Introduction

Moloney murine leukemia virus (MoMuLV)-TB is a murine type C retrovirus that induces T- cell lymphomas in susceptible strains of mice (Gardner, 1978; Peters et al, 1973; Yuen and Szurek, 1989). In particular, its temperature-sensitive mutant ts1, a lymphotropic, neurotropic strain of MoMuLV having a single point mutation in the envelope gene, seems to cause loss of T cells and motor neurons in susceptible hosts (Szurek et al, 1990; Wong et al, 1989, 1991). MoMuLV-ts1 infection of the brain causes a progressive neurodegenerative disease, manifested morphologically as spongiform polioencephalomyelopathy. The characteristic pathology of this condition includes astrogliosis, myelin pallor, and neuronal loss (Stoica et al, 1993). Productive ts1 infection in the brain occurs in endothelial cells, microglia, oligodendrocytes, and astrocytes but not in neurons (Stoica et al, 1993), implicating indirect mechanisms in ts1-mediated neuronal degeneration. In this context, neuronal degeneration may be a result of the proinflammatory cytokines and/or nitric oxide produced by ts1-infected surrounding glial cells (Choe et al, 1998; Kim et al, 2001). Studies of the ts1-induced neuropathology in this defined mouse model should provide new insights into the pathogenesis of retrovirus-induced neurodegeneration.

Virus infections affect cell division by interfering with cell cycle regulation or altering cellular gene expression. In turn, these perturbations trigger a variety of events ranging from apoptosis to transformation (Chretien et al, 2000; Genini et al, 2001; Roulston et al, 1999; Watanabe et al, 2000). Many of these changes involve alterations in p53 functions and activation of various cellular genes (Akagi et al, 1996; Cereseto et al, 1996; Yang and Prayson, 2000). Particularly, overexpression of p53 is a consistent feature of several viral infections in the central nervous system (Chretien et al, 2000; Ehsan et al, 2000; Jordan-Sciutto et al, 2000).

p53 is a transcriptional factor that plays a critical role in the regulation of growth arrest, apoptosis, and differentiation (Haffner and Oren, 1995; Levine, 1997; Liu et al, 1994). Regulation of the p53 protein level is achieved primarily via posttranslational mechanisms, although it may also be controlled at the transcriptional (Sun et al, 1995) and translational levels (Mosner et al, 1995). Also, p53 accumulation activates transcription of target genes, including p21, Mdm2, Gadd45, and bax-α, leading to growth arrest or apoptosis. Studies with the human papilloma virus E6 protein and the cellular oncogene Mdm2, which interact with and lead to the degradation of p53, have shown that p53 is degraded by the ubiquitin-mediated proteolytic pathway (Haupt et al, 1997; Kubbutat et al, 1997; Storey et al, 1998). Although the signals that target p53 for degradation are not yet fully understood, it is generally accepted that p53 phosphorylation may be involved, via the mitogen-activated protein kinase (MAPK) pathway (Fuchs et al, 1998a; Keller et al, 1999; Song et al, 1998). However, a number of studies have shown that phosphorylation of p53 may contribute to its accumulation by protecting it from ubiquitination and proteolysis (Persons et al, 2000; Shieh et al, 1997; Siliciano et al, 1997).

MAPKs are cellular signaling kinases that are activated by phosphorylation of specific tyrosine and threonine residues in response to various external and internal stimuli (Dhanasekaran and Reddy, 1998; Robinson and Cobb, 1997). In mammalian cells, three general groups of MAPKs have been identified: extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38/Hog. Activated MAPKs can phosphorylate numerous substrates, including a variety of transcription factors. In these factors, MAPK-mediated phosphorylation is a common mechanism used by the cell to induce gene expression (Lewis et al, 1998; Weitzman and Yaniv, 1998). Through their ability to regulate transcription factors, MAPKs regulate changes in the cell ranging from cell growth to apoptosis to senescence.

In cells infected by numerous viruses, one or more of the MAPKs is usually activated. For example, simian virus 40 activates ERK1/2, herpes simplex virus activates JNK and p38, and simian immunodeficiency virus activates all three MAPKs (McLean and Bachenheimer, 1999; Popik and Pitha, 1998; Yang et al, 1991; Zachos et al, 1999). MAPK activation can be induced through viral binding to the host cell, as in simian immunodeficiency virus activation of ERK1/2, JNK, and p38 kinase, or it can require viral protein synthesis, as in herpes simplex virus activation of JNK (Popik and Pitha, 1998). However, even where MAPK activation has a clear function in viral infection, little is known about how MAPK mediates this effect, and even less is known about the cellular proteins involved. An understanding of these points is therefore of great interest, because it will allow understanding of MAPK's activities in viral infection and will disclose how viruses regulate host cell machinery during infection.

In the present study, we addressed the question of whether the level of p53 is changed in astrocytes by ts1 infection, and, if so, which mechanisms are responsible. Our results show that p53 and bax-α levels are increased in the brain stem of ts1-infected mice. In an immortalized C1 astrocyte cell line (Lin et al, 1997), p53 accumulated via activation of the ERK pathway, rather than through p38 kinase activation, and p53-dependent gene expression was increased. Inhibition of ERK by the MAPK pathway inhibitor enhanced p53 protein degradation and blocked expression of p53-dependent genes. These results strongly implicate MAPK pathways in ts1-induced p53 accumulation in ts1-infected astrocytes.

Results

Increased Expression of p53 and bax-α in the Brain Stem of ts1-Infected Mice

We used Western immunoblotting to assess p53 expression in brain stem homogenates from control and ts1-infected mice. As shown in (Figure 1 (A and B), p53 was more abundant in ts1-infected brain stem tissues, relative to control mice, at 20 and 25 days postinfection (dpi) (p < 0.01). In addition, to localize p53 in the brain stem, we performed immunohistochemistry for p53 (Fig. 2). In control mice, many neurons in the brain stem were labeled, but p53 protein expression was absent or weak in glial cells (Fig. 2, A and C). By contrast, almost all of the glial cells, which were mostly astrocytes in affected areas in ts1-infected mice, showed moderate to strong nuclear staining for p53 (Fig. 2, B and D). In ts1-infected mice, no immunoreactivity for p53 was found in glial cells in other parts of the central nervous system (data not shown).

Figure 1
figure 1

Expression of p53 in the brain stem of control and ts1-infected mice. A, Increased p53 protein in brain stem homogenates from ts1-infected mice. At 20 and 25 days postinfection (dpi), tissue homogenates were prepared from the brain stem of control and ts1-infected mice and then subjected to immunoblotting analysis. B, Levels of p53 in the brain stem of ts1-infected mice versus controls. Protein levels for bands were compared by densitometric analysis. At 20 and 25 dpi, p53 protein from brain stem tissues of ts1-infected mice was increased 80.1% ± 18.4% and 63.7% ± 8.3%, respectively, over levels in control mice (p < 0.01). The results represented in the histogram are the mean ± se from tissues from three control and three ts1-infected mice.

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Figure 2
figure 2

Expression of p53 protein in brain stem glial cells of control and ts1-infected mice at 25 dpi. A, p53 immunoreactivity (red) in the nuclei of brain stem glial cells and neurons from a control mouse. B, p53 immunoreactivity in the nuclei of glial cells and neurons of the ts1-infected mouse brain stem tissue. C, Higher magnification view of the glial and neuronal cells in panel A. Note the absent or weak nuclear staining in astrocytes (arrow) and neurons (arrowhead). D, Higher magnification views of the glial and neuronal cells in panel B. Note the strong staining in astrocytes (arrow) and neurons (arrowhead). Bars indicate 50 μm.

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To determine whether the increased level of brain stem p53 protein occurs together with increased expression of bax-α, a p53-reponsive gene, the level of bax-α was determined by immunoblotting of brain stem homogenates of control and infected mice (Fig. 3). (Figure 3 (A and B) shows that ts1 infection induced a significant increase in bax-α protein level at 20 and 25 dpi (p < 0.01). To localize bax-α in infected brain stem tissues, immunostaining for bax-α was performed (Fig. 4). No immunoreactivity was evident in brain stem neurons of control mice, and expression was absent or weak in a few glial cells in these mice (Fig. 4, A and C). However, glial cells including astrocytes scattered throughout affected areas in ts1-infected mice showed strong cytoplasmic staining (Fig. 4, B and D).

Figure 3
figure 3

Changes in bax-α protein level in brain stem homogenates of ts1-infected mice. A, Increased abundance of bax-α protein in brain stem homogenates of ts1-infected mice. At 20 and 25 dpi, tissue homogenates were prepared from the brain stem of control and ts1-infected mice, and tissue homogenates (20 μg) were then subjected to immunoblot analysis. B, Relative increases in the level of bax-α protein in the brain stem of ts1-infected mice. Protein levels for bands were compared by densitometric analysis. At 20 and 25 dpi, the levels of bax-α protein in the brain stem of ts1-infected mice were increased 18.5% ± 9.4% and 22.1% ± 7.8%, respectively, over levels in control mice (p < 0.05). The results represented in the histogram are the mean ± se from tissues from three control and three ts1-infected mice.

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Figure 4
figure 4

Localization of bax-α in brain stem glial cells of control and ts1-infected mice at 25 dpi. A, Cytoplasmic bax-α immunoreactivity (brown) in brain stem glial cells of a control mouse. B, Cytoplasmic bax -α immunoreactivity in brain stem glial cells of ts1-infected mouse. C, Higher magnification view of the glial and neuronal cells in panel A. Note the weak cytoplasmic staining in astrocytes (arrow) and neurons (arrowhead). D, Higher magnification view of the glial and neuronal cells in panel B. Note the strong cytoplasmic staining in astrocytes (arrow) and neurons (arrowhead). Bars indicate 50 μm.

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Increased Expression of p53 and Its Responsive Genes, bax-α and p21, in ts1-Infected C1 Astrocytes

To determine whether p53 protein also accumulates in ts1-infected C1 astrocytes, the level of p53 protein was determined. (Figure 5 (A and B) shows that the level of p53 protein was increased in infected cells, relative to mock-infected controls at 24, 48, and 72 hours after infection (p < 0.01). In both mock-infected and ts1-infected astrocytes, the level of p53 protein decreased over time. To determine whether increased p53 levels might affect p53-responsive genes, we then examined the expression of bax-α and p21 in mock-infected and ts1-infected astrocytes by immunoblotting analysis. (Figure 5C, D, E, and F) shows that the levels of bax-α and p21 protein in ts1-infected C1 astrocytes were higher than those in the mock-infected astrocytes at 24 and 48 hours after infection (p < 0.01).

Figure 5
figure 5

ts1 infection induced increases in the level of p53, bax-α, and p21 protein in C1 astrocytes. A, Time course of p53 expression in C1 astrocytes after ts1 infection. C1 astrocytes were mock or ts1 infected and whole cell lysates prepared at 24, 48, and 72 hours after infection for immunoblotting. B, Relative increases in the level of p53 protein in ts1-infected C1 astrocytes. The protein level was expressed relative to that in controls at each time point to compare ts1 infection–induced changes in the level using data obtained from densitometric analysis of autoradiographs. C, Time course of bax-α expression in C1 astrocytes after ts1 infection. C1 astrocytes were mock or ts1 infected and whole cell lysates prepared at 24 and 48 hours after infection for immunoblotting. D, Relative increases in the level of bax-α protein in ts1-infected C1 astrocytes. E, Time course of p21 expression in C1 astrocytes after ts1 infection. C1 astrocytes were mock or ts1 infected and whole cell lysates prepared at 24 and 48 hours after infection for immunoblotting. F, Relative increases in the level of p21 protein in ts1-infected C1 astrocytes. The protein level was expressed relative to that in controls at each time point to compare ts1 infection–induced changes in the level using data obtained from densitometric analysis of autoradiographs. The results are the mean ± sd from three independent experiments carried out in triplicate (n = 9) and are representative of those from three independent experiments. *Significantly different from controls (p < 0.01).

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Normal Degradation of p53 in ts1-Infected C1 Astrocytes by the Calpain and Ubiquitin-Proteasome Pathways

To determine how proteolytic pathways involved in p53 degradation might be affected after ts1 infection, we measured the p53 level in mock-infected and ts1-infected C1 astrocytes after treatment with calpeptin, a specific calpain inhibitor, or MG-132, a potent proteasome inhibitor. Figure 6 shows that both agents caused increases in the level of p53 protein compared with ts1-infected cells without treatment. MG-132 also increased the level of p53 protein in mock-infected cells (p < 0.01), but calpeptin did not. These results indicate that these two proteolytic pathways are active in the degradation of p53 in ts1-infected cells.

Figure 6
figure 6

Proteolytic pathways involved in p53 degradation in C1 astrocytes. A, Effects of calpain and proteasome inhibitors on degradation of p53 proteins in ts1-infected C1 astrocytes. At 48 hours after infection, C1 astrocytes were treated with various protease inhibitors for 4 hours before cell lysate preparation. Whole cell lysates (20 μg of protein) were then separated by SDS-PAGE and analyzed by immunoblotting. B, Accumulation of p53 protein in ts1-infected C1 astrocytes after treatment with either MG-132, a proteasome inhibitor, or calpeptin, a calpain inhibitor. Compared with cells infected with ts1 alone, p53 protein proteolysis was significantly blocked in ts1-infected C1 astrocytes by treatment with MG-132 (54.3% ± 9.1%). *Significantly different from controls (p < 0.01); +significantly different from ts1 infection alone (dimethyl sulfoxide; p < 0.01). The results are the mean ± sd from three independent experiments carried out in triplicate (n = 9) and are representative of those from three independent experiments.

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Activation of ERK in C1 Astrocytes by ts1 Infection and Blockade of ts1 Infection–Induced p53 Accumulation by Inhibition of ERK Activity

Both ERKs and p38 kinase seem to participate in stabilization of p53 protein. To determine whether ERK or p38 kinase activity is altered after ts1 infection, we assessed the phosphorylation status of ERK 1/2 and p38 kinase in mock-infected and ts1-infected C1 cells. Figure 7A shows that phosphorylated ERK1/2 levels were increased after ts1 infection, whereas phosphorylated p38 kinase level is unchanged. Also, to determine whether accumulation of p53 requires similar changes in ERKs or p38 kinase, we used two specific inhibitors to inactivate ERK and p38 kinases. PD98059, a specific inhibitor of MEK1, acts by inhibiting activation of ERKs. SB202190 is a specific inhibitor of p38 kinase. Both were tested for their activity to modify ts1 infection–mediated p53 accumulation. Figure 7B shows that treatment with 20 μm of PD98059 decreased the level of p53 in parallel with decreases in the level of phosphorylated ERKs and in expression of p21, a p53-dependent gene. By contrast, treatment with 10 μm of SB202190 did not inhibit p53 accumulation in ts1-infected C1 astrocytes. These data pinpoint ERKs as upstream modifiers of p53 accumulation in ts1-infected C1 astrocytes.

Figure 7
figure 7

Activation of ERKs in ts1-infected C1 astrocytes. A, Increases in the level of phosphorylated ERKs in ts1-infected C1 astrocytes. After C1 astrocytes were mock or ts1 infected, whole cell lysates were prepared at 24 and 48 hours after infection and then subjected to immunoblotting for detection of phosphorylated ERK1/2 (p-ERKs), ERKs, and phosphorylated p38 kinase as described in “Materials and Methods.” B, Effect of the mitogen-activated protein kinase inhibitors PD98059 and SB202190 on the p53 protein level in ts1-infected C1 astrocytes. After ts1 infection, C1 cells were incubated with DMSO, PD98059, or SB202190. After 16 hours of incubation, the cells were lysed, and immunoblot analysis of p-ERK, p53, and p21 protein was performed. β-actin acted as a loading control (bottom row). The results are representative of those from three independent experiments.

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Discussion

The data clearly indicate that MoMuLV-ts1 infection increases the expression and accumulation of p53 protein and its dependent genes in astrocytes. Overexpression of p53 and bax-α is known to lead to cell cycle arrest or apoptosis (Haffner and Oren, 1995; Levine 1997; Liu et al, 1994; Miyashita and Reed, 1995), but whether the two events also occur in the brain stem of ts1-infected mice remains to be determined because most bax-α immunoreactive glial cells in brain stem lesions do not have the characteristic apoptotic morphology (Fig. 4). Because bax-α binds to bcl-2 and antagonizes its ability to block apoptosis, a p53-dependent increase in bax-α synthesis could tip the scales toward apoptosis. However, it is also possible that there is counteracting regulation of specific antiapoptotic molecules such as bcl-2 or bcl-XL. Recent reports suggest that bax-α–induced cell death is different from typical apoptotic processes, involving interference with mitochondrial functions and generation of reactive oxygen species (Pastorino et al, 1998; Xiang et al, 1996). Although the activities of the bcl-2/bax-α family are unclear in this model, they may be central to our understanding of this important aspect of ts1-mediated pathogenesis.

To examine changes in the level of p53 protein after ts1 infection and how ts1 affects p53 levels in astrocytes, we used a well-defined in vitro system. We first determined whether two well-known proteolytic pathways for p53, the ubiquitin-proteasome pathway (Kubbutat et al, 1997; Maki et al, 1996) and the calpain pathway (Pariat et al, 1997), are functional in ts1-infected C1 astrocytes. The data suggest that the ubiquitin-proteasome pathway plays the dominant role in the degradation of p53 in both mock-infected and ts1-infected C1 cells and that this pathway remains functional after ts1 infection. Interestingly the calpain pathway participates in p53 degradation in ts1-infected C1 astrocytes but not in mock-infected cells. This is consistent with our previous observations showing no significant changes in ubiquitin-conjugating activity but activation of the calpain pathway in ts1-infected C1 astrocytes (Kim et al, 2001). The increased p53 in ts1-infected C1 astrocytes apparently is not a consequence of proteasome malfunction.

p53 accumulation after ts1 infection is accompanied by increased ERK1/2 activity. ERK1/2 activation is inhibited by PD98059, a synthetic inhibitor of MEK1 (the immediate upstream kinase of ERK1/2) that prevents activation of MAPK and subsequent phosphorylation of MAPK substrates (Alessi et al, 1995; Dudley et al, 1995). Inhibition of ts1-induced ERK1/2 activation by PD98059 decreased p53 accumulation and p53 function, as demonstrated by a lower level of the p53-dependent p21WAF-1. These findings indicate that ERK1/2 activation is required for maximum accumulation and activation of p53 in ts1-infected C1 astrocytes. Decreased accumulation of p53 could conceivably be secondary to a shorter protein half-life; however, because PD98059 does not completely inhibit accumulation of p53 protein, additional upstream signaling pathways may contribute to the stability of p53 after ts1 infection.

The mechanisms responsible for the activation of ERKs in ts1-infected astrocytes may be diverse. One potential mechanism is that multiple Ca2+-dependent PKC isoforms may participate in ERK activation after ts1 infection as supported by a recent report (Monick et al, 2001) and our preliminary results (data not shown). Another mechanisms may be initiated by the endoplasmic reticulum (ER). Specially, the viral envelope protein plays an important role in both HIV- and ts1-mediated neuropathology. A single mutation in the ts1 env gene results in a protein called gPr80env, which is inefficiently processed and transported to the plasma membrane of infected astrocytes and T cells, leading to accumulation of gPr80env protein in the ER. In a previous study, we showed that the cytopathic effects of ts1 on primary astrocytes in vitro closely correlated with the accumulation of ts1 precursor envelope protein in the ER (Shikova et al, 1993). Therefore, the ER may be the primary intracellular organelle in which ts1 envelope protein-mediated signaling pathways responsible for ERK activation are generated. Several studies have demonstrated that alterations in protein folding and/or trafficking through the ER could activate PKR-like ER kinase (PERK) and protein kinase RNA-regulated (PKR) (Iwawaki et al, 2001; Kaufman, 1999), which in turn activate downstream mediators such as ERKs, leading to the observation that we detected in the present study.

The JNK and p38 kinase pathways are associated with increased apoptosis, whereas the ERK pathway has been shown to suppress apoptosis (Xia et al, 1995). Increased expression of upstream activators of p38 kinase and JNK such as apoptosis signal-regulating kinase 1 (ASK1) (Ichijo et al, 1997) and MAP kinase kinase kinase 1 (MEKK1) (Fuchs et al, 1998b) also triggers apoptosis. Therefore, specific activation of astrocyte ERKs may be a positive response to viral infection. Alternatively, phosphorylation of ERKs may be related to both induction of apoptosis and proliferation of astrocytes (Neary et al, 1998). It was recently reported that activation of ERK also regulates the expression of inducible nitric oxide synthase in astrocytes (Bhat et al, 1998). Together these observations show that the outcome of ERK activation in ts1-infected astrocytes is complex. We suspect that “bystander” effects of the activated MAPK pathway caused by ts1 infection could exert additional effects on astrocytes themselves or surrounding neurons.

Additionally, p53 protein plays a critical role in cellular response to environmental stress. Activation of p53 protein in response to stress involves posttranslational modification of the protein, including specific phosphorylation at serine residues within the N-terminal transactivation domain (Fuchs et al, 1998a; Giaccia and Kastan, 1998). Recently, other members of the MAP kinase family, including JNK and p38 kinase, have also been shown to phosphorylate p53 protein (Fuchs et al, 1998a; She et al, 2000). Activated JNK phosphorylates mouse p53, thereby affecting MDM2 binding to p53 (Fuchs et al, 1998b). This supports the idea that different upstream signal transducers activate p53 in response to different forms of cellular stress. Further analysis will be required to identify specific amino acids phosphorylated on p53 after activation of ERKs in ts1-infected astrocytes.

In conclusion, the data reported here show that the brain stem neuropathology of MoMuLV-ts1 infection involves changes in p53 protein levels and bax-α expression in astrocytes. They also suggest that p53 is accumulated in ts1-infected astrocytes as a consequence of ERK activation in vitro and that this results in expression of p53-dependent genes. Our results have possible implications for other retrovirus-induced neurodegeneration in animals and humans.

Materials and Methods

Sodium orthovanadate (Na3VO4), sodium β-glycerophosphate, sodium fluoride (NaF), dithiothreitol, and phenylmethylsulfonyl fluoride were purchased from Sigma Chemical Company (St. Louis, Missouri). Leupeptin, pepstatin, and aprotinin were purchased from Boehringer Mannheim (Indianapolis, Indiana). Iodoacetamide (an isopeptidase inhibitor) was obtained from Fluka Chemical Corp. (Milwaukee, Wisconsin). Phospho-specific ERK1/2 and p38 kinase antibodies, ERK1/2 antibody, and p38 kinase antibody were purchased from New England BioLabs (Beverly, Massachusetts). Monoclonal mouse IgG antibody against p53 (antibody-1) was obtained from Oncogene Research Products (Cambridge, Massachusetts). Polyclonal rabbit IgG antibody against bax-α (SC-526) and polyclonal rabbit IgG antibody against p21 (SC-471) were purchased from Santa Cruz Biotechnology (Santa Cruz, California). Monoclonal mouse IgG antibody against β-actin (A5441) was purchased from Sigma. A MEK1-specific inhibitor, PD98059, and a p38 kinase inhibitor, SB202190, were obtained from Calbiochem (San Diego, California). DMEM and fetal bovine serum (FBS) were obtained from Life Technologies, Inc. (Rockville, Maryland). PD98059, SB202190, MG-132, and calpeptin were dissolved in dimethyl sulfoxide and stored at −80° C.

Virus

ts1, a spontaneous temperature-sensitive mutant of MoMuLV, was propagated in TB cells, a thymus-bone marrow cell line. Virus titers were determined using a modified direct focus assay in the 15F cell line, a murine sarcoma–positive, leukemia-negative cell line, as described previously (Wong et al, 1981).

Animals and Virus Inoculation

FVB/N mice were obtained from Taconic Farms (Germantown, New York). Mice were maintained in sterilized microisolators and supplied with autoclaved food and water ad libitum. Newborn FVB/N mice were inoculated intraperitoneally with 0.1 ml of ts1 viral suspension containing 106 to 107 infection units/ml, as described previously (Stoica et al, 2000); control mice were inoculated with medium only. The mice were then observed daily for clinical signs of disease and killed at 25 dpi. The experimental protocol was approved by the Texas A&M University Institutional Animal Care and Use Committee.

Tissue Preparation

For histopathology and immunohistochemistry studies, ts1-infected (n = 5) and control (n = 5) mice were anesthetized using an intraperitoneal injection of pentobarbital (150 mg/kg) and transcardially perfused with 10% buffered formalin as a fixative, using a peristaltic pump. After 12 hours of fixation, each mouse's brain was dissected, with the brain stem segments separated for further processing. For Western immunoblotting analysis of p53 and bax-α protein in the brain stem, ts1-infected (n = 3) and control (n = 3) mice were killed and their brains were dissected; brain stem tissues were snap-frozen in liquid nitrogen and stored at −80° C.

Immunohistochemistry

Immunohistochemistry was performed to localize p53 and bax-α protein on brain stem tissue sections. Briefly, paraffin-embedded sections (6 μm) were deparaffinized and washed with 0.4% Triton X-100 in Tris-buffered saline (100 mm Tris, 150 mm NaCl, pH 7.4) for 20 minutes at room temperature. Potential nonspecific binding sites were blocked with 2% bovine serum albumin, after which the tissue sections were incubated with either monoclonal anti-p53 antibody or rabbit anti-bax-α antibody at a dilution of 1:100 (2.0 μg/ml) for 2 hours. After three 5-minute washes in Tris-buffered saline, sections were incubated with biotin-conjugated secondary anti-mouse or anti-rabbit IgG (Pierce, Rockford, Illinois) for 30 minutes at room temperature. Either a Vectastain ABC-alkaline phosphatase kit (Vector Laboratories, Burlingame, California) or a Vecta-Elite streptavidin-peroxidase kit with a benzidine substrate was used for color development. Sections were counterstained with 1% methyl green or diluted hematoxylin. Sections that were not incubated with a primary antibody served as negative controls.

Cell Culture and Virus Infection

Immortalized murine C1 astrocytes were maintained in DMEM supplemented with 10% FBS and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin) (Lin et al, 1997). All cells were grown at 37° C in a humidified incubator containing 5% CO2. Cells were passaged biweekly and used for experiments while in the exponential growth phase. For virus infection, C1 cells (4 × 105) in DMEM containing 2% FBS and 4 μg/ml polybrene were seeded in 100-mm tissue culture dishes. After culturing overnight, the medium was removed; the cells were infected with ts1 virus at a multiplicity of infection of 10 in DMEM containing 2% FBS and 4 μg/ml polybrene and incubated for 1 hour at 37° C under 5% CO2. After virus adsorption, the medium containing the virus was removed and DMEM containing 2% FBS added to the culture. The plates were then incubated for various times at 38.5° C (Lin et al, 1997). For mock infection, C1 astrocytes were treated identically, except that the medium used was DMEM containing polybrene only. For studies of proteasome and calpain proteolysis inhibition, MG-132 (20 μm), calpeptin (20 μm), or dimethyl sulfoxide only (less than 0.1%) was added to the culture medium of ts1-infected and mock-infected cells, and the cells were incubated for an additional 4 hours before cell lysate preparation, as described previously (Kim et al, 2001). For studies of MAPK inhibition, PD98059 (20 μm), SB202190 (10 μm), or dimethyl sulfoxide only (less than 0.1%) was added to the culture medium of ts1-infected and mock-infected cells at 4 hours postinfection, and the cells were incubated for an additional 16 hours before cell lysate preparation.

Tissue and Cell Extracts

Brain stem tissue lysates were prepared by homogenization of frozen tissues in 10 volumes of lysis buffer containing 50 mm Tris-HCl (pH 7.9), 150 mm NaCl, 1 mm EDTA, 1 mm Na3VO4, 30 mm sodium β-glycero-phosphate, 50 mm NaF, 10 mm iodoacetamide, 1 mm dithiothreitol, 1% NP-40, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml pepstatin, 10 μg/ml leupeptin, and 5 μg/ml aprotinin. Lysates were cleared by centrifugation at 13,000 × g at 4° C for 20 minutes and the resulting supernatants frozen at −80° C. Also, whole cell extracts from C1 cells were prepared as follows. Briefly, cells were washed twice with cold phosphate-buffered saline (pH 7.4) and lysed in lysis buffer. After incubation on ice for 15 minutes, the cell lysates were cleared by centrifugation at 13,000 × g for 20 minutes at 4° C and the supernatants kept frozen at −80° C. The protein content of the lysates was determined using the Bradford assay (Bio-Rad, Hercules, California), with bovine serum albumin as the standard.

Western Immunoblotting Analysis

Proteins (20 μg) were separated by 8% to 12% SDS-PAGE and then transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, New Hampshire). Kaleidoscope prestained standards (Bio-Rad) were used to determine molecular weight. The membranes were incubated for 1 hour in blocking buffer (20 mm Tris-HCl–buffered saline containing 5% nonfat milk powder and 0.1% Tween 20) at room temperature and then probed with appropriate antibodies in 20 mm Tris-HCl–buffered saline containing 2% nonfat milk powder and 0.1% Tween 20 overnight at 4° C. Normal mouse or rabbit IgGs at the same dilutions were used as controls. The blots were then incubated with anti-mouse or anti-rabbit IgG-peroxidase conjugate (1:10,000 dilution; Kirkegaard Perry Laboratories, Gaithersburg, Maryland) and developed using the enhanced chemiluminescence method (Amersham Life Science, Arlington Heights, Illinois). After stripping, the blots were then incubated with a mouse monoclonal anti-β-actin antibody to confirm equal protein loading and normalize the loading. Densitometric analysis of autoradiographs was performed using a densitometer (Model GS-690; Bio-Rad) equipped with the Multi-Analyst software program (version 1.01; Bio-Rad).

Statistical Analysis

Data obtained from densitometric analysis of Western immunoblots were analyzed by paired t test or ANOVA. Additionally, statistical significance between groups was determined by Dunnett's multiple comparison analysis. P values < 0.05 were considered statistically significant.