Analysis of Events Leading to Neuronal Death after Infection with E1-Deficient Adenoviral Vectors

MCN

Molecular and Cellular Neuroscience 11, 334–347 (1998) Article No. CN980690

Analysis of Events Leading to Neuronal Death after Infection with E1-Deficient Adenoviral Vectors Rachael M. Easton, Eugene M. Johnson, Jr., and Douglas J. Creedon Department of Neurology and Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110-1031

Although recombinant adenoviral vectors are being widely used to target genes to the nervous system, the cellular and genetic effects of recombinant adenoviral infection on neuronal function have not been well characterized. Using sympathetic neuronal cultures, we analyzed the effect of adenoviral infection on viral and neuronal gene expression and on neuronal function and viability. While a delayed cytotoxicity occurred 5 days after infection, numerous biochemical and genetic perturbations occurred within the infected cell prior to this time. This study demonstrates that numerous cellular alterations were produced by recombinant adenoviral vectors and, therefore, emphasizes the need for an analysis of the effects of these viral vectors on neuronal function in the interpretation of data regarding transgene expression induced by these vectors in neurons. It also suggests that continued improvements made to the viral vectors themselves might decrease this direct cytotoxicity and lead to improved safety and function of recombinant adenovirus in vivo.

INTRODUCTION Postmitotic neurons are resistant to standard methods of transfection and cannot be infected by commonly used retroviral constructs. Thus, the neuroscience community has investigated the potential use of DNA viruses for expressing foreign genes in neurons. Since the initial finding that adenovirus, a DNA virus that naturally targets the respiratory epithelium, is also capable of infecting neurons (Le Gal La Salle et al., 1993), adenoviral vectors have been used to express foreign genes in a number of neuronal populations both in vitro and in vivo. To date, recombinant adenoviral vectors are being investigated for their use in studying gene function, delineating neuronal connections (Moriyoshi et al., 1996), promoter analysis (Robert et al., 1997), and gene

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therapy [reviewed in Davidson and Bohn (1997) and Horellou et al. (1997)]. The most widely used recombinant adenoviral vectors lack the early region 1 genes, E1A and E1B, which are required for viral replication, and/or the early region 3 (E3) genes, which are not essential for viral replication. In our analysis, we sought to characterize E1-deleted recombinant adenoviral vectors that also contain a partial deletion in the E3 region in sympathetic neuronal cultures. We chose this recombinant adenoviral vector because of the availability of this system and the relative ease of production of high-titer recombinant viral stocks. For these same reasons, the E1-deleted recombinant adenoviral vectors are among the most widely used vectors by the neuroscience community. Our goal was to use recombinant adenovirus to perform a populationbased analysis of foreign gene expression on programmed cell death. Therefore, we determined the conditions whereby these adenoviral vectors infect and induce gene expression in the majority of neurons in the culture. Early in our studies we observed cytotoxicity with the vectors that could not be overcome by modification of the methods of preparation or purification. Similar toxicity was seen with multiple adenoviral preparations produced by different laboratories. Therefore, we set out to define the limits of the usefulness of these vectors in our paradigm and to elucidate the mechanism of neurotoxicity. We report that well before signs of overt toxicity in the neurons, a sequence of events occurred involving the expression of viral genes, induced expression of cellular genes, evidence of a cellular stress response, and decreases in protein synthesis. Our analysis indicates that these viral vectors exert time-dependent deleterious effects in sympathetic neurons. Although our analysis involved only a single neuronal type, it suggests that similar effects are pro1044-7431/98 $25.00 Copyright r 1998 by Academic Press All rights of reproduction in any form reserved.

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duced in other neurons and that these perturbations complicate the interpretation of data regarding transgene function driven by the use of these vectors.

RESULTS Recombinant Adenovirus Efficiently Infects Sympathetic Neuronal Cultures Because neurons are resistant to standard methods of transfection, we turned to recombinant viral vectors to express foreign genes in sympathetic neurons in vitro. Our goal was to find a vehicle that would allow delivery of the foreign gene to the majority of neurons in the culture and thereby permit a population-based analysis of gene function. We tried recombinant adenovirus because of its ability to infect nondividing cells and the relative ease of production of recombinant vectors. To determine the utility of adenovirus in our culture paradigm, we began by defining the possible range of neuronal infection. Sympathetic neurons, which had been maintained for 5 days in vitro, were infected with different dilutions of adenovirus and stained 3 days after infection. Based on the amount of virus used for the infection and the number of neurons in the culture, a multiplicity of infection (m.o.i.) was calculated. m.o.i. refers to the number of viral plaque-forming units (PFU) present per neuron in the culture. The neuronal cultures were stained for a fixed amount of time to compare infections with different m.o.i.s. The amount of infection increased linearly at low m.o.i.s, but leveled off above an m.o.i. of 100 (Fig. 1A). Over an m.o.i. of 100, the intensity of staining increased with increasing amounts of virus. For example, at an m.o.i. of 400, the transgene filled the neurites and the staining of the cell bodies appeared darker (data not shown). Although m.o.i. is often used to report the amount of virus used for the infection, it most likely does not adequately represent the number of viral particles infecting, or even encountering, each neuron. m.o.i. is calculated based on the infection of confluent 293 cells, which are plated on plastic tissue culture dishes. In our neuronal cultures, however, the neurons are sparsely plated only in the center of a tissue culture dish, which is coated with a layer of collagen. Because of this marked difference in culture geometry and substratum, traditional viral m.o.i.s deduced from infection of a confluent or nearly confluent monolayer on plastic are not directly predictive of a given m.o.i. to infect cultured neurons. Therefore, rather than rely on a calculated m.o.i. to determine the amount of virus to use for each infection, we have used an empirical method for this determina-

FIG. 1. Adenoviral toxicity occurs with less than maximal infections. (A) Neuronal cultures infected with an m.o.i. range of 1 to 400 were stained with X-gal 3 days after infection; the percentage of X-galpositive neurons (stained blue) was determined. Strips were counted through the center of the culture (approximately 300 neurons/strip) to assess the percentage of X-gal-positive neurons. Three wells were counted for each m.o.i. Mean 6 standard deviation is plotted. In parallel, infected cultures were maintained for an additional 4 days in vitro and then labeled for 4 h with [ 35S]methionine on day 7 postinfection. The cell lysate was collected and incorporated radioactivity measured as described under Experimental Methods. The nonneuronal background was subtracted and the infected cultures are expressed as a percentage of uninfected neurons maintained in NGF. The graph depicts one experiment in which three wells were measured for each condition. Mean 6 standard deviation is shown. Similar results have been obtained in two separate experiments with Ad5CMV.lacZ, as well as Ad5RSVntlacZ. (B) The data shown in (A) are replotted as protein synthesis rate versus percentage neurons infected. The linear regression and correlation coefficient (R) are shown.

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tion. For each experiment, a range of viral dilutions was tested on neurons after 3 days in vitro. After a 48-h infection, the neurons were stained for b-galactosidase. The dilution of adenovirus that yielded approximately 70–90% X-gal-positive neurons was then used to infect parallel cultures at 5 days in vitro. We found that this functional definition of infection level guarded against neuronal culture variations, any variations in titering methods, or changes in titer that may have occurred because of numerous freeze/thaw cycles. In the following experiments, we have used this functional definition to determine the amount of virus to use in each assay, unless otherwise stated.

Adenoviral-Infected Sympathetic Neurons Demonstrate Toxicity 5 Days after Infection Although neurons infected for 48 h appeared healthy, 6 to 7 days after infection morphological changes, including shrunken cell bodies and fragmented neurites, were apparent by phase-contrast microscopy (Fig. 2A) and, at later time points, these neurons went on to die. We observed this delayed neurotoxicity in several different adenoviral preparations, including adenoviral vectors expressing genes other than b-galactosidase and viral preparations produced from crude cell lysates rather than purified with a cesium chloride gradient (data not shown). We chose to study the effects of the cesium chloride-purified, b-galactosidase vector in more detail because it is commonly used in the literature as a control vector. In addition, evidence indicates that a more purified viral stock is less toxic (Caillaud et al., 1993). To analyze the effect of this recombinant viral vector on cellular function and to determine whether even earlier effects were produced by viral infection, we measured protein synthesis rates; protein synthesis is affected in many cell types by infection with a number of different viruses, including adenovirus (Bello and Ginsberg, 1976; Beltz and Flint, 1979). Upon analysis of protein synthesis rates, we found a toxic effect after 5 days of infection. At this time, protein synthesis rates were decreased by 40% compared to control. To ensure that this toxicity was not caused by the methods employed by our laboratory to produce the recombinant viral vectors, we tested recombinant adenovirus from an off-site viral facility (Gene Transfer Vector Core, University of Iowa College of Medicine). This adenoviral vector (Ad5RSVntlacZ) drove expression of lacZ from a Rous sarcoma virus (RSV), rather than a cytomegalovirus (CMV) promoter. Seven days after infection with Ad5RSVntlacZ, the protein synthesis rates in the in-

FIG. 2. Adenoviral infected SCG neurons demonstrate toxicity 5 days after infection. (A) At 5 days in vitro, neurons were infected with Ad5CMV.lacZ to yield 70–80% X-gal-positive neurons after a 48-h infection. Infected cultures were fixed and stained for b-galactosidase on the third (d3), fifth (d5), or seventh day (d7) after infection. Arrows indicate shrunken neuronal cell bodies expressing b-galactosidase. Uninfected cells (ctrl) were stained in parallel. Shown here are uninfected neurons stained on day 3 (ctrl) (bar, 50 µm). (B) At 5 days in vitro neurons were infected with Ad5CMV.lacZ to produce 81% X-gal-positive neurons at day 4 after infection. Protein synthesis assays were performed 3, 4, 5, 6, or 7 days after infection. Four wells were measured for each condition. The nonneuronal background was determined by measuring the protein synthesis rates of cultures that had been deprived of NGF since day 3 in vitro. The nonneuronal background (8.9 6 4.8% of total) was then subtracted from both the infected and the uninfected (control) cultures. The wells were averaged, and the protein synthesis of the infected cells is expressed as a percentage of the protein synthesis of the control cultures. The mean 6 the standard deviation is shown. Similar results were obtained in at least three separate experiments.

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fected cultures were measured and compared to control cultures. Similar to the Ad5CMV.lacZ vector prepared in our laboratory, infection with Ad5RSVntlacZ produced a decrease in protein synthesis in the infected neuronal cultures. The infected neurons had protein synthesis rates that were 20 6 4% of control values. The Ad5RSVntlacZ stock tested above had the same particleto-PFU ratio as the Ad5CMV.lacZ viral stock. We therefore tested additional adenoviral preparations to determine whether the particle-to-PFU ratio influenced this delayed cytotoxicity. We compared viral preparations with particle-to-PFU ratios of 25, 50, and 100 and found no significant difference in their effect on the neuronal cultures. These preparations all produced decreased protein synthesis at 7 days after infection; the protein synthesis rates of neuronal cultures infected with adenoviral stocks having a particle ratio of 25, 50, or 100 were 22 6 2%, 30 6 5%, and 9 6 4% of control, respectively. To control for the possibility that the toxicity observed with the lacZ vectors was due to the expression of lacZ, we tested an adenoviral vector that expressed NGF rather than b-galactosidase. As observed with a similar m.o.i. of the lacZ vector, the NGF-expressing vector produced a decrease in protein synthesis. Protein synthesis fell to 22 6 2% of control at 7 days after infection. We conclude that the transgene itself was not the cause of the toxicity. In the following experiments, we further characterize this observed toxicity using the b-galactosidase-expressing virus. We have chosen to characterize this virus because of the long history of use of b-galactosidase as a marker gene and the ease and sensitivity of detection of infected neurons afforded by this marker. To determine whether a decrease in protein synthesis at earlier time points may have been masked by an increase in the production of adenoviral or foreign proteins, neurons were labeled with [ 35S]methionine at 3, 5, or 7 days after infection, and the cellular lysate was separated on a gradient gel to visualize individual protein levels (Fig. 3). A global decrease in protein synthesis was evident in neurons infected with the higher m.o.i. by 5 days postinfection and in the lower m.o.i. by 7 days.

Toxicity Occurs with Lower Amounts of Virus One of the aims of functionally assessing the titer of virus required to infect the neurons from a particular plating was to avoid supermaximal infections, which might overwhelm the neurons and produce toxicity. Neurons were infected with a range of dilutions and the protein synthesis was measured 7 days after infection, a time when we had previously observed a large decrease

FIG. 3. Adenoviral infection produces a decrease in global protein synthesis. After 5 days in vitro, SCG cultures were mock-infected (A) or infected with Ad5CMV.lacZ at an m.o.i. of 10 (B) or an m.o.i. of 40 (C). Three, five, or seven days postinfection (dpi), the cultures were pulsed with Tran [ 35S]methionine for 90 min and the labeled proteins were separated on a 5–20% SDS–PAGE gradient gel. Protein sizes in kDa are shown on the left.

in protein synthesis (Fig. 2). As the number of infected neurons increased, protein synthesis decreased correspondingly (Fig. 1). We found a linear relationship between the amount of virus and the inhibition of protein synthesis (Fig. 1B). Although the relationship was linear, the toxicity was not necessarily produced by the recombinant virus itself; any toxic particle that covaries with the amount of virus, such as a toxic substance produced by the vector itself, a contaminant in the cesium preparation, or a revertant wild-type adenovirus, could have been the source of the toxicity. To examine the possibility that the particular vector was producing a toxic substance, we tested a recombinant virus generated from an independent construct in which the RSV promoter drove b-galactosidase expression (Ad5RSVntlacZ). Similar to what had been observed with the Ad5CMV.lacZ virus, neurons infected with Ad5RSVntlacZ also showed a decrease in protein synthesis that correlated with the level of infection (data not shown).

Toxicity Is Not Due to a Contamination of the Recombinant Virus A contaminant from the cesium preparation is another possible source of the toxicity. To check for the presence of such a contaminant, we analyzed the pro-

338 tein synthesis rates of neurons that had been infected with a heat-inactivated Ad5CMV.lacZ. Heat inactivation by heating the virus at 65°C for 10 min will render the virus incapable of infecting the host cell. This was verified by staining these neurons for b-galactosidase (Fig. 4A). Seven days after a 48-h treatment with heat-inactivated virus, neuronal protein synthesis had not decreased (Fig. 4B). In the same experiment, neurons

FIG. 4. Toxicity is not caused by a contaminant from the viral preparation. After 5 days in vitro, SCG neurons were infected with Ad5CMV.lacZ to yield approximately 80% X-gal-positive neurons after a 48-h infection (INF). In parallel, neuronal cultures were infected with a similar amount of virus that had been heat-inactivated by treatment at 65°C for 10 min (H.I.) or mock-infected (CTRL). (A) Seven days after infection neuronal cultures were fixed and stained with X-gal. X-gal-positive neurons appear darker than control. The neurons infected with the heat-inactivated virus have no staining; compare to the dark staining in the infected cultures. (2003 magnification) (B) In parallel, neurons were labeled with [ 35S]methionine for 4 h and the radioactivity in the cell lysate was determined as described under Experimental Methods. The nonneuronal background (8.3% of total) was subtracted and the infected cultures are expressed as a percentage of the uninfected neurons maintained in NGF. The graph depicts the results of one experiment for which three wells represent each condition. The mean 6 standard deviation is depicted. Similar results were obtained in a separate experiment.

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FIG. 5. Toxicity is not caused by contamination by wild-type adenovirus. Viral preparations were screened for possible contamination by wild-type adenovirus using PCR as described under Experimental Methods. The PCR was then separated on a 5% polyacrylamide gel, dried, and analyzed by using a PhosphorImager. The following samples are shown: lane (1) 106 PFU Ad5CMV.lacZ, (2) 107 PFU Ad5CMV.lacZ, (3) H2O, (4) 10 PFU Ad5 wild-type, (5) 25 PFU Ad5 wild-type. This analysis was performed at least twice for each viral preparation used.

infected with a similar dilution of untreated virus showed a 62% reduction in protein synthesis (Fig. 4B). Heat inactivation rules out the possible contamination by cesium chloride or glycerol, but does not eliminate the possibility of a revertant wild-type adenovirus. Viral stocks were assessed for wild-type adenovirus contamination by using both a PCR-based analysis and growth on A549 cells, a human carcinoma cell line sensitive to infection by wild-type, but not recombinant, adenovirus. No wild-type adenovirus was detected by PCR in 107 PFU of recombinant adenovirus. The same PCR detected as little as 10 PFU of the wild-type adenovirus type 5 stock (Fig. 5). This PCR analysis was performed at least twice for each adenoviral preparation. In an independent assay for wild-type adenovirus, A549 cells were exposed to dilutions of recombinant adenovirus stock (1023 to 1025 ), dilutions of wild-type adenovirus type 5, or mock-infected. After 48 h, the virus-containing medium was removed and fresh medium was added. Every 4 to 6 days, two-thirds of the medium was removed and replaced with fresh medium. The cells were followed for 3–4 weeks. Within this time, 10 PFU of wild-type virus had lysed the A549 cells, but no lysis was observed on either the recombinant or the mock-infected cells. This was repeated at least twice for each viral preparation. Therefore, contamination of the vector with wild-type virus cannot account for the neurotoxicity.

Toxicity Depends on Neuronal Age in Vitro As superior cervical ganglion (SCG) neurons age in culture, they become more resistant to many different insults, including trophic factor deprivation (Lazarus et al., 1976; Chun and Patterson, 1977) and 6-hydroxydopa-

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48, 72, or 96 h postinfection, neurons were fixed and stained with an antibody directed against phosphorylated c-Jun. Only a background, diffuse staining was present in mock-infected neurons (Fig. 7a). At 48 h, little staining was evident in the infected neurons; however, by 72 h, many nuclei were brightly stained (Fig. 7b). Positive nuclei were also observed at 96 h postinfection (data not shown). This same response was seen with AdRSVntlacZ (Fig. 7c). Therefore, the neurons infected with recombinant adenovirus manifest a ‘‘stress response,’’ as evidenced by the phosphorylation of c-Jun.

Adenoviral Genes Are Expressed in Infected Neurons FIG. 6. Onset of toxicity depends on neuronal age in vitro. After 14 days in vitro, sympathetic neurons were infected with a dilution of Ad5CMV.lacZ that produced approximately 80% infected cells after a 48-h infection. Protein synthesis rates were determined as stated under Experimental Methods. The graph represents the mean and range of two separate experiments in which three or four wells/ condition were assayed. Nonneuronal background has been subtracted; the protein synthesis of the infected cultures is expressed as a ratio of the uninfected controls.

Others have demonstrated that viral genes are transcribed and translated in nonneuronal cells infected with adenoviral vectors (Nevins, 1981; Rich et al., 1993; Yang et al., 1994a, b). Viral gene expression by recombinant adenoviral constructs has not been examined in neurons and could be one potential cause of the toxicity in our neuronal cultures. The study of the adenoviral genome has revealed that adenoviral genes can be toxic

mine treatment (Thoenen and Tranzer, 1968; Tranzer and Thoenen, 1968; Tranzer et al., 1969). Therefore, we tested whether neurons became more resistant to adenoviral-induced toxicity as they aged in vitro. Neurons that had been maintained in culture for 14 days were infected with Ad5CMV.lacZ to produce a 70–90% infection after 48 h, and protein synthesis was measured 3, 5, 7, and 10 days after infection (Fig. 6). Although mature neurons were equally well infected with adenovirus, the older neurons were moderately more resistant to the toxicity of adenovirus. The older neurons did, however, eventually manifest toxicity as indicated by decreased protein synthesis rates (Fig. 6) and visual inspection (data not shown). Thus, for SCG neurons, the onset of toxicity was dependent on the age in culture.

Recombinant Adenoviral Infection Activates c-Jun During many cellular stress responses, c-Jun becomes activated by phosphorylation of serine residues 63 and 73 within the transactivation domain [reviewed in Karin (1995) and Karin and Hunter (1995)]. We examined whether infection with a recombinant adenoviral vector could activate c-Jun by staining with antibodies directed against c-Jun protein that had been phophorylated on serine residue 63. Neurons were infected with a dilution of adenovirus that produced 50% infection after 48 h. At

FIG. 7. Infection with a recombinant adenoviral vector triggers c-Jun phosphorylation in infected neurons. After 5 days in vitro, sympathetic neurons were infected with Ad5CMV.lacZ (b, e) or Ad5RSVntlacZ (c, f) to produce a 50% infection after 48 h. Infected cultures were fixed at 72 h after the initial infection and stained with an antibody directed against the phosphorylated form of c-Jun. Phospho c-Jun immunoreactivity is observed in the infected neurons (b, c), but not in the mock-infected controls (a). The nuclei for the mock-infected (d) and infected (e, f) neurons were stained with Hoechst 33258. The phospho c-Jun protein localizes to the nucleus of the infected neuron and, by Hoechst 33258, changes can be detected in the nuclear morphology in the infected cultures (e) (bar, 20 µm).

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when expressed in trans (Klessig et al., 1984). Therefore, we examined the expression of genes encoded by the E3 and E4 regions of adenovirus to determine whether they were expressed and, if so, whether the time course of expression correlated with the cellular stress response, which was indicated by the detection of phosphorylated c-Jun in the neuronal nuclei. Indeed, we found that infected neurons expressed these adenoviral genes; 16 h after infection, mRNA transcripts produced by the adenoviral regions E3 and E4 were detected by reverse transcription (RT)-PCR analysis (Fig. 8). The expression of E4 mRNA reached maximal levels 36 h postinfection, whereas E3 mRNA increased with slower kinetics and reached maximal levels by 72 h postinfection. The induction of the adenoviral genes E3 and E4 occurred prior to the phosphorylation of c-Jun and the decrease in protein synthesis (Fig. 10). Thus, based on the time course of events, viral gene production may have produced the toxicity observed in the infected neuronal cultures.

Perturbations in Cellular Gene Expression Occur in Infected Neurons Viral gene products interact with the host proteins to provide a suitable environment for viral replication. For example, the adenoviral gene product E4orf6/7 interacts with the cellular transcription factor E2F to activate transcription of both adenoviral genes and cellular genes important for DNA replication (Adams and Kaelin, 1996; Cress and Nevins, 1996). During recombinant adenoviral infection, transcription from the E4 region occurs, as evinced by expression of E4orf1. Presumably, several E4 proteins, including E4orf6/7, could be produced in infected neuronal cultures and alter cellular function. We, therefore, examined whether cellular gene expression changed in infected neuronal cultures and, if

FIG. 8. Adenoviral genes are expressed early after infection with a recombinant adenoviral vector. After 5 days in vitro, SCG neurons were infected with Ad5CMV.lacZ. The neurons were lysed at various times (0, 4, 6, 9–120 h) after infection. The mRNA was isolated, reverse transcribed to produce cDNA, and analyzed by PCR with primers specific to the E3 and E4 regions of the adenoviral genome. No product is amplified from an uninfected neuronal culture (U). In addition, no product is amplified from non-reverse-transcribed RNA isolated from infected cultures (R), indicating that the isolated mRNA is not contaminated by viral DNA.

FIG. 9. Cellular genes are induced after adenoviral infection. After 5 days in vitro, SCG neurons were infected with Ad5CMV.lacZ. The neurons were lysed at various times (0, 4, 6, 9–120 h) after infection. The mRNA was isolated, reverse transcribed to produce cDNA, and analyzed by PCR with primers specific to hsp70, c-myb, or cyclin D1. Quantitation of the PhosphorImager data is shown in the graph. mRNA levels of these genes do not change in mock-infected neurons (data not shown).

so, whether the changes in gene expression occurred with a time course consistent with induction by viral genes. We found that c-myb and cyclin D1, which are regulated by E2F (Adams and Kaelin, 1996), were induced in the infected neurons. Induction of these genes occurred between 32 and 56 h postinfection (Fig. 9), a time after the induction of E4 (Fig. 10). The changes we observed in cellular gene expression were not caused by the presence of an undetected revertant wild-type adenovirus because infection with the recombinant virus produced a pattern of genetic changes different from that which has been observed previously in wild-type adenoviral infections. For example, hsp70 is induced after wild-type adenoviral infection and the induction of hsp70 is dependent upon the adenoviral E1A gene (Nevins, 1982; Kao and Nevins, 1983). In neuronal cultures that were infected with a recombinant adenovirus lacking the E1 region, no induction of hsp70 mRNA occurred over the same time course in which we observed the substantial induction of c-myb (Fig. 10). The level of hsp70 mRNA in infected cells was the same as that seen for mock-infected neurons (data not shown).

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FIG. 10. Summary of alterations in sympathetic neurons after infection with adenoviral vectors. Adenoviral vectors produce a series of changes in sympathetic neurons. Sequential observations are: (A) induction of the viral genes E4 and E3 (Fig. 8) followed by the expression of cellular genes cyclin D1 and c-myb (Fig. 9); (B) evidence of a cellular stress response as indicated by the appearance of phospho c-Jun in neuronal nuclei (Fig. 7); (C) decrease in global protein synthesis rate (Fig. 2); (D) obvious cytopathic effect observable by phase-contrast microscopy (Fig. 2), and, ultimate cell death. These changes are shown diagrammatically (E), suggesting a functionally linked series of events leading to the demise of the infected neuron.

DISCUSSION Toxicity of Recombinant Adenovirus Limits the Applicability of This Vector in Sympathetic Neurons in Vitro The ability of adenovirus to infect sympathetic neurons efficiently in vitro offers promise for the analysis of gene function in these neuronal cultures. This promise, however, is limited by the effects of the viral infection on neuronal function. Even before any change in neuronal viability was observed by light microscopy, marked perturbations occurred in cellular gene expression, in the phosphorylation status of c-Jun indicative of a cellular stress response, and in the rates of protein synthesis (Fig. 10). We presume that many other alter-

ations of neuronal function would be detected if examined. Therefore, these results indicate that one must consider the effects of the recombinant adenoviral vector itself in the interpretation of data regarding the function of a foreign gene expressed via these vectors. We observed that the deleterious effects of adenovirus occurred over time after the initial infection. Therefore, a time frame may exist in which the cell is not severely affected by the viral infection. From our analysis, no cellular alterations were detected within the first 48 h after infection, suggesting that a short-term analysis of foreign gene location or function could probably be done with confidence with these recombinant vectors. For our purposes, however, we were interested in a relatively long-term analysis of the effects of ectopic

342 gene expression on neuronal cell death. In the paradigm routinely used for such studies (Martin et al., 1988; Deckwerth and Johnson, 1993), neurons are infected at 5 days in vitro (div) and allowed to express the gene for 2 days prior to the induction of cell death. The effect of the foreign gene on the cell death process is determined 2–3 days after the induction of cell death, when detrimental effects of the viral vector itself are becoming manifest. Thus, we conclude that the E1-deficient adenoviral vector is not a reliable tool for the study of the effects of foreign gene expression in this neuronal cell death paradigm. Similar to our observations, a recent study by Slack and colleagues examined the function and viability of infected sympathetic neurons by electron microscopy, electrophysiological measurements, and MTT assay and concluded that the use of recombinant viral vectors is limited (Slack et al., 1996). Although we reached the same general conclusion, our biochemical and genetic analyses of recombinant adenoviral infection indicate a narrower window of opportunity for the study of foreign gene expression. Rather than concentrating on late time points, we assessed neuronal function over an extended time course after infection. This allowed the detection of early changes in neuronal function, including decreases in protein synthesis rates and changes in cellular and viral gene expression, not assessed in the previous characterization. The MTT assay, which reveals a 10–15% decrease in neuronal viability by 10 days postinfection (Slack et al., 1996), is a late indicator of cell dysfunction or loss and, thus, probably does not serve as a measure of the early changes in function that were detected by protein synthesis measurements (Fig. 2). For example, in neurons undergoing programmed cell death, protein synthesis rates fall well before decreases in MTT are observed (Deckwerth and Johnson, 1993). The window of opportunity for the study of a foreign gene product introduced by adenovirus is likely to vary depending on the neuronal culture being utilized. For example, although infected as quickly and to the same degree as young neurons, sympathetic neurons that had been maintained in culture for 2 weeks did not experience adenoviral-induced toxicity as rapidly as younger neurons. Although the time period when the neurons are relatively unaffected by adenoviral vectors may vary depending on the neuronal population, the eventual toxicity we observed in sympathetic neuronal cultures is likely to be a general phenomenon occurring with adenoviral infection of many neuronal types. For example, protein content and cell number decrease after recombinant adenoviral infection of dissociated co-cultures of spinal cord and dorsal root ganglia (Durham et al., 1996).

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In our analysis, we found that delayed toxicity also occurred with an adenoviral construct expressing NGF rather than b-galactosidase, strongly arguing that the transgene itself is not the cause. The study by Durham et al. implicated high expression of b-galactosidase as a partial cause of the toxicity observed with adenoviral vectors because an empty vector produced a lesser degree of toxicity. However, it is also possible that a decreased level of infection by the empty vector accounted for the lesser toxicity, since toxicity of the vector is proportional to the infectivity (Fig. 1). The level of infection produced by the empty vector was not determined in that study due to the lack of marker gene expression. Based on our result of a similar toxicity of NGF-expressing and lacZ-expressing vectors, we would argue that the expression of viral genes and the perturbation of cellular gene expression are direct consequences of the adenoviral vector backbone and are not caused by overexpression of the inserted transgene b-galactosidase. E1-deficient adenoviral vectors have also been used in a number of neuronal populations in vivo (Lisovoski et al., 1994; Castel-Barthe et al., 1996; Choi-Lundberg et al., 1997). Although the effects of adenovirus on neurons have not been well characterized in vivo, many researchers have observed a decrease in foreign gene expression over time. This has been proposed to be caused by promoter shut-off, as well as clearance of infected neurons by the immune system (Peltekian et al., 1997). Based on our study, we suggest that another possible explanation is the failure of protein synthesis and ultimate death of the infected neurons as a direct consequence of infection by E1-deficient adenovirus.

Production of Adenoviral Genes Is an Early Event after Infection and May Trigger Downstream Cellular Changes From our analysis, a likely explanation for the observed toxicity was the transcription of adenoviral gene products, which occurred despite the removal of early region E1. Adenoviral genes encoded by the E3 and E4 regions were induced prior to any detectable changes in neuronal gene expression or function and preceded a series of changes that ultimately ended in the demise of the infected cell (Fig. 10). Although this is the first demonstration that recombinant adenoviral constructs express viral genes in neurons, several in vivo studies have found that adenoviral constructs drive viral gene expression in the liver and lung and produce cell loss that is accompanied by an inflammatory reaction (Yang et al., 1994a, b). The toxicity in these in vivo analyses may

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be a result of the viral genes activating the immune response, which then clears the infected cells. Such a mechanism was not operative in our in vitro paradigm, which eliminated the role of the immune system and allowed investigation of the direct toxicity produced by the vector on the infected cell. The adenoviral gene products may produce toxicity by interacting with cellular proteins and altering the basal state of the cell. The E4orf6/7 protein, which is encoded by the adenoviral E4 region, can bind to the transcription factor E2F and modulate its ability to bind to specific promoters (Shenk and Flint, 1991; Cress and Nevins, 1996). E2F regulates the production of many genes involved in cell cycle regulation and DNA synthesis, including c-myb and cyclin D1 (Adams and Kaelin, 1996). These genes were induced in the infected neuronal cultures. We and others have proposed that an attempt by postmitotic cells to reenter the cell cycle may produce conflicting signals and trigger cell death (Ucker, 1991; Heintz, 1993; Freeman et al., 1994; Farinelli and Greene, 1996). The demonstration that cell cycle-related genes are induced by recombinant adenoviral infection may explain how recombinant adenoviral infection alters the cell cycle of epithelial cells (Teramoto et al., 1995). Induction of c-myb and cyclin D1 may also be an indication that the cell is triggering a programmed cell death pathway since these genes are increased after NGF deprivation of sympathetic neurons (Estus et al., 1994; Freeman et al., 1994). Signs of apoptosis after adenoviral infection are observed in nonneuronal cells in vitro (Yang et al., 1994a; Teramoto et al., 1995). We also observed chromatin condensation in infected neuronal cultures (Fig. 7).

Recombinant Adenoviral Vectors with More Extensive Deletions of Viral Genes May Produce Less Toxicity Further improvements to the first-generation vectors include the inactivation or deletion of additional adenoviral genes. These vectors are not as easily produced in high titers as the widely used E1-deficient vectors; however, such vectors may have decreased toxicity. In nonneuronal cells, less viral gene production is observed when vectors with additional viral gene deletions are used (Goldman et al., 1995; Gorziglia et al., 1996; Lieber et al., 1996). In this study, we have defined a series of changes that occurred after infection of sympathetic neuronal cultures with E1-deficient adenoviral vectors (Fig. 10). The first occurrence in this cascade was the production of viral genes, suggesting that the production of adenoviral genes may have led to the observed

toxicity. Thus, the continued development of recombinant adenoviral vectors by removal of more of the viral genome may produce recombinant vectors with decreased toxicity (Wang and Finer, 1996). We suggest, however, that all of these vectors should be carefully assessed in a controlled in vitro paradigm to assess the direct effects of the recombinant virus on neuronal viability and function.

EXPERIMENTAL METHODS Materials Reagents were purchased from Sigma (St. Louis, MO) unless otherwise stated. Timed pregnant Sprague– Dawley rats were obtained from Harlan Sprague– Dawley (Indianapolis, IN). Collagenase and trypsin were purchased from Worthington Biochemical Corp. (Freehold, NJ). Neuronal medium consisted of Eagle’s MEM with Earle’s salts (Life Technologies, Gaithersburg, MD) supplemented with 10% fetal bovine serum (Sigma), 100 U/ml penicillin, 100 µg/ml streptomycin, 1.4 mM L-glutamine, 20 µM fluorodeoxyuridine, 20 µM uridine, and 50 ng/ml mouse 2.5S NGF (Harlan Bioproducts, Indianapolis, IN). In addition to adenovirus produced in our laboratory, the recombinant adenovirus Ad5RSVntlacZ (Davidson et al., 1994), which expresses a nuclear targeted b-galactosidase gene under the RSV promoter, was purchased from the Gene Transfer Vector Core facility at the University of Iowa College of Medicine (Iowa City). This adenoviral construct contains the b-galactosidase gene in place of the adenoviral E1 region, thereby deleting E1A and E1B, and contains a partial deletion in the E3 region. The stock of Ad5RSVntlacZ used for the majority of studies had a titer of 1010 PFU/ml and 1012 particles (pt)/ml. We also tested an Ad5RSVntlacZ stock with a titer of 2 3 1010 PFU/ml and 1012pt/ml. Adenovirus expressing nerve growth factor (AdNGF), which was prepared similarly to Ad5RSVntlacZ, was also obtained from the Gene Transfer Vector Core facility at the University of Iowa College of Medicine. The AdNGF stock had a titer of a titer of 4 3 1010PFU/ml and 1012pt/ml. Wild-type adenovirus type 5, 293 cells, and A549 cells were obtained from ATCC (Rockville, MD).

Cell Culture Primary sympathetic neuronal cultures were established from SCG of embryonic day 21 rats by using a modification of a previously described method (Johnson

344 and Argiro, 1983). After dissection, ganglia were treated with 1 mg/ml collagenase for 30 min at 37°C followed by 2.5 mg/ml trypsin for 30 min at 37°C. The ganglia were then triturated with a flame-polished Pasteur pipette and filtered through a size 3-20/14 Nitex filter (Tetko Inc., Elmsford, NY). The number of viable cells was determined by trypan blue exclusion, and cells were plated in a drop of medium on ammoniated, air-dried collagen-coated tissue culture dishes. For protein synthesis measurements, 3000–5000 cells were plated on 24-well tissue culture dishes (Costar, Cambridge, MA). For cDNA preparation, approximately 100,000 cells were plated in 35-mm tissue culture dishes (Corning, Corning, NY). For immunohistochemistry, 2000 viable cells were plated in 2-well chamber slides (Nunc, Naperville, IL). After the cells were allowed to attach for 1–3 h at 37°C, additional medium was added to the wells.

Construction of Recombinant Adenoviral Vectors The E1-deficient recombinant adenovirus expressing the b-galactosidase gene under the CMV promoter Ad5CMV.lacZ was constructed by cotransfection of 293 cells with the plasmids pJM17 (generously provided by Christopher B. Newgard; University of Texas Southwestern Medical Center, Dallas, TX) and pACCMV.lacZ according to the protocol detailed in Becker et al. (1994). This method produces an adenoviral construct that contains the b-galactosidase gene in place of the viral E1 region, thereby deleting viral genes E1A and E1B. In this vector, the E3 region is similar to the Ad5RSVntlacZ vector, containing a deletion/insertion in the E3 region that eliminates some E3 transcripts while others may still be produced (Bett et al., 1995). The pACCMV.lacZ plasmid was constructed by inserting the b-galactosidase gene into the BamHI site of the pACCMV.pLpA plasmid (obtained from Christopher B. Newgard). The cell lysate produced by the cotransfection was plaquepurified twice (Becker et al., 1994). The clonal virus was then amplified by growth on 10–15 T150 flasks (Falcon, Lincoln Park, NJ) of 293 cells and purified over a cesium chloride step gradient (Leber et al., 1996) to yield a stock of 1010 PFU/ml and 1012 particles/ml.

Adenoviral Titer Viral titer was determined by a modification of the plaque assay that has previously been described (Graham and Prevec, 1991). Six-well plates of 293 cells at 80% confluency were infected for 1 h at 37°C with dilutions (1023 to 1029 ) of viral stock. After infection, the virus was aspirated, the cells were rinsed twice with sterile PBS

Easton, Johnson, and Creedon

(137 mM NaCl, 2.7 mM KCl, 5.4 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) and then overlaid with 293 medium [Dulbecco’s minimal essential medium (Life Technologies) containing 10% fetal bovine serum, 2 mM L-glutamine, 100 µg/ml streptomycin, 100 U/ml penicillin] containing 0.65% agarose. Four days after infection, plaques were counted on the dilution that had 50–100 distinct plaques. Four replicates were counted for each viral preparation; the final titer is an average of the four. Particle number was determined by the absorbance at a wavelength of 260 nm (1 OD260 5 1012 particles/ml).

PCR Analysis for Wild-type Adenovirus In a 25-µl PCR, 106–107 PFU of recombinant virus was used in a solution containing 50 µM dCTP, 100 µM dGTP, 100 µM dATP, and 100 µM dTTP, 10 µCi [a-32P]dCTP (3000 Ci/mmol; ICN, Costa Mesa, CA), 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris (pH 9.0), 0.1% Triton X-100, 1 µM each primer, and 1 U Taq polymerase (Life Technologies). The primer sequences designed to the adenovirus E1 region (kindly provided by the Gene Transfer Vector Core, University of Iowa Medical Center) are: AdE1 forward, 58-CAAGAATCGCCTGCTACTGTTGTC-38 AdE1 reverse, 58-CCTATCCTCCGTATCTATCTCCACC-38. PCR conditions (supplied by the Gene Transfer Vector Core, University of Iowa Medical Center) were 25 cycles of 30 s at 92°C, 30 s at 57°C, 30 s at 72°C followed by a 10-min extension at 72°C. The PCR was separated on a 5% acrylamide gel that was dried and visualized with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Wild-type adenovirus produced a 561-bp fragment.

Neuronal Infection Paradigm For most studies, the amount of virus (AdCMV.lacZ) to be used in the infection was determined by infecting neuronal cultures at 3 div with dilutions of viral stock. After a 48-h infection, the neurons were stained with X-gal and the dilution that gave 70–90% X-gal-positive cells was used to infect the corresponding cultures at 5 div. For X-gal staining, neuronal cultures were rinsed once with PBS, fixed in 0.5% formaldehyde, 0.2% glutaraldehyde for 5 min at room temperature, rinsed with PBS, and stained with 2 mM magnesium chloride, 4 mM potassium ferrocyanide, 4 mM potassium ferricyanide, 0.5 mg/ml 5-bromo-4-chloro-3-indolyl-b-D-galactopyra-

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noside (X-gal; Boehringer Mannheim, Indianapolis, IN) in PBS for 1 h at 37°C. Once the appropriate dilution of virus was determined, the 5 div neuronal cultures were infected with AdCMV.lacZ diluted in standard culture medium (400 µl/well). The infection was allowed to proceed for 48 h at 37°C. The virus-containing medium was then removed and fresh medium was added.

Protein Synthesis Assay The rate of protein synthesis was determined by using a modification of Deckwerth and Johnson (1993). Neuronal cultures were rinsed once with PBS and then labeled for 4 h at 37°C with 5–10 µCi/well Tran 35S-label (ICN, Costa Mesa, CA) in modified medium lacking cysteine and methionine. Cultures were rinsed once with 500 µl PBS and lysed with 500 µl of 0.5% N-lauryl sarcosine, 1 mM EDTA, 10 mM Tris–HCl, pH 7.5. The protein was precipitated for 1 h in 10% trichloroacetic acid at 0°C and collected by filtration through a nitrocellulose filter (0.45-µm pore size, BA-85; Schleicher & Schuell, Keene, NH), and its radioactivity was measured in a liquid scintillation counter (Beckman, Fullerton, CA). Protein synthesis rates for infected and control cultures have the nonneuronal background subtracted. Nonneuronal background was determined by measuring the protein synthesis rates of cultures that had been treated with neutralizing antibodies against NGF (anti-NGF) at the time of adenoviral infection; anti-NGF will kill all the neurons in the culture and leave only the nonneuronal cells. Nonneuronal cells account for a very small fraction of the total cells in the culture, and protein synthesis rates attributed to the nonneuronal cells alone accounted for less than 10% of the total protein synthesis rates. Therefore, any possible effects of infection on nonneuronal protein synthesis would not influence the interpretation of these results. The protein synthesis rates of the infected cultures are expressed as a percentage of control cultures that have been maintained in NGF. For NGF-maintained control cultures, total counts incorporated into TCA precipitates were in the range of 150,000–300,000 cpm/culture.

Phospho c-Jun Immunohistochemistry Phosphorylated c-Jun was detected in sympathetic neurons as described (Easton et al., 1997). Briefly, neuronal cultures were washed once with PBS, pH 7.4, before a 30-min fixation with 4% paraformaldehyde in PBS at 4°C. Neuronal cultures were washed three times with TBS (100 mM Tris–HCl, 0.9% NaCl, pH 7.6), exposed to blocking solution (TBS containing 5% goat serum and

0.3% Triton X-100) for 30 min at room temperature, and then incubated overnight at 4°C in the rabbit polyclonal anti-phospho c-Jun (Ser63) antibody (New England Biolabs, Beverly, MA) diluted 1:200 in TBS, 1% goat serum, 0.3% Triton X-100. Neuronal cultures were then washed three times with TBS and incubated at 4°C overnight in Cy3-donkey anti-rabbit IgG antibody (1.5 mg/ml; Jackson Immunochemicals, Westgrove, PA) diluted 1:400 in TBS, 1% goat serum, 0.3% Triton X-100. Cultures were washed twice in TBS and then stained with 1 µg/ml Hoechst 33258 (Molecular Probes, Eugene, OR) for 20 min at room temperature. After being rinsed with TBS, cultures were coverslipped and examined by fluorescence microscopy.

RT-PCR Analysis of Viral Gene Expression RT-PCR analysis of SCG neuronal cultures has been previously described by Estus et al. (1994) and Freeman et al. (1994). The rationale and methodology for this procedure have been detailed in Estus (1997). In summary, sympathetic neurons were infected after 5 days in vitro and mRNA was isolated at specified times after the start of infection by using an oligo(dT)–cellulose mRNA purification kit (QuickPrep Micro kit; Pharmacia, Piscataway, NJ) according to the manufacturer’s instructions. Half of the mRNA was converted into cDNA by reverse transcription with Moloney murine leukemia virus reverse transcriptase and random hexamers (16 µM) as primers. For PCR analysis, 1% of the cDNA was used in a 50-µl PCR, half of the PCR was separated on a 10% polyacrylamide gel, and the PCR product was visualized with a PhosphorImager. The primer sequences were as follows: E3(11), 58-CAGCTTTTTAAACGCTGGG-38; E3(2133), 58-CATTACAGGCTGGCTCCTTA-38; E4(1395), 58-TAAGCATAAGACGGACTACG-38; E4(2544), 58-CACTGACCGATGTGAATCAACC-38; hsp70(11106), 58-GCCCAAGGTGCAGGTGAACT-38; hsp70(21240), 58-CGTGATCACCGCGTTGGTCA-38; c-myb(12555), 58-AAAGCCTTTACCGTACCTAA-38; c-myb(22712), 58-ACCAGAGTTCGAGCTGAGAA38; cyclinD1(1336), 58-GCGAATTCGATGAAGGAGACCATTCCCT-38; cyclinD1(2529), 58-GGGGATCCTCTGCTTGTTCTCATCCGC-38. The primers designed against the E3 region recognize the transcript encoding the E3 gp19K protein; this transcript is unaffected by the E3 deletion in the vectors

346 used in this study (Bett et al., 1995). The E3 PCR fragment was amplified by using 23 cycles and E4 by using 19 cycles. The PCR products amplified were confirmed by DNA sequence analysis (data not shown). The hsp70 PCR fragment was amplified by using 23 cycles, c-myb was amplified by using 28 cycles, and cyclin D1 was amplified by using 22 cycles. The hsp70, c-myb, and cyclin D1 PCR products have been previously confirmed by sequence analysis (Estus et al., 1994; Freeman et al., 1994).

ACKNOWLEDGMENTS We thank Beverly L. Davidson and Richard Anderson at the Gene Transfer Vector Core, University of Iowa College of Medicine, for their assistance and expertise. We also thank Christopher B. Newgard and Thomas C. Becker at the University of Texas Southwestern Medical Center for their technical advice and generous provision of plasmids for the construction of recombinant adenoviral vectors. We are grateful to Patricia A. Osborne for critical evaluation of the manuscript. This work was supported by NIH Grants NS24679 and AG12947 and by Alzheimer’s Disease Research Center Grant 5P50AG05681.

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