Keywords: Moloney murine leukemia virus -ts1, Minocycline, Oxidative stress, Inflammation, Apoptosis, Nrf2

2.1 Minocycline not only delays paralysis and death, but also attenuates astrogliosis lesion ints1-infected FVB/N mice

The paralysis/survival curves inFigure 1A show that untreatedts1 -infected (ts1-only) mice became paralyzed at around 35 dpi, whilets1 -infected minocycline-treated (ts1-mino) mice lived longer, and became paralyzed much later (p<0.001). Brainstem and spinal cord tissues were prepared from uninfected,ts1-only andts1-mino mice, and stained with hematoxylin and eosin (H&E). The sections from uninfected mice showed normal neuropil in the brainstem, while those fromts1-only mice contained many spongiform lesions, with large vacuoles. By contrast, sections fromts1-mino mice had nearly normal cellular organization, with a few small vacuoles, but without discrete spongiform foci (Fig.lB). When we stained thets1-only brainstem tissues with astrocyte marker GFAP, we observed an increased in number and brightness of GFAP-expressing astrocytes when compared with uninfected brainstem sections, which indicates astrogliosis was induced ints1-only brainstems. However, nearly normal GFAP expression is displayed ints1-mino brain sections (Fig. 1C), indicating that astrogliosis lesion is prevented by minocycline treatment.

2.2 Minocycline prevents cell death ints 1-infected C1 cells

To assess the effects of minocycline on CNS cells, we examined its cytoprotective effect forts1 -infected C1 astrocytes.Figure 2 shows that at 48 and 72 h,ts1-only C1 cells underwent significant cell death, when compared to uninfected cells. When minocycline was added to these cultures, however, many of the infected cells remained alive (Fig. 2).

2.3 Minocycline reduces viral titer in the CNS

To determine whether minocycline reduces virus titers in the CNS, brainstems and spinal cords were removed fromts1-only andts1-mino mice at 30 dpi, and subjected to the viral titer protocol described in Experimental procedures.Figure 3A shows thatts1-mino CNS tissues had reduced viral titers, relative to those ofts1-only mice. To determine whether reduction of virus titers by minocycline is specific to the CNS, we also collected thymi and spleens fromts1-only andts1-mino mice at 30 dpi, and measured viral titers in these organs. Notably, we found that no differences in virus titers in thymi and spleens fromts1-mino mice vs.ts1-only mice (Fig. 3A). These data show that minocycline reduces virus titer in CNS tissues ofts1-mino mice, but not in thymi and spleens. To determine whether minocycline has a direct antiviral effect, we assessed the effects of minocycline on virus replication in cultured C1 astrocytes. Surprisingly, virus titers in the culture medium ofts1-mino cells were similar to those ofts1-only cells (Fig. 3B). These data indicate that minocycline at the concentration tested in this study do not directly affect virus replication in vitro, although minocycline prevents cell death ints1 -infected C1 cells. Thus virus replication is not the primary target for the cytoprotection by minocycline in cultured astrocytes.

2.4 Minocycline inhibits oxidative stress ints 1-infected-C1 astrocytes, through direct radical scavenging activity and upregulating Nrf2-mediated antioxidant defense

To find out whether protection of C1 cells by minocycline involves direct antioxidant activity, we treated uninfected C1 cells with 100 µM of hydrogen peroxide (H₂O₂) for 1 h, and then measured their relative ROS levels using CM-H₂DCFDA fluorescence.Figure 4A shows that ROS levels increased about 4-fold in H₂O₂-treated cells in comparison with untreated cells. When increasing doses of minocycline were added immediately after addition of H₂O₂, however, amounts of intracellular ROS were reduced in a minocycline dose-dependent fashion. This observation shows that minocycline has direct and rapid antioxidant effects in C1 cells.

To determine whether minocycline treatment has similar antioxidant effects onts1-infected C1 cells, we replaced H₂O₂ treatment withts1 infection, by adding virus to the cell cultures, again following their ROS levels with CM-H₂DCFDA.Figure 4B shows that ROS levels are significantly increased ints1-only C1 cells relative to uninfected control cultures, but that treatment of the infected cells with 25 and 50µM minocycline decreased ROS levels in a minocycline dose-dependent manner.Figure 4C shows that adducts of the oxidative marker malondialdehyde (MDA), the end product of lipid peroxidation, are also increased ints1-only C1 cells relative to uninfected cultures, but not ints1-mino cells.

Finally, we asked whether the antioxidant effects of minocycline also involve activation of endogenous antioxidant defenses pathways ints1-infected C1 cells.Figure 4D shows that amounts of the antioxidant defense-related protein Nrf2 are reduced ints1-only C1 cells, relative to uninfected cultures, while levels of Nrf2 are upregulated ints1-mino cells. Together these data show that minocycline provides antioxidant protection to H₂O₂ -treated orts1-infected C1 cells, and that the mechanisms for this defense include both radical scavenging and Nrf2 related antioxidant defense.

2.5 Minoycline reduces markers of inflammation in thets1-infected brainstem

Ints1-infected astrocytes or animals, NF-κB and COX-2 are upregulated, while the NF-κB inhibitor IκBoc is reduced ints1 infected astrocytes or animals (Kim et al., 2001;Kim et al., 2005). To determine whether minocycline treatment regulate levels of NF-kB and COX-2, cell lysates were prepared from cultured uninfected cells,ts1-only orts1-mino C1 cells and were subjected to Western blotting for levels of IκBα, nuclear NF-κB p65 and COX-2.Figure 5A shows that levels of nuclear NF-κB p65 were increased ints1-only C1 cells when compared to levels in uninfected C1 cells, while amounts of the NF-κB inhibitor IκBα are downregulated ints1-only C1 cells, in association with translocation of NF-κB from the cytoplasm to the nuclei of the cells. These data are consistent with the report from this laboratory (Kim et al., 2001). Ints1-mino C1 cells, however, levels of nuclear NF-κB p65 are reduced, relative to the elevations inte1-only cells, while amounts of IκBα are markedly upregulated. These results are also in agreement with a report from other group indicating that minocycline inhibits the degradation of IκBα in microglia (Nikodemova et al., 2006). Our data also show that COX-2 levels are elevated in culturedts1-only C1 cells, but that this elevation is reduced ints1-mino C1 cells.

To determine whether in vivo minocycline protection of thets1-infected CNS involves reduction of inflammation in the tissue, we stained paraffin sections of brainstem fromts1-only vs.ts1-mino mice, sacrificed at 30 dpi, for the inflammation marker COX-2. We have shown previously that COX-2 levels are upregulated in thets1-only brainstem (Kim et al., 2005), The same result is evident in the anti-COX-2-stainedts1-only brainstem sections (Fig 5B). Strong COX-2 immunoactivity (red), is especially evident in astrocytes (brown color). By contrast, thets1-mino brainstem sections had no spongiform lesions, and showed weak COX-2 staining when compared tots1-only brainstem sections. Sincets1 -infected CNS showed activation of microglia (Stoica et al., 1993;Zachary et al., 1997), and that minocycline inhibited microglia activation in various neurodegeneration models (Dheen et al., 2007;Zachary et al., 1997;Zink et al., 2005), we ask whether minocycline has inhibition effects on microgllia activation ints1 -infected brainstems. To address this question, we performed immunofluorescence staining to examine expression of CD68, a marker of activated microglia or macrophage (Zink et al., 2005), using frozen brainstem sections.Figure 5C shows that strong immunoactivity of CD68 is evident onts1-only brainstem sections, especially near the area where spongiform lesion was severe. By contrast, CD68 positive cell is not visible on eitherts1-mino or uninfected brain sections. These data indicate that minocycline attenuates inflammatory responses induced byts1 infection both in vitro and in vivo. This effect may also in part contribute to the cytoprotective effect of minocycline in infected C1 astrocytes, and to astrocyte and neuronal protection in the brain.

2.6 Minocycline prevents apoptosis ints1-infected astrocytes

To determine whether minocycline prevents apoptotic signaling in the infected cells, we examined levels of apoptotic-related proteins using (a) the early markers of apoptosis (phospho-p53 and p21), (b) levels of the anti-apoptotic protein Bcl2, and (c) the late executioner of apoptosis, which is cleaved caspase 3 (Wilson, 1998).Figure 6 shows thatts1-only C1 cells contained increased phospho-p53 (Ser15) and p21 at 24, 48 and 72 h of culture, whilets1-mino C1 cells had significantly less phospho-p53 (Ser15) and p21 at 48 and 72 h. At 72ts1-only C1 cells showed pronounced cleaved caspase 3 elevations, whilets1-mino cells had much lower levels. Finally, althoughts1-only C1 cells showed decreasing amounts of Bcl2 at all three timepoints,ts1-mino C1 cells maintained close-to-normal levels of Bcl2 throughout (Fig. 6).

2.7 Minocycline affects levels of Nrf2, COX-2 and phospho-p53 ints1-infected primary astrocytes

To determine whether minocycline has similar effects on primary astrocytes to those onts1-infected C1 astrocytes, we examined the expression patterns of three major proteins from minocycline-treatedts1-infected primary astrocytes. Similar data regarding to levels of Nrf2 were obtained from primary cultured astrocytes (Fig 7). In addition,Figure 7 also shows that similar COX-2 and phospho-p53 expression patterns were observed by Western blot from primary astrocytes with the similar treatment as C1 cells. These data indicates that effects of minocyclinets1-infected primary astrocytes are similar to those on onts1-infected C1 astrocytes.

2.8 Minocycline protects primary neurons cocultured withts1-infected C1 cells, or neurons exposed to spent medium from infected C1 cell cultures

To determine whether primary neurons are affected when they are exposed tots1-only C1 cells, we first set up cocultures of neurons (bottom wells) with uninfected orts1-only C1 cells (top inserts).Figure 8A shows that the neurons cocultured withts1-only C1 cells die. This does not occur when minocycline is added to these cocultured neurons. We next asked whether spent medium fromts1-only C1 cells could also harm primary neurons, and we found that it does.To find out how spent medium from infected cells kills neurons, we compared intracellular ROS levels in the minocycline treated vs. untreated neuron cultures exposed to spent medium from te1 -infected C1 cells.Figure 8B shows that intraneuronal ROS levels are elevated in neurons exposed to te1-only C1 cell spent medium, relative to those of neuronal cultures exposed to medium from uninfected C1 cells. These results identify induced oxidative stress as one of the pathways, by which neurons are killed by nearbyts1 -infected astrocytes.Figure 8B also shows that minocycline treatment of the neurons, given at the time when spent medium fromts1-only C1 cultures is added, reduces intraneuronal ROS levels, in a minocycline dose-dependent manner.Figure 8C shows that neuronal levels of Nrf2, which are reduced after addition ofts1-only C1 cell spent medium, are elevated by minocycline treatment. Neuronal protection by minocycline thus appears to involve antioxidant effects that include both direct radical scavenging and activation of endogenous cellular antioxidant pathways.

4.1 Animals and infection

FVB/N mouse pups were injected intraperitoneally (i.p.) with 0.1 ml/pupts1 virus suspension containing 1×10⁷ infectious units (IU)/ml ofts1, at 3 days after birth. The infected animals were then separated into groups.ts1 -infected minocycline-treated mice (ts1-mino, n=8) were treated with minocycline (Sigma), delivered intraperitoneally at 10 mg/kg/day at 5 days after birth (2 days post-infection, or dpi) continuously, until the infected untreated mice began to become paralyzed and then die. Untreatedts1 -infected mice (ts1l–only, n=10) were treated on the same schedule with an identical volume of normal saline. The two groups of mice were then followed for the development of paralysis and death. In mice infected at birth with this dose ofts1, early stages of paralysis are evident by about 30 dpi (with hindlimb drop, scored as Stage I, followed by tremor and loss of mobility at Stages II and III, and ended by the abrupt onset of Stage IV paralysis, which is brief and immediately precedes death). Mice are therefore sacrificed at Stage IV, and the time of sacrifice used as a proxy measurement for the time of survival.

4.2 Cells and minocycline treatment

Immortalized astrocytic cells of the C1 line, developed in our laboratory(Lin et al., 1997), were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% Fetal bovine serum (FBS). Primary astrocyte cultures were isolated from 1- to 2-day-old FVB/N mice as described previously (Shikova et al., 1993). Briefly, the cells were seeded into poly-L-lysine-coated plates and grown in DMEM-F-12 medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2.5 µg/ml fungizone, and the medium was changed every 3 days. After reaching confluence, the cells were passed four or five times and used before reaching confluence again. At this point, more than 95% of the cells in the cultures were positive for the astrocyte-specific glial fibrillary acid protein (GFAP) marker. For infection, 10⁶ cells were plated in each 100 mm plate with DMEM medium containing 1% FBS and 3 µg/ml polybrene the day before infection. Thets1 virus was diluted in the same medium and added to the cells at a multiplicity of infection (MOl) of 10 for 40 min, followed by washing of the cells and adding DMEM medium containing 10% FBS. Minocycline was added to the medium, to a final concentration of 50 µM, immediately afterts1 infection. Infected C1 astrocytes were tested for effects of minocycline on their survival in culture. Infected astrocytes were also cocultured with primary neurons (in dual-well chambers that prevent virus movement into the neuron-culturing medium) or were used to provide spent medium for addition to the neuron cultures. Because neurons are killed in both contexts, we tested minocycline for its ability to protect the neurons, either in cocultures or after their exposure to spent medium from the infected astrocyte cultures. 15F cells are used for virus titer assay as described previously (Jiang et al., 200a).

4.3 Virus titer determination

ts1 virus titers, in tissues and cell culture supernatants, were performed as described previously (Wong et al., 1985). Brain stems and spinal cords were taken at 30 dpi, weighed, and homogenized in 2 ml DMEM medium/sample. 15F cells were plated in DMEM medium containing 1% FBS and 3µg/ml polybrene, and were incubated with diluted tissue filtrates for 40 min to allow attachment of virus to the 15F cells. The medium was then replaced by DMEM containing 10% FBS. The medium was changed on the 3rd day after infection. On the fifth or sixth day after infection, the foci (infectious centers) were counted and the virus titers calculated and expressed as mean log₁₀ lU/g tissue ± Standard error for tissue, mean log₁₀ lU/ml for cell culture supernatants.

4.4 Primary neuron cultures

Primary cortical neurons were isolated and cultured in 6-well plates as described previously (Xiang et al., 1998), with minor modifications. Briefly, cortical brain tissues from newborn FVB/N mouse pups was excised, and individual cells were dissociated initially by trypsinization (0.125% in HBSS, Ca²⁺- and Mg²⁺-free) for 25 min at 37°C. After washing the cells with HBSS containing Ca²⁺ and Mg²⁺, the enzyme was inactivated with Neurobasal medium (Invitrogen, Carlsbad, CA) containing 10% FBS. The cells were then dissociated further in serum-free Neurobasal medium plus B27 supplement (Invitrogen, Carlsbad, CA) by sequential trituration. The dissociated cells were then plated on poly-D-lysine-coated 6 well plates in serum-free Neurobasal medium plus B27 supplement, and maintained at 37°C of CO₂ incubator.

4.5 Astrocyte-neuron cocultures

C1 astrocytes were cultured on 25 mm tissue culture inserts (Nunc), whose bottoms are composed of 0.02 µm Anopore membranes. These membranes do not allow thets1 virus to pass from the insert chamber. For coculturing experiments, uninfected orts1-infected astrocytes, cultured in Anopore inserts, were placed into receiving wells containing primary neurons cultured as described above. Both cell types were then followed over time, by microscopic evaluation, for cell death.

4.6 Western blotting analysis

C1 astrocytes, primary astrocyes or primary neurons from different treatment groups were washed with PBS and lysed in RIPA buffer as described previously(Kuang et al., 2005). Tissue lysates were cleared by centrifugation at 13,000×g for 20 min at 4°C. Protein concentrations were determined using the Bio-Rad Dc Protein Assay Reagent (Bio-Rad Laboratories, Hercule, CA). The lysates (30–50 µg total protein per sample) were separated on SDS-PAGE gels, transferred to PVDF membranes (Millipore Corp., Bedford, MA) and immunoblotted with primary antibodies. Nuclear and cytoplasmic extraction was performed using NE-PER Nuclear and cytoplasmic extraction kit (PIERCE) according to the manufacturer’s instruction.

The primary antibodies used were anti-cleaved caspase 3, phospho-p53 (Ser15), COX-2 (all from Cell signaling), anti-malondialdehyde, or MDA (GeneTex), anti-Nrf2 (R&D), anti-Bcl2, anti-Bax, anti-p21, IκBα and NF-κB p65 (all from Santa Cruz), followed by species-specific secondary antibodies. Immune complexes were detected on the membranes using enhanced chemiluminescence (NEN Life Science Products, Boston, MA) according to the manufacturer’s instructions. A monoclonal anti-β-actin antibody (Sigma) was used as a control for protein loading.

4.7 Intracellular reactive oxygen species (ROS) assay

For measurement of ROS levels in C1 astrocytes, 1.5 ×10⁴ /C1 cells per well were plated in 96- well plates with DMEM medium, containing 1% FBS and 3 µg/ml polybrene, the day before infection. The cells were then infected at a MOI of 5 for 40 min, and either left untreated or treated with minocycline at stepped concentrations. After 4 h, the cells were washed with PBS, and the cells were loaded with 20 µM of the fluorescent probe 5-(and-6)-chloromethyl-2’,7’-dichlorodihydrofluorescein diacetate acetyl ester (CM-H₂DCFDA; Molecular Probes) for 30 min at 37 °C, followed by washing with PBS. ROS levels in the cells were measured with a fluorescent plate reader (BioTek, Winoosk, Vermont) at an excitation/emission setting of 488/520 nm.

For measurement of ROS levels in neurons, primary neurons (2.5 × 10⁵ cell/well) were plated on poly-D-lysine coated 96 well plate for 5 days. These cells were then treated with spent medium from uninfected orts1 -infected astrocyte cultures, in two different ratios relative to medium already in the well: 1/15 and 1/7.5. After culturing with spent medium, some cultures were left untreated, while others were treated with minocycline. 4 h later, the medium-treated neurons were washed with PBS and loaded with 20µM CM-H₂DCFDA for 20 min. Intracellular relative ROS levels were detected as described previously.

4.8 Immunohistochemistry and immunocytochemistry

Brainstem or spinal cord tissues from uninfected,ts1-only andts1-mino animals were snap-frozen in Tissue-Tek OCT embedding medium (Electron Microscopy Sciences, Hatfield, PA) in liquid nitrogen, and cut as 5 µm sections, stained with hematoxylin and eosin (HE) for histo-pathological analysis. For immunofluorescence staining, the sections were thawed, fixed in acetone for 5 minutes, and then incubated first in 10% donkey serum for 15 minutes, washed, and then incubated in primary antibodies rabbit anti-GFAP (sigma), or rabbit anti-CD68 (Santa Cruz), at 1:200 overnight. After incubation and washing, the sections were then incubated in anti-rabbit IgG secondary antibodies conjugated with fluorescein (anti-goat IgG; 1:200) or Texas Red from Jackson Immunnoresearch, West Grove, PA. Control sections were incubated with IgGs from rabbit serum prior to incubation in secondary antibodies, or were incubated in secondary antibodies alone. No nonspecific staining was observed on control sections (data not shown).

Immunohistochemistry with paraffin sections was performed as described previously (Kim et al., 2005). Briefly,ts1-only (n=5) andts1-mino (n=5) mice were anesthetized at 30 dpi by intraperitoneal injection of pentobarbital (150 mg/kg), and then were transcardially perfused with 10% buffered formalin, using a peristaltic pump. After 12 h of fixation, each mouse’s brain was dissected, with the brainstem segments separated for further processing. The sections (6 µm) were deparaffinized and washed with Tris-buffered saline (TBS; 100 mM Tris, 150 mM NaCI, pH 7.4) for 20 min at room temperature. The sections were then subjected to an antigen retrieval protocol, in which they were heated in 10 mM citrate buffer (pH 6.0) for 10 min, blocked with 5% normal goat serum in TBS, and then incubated overnight at 4°C with rat anti-COX-2 antibody (Chemicon International) at a dilution of 1:100, or with rabbit anti- GFAP (Sigma; at 1:200). After three 5-min washes in TBS, the sections were then incubated with biotin-conjugated anti-rabbit or anti-goat immunoglobulin G (IgG) for 30 min at room temperature, and treated with reagents from a Vecta-Elite streptavidin peroxidase kit, with a benzidine substrate for color development. The sections were counterstained with 1% methyl green or diluted hematoxylin. Sections not incubated with a primary antibody served as negative controls.

4.9 Statistical analysis

Data are presented as means ± standard error (SEM). Cell culture experiments were conducted in triplicate or duplicate wells, with the means from three to four individual experiments used for statistical analysis. Statistical significance of the results was determined by analysis of variance (ANOVA) or Student’st-test.p values of < 0.05 were considered statistically significant. The cumulative incidence of hindlimb paralysis/death in infected mice was determined by analysis of covariance comparing slopes of curves forts1-infected mice to those for infected mice treated with minocycline.

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