Suppressing HIF-2α expression induces death in NGF-maintained neurons. A, sympathetic neurons were microinjected with a plasmid expressing shHIF2α or shLuc along with a plasmid expressing GFP-histone. After 12 h, the baseline number of healthy injected cells was determined, after which the medium was replaced with fresh NGF-containing medium. After an additional 48 h, the percentage of injected neurons that remained healthy was determined. B, neurons were micro-injected as outlined above, and 12 h later, the medium was replaced with fresh NGF-containing medium, either with or without DMOG (1 mM) or DHB (800 μM). Phase-contrast and corresponding fluorescence images of injected cells were captured 60 h after microinjection. Results with the shHIF2α(3) vector are shown. Magnification, 400×; scale bar, 20 μM. C, neurons were microinjected with shLuc or shHIF2α(3) as described above, and survival was assessed 60 h later. Results represent means ± S.E.M. from a total of six to eight dishes for each condition with 100 to 150 neurons injected per dish. Asterisks indicate a significant difference from control cells injected with shLuc (p < 0.01, ANOVA followed by Dunnett's post hoc test). Differences between the untreated shHIF2α-injected neurons and those treated with DMOG or DHB were not significant (p > 0.05, ANOVA followed by Bonferroni post hoc tests).

DMOG and DHB stimulate glucose uptake in NGF-deprived neurons. A, sympathetic neurons were maintained in the presence of NGF or deprived of NGF for 6, 12, or 24 h. NGF deprivation was carried out in the presence (▴) or absence (▪) of DMOG (1 mM). For the final 10 min of each treatment period, the cells were incubated in serum-free medium containing [³H]2-deoxy-d-glucose (2DOG) at 37°C. After several rinses, cells were lysed, and the amount of [³H]2DOG present in each lysate was determined by scintillation counting. Data were corrected for the 2DOG uptake measured in parallel cultures devoid of neurons and then normalized to the values from cultures maintained in the presence of NGF at each time point. B, sympathetic neurons were maintained in the presence of NGF or deprived of NGF for 24 h in the presence or absence of DHB (800 μM). At the end of the treatment period, cultures were incubated in serum-free medium containing [³H]2DOG for 10 min at 37°C, rinsed several times, and then lysed. 2DOG uptake was determined as described above. Results in A and B represent means ± S.E.M. from three independent experiments. Asterisks indicate a significant difference in the mean (p < 0.05) from corresponding NGF-deprived controls.

DMOG and DHB promote death in the presence of NGF in neurons starved of glucose. Sympathetic neurons were maintained in standard culture medium containing 5 mM glucose and 50 ng/ml NGF for their first 5 days in vitro. On day 5, the culture medium was replaced with fresh medium containing glucose and NGF, with or without DMOG (1 mM) or DHB (800 μM). After 48 h, one well from each treatment group was stained with Hoechst 33342, and images were captured under phase-contrast and fluorescence microscopy. A second well was stained with trypan blue (TB) and imaged under bright-field microscopy. B, sympathetic neurons were treated as described above except that on day 5, glucose-depleted culture medium containing NGF with or without DMOG or DHB was added to the cells. Magnification, 100× for all images. Scale bar, 60 μM. The images shown are representative of results obtained in three to four independent experiments. C, neurons were treated as described above, and cell survival was quantified 48 h later by staining the cultures with trypan blue and determining the fraction of cells that excluded the dye. Results represent the means ± S.E.M. from three independent experiments; asterisks indicate significant differences compared with the corresponding NGF-maintained control (p < 0.01, ANOVA followed by Dunnett's post hoc test).

DMOG and DHB rescue neurons from cell death caused by NGF withdrawal. Sympathetic neurons from neonatal mouse SCG were maintained in culture in the presence of NGF for 5 days before initiating NGF deprivation. Additional control cultures were maintained continually in the presence of NGF. Treatments with the indicated agents were initiated at 0, 6, 12, 18, and 24 h after the withdrawal of NGF. A, NGF (50 ng/ml); B, cycloheximide (CHX, 1 μg/ml); C, DMOG (1 mM); D, DHB (800 μM). Forty-eight hours after initiating NGF deprivation, cells were fixed and stained with Hoechst 33342 to permit visualization of their nuclear morphology. The percentage of survival represents the fraction of neurons exhibiting a rounded and diffusely stained nucleus, intact nuclear membrane, one or more prominent nucleoli, and a round and refractile cell body when viewed under epifluorescence and phase-contrast microscopy. Cells with condensed, fragmented, or otherwise irregularly shaped chromatin and nuclei were scored as unhealthy. Graphs depict the means ± S.E.M. of the data from three independent experiments. Means significantly different from the NGF-deprived control cultures are indicated with asterisks (p < 0.05, ANOVA followed by Dunnett's post hoc test).

DMOG and DHB prevent the accumulation of ROS after NGF withdrawal. A, sympathetic neurons were maintained in the presence of NGF or deprived of NGF for 24 h. Where indicated, DMOG or DHB was added to the media at the onset of NGF deprivation. At the end of the 24-h treatment period, cells were loaded with the redox-sensitive dye CM-H₂DCFDA for 20 min, and relative-fold changes in ROS compared with neurons maintained continuously in the presence of NGF were determined as described under Materials and Methods (n = 110-245 neurons). B, neurons were maintained in the presence of NGF or deprived of NGF for 24 h. At the end of the 24-h period, DMOG (1 mM) or DHB (800 μM) was added where indicated for a total of 20 min, coinciding with the CM-H₂DCFDA loading period. Relative changes in ROS were determined in 39 to 82 neurons. C, NGF-deprived neurons were treated with either 1 mM DMOG (▪) or 800 μM DHB (▴) at 6, 12, and 18 h after removal of NGF from the culture medium. The data for the addition of DMOG and DHB at 0 and 24 h after NGF withdrawal are from the graphs shown in A and B, respectively. The average change in intensity measured after 24 h of NGF deprivation in the absence of either drug (•, -NGF) is shown for comparison. All cultures were loaded with CM-H₂DCFDA for 20 min at the end of the 24 h of NGF withdrawal; ROS were measured in 70 to 319 neurons. For each graph in A to C, -fold change in CM-H₂DCFDA intensity represents the change from the intensity of the dye measured in sibling cultures of neurons maintained continuously in the presence of NGF (n = 39-71 neurons). Error bars indicate S.E.M. Asterisks indicate significant differences from the NGF-deprived control (A) or the NGF-maintained control (B) (p < 0.01, ANOVA followed by Dunnett's post hoc test).

Effects of disrupting HIF-1α expression on survival promoted by DMOG and DHB. A, on the fourth day in vitro, sympathetic neurons from HIF-1α(f+/f+)::ESRCre mice were treated with or without 4-HT (1 μM) for 24 h. RNA was then isolated from the cultures, reverse-transcribed to cDNA, and analyzed by PCR with primers that span exon 2 in HIF-1α: lane 1, no cDNA control reaction; lane 2, without 4-HT; lane 3, with 4-HT; M, molecular weight markers. In the absence of 4-HT, the majority of the HIF-1α amplification product corresponded to a 481-base pair cDNA that includes exon 2. After 4-HT treatment, the only detectable HIF-1α amplification product was the 290-base pair cDNA lacking exon 2; this transcript does not result in HIF-1α protein expression (Ryan et al., 2000; Vangeison et al., 2008). Note that a small amount of the 290-base pair amplification product was sometimes seen in the absence of 4-HT, presumably due to leaky Cre recombinase activity in these cells. B, neurons from HIF-1α(f+/f+)::ESRCre mice were treated with or without 4-HT for 24 h and then maintained in the presence of NGF or deprived of NGF, either with or without DMOG (1 mM) or DHB (800 μM). After 48 h of NGF deprivation, cells were fixed and stained with Hoechst dyes. Survival was assessed as described in Fig. 1. Results represent the means ± S.E.M. from three independent experiments. Asterisks indicate significant differences compared with the corresponding NGF-deprived controls (p < 0.05, ANOVA followed by Dunnett's post hoc test).

DMOG and DHB fail to inhibit cell death in glucose-starved neurons. A, sympathetic neurons were maintained in standard culture medium containing 5 mM glucose and 50 ng/ml NGF for their first 5 days in vitro. On day 5, the culture medium was replaced with fresh NGF-containing medium or with medium lacking NGF and supplemented with neutralizing anti-NGF antiserum. Where indicated, DMOG (1 mM), DHB (800 μM), and cpt-cAMP (400 μM) were added at the time of NGF withdrawal. After 48 h, one well from each treatment group was stained with Hoechst 33342, and images were captured under phase-contrast and fluorescence microscopy. A second well was stained with trypan blue (TB) and imaged under bright-field microscopy. B, sympathetic neurons were treated as described above except that on day 5, glucose-depleted culture medium containing NGF, or lacking NGF and supplemented with neutralizing anti-NGF antiserum, was added to each well. DMOG, DHB, and cpt-cAMP were also included at the time of NGF withdrawal where indicated. All images were captured at a magnification of 100×. Scale bar, 60 μM. The images shown are representative of results obtained in three to four independent experiments. C, neurons were treated as described above, and cell survival was quantified 48 h later by staining the cultures with trypan blue and determining the fraction of cells that excluded the dye. Results represent the means ± S.E.M. from three independent experiments; asterisks indicate significant differences compared with the NGF-maintained controls (p < 0.01, ANOVA followed by Dunnett's post hoc test).

Suppressing HIF-2α expression blocks DMOG-mediated protection and partially reduces DHB-mediated protection from NGF deprivation-induced death. A, COS-7 cells were transfected with expression plasmids for either mouse HIF-2α (top, lanes 2-6) or HIF-1α (bottom, lanes 2-6). Some cells were cotransfected with an shRNA vector targeting luciferase (shLuc, lane 3) or with one of three different shRNAs targeting HIF-2α: shHIF2α(1), lane 4; shHIF2α(2), lane 5; and shHIF2α(3), lane 6. The next day, cell lysates were prepared and analyzed by immunoblotting with antibodies against HIF-2α or HIF-1α. Lane 1 represents untransfected control lysates. Equivalent amounts of each lysate were also immunoblotted for α-tubulin as a control for protein loading. B, sympathetic neurons were microinjected with an expression plasmid for mouse HIF-2α together with plasmids expressing shHIF2α(1), shHIF2α(3), or shLuc. Each injection solution also contained an additional plasmid expressing a GFP-histone fusion protein to aid in the visualization of injected cells and in the analysis of nuclear morphology. After 24 h, cells were fixed and processed for immunofluorescence with an antibody against HIF-2α. Representative phase-contrast and epifluorescence images show readily detectable HIF-2α immunofluorescence in cells coinjected with shLuc but not in cells coinjected with shHIF2α(1) or shHIF2α(3). Scale bar, 20 μM. C and D, sympathetic neurons were microinjected with shLuc, shHIF2α(1), or shHIF2α(3) vector along with a plasmid expressing GFP-histone. The baseline number of healthy injected cells was determined 12 h later. At that time, NGF deprivation was initiated in the presence or absence of 1 mM DMOG (C) or 800 μM DHB (D). The number of injected cells that remained healthy after 24 h of NGF deprivation was expressed as a percentage of the total number of cells injected. Results are expressed as means ± S.E.M. from 4 to 11 dishes that were injected for each condition with 100 to 150 neurons injected per dish. The survival of neurons injected with either of the shHIF2α vectors was significantly less than that of neurons injected with shLuc (^*, p < 0.01). DMOG treatment resulted in an increase in the survival of shLuc-injected neurons (#, p < 0.001), but it had no significant effect on neurons injected with shHIF2α(1) or shHIF2α(3) (p > 0.05). For every shRNA injected, DHB treatment resulted in significantly greater survival compared with the corresponding untreated control (#, p < 0.001), although the level of protection by DHB in shHIF2α(1)- and shHIF2α(3)-expressing neurons was less than that seen in control shLuc-injected neurons (p < 0.01). Data were analyzed using ANOVA and Bonferroni post hoc tests.