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. 1997 Dec 15;17(24):9656-66.
doi: 10.1523/JNEUROSCI.17-24-09656.1997.

Analysis of the mechanism of loss of trophic factor dependence associated with neuronal maturation: a phenotype indistinguishable from Bax deletion

R M Easton  1 , T L Deckwerth, A S Parsadanian, E M Johnson Jr
Affiliations

Analysis of the mechanism of loss of trophic factor dependence associated with neuronal maturation: a phenotype indistinguishable from Bax deletion

R M Easton et al. J Neurosci. .

Abstract

During development, sympathetic neurons are critically dependent on nerve growth factor (NGF) for survival. Neurons isolated from the superior cervical ganglia (SCG) of embryonic rodents and maintained for 1 week in vitro undergo programmed cell death in response to NGF deprivation. As the cells mature in vitro and in vivo, however, these neurons develop a resistance to NGF deprivation and become much less acutely dependent on NGF for survival. Using an in vitro model of neuronal maturation, we confirmed that SCG neurons maintained in culture for 3-4 weeks did not experience a dramatic loss in viability after NGF removal, yet they did undergo the initial biochemical and genetic changes elicited by NGF deprivation of young neurons. NGF deprivation of mature neurons produced rapid decreases in glucose uptake and protein and RNA synthesis rates, increased phosphorylation of c-Jun, and an increase in c-jun mRNA. Mature neurons, however, experienced a block in the cell death program before the final stages of the pathway activated in young neurons, which includes the induction of c-fos mRNA and characteristic apoptotic nuclear changes. This maturation-induced block was indistinguishable by these criteria from the block produced by Bax deficiency. Expression of Bax in mature neurons restored the apoptotic pathway, such that after NGF removal, Bax-overexpressing mature neurons resumed the apoptotic program, including the induction of c-Fos and passage through a caspase checkpoint. Thus, a block in the apoptotic program at or near the BAX checkpoint accounts for the decreased dependence of mature neurons on neurotrophic factor to maintain survival.

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Figures

Fig. 1.
Fig. 1.
Mature sympathetic neurons are resistant to NGF deprivation. Neurons that had been maintained for 23 d in vitro (DIV) were either continued in AM50 (A) or deprived of NGF (B) for 16 d, and then fixed and stained with crystal violet, as described in Materials and Methods. In parallel, six DIV neurons were either maintained in AM50 (C) or deprived of NGF (D) for 2 d before staining with crystal violet. Viable cells were counted as described in Materials and Methods. The number of neurons remaining in the NGF-deprived cultures is expressed as a percentage of the number of viable neurons in the NGF-maintained controls (E). Data from four neuronal cultures are shown as mean ± SD. Mature neurons, 23 DIV, were either maintained in NGF (F, H, J) or deprived of NGF for 16 d (G, I, K) and then stained with Hoechst 33258 (F, G) or calcein AM (J, K). Phase-contrast microscopy is shown inH and I. Scale bars: A–D, 50 μm; F and G, 20 μm;H–K, 50 μm).
Fig. 2.
Fig. 2.
Mature sympathetic neurons undergo early degenerative changes when deprived of NGF. A, Soma diameter was measured in 23 DIV neurons that were either maintained in NGF (open bar) or deprived of NGF (filled bar) for 16 d. The diameters of 30 neurons were measured for each condition. B, Total neuronal protein was measured in young and mature neurons after 1, 2, 4, or 7 d after NGF deprivation. The total neuronal protein of deprived neurons is expressed as a percentage of NGF-maintained cultures. All data are shown as mean ± SD.
Fig. 3.
Fig. 3.
Mature sympathetic neurons undergo the early metabolic changes when deprived of NGF. Neurons maintained in vitro for 3–4 weeks were deprived of NGF for the indicated amounts of time. Protein synthesis (▴), RNA synthesis (▪), and glucose uptake (○) were determined as described in Materials and Methods. Each plot depicts the mean and range of two experiments. Within each experiment, two to five neuronal cultures were analyzed per time point.
Fig. 4.
Fig. 4.
The phosphorylated form of c-Jun increases after trophic factor deprivation. After 4 weeks in vitro, SCG neurons were deprived of NGF for a given amount of time, fixed in 4% paraformaldehyde, and stained with an antibody against the phosphorylated form of c-Jun. Phospho-c-Jun immunoreactivity (A, C, E) and Hoechst 33258 staining (B, D, F) are shown. c-Jun became phosphorylated by 6 hr after deprivation and was localized to the nucleus (C, D). An increase in phospho-c-Jun did not occur with medium change alone (A, B). Twelve hours after NGF deprivation all neurons in the culture were brightly stained (data not shown) and remained positive out to 48 hr (E, F). Similar results were observed in two separate experiments. Scale bar, 20 μm.
Fig. 5.
Fig. 5.
Mature sympathetic neurons experience a block between the first and second phases of gene induction after NGF deprivation. After one (filled bar) or four (open bar) weeks in vitro, neuronal cultures were deprived of NGF, and cDNA was prepared at specific times after deprivation. PCR analysis was conducted, as described in Materials and Methods, and the representative genes are depicted.A, cyclophilin has been demonstrated previously to decrease in young SCG neurons deprived of NGF.B, c-jun and mkp-1 are genes induced during the first phase of gene induction (by 6 hr) after NGF deprivation. Quantitation for c-jun is shown in the graph. C, c-fos and fos Bare among the late genes that increase by 10–15 hr after NGF deprivation. This same increase was seen in our young neurons (1 weekin vitro) deprived of NGF but not in the matched mature cultures (4 weeks in vitro). Quantitation for c-fos is shown in the graph. The data shown are from one preparation of neuronal cultures. Data are expressed as percentages of the NGF-maintained culture. These results have been confirmed in an independent time course.
Fig. 6.
Fig. 6.
BAX and BCLxL protein levels do not change over time in vitro. After 1–5 weeks in culture, neurons were lysed in reducing sample buffer and stored at −70°C. After completion of the time course, the amount of protein was quantified by dotMETRIC analysis, and an equal amount of protein was loaded in each lane. After SDS-PAGE and transfer, the blot was probed with a rabbit polyclonal antibody against BAX, as described in Materials and Methods. The same blot was stripped and probed again with an antibody against BCLxL. After each primary antibody, the blot was probed with a secondary antibody conjugated to alkaline phosphatase, developed with CDP-Star, and exposed to autoradiography film. The film was scanned and quantified by using ImageQuant. Data shown in the graph are expressed as a percentage of the amount of protein after 1 week in culture. Similar results were observed in four independent experiments.
Fig. 7.
Fig. 7.
Bax expression in mature sympathetic neurons restores NGF dependence. A, Primary SCG neurons that had been maintained in vitro for 26 d were co-injected with pGreenLantern-1 and either pcDNA3 vector (•, ○; n = 1271) or FLAG-Bax (▾, ▿; n = 750). Sixteen to 24 hr after injection, microinjected cells were counted on the fluorescent microscope, and this number was used as the baseline. Scored neurons were GFP-positive (fluorescent green) and phase-bright. Half of the injected cultures was maintained in NGF (•, ▾), whereas the other half was switched to medium containing a neutralizing antibody against NGF (○, ▿). At 24 and 48 hr after treatment, the GFP-positive, phase-bright neurons were counted by a naive observer. The numbers of neurons scored at 24 and 48 hr are expressed as a percentage of the baseline count. The mean and SE for six experiments are shown. In each experiment, 39–196 neurons were injected for each condition. Comparison betweenBax-injected and control-injected neurons revealed that in the absence of NGF, the two conditions are significantly different at 24 and 48 hr (p < 0.05).Bax-injected and control-injected neurons in the presence of NGF have no statistically significant difference at 24 and 48 hr. Data passed tests for normality and equivalency, so statistical significance was assessed by using a one-way ANOVA and Tukey test. To confirm the production of the transgene, neurons were microinjected with FLAG–Bax, maintained in NGF for 16–24 hr (B–D), and then fixed and stained with an anti-FLAG antibody (C). GFP (B) marks the microinjected neurons, and Hoechst 33258 staining (D) reveals healthy nuclei. Scale bar, 20 μm.
Fig. 8.
Fig. 8.
BAX restores the apoptotic program in mature sympathetic neurons deprived of NGF. Twelve hours after NGF deprivation, injected neurons were fixed and stained with an antibody against c-Fos. Neurons injected with Bax were marked by GFP fluorescence and showed nuclear c-Fos immunoreactivity. Nuclear staining with Hoechst 33258 demonstrated that nuclei that were c-Fos-positive displayed abnormal morphology. The nucleus of the injected cell was condensed and smaller than its uninjected neighbor. c-Fos immunoreactivity was not seen in pcDNA3 (vector alone)-injected neurons after NGF deprivation.
Fig. 9.
Fig. 9.
Cell death induced by NGF deprivation of BAX-expressing mature neurons is inhibited by the caspase inhibitor BAF. Twenty-six DIV SCG neurons were co-injected with GFP and either pcDNA3 vector (•) or FLAG-Bax (▪, ▴). Sixteen to 24 hr after injection, microinjected cells were counted on the fluorescent microscope, and this number was used as the baseline. Scored neurons were GFP-positive (fluorescent green) and phase-bright. After this baseline count, the neurons were switched to medium containing neutralizing antibodies to NGF with (▴) or without 30 μm BAF (•, ▪). At 24 and 48 hr after treatment, the GFP-positive, phase-bright neurons were counted by a naive observer. The numbers of neurons scored at 24 and 48 hr after treatment are expressed as a percentage of the baseline count. The mean and range of two experiments are depicted.
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