Role of BCL-2 in the Survival and Function of Developing and Mature Sympathetic Neurons · 2016....

Neuron, Vol. 15, 649-661, September, 1995, Copyright © 1995 by Cell Press

Role of BCL-2 in the Survival and Function of Developing and Mature Sympathetic Neurons

Laura J. S. Greenlund,* Stanley J. Korsmeyer,~r and Eugene M. Johnson, Jr.* *Department of Neurology Department of Molecular Biology and Pharmacology tHoward Hughes Medical Institute Washington University School of Medicine St. Louis, Missouri 63110

Summary

Sympathetic neurons, when placed in culture during the period of naturally occurring cell death, will die by apoptosis when deprived of nerve growth factor (NGF). In this system, the mRNA levels of the BCL-2 family members decrease after NGF deprivation and during apoptosis. Sympathetic neurons from BCL-2-deficient mice died more rapidly after NGF deprivation than neurons from wild-type littermates. Sympathetic neurons of adult animals are relatively independent of NGF for survival. If sympathetic neurons are maintained in vitro for several weeks, loss of acute trophic factor dependence develops with a time course similar to that seen in the intact animal. Examination of neurons from BCL-2-deficient mice showed that BCL-2 expression is not required for the development of trophic factor independence. Therefore, BCL-2 is an important regulator of the survival of sympathetic neurons after NGF deprivation during the period of naturally occurring programmed neuronal death, but BCL-2 is not involved in the development of trophic factor independence in mature sympathetic neurons.

Introduction

The development of the nervous system begins with the birth of about twice as many neurons as are finally present in the adult. In the sympathetic nervous system, the immature neuronal precursors are not dependent on nerve growth factor (NGF) for survival (Coughlin and Collins, 1985; Birren et al., 1993; DiCicco-Bloom et al., 1993). Sym- pathetic neurons then become acutely dependent on NGF for survival, and neurons that do not receive sufficient neurotrophic factor undergo programmed neuronal death. In the rat, the period of NGF dependence begins about embryonic day 19.5 (Birren et al., 1993). Following the period of programmed neuronal death, trophic factor dependence decreases progressively, so that adult sympathetic neurons are much tess acutely dependent on NGF for survival, but still respond to NGF with increased growth and will die only upon prolonged periods of trophic factor deprivation (Gorin and Johnson, 1980). This developmental process allows for selecting the appropriate number of neurons to match the target size in the developing animal, for ridding the nervous system of inappropriate connections (Oppenheim, 1991), and later, for stabilizing

the nervous system of the adult animal. Since most neurons are irreplaceable in the adult, it is imperative that they survive injury, such as peripheral nerve axotomy, so that regeneration can occur.

The death of sympathetic neurons after NGF deprivation (Martin et al., 1988; Deckwerth and Johnson, 1993; Ed- wards and Tolkovsky, 1994) is characterized by changes that are apoptotic (Kerr et al., 1972), including fragmenta- tion of DNA into oligonucleosomes, shrinkage of the cell with preservation of the organelles, and blebbing in the cytoplasm. In many cases, apoptosis appears to be an active process wherein the expression of certain genes is required for cell death, because in sympathetic neurons (Martin et al., 1988) and in other cell types (Tata, 1966; Pratt and Greene, 1976; Cohen and Duke, 1984), the process can be blocked by inhibitors of RNA and protein synthesis.

BCL-2, a mitochondrial and perinuclear membrane protein (Hockenbery et al., 1990; Monaghan et al., 1992; Ja- cobson et al., 1993), and its family members BAX (Oltvai et al., 1993) and BCL-X (long and short forms; Boise et al., 1993) modulate the sensitivity of cells to death. BCL-2 overexpression in the interleukin-3 (IL-3)-dependent B cell line, FL5.12, blocks death induced by trophic factor deprivation; however, if, in addition, BAX is overexpressed, the death repressor activity of BCL-2 is antagonized. If BAX is overexpressed independently, death induced by IL-3 deprivation is accelerated (Oltvai et al., 1993). The BCL-2- related protein, BCL-X, is produced in a long or a short form by alternative mRNA splicing. FL5.12 cells are protected from death induced by trophic factor deprivation if BCL-XL (the long form of BCL-X) is overexpressed. In contrast, the short form of BCL-X, BCL-Xs, acts much like bax and can antagonize the protective effect of BCL-2 overexpression (Boise et al., 1993).

Dimerization is important for the function of BCL-2; BCL-2 can form homodimers with itself or can form hetero- dimers with BAX (Oltvai et al., 1993). Oltvai et al. (1993) propose that BCL-2/BCL-2 homodimers protect cells from death, and that BAX titrates the levels of these homodi- mere, rendering cells more susceptible to death. In addition, BCL-2 binds to a number of other proteins including R-Ras (Fernandez-Sarabia and Bischoff, 1993); interaction with ras signal transduction pathways may be one mode of BCL-2 action. At least three other proteins have been identified that bind to BCL-2, including Nip1, Nip2, and Nip3 (Boyd et al., 1994). Nip1 shows homology with the Ca2÷/calmodulin-dependent phosphodiesterases, Nip2 shows homology with the human GTPase-activating protein RhoGAP, and Nip3 is a novel protein. The interaction of BCL-2 with Nip1, Nip2, and Nip3 may be another means to modify intracellular signaling processes (Boyd et al., 1994).

Although all of the mechanisms by which BCL-2 protects cells from death remain to be elucidated, a number of functions have been suggested. Two groups have shown

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that BCL-2 has antioxidant properties. Hockenbery et al. (1993) have reported that BCL-2 overexpression in a T cell line blocks lipid peroxidation induced by dexamethasone treatment. Kane et al. (1993) have demonstrated that BCL-2 overexpression in a hypothalamic neural cell line inhibits the generation of reactive oxygen species after glutathione depletion with diethylmaleate. BCL-2 may also alter Ca 2+ fluxes. Lam et al. (1994) examined a lymphocyte cell line treated with thapsigargin, which inhibits the endoplasmic reticulum Ca 2+ pump. Cells that overexpressed BCL-2 released much less Ca 2÷ into the cytoplasm, indicating that BCL-2 reduces Ca 2÷ efflux through the endoplasmic reticulum membrane.

In addition, genetic evidence from the nematode Caenor- habditis elegans suggests that the worm homolog of BCL- 2, ced-9 (Hengartner and Horvitz, 1994), functions to block, either directly or indirectly, the function of the puta- tive cysteine protease, ced-3. Functional ced-3, a protein that shares homology with the human cysteine protease IL-lJ~-converting enzyme (Yuan et al., 1993) and with human ich-1 (Wang et al., 1994), is required for programmed cell death in C. elegans (Yuan and Horvitz, 1990, 1992); however, increased ced-9 activity overrides ced-3 activity and blocks cell death (Hengartner et al., 1992). The human Bcl-2 gene can functionally replace C. elegans ced-9 to block cell death in the worm (Vaux et al., 1992; Hengartner and Horvitz, 1994). Thus, BCL-2 either directly or indirectly blocks the action of ced-3.

The developmental expression patterns of Bcl-2 in the CNS have been analyzed by a number of groups. Data at both the RNA and protein levels indicate that Bcl-2 is expressed at its highest levels in the prenatal brain, but postnatal and adult animals, including humans, express lower levels of Bcl-2 in the CNS (Abe-Dohmae et al., 1993; Castren et al., 1994; Ferrer et al., 1994; Merry et al., 1994). Very few studies have analyzed the expression of Bcl-2 in the PNS. Merry et al. (1994) reported that BCL-2 protein expression in the prenatal mouse dorsal root ganglion (DRG) is retained into adulthood and that postnatal superior cervical ganglion (SCG) neurons also express BCL-2 protein even into adulthood. Martinou et al. (1994b) demonstrated that SCGs and DRGs from 10 week human fe- tuses also express BCL-2 protein. Currently, data are sparse regarding Bcl-2 expression during development of the PNS or the importance of this expression in regulating neuronal survival.

Overexpression of BCL-2 in trigeminal neurons (Alisopp et al., 1993) or sympathetic ganglion neurons (Garcia et al., 1992; Greenlund et al., 1995) delays apoptosis induced by trophic factor deprivation. In contrast, ciliary ganglion neurons are not protected by BCL-2 overexpression from apoptosis induced by ciliary neurotrophic factor (CNTF) deprivation, suggesting that more than one cell death pathway exists within PNS neurons (AIIsopp et al., 1993). PC12 cells, a sympathetic neuron-like cell line, stably transfected with Bcl-2 are also more resistant to apoptosis in response to various insults (Batistatou et al., 1993; Mah et al., 1993). Endogenous expression of BCL-2 in sympathetic neurons clearly occurs, and overexpression of

BCL-2 renders the neurons more resistant to death. How- ever, very little evidence is apparent that BCL-2 is important for the normal regulation of sympathetic neuronal survival, either in the embryo, during the period of naturally occurring cell death, or in the adult, when neurons have become more resistant to trophic factor deprivation-induced death.

We used an in vitro model (Martin et al., 1988) for the developmental programmed neuronal death of rat and mouse sympathetic neurons to examine the mRNA levels of the Bcl-2 family members during apoptosis. We hypothesized that immature sympathetic neurons may initiate apoptosis after NGF deprivation either by up-regulating the expression of bax or BcI-Xs or by down-regulating the expression of Bcl-2 or Bcl-XL. We also examined sympathetic neurons from BCL-2-deficient mice to determine whether BCL-2 is a regulator of neuronal survival after trophic factor deprivation.

An in vitro model for the loss of acute trophic factor dependence that occurs with maturation in sympathetic neurons was used to examine the importance of BCL-2 in the development of this independence. We hypothesized that mature neurons could down-regulate the expression of bax and BcI-Xs or up-regulate the expression of Bcl-2 and Bcl-XL to become more resistant to apoptosis. Sympa- thetic neurons from BCL-2-deficient animals were used to examine whether BCL-2 expression is important for the development of trophic factor independence.

Lastly, we used microinjection to overexpress BCL-2 in sympathetic neurons to determine the time course of survival after NGF deprivation and to examine whether BCL-2 overexpression could mimic the effects of NGF as evidenced by blocking the early fall in protein synthesis rates that occurs after NGF deprivation of sympathetic neurons (Deckwerth and Johnson, 1993).

Results

Immunostaining Shows BCL-2 Protein Is Present in Both Neurons and Nonneuronal Cells BCL-2 is localized in the mitochondria of lymphoid cells (Hockenbery et al., 1990) and in the perinuclear membrane and endoplasmic reticulum of lymphoid and other cell types (Chen-Levy et al., 1989; Monaghan et al., 1992; Jacobson et al., 1993). Merry et al. (1994), using the monoclonal anti-BCL-2 antibody 3F11, have shown that in vivo both neurons and supporting cells in the SCG of postnatal day 20 (P20) mice express BCL-2. The pattern of staining in neurons is heterogeneous, and nuclear staining is observed. We first determined how the pattern of BCL-2 expression in the in vitro system compared to the pattern of BCL-2 expression in vivo. In this system, cultures of dissociated rat SCG cells are prepared the day before birth of the animal, just before the period of naturally occurring neuronal death in the rat SCG (Wright et al., 1983). For immunostaining, dissociated SCG cells that were not preplated to remove nonneuronal cells (see Experimental Procedures for details) were used so that Schwann cells and fibroblasts would be more numerous and could be

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Figure 1. Immunohistochemistry for BCL-2 on SCG Neurons and Supporting Cells in Culture Photomicrographs illustrating the pattern of BCL-2 expression in vitro for SCG neurons, Schwann cells, and fibrobiasts. After 1 week in culture, SCG neurons and supporting cells were stained with the 3F11 antibody or a control hamster monoclonal antibody. (A-C) Neurons maintained in the presence of NGF and incubated with the control antibody show no staining (A). Neurons stained with the 3Fll anti-BCL-2 antibody show both cytoplasmic and nuclear staining with exclusion of the nucleoli (B). After 15 hr of NGF deprivation, neurons incubated with the 3F11 antibody show significantly less intense staining in both the nucleus and cytoplasm (C). (D-F) Lower power photomicrographs of in vitro SCG cells, each showing two fields of cells. Neurons (open arrows) and nonneuronal cells incubated with a control hamster monoclonal antibody show no staining (D). In the presence of NGF, Schwann cells (small arrows), fibroblasts (large arrows), and neurons stained with 3Fl l (E). Neurons deprived of NGF for 15 hr show much less intense staining with 3Fl l (F); however, nonneuronal cells stained with the same intensity as in the presence of NGF. Bars, 15 I~m.

clearly visualized. Rat neuronal cultures that had been maintained in NGF for I week were stained with the 3F11 antibody. Immunohistochemistry showed light staining in both the neurons and nonneuronal support ing cells. The staining in the neurons was both cytoplasmic and nuclear; however, the nucleoli appeared to be excluded (Figures 1A and 1B). Both Schwann cells and fibroblasts (Figures 1D and 1 E) were also positive for BCL-2; however, staining was exclusively cytoplasmic in these cells. The intensity of staining was similar in the neurons and support ing cells, suggesting that BCL-2 protein levels are similar. The neuronal staining appeared uniform in the cultures; this is the only notable difference between the in vitro pattern and the in vivo pattern described by Merry et al., which shows heterogeneous neuronal staining. The in vitro system, therefore, closely mimics the in vivo situation.

To determine how the BCL-2 protein levels or localization changed after NGF deprivation, sympathet ic neurons were deprived of NGF for 15 hr and stained. By 15 hr after NGF deprivation, DNA fragmentat ion begins (Deckwerth and Johnson, 1993) and peak levels of gene induction

occur for several apoptosis-associated genes (see below) (Freeman et ai., 1994; Estus et al., 1994). After 15 hr of NGF deprivation, the intensity of BCL-2 staining in the neurons was significantly reduced, and the nuclei no longer appeared to stain (Figure 1C). This is consistent with the observation that neuronal protein synthesis rates decline rapidly after trophic factor deprivation (Deckwerth and Johnson, 1993). No reduction in the intensity of staining was seen in the nonneuronal cells after NGF deprivation (Figure 1F). These results suggest the possibil ity that loss of BCL-2 protein, subsequent to NGF deprivation, plays a role in the neuron reaching the point that it under- goes apoptosis.

Expression of the BCL-2 Family Members Is Decreased during Apoptosis Since apoptosis of sympathet ic neurons can be com.- pletely blocked by the addition of protein synthesis or RNA synthesis inhibitors, the expression of certain genes is probably important for apoptosis to proceed. Recently, several genes have been identif ied by RT-PCR that are

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Figure 2. RT-PCR Analysis of mRNAs for BCL-2 Family Members during Neuronal Apoptosis In three independent experiments, preplated SCG neurons, after 1 week in culture, were deprived of NGF for the indicated number of hours, mRNA was isolated, and cDNA was prepared. Messages were analyzed by PCR that included radiolabeled dCTP. Products were separated by SDS-PAGE and visualized by autoradiography; band intensity was quantified by Phosphorimager analysis. A representative autoradiograph is shown for each message, and the mean and SEM for band intensity are plotted for the three experiments (single asterisk, p < .05; double asterisk, p < .01; ANOVA with Dunnett multiple comparisons test).

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markedly~ induced at the transcriptional level in dying sympathetic neurons (Freeman et al., 1994; Estus et al., 1994). These include, among others, the G 1 to S transition regulator, cyclin D1, several Fos- and Jun-family transcription factors, and the transcription factor NGFI-A. We hypothesized that the BCL-2 family members may be regulators of the apoptotic process in sympathetic neurons. To evaluate gene expression in a small number of cultured sympathetic neurons, we used the semiquantitative RT-PCR assay characterized by Estus et al. (1994) and Freeman et al. (1994). A group of individual preplated cultures, each containing the same number of neurons, was prepared at a single time. These neurons were maintained in NGF for 6 days and then deprived of NGF for a specified number of hours before mRNA was harvested. We began the analysis of the BCL-2 family members by determining the number of PCR cycles and the amount of input cDNA required for each set of primers to yield a linear amount of radiolabeled PCR product (data not shown). Using these conditions, the relative mRNA abundance of the BCL-2 family members during apoptosis was analyzed. In three independent experiments, analysis of cDNA harvested from neuronal cultures at given times after NGF deprivation showed that RNA levels for all Bcl-2 family members de- clined after NGF deprivation (Figure 2). The general pattern was for levels to be maintained for the first 10-15 hr and then decline. BcI-Xs was near the limit of detection in the linear range of amplification, so reliable quantified

results could not be obtained. By 48 hr after deprivation, most neurons were dead and no tyrosine hydroxyiase message was detected; however, significant levels of Bcl-2 message, in particular, remained detectable. This is in agreement with the immunostaining for BCL-2 that showed a significant amount of the protein in the cultured nonneuronal cells. These nonneuronal cells are not sensitive to NGF deprivation and are the only cells left in the culture after 48 hr of NGF deprivation. It is noteworthy that the majority of messages analyzed after NGF deprivation of sympathetic neurons fall within similar time courses (Freeman et al., 1994; Estus et al, 1994; Greenlund et al., 1995). This suggests that both decreased RNA synthesis and active RNA degradation may be important for neuronal apoptosis (Freeman et al., 1994; Estus et al., 1994) since under normal circumstances the mRNAs would be expected to have different half-lives. The decreased neuronal synthesis of the protective proteins, BCL-2 and BCL- XL, during apoptosis may increase the susceptibility of neurons to death and contribute to making the process irreversible.

Sympathetic Neurons from BCL-2-Deficient Mice Die More Rapidly Than Neurons from Wild-Type Mice To determine unequivocally whether BCL-2 is important in regulating neuronal survival during the period of naturally occurring cell death, we examined neuronal cultures from


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Figure 3. TimeCourseof Apoptosis in Neurons from BCL-2-Deficient, Heterozygous, and Wild-Type Mice After 1 week in culture, sympathetic neurons from wild-type (n = 5 animals), heterozygous (n = 7 animals), or BCL-2-deficient (n = 8 animals) mice were either maintained in NGF or deprived of NGF for 24 or 32 hr. The live neurons in each culture were counted, and this number was normalized to the NGF-maintained control cultures prepared from the same mouse. After 24 hr of NGF deprivation, significantly fewer neurons are alive in cultures prepared from BCL-2- deficient mice and heterozygous mice compared with cultures prepared from wild-type littermates. The data show the mean and SEM for each set of animals (single asterisk, p < .05; double asterisk, p < .01 ; triple asterisk, p < .001; ANOVA with Bonferroni multiple comparisons test).

mice that lack BCL-2 (Veis et al., 1993). BCL-2-deficient mice are the result of a mating between 2 heterozygous animals. Litters produced by this mating have wild-type, heterozygous, and BCL-2-deficient animals in them. These animals were sacrificed at P1 or P2 and dissected; tail clips were taken at the time of dissection for later genotyping. Since the genotype of each animal was not known at the time of dissection, pairs of SCGs were removed from each animal, individually dissociated, and then maintained separately for 1 week in the presence of NGF. At the time of dissociation, there was no statistical difference in the number of SCG neurons recovered from animals of the three genotypes (p > .1, ANOVA); however, after 1 week in vitro in the presence of NGF, SCG cultures from BCL-2-deficient mice contained significantly fewer neurons than cultures from wild-type littermates (p < .05, ANOVA with Bonferroni multiple comparisons test). At 1 week in vitro, SCG cultures were deprived of NGF for 24 or 32 hr, then fixed and stained; the number of neurons was then counted. Crystal violet positivity and cellular mor- phology were used as criteria for neuronal viability (Deck-

werth and Johnson, 1993; Franklin et al., 1995). At least two cultures were counted for every t ime point for each animal indicated. The data in Figure 3 show that BCL-2- deficient neurons (n = 8 animals) die more quickly than wild-type neurons (n = 5 animals), with heterozygotes (n = 7 animals) having an intermediate rate. Although the BCL-2-deficient animals do not show gross defects in the development of the nervous system (Veis et al., 1993), the data demonstrate that BCL-2 is an important regulator of neuronal survival under conditions of complete trophic factor deprivation.

BCL-2 Family Expression and Maturation-Acquired Trophic Factor Independence To determine whether the BCL-2 family members are important in the decreased acuteness of trophic factor dependence associated with maturation, we began by comparing the mRNA levels of the BCL-2 family members from young neurons, which were still sensitive to NGF deprivation, with those of mature neurons. Sympathetic neurons that are maintained for 4 weeks in the presence of NGF show dramatic decreases in trophic factor dependence, much as they would in vivo. This in vitro model of maturation provides a controlled situation that allows a careful analysis of changes in gene expression as maturation pro- ceeds, in order that 1- and 4-week-old neurons could be directly compared and that the number of neurons being analyzed was constant, a set of individual cultures was prepared at one time and divided into two groups. One group of cultures was maintained in NGF for I week before harvesting RNA; the second group of cultures was maintained in NGF for 4 weeks. The mature neuronal cultures were deprived of NGF for specific times before RNA was harvested. Figure 4 is a representative experiment and shows that the RNA messages of most of the BCL-2 family members are not significantly altered as neurons decrease in trophic factor dependence. The most notable change is the decrease in Bcl-2 mRNA in 4 week in vitro neurons compared with I week in vitro neurons. In contrast to young neurons (see Figure 2), mRNA abundance in mature neurons does not decrease dramatically after NGF deprivation. That Bcl-2 mRNA is decreased and that the expression of the other Bcl-2 family members is unaltered as trophic factor independence develops indicates that modulation of the expression of these genes is not the molecular mechanism of trophic factor independence. In addition, the data show that in older neurons, no longer acutelydependent on trophic factor, NGFdeprivation does not result in decreased mRNA abundance. This further correlates the apparent degradation and decreased levels of mRNA with apoptosis.

BCL-2 Protein Is Not Up-Regulated during Maturation and the Acquisition of Trophic Factor Independence Using in situ hybridization in combination with immunohistochemistry, Kondo et al. (1992) and Chleq-Deschamps et al. (1993) have shown that BCL-2 expression may be regulated at the translational level. For example, germinal center lymphocytes express large amounts of BCL-2 mes-

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Neurons were maintained in culture for either 1 week or 4 weeks. The 4 week cultures were deprived of NGF for the indicated number of hours, mRNA was isolated from neurons and cDNA was prepared. SDS-PAGE was used to separate radiolabeled PCR products, and autoradiography was used to view the products. The number of amplification cycles done for each message is indicated. Note that there is little or no decrease in the abundance of tyrosine hydroxylase message or in the messages for the BCL-2 family members after NGF deprivation.

sage but little detectable BCL-2 protein, and mantle lymphocytes express both message and protein (Kondo et al., 1992). To examine whether BCL-2 protein was increased even though the mRNA was decreased, neurons maintained 1 or 4 weeks in NGF were immunosta ined for BCL-2 protein. The intensity of staining was very similar in the two sets of cultures (data not shown), suggest ing that the amount of BCL-2 protein did not change with maturat ion. In addit ion, immunoprec ip i ta t ion fo l lowed by Western blot- t ing was used to determine whether BCL-2 protein increased as neurons matured. We conf i rmed that the anti- mouse BCL-2 ant ibody, 3 F l l , immunoprec ip i ta ted rat BCL-2 by isolat ing both rat and mouse thymocytes, immu- noprecipi tat ing with 3F11, and separat ing by SDS-PAGE. A Western blot of thymocyte lysates from both species showed a 26 kDa band (data not shown). To compare the amount of BCL-2 in NGF-sensit ive neurons with amounts in mature neurons, cultures contain ing the same number

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Figure 5. Western Blot Comparing BCL-2 Protein in Young and Ma- ture Sympathetic Neurons Neuronal cultures were maintained in NGF for either 1 week or 4 weeks before harvesting. Following immunoprecipitation with 3F11 antibody, samples were separated by SDS-PAGE and then transferred. The Western blot shows an immunoprecipitation containing the 3F11 antibody alone and immunoprecipitations containing 3Fl l antibody in combination with 1 week or 4 week neuronal lysates. Both the 1 week and 4 week neuronal lysates show a single 26 kDa BCL-2 band of similar intensity relative to the 3Fl l band. This was repeated with similar results.

of preplated neurons (the equivalent of 19 rats/culture) were prepared at a single t ime and divided into two sets. The first set of cultures was maintained in NGF for 1 week before harvest ing, and the second set was mainta ined in NGF for 4 weeks before harvesting. An immunoprec ip i ta- tion and Western blot with 3 F l l of both sets of lysates showed a 26 kDa BCL-2 band of s imi lar or decreased intensity (Figure 5) in older neurons. This indicates that an increase in BCL-2 protein is not a factor in the loss of acute trophic factor dependence.

Neurons from BCL-2-Deficient Mice Become Less Acutely Trophic Factor Dependent with Maturation Although BCL-2 levels are not increased during maturation, it is possible that the basal expression levels are necessary and suff icient for the loss of acute trophic factor dependence. To examine this possibil ity, individual pairs of gangl ia from BCL-2-deficient, heterozygous, and wild- type mice were independent ly dissociated and mainta ined separately in NGF for 3 weeks. Cultures were then depr ived of NGF for 72 hr, f ixed, and stained. Neurons were counted using crystal v iolet posit ivity and cell morpho logy as criteria for viabi l i ty (Figure 6). At least two cultures per animal indicated were counted for each t ime point. No neuronal death occurred in any of the cultures after 3 days of NGF depr ivat ion, regardless of genotype. Therefore, BCL-2 expression inf luences neuronal survival in response to t rophic factor depr ivat ion at the t ime of natural ly occurr ing p rogrammed neuronal death but is not required for the loss of acute trophic factor dependence in sympathetic neurons.


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Figure 6. AnalysisofTrophicFactorlndependencein BCL-2-Deficient Mouse Neuronal Cultures After 3 weeks in culture, sympathetic neurons from wild-type (n = 4 animals), heterozygous (n = 4 animals), or BCL-2-deficient (n = 5 animals) mice were either maintained in NGF or deprived of NGF for 72 hr. The live neurons in each culture were counted, and this number was normalized to the NGF-maintained control culture prepared from the same mouse. The data show the mean and SEM (p > .05 for all comparisons; ANOVA with Bonferroni multiple comparisons test).

Overexpression of BCL-2 in Sympathetic Neurons Delays Death but Does Not Preserve Cellular Function Overexpression of BCL-2 has been reported to inhibit or prevent the death of many cell types in response to a number of insults (Hockenbery et at., 1990; Garcia et al., 1992; Allsopp et al., 1993; Batistatou et al., 1993; Kane et al., 1993; Zhong et al., 1993). However, closer examination of the data suggests that BCL-2 does not block death completely, but rather slows the inevitable process. To examine whether overexpression of BCL-2 would block or just delay the death of sympathetic neurons, we microinjected a human Bcl-2 expression vector or a control lacZ expression vector into the nucleus of cultured sympathetic neurons. Immunohistochemistry for human BCL-2 or X-Gal histochemistry for lacZ 24 hr after injection confirmed that 96% (n = 26) of the neurons injected with the Bcl-2 expression vector and 90% (n = 37) of the neurons injected with the lacZ vector were positive for the particular protein. To examine the effect on survival, neurons were injected with the Bcl-2 or lacZ expression vectors and then maintained in NGF for 24 hr to allow time for sufficient gene expression. Neurons were then deprived of NGF, and the number of viable, injected neurons was determined at 24, 48, and 72 hr after NGF deprivation. In five experiments, the number of viable neurons was significantly increased by over-

expression of BCL-2 but not by lacZ expression, although the Bcl-2 vector-injected neurons died over a period of days (Figure 7A).

To explore further why the protection imparted by BCL-2 was temporary, we examined whether BCL-2 would block the loss of cellular functions after NGF deprivation. The rate of neuronal protein synthesis, as assayed by [3~S]methionine incorporation, decreases rapidly, to 20% of NGF control in 16-18 hr after NGF deprivation; this decrease can largely be prevented by K ÷ depolarization or increased cAMP (T. L. Deckwerth, unpublished data), either of which prevents apoptosis of the neurons (Koike et al., 1989; Ry- del and Greene, 1988). We tested whether BCL-2 overexpression would similarly block this fall in protein synthesis. We developed an in situ protein synthesis assay that al- lowed the comparison of [35S]methionine/cysteine incorporation in individual neurons. Using this assay, neurons that were injected and expressing the foreign gene product were compared with uninjected neighboring neurons. Cells were injected with the Bcl-2 or lacZ expression vectors, maintained in NGF-containing medium for 24 hr, and then deprived of NGF. After 14 hr of NGF deprivation, neuronal cultures were pulsed for 4 hr with [3~S]methionine/cysteine. After exposure to emulsion, the number of grains overlaying neurons was counted under dark-field microscopy (Figures 7B and 7C). When injected neurons, maintained in NGF, were compared with uninjected neighboring cells, the number of grains overlaying the neurons did not differ (lowest p > .2, Student's t test; Figure 7C), irrespective of the vector injected. Thus, the rate of [3sS]methionine/cysteine incorporation and overall rates of protein synthesis were unaffected by the injection process. Neurons deprived of NGF (18 hr total) had, on average, 79% fewer silver grains over them than those maintained in NGF (Figure 7C). In NGF-deprived cultures, neurons overexpressing BCL-2 (Figure 7B) or LacZ (data not shown) had no difference in the number of grains overlaying them compared with uninjected neighboring neurons (lowest p > .4, Student's t test; Figure 7C).. Therefore, the dramatic decrease in protein synthesis that occurs with NGF deprivation is not prevented by BCL-2 overexpression. Because this crucial cellular function is not maintained by the expression of BCL-2, it is not surprising that injected neurons do not remain viable for a prolonged period. The failure of BCL-2 to maintain protein synthesis rates indicates that, although the loss of viability is re- tarded, BCL-2 does not prevent the loss of cellular function associated with NGF deprivation-induced death in sympathetic neurons.

Discussion

Overexpression of BCL-2 in neurons or neuron-like cell lines protects cells from apoptosis in response to a number of death-inducing stimuli (Garcia et al., 1992; AIIsopp et al., 1993; Mah et al., 1993). Recently, Martinou et al. (1994a) have produced transgenic mice that overexpress BCL-2 in the nervous system. These mice have a reduced number of neuronal deaths in both the retina and facial nucleus during the period of programmed neuronal death,

Neuron 656

0 0 2 0 4 0 6"0 8 '0

bcl-2 Iz

Hours After NGF Deprivation

B

C 1 2 C

I,I.

Z 10( +

¢o

2 8¢

Z

6C

O 0 4C

0 0 3 2e a .

T i !

Ave Per Total Neuron Grains

+NGF 61 104& BCL-2 +NG~ 56 927

-NGF 14 390 ]BCL-2 -NG~ 11 321

+NGF BCL-2 -NGF BCL-2 +NGF -NGF

phase cont rast f l u o r e s c e n c e In situ

bcl-2 +NGF

bcl-2 -NGF

Figure 7. BCL-20verexpression Delays Neuronal Apoptosis but Does Not Block the Fall in Protein Synthesis Rates That Occurs after NGF Deprivation

(A) After 5-6 days in culture, sympathetic neurons were injected with the Bcl-2 (n = 300) or lacZ (n = 382) expression vector and maintained in NGF for 24 hr. All cultures were then deprived of NGF for the indicated number of hours. The mean and SEM for five experiments are shown. (B) The left column shows photomicrographs of phase-contrast images of injected (large arrows) and noninjected (small arrows) neurons in the presence and absence of NGF. The middle column shows fluorescent images of the same fields of neurons; injected neurons were identified by the presence of the fluorescent label within the cell (identified by large arrows). The right column shows the in situ protein synthesis assay with silver grains overlaying neurons. Note that expression of BCL-2 had no effect on methionine incorporation in either the presence of absence of NGF. Bar, 25 pm. (C) Quantification of grains over injected versus uninjected neurons in the presence or absence of NGF. There is no statistical difference in the number of grains overlaying neurons overexpressing BCL-2 (hatched bars; n = 17) or uninjected neurons (closed bars; n = 17) in the presence of NGF (p > .2, Student's t test). The inset shows the average number of grains counted per neuron for each condition and the total number of grains counted. The data are normalized to the NGF-maintained cells, injected or uninjected. After a total of 18 hr of NGF deprivation, the number of grains over neurons is decreased by an average of 79%. BCL-2-overexpressing neurons (n = 28) show the same decrease in grain density as uninjected neighboring neurons (n = 28). The data are the mean grain counts and SEM of all neurons in two independent experiments.


as well as reduced neuronal death after experimental stroke. Although BCL-2 overexpression clearly affects neuronal survival, the importance of endogenous BCL-2 has not been assessed. We examined, in a well-characterized in vitro system, the physiological importance of endogenous BCL-2 in regulation of neuronal survival, function, and maturation.

BCL-2 and Maturation-Induced Trophic Factor Independence Using RT-PCR, we determined whether the loss of acute trophic factor dependence in mature neurons was corre- lated with changes in BCL-2 family member gene expression. The mRNA levels for most Bc/-2 family members were not significantly altered, and there was a decrease in Bc/-2 message abundance with the development of trophic factor independence. In addition, immunostaining and Western blots show no increase in BCL-2 protein with maturation. Examination of neurons from BCL,2-deficient mice demonstrates that BCL-2 expression was not required for the loss of acute trophic factor dependence. Thymocyte (Moore et al., 1994) and B cell (Merino et al., 1994) development each involve stage-specific regulation of BCL-2 expression. Pro-B cells express high levels of BCL-2, but pre-B and immature B cells express only low levels. Those B cells that reach maturity express high levels of BCL-2 (Merino et al., 1994), which is important for maintaining memory B cells for prolonged periods (NuSez et al., 1991). The susceptibility of B cells at various stages of development to apoptosis-inducing stimuli, such as glucocorticoid treatment, correlates positively with the levels of BCL-2 expression (Merino et al., 1994). A similar developmental process in which the BCL-2 family members regulate the sensitivity of sympathetic neurons to NGF deprivation does not appear to occur. It is noteworthy that in mature neurons the decrease in mRNA abundance after NGF deprivation does not occur, indicating that the initia- tion of degradative events after NGF deprivation is greatly slowed in mature neurons. The molecular mechanism un- derlying loss of acute trophic factor dependence has yet to be elucidated. Understanding the mechanisms of resistance to apoptosis in the absence of trophic factor may prove to be important in understanding both apoptosis and disease states in which neurons are inappropriately susceptible to death.

BCL-2 and Neuronal Survival during the Period of Programmed Neuronal Death Again by using RT-PCR, we examined mRNA for the BCL-2 family members in neurons undergoing apoptosis. Bcl-2 and Bcl-Xt_ mRNA levels decreased after 15 hr of NGF deprivation; at 15 hr, neurons can be rescued from death by addition of NGF (Deckwerth and Johnson, 1993). Thus, this fall in rnRNA levels was not caused by the loss of neurons and most likely reflects active RNA degradation combined with decreased RNA synthesis. Immunostain- ing of NGF-deprived neurons also showed a significant decrease in BCL-2 protein by 15 hr after NGF deprivation. The reported half-life for BCL-2 protein is 10 hr (Merino et al., 1994); since protein synthesis rates decline rapidly

after NGF deprivation, at least half of the BCL-2 protein would be expected to be gone by 15 hr after NGF deprivation, an observation consistent with the immunohisto- chemical data. The decreased levels of BCL-2 and BCL-X, after NGF deprivation, coupled with increased levels of cyclin D1 and several Fos/Jun family transcription factors, may be important in creating a cellular environment that irreversibly commits a neuron to die. Experiments with neurons from BCL-2-deficient mice demonstrate that elim- ination of BCL-2 expression significantly increased the rate at which neurons die in response to trophic factor deprivation. Similarly, when Nakayama et al. (1993) and Veis et al. (1993) evaluated the effect of the death-inducing stimuli, dexamethasone and ~, irradiation, on T cells of BCL-2 homozygous mutant mice (chimeras and complete knockouts, respectively), they observed an increased susceptibility to death. The BCL-2-deficient mice do not show gross developmental defects In the nervous system (Veis et al., 1993). This may be explained by the redundant action of BCL-XL or similar molecules. In addition, in this in vitro paradigm, neurons were deprived completely of trophic factor. Under these extreme conditions, the lack of BCL-2, or the presence of only a single copy, increases the rate of death and may increase the susceptibility to death induced by a variety of insults. The in vitro conditions used in these experiments may be more akin to axotomy or other injury paradigms in which neurons are more completely deprived of trophic factor. During development, neurons receive some target-derived or other trophic support and are not completely deprived of trophic factor; under these conditions, molecules in addition to BCL-2, such as cell-cell/matrix contacts, and electrical activity may all function to support neuronal viability. Under more extreme conditions, the lack of one or more of these could lead to increased susceptibility to death. Based on the in vitro results, one would predict that ganglia of BCL-2- deficient mice would have more neuronal death in response to axotomy or target removal. That endogenous BCL-2 levels decrease shortly before neurons are irreversibly committed to die and that neurons from BCL-2- deficient mice die much more rapidly suggest that a decrease in endogenous neuronal BCL-2 levels increases the susceptibility of neurons to death, and as such, this decrease is an important factor in the neuron becoming committed to die. These observations support previous speculations (Estus et al., 1994) that the "program" in programmed neuronal death involves not only increased expression of certain genes but also decreased expression of others.

Where Does BCL-2 Act to Block Programmed Neuronal Death? Overexpression of BCL-2 in sympathetic neurons significantly delayed, but did not block, apoptosis after NGF deprivation; nor did it block the early fall in protein synthesis rates that occurs in NGF deprivation-induced apoptosis. This is in contrast to other agents, K + depolarization or increased cAMP, that maintain viability after trophic factor deprivation. Since protein synthesis rates are not maintained by BCL-2, the expression of BCL-2 itself

Neuron 658

would be expected to fall, and BCL-2 would no longer be overexpressed. This loss of BCL-2 protein, and the global decrease in cel lular protein synthesis, contr ibutes to making BCL-2 only a temporary saving agent.

Raft and col leagues (Jacobsen et al., 1994) propose that apoptosis is a mult istep process consist ing of an activation phase, an effector phase, and a degradat ion phase. We would suggest a somewhat modif ied scheme of pro- g rammed cell death involving an act ivat ion phase, followed by a propagat ion phase, a terminal apoptot ic phase, and, at least in vivo, a degradat ion phase. In sympathet ic neurons, the act ivat ion phase of NGF depr iva t ion- induced death involves the expected dephosphory la t ion of the TrkA receptor (C. Sanz-Rodr iguez, unpubl ished data), decreased MAP kinase act iv i ty (D. J. Creedon, submitted), and the increased format ion of react ive oxygen species (Greenlund et al., 1995). These events t r igger processes in

the propagat ion phase that include posit ive and negat ive regulat ion of gene expression and other biochemical changes within the cell. RNA levels for genes including c-jun, c-fos, and cyclin D1 are markedly increased, whi le RNA levels for Bcl-2 and Bcl-XL are decreased during the propagat ion phase. A port ion of the negat ive regulat ion of RNA levels may be exp la ined by an act ive degradat ion of RNA. In addit ion, the synthesis of new protein and RNA is dramatical ly decreased during the propagat ion phase. The terminal apoptot ic phase includes events that abso- lutely commit a neuron to die, i.e., the f ragmentat ion of DNA into o l igonucleosomal f ragments and, potential ly, the act ivat ion of a ced-3-1ike cyste ine protease (Yuan et al., 1993).

To what extent BCL-2 is capable of reversing the events associated with the act ivat ion and propagat ion phases out l ined above is not known. Our data suggest that, since BCL-2 overexpression does not block the fall in neuronal protein synthesis rates (even though it c lear ly prolongs viability), BCL-2 may be an effect ive b locker of the terminal apoptot ic phase during which kil l ing actual ly takes place but not of events in the act ivat ion or propagat ion phase. Albrecht et al. (1994) observed a similar phenomenon in analyzing BCL-2 protect ion from tumor necrosis factor (TNF)- induced apoptosis. TNF t reatment normal ly stimu-

lates the translocat ion of NF-KB to the nucleus of L929 cells before apoptot ic changes are evident. Al though L929 cells that overexpress BCL-2 are protected from death induced by TNF, NF-KB is still t ranslocated after TNF treatment. Whether overexpression of BCL-2 in sympathet ic neurons would block the al terat ions in gene expression of the propagat ion phase is not known. However, the abil i ty of BCL-2 to block apoptosis induced by a myr iad of stimuli, including those showing no lag phase and no requirement for ongoing macromolecu lar synthesis (e.g., serum depri- vat ion of PC12 cells; Bat istatou et al., 1993), is most consistent with an act ion at, or immediate ly prior to, the terminal apoptot ic phase. This may include a direct or indirect interact ion of BCL-2 with a ced-3-1ike molecule. Under- standing the events in this process will provide means not only for prolonging neuronal viabi l i ty in injury or disease but also for preserving neuronal function.

Experimental Procedures

Rat Sympathetic Neuronal Culture Primary cultures of SCG neurons were prepared by a modification (Martin et al., 1988) of the method of Johnson and Argiro (1983). For preplating, dissociated SCG cells were plated on untreated 60 or 100 mm Falcon (Becton Dickinson Labware, Lincoln Park, N J) tissue culture dishes for 90 min, enough time for most of the nonneuronal cells to attach to the plastic; however, neurons require a collagen (or other) substratum for attachment and remained floating. The floating neuronal cells were carefully washed off the plastic dish, filtered through Nitex (size 3-20/14; Tetko Inc., Gaithersburg, MD) to remove aggre- gates of neurons, and then plated. Neurons were plated in the center of collagen-coated 35 mm dishes and maintained in NGF-containing medium (AM50). AMS0 was Eagle minimal with Earle's salts (MEM; Life Technologies, Gaithersburg, MD), with the addition of 50 ng/ml NGF (prepared by the method of Bocchini and Angeletti [1969]), 10% fetal bovine serum (Hyclone, Logan, UT), 2 mM L-glutamine, 100 p.g/ ml penicillin, 100 p.g/ml streptomycin, 20 ~M fluorodeoxyuridine (an antimitotic), and 20 ~M uridine. Neurons were deprived of NGF by incubation in the same medium, but without NGF (AM0) and with goat polyclonal anti-mouse NGF antiserum added.

Immunostaining To stain for endogenous BCL-2, neurons were fixed in fresh 4O/o paraformaldehyde in phosphate-buffered saline (PBS) at room temperature for 30 rain, permeabilized for 20 min in Tris-buffered saline (TBS) containing 0.1% Triton X-100, 1% BSA, and 1% normal goat serum, and then incubated with the primary antibody, 3F11 hamster monoclonal anti-mouse BCL-2 (1:20; 50 i~g/ml final concentration) (Merry et al., 1994), or a control 6C8 hamster monoclonal anti-human BCL-2 antibody (1:20; 50 ~g/ml final concentration) (Hockenbery et al., 1990) diluted in the permeabilization solution. Incubation with the primary antibody was for 1.5 hr at room temperature. Samples were rinsed twice and then washed three times for 10 rain in permeabilization solution. The biotinylated goat anti-hamster secondary antibody (South- ern Biotechnology, Birmingham, AL) was diluted 1:100 (5 t~g/ml final concentration) in permeabilization solution and incubated for 30 min at room temperature with the samples. After three 10 rain washes in PBS, samples were incubated for 30 rain in ABC reagent (avidin-HRP; Vector Laboratories, Burlingame, CA). After three 15 rain washes in PBS, samples were stained for 5-7 min with 3,3'-diaminobenzidine (Vector Laboratories). To detect overexpressed human BCL-2, neurons were stained as described above, but the primary antibody was 1:100 (10 t~g/ml) 6C8 hamster monoclonal anti-human BCL-2. The biotinylated secondary antibody and staining procedures were the same as those described above.

cDNA Preparation This method and its validation have been described previously (Estus et al., 1994). Primary cultures (-25,000 neurons/dish) that had been preplated on plastic were maintained in AM50 for 6 days. Cultures were deprived of NGF for the indicated times, and then RNA was harvested. Polyadenylated RNA was isolated using an oligo(dT)- cellulose mRNA purification kit as directed by the manufacturer (QuickPrep Micro kit, Pharmacia, Piscataway, N J). Half of the poly(A) RNA was converted to cDNA by reverse transcription with Moloney murine leukemia virus reverse transcriptase (Superscript, Life Tech- nologies) with random hexamers (16 I~M) as primers. The 30 p.I reaction contained 50 mM Tris (pH 8.3), 40 mM KCI, 6 mM MgCI2, 1 mM dithiothreitol, 500 I~M dATP, 500 p.M dTTP, 500 I~M dCTP, 500 I~M dGTP, and 20 U of RNasin (Prornega, Madison, WI). After 10 min at 20°C, the samples were incubated for 50 min at 42°C; the reaction was terminated by adding 70 p.I of water and heating to 94°C for 5 rain.

PCR Analysis For a more detailed description of this method, see Freeman et al., 1994; Estus et al., 1994; or Greenlund et al., 1995. Oligonucleotide primers were synthesized by the Washington University Protein Chem- istry Laboratory. Reactions for PCR amplification of specific cDNAs were prepared on ice. Each reaction contained 50 I~M dCTP, 100 p.M dGTP, 100 I~M dATP, 100 ~M dTTP, 15 p.Ci of [~-32P]dCTP (3000 Ci/


mmol), 1.5 mM MgCI~, 50 mM KCI, 10 mM Tris (pH 9.0), 0.1% Triton X-100, 1 mM each primer, 1 U of Taq polymerase (Life Technologies), and 1% of the cDNA synthesized in the reverse transcription reaction. Each reaction was run for cycles of 1 min at 94°C, 1 min at 55°C, and 2 min at 72°C in a Perkin-Elmer Cetus (Norwalk, CT) thermocycler. After amplification, products were separated on a 10% polyacrylamide gel, which was dried and visualized with ImageQuant software on a Phosphorlmager (Molecular Dynamics, Sunnyvale, CA). The Bcl-2 forward primer sequence was 5'-CTTTGTGGAACTGTACGGCCC- CAGCATGCG-3'; the reverse was 5'-ACAGCCTGCAGCTTTGTTT- CATGGTACATC-3' (the pair generated a 231 bp fragment). The bax forward primer sequence was 5'-GGGAATTCTGGAGCTGCAGAG- GATGATT-3'; the reverse was 5'-GCGGATCCAAGTTGCCATCAG- CAAACAT-3' (the pair generated a 96 bp fragment). The rat Bcl-X forward primer was 5'-AGGCTGGCGATGAGTTTGAA-3'; the reverse was 5'-CGGCTCTCGGCTGCTGCATT-3' (the pair generated a 337 bp fragment for the long-form cDNA and a 150 bp fragment for the short-form cDNA). The tyrosine hydroxylase forward primer was 5'-TTCAGAAGGGCCGTCTCAGA-3'; the reverse was 5'-CCGCTGCT- GCTGCTGCAGCT-3' (the pair generated a 129 bp fragment). The Bcl-2 and bax primers were based on mouse sequence, so the products were subcloned and sequenced to confirm their identity. The Bcl-X primers were based on rat sequence and generated only two products, both of the expected size. The decrease in message abundance after NGF deprivation was tested in at least three independent sets of cDNA for all messages. The analysis of message abundance with maturation was tested in four sets of cDNA for BcI-X (five independent experiments), two sets for Bcl-2 (five independent experiments), and one set for bax (two independent experiments).

Preparation of SCG Cultures and Genotyping from BCL-2-Deficient Litters Mice (Veis et al., 1993) were dissected on P1 or P2. The geneotype of each animal was unknown at the time of dissection, so the pairs of ganglia from each animal were individually dissociated and plated on collagen-coated glass chamber slides. At least two cultures were prepared for every data point for each animal. The final cultures contained - 1000 neurons/well. The medium and culture conditions were the same as those described above for rat neuronal cultures. Geneo- typing was done by digesting the tail of each animal overnight at 55°C in 250 I11 of a solution containing 50 mM KCI, 10 mM Tris-HCI, 1 mM EDTA (pH 8.0), 0.5% SDS, and 2 mg/ml proteinase K (Boehringer Mannheim, Indianapolis, IN). Samples were centrifuged to remove large debris, and 150 ~1 of the supernatant was transferred to fresh tubes. After 250 Id of a solution containing 100 mM NaCI, 10 mM Tris- HCI (pH 8.0), 1 mM EDTA (pH 8.0), 0.5% SDS, and 1 mg/ml RNase A (Boehringer Mannheim) was added to the tail digest samples, they were incubated at 37°C for 1 hr. DNA was extracted twice with phenol/ chloroform and once with chloroform and was then precipitated with ethanol. The resulting DNA was diluted 1:10 and 1 I11 was used in a PCR reaction to determine geneotype. Reactions for PCR amplification of specific cDNAs were prepared on ice. Each reaction contained 50 I~M dCTP, 100 I~M dGTP, 100 pM dATP, 100 ~M dTTP, 1.5 mM MgCI2, 50 mM KCI, 10 mM Tris (pH 9.0), 0.1% Triton X-100, 1 mM each primer, 1 U of Taq polymerase (Life Technologies), and 1 i11 of the dituted tail DNA. Each reaction was run for 35 cycles of 1 min at 94°C, 1 min at 55°C, and 2 rain at 72°C. The primers used to detect Bcl-2 were the same as those stated above. The forward primer for the detection of the neomycin resistance gene was: 5'-GGATCGGCCATTGAACAA- GATG-3'; reverse primer was 5'-CCGGGCGCCCCTGCGCTGACAGC- 3' (the pair generated a 141 bp fragment).

Crystal Violet Staining and Neuronal Counting for Transgenic Mouse Experiments This method is established as a reliable assay of viability (Deckwerth and Johnson, 1993; Franklin et al., 1995). Neuronal cultures on glass chamber slides were fixed with freshly prepared 4% paraformaldehyde in PBS overnight at 4°C, stained with 1% crystal violet (EM Diagnostic, Gibbstown, NY), destained in water, dehydrated in increasing ethanol concentrations, transferred to toluene, and mounted in a toluene- based mounting solution (Pro-Texx; Baxter Diagnostics, Deerfield, IL). Neurons were scored as viable if they had a defined cellular outline

and visible nucleolus. Slides were coded and counts were done by an independent observer.

Immunoprecipitation and Western Blots Preplated cultures of rat SCG neurons were prepared as described. The equivalent of 38 ganglia (3.7 x 10 s preplated neurons) were plated per 60 mm dish, and one dish was used per immunoprecipitation. After 1 or 4 weeks in culture, neurons were lysed in 250 i~1 of buffer containing 1% Triton X-100 (Sigma Chemicals, St. Louis, MO), 0.15 M NaCI, 10 mM Tris (pH 7.4), 50 p.g/mt phenylmethylsulfonyl fluoride (Boehringer Mannheim), 10 i~g/ml aprotinin (Boehringer Mannheim), and 10 I~g/ml leupeptin (Boehringer Mannheim). The plates were incubated on ice for 30 min followed by scrapping to maximize recovery. Lysates were centrifuged at 13,000 rpm for 5 min to remove large cellular debris and nuclei. For each immunoprecipitation, 3 ~1 of 1 mg/ ml 3F11 antibody was used. Samples were incubated for 1 hr at 4°C on a rocker with the antibody. Protein G Sepharose in PBS (50 i11) was added to each sample followed by another 1 hr incubation at 4°C on a rocker. The beads were then washed five times in the lysis buffer to reduce nonspecific binding. On the last wash, all buffer was removed and reducing sample buffer containing 50 mM Tris-CI (pH 6.8), 100 mM dithiothreitol, 2% SDS, 0.1% bromphenol blue, and 10% glycerol was added to each reaction. Samples were boiled and loaded onto a 15% SDS-polyacrylamide gel. After electrophoresis, gels were transferred to Immobilon-P (Millipore, Bedford, MA). Blots were blocked overnight in TBS plus 5% nonfat dry milk, washed in TBS plus 0,05% Tween-20 (TBST), and then incubated with a 1:1000 (1 t~g/mt final concentration) dilution of 3F11 antibody in TBST for 45 min at room temperature. After thorough washing, blots were incubated with 1: 1000 (0.5 t~g/ml) biotinylated goat anti-hamster antibody (Southern Biotechnology) in TBST for 30 min at room temperatu re. After washing, blots were incubated with 1:5000 avidin-HRP (Zymed, San Francisco, CA) in TBST for 15 rain at room temperature. Blots were developed by enhanced chemiluminescence according to the manufacturer's in- structions (Amersham, Arlington Heights, IL).

Construction and Injection of Expression Vectors The base expression vector was constructed by cloning into pUC19 a 695 bp hCMV IE-1 promoter (620 bp upstream of the transcription start site and 75 bp downstream; Boschart et al., 1985) and a 355 bp portion of the mouse protamine-1 gene (Kleene et al., 1985) including the polyadenylation signal. Specific expression vectors were constructed by cloning cDNAs into the BamHI site between the promoter and polyadenylation signal of the base vector. The Bcl-2 cDNA was a 1.9 kb fragment, clone #58 (Seto et al., 1988), containing the entire coding region for human BCL-2. DNAwas prepared by column purification (Qiagen, Chatsworth, CA) and resuspended at 0.18 mg/ml in ster- ile-filtered, deionized water. For injections, DNA was combined 1:1 with a dye solution containing 8 mg/ml rhodamine dextran, 100 mM KCI, and 10 mM K3PO4, so that the final DNA concentration injected was 0.09 mg/ml. Neuronal culture medium was changed to Leibovitz's L-15 (Life Technologies), and injections were made into the nucleus of each neuron. Concentrations of DNA above 0.5 mg/ml produced a delay in apoptosis irrespective of the cDNA in the vector. We demonstrated by the in situ protein synthesis assay (see below) that injection of DNA at concentrations as low as 0.2 mg/ml occasionally caused an inhibition of protein synthesis in the presence of NGF, which pre- sumably explains the delay in death. Experiments were scored by an independent observer.

In Situ Protein Synthesis Assay This method has been described previously (Greenlund et al., 1995). In brief, neurons were injected with solutions containing 0.09 mg/ml expression vector DNA, 2.25 mg/ml Cy3-1abeled donkey anti-sheep antibody as a fixable tracer (Chemicon, Temecula, CA), 50 mM KCI, and 5 mM K3PO4 and then maintained in AM50 for 24 hr. Cultures were deprived of NGF for 14 hr before beginning a 4 hr labeling period. During the labeling period, neurons were incubated at 35°C in Eagle medium with Earle's salts and 10% fetal bovine serum containing 10 ~M unlabeled L-methionine and cysteine, 10 ~Ci/ml Tran[3SS]-Iabel (ICN, Irving, CA), and 50 ng/ml NGF or polyclonal goat anti-NGF antibody. After labeling, cultures were washed and fixed in 4% paraformal-

Neuron 66O

dehyde. Cells were dehydrated and dipped in Kodak emulsion (Kodak, Rochester, NY). After an overnight exposure, dishes were developed in D-19 developer (Kodak), fixed, and rinsed. Grains lying within the borders of the cell body of each neuron were counted by an independent observer under dark-field microscopy.

Acknowledgments

All correspondence should be addressed to E. M J, We thank P. A. Lampe, J. L. Colombo, and P, A. K. Osborne for their expert technical assistance and the Johnson Lab members for reviewing the manu- script, This work was supported by the Renald McDonald Children's Foundation, the Ataxia Telangiectasia Children's Project, and the Washington University Alzheimer's Disease Research Center (P50- AG05681).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC Section 1734 solely to indicate this fact.

Received February 8, 1995; revised May 16, 1995.

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Role of BCL-2 in the Survival and Function of Developing and Mature Sympathetic Neurons · 2016....

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