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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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HIF-1αPP→AG inhibits NGF deprivation–induced death by acting at a point downstream to cytochrome c release. Sympathetic neurons were infected with Ad-EGFP or Ad-HIF-1αPP→AG/EGFP virus (MOI = 100) or left uninfected. The next day, the cells were refed with fresh NGF-containing medium or with medium lacking NGF and cultured for an additional 24 or 48 h. (A) The images show Ad-EGFP and Ad-HIF-1αPP→AG/EGFP-infected neurons 24 h after NGF withdrawal. Top panels show EGFP or HIF-1αPP→AG/EGFP fluorescence, whereas the bottom panels show the corresponding Hoechst-stained nuclei in the same field of view (marked by arrowheads). Bar, 15 μm. (B) Cell survival (mean ± SEM, n = 3) was quantified in neurons expressing EGFP or HIF-1αPP→AG/EGFP using the criteria described in Materials and methods. (C) MTT assays were performed on infected cultures 24 and 48 h after NGF withdrawal. Data represent mean ± SEM (n = 3) and are reported as a percentage of the activity in uninfected NGF-maintained neurons. (D) Neurons were infected with Ad-HIF-1αPP→AG/EGFP as above. After an additional 20 h in the presence or absence of NGF, the cells were analyzed for cytochrome c immunofluorescence. The images show Ad-HIF-1αPP→AG/EGFP fluorescence (top), Hoechst-stained nuclei (middle), and cytochrome c immunofluorescence (bottom) for a single field of view for each treatment. Bar, 20 μm. (E) Neurons were infected with Ad-HIF-1αPP→AG/EGFP and then treated with or without NGF as outlined in D. A set of uninfected neurons was transferred to an incubator equilibrated to 1% O2, whereas a second set of uninfected cells and the cells infected with Ad-HIF-1αPP→AG/EGFP remained at 20% O2. After 20 h the cells were analyzed for cytochrome c immunofluorescence. Data represent the percentage of cells with punctate cytochrome c and are the mean ± SEM from three experiments. (F) Caspase assays were performed using the substrate DEVD-AFC and lysates prepared from uninfected neurons or neurons infected with Ad-HIF-1αPP→AG/EGFP or Ad-EGFP. NGF deprivation was for 24 h. Caspase activity is reported as relative fluorescence units/microgram protein (mean ± SEM, n =3).
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fig7: HIF-1αPP→AG inhibits NGF deprivation–induced death by acting at a point downstream to cytochrome c release. Sympathetic neurons were infected with Ad-EGFP or Ad-HIF-1αPP→AG/EGFP virus (MOI = 100) or left uninfected. The next day, the cells were refed with fresh NGF-containing medium or with medium lacking NGF and cultured for an additional 24 or 48 h. (A) The images show Ad-EGFP and Ad-HIF-1αPP→AG/EGFP-infected neurons 24 h after NGF withdrawal. Top panels show EGFP or HIF-1αPP→AG/EGFP fluorescence, whereas the bottom panels show the corresponding Hoechst-stained nuclei in the same field of view (marked by arrowheads). Bar, 15 μm. (B) Cell survival (mean ± SEM, n = 3) was quantified in neurons expressing EGFP or HIF-1αPP→AG/EGFP using the criteria described in Materials and methods. (C) MTT assays were performed on infected cultures 24 and 48 h after NGF withdrawal. Data represent mean ± SEM (n = 3) and are reported as a percentage of the activity in uninfected NGF-maintained neurons. (D) Neurons were infected with Ad-HIF-1αPP→AG/EGFP as above. After an additional 20 h in the presence or absence of NGF, the cells were analyzed for cytochrome c immunofluorescence. The images show Ad-HIF-1αPP→AG/EGFP fluorescence (top), Hoechst-stained nuclei (middle), and cytochrome c immunofluorescence (bottom) for a single field of view for each treatment. Bar, 20 μm. (E) Neurons were infected with Ad-HIF-1αPP→AG/EGFP and then treated with or without NGF as outlined in D. A set of uninfected neurons was transferred to an incubator equilibrated to 1% O2, whereas a second set of uninfected cells and the cells infected with Ad-HIF-1αPP→AG/EGFP remained at 20% O2. After 20 h the cells were analyzed for cytochrome c immunofluorescence. Data represent the percentage of cells with punctate cytochrome c and are the mean ± SEM from three experiments. (F) Caspase assays were performed using the substrate DEVD-AFC and lysates prepared from uninfected neurons or neurons infected with Ad-HIF-1αPP→AG/EGFP or Ad-EGFP. NGF deprivation was for 24 h. Caspase activity is reported as relative fluorescence units/microgram protein (mean ± SEM, n =3).

Mentions: To determine whether HIF-1αPP→AG can influence trophic factor deprivation–induced cell death, neurons were infected with Ad-HIF-1αPP→AG/EGFP or Ad-EGFP and the next day deprived of NGF. By 24 h of NGF deprivation, about half of the Ad-EGFP infected and uninfected neurons showed signs of chromatin condensation and nuclear fragmentation (Fig. 7, A and B). In contrast, the nuclei of nearly all HIF-1αPP→AG/EGFP expressing neurons appeared healthy, closely resembling those of NGF-maintained neurons. By 48 h, over 80% of neurons expressing HIF-1αPP→AG/EGFP remained healthy compared with just 25% of the control cells. Survival was also assessed using a MTT metabolic activity assay. NGF-deprived neurons infected with Ad-HIF-1αPP→AG/EGFP retained significantly more MTT reducing activity than control neurons infected with Ad-EGFP (Fig. 7 C). Thus, expression of a stabilized, prolyl hydroxylase-resistant form of HIF-1α protects neurons, at least transiently, from apoptosis caused by 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)

HIF-1αPP→AG inhibits NGF deprivation–induced death by acting at a point downstream to cytochrome c release. Sympathetic neurons were infected with Ad-EGFP or Ad-HIF-1αPP→AG/EGFP virus (MOI = 100) or left uninfected. The next day, the cells were refed with fresh NGF-containing medium or with medium lacking NGF and cultured for an additional 24 or 48 h. (A) The images show Ad-EGFP and Ad-HIF-1αPP→AG/EGFP-infected neurons 24 h after NGF withdrawal. Top panels show EGFP or HIF-1αPP→AG/EGFP fluorescence, whereas the bottom panels show the corresponding Hoechst-stained nuclei in the same field of view (marked by arrowheads). Bar, 15 μm. (B) Cell survival (mean ± SEM, n = 3) was quantified in neurons expressing EGFP or HIF-1αPP→AG/EGFP using the criteria described in Materials and methods. (C) MTT assays were performed on infected cultures 24 and 48 h after NGF withdrawal. Data represent mean ± SEM (n = 3) and are reported as a percentage of the activity in uninfected NGF-maintained neurons. (D) Neurons were infected with Ad-HIF-1αPP→AG/EGFP as above. After an additional 20 h in the presence or absence of NGF, the cells were analyzed for cytochrome c immunofluorescence. The images show Ad-HIF-1αPP→AG/EGFP fluorescence (top), Hoechst-stained nuclei (middle), and cytochrome c immunofluorescence (bottom) for a single field of view for each treatment. Bar, 20 μm. (E) Neurons were infected with Ad-HIF-1αPP→AG/EGFP and then treated with or without NGF as outlined in D. A set of uninfected neurons was transferred to an incubator equilibrated to 1% O2, whereas a second set of uninfected cells and the cells infected with Ad-HIF-1αPP→AG/EGFP remained at 20% O2. After 20 h the cells were analyzed for cytochrome c immunofluorescence. Data represent the percentage of cells with punctate cytochrome c and are the mean ± SEM from three experiments. (F) Caspase assays were performed using the substrate DEVD-AFC and lysates prepared from uninfected neurons or neurons infected with Ad-HIF-1αPP→AG/EGFP or Ad-EGFP. NGF deprivation was for 24 h. Caspase activity is reported as relative fluorescence units/microgram protein (mean ± SEM, n =3).
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fig7: HIF-1αPP→AG inhibits NGF deprivation–induced death by acting at a point downstream to cytochrome c release. Sympathetic neurons were infected with Ad-EGFP or Ad-HIF-1αPP→AG/EGFP virus (MOI = 100) or left uninfected. The next day, the cells were refed with fresh NGF-containing medium or with medium lacking NGF and cultured for an additional 24 or 48 h. (A) The images show Ad-EGFP and Ad-HIF-1αPP→AG/EGFP-infected neurons 24 h after NGF withdrawal. Top panels show EGFP or HIF-1αPP→AG/EGFP fluorescence, whereas the bottom panels show the corresponding Hoechst-stained nuclei in the same field of view (marked by arrowheads). Bar, 15 μm. (B) Cell survival (mean ± SEM, n = 3) was quantified in neurons expressing EGFP or HIF-1αPP→AG/EGFP using the criteria described in Materials and methods. (C) MTT assays were performed on infected cultures 24 and 48 h after NGF withdrawal. Data represent mean ± SEM (n = 3) and are reported as a percentage of the activity in uninfected NGF-maintained neurons. (D) Neurons were infected with Ad-HIF-1αPP→AG/EGFP as above. After an additional 20 h in the presence or absence of NGF, the cells were analyzed for cytochrome c immunofluorescence. The images show Ad-HIF-1αPP→AG/EGFP fluorescence (top), Hoechst-stained nuclei (middle), and cytochrome c immunofluorescence (bottom) for a single field of view for each treatment. Bar, 20 μm. (E) Neurons were infected with Ad-HIF-1αPP→AG/EGFP and then treated with or without NGF as outlined in D. A set of uninfected neurons was transferred to an incubator equilibrated to 1% O2, whereas a second set of uninfected cells and the cells infected with Ad-HIF-1αPP→AG/EGFP remained at 20% O2. After 20 h the cells were analyzed for cytochrome c immunofluorescence. Data represent the percentage of cells with punctate cytochrome c and are the mean ± SEM from three experiments. (F) Caspase assays were performed using the substrate DEVD-AFC and lysates prepared from uninfected neurons or neurons infected with Ad-HIF-1αPP→AG/EGFP or Ad-EGFP. NGF deprivation was for 24 h. Caspase activity is reported as relative fluorescence units/microgram protein (mean ± SEM, n =3).
Mentions: To determine whether HIF-1αPP→AG can influence trophic factor deprivation–induced cell death, neurons were infected with Ad-HIF-1αPP→AG/EGFP or Ad-EGFP and the next day deprived of NGF. By 24 h of NGF deprivation, about half of the Ad-EGFP infected and uninfected neurons showed signs of chromatin condensation and nuclear fragmentation (Fig. 7, A and B). In contrast, the nuclei of nearly all HIF-1αPP→AG/EGFP expressing neurons appeared healthy, closely resembling those of NGF-maintained neurons. By 48 h, over 80% of neurons expressing HIF-1αPP→AG/EGFP remained healthy compared with just 25% of the control cells. Survival was also assessed using a MTT metabolic activity assay. NGF-deprived neurons infected with Ad-HIF-1αPP→AG/EGFP retained significantly more MTT reducing activity than control neurons infected with Ad-EGFP (Fig. 7 C). Thus, expression of a stabilized, prolyl hydroxylase-resistant form of HIF-1α protects neurons, at least transiently, from apoptosis caused by 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