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Originally published In Press as doi:10.1074/jbc.M709065200 on December 17, 2007

J. Biol. Chem., Vol. 283, Issue 10, 6546-6560, March 7, 2008
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Co-administration of Ciliary Neurotrophic Factor with Its Soluble Receptor Protects against Neuronal Death and Enhances Neurite Outgrowth*Formula

Mark A. Ozog1, Geetanjalee Modha, John Church, Rayne Reilly, and Christian C. Naus2

From the Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada

Received for publication, November 5, 2007 , and in revised form, December 11, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Attempts to promote neuronal survival and repair with ciliary neurotrophic factor (CNTF) have met with limited success. The variability of results obtained with CNTF may, in part, reflect the fact that some of the biological actions of the cytokine are mediated by a complex formed between CNTF and its specific receptor, CNTFR{alpha}, which exists in both membrane-bound and soluble forms. In this study, we compared the actions of CNTF alone and CNTF complexed with soluble CNTFR{alpha} (hereafter termed "Complex") on neuronal survival and growth. Although CNTF alone produced limited effects, Complex protected against glutamate-mediated excitotoxicity via gap junction-dependent and -independent mechanisms. Further examination revealed that only Complex promoted neurite outgrowth. Differential gene expression analysis revealed that, compared with CNTF alone, Complex differentially regulates several neuroprotective and neurotrophic genes. Collectively, these findings indicate that CNTF exerts more robust effects on neuronal survival and growth when applied in combination with its soluble receptor.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Ciliary neurotrophic factor (CNTF),3 a member of interleukin (IL)-6 cytokine family, was originally identified by its ability to support the in vitro survival of parasympathetic neurons from the chick ciliary ganglion (1). CNTF has been termed a "brain injury" cytokine based on several observations. First, humans and mice lacking CNTF appear normal, indicating that the cytokine is not essential for normal development or maintenance (2). Second, the level of CNTF in astrocytes increases dramatically in response to injury (3, 4). Third, CNTF is normally produced as a nonsecreted cytokine and is only released when the membrane integrity of astrocytes or Schwann cells becomes compromised, e.g. in response to injury (5-7). Fourth, although the expression of the specific receptor for CNTF, termed CNTFR{alpha}, is normally restricted to the plasma membrane of neurons (8), surviving astrocytes in the penumbra of an injury also begin to express CNTFR{alpha} (9, 10) suggesting a mechanism by which the nervous system responds to sublethal environmental stress or injury.

Although the above considerations have prompted interest in CNTF as a therapeutic agent against neuronal injury and neurodegenerative diseases, it has become apparent that the administration of exogenous CNTF elicits variable and, in any case, only weak neuroprotective and neurotrophic effects in experimental animals and humans (11-13). The limited therapeutic efficacy of CNTF as a neuroprotective and neuroregenerative agent may be consequent upon the administration of the cytokine without the soluble form of its specific receptor. Although CNTFR{alpha} is normally localized to the neuronal plasma membrane, it is anchored there by a glycosylphosphatidylinositol linkage that is sensitive to proteolysis and phospholipase-C-mediated cleavage, and following injury, it is released into the extracellular space as a soluble component (14, 15). Indeed, soluble CNTFR{alpha} has been identified in proximity to injured tissue and in the urine of patients with amyotrophic lateral sclerosis (4, 16). Following injury-induced release, CNTF and soluble CNTFR{alpha} together form a heterodimer (hereafter referred to as "Complex") that can interact with the ubiquitously expressed β-receptor subunits, glycoprotein 130 (gp130) and leukemia inhibitory factor receptor β (LIFRβ) (14, 17, 18). The subsequent heterodimerization of gp130 and LIFRβ activates several signaling cascades, including the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway (19). In addition, we have recently found that Complex increases both the expression of the gap junctional constituent connexin43 and intercellular coupling in glia via trans-signaling (17, 18), the term used to describe the ability of a ligand to bind a soluble receptor ({alpha} subunit), which subsequently associates with the β subunits of the receptor on cells that do not normally express the {alpha}-receptor of the ligand (20).

In light of the limited efficacy of CNTF alone as a neuroprotective and neurotrophic agent, and increasing evidence that connexins and gap junctions elicit neuroprotective and neurotrophic effects (21-26), in this study we examined the effects of exogenous CNTF, applied alone or in combination with its soluble receptor CNTFR{alpha}, on excitotoxic cell death, neuronal survival, and neuronal development.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Neuron/Astrocyte Co-cultures
Primary co-cultures of murine cortical astrocytes and neurons were prepared as described previously (25). Briefly, astrocyte cultures were established from the cortices of 1-day-old CD-1 mouse pups and plated onto poly-D-lysine-coated (50 µg/ml) 6-well plates. Culture medium was replaced every 3 days. Because reactive and newly plated (reactive-like) astrocytes express CNTFR{alpha}, we matured our astrocytes in vitro for 6 weeks to obtain quiescent cultures (18). Neurons, obtained from the neocortices of embryonic CD-1 mice aged 16 days gestation, were then seeded on top of the astrocytes and allowed to settle for 2 h before replacing the medium with Medium I (54 ml of Neurobasal Medium, 36 ml of Dulbecco's modified Eagle's medium/F-12, 1 ml of B-27 supplements; Invitrogen). On day 4, 2 µM cytosine arabinoside was added during a fresh medium change for 24 h. Thereafter, two-thirds of the conditioned medium was replaced every 3 days with fresh Medium I. Co-cultures were maintained for 2 weeks prior to experiments to ensure expression of functional glutamate receptors by the neurons (27).

Neuron-enriched Cultures
In a manner similar to that described above, neurons were seeded directly onto either PDL (50 µg/ml)/laminin-coated (100 µg/ml) transwells (neurite outgrowth quantification assay kit; Chemicon, Temecula, CA) or PDL/laminin-coated 2-well chamber slides (BD Biosciences/Fisher) at 2.5 x 105 cells per chamber. Two h after cell plating, the medium was replaced with serum-free Medium I. Cultures maintained for 13 days in vitro (DIV) were treated with cytosine arabinoside as described above. Astroglial contamination of the cultures was <5%, as assessed by direct morphological analysis and glial fibrillary acidic protein immunoreactivity.

PC12 Cells
Rat pheochromocytoma cells (PC12; ATCC, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% horse serum, 5% fetal bovine serum, 10 units/ml penicillin, and 10 µg/ml streptomycin. Differentiation of the PC12 cells was induced by exposure to serum-reduced medium (1% horse serum, 1% fetal bovine serum) supplemented with NGF (50 ng/ml; Sigma) for 72 h.

Test Treatments
Prior to the application of glutamate, neuron-astrocyte co-cultures were pretreated with vehicle (PBS), CNTF (20 ng/ml; R & D Systems, Minneapolis, MN), soluble CNTFR{alpha} (200 ng/ml; R & D Systems), or Complex (20 ng/ml CNTF + 200 ng/ml CNTFR{alpha}, an ~1:5 molar ratio to favor the association of CNTF with soluble CNTFR{alpha}) (18) with a fresh medium change every 24 h for 72 h. In some experiments, 24 h following the final pretreatment, the gap junction blocker carbenoxolone (CBX; 25 µM; Sigma), its inactive analogue glycyrrhizic acid (GZA; 25 µM; Sigma), or solvent (H2O) was added to the co-cultures for 1 h as described previously (25). The co-cultures were then bathed in Earle's balanced salt solution (Invitrogen) containing glutamate (1 mM) for 3 h. For a comparative control, selected sister cultures were treated similarly but with the omission of glutamate. Cells were then maintained in fresh Medium I for 24 h and subsequently assessed for cell viability (see below).

Neuron-enriched cultures were exposed to vehicle, CNTF, CNTFR{alpha}, or Complex for 3 days starting at either 2 h (hereafter termed "3 DIV" cultures) or 10 days ("10 + 3 DIV" cultures) after initial plating. A fresh medium change was performed every 24 h during treatments.

The JAK/STAT, MAPK/ERK, and PI3K/Akt signaling cascades were inhibited with 50 µM AG490 (Calbiochem), 10 µM U0126 (Promega, Madison, WI), and 20 µM LY294002 (Calbiochem), respectively. The inhibitors were applied 45 min prior to and throughout the treatment of neurons with vehicle or Complex. The inhibitors were applied at concentrations commonly used by others on neuronal cells in vitro and adequately blocked the signaling pathways as assessed by substrate phosphorylation detection on control immunoblots (data not shown). To examine if CNTF, CNTFR{alpha}, or Complex induced or enhanced neuronal differentiation, undifferentiated PC12 cells were exposed to the test agent in place of NGF for 3 days or were co-treated with NGF and the test agent for 24 h, respectively.

Cell Viability Assays
Cell survival was determined by measuring LDH content (Sigma) within the conditioned medium, the ability of the cells to exclude propidium idodide (PI), as well as the absence of cleaved caspase3 (see below) and/or TUNEL (DeadEnd Fluorometric TUNEL System; Promega).

RNA Isolation, Microarray Hybridization, and PCR
RNA Isolation—Cytoplasmic RNA was isolated from cells using TRIzol reagent (Invitrogen) and stored at -80 °C until used for microarray hybridization, real time PCR, or reverse transcription (RT)-PCR experiments.

Microarray Hybridization—RNA samples were processed for microarray hybridization as described previously by Treister et al. (28). Briefly, 2 µg of RNA was used to synthesize cDNA with the aid of CodeLink expression assay reagent kits (Amersham Biosciences). Target cRNA product was subsequently acquired using RNeasy kits (Qiagen), fragmented, and applied to a Codelink Mouse Uniset I microarrays (Amersham Biosciences) containing ~10,000 mouse oligonucleotide gene probes. Positive signals on the arrays were detected with streptavidin-Alexa 647 and scanned using ScanArray Express software and a ScanArray Express HT scanner. Microarray images were analyzed using CodeLink image and data analysis software. Experiments were carried out in duplicate, with consistent results. Values presented consist of the mean ratio of normalized intensities of the different treatment regimens as described, and experiments were carried out in duplicate. The Ingenuity Pathways Knowledge Base (Ingenuity Systems, Inc., Redwood City, CA) was used to assess gene expression in the context of regulatory clusters.


Figure 1
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  		FIGURE 1.
Complex reduces LDH release from neuron-astrocyte co-cultures treated with 1 mM glutamate for 3 h. Compared with controls (sham insult), significant increases in released LDH levels were seen 24 h after the glutamate insult in co-cultures pretreated with vehicle (PBS), CNTFR{alpha} (200 ng/ml), or CNTF (20 ng/ml). Pretreatment of the cultures with Complex (CNTFR{alpha} + CNTF), however, prevented a significant increase in the release of LDH (p > 0.05 sham insult with Complex). Addition of the gap junction blocker CBX during glutamate application further increased the levels of LDH released under all treatment conditions. Pretreatment of the cultures with either CNTF or Complex, however, significantly reduced LDH release following the glutamate insult with CBX, compared with vehicle and CNTFR{alpha} pretreatments. #, p < 0.05; ^, p < 0.01; and *, p < 0.001 compared with other treatments as indicated. NS, not significant.

 
Real Time PCR—Real time PCR was used to verify the differential expression of selected genes in a manner similar to that described by James et al. (29). Briefly, specific target primers and probe sets (Taqman Assays-on-Demand, Applied Biosystems), their corresponding probes, and template RNA were combined with TaqMan (Universal PCR Mastermix, No AmpErase UNG, Applied Biosystems). Amplified target sequences were detected with the ABI Prism 7500 sequence detector (Applied Biosystems). Gene expression values were corrected using 18 S RNA as the endogenous control.

RT-PCR—Isolated RNA was subjected to RT-PCR as described previously (18). The RT product was amplified in the presence of primers for CNTF (30), CNTFR{alpha} (30), gp130 (31), and LIFRβ (32). Control experiments were performed in parallel in the absence of the reverse transcriptase enzyme and did not produce any products.

Immunocytochemistry
Cells were fixed with paraformaldehyde, processed as described previously (18), and labeled with antibodies against MAP2 and neurofilament 200 (Sigma), pSTAT3 (Tyr-705; Cell Signaling, Beverly, MA), or cleaved caspase3 (Cell Signaling), with subsequent Alexa- and fluorescein-conjugated secondary IgG antibody application (Molecular Probes Inc., Eugene, OR). Immunolabeled cells were mounted in ProLong Gold containing DAPI (Molecular Probes Inc.), viewed on a Zeiss Axioskop microscope, and analyzed using AxioVision 4.2 software. Specificity of antibody labeling was assessed by omitting the primary antibody from the labeling protocol. PC12 cells were stained with fluorescently tagged wheat germ agglutinin (Molecular Probes) to define cell limits, fixed in paraformaldehyde, and processed as described above.

Protein Isolation and Immunoblot Analysis
Neuron-enriched cultures were seeded in 6-well PDL/laminin-coated culture plates (BD Biosciences) and were maintained in culture for 24 h prior to a 15-min exposure to vehicle or Complex. Protein was harvested and processed as described previously (18), electrotransferred onto a nitrocellulose membrane, and immunoblotted against pSTAT3 (Tyr-705; Cell Signaling). To normalize protein loading, bound antibodies were stripped, and the blot was probed for total STAT3 (Cell Signaling). Densitometric analyses of immunoblot signals on Kodak X-Omat x-ray film were performed using Scion Image software (Scion, Frederick, MD).

Neurite Outgrowth Assays
Neurite outgrowth was analyzed in two ways. Indirect measurements were performed on neurons treated with agents for 3 days using the neurite outgrowth quantification assay kit (Chemicon) (33). Direct morphometric analyses were conducted on individual MAP2-immunolabeled neurons. Because MAP2 immunoreactivity can be observed in both dendrites and axons in developing neurons (34), no attempt was made to differentiate dendrites from axons; neurofilament 200 immunolabeling was employed only to confirm that axons of the neurons used for analyses were not in contact with neighboring cells. The total number of neurites was estimated by counting the tip ends of all MAP2-positive neurites elaborated by a single neuron. The cumulative length of primary neurites was the sum of the lengths of all neurites ≥8 µm long that extended directly from the soma. A branching point was defined as a point at which a neurite ≥8 µm in length extended from another neurite; the ratio of the sum of the number of branching points on a single neuron to the sum of the number of primary neurites on that neuron defined the branching ratio.

Data Analyses
Each experiment was performed on at least four different batches of PC12 cells or four different culture preparations obtained from different litters of mice. Neurite outgrowth measurements were performed on at least 16 neurons per treatment group from each culture preparation, for a total of at least 64 neurons per treatment group. For assessment of PI, TUNEL, and cleaved caspase3 labeling, at least eight randomly selected areas from each culture per treatment group were used. Data are presented as means ± S.E., and the results were analyzed by one-way analysis of variance followed by the Student-Newman-Keuls post hoc test. A p value of less than 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Complex Induces Neuroprotection in Vitro—We have reported previously that gap junctions exert a neuroprotective effect against glutamate excitotoxicity and that Complex, but not CNTF alone, up-regulates functional gap junctional coupling (18, 25). Initially, therefore, we sought to determine whether pretreating neuron-astrocyte co-cultures with Complex can reduce glutamate-induced neurotoxicity and whether this effect is dependent on gap junctional communication. As glutamate-mediated cell death can proceed by both necrosis and apoptosis (35), the effects of Complex on neuronal viability were assessed by measuring the release of LDH into the extracellular space, the ability of cells to exclude the cationic dye PI, the presence of cleaved caspase3, and TUNEL.


Figure 2
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  		FIGURE 2.
Complex reduces both propidium iodide staining and TUNEL in neuron-astrocyte co-cultures treated with 1 mM glutamate for 3 h. A, representative photomicrographs of co-cultures pretreated for 3 days with vehicle (PBS), CNTFR{alpha} (200 ng/ml), CNTF (20 ng/ml), or Complex (CNTFR{alpha} + CNTF) following the sham insult or challenged with glutamate in the absence or presence of the gap junction blocker CBX, and subsequently stained with PI (red) and then labeled with TUNEL (green). Several cells can be identified as being labeled for both markers (overlaid in yellow). Nuclei were stained with DAPI (blue). Bars = 100 µm. A reduction in glutamate-induced TUNEL can be seen in CNTF pretreated cultures compared with vehicle or CNTFR{alpha} pretreatments in both the absence and presence of CBX. Complex further reduced PI staining and TUNEL under each condition. B, when quantified, CNTF significantly reduced TUNEL but not PI staining in glutamate-treated cultures in both the absence and presence of CBX, as compared with vehicle and CNTFR{alpha} pretreatments. Pretreatment with Complex in the absence of CBX significantly reduced glutamate-mediated PI staining compared with vehicle, CNTFR{alpha}, and CNTF pretreatments and significantly reduced TUNEL compared with vehicle and CNTFR{alpha} pretreatments. In the presence of CBX, Complex significantly reduced both PI staining and TUNEL compared with all other agents. The number of PI-stained and TUNEL-labeled cells is expressed as a percentage of total cells within the same field examined. #, p < 0.05; ^, p < 0.01; and *, p < 0.001 compared with other treatments as indicated. NS, not significant.

 
Under control conditions, pretreatment of neuron-astrocyte co-cultures with CNTFR{alpha}, CNTF, or Complex did not significantly alter LDH release (Fig. 1), PI staining (Fig. 2), or TUNEL (Fig. 2), compared with vehicle pretreatment alone. As expected, application of 1 mM glutamate to the co-cultures for 3 h resulted in substantial increases in LDH release (p < 0.001; Fig. 1) as well as PI staining and TUNEL (p < 0.01 in both cases; Fig. 2) at 24 h post-insult. Whereas pretreatment of co-cultures with CNTFR{alpha} alone failed to significantly alter glutamate-induced cell death (Figs. 1 and 2), pretreatment with CNTF alone significantly reduced TUNEL (p < 0.001; Fig. 2) but not LDH release (p > 0.05; Fig. 1) or PI staining (p > 0.05; Fig. 2). In contrast, Complex pretreatment significantly reduced glutamate-induced cell death as measured in all three cell viability assays (p < 0.01 or less in all cases; Figs. 1 and 2). Together, these results indicate that Complex affords greater protection against glutamate-induced cytotoxicity than either CNTF or CNTFR{alpha} alone.

Functional Gap Junctions Contribute to Complex-mediated Neuroprotection—Substantial evidence supports a neuroprotective role for gap junctions and their constituent proteins, the connexins (see under "Discussion"). Previously, we reported that Complex, but not CNTF alone, enhances gap junctional communication and connexin43 expression in astrocytes (18), suggesting a possible mechanism by which this agent could elicit neuroprotection. Therefore, in the next series of experiments, we applied glutamate to neuron-astrocyte co-cultures pretreated with CNTFR{alpha}, CNTF, or Complex in the absence or presence of the gap junction blocker CBX or the inactive analogue GZA. Consistent with our previous report (25), neither CBX nor GZA induced cytotoxic effects in the sham-insulted cultures, and GZA failed to modulate any effects of CNTFR{alpha}, CNTF, or Complex on glutamate-induced excitotoxicity (data not shown). In contrast, the addition of CBX increased glutamate-induced cell death, an effect that was not inhibited by pretreatment with CNTFR{alpha} alone (Figs. 1 and 2). Although CNTF significantly reduced LDH release (Fig. 1) and TUNEL (Fig. 2) following glutamate exposure in the presence of CBX, it failed to significantly reduce PI staining (p > 0.05; Fig. 2). Finally, Complex significantly reduced not only the increases in LDH release and TUNEL evoked by glutamate in the presence of CBX (Figs. 1 and 2) but also the increase in PI staining (Fig. 2). Interestingly, whereas the ability of Complex to inhibit LDH release was not significantly different to that of CNTF alone (Fig. 1), the reductions in PI staining and TUNEL were significantly greater with Complex versus CNTF (Fig. 2). Together, these results are consistent with the possibility that the neuroprotective effects of Complex are mediated by gap junction-dependent and -independent mechanisms.


Figure 3
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  		FIGURE 3.
Primary cortical neurons respond to Complex in vitro. A, MAP2-positive neurons maintained in culture for 24 h and treated with Complex (200 ng/ml CNTFR{alpha} + 20 ng/ml CNTF) or with CNTF for 15 min are immunoreactive for Tyr-705-phosphorylated STAT3 (PSTAT3). Immunoreactivity to PSTAT3 can be seen in both the somatic (yellow in merged image) and nuclear (pale blue in merged image) regions. Bar = 20 µm. B, treatment of similarly aged neuronal cultures with Complex elicited a dose-responsive increase in PSTAT3 levels. When normalized to total STAT3 (TSTAT3) levels, significant PSTAT3 levels were induced in Complex concentrations above 22 ng/ml media. *, p < 0.001 compared with vehicle, 0.22 µl of Complex, and 2.2 µl of Complex treatments.

 
CNTF and Complex Induce Differential Gene Expression—Although equal amounts of CNTF were present during the CNTF and Complex pretreatments, the protective effects of Complex against glutamate-induced reductions in neuronal viability were more robust than those afforded by CNTF (or CNTFR{alpha}) alone. These findings suggest that Complex and CNTF must have differential effects on the many processes that contribute to excitotoxic cell death, at least some of which are gap junction-independent (see above). Therefore, we examined potential differences in gene regulation by CNTF and Complex in the neuron-astrocyte co-cultures by performing cDNA array analysis on RNA isolated from treated co-cultures using Codelink Mouse Uniset I microarrays.

Focusing on ratio differences of at least 3-fold, the microarray findings revealed that out of 10,000 genes examined, Complex up-regulated 29 genes and down-regulated 18 genes compared with CNTF alone (Table 1). Real time PCR on three genes (one differentially up-regulated by Complex (Apod), one approximately equal in expression (Plat), and one differentially down-regulated by Complex (Cklf)) substantiated the microarray results (Table 1); differences between the microarray and real time PCR results may reflect differences in the sensitivities of the two methods. Interestingly, a number of the genes differentially up-regulated by Complex have been reported to be directly up-regulated in neurons in response to injury and are thought to represent a neuroprotective response (see "Discussion"). These findings suggest that in addition to its established effects on astrocytes (18), Complex may exert cytoprotective effects directly on neurons different from those of CNTF alone. To examine this possibility, in the next series of experiments CNTF and Complex (as well as CNTFR{alpha}) were applied to neuron-enriched (>95%) cultures.


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  		TABLE 1
Analysis of differential gene expression induced by CNTF or Complex in neuron-astrocyte co-cultures

Differential expression was assessed on RNA reverse-transcribed and applied to either CodeLink Mouse Uniset I microarrays (Amersham Biosciences) containing approximately 10,000 mouse oligonucleotide gene probes or real time PCR using TaqMan and an ABI Prism 7500 sequence detector (Applied Biosystems).

 
Complex Directly Enhances Neuronal Viability—Activation of the JAK/STAT pathway mediates, to a great extent, the neuroprotective effects of IL-6 family member cytokines, including CNTF (19). To examine whether Complex can activate this pathway in neurons (as it does in astrocytes) (18), we assessed the effects of Complex on the levels of tyrosine 705-phosphorylated STAT3 (pSTAT3) in our neuron cultures. Whereas serine 727 phosphorylation is necessary for STAT3 activation, phosphorylation of tyrosine 705 is required for the transcription factor to translocate into the nucleus and subsequently alter gene expression. As depicted in Fig. 3A, whereas vehicle-treated neurons showed limited activation of the JAK/STAT pathway, CNTF and Complex induced extensive STAT3 phosphorylation at the somatic level with lesser immunostaining in the neurites; the subcellular localization of Complex-induced pSTAT3 is entirely consistent with that observed following the injury-induced activation of the CNTF receptor system in neurons in vivo (36). In addition, the ability of Complex to activate the JAK/STAT pathway was concentration-dependent, with maximal STAT3 phosphorylation being observed at 220 ng of Complex/ml of media (Fig. 3B). Thus, 20 ng/ml CNTF was employed in subsequent experiments as it elicited maximal STAT3 phosphorylation (in Complex form) and is equivalent to the amount used in our preceding experiments.


Figure 4
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  		FIGURE 4.
Complex reduces LDH release from newly plated neocortical neurons in vitro. Treatment of neuron-enriched cultures 2 h post-plating with either CNTFR{alpha} (200 ng/ml) or CNTF (20 ng/ml) alone did not significantly reduce necrosis, as assessed by LDH release, when compared with vehicle (PBS) treatment. Treatment of the cultures with Complex (CNTFR{alpha} + CNTF), however, significantly reduced LDH release during the first 24 and 48 h. #, p < 0.05 compared with other treatments as indicated.

 
The survival of neurons placed in primary culture is compromised in the absence of growth factors or glial cells, with the majority of cells dying within the first 3-5 days (27, 37). Given the broad effects of Complex to promote cell survival, we tested the ability of the agent to promote or enhance the survival of neuron-enriched cultures.

To examine the effects of CNTFR{alpha}, CNTF, and Complex directly on neuronal viability, the agents were applied for 3 days starting at either 2 h (hereafter termed 3 DIV cultures) or 10 days (10 + 3 DIV cultures) after plating with subsequent analysis of cell mortality. Neurons maintained in culture for two different periods of time were employed to assess the effects of cytokine treatment on developing neurons and on neurons of a more mature state (27). At no time point examined did CNTFR{alpha} significantly alter LDH release, compared with vehicle treatment, in either the 3 DIV (Fig. 4) or 10 + 3 DIV (not shown) cultures. Although CNTF treatment appeared to reduce LDH release from 3 DIV cultures in the first 24 h, LDH release at this time point and all others examined were not significantly different (p > 0.05) from vehicle treatment (Fig. 4). Treatment of the 3 DIV neurons with Complex, however, significantly reduced LDH release at the 24- and 48-h time points (Fig. 4). Neither CNTF nor Complex significantly influenced LDH release from the 3 DIV cultures at the 72-h time point (Fig. 4) or at any of the time points examined in the 10 + 3 DIV neuronal cultures (data not shown).

Next, cleaved caspase3 and TUNEL were assessed following treatment of the neuron-enriched cultures with CNTFR{alpha}, CNTF, or Complex. Treatment of 3 DIV or 10 + 3 DIV neurons with CNTFR{alpha} alone had no effect on the number of cells immunoreactive for cleaved caspase3 (Fig. 5B) or labeled with TUNEL (Fig. 5D), compared with vehicle treatment. Although CNTF alone failed to significantly affect the number of cleaved caspase3-positive cells in 3 DIV cultures, it significantly reduced the number of immunopositive cells in 10 + 3 DIV cultures (Fig. 5B) and significantly decreased TUNEL staining in both 3 DIV and 10 + 3 DIV cultures (Fig. 5D). In contrast, Complex significantly reduced the number of neurons exhibiting cleaved caspase3 (Fig. 5, A and B) and TUNEL staining (Fig. 5, C and D) in both 3 DIV and 10 + 3 DIV cultures; the reduction in cleaved caspase3 and TUNEL staining by Complex was in all cases significantly greater than that observed with CNTF alone.

In summary, although soluble CNTFR{alpha} had no effect on the survival of neurons in vitro and CNTF caused somewhat variable reductions in necrosis and apoptosis, Complex significantly reduced both necrosis and apoptosis in 3 DIV neurons and apoptosis in neurons maintained in culture for a longer duration (i.e. 10 + 3 DIV).

Complex, but Not CNTF, Induces Neurite Outgrowth—In addition to its involvement in neuroprotection, CNTF-mediated activation of the JAK/STAT pathway is known to promote neurite outgrowth (38, 39). To examine potential trophic effects elicited by Complex, which may contribute to its prosurvival effects described above, we applied CNTF and CNTFR{alpha}, individually or in combination, to cortical neurons seeded on transwells (33). Compared with vehicle, 3-day treatments with CNTF or soluble CNTFR{alpha} failed to significantly affect neurite outgrowth in either 3 DIV or 10 + 3 DIV neocortical neurons into the transwells (Fig. 6A). In contrast, treatment of 3 DIV or 10 + 3 DIV neocortical neurons with Complex induced statistically significant increases in the outgrowth of neurites into the transwells (Fig. 6A).

To examine morphological changes more closely, 3 DIV neocortical neurons were cultured at low density on chamber slides and treated with the agents. Consistent with the transwell results, treatment of 3 DIV neurons with either CNTF or CNTFR{alpha} failed to significantly affect neurite outgrowth, as measured by total neurite number, primary neurite lengths, or branching, compared with vehicle treatment (Fig. 6C). In contrast, treatment with Complex resulted in significant increases in all measured parameters (Fig. 6, B and C).

When maintained for greater than 1 week in vitro, the neurites of neocortical neurons become long, highly branched, and overlap with each other and with those of neighboring cells (Fig. 7). Although the extensive neuritic arbors of 10 + 3 DIV neurons precluded accurate morphometric analyses on individual neurons, treatment of 10 + 3 DIV neurons with Complex, but not CNTF or CNTFR{alpha}, induced an observable increase in neurite outgrowth, including longer and more branched processes (Fig. 7). In addition, and in agreement with the ability of Complex to enhance neuronal survival, fewer pyknotic nuclei were identified in both 3 DIV (not shown) and 10 + 3 DIV (Fig. 7) neuronal cultures treated with Complex than in those treated with CNTF or CNTFR{alpha} alone. Similar results, both quantitatively and qualitatively, were obtained in 3 DIV (Fig. 8, A and B) and 10 + 3 DIV (Fig. 8C) basal ganglion neurons, respectively, indicating that the ability of Complex to promote neurite outgrowth is not restricted to neocortical neurons.


Figure 5
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  		FIGURE 5.
CNTF and Complex reduce apoptosis in neocortical neurons in vitro. A, representative photomicrographs of neurons treated with vehicle (PBS) or Complex every 24 h for 3 days, starting 2 h (3 DIV) or 10 days (10 + 3 DIV) after plating, and immunolabeled against cleaved caspase3 (red). Nuclei were stained with DAPI (blue). Bars = 100 µm. B, CNTF significantly reduced the number of cells exhibiting cleaved caspase3 in 10 + 3 DIV but not in 3 DIV cultures. Complex, however, significantly reduced the number of caspase3-positive cells in both 3 DIV and 10 + 3 DIV cultures. *, p < 0.001 compared with all other treatments or as indicated; ^, p < 0.01 compared with other treatments as indicated. C, representative photomicrographs of 3 DIV and 10 + 3 DIV neuronal cultures treated with vehicle or Complex and labeled with TUNEL (green) and DAPI (blue). Bars = 50 µm. D, CNTF significantly reduced the number of TUNEL-positive neurons in both 3 DIV and 10 + 3 DIV cultures, an effect accentuated by Complex treatment. *, p < 0.001; ^, p < 0.01; and #, p < 0.05 compared with other treatments as indicated.

 


Figure 6
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  		FIGURE 6.
Complex increases neurite outgrowth in mouse neocortical neurons in vitro. A, neurons plated onto transwells of neurite outgrowth quantification assay kits and treated with agents every 24 h for 3 days, starting 2 h (3 DIV) or 10 days (10 + 3 DIV) after plating, reveal that Complex, but not CNTFR{alpha} or CNTF alone, promotes neurite formation and/or growth. ^, p < 0.01 compared with vehicle and CNTFR{alpha} treatments; #, p < 0.05 compared with CNTF treatment. B, representative photomicrographs of 3 DIV neocortical neurons that had been seeded onto chamber slides, treated with vehicle (PBS) or Complex, and immunolabeled against MAP2. Bars = 25 µm. C, direct morphometric analyses of individual 3 DIV neurons indicates that although neither CNTFR{alpha} nor CNTF alone promoted neurite outgrowth, Complex significantly enhanced the total number of neurites, cumulative primary neurite length, and neurite branching. *, p < 0.001 compared with all other treatments.

 
The neuroprotective and growth-promoting effects of CNTF have been shown previously to involve the JAK/STAT, PI3K/Akt, and MAPK/ERK signaling pathways. To determine whether these pathways also contributed to the effects of Complex on neurite outgrowth in our model system, neocortical neurons in primary culture were maintained in the absence or presence of pathway inhibitors. All inhibitors were dissolved in Me2SO (0.02% v/v), which did not elicit any effects on neurite outgrowth (Fig. 9).

In other neuronal models, the JAK/STAT, PI3K/Akt, and MAPK/ERK pathways elicit varying effects on neurite outgrowth. Thus, basal effects of the pathway inhibition were initially examined in our model system. Inhibition of the JAK/STAT pathway with AG490 induced notable cell death within the 3 DIV cultures but no apparent cell death in 10 + 3 DIV cultures (not shown). MAP2-positive 3 DIV neurons that remained viable in the presence of AG490 possessed few neurites that were restricted in both length and branching (Fig. 9). Treatment of sister cultures with LY29400 to inhibit the PI3K/Akt pathway did not result in any changes in neurite outgrowth (Fig. 9). However, inhibition of the MAPK/ERK pathway with U0126 significantly increased neurite branching, which contributed to an overall increase in neurite number but did not affect neurite lengths (Fig. 9). Taken together, under cytokine-free conditions, neurite outgrowth in neocortical neurons maintained in primary culture for 72 h was positively regulated by the JAK/STAT pathway, uninfluenced by the PI3K/Akt pathway, and negatively regulated by the MAPK/ERK pathway.

To determine which signaling pathway(s) mediated the effects of Complex on neurite extension and elaboration, cortical neurons were exposed to AG490, LY294002, or U0126 prior to and throughout treatment with Complex. In cultures exposed to AG490, Complex did not rescue the neurons from the inhibitor-associated cell death (not shown) or decrease in neurite outgrowth in MAP2-positive viable neurons (Fig. 9). Inhibition of the PI3K pathway with LY294002 reduced the effects of Complex to increase the total number of neurites and the cumulative length of primary neurites but had little effect on the ability of Complex to increase neurite branching (Fig. 9). Application of U0126 to inhibit the MAPK/ERK pathway did not influence the ability of Complex to increase the total number of neurites per cell but did significantly reduce the Complex-induced increase in the cumulative length of primary neurites (Fig. 9). In summary, the increase in neurite outgrowth induced by Complex in neocortical neurons appeared to be mediated predominantly by the JAK/STAT and PI3K pathways and to a lesser degree by the MAPK/ERK pathway.


Figure 7
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  		FIGURE 7.
Complex increases neurite outgrowth in 10 + 3 DIV mouse neocortical neurons. Cortical neurons maintained in vitro for 10 days were treated with the agents every 24 h for 3 days and subsequently immunoprocessed for MAP2 (green) and stained with DAPI (blue). Although fewer cells are represented in the Complex panel, extensive MAP2-positive neurites and limited pyknotic nuclei (condensed chromatin stained with DAPI; arrows) are seen in the image compared with all other treatments. Bars = 100 µm.

 
Complex Does Not Induce Differentiation of PC12 Cells—The effects of Complex to promote neurite outgrowth detailed above may have reflected an ability to induce the differentiation of immature neurons within the cultures. Indeed, both CNTF and a modified version of Complex (hyper-CNTF) have been reported to induce the differentiation of neuronal precursors and some cell lines, including PC12 cells, although results to date have been inconsistent (40-42). Therefore, in the final part of the study we performed a limited series of experiments to examine the ability of Complex to induce differentiation in PC12 cells.

PC12 cells exhibit genetic drift, resulting in the production of clonal variants that may contribute to conflicting reports of cytokine-induced differentiation (43). Initially, therefore, we characterized the endogenous expression of CNTF and the tri-partite receptor subunits in our preparation of PC12 cells. RT-PCR analysis was performed on RNA isolated from the cell line prior to, and following, differentiation with NGF. In agreement with previous reports (44), CNTF, CNTFR{alpha} and gp130 transcripts were expressed in both undifferentiated and NGF-differentiated PC12 cells (Fig. 10A). In comparison with the transcript levels of the other receptor subunits examined, but consistent with previous reports (45), LIFRβ expression was low in both undifferentiated and NGF-differentiated PC12 cells (Fig. 10A). Together, these results indicate that the PC12 cells used in the present study have the potential to respond to both CNTF and Complex.

A 3-day treatment of undifferentiated PC12 cells with CNTF, CNTFR{alpha}, or Complex neither induced observable morphological alterations nor halted cell division (data not shown). The failure of CNTF or Complex to induce the differentiation of our PC12 cells may have been a consequence of low LIFRβ expression (see above). To examine receptor activation, undifferentiated PC12 cells were exposed to the cytokines for 15 min followed by immunoblotting cell lysates against pSTAT3 (Tyr-705). A long exposure time of the immunoblot to the x-ray film was required to detect pSTAT3, indicative of low levels, with neither CNTFR{alpha}, CNTF nor Complex altering pSTAT3 levels in the undifferentiated PC12 cells (Fig. 10B).

Finally, to determine whether Complex might enhance rather than initiate differentiation, the cytokines were applied in combination with NGF to undifferentiated PC12 cells for 24 h. Neither CNTF, CNTFR{alpha}, nor Complex altered the percentage of NGF-stimulated cells bearing processes (Fig. 10C). However, cells undergoing differentiation within the NGF-stimulated cultures exhibited longer and more branched neurites when co-treated with Complex but not with either CNTFR{alpha} or CNTF (Fig. 10, D and E). In addition, and in agreement with results obtained in neocortical neurons (see above), both CNTF and Complex reduced the number of pyknotic nuclei in NGF-differentiated PC12 cells; the percentages of pyknotic nuclei in cells treated with vehicle, CNTFR{alpha}, CNTF, or Complex were 13 ± 1, 13 ± 1, 8 ± 1 (p < 0.05 compared with vehicle-treated controls), and 6 ± 1% (p < 0.001), respectively.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Despite considerable interest in CNTF as a therapeutic agent against neuronal injury and neurodegenerative diseases, applied alone the cytokine has been found to exhibit only limited efficacy. The results of this study support these findings. Thus, CNTF alone failed to significantly promote neurite outgrowth in both 3 and 10 + 3 DIV neocortical and basal ganglion neurons, as well as in NGF-stimulated PC12 cells. In addition, although CNTF elicited some anti-apoptotic effects, it failed to demonstrate significant anti-necrotic properties in our study. Taken together, these findings suggest that the neuroprotective actions of CNTF are mediated predominantly by an anti-apoptotic effect which, in turn, may reflect the ability of CNTF to up-regulate inhibitors of apoptosis genes via the JAK/STAT pathway (46).

In contrast to the variable results obtained with CNTF alone, Complex elicited robust neuroprotective and neurotrophic actions in all of the assays employed in the present experiments. Thus, pretreatment with Complex (i) limited glutamate-induced cell death in neuron-astrocyte co-cultures, (ii) maintained neuron enriched cultures to a greater extent than observed with CNTF alone, and (iii) promoted neurite outgrowth.

The facts that Complex and CNTF treatments involved equivalent amounts of CNTF and yet elicited markedly different pro-survival effects suggest that CNTF in combination with its soluble receptor must possess additional actions to those exerted by CNTF alone. This possibility received support from the microarray analyses, which indicated that, compared with CNTF alone, Complex differentially affected the expression of 47 genes, several of which are known to contribute to neuroprotection (e.g. Apod (47), Dcn (48), Dspg3 (49), Mdm2 (50), Plg (51), Sgk2 (52), and Tnfrsf18 (53)) and/or neuronal development (e.g. Apod (54), Dcn (54, 55), Dspg3 (49, 56), Plg (57), and Plxnb3 (58)). To identify pathways and/or biological processes impacted by Complex treatment, we used the Ingenuity Pathways Knowledge Base to assess relationships between these differentially expressed genes. From these analyses, three pathways were highlighted that involved 9 or more genes that were differentially regulated by Complex. We found that 11 genes mapped to the cell signaling/cardiovascular disease pathway (supplemental Fig. S1), 10 to the cell death/cell development pathway (supplemental Fig. S2), and 9 to cell cycle pathways (supplemental Fig. S3). In addition, several of these genes are associated with biological functions relevant to our results (see supplemental Table S1). It can be appreciated that the neuroprotective and neurotrophic effects of Complex are likely influenced, independently or collectively, by several signaling pathways and the numerous genes downstream of these pathways.


Figure 8
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  		FIGURE 8.
Complex increases neurite outgrowth in basal ganglion neurons in vitro. A, representative photomicrographs of 3 DIV basal ganglion neurons that had been treated with vehicle (PBS) or Complex and immunolabeled against MAP2. Bars = 50 µm. B, direct morphometric analyses of individual neurons indicates that Complex, but not CNTFR{alpha} or CNTF alone, significantly enhanced the total number of neurites, cumulative primary neurite length, and neurite branching. *, p < 0.001 compared with all other treatments. C, representative photomicrograph of 10 + 3 DIV basal ganglia neurons treated with vehicle or Complex and immunolabeled against MAP2. Bars = 100 µm.

 
Because the microarray analyses were performed on neuron-astrocyte co-cultures, we are unable to provide a definitive statement as to whether Complex differentially affected gene expression in neurons and/or astrocytes. Nevertheless, the neuroprotective effects of Complex appeared to reflect both indirect actions, likely mediated via astrocytes, and direct actions on neurons themselves. The ability of Complex, but not CNTF, to trans-signal the astrocytes (18) could also contribute to the differential effects of Complex and CNTF on neuroprotection. As evidenced by the ability of CBX to attenuate the neuroprotective effect of the Complex, the indirect actions of Complex could be mediated by increases in connexin43 expression and thus functional gap junctional coupling between astrocytes. The possibility that Complex also exerts direct protective effects on neurons is supported by the observations that the neuroprotective effects of Complex were not occluded by inhibiting gap junctional communication with CBX and that Complex, but not CNTF, enhanced neuronal viability in glial cell-deficient, neuron-enriched cultures and NGF-differentiated PC12 cells (which are known to be gap junction communication-deficient) (59). Taken together, although the ability of Complex, but not CNTF, to trans-signal astrocytes could contribute to the differential neuroprotective effects of Complex versus CNTF in the neuron-astrocyte co-cultures, the differential protective effects of the agents on neurons in neuron-enriched cultures cannot be explained by trans-signaling.


Figure 9
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  		FIGURE 9.
The neurotrophic effect of Complex to enhance neurite outgrowth is mediated by the MAPK/ERK, JAK/STAT, and PI3K/Akt pathways. Cortical neurons were treated with vehicle or Complex in the absence or presence of solvent (Me2SO (DMSO)), AG490 (50 µM), LY294002 (20 µM), or U0126 (10 µM) every 24 h for 3 days. Total neurite number, neurite length, and neurite branching were affected to various degrees by the presence of the different pathway inhibitors. *, p < 0.001; ^, p < 0.01; and #, p < 0.05 compared with same treatment with no inhibitors. a, p < 0.001; b, p < 0.01; and c, p < 0.05 compared with vehicle treatment with same inhibitor.

 
That Complex is able to exert direct effects on neurons is also evidenced by the effects of the agent on neuronal development. In this study, CNTF failed to affect neurite number, lengths, and branching in neocortical and basal ganglion neurons as well as in NGF-treated PC12 cells, suggesting that the tendency of CNTF to enhance neurite outgrowth in the transwell experiments likely reflected enhanced cell survival, a finding seen by others (60). In contrast to CNTF, Complex consistently promoted neurite outgrowth in neocortical neurons, basal ganglion neurons, and NGF-treated PC12 cells by increasing neurite number, lengths, and branching. Although dendritic branching can result from alternating outgrowth and retraction, the ability of Complex to exert its effects on developing (3 DIV neurons and differentiating PC12) cells is consistent with the possibility that its actions are mediated, at least in part, by promoting neurite formation and extension. Complex also enhanced neurite outgrowth in 10 + 3 DIV cortical and basal ganglion neurons, suggesting that its effects are not restricted to a specific neuron subtype or maturational state. Finally, the effects of Complex to promote neurite outgrowth is likely not explained by neuronal differentiation because Complex failed to induce the differentiation of PC12 cells and failed to differentiate murine neuroblastoma Neuro-2a and human embryonal NTERA-2/D1 cell lines.4 The consistent effects of Complex on neurite outgrowth contrast with the variable effects of CNTF observed here and reported in other studies (Table 2).


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  		TABLE 2
Varying effects of exogenously administered CNTF on neurite outgrowth in different neuron models

 
The ability of Complex, but not CNTF, to consistently promote neuronal survival and development may reflect differences in receptor affinities, illegitimate receptor activation, and/or the expression of the membrane-bound form of CNTFR{alpha}. First, the biological activity of CNTF is increased 30-fold when the cytokine signals through soluble CNTFR{alpha} as compared with the membrane-bound form of the receptor (61). In a similar manner, IL-6 combined with its soluble receptor enhances neuronal survival and neurite extension to a greater extent than IL-6 alone, despite the neuronal expression of IL-6 receptors (62). Second, CNTF may induce nonspecific actions by illegitimately binding to IL-6 receptors and LIFRβ (63) producing an effect that, in turn, could offset the specific actions of CNTF. Applying excess soluble CNTFR{alpha} to favor the formation of the CNTF-CNTFR{alpha} heterodimer may prevent nonspecific binding of CNTF to other receptors. Third, in contrast to CNTF, the biological effects of Complex are not dependent on the membrane-bound form of CNTFR{alpha}, the expression of which varies with neuron subtypes, subcellular localizations, and developmental stage (64, 65). Although CNTFR{alpha} expression commences early during neuronal development, it cannot be discounted that a limited or insufficient level of CNTFR{alpha} expression in our "young" neurons may have limited the response to CNTF alone. However, this point only further strengthens the potential efficacy of Complex as a therapeutic intervention following injury and disease.


Figure 10
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  		FIGURE 10.
Complex enhances, rather than induces, the differentiation of PC12 cells. A, RT-PCR performed on both undifferentiated and NGF-differentiated PC12 cells indicates endogenous expression of transcripts for CNTF and its receptor subunits, independent of differentiation state. Standards (Stnd) represent 500 (top band), 400, 300, and 200 bp. B, immunoblotting of protein lysate from undifferentiated PC12 reveals that neither CNTFR{alpha}, CNTF, nor Complex induces significant increases in PSTAT3 levels. C, co-treatment of NGF-stimulated PC12 cells with CNTFR{alpha}, CNTF, or Complex failed to significantly affect the number of cells exhibiting processes. D, representative differential interference contrast images of NGF-treated PC12 cells show enhanced neurite outgrowth when co-treated with Complex. Bars = 20 µm. E, co-treatment of PC12 cells with NGF and Complex, but not CNTFR{alpha} or CNTF, significantly increased neurite number, lengths and branching. *, p < 0.001 compared with all other treatments; ^, p < 0.01 compared with vehicle and CNTF treatments; {diamondsuit}, p < 0.001 compared with CNTFR{alpha} treatment.

 
Depending on the neuronal model and the absence or presence of exogenous cytokines, the JAK/STAT, PI3K/Akt, and MAPK/ERK signaling pathways can stimulate, inhibit, or modulate neurite outgrowth. In our study, inhibition of the JAK/STAT pathway with AG490 resulted in extensive cell death in acutely isolated neurons but not in neurons matured in vitro. This finding may be explained by the critical requirement of functional JAK/STAT signaling in development and by its prominent role in cell survival (19, 66). The small population of young MAP2-positive neurons that remained viable in the presence of AG490 exhibited fewer, shorter, unbranched neurites. Although it is appreciated that the reduction in neurite outgrowth may have been a consequence of compromised viability of the neurons in the presence of AG490, this finding is in agreement with inhibition of STAT3 activation limiting neurite outgrowth in other nerve models (38). That Complex-mediated enhancement of neurite outgrowth was blocked by AG490 suggests the actions of cytokine are highly dependent on the JAK/STAT pathway.

Although not required for inherent neurite outgrowth in vitro, we found that functional PI3K/Akt pathway signaling also contributes to cytokine-mediated neurite development and extension, a conclusion shared by Gerecke et al. (67). Application of the PI3K/Akt pathway inhibitor LY294002 had no effect on neurite development or outgrowth in our cortical neurons under basal conditions. However, LY294002 reduced Complex-mediated enhancement of neurite outgrowth, demonstrating a dependence of Complex on the PI3K pathway. Complementing our finding, inhibition of the PI3K/Akt pathway restricts neurite outgrowth mediated by other growth factors and cytokines, including hepatocyte growth factor, brain-derived neurotrophic factor, NGF, NRG-1β, and BMP-7 (67-71).

The MAPK/ERK pathway acted to limit neurite outgrowth in the absence of exogenous cytokine stimulation in vitro but weakly promoted neurite outgrowth during Complex stimulation. In the absence of Complex, inhibition of the MAPK/ERK pathway with U0126 significantly increased neurite formation, mainly via novel neurite branching demonstrating a negative influence of the MAPK/ERK pathway on neurite outgrowth (this study and see Ref. 70). In contrast, activation of the MAPK/ERK pathway has also been shown to promote neurite (mainly axonal) growth in other models (70, 72). Inhibition of the MAPK/ERK pathway did not alter Complex-mediated increases in neurite number but did limit the ability of Complex to increase the lengths of the neurites. This finding suggests that Complex-mediated activation of the MAPK/ERK pathway contributes to Complex-induced increases in neurite extension but not in neurite formation. Similarly, Bonnet et al. (69) have shown that inhibition of the MAPK/ERK pathway by U0126 blocks brain-derived neurotrophic factor-induced neurite elongation in retinal ganglion cells.

In summary, we find that CNTF exhibits greater efficacy as a neuroprotective and neurotrophic agent when applied in combination with its soluble receptor. Taken together, our findings suggest that Complex may possess a more favorable therapeutic profile than CNTF alone. Furthermore, the ability of Complex to influence both CNTFR{alpha}-expressing and -deficient cells suggests that this agent may have therapeutic implications beyond the nervous system.


   FOOTNOTES
 
* This work was supported by the Heart and Stroke Foundation of British Columbia and Yukon. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S3 and Table S1. Back

1 Supported by both Killam and Natural Sciences and Engineering Research Council of Canada postdoctoral awards. Back

2 Recipient of a Canada Research Chair. To whom correspondence should be addressed: Dept. of Cellular and Physiological Sciences, University of British Columbia, 2350 Health Sciences Mall, Vancouver, British Columbia V6T 1Z3, Canada. Tel.: 604-822-2578; Fax: 604-822-2316; E-mail: cnaus{at}interchange.ubc.ca.

3 The abbreviations used are: CNTF, ciliary neurotrophic factor; CBX, carbenoxolone; CNTFR{alpha}, ciliary neurotrophic factor receptor {alpha}; DIV, days in vitro; GZA, glycyrrhizic acid; gp130, glycoprotein 130; IL-6, interleukin-6; JAK/STAT, Janus kinase/signal transducers and activators of transcription; LDH, lactate dehydrogenase; LIFRβ, leukemia inhibitory factor receptor β; MAP2, microtubule-associated protein 2; MAPK/ERK, mitogen-activated protein kinase/extracellular signal-regulated kinase; NGF, nerve growth factor; PBS, phosphate-buffered saline; PDL, poly-D-lysine; PI, propidium iodide; PI3K, phosphatidylinositol 3-kinase; pSTAT3, Tyr-705-phosphorylated STAT3; RT-PCR, reverse transcription-PCR; STAT3, signal transducers and activators of transcription 3; TUNEL, terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick-end labeling; DAPI, 4,6-diamidino-2-phenylindole. Back

4 M. Ozog, unpublished observations. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Adler, R., Landa, K. B., Manthorpe, M., and Varon, S. (1979) Science 204, 1434-1436[Abstract/Free Full Text]
  2. Masu, Y., Wolf, E., Holtmann, B., Sendtner, M., Brem, G., and Thoenen, H. (1993) Nature 365, 27-32[CrossRef][Medline] [Order article via Infotrieve]
  3. Ip, N. Y., Wiegand, S. J., Morse, J., and Rudge, J. S. (1993) Eur. J. Neurosci. 5, 25-33[Medline] [Order article via Infotrieve]
  4. Lee, M. Y., Deller, T., Kirsch, M., Frotscher, M., and Hofmann, H. D. (1997) J. Neurosci. 17, 1137-1146[Abstract/Free Full Text]
  5. Nieto-Sampedro, M., Lewis, E. R., Cotman, C. W., Manthorpe, M., Skaper, S. D., Barbin, G., Longo, F. M., and Varon, S. (1982) Science 217, 860-861[Abstract/Free Full Text]
  6. Rudge, J. S., Alderson, R. F., Pasnikowski, E., McClain, J., Ip, N. Y., and Lindsay, R. M. (1992) Eur. J. Neurosci. 4, 459-471[CrossRef][Medline] [Order article via Infotrieve]
  7. Stockli, K. A., Lottspeich, F., Sendtner, M., Masiakowski, P., Carroll, P., Gotz, R., Lindholm, D., and Thoenen, H. (1989) Nature 342, 920-923[CrossRef][Medline] [Order article via Infotrieve]
  8. Davis, S., Aldrich, T. H., Valenzuela, D. M., Wong, V. V., Furth, M. E., Squinto, S. P., and Yancopoulos, G. D. (1991) Science 253, 59-63[Abstract/Free Full Text]
  9. Park, C. K., Ju, W. K., Hofmann, H. D., Kirsch, M., Ki, K. J., Chun, M. H., and Lee, M. Y. (2000) Brain Res. 861, 345-353[CrossRef][Medline] [Order article via Infotrieve]
  10. Rudge, J. S., Pasnikowski, E. M., Holst, P., and Lindsay, R. M. (1995) J. Neurosci. 15, 6856-6867[Abstract/Free Full Text]
  11. Bloch, J., Bachoud-Levi, A. C., Deglon, N., Lefaucheur, J. P., Winkel, L., Palfi, S., Nguyen, J. P., Bourdet, C., Gaura, V., Remy, P., Brugieres, P., Boisse, M. F., Baudic, S., Cesaro, P., Hantraye, P., Aebischer, P., and Peschanski, M. (2004) Hum. Gene Ther. 15, 968-975[CrossRef][Medline] [Order article via Infotrieve]
  12. Emerich, D. F., and Winn, S. R. (2004) Cell Transplant. 13, 253-259[Medline] [Order article via Infotrieve]
  13. Ogata, N., Ogata, K., Imhof, H. G., and Yonekawa, Y. (1996) Acta Neurochir. 138, 580-583[CrossRef][Medline] [Order article via Infotrieve]
  14. Davis, S., Aldrich, T. H., Ip, N. Y., Stahl, N., Scherer, S., Farruggella, T., DiStefano, P. S., Curtis, R., Panayotatos, N., and Gascan, H. (1993) Science 259, 1736-1739[Abstract/Free Full Text]
  15. Ip, N. Y., and Yancopoulos, G. D. (1992) Prog. Growth Factor Res. 4, 139-155[CrossRef][Medline] [Order article via Infotrieve]
  16. Rudge, J. S., Li, Y., Pasnikowski, E. M., Mattsson, K., Pan, L., Yancopoulos, G. D., Wiegand, S. J., Lindsay, R. M., and Ip, N. Y. (1994) Eur. J. Neurosci. 6, 693-705[CrossRef][Medline] [Order article via Infotrieve]
  17. Ozog, M. A., Bechberger, J. F., and Naus, C. C. (2002) Cancer Res. 62, 3544-3548[Abstract/Free Full Text]
  18. Ozog, M. A., Bernier, S. M., Bates, D. C., Chatterjee, B., Lo, C. W., and Naus, C. C. (2004) Mol. Biol. Cell 15, 4761-4774[Abstract/Free Full Text]
  19. Alonzi, T., Middleton, G., Wyatt, S., Buchman, V., Betz, U. A., Muller, W., Musiani, P., Poli, V., and Davies, A. M. (2001) Mol. Cell. Neurosci. 18, 270-282[CrossRef][Medline] [Order article via Infotrieve]
  20. Rose-John, S., and Heinrich, P. C. (1994) Biochem. J. 300, 281-290[Medline] [Order article via Infotrieve]
  21. Blanc, E. M., Bruce-Keller, A. J., and Mattson, M. P. (1998) J. Neurochem. 70, 958-970[Medline] [Order article via Infotrieve]
  22. Lin, J. H., Yang, J., Liu, S., Takano, T., Wang, X., Gao, Q., Willecke, K., and Nedergaard, M. (2003) J. Neurosci. 23, 430-441[Abstract/Free Full Text]
  23. Nakase, T., Fushiki, S., and Naus, C. C. (2003) Stroke 34, 1987-1993[Abstract/Free Full Text]
  24. Nakase, T., Sohl, G., Theis, M., Willecke, K., and Naus, C. C. (2004) Am. J. Pathol. 164, 2067-2075[Abstract/Free Full Text]
  25. Ozog, M. A., Siushansian, R., and Naus, C. C. (2002) J. Neuropathol. Exp. Neurol. 61, 132-141[Medline] [Order article via Infotrieve]
  26. Theis, M., Jauch, R., Zhuo, L., Speidel, D., Wallraff, A., Doring, B., Frisch, C., Sohl, G., Teubner, B., Euwens, C., Huston, J., Steinhauser, C., Messing, A., Heinemann, U., and Willecke, K. (2003) J. Neurosci. 23, 766-776[Abstract/Free Full Text]
  27. Dugan, L. L., Bruno, V. M., Amagasu, S. M., and Giffard, R. G. (1995) J. Neurosci. 15, 4545-4555[Abstract]
  28. Treister, N. S., Richards, S. M., Lombardi, M. J., Rowley, P., Jensen, R. V., and Sullivan, D. A. (2005) J. Dent. Res. 84, 160-165[Abstract/Free Full Text]
  29. James, C. G., Appleton, C. T., Ulici, V., Underhill, T. M., and Beier, F. (2005) Mol. Biol. Cell 16, 5316-5333[Abstract/Free Full Text]
  30. Malgrange, B., Rogister, B., Lefebvre, P. P., Mazy-Servais, C., Welcher, A. A., Bonnet, C., Hsu, R. Y., Rigo, J. M., Van De Water, T. R., and Moonen, G. (1998) Neurochem. Res. 23, 1133-1138[CrossRef][Medline] [Order article via Infotrieve]
  31. Zaheer, A., Zhong, W., Uc, E. Y., Moser, D. R., and Lim, R. (1995) Cell. Mol. Neurobiol. 15, 221-237[CrossRef][Medline] [Order article via Infotrieve]
  32. Nakashima, K., Yanagisawa, M., Arakawa, H., and Taga, T. (1999) FEBS Lett. 457, 43-46[CrossRef][Medline] [Order article via Infotrieve]
  33. Smit, M., Leng, J., and Klemke, R. L. (2003) BioTechniques 35, 254-256[Medline] [Order article via Infotrieve]
  34. Dehmelt, L., and Halpain, S. (2004) J. Neurobiol. 58, 18-33[CrossRef][Medline] [Order article via Infotrieve]
  35. Dirnagl, U., Iadecola, C., and Moskowitz, M. A. (1999) Trends Neurosci. 22, 391-397[CrossRef][Medline] [Order article via Infotrieve]
  36. Lee, N., Neitzel, K. L., Di Marco, A., Laufer, R., and Maclennan, A. J. (2005) Neurosci. Lett. 374, 161-165[CrossRef][Medline] [Order article via Infotrieve]
  37. Banker, G. A., and Cowan, W. M. (1977) Brain Res. 126, 397-420[CrossRef][Medline] [Order article via Infotrieve]
  38. Qiu, J., Cafferty, W. B., McMahon, S. B., and Thompson, S. W. (2005) J. Neurosci. 25, 1645-1653[Abstract/Free Full Text]
  39. Xia, X. G., Hofmann, H. D., Deller, T., and Kirsch, M. (2002) Mol. Cell. Neurosci. 21, 379-392[CrossRef][Medline] [Order article via Infotrieve]
  40. Lawrance, G., Rylett, R. J., Richardson, P. M., Dunn, R. J., Dow, K. E., and Riopelle, R. J. (1995) J. Neurochem. 64, 1483-1490[Medline] [Order article via Infotrieve]
  41. Emsley, J. G., and Hagg, T. (2003) Exp. Neurol. 183, 298-310[CrossRef][Medline] [Order article via Infotrieve]
  42. Wu, Y. Y., and Bradshaw, R. A. (1996) J. Biol. Chem. 271, 13023-13032[Abstract/Free Full Text]
  43. Sterneck, E., Kaplan, D. R., and Johnson, P. F. (1996) J. Neurochem. 67, 1365-1374[Medline] [Order article via Infotrieve]
  44. Zhong, W., Zaheer, A., and Lim, R. (1994) Brain Res. 661, 56-62[CrossRef][Medline] [Order article via Infotrieve]
  45. Ng, Y. P., He, W., and Ip, N. Y. (2003) J. Biol. Chem. 278, 38731-38739[Abstract/Free Full Text]
  46. Wiese, S., Digby, M. R., Gunnersen, J. M., Gotz, R., Pei, G., Holtmann, B., Lowenthal, J., and Sendtner, M. (1999) Nat. Neurosci. 2, 978-983[CrossRef][Medline] [Order article via Infotrieve]
  47. Belloir, B., Kovari, E., Surini-Demiri, M., and Savioz, A. (2001) J. Neurosci. Res. 64, 61-69[CrossRef][Medline] [Order article via Infotrieve]
  48. Stichel, C. C., Kappler, J., Junghans, U., Koops, A., Kresse, H., and Muller, H. W. (1995) Brain Res. 704, 263-274[CrossRef][Medline] [Order article via Infotrieve]
  49. Matsui, F., Kakizawa, H., Nishizuka, M., Hirano, K., Shuo, T., Ida, M., Tokita, Y., Aono, S., Keino, H., and Oohira, A. (2005) J. Neurosci. Res. 81, 837-845[CrossRef][Medline] [Order article via Infotrieve]
  50. Tu, Y., Hou, S. T., Huang, Z., Robertson, G. S., and MacManus, J. P. (1998) J. Cereb. Blood Flow Metab. 18, 658-669[CrossRef][Medline] [Order article via Infotrieve]
  51. Nagai, N., De Mol, M., Lijnen, H. R., Carmeliet, P., and Collen, D. (1999) Circulation 99, 2440-2444[Abstract/Free Full Text]
  52. Imaizumi, K., Tsuda, M., Wanaka, A., Tohyama, M., and Takagi, T. (1994) Brain Res. Mol. Brain Res. 26, 189-196[Medline] [Order article via Infotrieve]
  53. Kim, B. J., Li, Z., Fariss, R. N., Shen, D. F., Mahesh, S. P., Egwuagu, C., Yu, C. R., Nagineni, C. N., Chan, C. C., and Nussenblatt, R. B. (2004) Investig. Ophthalmol. Vis. Sci. 45, 3170-3176[Abstract/Free Full Text]
  54. Del Signore, A., De Sanctis, V., Di Mauro, E., Negri, R., Perrone-Capano, C., and Paggi, P. (2006) Eur. J. Neurosci. 23, 65-74[CrossRef][Medline] [Order article via Infotrieve]
  55. Davies, J. E., Tang, X., Denning, J. W., Archibald, S. J., and Davies, S. J. (2004) Eur. J. Neurosci. 19, 1226-1242[CrossRef][Medline] [Order article via Infotrieve]
  56. Faissner, A., Clement, A., Lochter, A., Streit, A., Mandl, C., and Schachner, M. (1994) J. Cell Biol. 126, 783-799[Abstract/Free Full Text]
  57. Nagata, K., Nakajima, K., Takemoto, N., Saito, H., and Kohsaka, S. (1993) Int. J. Dev. Neurosci. 11, 227-237[CrossRef][Medline] [Order article via Infotrieve]
  58. Hartwig, C., Veske, A., Krejcova, S., Rosenberger, G., and Finckh, U. (2005) BMC Neurosci. 6, 53[CrossRef][Medline] [Order article via Infotrieve]
  59. Cushing, P., Bhalla, R., Johnson, A. M., Rushlow, W. J., Meakin, S. O., and Belliveau, D. J. (2005) J. Neurosci. Res. 82, 788-801[CrossRef][Medline] [Order article via Infotrieve]
  60. Ip, N. Y., Li, Y. P., van de Stadt, I., Panayotatos, N., Alderson, R. F., and Lindsay, R. M. (1991) J. Neurosci. 11, 3124-3134[Abstract]
  61. Saggio, I., Gloaguen, I., Poiana, G., and Laufer, R. (1995) EMBO J. 14, 3045-3054[Medline] [Order article via Infotrieve]
  62. Hirota, H., Kiyama, H., Kishimoto, T., and Taga, T. (1996) J. Exp. Med. 183, 2627-2634[Abstract/Free Full Text]
  63. Gearing, D. P., Ziegler, S. F., Comeau, M. R., Friend, D., Thoma, B., Cosman, D., Park, L., and Mosley, B. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 1119-1123[Abstract/Free Full Text]
  64. Ip, N. Y., McClain, J., Barrezueta, N. X., Aldrich, T. H., Pan, L., Li, Y., Wiegand, S. J., Friedman, B., Davis, S., and Yancopoulos, G. D. (1993) Neuron 10, 89-102[CrossRef][Medline] [Order article via Infotrieve]
  65. Maclennan, A. J., Vinson, E. N., Marks, L., McLaurin, D. L., Pfeifer, M., and Lee, N. (1996) J. Neurosci. 16, 621-630[Abstract/Free Full Text]
  66. Takeda, K., Kaisho, T., Yoshida, N., Takeda, J., Kishimoto, T., and Akira, S. (1998) J. Immunol. 161, 4652-4660[Abstract/Free Full Text]
  67. Gerecke, K. M., Wyss, J. M., and Carroll, S. L. (2004) Mol. Cell. Neurosci. 27, 379-393[Medline] [Order article via Infotrieve]
  68. Thompson, J., Dolcet, X., Hilton, M., Tolcos, M., and Davies, A. M. (2004) Mol. Cell. Neurosci. 27, 441-452[CrossRef][Medline] [Order article via Infotrieve]
  69. Bonnet, D., Garcia, M., Vecino, E., Lorentz, J. G., Sahel, J., and Hicks, D. (2004) Brain Res. 1007, 142-151[CrossRef][Medline] [Order article via Infotrieve]
  70. Kim, I. J., Drahushuk, K. M., Kim, W. Y., Gonsiorek, E. A., Lein, P., Andres, D. A., and Higgins, D. (2004) J. Neurosci. 24, 3304-3312[Abstract/Free Full Text]
  71. Higuchi, M., Onishi, K., Masuyama, N., and Gotoh, Y. (2003) Genes Cells 8, 657-669[Abstract]
  72. Desbarats, J., Birge, R. B., Mimouni-Rongy, M., Weinstein, D. E., Palerme, J. S., and Newell, M. K. (2003) Nat. Cell Biol. 5, 118-125[CrossRef][Medline] [Order article via Infotrieve]
  73. Burdick, J. A., Ward, M., Liang, E., Young, M. J., and Langer, R. (2006) Biomaterials 27, 452-459[CrossRef][Medline] [Order article via Infotrieve]
  74. Holm, N. R., Christophersen, P., Hounsgaard, J., Gammeltoft, S., and Olesen, S. P. (2002) J. Neurochem. 82, 495-503[CrossRef][Medline] [Order article via Infotrieve]
  75. Cui, Q., Yip, H. K., Zhao, R. C., So, K. F., and Harvey, A. R. (2003) Mol. Cell. Neurosci. 22, 49-61[CrossRef][Medline] [Order article via Infotrieve]
  76. Avwenagha, O., Campbell, G., and Bird, M. M. (2003) J. Neurocytol. 32, 1055-1075[CrossRef][Medline] [Order article via Infotrieve]
  77. Guo, X., Metzler-Northrup, J., Lein, P., Rueger, D., and Higgins, D. (1997) Brain Res. Dev. Brain Res. 104, 101-110[CrossRef][Medline] [Order article via Infotrieve]
  78. Zhang, J., Lineaweaver, W. C., Oswald, T., Chen, Z., Chen, Z., and Zhang, F. (2004) J. Reconstr. Microsurg. 20, 323-327[CrossRef][Medline] [Order article via Infotrieve]
  79. Simon, R., Thier, M., Kruttgen, A., Rose-John, S., Weiergraber, O., Heinrich, P. C., Schroder, J. M., and Weis, J. (1995) Neuroreport 7, 153-157[Medline] [Order article via Infotrieve]
  80. Kelleher, M. O., Myles, L. M., Al Abri, R. K., and Glasby, M. A. (2006) Acta Neurochir. 148, 55-61[CrossRef][Medline] [Order article via Infotrieve]
  81. Shuto, T., Horie, H., Hikawa, N., Sango, K., Tokashiki, A., Murata, H., Yamamoto, I., and Ishikawa, Y. (2001) Neuroreport 12, 1081-1085[CrossRef][Medline] [Order article via Infotrieve]
  82. McCallister, W. V., McCallister, E. L., Dubois, B., and Trumble, T. E. (2004) J. Reconstr. Microsurg. 20, 473-481[CrossRef][Medline] [Order article via Infotrieve]
  83. Gupta, S. K., Haggarty, A. J., Carbonetto, S., Riopelle, R. J., Richardson, P. M., and Dunn, R. J. (1993) Eur. J. Neurosci. 5, 977-985[CrossRef][Medline] [Order article via Infotrieve]
  84. Heymanns, J., and Unsicker, K. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 7758-7762[Abstract/Free Full Text]

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