2 and transcriptional factors CREB and NF-κB in hippocampal neuronal cell culture

Journal of Neuroimmunology 195 (2008) 36 – 46 www.elsevier.com/locate/jneuroim

Chronic CXCL10 alters the level of activated ERK1/2 and transcriptional factors CREB and NF-κB in hippocampal neuronal cell culture Hilda Bajova, Thomas E. Nelson, Donna L. Gruol ⁎ Molecular and Integrative Neurosciences Department, The Scripps Research Institute, La Jolla, CA 92037, USA Received 26 October 2007; received in revised form 21 December 2007; accepted 8 January 2008

Abstract Signal transduction pathways may be important targets of chemokines during neuroinflammation. In the current study, Western blot analyses show that in rat hippocampal neuronal/glial cell cultures chronic CXCL10 increases the level of protein for ERK1/2 as well as for the transcriptional factors CREB and NF-κB. Bcl-2, an anti-apoptotic protein whose expression can be regulated by a pathway involving ERK1/2, CREB and NF-κB, was also increased in the CXCL10 treated cultures. These results implicate a role for ERK1/2, CREB and NF-κB in effects of CXCL10 on hippocampal cells and suggest that chronic CXCL10 may have a protective role during certain neuroinflammatory conditions. © 2008 Elsevier B.V. All rights reserved. Keywords: Chemokines; CNS; Neuroinflammation; Neuroprotection

1. Introduction Altered patterns of chemokine expression by CNS cells are the typical hallmarks of many CNS inflammatory and neurodegenerative diseases, suggesting a role for these factors during CNS neuroinflammation. The CXC chemokine ligand 10 (CXCL10) is thought to play an important role in neuroinflammatory diseases and its action may involve neuronal cells (van Marle et al., 2004). During CNS neuroinflammation expression of CXCL10 is elevated several fold (Franciotta et al., 2001; Kieseier et al., 2002; Kolb et al., 1999; Letendre et al., 1999; Sorensen et al., 1999; Xia et al., 2000) and occurs in a variety of CNS cells including astrocytes, microglia, Abbreviations: BSA, bovine serum albumin; CREB, cAMP response element binding protein; CXCL10, CXC chemokine ligand 10; DIV, days in vitro; ERK1/2, Extracellular-signal related kinase 1 and 2; GFAP, glial fibrillary acidic protein; HAD, human immunodeficiency virus-associated dementia; MAPK, mitogen-activated protein kinase; MS, multiple sclerosis; NF-κB, nuclear factor-kappa B; NSE, neuron specific enolase. ⁎ Corresponding author. Molecular and Integrative Neurosciences Department, SP30-1170, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA. Tel.: +1 858 784 7060; fax: +1 858 784 7393. E-mail address: [email protected] (D.L. Gruol). 0165-5728/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2008.01.003

and neurons (Asensio and Campbell, 1999; Carter et al., 2007; Kutsch et al., 2000; Ransohoff et al., 1993; Rossi and Zlotnik, 2000; Shen et al., 2006; Simpson et al., 2000; Wang et al., 1998). CXCL10 exerts its biological activity by binding to the Gprotein coupled receptor CXCR3, which is expressed on CNS neurons, astrocytes and microglia (Coughlan et al., 2000; Xia et al., 2000). In hippocampal neurons, exogenously applied CXCL10 alters synaptic plasticity (Vlkolinsky et al., 2004), activity-dependent intracellular Ca2+ signaling and neuronal and synaptic activity (Nelson and Gruol, 2004), suggesting that CXCR3 can regulate pathways that modulate neuronal function. However, CXCL10 can also have neurotoxic effects and induce neuronal apoptosis under conditions of virally induced neuroinflammation (Sui et al., 2004; Sui et al., 2006; van Marle et al., 2004). Thus, CXCL10 can have diverse effects on the CNS during neuroinflammatory conditions. The mechanisms mediating the effects of CXCL10 on CNS neurons have yet to be identified. Binding of CXCL10 to CXCR3 can activate a variety of intracellular signal transduction pathways (Bonacchi et al., 2001; Moser and Loetscher, 2001; Xia et al., 2000), including the Ras/ERK signaling pathway, a pathway that is known to play an important role in functions such as growth, differentiation, survival, and long-

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term memory (Bahr et al., 2002; Bailey et al., 1997; Fukunaga and Miyamoto, 1998; Hetman and Gozdz, 2004; Kornhauser and Greenberg, 1997; Vaudry et al., 2002; Waetzig and Herdegen, 2003). However, prolonged activation of ERK has been reported to participate in neuronal death induced by various stimuli (Alessandrini et al., 1999; Murray et al., 1998; Runden et al., 1998). During chronic neuroinflammatory conditions (e.g., HIV infection) CNS cells are likely to be exposed to CXCL10 for prolonged periods and it is unknown if the ERK pathway is affected by CXCL10 under such conditions. In the current study, we addressed this question using cultured rodent hippocampal cells chronically treated with CXCL10 at pathophysiological concentrations. Because the MAPK/ERK pathway can regulate the activity of various transcription factors including CREB (cAMP response element binding protein) (Gutkind, 1998) and NF-κB (Lee and Burckart, 1998; Takeuchi and Fukunaga, 2003) and subsequent gene expression, we also determined if the levels of these transcription factors were affected by chronic CXCL10. Results from Western blot analyses showed that in the culture model, exposure to pathophysiological levels of chronic CXCL10 results in altered levels of total protein and/or activated protein for ERK and the downstream transcription factors CREB and NF-κB. In addition, chronic CXCL10 results in increased expression of markers of gliosis and neuroprotection. 2. Materials and methods The animal procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Animal facilities and experimental protocols were in accordance with the Association for the Assessment and Accreditation of Laboratory Animal Care. 2.1. Cell cultures Primary cultures were prepared from rat hippocampus (Sprague–Dawley; Charles River, Wilmington, MD, USA) and maintained in vitro as described previously (Nelson and Gruol, 2004). Briefly, hippocampal were isolated at embryonic day 20, minced, and briefly triturated in Ca2+- and Mg2+-free saline containing (in mM): 137 NaCl, 5.4 KCl, 0.17 Na2HPO4, 0.22 KH2PO4, 27.7 glucose, 43.8 sucrose, 10 HEPES–NaOH (pH 7.3 with NaOH). Enzymatic treatment was not used. The plating suspension consists primarily of small pieces of tissue and clusters of cells. During the culture period the plated tissue spreads and thins and eventually forms a monolayer of neurons that grow on top of a glial cell layer. For immunocytochemistry, a plating density of 1 hippocampus per glass bottom culture dish (MatTek, Ashland, MA, USA) coated with Matrigel (BD Biosciences, Bedford, MA, USA) was used. For Western blotting, a plating density of 2.5 hippocampi per 35-mm tissue culture dish, also coated with Matrigel, was used. The plating medium contained minimal essential medium with Earle's salts and L-glutamine (Gibco-Invitrogen, Carlsbad, CA, USA), 10% heat-inactivated horse serum (Gibco-Invitrogen), and 10% heat-

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inactivated fetal calf serum (Gibco-Invitrogen), and was supplemented with D-glucose to a final concentration of 5.0 g/ l. Medium was exchanged twice weekly with medium having a similar composition as above, except that the fetal calf serum was omitted. Cultures were incubated at 37 °C in a 5% CO2 humidified atmosphere. Brief treatment with the anti-mitotic agent 5-fluorodeoxyuridine (20 μg/ml, days 2–6 in vitro; Sigma, St. Louis, MO, USA) limited the number of nonneuronal cells in culture. Antibiotics were not used. 2.2. CXCL10 treatment Cultures from each dissection were divided into control and chronic CXCL10 treatment groups. Human recombinant CXCL10 (BSA- and endotoxin-free; R&D Systems, Minneapolis, MN, USA), which is active at rat receptors (recombinant rat CXCL10 is not commercially available), was reconstituted in 0.1% bovine serum albumin (BSA; fatty acid-free) to a final concentration of 50 μM and stored at − 20 °C. CXCL10 treatment was started on the second day in vitro (DIV) by adding CXCL10 to the cultures. To the control cultures, instead of CXCL10, the same amount of 0.1% BSA (fatty acid-free) was added. All treatments lasted for 9 days with CXCL10 reapplied every third day during normal media changes. Results for each study were obtained from at least 3 different culture sets. The ED50 for physiological actions of the CXCL10 used in our studies ranges from 15 to 75 ng/ml (∼ 1–5 nM) in transfected immune cells (R&D systems; measured by the ability to chemoattract mouse BaF/3 cells transfected with CXCR3). However, our previous studies showed that in rat hippocampal cultures neurophysiological actions of the CXCL10 required a higher concentration, 100 nM CXCL10 (Nelson and Gruol, 2004), a concentration similar to that reported for physiological actions of other chemokines on neurons and glia (Biber et al., 2001; Liu et al., 2003; Meucci et al., 1998; Oh et al., 2002). Therefore, we focused on a concentration range known to have biological activity in our system and CXCL10 was tested at 50, 100 and 250 nM. These concentrations are likely to reflect pathophysiological conditions because CXCL10 levels in the normal brain are low or undetectable (Mennicken et al., 1999). For example, CXCL10 levels in the CSF of humans under normal conditions are around 100 pg/ml (Galimberti et al., 2006) but can increase as much as 50 fold during pathophysiological conditions such as CNS inflection (Cinque et al., 2005). 2.3. Western blot assays At 11 DIV the cultures were processed for Western blot assay. Samples were placed on ice, washed 3 times with ice cold phosphate-buffered saline (PBS), harvested and homogenized in the Lysis buffer: 50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.5% NP-40, a Protease Inhibitor Cocktail Tablet (Boehringer Mannheim, Indianapolis, IN, USA), and a cocktail of phosphatase inhibitors (4.5 mM sodium pyrophosphate, 10 mM β-glycerophosphate, 1 mM sodium fluoride, 1 mM sodium orthovanadate). The protein

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concentrations were determined using the Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, CA, USA). The samples (in duplicate) were separated by NuPAGE® gels (Invitrogen, Carlsbad, CA, USA), transferred onto Immobilon P membranes (Millipore, Billerica, MA, USA) and stained with Ponceau S staining to confirm uniform transfer. After destaining, the membranes were transferred to PBS or Tris buffered saline (TBS) containing 0.1% Tween-20 and 5% of non-fat dry milk or casein for blocking, and then incubated in one of the following primary antibodies: polyclonal rabbit anti-MAP Kinase (ERK1/2) (1:5000, #61-7400, Zymed, Carlsbad, CA, USA), p-ERK1/2 mouse monoclonal (1:1000, sc-7383, Santa Cruz, Santa Cruz, CA, USA), CREB rabbit polyclonal antibody (1:1000, #9192, Cell Signaling, Danvers, MA, USA), pCREB (Ser133) mouse monoclonal antibody (1:1000, #9196, Cell Signaling), monoclonal anti β-actin clone AC-15 (1:5000, #A5441, Sigma, St. Luis, MO, USA), monoclonal antibody to glial fibrillary acidic protein (GFAP; 1:5000, MAB360, Chemicon, Temecula, CA, USA), mouse monoclonal anti-neuron specific enolase antibody (NSE; 1:5000, MAB314, Chemicon), NF-κB p65 rabbit polyclonal antibody (1:1000, sc-372, Santa Cruz), pNF-κB p65 (Ser 536) rabbit polyclonal antibody (1:1000, #3031, Cell Signaling), IκB-alpha rabbit polyclonal antibody (1:1000, Cell Signaling, #9242), pIκB-α (Ser32) rabbit polyclonal antibody (1:1000, Cell Signaling, #9241), Bcl-2 mouse monoclonal antibody (1:500, sc7382, Santa Cruz), SOD2 rabbit polyclonal antibody (1:5000, sc30080, Santa Cruz) followed by secondary antibodies: goat antirabbit or goat anti-mouse IgG (H + L) (1:10,000, Southern Biotech, Birmingham, AL, USA). For visualization, the ECL system (Amersham, Piscataway, NJ, USA) and Kodak Biomax ML films (Kodak, Rochester, NY, USA) were used. We measured the absorbencies of the immunoreactive bands using the NIH Image software program (http://rsb.info.nih.gov/nih-image). All membranes were stripped and reblotted for β-actin. All treatment groups were run on the same gel to facilitate comparison of data. To adjust for variability that can occur between different culture sets, the data were normalized. For each gel, density measurements were first normalized to density measurements for β-actin in the same lane of the gel and then the normalized values for each treatment group were normalized to the normalized value for the control treatment group run on the same gel. Normalization to β-actin, a structural protein expressed by both neurons and glia, minimized potential effects of any loading errors on results. In addition, normalization to βactin adjusted for potential generalized effects of CXCL10 on cell growth, which could complicate identification of selective effects of CXCL10 on specific cellular proteins. For statistical analysis, normalized data from different culture sets were combined. Because the normalization protocol resulted in values of 1.0 for the control treatment groups and no variance in the mean of control values, the one group t-test was used to test for statistical significance. Statistical significance was set at p b 0.05. The one group t-test tests the hypothesis that the difference between the normalized mean of experimental samples and the hypothesized population mean (1.0) is statistically significant. This test is useful when experimental values are expressed in relative terms with respect to control values. Although this test

minimizes the problem of variability between culture sets, it is less robust than a 2-sample t-test or ANOVA. 2.4. Immunocytochemistry Immunocytochemical staining of the hippocampal cultures was performed according to previously published methods (Nelson and Gruol, 2004) using the same primary antibodies as for the Western blotting (listed above). In brief, cultures (9–14 DIV) were rinsed with serum-free MEM, fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS, 100 mM, pH 7.3) for 15 min, and permeablized with 0.05% Triton X-100 in PBS for 30 min. The cultures were then incubated overnight (4 °C) in PBS containing the primary antibodies (listed above) in dilutions 1:50–500 and 0.05% BSA as a blocking agent. Immunoreactivity was detected with the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA, USA). As a control for nonspecific staining, prior to the immunostaining procedure the primary antibodies were co-incubated with the respective antigenic peptides (1:10 dilution) used for production of the primary antibodies. Non-specific staining was not observed for any of the antibodies used for immunocytochemical studies. 3. Results 3.1. Chronic CXCL10 effects on culture characteristics Mixed neuronal/glial cultures prepared from embryonic rat hippocampus were exposed to CXCL10 (50, 100 and 250 nM) starting at 2 days in vitro (DIV) and continuing until 11 DIV. Microscopic examination of the cultures at the end of the treatment period (11 DIV) did not reveal any prominent differences in culture characteristics or the morphological features of neuronal and glial cells between control and CXCL10-treated cultures at any of the CXCL10 concentrations tested (Fig. 1A, C). Consistent with these results, measurement of total protein levels in the cultures showed no significant difference between control and chronic CXCL10 treated cultures (Fig. 1B), suggesting that the overall culture growth and viability were not altered by the treatment paradigm. However, Western blot analyses of the relative level of specific cellular proteins showed a significant effect of chronic CXCL10 exposure. The relative level of glial fibrillary acidic protein (GFAP) was prominently increased in the chronic CXCL10-treated cultures in a dose-dependent manner (Fig. 1D, G). The relative levels of the neuron specific protein enolase and the housekeeping protein β-actin were also significantly increased but only at the highest concentration of CXCL10 tested (250 nM) (Fig. 1E–G). These results show that chronic exposure of hippocampal neurons and glial cells to CXCL10 can result in a significant alteration in the levels of important cellular proteins. 3.2. Chronic CXCL10 increases the level of activated ERK1/2 Chemokines have been reported to produce cellular actions by activating a variety of signal transduction pathways (Cartier et al., 2005). Our previous studies showing that acute exposure

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to CXCL10 alters neuronal excitability and synaptic transmission in hippocampal cultures (Nelson and Gruol, 2004) provides evidence for the expression of a CXCL10 signaling pathway in the hippocampal cells. Our current studies showing that chronic exposure to CXCL10 alters the levels of cell-specific proteins in the hippocampal cultures also provides evidence for a CXCL10 signaling pathway in the hippocampal cells. Although limited information is available on the signaling pathway activated by CXCL10 in CNS neurons and glia, it has been reported that acute exposure to CXCL10 can activate the ERK1/2 pathway in neurons (Bonacchi et al., 2001; Xia et al., 2000), a pathway that mediates a number of neuronal functions including synaptic plasticity, neuronal growth, differentiation, and survival (Bahr et al., 2002; Bailey et al., 1997; Fukunaga and Miyamoto, 1998; Hetman and Gozdz, 2004; Kornhauser and Greenberg, 1997; Vaudry et al., 2002; Waetzig and Herdegen, 2003). In the hippocampal cultures, acute exposure to CXCL10 increased the level of activated (i.e., phosphorylated) ERK1/2 (no effect on total ERK1/2), consistent with an involvement of the ERK1/2 pathway in the actions of acute CXCL10 on the hippocampal cells (Nelson and Bajova, 2007). However, it is unknown if the ERK1/2 pathway is involved in actions of CXCL10 in the hippocampal cultures under conditions of chronic CXCL10

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exposure. To address this question, we determined the relative level of the activated form of ERK1/2 protein using Western blot analysis in control cultures and cultures exposed to 100 and 250 nM CXCL10 for 9 days. ERK1/2 is activated by phosphorylation at residues 183 and 185. Analysis of Western blots probed with an antibody specific for the phosphorylated form of ERK1/2 (pERK1/2) protein showed a significant, dose-dependent increase in the level of pERK1/2 in cultures exposed chronically to CXCL10 at 100 nM (30% increase) or 250 nM (78% increase) above pERK1/2 levels in control cultures (Fig. 2A). The level of total ERK1/2 protein was also increased by the chronic CXCL10treatment in a dose-dependent manner with a significant increase at 250 nM CXCL10 (50% increase) compared with ERK1/2 levels in control cultures (Fig. 2B). When the level of pERK1/2 was expressed relative to total ERK1/2, there was no significant difference in the ratio of pERK to total ERK between control and CXCL10-treated cultures at either of the CXCL10 concentrations tested (Fig. 2C). These results show that chronic exposure CXCL10 alters the ERK1/2 pathway by increasing both the level of total ERK1/2 and the level of the activated form (pERK1/2). The increase in level of the activated form of ERK (∼25–70%) observed in the CXCL10-treated cultures is

Fig. 1. Chronic CXCL10 treatment alters the level of enolase and GFAP in hippocampal cultures. (A) Digitized phase contrast images of live control and CXCL10treated hippocampal cultures. Clusters of phase bright neurons and their phase dark processes are evident under all treatment conditions. The morphological features of the cultures and neurons were similar in all treatment groups. (B) Graph showing mean normalized values (± SEM) for total protein levels in control and CXCL10treated cultures. Protein levels in CXCL10-treated cultures were normalized to levels in control cultures. Control values are equal to one and represented by the dotted line. (C). Digitized images (Hoffman optics) of control and CXCL10-treated hippocampal cultures immunostained with an antibody to GFAP. The cultures were plated at low neuronal density so that the astrocyte population was clearly visible. The astrocyte layer forms the substratum upon which the neurons grow and is masked by the neuronal population in the denser plated cultures used for protein analyses. The white arrow in the images indicates one of the neurons. The black arrow indicates one of the astrocytes. The calibration bar also applies to A. (D–F) Graphs showing mean values (±SEM) for the relative level of several proteins assessed by Western blot in control (n = 6) and chronic CXCL10-treated cultures (n = 6 for each CXCL10 concentration). CXCL10 at 250 nM increased the levels of GFAP (D), neuron specific enolase (E) and β-actin (F). The results for β-actin are presented as the ratio of CXCL10-treated to control values. Measured values for neuron specific enolase and GFAP were normalized to the β-actin level in the same lane and then the normalized value for each treatment group was normalized to the normalized value for the control group run on the same gel (see Materials and methods; 50 nM, striped bar; 100 nM, white bar; 250 nM, black bar). The dashed lines represent control levels, which are equal to 1. Statistical significance (⁎), calculated by one group t-test, was set at p ≤ 0.05. (G) Representative Western blots of β-actin, neuron specific enolase and GFAP.

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cell membrane to the nucleus through regulation of the functional activity of various transcription factors with subsequent regulation of gene expression. The activity of the transcription factor CREB is regulated by phosphorylation that is mediated by several kinases, including ERK1/2. Therefore, it was of interest to determine if chronic CXCL10 treatment of the cultures also affected the status of CREB. Using Western blot analysis we determined the level of the phosphorylated (activated) form of CREB (pCREB) and total levels of CREB following chronic exposure of the hippocampal cultures to 100 and 250 nM CXCL10. Chronic CXCL10 at 100 nM did not significantly alter the level of pCREB (Fig. 3A), whereas at 250 nM, chronic CXCL10 produced a significant increase in the level of pCREB (∼ 75% increase) compared with levels in control cultures (Fig. 3A). The level of total CREB protein was reduced (24% Fig. 2. Chronic CXCL10 alters the levels of phosphorylated and total ERK. (A–C) Graphs showing mean normalized values (±SEM) for the relative level of pERK (A), ERK (B) (normalized to β-actin), and pERK/ERK (C) assessed by Western blot in control (n = 6) and chronic CXCL10 treated cultures (n = 6 for each CXCL10 concentration; 100 nM, white; 250 nM, black). The dashed lines represent control levels. Statistical significance (⁎), calculated by one group t-test, was set at p ≤ 0.05. (D) Representative Western blots of pERK and ERK.

comparable to the increase produced by high frequency stimulation paradigms used to induce long-term potentiation (LTP) in the hippocampus, a cellular model of memory and learning (Kanterewicz et al., 2000). Therefore, the effects of CXCL10 are in a biologically relevant range. 3.3. Chronic CXCL10 increases the level of activated CREB Although ERK1/2 is involved in a number of cell functions, a primary role is the transduction of extracellular signals at the

Fig. 3. Chronic CXCL10 alters the levels of phosphorylated and total CREB. (A–C) Graphs showing mean normalized values (±SEM) for the relative level of phosphorylated and total CREB assessed by Western blot in control (n = 6) and chronic CXCL10 treated cultures (n = 6 for each CXCL10 concentration; 100 nM, white bar; 250 nM, black bar). The dashed lines represent control levels. Statistical significance (⁎), calculated by one group t-test, was set at p ≤ 0.05. (D) Representative Western blots of pCREB and CREB.

Fig. 4. Chronic CXCL10 increases the level of activated NF-κB. (A–C, E–G) Graphs showing mean normalized values (±SEM) for the relative level of phosphorylated NF-κB (A), total NF-κB (B), the ratio pNF-κB/NF-κB (C), pIκB (E), total IκB (F), and the ratio of pIκB/IκB (G) assessed by Western blot in control (n = 6) and chronic CXCL10 treated cultures (n = 6 for each CXCL10 concentration; 50 nM, striped bar; 100 nM, white bar; 250 nM, black bar). The dashed lines represent control levels. Statistical significance (⁎), calculated by one group t-test, was set at p ≤ 0.05. Representative Western blots of pNF-κB and NF-κB (D); pIκB and IκB (H).

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Fig. 5. Immunostaining for signal transduction proteins. (A) Digitized images of microscopic fields after immunostaining for ERK (A, top panel,) pERK (A, bottom panel), CREB (B, top panel), pCREB (B, bottom panel), NF-κB (C, top panel) and pNF-κB. Immunostaining is shown under Hoffman and phase contrast optics. Immunostained cells are evident under Hoffman optics, whereas all cells present in the field are evident under phase contrast optics. The prominent immunostaining of neurons with little or no staining of the surrounding glial cells was observed. The neuronal cells are visible under both Hoffman and phase contrast optics, whereas glial cells are visible only under phase contrast optics. Scale bar = 20 μm and applies to all images. White arrows indicate representative neurons and black arrows indicate representative glial cells.

reduction) at both 100 nM and 250 nM concentrations of CXCL10 and this reduction was significant for 250 nM CXCL10 (Fig. 3B). When the level of activated CREB was expressed as a ratio of activated CREB to total CREB (pCREB/ CREB ratio) the pCREB/CREB ratio was significantly increased (135%) in cultures treated with 250 nM chronic CXCL10 relative to pCREB/CREB ratio for control cultures (Fig. 3C). The increase in the pCREB/CREB ratio (86% increase) at 100 nM CXCL10 did not reach significance. These results show that chronic CXCL10 results in an increase in the level of the activated form of the transcription factor CREB, but unlike ERK1/2, where the increased level of pERK1/2 was associated with an increased level of total ERK1/2 protein, the increased level of pCREB was not associated with an increase in total CREB protein. The effect of CXCL10 on both ERK1/2 and

CREB was greatest at the 250 nM concentration of CXCL10, a result that may implicate the increased level of activated ERK1/ 2 in the increased level of activation of the downstream transcription factor CREB. 3.4. Chronic CXCL10 increases the level of activated NF-κB NF-κB (Nuclear factor-kappa B) is a transcription factor involved in several different signaling pathways including the MAPK/ERK pathway (Lee et al., 1998; Takeuchi and Fukunga, 2004; Mattson, 2005). Like ERK1/2 and CREB, NF-κB is activated by phosphorylation (Neumann and Naumann, 2007). To determine if the level of NF-κB was also altered by chronic CXCL10, we determined the relative level of the phosphorylated form (pNF-κB) and total protein (NF-κB) in the hippocampal

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cultures. For these studies, the cultures were treated chronically with CXCL10 at 50, 100 and 250 nM concentrations. Although chronic CXCL10 at 50 and 100 nM did not alter the level of pNF-κB, 250 nM CXCL10 produced a significant increase (154% increase) compared with the level of pNF-κB in control cultures (Fig. 4A). Total NF-κB was not significantly altered at any of the CXCL10 concentrations tested (Fig. 4B). The ratio of pNF-κB to total NF-κB (pNF-κB/NF-κB) was significantly increased in cultures treated with 250 nM CXCL10 (132%) compared with control cultures but the ratio was not significantly altered in the cultures chronically treated with 50 nM or 100 nM (Fig. 4C). NF-κB is maintained in an inactive form in the cytoplasm by the formation of a complex with inhibitory proteins, the IκBs (inhibitory kappa B). Proteolytic degradation of IκB is required for NF-κB translocation into the nucleus (Memet, 2006). This irreversible step in the signaling pathway constitutes a commitment to transcriptional activation. Because activation of NF-κB requires phosphorylation of inhibitory κB (I-kB) and its release from the inactive complex NF-κB/I-κB, leading to a subsequent phosphorylation of pNF-κB and degradation of pIκB, we also compared the levels of phosphorylated (pI-κB) and total protein I-κB between control and CXCL10 treated cultures. Similar to pNF-κB, lower concentrations of CXCL10 (50 and 100 nM) had no significant effects on the levels of pI-κB, as well as the ratio pI-κB/I-κB (Fig. 4E, F, G). The levels of pI-κB were also unchanged by treatment of the cultures with 250 nM CXCL10. However, the total protein levels of I-κB were significantly decreased in cultures treated with 250 nM CXCL10 (28%) and the ratio of pI-κB/I-κB in cultures treated with 250 nM chronic CXCL10 was significantly increased (59%) above levels in control cultures (Fig. 4G). Thus, the results from both NF-κB as well as I-κB are consistent with a significant elevation in the level of activated NF-κB by chronic CXCL10 treatment at a concentration of 250 nM.

proteins normally found in neurons (e.g., NMDA receptor) showed that the neurons and glia in our cultures could be readily identified under phase contrast optics based on morphological criteria (e.g., Fig. 1A, C). Immunoreactivity for pERK1/2 and total ERK1/2, pCREB and total CREB or pNF-κB and NF-κB was detected in both neurons and glial cells in the CXCL10treated cultures (250 nM CXCL10). However, immunoreactivity was prominent in the neuronal cells for all signal transduction molecules studied, whereas the glial cells showed only light staining (Fig. 5). This result suggests that neurons are the primary cell type involved in the CXCL10-induced changes in the level of the signal transduction proteins observed in our Western blot analyses. 3.6. Chronic CXCL10 increases expression of anti-apoptotic proteins Bcl-2 and SOD2 Activation of ERK1/2, CREB, and NF-κB can enhance antiapoptic actions by inducing expression of anti-apoptotic Bcl-2 proteins and antioxidant enzymes such as manganese superoxide dismutase (SOD2) that protect against superoxide radicals (Hetman and Gozdz, 2004; Hinerfeld et al., 2004; Martinou et al., 1994; Mattson et al., 1997; Mattson and Meffert, 2006). The increased levels of ERK1/2, CREB, and NF-κB coupled with the increased levels of the neuronal protein enolase and glial protein GFAP suggested that these downstream neuroprotective proteins could also be altered by chronic CXCL10. To test this possibility, we determined the relative levels of Bcl-2 and SOD2 hippocampal culture using Western blot analysis. The levels of Bcl-2 protein in the hippocampal cultures were increased in a dose-dependent manner by chronic CXCL10 (Fig. 6A, B). Cultures treated chronically with 50 nM CXCL10 showed a non-significant increase (41% increase), but the two higher concentrations of CXCL10 tested produced significant increases in Bcl-2 levels (100 nM, 145% increase; 250 nM, 82%

3.5. Neurons and glial cells express the signal transduction pathways The hippocampal cultures contain both neurons and glial cells, and both cell types have been shown to express CXCR3, the receptor for CXCL10 (Coughlan et al., 2000; Nelson and Gruol, 2004; Xia et al., 2000). Our immunohistochemical studies showed that in the hippocampal cultures neurons are strongly immunoreactive for CXCR3, whereas the astrocytes are only weakly immunoreactive (Nelson and Gruol, 2004). These results implicate neurons as the primary cell type involved in the altered expression of the signal transduction molecules studied in Western blots of the CXCL10-treated hippocampal cultures. To confirm that neurons expressed the signal transduction molecules, we immunostained the CXCL10-treated cultures (250 nM) using antibodies specific for pERK1/2 and total ERK1/2, pCREB and total CREB or pNF-κB and NF-κB and visually identified the cell types showing immunoreactivity using Hoffman optics. A separate set of immunocytochemical studies using cell-specific antibodies (GFAP and neuron specific enolase) and antibodies for

Fig. 6. Chronic CXCL10 increases Bcl-2 and SOD2. (A, C) Graph showing the mean normalized value (±SEM) for the relative level of Bcl-2 (A) and SOD2 (tetramer) (C) assessed by Western blot in control (n = 6) and chronic CXCL10 treated cultures (n = 6 for each CXCL10 concentration; 50 nM, diagonal; 100 nM, white; 250 nM, black). The dashed lines represent control levels. Statistical significance (⁎), calculated by one group t-test, was set at p ≤ 0.05. (B, D). Representative Western blot of Bcl-2 (B) and SOD2 (D).

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increase), suggesting an activation of anti-apoptotic mechanisms in the hippocampal cultures chronically treated with CXCL10. Levels of SOD-2 protein were also increased in a dose-dependent manner in the hippocampal cultures chronically treated with CXCL10 with a significant increase at 250 nM CXCL10 (60% increase). 4. Discussion Emerging research showing increased levels of the chemokine CXCL10 in the CNS during neuroinflammatory diseases has stimulated interest the consequences of the increased levels to CNS biology. Recent studies show that in cultured mouse CNS neurons acutely applied CXCL10 (25 nM) can result in activation of the ERK1/2 pathway (Xia et al., 2000), a pathway that plays a central role in CNS synaptic functions and cell growth and survival (Bahr et al., 2002; Bailey et al., 1997; Fukunaga and Miyamoto, 1998; Hetman and Gozdz, 2004; Kornhauser and Greenberg, 1997; Vaudry et al., 2002; Waetzig and Herdegen, 2003). Little is known about the signal transduction pathways activated in CNS cells by chronic exposure to CXCL10. Our results show that the level of the activated (i.e., phosphorylated) form of ERK as well as the level of total ERK1/2 protein is increased in rat hippocampal cultures in response to chronic CXCL10. In addition, the levels of the activated form of the transcription factors CREB and NF-κB, which are regulated by ERK1/2 (Gutkind, 1998; Lee and Burckart, 1998; Takeuchi and Fukunaga, 2003), are also altered. The level of the activated (i.e., phosphorylated) form of CREB (pCREB) was increased by CXCL10 treatment compared with control cultures although the level of total CREB protein was reduced. The level of phosphorylated NF-κB was also increased relative to the level in control cultures with no change in the level of total NF-κB. Although we did not carry out quantitative immunocytochemical studies, immunostaining for these proteins was intense in the cultured neurons with only minor or no staining in the glial population, suggesting that the changes observed in the Western blots primarily occurred in the neuronal population. Our previous immunohistochemical studies (Nelson and Gruol, 2004) showed that the cultured hippocampal neurons are strongly immunoreactive for CXCR3, the receptor for CXCL10, whereas astrocytes are only weakly immunoreactive, consistent with higher level of CXCR3 in neurons than in glia and the effects of CXCL10 on signal transduction molecules occurring primarily in the neuronal population. ERK1/2 activation plays a key role in promoting neuronal growth, differentiation, survival, as well as in long-term memory (Bailey et al., 1997; Fukunaga and Miyamoto, 1998; Hetman and Gozdz, 2004; Kornhauser and Greenberg, 1997). However, sustained ERK1/2 activation has been associated with neuronal death (Alessandrini et al., 1999; Murray et al., 1998; Runden et al., 1998). Abnormal activation of the ERK1/2 pathway has also been implicated in the pathogenesis of Alzheimer's disease (Alessandrini et al., 1999; Drewes et al., 1992; Knowles et al., 1999; Perry et al., 1999; Trojanowski et al., 1993; Veeranna et al., 1998). In our studies, the level of activated and total ERK1/2 was increased but there was no

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evidence supporting a negative effect of the elevated levels on the hippocampal cultures. Thus, the characteristics of the hippocampal cultures and morphological features of the neurons and glial cells in the CXCL10-treated cultures were similar to that observed in control cultures. Moreover, chronic CXCL10 increased the level of neuron specific enolase, the anti-apoptotic protein Bcl-2 and the protective enzyme SOD2. These results are suggestive of a survival promoting effect of chronic CXCL10 under the conditions used in our studies. Activation of ERK1/2 can result in rapid translocation of these enzymes to the nucleus, where they regulate the functional activity of various transcription factors (Treisman, 1996). In our studies the levels of two transcription factors that are regulated by the ERK pathway, CREB and NF-κB, were also altered by chronic CXCL10 treatment. CREB and its close relatives are widely accepted as prototypical stimulus-inducible transcription factors and are activated in response to a vast array of physiological stimuli (Mayr and Montminy, 2001). Chronic CXCL10 increased the level of activated CREB and decreased the level of total CREB. The effects of CXCL10 on the level of activated CREB (pCREB) suggest a possible role for increased CREB-mediated gene expression in the hippocampal neurons following chronic exposure to CXCL10. Such an effect could significantly alter hippocampal function. Several studies have shown that CREB-mediated gene expression is important for the survival of a variety of neuronal types (Bonni et al., 1999; Riccio et al., 1999; Walton et al., 1999). In addition, CREB induced gene expression plays an important role in learning, memory and synaptic plasticity (Bito et al., 1996; Hu et al., 1999). Because CREB regulation of cellular responses may be mediated through the expression of CREB itself, in addition to activation by phosphorylation (Walker et al., 1995), the CXCL10-dependent decrease in the level of total CREB protein observed in our studies may indicate an attempt of neuronal cells to down-regulate CREB-mediated gene expression perhaps as a neuroadaptive response to the chronic activation of the ERK pathway. The transcriptional activator NF-κB is a key regulator of the synthesis of cytokines and many other immunoregulatory gene products, and is widely distributed in the CNS (Meffert and Baltimore, 2005; Memet, 2006). Activation of NF-κB has been shown to be triggered by a wide variety of stimuli, such as glutamate depolarization, increase in intracellular Ca2+, neuropeptides, neural cell adhesion molecule, and cytokines (e.g., interleukin-1) (Freudenthal et al., 2004). NF-κB is activated in neurons and glial cells in a variety of acute and chronic neurodegenerative conditions (Epstein and Gelbard, 1999; Mattson, 2005). NF-κB influences the neurodegenerative process directly by affecting gene expression in neurons and indirectly by regulating gene expression in glial cells (O'Neill and Kaltschmidt, 1997). Activation of NF-κB in neurons promotes their survival and plasticity; on the other hand its activation in glial cells may play a major role in inflammatory processes that can damage and kill neurons (Mattson, 2005). Our Western blot studies show that chronic CXCL10 at 250 nM can significantly increase the levels of activated NF-κB and our results from immunocytochemistry showed intense pNF-κB

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staining in neurons with only light or no staining in glial cells. The intense pNF-κB staining in neurons but not glia is consistent with a neuroprotective role for the increased level of activated NF-κB in the CXCL10-treated cultures. A neuroprotective role of the increased levels of activated ERK1/2 and the transcription factors CREB and pNF-κB in the hippocampal cultures was supported by our studies on the effect of chronic CXCL10 on the level of the anti-apoptotic protein Bcl-2 and SOD2. The level of both proteins was increased by chronic CXCL10. Bcl-2 prevents activation of caspases involved in apoptosis, including caspase-3 and -9, by inhibition of cytochrome c release and by binding to apoptotic protease activating factor 1 (Murphy, 1999). SOD2, is a mitochondrial enzyme that protects against superoxide radicals that are normal by-products of cellular metabolism but can produce cell damage (Hinerfeld et al., 2004). A recent study showed that exposure to low concentrations of exogenous CXCL10 (10–100 ng/ml) for 18 h has pro-apoptotic effects on human fetal neuronal cell cultures (Sui et al., 2006). Activation of caspase-3 and -9 was shown to play a crucial role in CXCL10-dependent neuronal apoptosis observed in human fetal neuronal cell cultures (Sui et al., 2006). In our studies using rat hippocampus obtained from embryos at an older age (equivalent to third trimester human hippocampus) and chronic exposure to higher concentrations of CXCL10 for 9 days there was no evidence of toxicity by CXCL10. Thus, examination of cultures revealed no obvious differences in the number of neurons and neuronal clusters in culture. Moreover, we observed increased levels of neuronal and glial proteins, consistent with growth and development rather than toxicity (Fig. 1). The hippocampal cultures also showed a CXCL10-dependent increase in the level of Bcl-2, which could prevent neuronal death by blocking the activation of caspase-3 and -9. The increased levels of Bcl-2 could explain a lack of neurotoxicity in the hippocampal cultures exposed to chronic CXCL10 treatment. Because Bcl-2 expression is regulated by ERK1/2-dependent CREB activation (Hetman and Gozdz, 2004) as well as by NFκB (Mattson and Meffert, 2006), the CXCL10 induced increase in the level of activated ERK1/2 and/or one or more of the downstream transcription factors may mediate the upregulation of Bcl-2 levels. The lack of a toxic effect of CXCL10 in the rat hippocampal cultures compared to the toxicity observed in cultured human fetal neurons studied by Sui et al. (2004) is likely to reflect differences in the sensitivity of neurons to CXCL10 due to age or cell type. Consistent with this possibility, studies of human cholinergic neurons (LAN-2) also show a resistance to CXCL10-induced toxicity (500 nM CXCL10; higher concentrations produced toxicity) (van Marle et al., 2004). Although our data indicate predominant effects of chronic CXCL10 on neurons in the hippocampal cultures, increased levels of GFAP in the CXCL10-treated cultures suggest that CXCL10 also affects astrocytes. In general, astrocyte responses to chemokines involve chemotaxis, cell proliferation and survival (Farina et al., 2007). Increased levels of GFAP may also indicate astrocyte activation in our cell cultures by chronic CXCL10 treatment. Activated astrocytes are known to produce soluble trophic and growth factors, and immunomodulatory cytokines that enhance the survival of adjacent neurons and glia

in the likely attempt to preserve tissue integrity (Liberto et al., 2004; Ridet et al., 1997; Schwartz et al., 1993). Therefore, effects of chronic CXCL10 on hippocampal neurons observed in our studies may also involve indirect pathways through activation of astrocytes and subsequent secretion of growth factors, cytokines and chemokines and act on the neurons. Further studies will be required to test this possibility. Taken together, our studies show that prolonged exposure of hippocampal neuronal/glial cultures to CXCL10 results in a modulation of the ERK1/2 signal transduction pathway and the activation downstream transcription factors CREB and NF-κB in the hippocampal neurons, an effect that could play an important role in the CNS actions of CXCL10 during conditions of chronic neuroinflammation. Our results suggest that CXCL10 could be neuroprotective under some neuroinflammatory conditions. Moreover, because ERK1/2, CREB and NFκB are important regulators of neuronal physiology (Fukunaga and Miyamoto, 1998), our studies raise the possibility that CXCL10 actions on these signal transduction molecules could lead to a significant alteration in neuronal physiology during neuroinflammatory conditions. Acknowledgements The authors wish to thank Ms. Suzanne Phong and Stephine Chow for technical assistance and Ms. Floriska Chizer for her administrative assistance. This work was supported by RO1 MH63712 and P30 MH62261. References Alessandrini, A., Namura, S., Moskowitz, M.A., Bonventre, J.V., 1999. MEK1 protein kinase inhibition protects against damage resulting from focal cerebral ischemia. Proc. Natl. Acad. Sci. U. S. A. 96, 12866–12869. Asensio, V.C., Campbell, I.L., 1999. Chemokines in the CNS: plurifunctional mediators in diverse states. Trends Neurosci. 22, 504–512. Bahr, B.A., Bendiske, J., Brown, Q.B., Munirathinam, S., Caba, E., Rudin, M., Urwyler, S., Sauter, A., Rogers, G., 2002. Survival signaling and selective neuroprotection through glutamatergic transmission. Exp. Neurol. 174, 37–47. Bailey, C.H., Kaang, B.K., Chen, M., Martin, K.C., Lim, C.S., Casadio, A., Kandel, E.R., 1997. Mutation in the phosphorylation sites of MAP kinase blocks learning-related internalization of apCAM in Aplysia sensory neurons. Neuron 18, 913–924. Biber, K., Sauter, A., Brouwer, N., Copray, S.C., Boddeke, H.W., 2001. Ischemia-induced neuronal expression of the microglia attracting chemokine Secondary Lymphoid-tissue Chemokine (SLC). Glia 34, 121–133. Bito, H., Deisseroth, K., Tsien, R.W., 1996. CREB phosphorylation and dephosphorylation: a Ca(2+)- and stimulus duration-dependent switch for hippocampal gene expression. Cell 87, 1203–1214. Bonacchi, A., Romagnani, P., Romanelli, R.G., Efsen, E., Annunziato, F., Lasagni, L., Francalanci, M., Serio, M., Laffi, G., Pinzani, M., Gentilini, P., Marra, F., 2001. Signal transduction by the chemokine receptor CXCR3: activation of Ras/ERK, Src, and phosphatidylinositol 3-kinase/Akt controls cell migration and proliferation in human vascular pericytes. J. Biol. Chem. 276, 9945–9954. Bonni, A., Brunet, A., West, A.E., Datta, S.R., Takasu, M.A., Greenberg, M.E., 1999. Cell survival promoted by the Ras-MAPK signaling pathway by transcriptiondependent and -independent mechanisms. Science 286, 1358–1362. Carter, S.L., Muller, M., Manders, P.M., Campbell, I.L., 2007. Induction of the genes for Cxcl9 and Cxcl10 is dependent on IFN-gamma but shows differential cellular expression in experimental autoimmune encephalomyelitis and by astrocytes and microglia in vitro. Glia 55, 1728–1739.

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