpha partially suppressed the protective effect of low O(2), whereas deletion of HIF-1alpha combined with forced BIM(EL) expression completely reversed the ability of low O(2) to inhibit cell death.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Physiology, University of Rochester School of Medicine, Rochester, NY 14642, USA.

ABSTRACT
Changes in O(2) tension can significantly impact cell survival, yet the mechanisms underlying these effects are not well understood. Here, we report that maintaining sympathetic neurons under low O(2) inhibits apoptosis caused by NGF deprivation. Low O(2) exposure blocked cytochrome c release after NGF withdrawal, in part by suppressing the up-regulation of BIM(EL). Forced BIM(EL) expression removed the block to cytochrome c release but did not prevent protection by low O(2). Exposing neurons to low O(2) also activated hypoxia-inducible factor (HIF) and expression of a stabilized form of HIF-1alpha (HIF-1alpha(PP-->AG)) inhibited cell death in normoxic, NGF-deprived cells. Targeted deletion of HIF-1alpha partially suppressed the protective effect of low O(2), whereas deletion of HIF-1alpha combined with forced BIM(EL) expression completely reversed the ability of low O(2) to inhibit cell death. These data suggest a new model for how O(2) tension can influence apoptotic events that underlie trophic factor deprivation-induced cell death.

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Targeted deletion of HIF-1α combined with ectopic BIMEL abolishes the neuroprotective effect of low O2. (A) Sympathetic neurons isolated from HIF-1αfl/fl mice were infected with either Ad-EGFP or Ad-Cre or left uninfected. 2 d later, RNA was extracted and analyzed by RT-PCR for expression of HIF-1α and an unrelated control gene (cyclophilin). (B) HIF-1αfl/fl neurons were microinjected with expression plasmids for Cre recombinase and BIMEL/EGFP, either alone or as an equimolar mixture. Uninjected neurons and neurons injected with an EGFP expression plasmid were included as controls. NGF deprivation was initiated the next day at which time the cells were transferred to an incubator equilibrated to 1% O2. After 48 h, the cells were stained with Hoechst dye and the fraction of injected neurons that remained healthy was determined. Results (mean ± range) were derived from two independent experiments.
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fig8: Targeted deletion of HIF-1α combined with ectopic BIMEL abolishes the neuroprotective effect of low O2. (A) Sympathetic neurons isolated from HIF-1αfl/fl mice were infected with either Ad-EGFP or Ad-Cre or left uninfected. 2 d later, RNA was extracted and analyzed by RT-PCR for expression of HIF-1α and an unrelated control gene (cyclophilin). (B) HIF-1αfl/fl neurons were microinjected with expression plasmids for Cre recombinase and BIMEL/EGFP, either alone or as an equimolar mixture. Uninjected neurons and neurons injected with an EGFP expression plasmid were included as controls. NGF deprivation was initiated the next day at which time the cells were transferred to an incubator equilibrated to 1% O2. After 48 h, the cells were stained with Hoechst dye and the fraction of injected neurons that remained healthy was determined. Results (mean ± range) were derived from two independent experiments.

Mentions: To determine whether expression of Cre recombinase in HIF-1αfl/fl sympathetic neurons results in a substantial loss of HIF-1α, we infected the neurons with an adenovirus that expresses Cre (Ad-Cre) or with the control Ad-EGFP virus. Immunofluorescence confirmed Cre expression in virtually all of the cells by 24 h after infection (unpublished data). RT-PCR analysis revealed greatly reduced HIF-1α expression specifically in Ad-Cre infected cultures (Fig. 8 A), suggesting that efficient deletion of the floxed HIF-1α gene had occurred. Loss of HIF-1α had no effect on the ability of NGF to promote survival for at least 4 d, and initial NGF deprivation experiments suggested that HIF-1α deletion did not significantly affect short-term protection afforded by low O2 as measured at 24 h after NGF withdrawal (unpublished data; see below).


Inhibition of NGF deprivation-induced death by low oxygen involves suppression of BIMEL and activation of HIF-1.

Xie L, Johnson RS, Freeman RS - J. Cell Biol. (2005)

Targeted deletion of HIF-1α combined with ectopic BIMEL abolishes the neuroprotective effect of low O2. (A) Sympathetic neurons isolated from HIF-1αfl/fl mice were infected with either Ad-EGFP or Ad-Cre or left uninfected. 2 d later, RNA was extracted and analyzed by RT-PCR for expression of HIF-1α and an unrelated control gene (cyclophilin). (B) HIF-1αfl/fl neurons were microinjected with expression plasmids for Cre recombinase and BIMEL/EGFP, either alone or as an equimolar mixture. Uninjected neurons and neurons injected with an EGFP expression plasmid were included as controls. NGF deprivation was initiated the next day at which time the cells were transferred to an incubator equilibrated to 1% O2. After 48 h, the cells were stained with Hoechst dye and the fraction of injected neurons that remained healthy was determined. Results (mean ± range) were derived from two independent experiments.
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Related In: Results  -  Collection

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fig8: Targeted deletion of HIF-1α combined with ectopic BIMEL abolishes the neuroprotective effect of low O2. (A) Sympathetic neurons isolated from HIF-1αfl/fl mice were infected with either Ad-EGFP or Ad-Cre or left uninfected. 2 d later, RNA was extracted and analyzed by RT-PCR for expression of HIF-1α and an unrelated control gene (cyclophilin). (B) HIF-1αfl/fl neurons were microinjected with expression plasmids for Cre recombinase and BIMEL/EGFP, either alone or as an equimolar mixture. Uninjected neurons and neurons injected with an EGFP expression plasmid were included as controls. NGF deprivation was initiated the next day at which time the cells were transferred to an incubator equilibrated to 1% O2. After 48 h, the cells were stained with Hoechst dye and the fraction of injected neurons that remained healthy was determined. Results (mean ± range) were derived from two independent experiments.
Mentions: To determine whether expression of Cre recombinase in HIF-1αfl/fl sympathetic neurons results in a substantial loss of HIF-1α, we infected the neurons with an adenovirus that expresses Cre (Ad-Cre) or with the control Ad-EGFP virus. Immunofluorescence confirmed Cre expression in virtually all of the cells by 24 h after infection (unpublished data). RT-PCR analysis revealed greatly reduced HIF-1α expression specifically in Ad-Cre infected cultures (Fig. 8 A), suggesting that efficient deletion of the floxed HIF-1α gene had occurred. Loss of HIF-1α had no effect on the ability of NGF to promote survival for at least 4 d, and initial NGF deprivation experiments suggested that HIF-1α deletion did not significantly affect short-term protection afforded by low O2 as measured at 24 h after NGF withdrawal (unpublished data; see below).

Bottom Line: Forced BIM(EL) expression removed the block to cytochrome c release but did not prevent protection by low O(2).Exposing neurons to low O(2) also activated hypoxia-inducible factor (HIF) and expression of a stabilized form of HIF-1alpha (HIF-1alpha(PP-->AG)) inhibited cell death in normoxic, NGF-deprived cells.Targeted deletion of HIF-1alpha partially suppressed the protective effect of low O(2), whereas deletion of HIF-1alpha combined with forced BIM(EL) expression completely reversed the ability of low O(2) to inhibit cell death.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Physiology, University of Rochester School of Medicine, Rochester, NY 14642, USA.

ABSTRACT
Changes in O(2) tension can significantly impact cell survival, yet the mechanisms underlying these effects are not well understood. Here, we report that maintaining sympathetic neurons under low O(2) inhibits apoptosis caused by NGF deprivation. Low O(2) exposure blocked cytochrome c release after NGF withdrawal, in part by suppressing the up-regulation of BIM(EL). Forced BIM(EL) expression removed the block to cytochrome c release but did not prevent protection by low O(2). Exposing neurons to low O(2) also activated hypoxia-inducible factor (HIF) and expression of a stabilized form of HIF-1alpha (HIF-1alpha(PP-->AG)) inhibited cell death in normoxic, NGF-deprived cells. Targeted deletion of HIF-1alpha partially suppressed the protective effect of low O(2), whereas deletion of HIF-1alpha combined with forced BIM(EL) expression completely reversed the ability of low O(2) to inhibit cell death. These data suggest a new model for how O(2) tension can influence apoptotic events that underlie trophic factor deprivation-induced cell death.

Show MeSH
Related in: MedlinePlus