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Cell Growth & Differentiation Vol. 11, 625-633, December 2000
© 2000 American Association for Cancer Research

Articles

Induction of DNA Replication in Adult Rat Neurons by Deregulation of the Retinoblastoma/E2F G1 Cell Cycle Pathway 1

Deanna S. Smith2, Gustavo Leone3, James DeGregori4, Mohamed Nagy Ahmed, Mazin B. Qumsiyeh and Joseph R. Nevins5

Departments of Genetics [D. S. S., G. L., J. D., J. R. N.] and Pathology [M. N. A., M. B. Q.], Howard Hughes Medical Institute [J. R. N.], Duke University Medical Center, Durham, North Carolina 27710

Abstract

In adult organisms, a range of proliferative capacities are exhibited by different cell types. Stem cell populations in many tissues readily enter the cell cycle when presented with serum growth factors or other proliferative cues, whereas "terminally" postmitotic cells, such as cardiac myocytes and neurons, fail to do so. Although they rarely show evidence of a proliferative capacity in vivo, there is accumulating evidence to suggest that DNA synthesis can be triggered in postmitotic cells. We now show that cultured adult rat sensory neurons can replicate DNA in response to ectopic expression of E2F1 or E2F2 and that this is augmented by expression of cyclin-dependent kinase activities. We also find that addition of serum and laminin inhibits the E2F-induced S-phase in neurons but not in nonneuronal cells in the same cultures. We conclude that, although terminally differentiated neurons possess the capacity to reinitiate DNA replication in response to G1 regulatory activities, they fail to do so in the presence of signals that do not inhibit S-phase in other cell types in the same cultures. This suggests the existence of cell type-specific inhibitory pathways induced by these signals.

Introduction

A variety of differentiated cell types maintain the capacity to reinitiate cell division in response to injury and other stimuli, whereas other cells in adult animals appear to have lost this capacity. In the adult nervous system for example, neurons show no potential for reinitiating the cell cycle to replenish injured or dying cells, whereas nonneuronal cells often proliferate after damage to the nervous system (1, 2, 3) . Elucidating the molecular basis of this profound difference in proliferative potential is critical in understanding the basic mechanisms controlling the relationship of cell cycle to cell fate and differentiation.

Despite the apparent inability of normal mature neurons to proliferate, there is evidence that pathologies of the adult nervous system may involve aberrant or abortive attempts of mature neurons to re-enter the cell cycle. For example, dying neurons in the brains of patients with neurodegenerative diseases express proteins associated with DNA synthesis and mitosis (4, 5, 6) . Additional evidence for unscheduled cell cycle events in neuronal populations has been provided by the analysis of two mouse neurological mutants, staggerer and lurcher, where there are elevated levels of several cell cycle proteins as well as increased BrdU6 incorporation in cerebellar granule cells (6) . Although these experiments point to the initiation of DNA replication in adult neurons, BrdU incorporation could also represent DNA repair.

Young neurons in developing nervous systems forced to express viral oncogenes or cell cycle regulatory proteins also show evidence of aberrant proliferative activity. This seems to be deleterious to developing neurons, which undergo apoptosis rather than complete a normal cell cycle. Expression of the oncogenic SV40 large T-antigen in newly born neurons of transgenic mice (7, 8, 9, 10) or targeted disruption of the Rb tumor suppressor locus (Rb) causes developing neurons to incorporate BrdU, express cell cycle markers, and exhibit increased apoptosis. Overexpression of E2F1, a transcription factor regulated by Rb, produces a similar phenotype in embryonic carcinoma cells after neuronal differentiation by retinoic acid (11) . Finally, overexpression of the adenovirus E1A oncogene or E2F1 reportedly stimulated BrdU incorporation in newly postmitotic cerebellar neurons (12) . A caveat to all of the above experiments is that it is difficult to determine whether the unscheduled cell cycle events represent the failure of a cycling neuronal precursor to exit the cell cycle or whether a fully mature neuron has re-entered the cell cycle in response to the stimulus.

Several findings suggest that overexpression of oncogenes or E2F family members can induce cell cycle reentry in fully mature postmitotic cells, including adult cardiac myocytes (13) and myotubes differentiated from C2C12 cells (14) . Additionally, cortical neurons in brains of 6-week-old rats have been reported to incorporate BrdU in response to overexpression of a combination of E1A and E2F1 by immunoliposome delivery (12) . In this study, however, the unequivocal identification of BrdU-positive cells as neurons was not demonstrated clearly. And again, BrdU incorporation alone is not an absolute indication of DNA replication but can also represent DNA repair.

On the basis of these intriguing reports, however, we felt it likely that adult neurons could be forced to enter the cell cycle, at least as far as S-phase, and were interested in determining the extent to which this would occur and to characterize the conditions under which it would occur. To circumvent some of the problems encountered using embryonic cultures that contain a mixture of recently postmitotic neurons and neuroblasts, we used as a model system primary sensory neurons from adult rats. This system was chosen because sensory neurons are one of the few types of adult neurons that can readily be maintained in dissociated culture, and because the large size and characteristic morphology of most DRG neurons make them readily distinguishable from other cell types in the cultures, such as Schwann cells and fibroblasts.

Because of previous findings implicating the accumulation of E2F activity as a critical, rate-limiting step for S-phase entry (reviewed in Refs. 15 and 16 ), we have explored the potential of E2F overexpression to induce S-phase in adult neurons. To overcome the problem of inefficient transfection normally associated with adult sensory neurons, we turned to an alternate system of gene delivery, the use of recombinant adenoviral vectors. Our results show that adult neurons can indeed be induced to replicate DNA in response to the accumulation of E2F and other cell cycle-regulatory proteins. Actual replication of DNA is demonstrated by FISH analysis. We also show that factors in the culture environment, such as serum factors and laminin, can play a strong inhibitory role on the outcome of E2F overexpression in neurons. This may shed light on the recalcitrance of neurons in the adult nervous system to reenter the cell cycle in the presence of stimuli that trigger proliferation in other cell types.

Results

Adult Sensory Neurons Do Not Replicate DNA in Tissue Culture.
To establish baseline levels of DNA synthesis prior to inducing ectopic E2F activity, dissociated DRG cultures were incubated with 10 µM BrdU for 48 h. Large, round DRG neurons (25–100 µm in diameter) were easily identified by DIC microscopy; these cells did not stain positively for BrdU (Fig. 1Citation A, red arrow). Nonneuronal cells plated in the absence of serum are spindle shaped and frequently were BrdU positive (Fig. 1Citation A, blue arrows). These results were confirmed using bright-field microscopy and double-labeling for BrdU and GAP-43, which is enriched in neurons (Fig. 1Citation B, red arrow). Nonneuronal cells in medium containing 10% horse serum (Fig. 1Citation B, blue arrows) were often less spindle shaped but frequently incorporated BrdU (brown). Fig. 1CCitation shows four fields in a GAP-43-stained culture at a lower magnification to show the distinction between cells that were scored as neuronal (red arrows) and nonneuronal (black arrows) when counting BrdU-positive cells.



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  		Fig. 1. Nonneuronal cells, but not neurons, proliferate and incorporate BrdU in dissociated cultures. A, BrdU-negative neuron (red arrow) in a dissociated DRG culture incubated with BrdU for 48 h appears more protuberant by DIC microscopy than nonneuronal cells (blue arrows), some of which are BrdU positive (brown). B, a BrdU-negative neuron (red arrow) is stained intensely for GAP-43 in both soma and processes (blue). In the same field are shown two nonneuronal cells (blue arrows), one of which is BrdU positive (brown). Bar: A and B, 20 µm. C, low magnification of 24–36 h cultures stained for GAP-43 using the 91E12 antibody. The antibody strongly labeled round cells with long processes that have been counted as neurons in the remainder of these studies (red arrows). The heterogeneous neurons in intact ganglia and in dissociated DRGs range in size from 5 to 50 µm in diameter. Flat cells with a shorter or absent processes that stained less intensely for GAP-43 were scored as nonneuronal cells (black arrows). Bar, 50 µm. D, prior sciatic nerve lesion (Ax.) and 10% horse serum (Ser) stimulated nonneuronal cell proliferation but was unable to stimulate DNA synthesis in neurons. Laminin and NGF were also unable to trigger BrdU incorporation in neurons. The data are expressed as means ± SD from four separate animals. All neurons and 400 nonneuronal cells in a 2-cm2 area were scored.

 
By these criteria, it was clear that neurons did not incorporate BrdU, whereas nonneuronal cells exhibited a varying propensity to do so, depending on culture conditions (Fig. 1D)Citation . When DRGs from naive animals were cultured in the absence of serum, laminin, or NGF, <5% of nonneuronal cells had incorporated BrdU (medium in these cultures was supplemented with N1 additives, a standard nutritional supplement for neurons consisting of insulin, putrescine, transferrin, sodium selenite, and progesterone). As expected, surgically injuring axons by sciatic nerve crush 2 days before culturing produced a proliferative response such that 25% of nonneuronal cells were BrdU positive. Finally, exchanging the N1 additives for 10% horse serum with NGF and plating cells on laminin caused close to 80% of nonneuronal cells to incorporate BrdU during the incubation period. Under none of these conditions did we observe BrdU in neuronal nuclei (Fig. 1D)Citation . These initial observations confirm the generally held belief that adult neurons, under a wide range of culture conditions, are terminally postmitotic.

Use of Recombinant Adenoviruses to Overexpress Cell Cycle Regulatory Proteins in Neurons and Nonneuronal Cells.
The E2F family of transcriptional regulators are important components of the machinery that controls entry into S-phase. E2F overexpression induces S-phase in quiescent cells in culture, probably by overcoming the effect of stoichiometric levels of inhibitory proteins in the Rb tumor suppressor family. To characterize the competence of adult sensory neurons to initiate DNA replication, we overexpressed two members of the E2F family, alone or together with other regulatory proteins, in the dissociated DRG cultures. Because adult neurons are difficult to transfect, we turned to recombinant adenoviruses as a means of introducing genes into cultured neurons. The same viruses have been used in this and other laboratories to produce a variety of proteins implicated in the G1 to S-phase transition (17, 18, 19, 20) .

We tested the efficiency of viral infection using a virus designed to express E2F1 (AdE2F1). Cultures were exposed to either AdE2F1 or a control virus lacking an insert (Ad Con) for 3 h. Three bands that represent different phosphoforms of E2F1 are discernable on a Western blot prepared from cultures infected with AdE2F1 (Fig. 2A)Citation . Because the cultures contained mixed cell types, we also performed immunostaining. E2F1 was barely detectable in cultures infected with the control virus (Fig. 2Citation B, AdCon), whereas after exposure to AdE2F1, most nonneuronal cells (blue arrows) and some neurons (red arrow) overexpressed detectable E2F1 (Fig. 2Citation B, AdE2F1). Cells infected with a virus carrying the E2F2 gene showed E2F2 overexpression by immunostaining (Fig. 2B)Citation . Other virus constructs carrying the DP1, cdk2, and cyclin E genes were also capable of infecting neurons and nonneuronal cells and overexpressing the products of these genes (data not shown).



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  		Fig. 2. Overexpression of E2F proteins in neurons and nonneuronal cells. A, DRG cultures plated on laminin in serum-containing medium were infected with a control adenovirus (AdCon) or a virus carrying the E2F1 cDNA (AdE2F1). Extracts were prepared 48 h later by lysing the cultures in SDS sample buffer. Samples were analyzed by Western blotting using an E2F1 monoclonal antibody. The three isoforms have been detected in other cell types after infection with Ad-E2F1 and represent phosphorylated forms of the protein. B, DRG cultures plated on laminin in serum-containing medium were infected with the control virus (AdCon, top panel) or AdE2F1 or AdE2F2 (bottom panels) and then immunostained 24 h later with an antibody specific to E2F1 or E2F2. Nonneuronal cells are identified by blue arrows and neurons by red arrows. C, DRG cultures primed by peripheral nerve crush in vivo were infected with Ad-E2F1 beginning 0, 6, 24, or 72 h after plating. E2F1 expression was assayed by immunoperoxidase histochemistry 24 h after infection. The percentage of neurons that stained positive for E2F1 increased as the delay between time of plating and time of infection increased so that by 72 h after plating, most of the neurons were positive for E2F1 expression. These results represent the means for four separate experiments; bars, SE. D, removal of serum and laminin had no impact on the number of infected neurons in these cultures. Neurons from axotomized animals were plated for 48 h and then infected with AdE2F1/AdDP1. After 24 h, cells were processed for E2F1 immunostaining. The percentage of E2F1-positive cells was determined for each condition. These results represent the means for three separate experiments; bars, SE.

 
We found that several manipulations increased the efficiency of neuronal infection, as measured by E2F1 immunostaining. The most effective factors were using a high viral titer and prior sciatic nerve crush in vivo (data not shown). Allowing 24–72 h plating period prior to addition of viruses also increased infection efficiency so that we routinely observed that 70–90% of neurons expressed detectable E2F1 1 day after infection (Fig. 2C)Citation . Plating neurons on polylysine instead of laminin and with defined media supplement instead of serum did not change the efficiency of infection of cultured neurons (Fig. 2D)Citation .

E2F1 or E2F2 Overexpression Can Stimulate DNA Synthesis in Neurons and Nonneuronal Cells in the Absence of Serum or Laminin.
Using the conditions under which adenovirus vectors efficiently infect both neurons and nonneuronal cells and stimulate the abundant expression of the exogenous proteins, we examined the effects of E2F1 and E2F2 overexpression on BrdU incorporation. Because E2F activity represents a heterodimer of an E2F protein and a DP protein, we always coinfected cells with a recombinant virus carrying the DP1 gene when using the Ad-E2F1 or Ad-E2F2 viruses so as to ensure maximal production of E2F activity.

When cultured in medium supplemented with 10% horse serum on a laminin substratum, many nonneuronal cells were BrdU positive by 24 h after exposure to the virus (Fig. 3Citation A, white arrows). In stark contrast, neurons did not incorporate BrdU under these conditions (Fig. 3ACitation , blue arrow), despite the fact that the neurons expressed substantial levels of E2F. Strikingly, in cultures plated on polylysine-coated dishes, E2F1/DP1 activity was able to trigger BrdU incorporation in both neurons (Fig. 3BCitation , blue arrow) and nonneuronal cells (Fig. 3Citation B, white arrows). Neurons plated on laminin in the absence of serum did not incorporate BrdU (Fig. 3Citation , and C and D), and a transient encounter with serum was sufficient to block E2F-induced DNA synthesis, suggesting that multiple pathways can trigger suppression of E2F-induced S-phase in neurons.



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  		Fig. 3. E2F-overexpressing neurons incorporate BrdU when plated in the absence of serum or laminin. A and B, dissociated cultures of DRG cells, 24 h after plating on polylysine in the presence or absence of 10% horse serum, were infected with AdE2F2/Ad-DP1. BrdU (to 10 µM) was added 12 h later, and the cells were fixed at 36–40 h after plating. Cultures were double-labeled by immunofluorescence with an antibody to neurofilament protein to label neurons (red) and a BrdU antibody to mark cells that have replicated DNA (green). Neurons are identified by the blue arrows and nonneuronal cells by the white arrows. Neurons that incorporate BrdU in response to E2F appear as yellow. Bar, 50 µm. C and D, E2F2/DP1 infected cultures grown on laminin (C) in which only nonneuronal cells are BrdU positive are compared with E2F2/DP1-infected cultures in the absence of laminin (D) showing a BrdU-positive neuron. Bar, 50 µm. E, the percentages of BrdU-positive neurons and nonneuronal cells were determined after immunoperoxidase staining with an antibody against BrdU. Cultures were plated on poly-D-lysine (poly) or laminin (lam) in the presence or absence of 10% horse serum (Serum). Some cultures were supplemented with 50 ng/ml NGF. The data are expressed as means from four separate animals; bars, SD. All neurons and 400 nonneuronal cells in a 2-cm2 area in the middle of the culture dish were scored. F, to examine the effects of viral infection of neuronal loss, we compared ratios between the number of surviving neurons in infected cultures and uninfected cultures at the time of fixation. Although all infected cultures had fewer neurons than uninfected cultures, the number of neurons in cultures infected with the control virus (AdCon) was not significantly different from that of cultures infected with the E2F-carrying viruses, demonstrating that the effect on neuronal loss was not attributable to E2F overexpression but rather to nonspecific toxicity of viral infection. All neurons in a 2-cm2 area in the middle of the culture dish were scored. The data are expressed as means from four separate animals; bars, SD.

 
Counting the number of BrdU-positive neurons and nonneuronal cells from several experiments revealed that in the absence of serum and laminin, ~20% of E2F1/DP1- or E2F2/DP1-overexpressing neurons incorporated BrdU during the labeling period (Fig. 3Citation E, poly). Supplementing the culture medium with NGF modestly increased this percentage, so that ~25% of neurons were BrdU positive (Fig. 3Citation E, NGF/poly). Neurons in control cultures (AdCon) or in cultures containing serum or laminin did not incorporate BrdU to any significant amount. In control cultures, ~20% of nonneuronal cells incorporated BrdU in the absence of serum or laminin. E2F1/DP1 or E2F2/DP1 overexpression increased the number to about 30–40%, and NGF augmented this effect so that up to half of the nonneuronal cells were BrdU positive. The effect of E2F overexpression on nonneuronal cells was masked by the effects of serum, which stimulated BrdU incorporation in 80–90% of nonneuronal cells.

Our results indicate that a factor or factors in serum, as well as signals provided by laminin substratum, override a proliferative response triggered by ectopic E2F activity. An alternative explanation for these results is that serum and laminin were particularly toxic to neurons that had entered S-phase in response to E2F overexpression. This does not seem likely, however, because at the time of analysis, there were in general slightly more total neurons/dish in the presence of serum (Fig. 3F)Citation .

Overexpression of E2F Induces DNA Replication in Neurons.
Although the data shown in Fig. 3Citation is consistent with an induction of DNA synthesis in neurons in response to E2F, it is not possible to conclude from these results that this represents true DNA replication as would occur during S-phase. For instance, the incorporation of BrdU could reflect repair synthesis. Previously, we have addressed this question in fibroblast cultures by analyzing the DNA content of cells by fluorescence-activated cell sorter analysis, showing that E2F overexpression could yield cells with a G2 DNA content (19) . Such an analysis is not possible for the neuronal cultures because the neurons are not a pure population, and we would not be able to distinguish whether the replicated DNA was derived from the neurons or from the nonneuronal cells.

As an alternative approach, we have used FISH to assay for replicated genes in interphase nuclei. It has been shown previously that specific probes produce singlet signals in quiescent cells, whereas cells that have recently undergone replication often yield doublet signals (21 , 22) . As shown in Fig. 4Citation , a cosmid probe specific for the human elastin gene locus served as an indicator for the state of this particular DNA locus. As can be seen in Fig. 4, ACitation and B, the locus was unreplicated in serum starved Ref52 fibroblasts (A) and replicated in serum-induced cells (B). In addition, E2F-induced DNA replication in the Ref52 fibroblasts was also evident in this assay (Fig. 4C)Citation . The same assay was then applied to the E2F2-expressing DRG cultures. After DAPI staining, neuronal nuclei were identified based on their larger size as compared with nonneuronal nuclei, and the neuronal cell types were further confirmed by DIC microscopy before the FISH procedure. In the presence of serum, doublet signals were seen in nonneuronal nuclei but never in the neuronal nuclei (data not shown), consistent with the lack of BrdU incorporation in neurons grown in serum. In contrast, doublet signals were readily observed in neurons overexpressing E2F2 in the absence of serum (Fig. 4D)Citation . A quantitative analysis of data from these cultures is summarized in Fig. 4ECitation . From these data, together with the analysis of BrdU incorporation, we conclude that the overexpression of E2F2 in neurons leads to true DNA replication.



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  		Fig. 4. FISH analysis of DNA replication. A Spectrum Orange-labeled Williams syndrome probe that detects the human elastin gene locus was used as a marker for both E2F2- and serum-induced replication in interphase Ref52 cells (A–C) and neurons (D). Nuclei were counterstained with DAPI. Insets, the singlet or doublet signals digitally enlarged. Bar, 5 µm. A, a singlet signal, representing a nonreplicated DNA locus, can be seen in quiescent, serum-starved Ref52 fibroblasts. B, doublet signals, indicative of replication of the locus, are identified in serum-stimulated Ref52 fibroblasts. C, doublet signals, indicative of replication, are detected in E2F2-expressing Ref52 fibroblasts that were serum starved. D, doublet signal detected in an E2F2-expressing neuron from a DRG culture. Because morphology was somewhat disrupted by the FISH procedure, the location of neurons in DRG cultures was marked prior to FISH analysis. Neuron identification was further confirmed by the larger size of DAPI-stained nuclei. E, quantitative analysis of the number and percentage of doublet signals based on the FISH assays in Ref52 cells and DRG cultures plated in the absence of serum or laminin. A–D, arrows indicate positive FISH signals.

 
Collaborative Effects of Cell Cycle Regulatory Proteins in the Induction of S-Phase in DRG Neurons.
Because only 20% of neurons were induced to incorporate BrdU in response to E2F expression, we tested whether other known cell cycle-regulatory proteins could coerce more neurons to enter S-phase. Progression of quiescent fibroblasts into S-phase requires the activity of Ras, and although Ras alone is not sufficient to stimulate DNA synthesis, coexpression of Ras with Myc allows the activation of cyclin E-dependent kinase activity and induction of S-phase (18) . In the developing nervous system, however, Ras signaling has been implicated in processes other than proliferation including the regulation of neuronal differentiation and survival in response to neurotrophins (23, 24, 25, 26) and synaptic function in the adult nervous system (27) . This dichotomy of function motivated an exploration of the effects of Myc and Ras proteins on E2F-induced DNA synthesis in adult neurons and nonneuronal cells. In addition, because cyclin E-dependent kinase activity alone is sufficient to induce S-phase in quiescent fibroblasts (28) and is thought to be greatly reduced in the adult nervous system, we have also examined the effects of exogenous kinase activity in addition to Myc and Ras in the neuronal S-phase assay. We again used recombinant adenoviruses to overexpress these proteins, alone or in combination with E2F/DP1. All experiments were carried out in the absence of serum or laminin and with N1 additives and 50 ng/ml NGF included in the culture medium.

Neither Myc nor Ras alone were able to stimulate S-phase in neurons (not shown). Together, however, they produced a modest amount of BrdU labeling in neurons (Fig. 5A)Citation . Cyclin E-dependent kinase expression (by coinfection with Adcdk2 and AdCycE viruses) could also stimulate S-phase in some neurons (5–10%), somewhat less than that achieved by E2F2/DP1 (20% of neurons). Neither Myc/Ras expression nor cyclin E-dependent kinase expression had an additive effect with E2F1. However, when Myc, Ras, cyclin E, and cdk2 were all expressed along with E2F1/DP1, we observed a substantial increase in the proportion of BrdU-labeled neurons; up to 40% had incorporated BrdU. The same results were obtained when these other proteins were coexpressed with E2F2/DP1 (not shown). Virus infection resulted in a loss of some neurons by the time of fixation (Fig. 5B)Citation , but because this was also the case for neurons expressing the control virus, the increase in BrdU-positive neurons by cell cycle regulatory proteins is probably not attributable to differential survival or recruitment of a stem cell population. Our results indicate that fully mature neurons in the absence of serum and laminin undergo DNA replication in response to forced expression of combinations of G1 regulatory proteins.



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  		Fig. 5. Expression of G1 cell cycle regulatory proteins enhances E2F-induced DNA synthesis in neurons. A, dissociated cultures of DRG cells grown in the absence of serum or laminin and in the presence of NGF were infected with recombinant adenoviruses carrying Myc (Admyc) Ras (Adras), cyclin E (AdcycE), cdk2 (Adcdk2), or E2F1 (AdE2F1) alone or in the combinations indicated below the columns. No Virus, no adenovirus was included in the cultures. AdCon, the control virus carrying no inserted gene. BrdU was added at 24 h after infection, and cells were fixed at 36 h after infection. The percentage of BrdU-positive neurons was determined by counting neuronal nuclei that were positive for immunoperoxidase staining. All of the neurons in a 2-cm2 area in the center of the dish were counted. The data are presented as the means of three experiments; bars, SE. A similar result was obtained with E2F2 substituted for E2F1. B, the number of neurons/cm2 reveals that in general there are fewer neurons after viral infection, but the numbers are not dramatically different between cultures infected with AdCon and cultures infected with the whole battery of gene-carrying viruses. This suggests that the loss of neurons again represents nonspecific toxicity from viral infection, not expression of ectopic proteins. The data are representative of three experiments; bars, SE.

 
Discussion

As cells take on a differentiated character, they exit the cell cycle and lose the ability to respond to proliferative signals, entering a state of apparent permanent quiescence. Yet, relatively little is known of the underlying mechanisms that create the state where cells no longer respond to proliferative signals. Perhaps the most extreme example can be seen in the nervous system, where terminally differentiated neurons are characterized as having lost all capacity to proliferate and thus fail to regenerate.

In recent years, several investigators have reported success in triggering the initiation of the cell cycle in postmitotic cells (8 , 9 , 11, 12, 13, 14 , 29 , 30) . Most of these reports involve directly or indirectly stimulating the activity of E2F transcriptional regulators. Unfortunately, the data for triggering replication in neurons is sparse, and most frequently involves experiments in which it is difficult to ascertain whether truly postmitotic neurons reentered the cell cycle or whether developing neurons or neuronal stem cells were prevented from exiting the cell cycle at appropriate times. In addition, it is not always clear whether BrdU incorporation represents actual DNA replication or repair synthesis. We sought to gain a better understanding of the molecular events associated with the quiescence of adult neurons, reasoning that such information could be of significance when considering approaches to stimulating neuronal division or blocking tumorigenesis.

Our observation that dissociated sensory neurons from adult rats were indeed responsive to the actions of E2F and other proteins support the theory that fully differentiated cells do possess the capacity to re-enter the cell cycle and replicate DNA. In some experiments, 40% of mature, differentiated neurons were able to replicate DNA (as shown by FISH analysis) in response to adenovirus-mediated overexpression of cell cycle proteins. Simple viral infection with an equal titer of control virus did not induce this response. We do not believe these results are attributable to differential survival or differentiation of a stem cell population because in general, similar numbers of neurons were present in control and E2F-expressing cultures. Furthermore, because staining with neuronal markers can label stem cells and newly differentiated neurons as well as adult neurons, we also used the size and morphology of adult DRG neurons as a guide for neuronal identity, because these characteristics are quite distinctive. We strongly believe the cells we were scoring as BrdU-positive cells were fully mature adult DRG neurons.

Although 40% of neurons showed evidence of initiating DNA synthesis in response to the combined activities of E2F/DP1, Myc, Ras, and cyclin E-dependent kinase, a smaller percentage were BrdU positive when subsets of these proteins were overexpressed. This may reflect the fact that DRG neurons are morphologically and functionally heterogeneous (31) and may have distinct responses to the cell cycle regulatory proteins used in these experiments. Alternatively, this may represent a quantitative effect whereby the extent of accumulation of key S-phase-promoting activities, such as E2F and cyclin E/cdk2, is enhanced. In addition, although it is clear from these studies that overexpression of E2F activity can stimulate S-phase in neuronal cells, it is not clear whether these cells can undergo a normal mitotic event. We have found no evidence for mitotic events or a new round of DNA synthesis in the E2F-overexpressing cells, suggesting that other molecular signals are responsible for sister chromatid separation and continued cycling. Because viruses themselves are toxic to neurons over time, our system will have to be modified to study the effects of deregulated E2F activity on entry into these later stages of the cell cycle.

Another important outcome of our investigations is the discovery that environmental factors have dramatic effects on the responsiveness of neurons to E2F activity. In control cultures, as expected, the majority of nonneuronal cells proliferated in response to 10% horse serum, which contains a variety of factors that stimulate proliferation in many cell types. Neurons in the same cultures remained quiescent. E2F activity could trigger DNA replication in neurons, but surprisingly, the presence of serum in the culture medium completely blocked this response. Indeed, if serum was included in the dissociation process for several minutes, neurons become refractory to S-phase induction by G1 regulatory proteins, suggesting that a transient association of the signaling factor(s) was sufficient to exert the effect. Although we do not know the nature of the inhibitory factor(s) present in serum, it is possible that the same serum growth factors that generally stimulate proliferation might produce a distinct response in neurons and other terminally differentiated cell types. Alternatively, neurons may be selectively responsive to inhibitory molecules present in serum. Indeed, there is accumulating data describing the control of proliferation in neuronal precursors by secreted factors, including fibroblast growth factors, bone morphogenic proteins, leukemia inhibitory factor, pituitary adenylate cyclase-activating polypeptide, sonic hedgehog, and a variety of neurotrophins (32, 33, 34, 35) .

Neuronal DNA synthesis was also blocked to a large extent by plating cells on a laminin substratum. Signaling through integrin receptors thus seems to intersect with and inhibit the effects of ectopic E2F activity in neurons but not in other cell types present in the cultures. Several recent reports demonstrate inhibitory effects of isoforms of the ß1 integrin subunit on cell cycle progression (35, 36, 37) , and ß1 has been implicated in regulating neuronal differentiation and axon growth (38 , 39) . The fact that, in our experiments, laminin does not inhibit S-phase in nonneuronal cells could reflect a difference in integrin subunit composition or in the downstream signaling pathways impinging on the changes produced by E2F activity.

Our results indicate that neurons must overcome several levels of regulation to replicate DNA. First they must accumulate sufficient levels of G1 regulatory factors to overcome the effect of inhibitory proteins designed to prevent transcription of genes important for S-phase progression. Several studies have pointed to the importance of known cdk-inhibitory proteins in maintaining a postmitotic state, and Rb family members undoubtedly also play a role. However, even with sufficient expression of E2Fs and cyclins, neurons will not replicate DNA unless extracellular inhibitory cues are removed from the environment. It will be important to determine the molecules responsible for these inhibitory cues and to uncover the signaling pathways that interfere with E2F-induced S-phase entry.

Materials and Methods

Sciatic Nerve Lesions.
Adult rats were anesthetized by i.p. injection of ketamine/chloral hydrate, and the sciatic nerves were exposed at the hip by blunt dissection and transected. This procedure interrupts approximately 70–80% of the axons emerging from the L4 and L5 dorsal root ganglia (40) . After nerve transection, rats were allowed to survive for 2–3 days before the axotomized DRGs were removed and cultured.

DRG Cultures.
DRG cultures were prepared as described previously (41) . Briefly, individual ganglia were snipped open with iris scissors, washed three times in F14 medium (Sigma), and exposed to 5 mg/ml collagenase (type XI; Sigma Chemical Co.) for 90 min. Ganglia were triturated through a flamed Pasteur pipette and then plated in tissue culture dishes coated with either poly-D-lysine (0.5 mg/ml; Sigma) or with poly-D-lysine and laminin (10 µg/ml; Upstate Biotechnologies, Inc.). Approximately 105 neurons were plated per 35-mm dish in F14 medium supplemented with either 10% horse serum or N1 additives (Sigma), with or without 50 ng/ml NGF. The number of nonneuronal cells per culture varied, but these cells were ~10 times more abundant than neurons prior to proliferation.

Recombinant Adenoviruses.
Viral stocks were created as described previously (42) . Infection efficiency in neurons was determined by immunostaining for the encoded gene product. We used a multiplicity of infection of 500-1000 fluorescence-forming units/ml as determined by immunofluorescence of the viral 72k protein in 293 cells. When multiple viruses were used together, the amount of individual viruses was decreased so that the total multiplicity of infection was the equivalent of 1000 fluorescence-forming units/ml in 293 cells. Cells were generally fixed 36 h after infection.

Immunostaining and Western Blotting to Determine Expression of Exogenous Proteins.
Cultures were fixed for 10 min at -20°C in methanol:acetone (1:1) and for an additional 10 min at room temperature in 4% paraformaldehyde. All antibodies used to detect overexpressed proteins were purchased from Santa Cruz and used at a dilution of 1:500 for immunostaining and 1:1000 for Western blotting. A monoclonal antibody to GAP-43 (gift of J. H. P. Skene, Duke University, Durham, NC), and a polyclonal IgG fraction of rabbit antiserum to neurofilament H (Sigma) were used to identify neurons. Immunoperoxidase labeling was carried out using an ABC amplification kit (Vector Labs). FITC and Texas Red conjugated sheep or mouse IgG was used for immunofluorescence. For Western blotting, DRG cultures were lysed in sample buffer, separated on a 10% acrylamide SDS gel, transferred to Immobilon-P (Millipore) nylon membrane, and probed for overexpression with specific antibodies.

BrdU Detection.
Cells were exposed to 10 µM BrdU for the indicated times then fixed for 10 min at -20°C in methanol:acetone (1:1) and for an additional 10 min at room temperature in 4% paraformaldehyde. After fixation, cells were exposed to 0.2 M HCl for 15 min. Incorporated BrdU was visualized by immunoperoxidase staining using an anti-BrdU antibody (Amersham) diluted 1:3 in blocking solution. Cells were counterstained with hematoxylin or antibodies to GAP-43, neurofilament H, or S100, and the percentage of each cell type (neurons and nonneuronal cells) that had incorporated BrdU during the labeling interval was determined by counting at x40 using DIC microscopy. A 2-cm2 area was scored in each dish.

FISH Assays for DNA Replication.
The DNA probe for Williams syndrome (maps to human 7q11.23) was used for this analysis. The probe contains three contiguous cosmids containing loci for elastin, LIMK1, and D7S613 (Ref. 43 ; Vysis, Inc., Downes Grove, MD). The signals (one on each homologue) are visible at interphase and appear as doublets when chromosomes have replicated the targeted sequence. DRG cultures on glass chamber slides were infected and then processed for FISH 36 h after infection. Cultures were fixed in 4% paraformaldehyde for 20 min and then in methanol:acetone (1:1) for 5 min at 4°C. At this point, the exact location of specific neurons was marked on the slide, because the denaturing conditions that follow this step are disruptive to normal cell morphology. Cells were denatured by treatment with 70% formamide at 75°C for 2 min and then dehydrated through 70, 85, and 100% cold ethanol and allowed to air dry. The probe was applied along with a coverslip. The hybridization was carried out for at least 14 h at 37°C in a humidified chamber. After stringency washes, the slides were mounted with a DAPI (fluoresces blue) in antifade solution. A Zeiss Axiophot epifluorescence microscope equipped with a cooled charge coupled device was used for image acquisition. All images were taken via x63 oil immersion lens. Sequential images of single interphase nuclei were acquired using an excitation wavelength appropriate to excite the fluorescent molecules (red and blue). The images were collected and processed using an image acquisition software (Vysis, Inc.).

Acknowledgments

We thank Kaye Culler for help with the preparation of the manuscript and James McNamara and Li-Huei Tsai for comments on the manuscript.

Footnotes

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 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Howard Hughes Medical Institute. D. S. S. was supported by an NIH postdoctoral fellowship, G. L. was supported by a fellowship from the MRC (Canada), and J. D. was supported by the Howard Hughes Medical Institute. Back

2 Present address: Department of Pathology, Harvard Medical School, Boston, MA 02110. Back

3 Present address: Division of Human Cancer Genetics, The Ohio State University, Columbus, OH 43210. Back

4 Present address: Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, Denver, CO 80262. Back

5 To whom requests for reprints should be addressed, at Department of Genetics, Durham University Medical Center, Box 3054, CARL Building, Room 268, Research Drive, Durham, NC 27710. Back

6 The abbreviations used are: BrdU, bromodeoxyuridine; Rb, retinoblastoma; DRG, dorsal root ganglion; FISH, fluorescence in situ hybridization; NGF, nerve growth factor; cdk, cyclin-dependent kinase; DAPI, 4,6-diamido-2-phenylenediamine; DIC, differential interference contrast. Back

Received for publication 6/ 2/00. Revision received 10/19/00. Accepted for publication 10/20/00.

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