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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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Low O2 delays cell death caused by NGF deprivation. Sympathetic neurons were either deprived of NGF or refed with fresh NGF-containing media and immediately transferred to 20%, 2%, or 1% O2 incubators. At the end of the treatment, the cells were immediately placed in fixative and stained with Hoechst 33,342. (A) Phase-contrast and epifluorescence images show a single field of view for each condition. Cultures were exposed to either 20% or 1% O2 and deprived of NGF for 24 h. Note the presence of several apoptotic nuclei in cultures exposed to 20% O2 (arrowheads) but not in those at 1% O2. Bar, 15 μm. (B) Cells were scored for viability as described in Materials and methods. Results represent the percentage of cells having uniformly stained chromatin, phase-bright cell bodies, and clearly discernible nuclear membranes (mean ± SEM, n = 3).
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fig1: Low O2 delays cell death caused by NGF deprivation. Sympathetic neurons were either deprived of NGF or refed with fresh NGF-containing media and immediately transferred to 20%, 2%, or 1% O2 incubators. At the end of the treatment, the cells were immediately placed in fixative and stained with Hoechst 33,342. (A) Phase-contrast and epifluorescence images show a single field of view for each condition. Cultures were exposed to either 20% or 1% O2 and deprived of NGF for 24 h. Note the presence of several apoptotic nuclei in cultures exposed to 20% O2 (arrowheads) but not in those at 1% O2. Bar, 15 μm. (B) Cells were scored for viability as described in Materials and methods. Results represent the percentage of cells having uniformly stained chromatin, phase-bright cell bodies, and clearly discernible nuclear membranes (mean ± SEM, n = 3).

Mentions: To assess whether reducing O2 tension affects the survival of sympathetic neurons, dissociated neurons that had been maintained in vitro for 5 d under standard culture conditions (i.e., 5% CO2 and 95% air, equal to 20% O2) were refed with fresh NGF-containing media or deprived of NGF and immediately placed in incubators equilibrated to 20%, 2%, or 1% O2. Survival was assessed after staining cells with the DNA-binding dye Hoechst 33,342 and examining neuronal nuclei for evidence of chromatin condensation. Exposing NGF-maintained neurons to 1–2% O2 for up to 4 d had no effect on their survival (unpublished data). On the other hand, low O2 exposure markedly increased the survival of NGF-deprived neurons, with the greatest effect seen at 1% O2 (Fig. 1, A and B). Under these conditions, 75% of neurons exhibited normal nuclear morphology as late as 48 h after NGF deprivation. Although slightly smaller, the cell soma also resembled those of NGF-maintained neurons, whereas the neuronal processes appeared less well protected. Based on these initial observations, we speculated that reducing O2 might interfere with certain aspects of the cell death pathway known to be activated during trophic factor deprivation.


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)

Low O2 delays cell death caused by NGF deprivation. Sympathetic neurons were either deprived of NGF or refed with fresh NGF-containing media and immediately transferred to 20%, 2%, or 1% O2 incubators. At the end of the treatment, the cells were immediately placed in fixative and stained with Hoechst 33,342. (A) Phase-contrast and epifluorescence images show a single field of view for each condition. Cultures were exposed to either 20% or 1% O2 and deprived of NGF for 24 h. Note the presence of several apoptotic nuclei in cultures exposed to 20% O2 (arrowheads) but not in those at 1% O2. Bar, 15 μm. (B) Cells were scored for viability as described in Materials and methods. Results represent the percentage of cells having uniformly stained chromatin, phase-bright cell bodies, and clearly discernible nuclear membranes (mean ± SEM, n = 3).
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Related In: Results  -  Collection

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fig1: Low O2 delays cell death caused by NGF deprivation. Sympathetic neurons were either deprived of NGF or refed with fresh NGF-containing media and immediately transferred to 20%, 2%, or 1% O2 incubators. At the end of the treatment, the cells were immediately placed in fixative and stained with Hoechst 33,342. (A) Phase-contrast and epifluorescence images show a single field of view for each condition. Cultures were exposed to either 20% or 1% O2 and deprived of NGF for 24 h. Note the presence of several apoptotic nuclei in cultures exposed to 20% O2 (arrowheads) but not in those at 1% O2. Bar, 15 μm. (B) Cells were scored for viability as described in Materials and methods. Results represent the percentage of cells having uniformly stained chromatin, phase-bright cell bodies, and clearly discernible nuclear membranes (mean ± SEM, n = 3).
Mentions: To assess whether reducing O2 tension affects the survival of sympathetic neurons, dissociated neurons that had been maintained in vitro for 5 d under standard culture conditions (i.e., 5% CO2 and 95% air, equal to 20% O2) were refed with fresh NGF-containing media or deprived of NGF and immediately placed in incubators equilibrated to 20%, 2%, or 1% O2. Survival was assessed after staining cells with the DNA-binding dye Hoechst 33,342 and examining neuronal nuclei for evidence of chromatin condensation. Exposing NGF-maintained neurons to 1–2% O2 for up to 4 d had no effect on their survival (unpublished data). On the other hand, low O2 exposure markedly increased the survival of NGF-deprived neurons, with the greatest effect seen at 1% O2 (Fig. 1, A and B). Under these conditions, 75% of neurons exhibited normal nuclear morphology as late as 48 h after NGF deprivation. Although slightly smaller, the cell soma also resembled those of NGF-maintained neurons, whereas the neuronal processes appeared less well protected. Based on these initial observations, we speculated that reducing O2 might interfere with certain aspects of the cell death pathway known to be activated during trophic factor deprivation.

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