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Ciliary Neurotrophic Factor May Activate Mature Astrocytes via Binding with the Leukemia Inhibitory Factor Receptor
Molecular and Cellular Neuroscience 17, 373–384 (2001) doi:10.1006/mcne.2000.0926, available online at http://www.idealibrary.com on
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Ciliary Neurotrophic Factor May Activate Mature Astrocytes via Binding with the Leukemia Inhibitory Factor Receptor Christelle Monville,* Muriel Coulpier, † Luciano Conti, ‡ Claudio De-Fraja, ‡ Patrick Dreyfus,* Christiane Fages,* Danielle Riche,* Marcienne Tardy,* Elena Cattaneo, ‡ and Marc Peschanski* ,1 *INSERM U421, IM3, Faculte´ de Me´decine, 94010 Cre´teil cedex, France; †Regeneron Pharmaceuticals, Inc., Tarrytown, New York 10591; and ‡Institute of Pharmacological Sciences, 20133 Milan, Italy
Ciliary neurotrophic factor (CNTF) acts on immature astrocytes that express its trimeric receptor. In contrast, mature astrocytes do not significantly express the specific CNTF␣ receptor subunit, yet they respond to CNTF administration in vivo. Here we show that this controversy may be solved by a shift in astroglial sensitivity to CNTF over time, related to a change in the type of receptor bound by the cytokine on mature astrocytes. A convergent set of results supports the hypothesis that the CNTF effect is due to the illegitimate binding on the leukemia inhibitory factor receptor (LIFR): (i) it requires high concentration of recombinant rat CNTF; (ii) it involves the Jak/Stat and Ras-MAPK pathways; (iii) it is preserved in CNTFR␣ⴚ/ⴚ cells; (iv) it is potentiated by soluble CNTFR␣ added to the medium; and (v) it is significantly decreased by a partial antagonist of LIFR. On these bases, we propose a mechanistic model in which, in the adult brain, a CNTF/LIFR interglial system may be modulated by neurons that synthesize CNTFR␣.
INTRODUCTION Synthesis of the ciliary neurotrophic factor (CNTF) and of all three components of its trimeric receptor is widespread in the adult central nervous system (Squinto et al., 1990; Sto¨ckli et al., 1991; Sendtner et al., 1994; MacLennan et al., 1996). Its neuroprotective ef1 To whom correspondence and reprint requests should be addressed at INSERM U421, IM3, Faculte´ de me´decine, 8 rue du Ge´ne´ral Sarrail, 94010 Cre´teil cedex, France. Fax: 33 1 49 81 37 09. E-mail: [email protected].
1044-7431/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
fects, together with the source of the factor in astrocytes, the location of its specific CNTFR␣ receptor subunit specifically on neurons in vivo, and its lack of a signal peptide, have led to the hypothesis that CNTF may act as an “injury molecule” (Thoenen, 1991; Sendtner et al., 1997), protecting neurons after release from astrocytes following injury. Recent demonstration of phenotypic changes in astrocytes after administration of CNTF into the adult brain (Winter et al., 1995; Levison et al., 1996; Clatterbuck et al., 1996; Lisovoski et al., 1997) has suggested that it may also play a role in inducing astroglial activation. The CNTF triggers astroglial differentiation very efficiently in glial precursors and immature astrocytes (Hughes et al., 1988; Lillien et al., 1988; Kahn et al., 1995; Bonni et al., 1997). This effect is likely related to the binding of the cytokine to its receptor, the three components of which are expressed by these precursors and immature cells (Bonni et al., 1997). In contrast, effects of CNTF on mature astrocytes have remained controversial. Astroglial activation in long-term cultures treated with CNTF has indeed been altogether weak or nonexisting (Meyer and Unsicker, 1994; Smith et al., 1996). In addition, despite the observation of CNTF effects in vivo, astrocytes in the adult brain have not been shown to express the specific ␣ subunit of the trimeric CNTF receptor (Squinto et al., 1990; MacLennan et al., 1996; Kordower et al., 1997), the association of which to the dimeric receptor of the leukemia inhibitory factor (LIF) is necessary to form the trimeric CNTF receptor (Ip et al., 1993; Davis et al., 1993b). Paradoxically, this expres-
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374 sion is observed by Western and Northern blotting in long-term astrocytic cultures in which the effects of CNTF are not solid (Rudge et al., 1994; Alderson et al., 1999). These data have led to different hypotheses, including the expression of an undetectable, though sufficient, amount of CNTFR␣ in adult astrocytes (Levison et al., 1996) or the need for an interaction with another cell type to obtain the CNTF-elicited astroglial activation (Kahn et al., 1997). The present study has explored an alternative hypothesis that these apparently discrepant data may reveal a shift in astroglial sensitivity to CNTF over time, related to a change in the type of receptor bound by the cytokine. This hypothesis was based upon studies that have demonstrated that CNTF may act on cells lacking the CNTFR␣ subunit via illegitimate binding with the dimeric LIF receptor, although at a much higher concentration (Schooltink et al., 1992; Gearing et al., 1994). The present results show that CNTF-induced astroglial activation can indeed be observed in longterm astroglial cultures and that the biological characteristics of this effect are compatible with the illegitimate binding of CNTF with the dimeric LIF receptor.
RESULTS Enriched astroglial cell cultures were readily obtained from cerebral hemispheres of Swiss mice (Iffa Credo, France), CNTF⫺/⫺ mice (BRL, Basel Switzerland), or CNTFR␣⫺/⫺ (Regeneron, Tarrytown, NY), and maintained for several weeks without signs of cell alteration. CNTF Effect on Long-Term Astrocytes: Dose Dependence and Intracellular Signaling Pathways In immature astrocytes (maintained 7 days in culture, not at confluence), addition of recombinant rat CNTF (rCNTF) to the medium at concentrations as low as 10 ng/ml (lowest concentration used) provoked an increase in the cellular content of glial fibrillary acid protein (GFAP) (Fig. 1a). Western blotting showed no endogenous CNTF in these short-term cultures either in the absence of treatment or 3 days after addition of rCNTF to the medium. In sharp contrast, rCNTF was inefficient at concentrations as high as 100 ng/ml in long-term cultures when astrocytes were maintained in culture before treatment until confluence was reached (around 14 days). An increase in the cellular content of GFAP was observed in a significant and reproducible fashion only
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when the highest concentration of rCNTF studied (250 ng/ml) was used (Fig. 1b). Similarly, cellular content of endogenous CNTF, which was quantifiable in these more mature astrocytes, was increased significantly above control levels only when 250 ng/ml of rCNTF was added to the medium (Fig. 1c) and not following treatment with lower doses of the cytokine. To eliminate an artifactual synergistic effect of the serum component TGF, a pan-TGF antibody was added to the culture medium. This treatment had no effect on the increase of CNTF intracellular content after addition of 250 ng/ml of rCNTF to the medium. A quantification of rCNTF, which would have been internalized by cultured cells, was excluded by measuring no CNTF in samples obtained from CNTF⫺/⫺ mice after treatment of mature astrocytes with 250 ng/ml of rCNTF. CNTF cellular content was then used as a marker of astroglial activation in following experiments, rather than GFAP, because this molecule is present in astroglial cultures only after 14 days, not at 7, and therefore may be a discriminant marker of mature astrocytes. Consequently, using CNTF as a marker allowed us to overcome the potential problem created by the persistence of a small subpopulation of astroglial precursors or immature cells—which express GFAP but not CNTF—in long-term cultures (see below). The two intracellular signaling systems studied, namely the Jak/ STAT and the Ras-MAPkinase pathways, exhibited rapid and sustained activation when long-term astroglial cultures were treated with 250 ng/ml of rCNTF. Stat3 (Figs. 2a and 2b) and Stat1 (Figs. 2c and 2d) were indeed strongly phosphorylated as soon as 5 min after treatment and up to 1 h. In parallel, the adapter protein Shc, ERK1, and ERK2, which are part of the Ras-MAPK pathway associated with the CNTF receptor, were activated as soon as 5 min after rCNTF application (Figs. 3a and 3b). For these elements of the Ras-MAPK pathway, the activation was still significant 240 min after application. In contrast, application of 30 ng/ml of rCNTF either was inefficient or induced only limited phosphorylation. Dependence of the Effects of CNTF on the Presence or Concentration of CNTFR␣ The requirement for the presence of CNTFR␣ in the astroglial effects of rCNTF at high concentration was assessed by adding the cytokine to the medium of cultures of mature astrocytes obtained from CNTFR␣ knock-out mice. In these cultures, addition of 250 ng/ml of rCNTF induced an increase in CNTF cellular content similar to that seen in wild-type cells (Fig. 4a), demonstrating that
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375 the specific ␣ receptor subunit was not required in the effects. The effect of providing CNTFR␣ to wild-type mature astrocytes was then assessed using the soluble compound myc-sCNTFR␣. Under these conditions, a potentiation of the effects was observed as a major shift toward values lower than the dose required for rCNTF effects on mature astrocytes. Increase of CNTF cellular content was indeed observed with the lowest concentration studied (10 ng/ml) and maintained at higher concentrations (Fig. 4b). All three CNTF receptor subunits, including CNTFR␣, were present in the long-term astroglial cultures (Fig. 6b). Immunocytochemical staining, however, demonstrated that CNTFR␣ was not evenly distributed in long-term cultures of astrocytes but was instead present only on a small subpopulation of small bipolar cells. The vast majority of cells which were large and displayed the typical flattened morphology of longterm cultured astrocytes were not decorated by antibodies against CNTFR␣ (Fig. 5). In contrast, these latter cells, which were the only ones to contain CNTF, expressed the two other subunits of the CNTF receptor, LIFR and gp130 (Fig. 5). To check that an indirect mechanism was not involved in the CNTF effects on mature astrocytes in which CNTFR␣ present on the small cells would be donated to the mature astrocytes, the concentration of CNTFR␣ was modified in two different experiments. A mechanical lesion was made in the cultures to provoke an increase in the concentration of CNTFR␣ (Fig. 6b). The lesion provoked an increase in CNTF cellular content at baseline but did not amplify the effects of rCNTF (Fig. 6a). Reciprocally, a phosphatidylinositol-specific phospholipase C (PIPLC) was used to cleave the glycosylphosphatidylinositol anchor that links CNTFR␣ to the cell membrane, and the medium was changed before the application of rCNTF to eliminate as much of the ␣ receptor subunit as possible. The PIPLC treatment, the biological activity of
FIG. 1. (a) Effect of rCNTF on GFAP cellular content in immature astrocytes. rCNTF provoked an increase of 44 ⫾ 9% (factorial ANOVA significant at 95%, t test ***P ⬍ 0.001, **P ⬍ 0.01, or *P ⬍ 0.05) at the lowest concentration used (10 ng/ml), compared to untreated cultures. This effect was maintained, without further in-
crease, at all other concentrations. (b) Effect of rCNTF on GFAP cellular content in mature astrocytes. Addition of 250 ng/ml rCNTF induced a statistically significant increase compared to untreated controls (⫹23 ⫾ 5.6%, factorial ANOVA significant at 95%, t test *P ⬍ 0.05). In contrast, application of rCNTF provoked a statistically significant decrease, compared to untreated controls, for 30 and 50 ng/ml (⫺24 ⫾ 8.5 and ⫺16 ⫾ 1.7%, respectively, factorial ANOVA significant at 95%, t test *P ⬍ 0.05). (c) Effect of rCNTF on CNTF cellular content in mature astrocytes. No change in CNTF cellular content was observed when rCNTF was added at 10, 30, 50, and 100 ng/ml concentrations. In contrast, a significant increase was consistently measured at 250 ng/ml (⫹39.6 ⫾ 10%, factorial ANOVA significant at 95%, t test **P ⬍ 0.01).
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FIG. 2. (a, b) Activation of the Jak/Stat pathway by rCNTF in mature astrocytes. Stat3 was phosphorylated with 30 ng/ml (⫹700 ⫾ 250% of control, factorial ANOVA significant at 95%, t test *P ⬍ 0.05), and more strongly with 250 ng/ml (⫹3400 ⫾ 1800% of control, factorial ANOVA significant at 95%, t test *P ⬍ 0.05) after 5 and 30 min (⫹297 ⫾ 86 and ⫹615 ⫾ 170% of control, factorial ANOVA significant at 95%, t test P ⬍ 0.01 in both cases). This activation was not observed after 60 min. (c, d) Stat1 was phosphorylated with 30 ng/ml (⫹140 ⫾ 90%, NS) and 250 ng/ml (⫹409 ⫾ 160% of control, factorial ANOVA significant at 95%, t test *P ⬍ 0.05) after 5 and 30 min (⫹192 ⫾ 100% with 30 ng/ml and ⫹321 ⫾ 170% with 250 ng/ml).
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a molecule that was shown to partially block the activation of VIP by CNTF (Vernallis et al., 1997), was added to the culture medium together with 250 ng/ml of rCNTF. Under these conditions, a significant decrease (of about 50%) in the effects of rCNTF on astrocytes was observed (Fig. 7).
DISCUSSION The main result of this study is that CNTF can provoke biochemical changes in mature astrocytes in vitro, as previously observed in immature glial cells. The receptor system is not, however, the complete tripartite CNTF receptor since it does not involve the specific ␣ subunit. A convergent body of facts suggests that CNTF may act on mature astrocytes by binding illegitimately to the dimeric receptor of the LIF. This demonstrates a developmental shift in astrocytes, since CNTF binds to its own full trimeric receptor system on immature astrocytes and glial precursors. In the mature brain, CNTF is synthesized by astrocytes and its specific CNTFR␣ subunit is synthesized—and potentially released in the extracellular space via cleavage of its GPI anchor— by neurons. Taking into account the present data, we propose a mechanistic model in which neurons may consequently play a role in the activation of mature astrocytes by modulating the efficacy of a CNTF/LIFR interglial signaling system. CNTF May Effect Biochemical Differentiation of Mature Astrocytes via Binding to the LIF Receptor
FIG. 3. (a, b) Activation of the Shc-Grb2-MAPKs pathway by rCNTF in mature astrocytes. ERK1 (p42MAPK) and mainly ERK2 (p44MAPK) were strongly activated with 250 ng/ml after 5 and 30 min (⫹470 ⫾ 60 and ⫹2133 ⫾ 490% of control, factorial ANOVA significant at 95%, t test P ⬍ 0.001 in both cases).
which was controlled by observing an increase in cholinesterase activity in the medium, did not alter the effects of rCNTF on mature astrocytes (Fig. 6a). Dependence of the Effects of CNTF on the Activity of the Dimeric LIF Receptor To test the hypothesis that CNTF effects on astrocytes require the activity of the dimeric LIF receptor, hLIF05,
CNTF provokes astroglial differentiation of glial precursors and strongly activates the synthesis of GFAP in immature astrocytes, in vivo as well as in vitro (Hughes et al., 1988; Lillien et al., 1988; Kahn et al., 1995, 1997; Bonni et al., 1997; Rajan and McKay, 1998). In contrast, an effect of CNTF on the synthesis of GFAP or endogenous CNTF has been observed in the adult only when the cytokine was administered into the brain of living rodents (Winter et al., 1995; Levison et al., 1996; Clatterbuck et al., 1996; Lisovoski et al., 1997) and not in purified astroglial cultures (Meyer and Unsicker, 1994; Smith et al., 1996). These paradoxical results had led to the hypothesis that activation of mature astrocytes by CNTF was indirect and followed an effect of the cytokine on neighboring cells in vivo (Meyer and Unsicker, 1994; Smith et al., 1996). The present results do not support this hypothesis since an activation of GFAP and endogenous CNTF synthesis was obtained in longterm purified astroglial cultures. In a previous report, in
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FIG. 4. (a) Effect of rCNTF on astrocytes obtained from CNTFR␣⫺/⫺ mice. Addition of 250 ng/ml induced an increase of CNTF cellular content in both wild-type and CNTFR␣-deficient mice (⫹85 ⫾ 21 and ⫹143 ⫾ 47%, respectively, factorial ANOVA significant at 95%, t test 䡠䡠P ⬍ 0.01 and *P ⬍ 0.05). (b) Addition of soluble CNTFR␣ provoked a major shift in the concentration of rCNTF required to observe glial effects. Addition of sCNTFR␣ at 200 ng/ml to the culture medium was followed by a major increase of CNTF cellular content with 10 ng/ml (⫹66.5 ⫾ 30%, factorial ANOVA significant at 95%, t test *P ⬍ 0.05). A similar increase was maintained, whatever the concentration of rCNTF used, between 30 and 250 ng/ml.
which astroglial activation was observed in the brain of adult rats following intracerebral injection of adenoviral vectors recombinant for a secretable form of CNTF
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(Lisovoski et al., 1997), the very restricted areas in which this activation was observed around sites of potential delivery of the cytokine had led us to hypothesize either that CNTF did not spread freely far away from these sites or that a high concentration of the cytokine was required. The present in vitro results strongly support the latter hypothesis since, in sharp contrast to the results obtained in short-term cultures (of immature astrocytes), there was no effect of CNTF observed at concentrations lower than 10 ⫺8 M. This dose requirement explains the failure of previous in vitro studies to observe the effects (Meyer and Unsicker, 1994; Smith et al., 1996). The existence of an effect of CNTF on mature astrocytes is paradoxical because all studies of the sites of synthesis of the specific ␣ subunit of the CNTF receptor, through visualization of mRNA (Ip et al., 1993) or protein (MacLennan, 1996; Kordower et al., 1997), have failed to reveal an astrocytic localization in the adult brain. CNTFR␣ is, essentially, if not exclusively, synthesized by neurons in the central nervous system. To explain the effects of CNTF on astrocytes, authors have hypothesized a low, undetectable level of CNTFR␣ expression (Levison et al., 1996) or the activation of its expression in response to inflammatory mechanisms (Rudge et al., 1994, 1995). The present results support an alternative hypothesis that CNTF may bind illegitimately to the dimeric LIF receptor. CNTF normally binds to a high-affinity receptor complex, which is composed of three subunits, CNTFR␣ and the two  subunits (LIFR and gp130), which together form the receptor of another cytokine, the leukemia inhibitory factor (Squinto et al., 1990; Davis et al., 1993b; Ip et al., 1993; Stahl et al., 1994). It has been shown, however, that CNTF can provoke biological effects on cells that express only the LIF receptor (Schooltink et al., 1992; Davis et al., 1993b; Gearing et al., 1993). The arguments in favor of the existence of such a mechanism in the present experiments with mature astrocytes are numerous: the astrocytic activation was observed at the same concentration of the cytokine when cells were taken from mice that do not express CNTFR␣ or from wild-type mice, indicating that this specific subunit is not required; although CNTFR␣ is present in long-term astroglial cultures (Rudge et al., 1994; Alderson et al., 1999), immunohistochemical analysis indicated that it was not expressed by mature astrocytes, and alteration of its concentration did not modify the conditions of the astroglial effects of CNTF; the binding of CNTF to the glial receptor provoked the phosphorylation of molecules that belong to the two intracellu-
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FIG. 5. Immunocytochemical staining showing that the CNTFR␣ subunit is only present in a subpopulation of smaller bipolar cells. The two  components of the LIF receptor (LIFR and gp130) as well as CNTF are, in contrast, present in large cells with a flat polygonal shape and several processes. Scale bar: 10 m.
lar signaling pathways linked to the LIF (and by extension the CNTF) receptor, namely the Jak/Stat and the (Shc-)Ras-MAPK pathways (see references and discussion in Stahl and Yancopoulos, 1994; Stahl et al., 1994; Boulton et al., 1994; Bonni et al., 1997; Rajan and McKay, 1998); these effects were characterized by the need for a much lower concentration of the cytokine when CNTFR␣ was added in a soluble form to the medium (at least 100-fold), a result comparable to those previously described for cells treated with CNTF that expressed only the LIF receptor
(Davis et al., 1993b; Panayotatos et al., 1994); and partial blockade of the binding of CNTF to the LIFR using a specific antagonist (hLIF05) significantly reduced the effects of CNTF on astrocytes. Altogether, the shift in response to CNTF observed during the maturation of astrocytes may be explained by the loss of expression of the CNTFR␣ subunit, and therefore of the high-affinity receptor present at early stages, and the consequent use of the two  subunits of the LIF receptor that form a low-affinity receptor for CNTF.
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FIG. 6. (a) Modulation of CNTFR␣ availability in astroglial cultures. A lesion, which increased the level of CNTFR␣, did not amplify the effects of rCNTF on CNTF cellular content (⫹72 ⫾ 10.8% of control, factorial ANOVA significant at 95%, t test **P ⬍ 0.01). PIPLC treatment in order to remove CNTFR␣ anchored at the cell membranes of the small bipolar cells did not alter the effects of rCNTF on the mature astrocytes (⫹97 ⫾ 21.4% vs ⫹84 ⫾ 47% of control, ANOVA significant at 95%, t test 䡠䡠䡠P ⬍ 0.001). (b) Western blot demonstrating the presence of the three components of the trimeric CNTF receptor (LIFR, 200K mol wt; gp130, 130K mol wt; CNTFR␣, 80K mol wt) in mature astroglial cultures from wild-type mice. NL, nonlesioned; L, lesioned.
An Interglial Signaling System Which Is Potentially under Neuronal Control in the Adult Brain The biological significance of such a low-affinity receptor system for CNTF on mature astrocytes is but a matter of speculation. It is intriguing, however, that
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astrocytes are the only known source of CNTF in the adult brain (Sto¨ckli et al., 1991; Sendtner et al., 1994). Release of large amounts of CNTF in the extracellular space will therefore affect astrocytes via a paracrine/ autocrine system in the same time it affects neighboring neurons that express CNTFR␣ (Ip et al., 1993; MacLennan, 1996; Kordower et al., 1997). Following injury to the brain parenchyma, release of CNTF by dying astrocytes will thus provoke activation of surviving astrocytes in the close vicinity of the lesion, as well as exert a neuroprotective role as hypothesized by several authors (Thoenen, 1991; Sendtner et al., 1997). The strong potentiation of the CNTF effects on astrocytes by soluble CNTFR␣ suggests the possibility of an additional type of signaling system. CNTFR␣ is synthesized by neurons but it is linked to their membrane by a GPI anchor (Davis et al., 1991), which can be cleaved even in the intact brain since it is detected in a soluble form in the cerebrospinal fluid (Davis et al., 1993a). The presence of CNTFR␣ in a soluble form in the brain will transform the low-affinity astroglial receptor system into a high-affinity one and, as a consequence, will allow much smaller amounts of CNTF to provoke biochemical changes in astrocytes. Our data therefore are compatible with the hypothesis of a three-partner system in which astroglial release of CNTF would affect neighboring astrocytes in a manner which would strongly depend upon neuronal release of CNTFR␣ (Fig. 8). A CNTF-based interglial signal could thus be modulated by neurons in the absence of a lesion provoking the release of massive amounts of the cytokine. The physiological conditions under which such a system may operate are, however, not known and their very existence may even be questioned. On the one hand, and although release of GPI-anchored CNTFR␣ has been demonstrated for skeletal muscle following denervation, physiological conditions under which neurons may modulate a similar release are not known. On the other hand, the issue of a potential physiological release of CNTF by astrocytes (i.e., not following injury) remains unsolved. CNTF lacks a signal peptide, indicating that it is not released via conventional exocytotic mechanisms (Thoenen, 1991; Sendtner et al., 1994). It could, however, be released via a nonconventional energy-dependent pathway (Kamiguchi et al., 1995) reminiscent of those demonstrated for the 18-kDa isoform of bFGF and IL-1 (Rubartelli et al., 1990; Florkiewicz et al., 1995). This has not been observed, however, in astroglial cultures, the conditioned medium of which does not support the survival of ciliary ganglion cells (Rudge et al., 1992) and does not provoke phosphorylation of appropriate intracellular signaling proteins
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FIG. 7. Effect of partial blockade of the LIFR on the effects of rCNTF on mature astrocytes. Addition of the partial LIFR antagonist hLIF05 to the culture medium provoked a 50% reduction of the increase of the intracellular CNTF levels provoked by the addition of 250 ng/ml of rCNTF in mature astrocytes (ANOVA significant at 95%, t test **P ⬍ 0.01).
(Rudge et al., 1995). The answer to that question, of a potential physiological release of CNTF by astrocytes in the mature brain, will determine whether the interglial signaling system defined in the present study is just another injury mechanism or whether it operates, under the control of neurons releasing CNTFR␣, under currently unknown physiological conditions.
EXPERIMENTAL METHODS Astroglial cultures were prepared from cerebral hemispheres of neonatal Swiss mice (Iffa Credo, France), CNTF knockout mice (BRL, Switzerland), and neonatal CNTFR␣ knockout mice (Regeneron Pharmaceutical Inc., U.S.A.). Cultures were either grown at confluence for 14 days, thus defining “mature” cultures, or maintained only for 7 days. In the latter case, identified as “immature” cultures, cells occupied less than 50% of the dish. Culture Conditions Astroglial cultures were prepared as previously described (Bardakdjian et al., 1979). After 7 or 14 days in vitro, the medium (minimal essential medium containing 2 mM glutamine, essential amino acids, 0.03% glucose, penicillin–streptomycin) was removed and replaced with the same medium but without fetal calf serum (FCS). Recombinant rat CNTF (Boehringer
Mannheim, Germany) (rCNTF) was then added to the medium at a concentration of 0, 10 (4.4 ⫻ 10 ⫺10 M), 30 (1.3 ⫻ 10 ⫺9 M), 50 (2.2 ⫻ 10 ⫺9 M), 100 (4.4 ⫻ 10 ⫺9 M), or 250 ng/ml (10 ⫺8 M). The cells were collected 3 days later. In preliminary studies, it was confirmed (Levison et al., 1996) using tritiated thymidine that CNTF does not provoke astroglial proliferation in in vitro cultures. Some of the mature astroglial cultures, prepared as described above, were mechanically lesioned using the fine tip of a plastic pipette 18 days after astroglial plating. After lesion, the medium was removed and cultures were treated with 250 ng/ml of rCNTF or left untreated. In other experiments, PIPLC (Boehringer) was added at the concentration of 1 U/ml and the medium was exchanged 24 h later. In half of the dishes, rCNTF was then applied as described above. PIPLC activity was checked by quantifying the acetylcholine esterase activity in the supernatant, according to the technique described by Ellman et al. (1964). In the experiments with soluble CNTFR␣, 200 ng/ml of mycsCNTFR␣ (kindly provided by Ralph Laufer, IRBM, Italy) was added to the medium without FCS after 14 days in vitro. Thirty minutes later, rCNTF was added at 0, 10, 30, 50, 100, and 250 ng/ml. The cells were collected 3 days later. To block a potential binding of CNTF on the dimeric LIFR, hLIF05 (kindly provided by Dr. Ann Vernallis, UK) was used. This compound is a specific antagonist of the LIF receptor that has been shown in other cell models to partially block the activation of the VIP gene
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FIG. 8. CNTF/LIF receptor as an interglial signaling system under neuronal control. This scheme presents a hypothesis in which the CNTF-based glial signaling system is open to a neuronal partner which can strongly amplify the binding efficiency of the ligand by providing the CNTFR␣ subunit and thus restore a high-affinity receptor system.
by CNTF (50% inhibition with 5 g/ml of hLIF05; Vernallis et al., 1997). hLIF05 was added 12 h before rCNTF (250 ng/ml), and the cells were collected 24 h after rCNTF treatment. In all cases, the medium was removed at the end of the experiments and each dish was rinsed three times with HBSS (Hanks’ balanced salt solution, Seromed, Germany). The cells were collected by scraping into 62.5 mM Tris–HCl (pH 6.8), 2 mM EDTA, 2 mM phenylmethylsulfonyl fluoride, 0.5% Triton X-100, and 2.3% sodium dodecyl sulfate. Biochemical Analysis Total protein content was determined by the method of Lowry et al. (1951) with bovine serum albumin as a standard. The proteins were analyzed by Western blotting under the conditions described by Laemmli (1970). Briefly, samples were boiled for 5 min after addition of 10% glycerol, 5% mercaptoethanol (or 5% dithiothreitol for the MAPK-P), and 5% bromophenol blue, and then
lysates were electrophoresed on 7.5% (LIFR, gp130, CNTFR␣, Stat3-P, and Stat1-P), 10% (GFAP and MAPKP), or 12% (CNTF) SDS–polyacrylamide gels. Gels were blotted on nitrocellulose, blocked for 1 h in 5% nonfat dry milk in TBS-T (20 mM Tris, pH 7.5/500 mM NaCl/ 0.1% Tween 20), and then probed overnight at 4°C with the following antibodies: polyclonal anti-CNTF (R&D Systems, UK, 1/500), polyclonal anti-CNTFR␣ (kindly provided by Dr. A. J. MacLennan, University of Florida, U.S.A., 1/1000), monoclonal anti-gp130 (Santa Cruz, U.S.A., 1/1000), polyclonal anti-LIFR (Santa Cruz, 1/500), monoclonal anti-p44/p42 MAP kinase (or antiphospho ERK1/ERK2, New England Biolabs, UK, 1/1000), polyclonal anti-ERK1 (Santa Cruz, 1/4000), polyclonal anti-phospho-specific Stat3 (Tyr705) (New England Biolabs, 1/1000), polyclonal anti-phospho-specific Stat1 (Tyr701) (New England Biolabs, 1/1000), monoclonal anti-Stat3 (Santa Cruz, 1/1000), and polyclonal anti-Stat1 (Santa Cruz, 1/1000). After washing with TBS-T, membranes were incubated with a horseradish peroxidase-conjugated donkey anti-rabbit sec-
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ondary antibody (for CNTFR␣, LIFR, ERK1, phosphospecific Stat3, phospho-specific stat1, and stat1), a mouse anti-goat antibody (for CNTF), or a sheep antimouse antibody (for gp130, p44/p42 MAP kinase, and Stat3), followed by the enhanced chemiluminescent reaction (Amersham, Sweden), according to the manufacturer’s instructions. For GFAP, the sheets were incubated with a polyclonal anti-GFAP antibody (Dakopatts, Denmark, 1/100) and revealed with antirabbit IgG coupled to 125I, according to a previously described technique (Andres-Barquin et al., 1994). The sheets were placed in contact with a RPN6 film (Amersham). The levels of CNTF and GFAP were measured by densitometry and brought back to the total protein laid down. To limit variations in their processing, extracts from the control and all experimental conditions were treated in parallel on a single sheet for each specific experiment. In addition, to evaluate the variability between specific experiments, control extracts were subsequently reloaded on a single sheet and processed together. Statistical analysis used one-factor ANOVA and unpaired t test. Immunohistochemical Analysis The medium was removed and cells were rinsed two times with PBS. They were then fixed with cold methanol (⫺20°C) and probed 1 h at room temperature with the following antibodies: polyclonal anti-CNTF (1/100), polyclonal anti-CNTFR␣ (1/50), monoclonal anti-gp130 (1/100), and polyclonal anti-LIFR (1/100). After washing with PBS, cells were exposed to secondary antibodies linked to TRITC.
ACKNOWLEDGMENTS These studies were supported by INSERM and Association Franc¸aise contre les Myopathies. The authors gratefully acknowledge help from Ralph Laufer (IRBM P. Angeletti, Rome), John McLennan (University of Florida College of Medicine, Gainesville, FL), Austin Smith (Center for Genome Research, Edimburg, UK), Ann Vernallis (School of Health and Life Sciences, Aston, UK), Tom De Chiara and George Yancopoulos (Regeneron, Tarrytown, NY), Sharon L. Juliano (USUHS, Bethesda, MD), Philippe Hantraye (CNRS, Orsay, Italy), and Brigitte Onte´niente (INSERM, Cre´teil, Italy).
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Ciliary Neurotrophic Factor May Activate Mature Astrocytes via Binding with the Leukemia Inhibitory Factor Receptor Christelle Monville,* Muriel Coulpier, † Luciano Conti, ‡ Claudio De-Fraja, ‡ Patrick Dreyfus,* Christiane Fages,* Danielle Riche,* Marcienne Tardy,* Elena Cattaneo, ‡ and Marc Peschanski* ,1 *INSERM U421, IM3, Faculte´ de Me´decine, 94010 Cre´teil cedex, France; †Regeneron Pharmaceuticals, Inc., Tarrytown, New York 10591; and ‡Institute of Pharmacological Sciences, 20133 Milan, Italy
Ciliary neurotrophic factor (CNTF) acts on immature astrocytes that express its trimeric receptor. In contrast, mature astrocytes do not significantly express the specific CNTF␣ receptor subunit, yet they respond to CNTF administration in vivo. Here we show that this controversy may be solved by a shift in astroglial sensitivity to CNTF over time, related to a change in the type of receptor bound by the cytokine on mature astrocytes. A convergent set of results supports the hypothesis that the CNTF effect is due to the illegitimate binding on the leukemia inhibitory factor receptor (LIFR): (i) it requires high concentration of recombinant rat CNTF; (ii) it involves the Jak/Stat and Ras-MAPK pathways; (iii) it is preserved in CNTFR␣ⴚ/ⴚ cells; (iv) it is potentiated by soluble CNTFR␣ added to the medium; and (v) it is significantly decreased by a partial antagonist of LIFR. On these bases, we propose a mechanistic model in which, in the adult brain, a CNTF/LIFR interglial system may be modulated by neurons that synthesize CNTFR␣.
INTRODUCTION Synthesis of the ciliary neurotrophic factor (CNTF) and of all three components of its trimeric receptor is widespread in the adult central nervous system (Squinto et al., 1990; Sto¨ckli et al., 1991; Sendtner et al., 1994; MacLennan et al., 1996). Its neuroprotective ef1 To whom correspondence and reprint requests should be addressed at INSERM U421, IM3, Faculte´ de me´decine, 8 rue du Ge´ne´ral Sarrail, 94010 Cre´teil cedex, France. Fax: 33 1 49 81 37 09. E-mail: [email protected].
1044-7431/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
fects, together with the source of the factor in astrocytes, the location of its specific CNTFR␣ receptor subunit specifically on neurons in vivo, and its lack of a signal peptide, have led to the hypothesis that CNTF may act as an “injury molecule” (Thoenen, 1991; Sendtner et al., 1997), protecting neurons after release from astrocytes following injury. Recent demonstration of phenotypic changes in astrocytes after administration of CNTF into the adult brain (Winter et al., 1995; Levison et al., 1996; Clatterbuck et al., 1996; Lisovoski et al., 1997) has suggested that it may also play a role in inducing astroglial activation. The CNTF triggers astroglial differentiation very efficiently in glial precursors and immature astrocytes (Hughes et al., 1988; Lillien et al., 1988; Kahn et al., 1995; Bonni et al., 1997). This effect is likely related to the binding of the cytokine to its receptor, the three components of which are expressed by these precursors and immature cells (Bonni et al., 1997). In contrast, effects of CNTF on mature astrocytes have remained controversial. Astroglial activation in long-term cultures treated with CNTF has indeed been altogether weak or nonexisting (Meyer and Unsicker, 1994; Smith et al., 1996). In addition, despite the observation of CNTF effects in vivo, astrocytes in the adult brain have not been shown to express the specific ␣ subunit of the trimeric CNTF receptor (Squinto et al., 1990; MacLennan et al., 1996; Kordower et al., 1997), the association of which to the dimeric receptor of the leukemia inhibitory factor (LIF) is necessary to form the trimeric CNTF receptor (Ip et al., 1993; Davis et al., 1993b). Paradoxically, this expres-
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374 sion is observed by Western and Northern blotting in long-term astrocytic cultures in which the effects of CNTF are not solid (Rudge et al., 1994; Alderson et al., 1999). These data have led to different hypotheses, including the expression of an undetectable, though sufficient, amount of CNTFR␣ in adult astrocytes (Levison et al., 1996) or the need for an interaction with another cell type to obtain the CNTF-elicited astroglial activation (Kahn et al., 1997). The present study has explored an alternative hypothesis that these apparently discrepant data may reveal a shift in astroglial sensitivity to CNTF over time, related to a change in the type of receptor bound by the cytokine. This hypothesis was based upon studies that have demonstrated that CNTF may act on cells lacking the CNTFR␣ subunit via illegitimate binding with the dimeric LIF receptor, although at a much higher concentration (Schooltink et al., 1992; Gearing et al., 1994). The present results show that CNTF-induced astroglial activation can indeed be observed in longterm astroglial cultures and that the biological characteristics of this effect are compatible with the illegitimate binding of CNTF with the dimeric LIF receptor.
RESULTS Enriched astroglial cell cultures were readily obtained from cerebral hemispheres of Swiss mice (Iffa Credo, France), CNTF⫺/⫺ mice (BRL, Basel Switzerland), or CNTFR␣⫺/⫺ (Regeneron, Tarrytown, NY), and maintained for several weeks without signs of cell alteration. CNTF Effect on Long-Term Astrocytes: Dose Dependence and Intracellular Signaling Pathways In immature astrocytes (maintained 7 days in culture, not at confluence), addition of recombinant rat CNTF (rCNTF) to the medium at concentrations as low as 10 ng/ml (lowest concentration used) provoked an increase in the cellular content of glial fibrillary acid protein (GFAP) (Fig. 1a). Western blotting showed no endogenous CNTF in these short-term cultures either in the absence of treatment or 3 days after addition of rCNTF to the medium. In sharp contrast, rCNTF was inefficient at concentrations as high as 100 ng/ml in long-term cultures when astrocytes were maintained in culture before treatment until confluence was reached (around 14 days). An increase in the cellular content of GFAP was observed in a significant and reproducible fashion only
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when the highest concentration of rCNTF studied (250 ng/ml) was used (Fig. 1b). Similarly, cellular content of endogenous CNTF, which was quantifiable in these more mature astrocytes, was increased significantly above control levels only when 250 ng/ml of rCNTF was added to the medium (Fig. 1c) and not following treatment with lower doses of the cytokine. To eliminate an artifactual synergistic effect of the serum component TGF, a pan-TGF antibody was added to the culture medium. This treatment had no effect on the increase of CNTF intracellular content after addition of 250 ng/ml of rCNTF to the medium. A quantification of rCNTF, which would have been internalized by cultured cells, was excluded by measuring no CNTF in samples obtained from CNTF⫺/⫺ mice after treatment of mature astrocytes with 250 ng/ml of rCNTF. CNTF cellular content was then used as a marker of astroglial activation in following experiments, rather than GFAP, because this molecule is present in astroglial cultures only after 14 days, not at 7, and therefore may be a discriminant marker of mature astrocytes. Consequently, using CNTF as a marker allowed us to overcome the potential problem created by the persistence of a small subpopulation of astroglial precursors or immature cells—which express GFAP but not CNTF—in long-term cultures (see below). The two intracellular signaling systems studied, namely the Jak/ STAT and the Ras-MAPkinase pathways, exhibited rapid and sustained activation when long-term astroglial cultures were treated with 250 ng/ml of rCNTF. Stat3 (Figs. 2a and 2b) and Stat1 (Figs. 2c and 2d) were indeed strongly phosphorylated as soon as 5 min after treatment and up to 1 h. In parallel, the adapter protein Shc, ERK1, and ERK2, which are part of the Ras-MAPK pathway associated with the CNTF receptor, were activated as soon as 5 min after rCNTF application (Figs. 3a and 3b). For these elements of the Ras-MAPK pathway, the activation was still significant 240 min after application. In contrast, application of 30 ng/ml of rCNTF either was inefficient or induced only limited phosphorylation. Dependence of the Effects of CNTF on the Presence or Concentration of CNTFR␣ The requirement for the presence of CNTFR␣ in the astroglial effects of rCNTF at high concentration was assessed by adding the cytokine to the medium of cultures of mature astrocytes obtained from CNTFR␣ knock-out mice. In these cultures, addition of 250 ng/ml of rCNTF induced an increase in CNTF cellular content similar to that seen in wild-type cells (Fig. 4a), demonstrating that
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375 the specific ␣ receptor subunit was not required in the effects. The effect of providing CNTFR␣ to wild-type mature astrocytes was then assessed using the soluble compound myc-sCNTFR␣. Under these conditions, a potentiation of the effects was observed as a major shift toward values lower than the dose required for rCNTF effects on mature astrocytes. Increase of CNTF cellular content was indeed observed with the lowest concentration studied (10 ng/ml) and maintained at higher concentrations (Fig. 4b). All three CNTF receptor subunits, including CNTFR␣, were present in the long-term astroglial cultures (Fig. 6b). Immunocytochemical staining, however, demonstrated that CNTFR␣ was not evenly distributed in long-term cultures of astrocytes but was instead present only on a small subpopulation of small bipolar cells. The vast majority of cells which were large and displayed the typical flattened morphology of longterm cultured astrocytes were not decorated by antibodies against CNTFR␣ (Fig. 5). In contrast, these latter cells, which were the only ones to contain CNTF, expressed the two other subunits of the CNTF receptor, LIFR and gp130 (Fig. 5). To check that an indirect mechanism was not involved in the CNTF effects on mature astrocytes in which CNTFR␣ present on the small cells would be donated to the mature astrocytes, the concentration of CNTFR␣ was modified in two different experiments. A mechanical lesion was made in the cultures to provoke an increase in the concentration of CNTFR␣ (Fig. 6b). The lesion provoked an increase in CNTF cellular content at baseline but did not amplify the effects of rCNTF (Fig. 6a). Reciprocally, a phosphatidylinositol-specific phospholipase C (PIPLC) was used to cleave the glycosylphosphatidylinositol anchor that links CNTFR␣ to the cell membrane, and the medium was changed before the application of rCNTF to eliminate as much of the ␣ receptor subunit as possible. The PIPLC treatment, the biological activity of
FIG. 1. (a) Effect of rCNTF on GFAP cellular content in immature astrocytes. rCNTF provoked an increase of 44 ⫾ 9% (factorial ANOVA significant at 95%, t test ***P ⬍ 0.001, **P ⬍ 0.01, or *P ⬍ 0.05) at the lowest concentration used (10 ng/ml), compared to untreated cultures. This effect was maintained, without further in-
crease, at all other concentrations. (b) Effect of rCNTF on GFAP cellular content in mature astrocytes. Addition of 250 ng/ml rCNTF induced a statistically significant increase compared to untreated controls (⫹23 ⫾ 5.6%, factorial ANOVA significant at 95%, t test *P ⬍ 0.05). In contrast, application of rCNTF provoked a statistically significant decrease, compared to untreated controls, for 30 and 50 ng/ml (⫺24 ⫾ 8.5 and ⫺16 ⫾ 1.7%, respectively, factorial ANOVA significant at 95%, t test *P ⬍ 0.05). (c) Effect of rCNTF on CNTF cellular content in mature astrocytes. No change in CNTF cellular content was observed when rCNTF was added at 10, 30, 50, and 100 ng/ml concentrations. In contrast, a significant increase was consistently measured at 250 ng/ml (⫹39.6 ⫾ 10%, factorial ANOVA significant at 95%, t test **P ⬍ 0.01).
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FIG. 2. (a, b) Activation of the Jak/Stat pathway by rCNTF in mature astrocytes. Stat3 was phosphorylated with 30 ng/ml (⫹700 ⫾ 250% of control, factorial ANOVA significant at 95%, t test *P ⬍ 0.05), and more strongly with 250 ng/ml (⫹3400 ⫾ 1800% of control, factorial ANOVA significant at 95%, t test *P ⬍ 0.05) after 5 and 30 min (⫹297 ⫾ 86 and ⫹615 ⫾ 170% of control, factorial ANOVA significant at 95%, t test P ⬍ 0.01 in both cases). This activation was not observed after 60 min. (c, d) Stat1 was phosphorylated with 30 ng/ml (⫹140 ⫾ 90%, NS) and 250 ng/ml (⫹409 ⫾ 160% of control, factorial ANOVA significant at 95%, t test *P ⬍ 0.05) after 5 and 30 min (⫹192 ⫾ 100% with 30 ng/ml and ⫹321 ⫾ 170% with 250 ng/ml).
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a molecule that was shown to partially block the activation of VIP by CNTF (Vernallis et al., 1997), was added to the culture medium together with 250 ng/ml of rCNTF. Under these conditions, a significant decrease (of about 50%) in the effects of rCNTF on astrocytes was observed (Fig. 7).
DISCUSSION The main result of this study is that CNTF can provoke biochemical changes in mature astrocytes in vitro, as previously observed in immature glial cells. The receptor system is not, however, the complete tripartite CNTF receptor since it does not involve the specific ␣ subunit. A convergent body of facts suggests that CNTF may act on mature astrocytes by binding illegitimately to the dimeric receptor of the LIF. This demonstrates a developmental shift in astrocytes, since CNTF binds to its own full trimeric receptor system on immature astrocytes and glial precursors. In the mature brain, CNTF is synthesized by astrocytes and its specific CNTFR␣ subunit is synthesized—and potentially released in the extracellular space via cleavage of its GPI anchor— by neurons. Taking into account the present data, we propose a mechanistic model in which neurons may consequently play a role in the activation of mature astrocytes by modulating the efficacy of a CNTF/LIFR interglial signaling system. CNTF May Effect Biochemical Differentiation of Mature Astrocytes via Binding to the LIF Receptor
FIG. 3. (a, b) Activation of the Shc-Grb2-MAPKs pathway by rCNTF in mature astrocytes. ERK1 (p42MAPK) and mainly ERK2 (p44MAPK) were strongly activated with 250 ng/ml after 5 and 30 min (⫹470 ⫾ 60 and ⫹2133 ⫾ 490% of control, factorial ANOVA significant at 95%, t test P ⬍ 0.001 in both cases).
which was controlled by observing an increase in cholinesterase activity in the medium, did not alter the effects of rCNTF on mature astrocytes (Fig. 6a). Dependence of the Effects of CNTF on the Activity of the Dimeric LIF Receptor To test the hypothesis that CNTF effects on astrocytes require the activity of the dimeric LIF receptor, hLIF05,
CNTF provokes astroglial differentiation of glial precursors and strongly activates the synthesis of GFAP in immature astrocytes, in vivo as well as in vitro (Hughes et al., 1988; Lillien et al., 1988; Kahn et al., 1995, 1997; Bonni et al., 1997; Rajan and McKay, 1998). In contrast, an effect of CNTF on the synthesis of GFAP or endogenous CNTF has been observed in the adult only when the cytokine was administered into the brain of living rodents (Winter et al., 1995; Levison et al., 1996; Clatterbuck et al., 1996; Lisovoski et al., 1997) and not in purified astroglial cultures (Meyer and Unsicker, 1994; Smith et al., 1996). These paradoxical results had led to the hypothesis that activation of mature astrocytes by CNTF was indirect and followed an effect of the cytokine on neighboring cells in vivo (Meyer and Unsicker, 1994; Smith et al., 1996). The present results do not support this hypothesis since an activation of GFAP and endogenous CNTF synthesis was obtained in longterm purified astroglial cultures. In a previous report, in
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FIG. 4. (a) Effect of rCNTF on astrocytes obtained from CNTFR␣⫺/⫺ mice. Addition of 250 ng/ml induced an increase of CNTF cellular content in both wild-type and CNTFR␣-deficient mice (⫹85 ⫾ 21 and ⫹143 ⫾ 47%, respectively, factorial ANOVA significant at 95%, t test 䡠䡠P ⬍ 0.01 and *P ⬍ 0.05). (b) Addition of soluble CNTFR␣ provoked a major shift in the concentration of rCNTF required to observe glial effects. Addition of sCNTFR␣ at 200 ng/ml to the culture medium was followed by a major increase of CNTF cellular content with 10 ng/ml (⫹66.5 ⫾ 30%, factorial ANOVA significant at 95%, t test *P ⬍ 0.05). A similar increase was maintained, whatever the concentration of rCNTF used, between 30 and 250 ng/ml.
which astroglial activation was observed in the brain of adult rats following intracerebral injection of adenoviral vectors recombinant for a secretable form of CNTF
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(Lisovoski et al., 1997), the very restricted areas in which this activation was observed around sites of potential delivery of the cytokine had led us to hypothesize either that CNTF did not spread freely far away from these sites or that a high concentration of the cytokine was required. The present in vitro results strongly support the latter hypothesis since, in sharp contrast to the results obtained in short-term cultures (of immature astrocytes), there was no effect of CNTF observed at concentrations lower than 10 ⫺8 M. This dose requirement explains the failure of previous in vitro studies to observe the effects (Meyer and Unsicker, 1994; Smith et al., 1996). The existence of an effect of CNTF on mature astrocytes is paradoxical because all studies of the sites of synthesis of the specific ␣ subunit of the CNTF receptor, through visualization of mRNA (Ip et al., 1993) or protein (MacLennan, 1996; Kordower et al., 1997), have failed to reveal an astrocytic localization in the adult brain. CNTFR␣ is, essentially, if not exclusively, synthesized by neurons in the central nervous system. To explain the effects of CNTF on astrocytes, authors have hypothesized a low, undetectable level of CNTFR␣ expression (Levison et al., 1996) or the activation of its expression in response to inflammatory mechanisms (Rudge et al., 1994, 1995). The present results support an alternative hypothesis that CNTF may bind illegitimately to the dimeric LIF receptor. CNTF normally binds to a high-affinity receptor complex, which is composed of three subunits, CNTFR␣ and the two  subunits (LIFR and gp130), which together form the receptor of another cytokine, the leukemia inhibitory factor (Squinto et al., 1990; Davis et al., 1993b; Ip et al., 1993; Stahl et al., 1994). It has been shown, however, that CNTF can provoke biological effects on cells that express only the LIF receptor (Schooltink et al., 1992; Davis et al., 1993b; Gearing et al., 1993). The arguments in favor of the existence of such a mechanism in the present experiments with mature astrocytes are numerous: the astrocytic activation was observed at the same concentration of the cytokine when cells were taken from mice that do not express CNTFR␣ or from wild-type mice, indicating that this specific subunit is not required; although CNTFR␣ is present in long-term astroglial cultures (Rudge et al., 1994; Alderson et al., 1999), immunohistochemical analysis indicated that it was not expressed by mature astrocytes, and alteration of its concentration did not modify the conditions of the astroglial effects of CNTF; the binding of CNTF to the glial receptor provoked the phosphorylation of molecules that belong to the two intracellu-
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FIG. 5. Immunocytochemical staining showing that the CNTFR␣ subunit is only present in a subpopulation of smaller bipolar cells. The two  components of the LIF receptor (LIFR and gp130) as well as CNTF are, in contrast, present in large cells with a flat polygonal shape and several processes. Scale bar: 10 m.
lar signaling pathways linked to the LIF (and by extension the CNTF) receptor, namely the Jak/Stat and the (Shc-)Ras-MAPK pathways (see references and discussion in Stahl and Yancopoulos, 1994; Stahl et al., 1994; Boulton et al., 1994; Bonni et al., 1997; Rajan and McKay, 1998); these effects were characterized by the need for a much lower concentration of the cytokine when CNTFR␣ was added in a soluble form to the medium (at least 100-fold), a result comparable to those previously described for cells treated with CNTF that expressed only the LIF receptor
(Davis et al., 1993b; Panayotatos et al., 1994); and partial blockade of the binding of CNTF to the LIFR using a specific antagonist (hLIF05) significantly reduced the effects of CNTF on astrocytes. Altogether, the shift in response to CNTF observed during the maturation of astrocytes may be explained by the loss of expression of the CNTFR␣ subunit, and therefore of the high-affinity receptor present at early stages, and the consequent use of the two  subunits of the LIF receptor that form a low-affinity receptor for CNTF.
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FIG. 6. (a) Modulation of CNTFR␣ availability in astroglial cultures. A lesion, which increased the level of CNTFR␣, did not amplify the effects of rCNTF on CNTF cellular content (⫹72 ⫾ 10.8% of control, factorial ANOVA significant at 95%, t test **P ⬍ 0.01). PIPLC treatment in order to remove CNTFR␣ anchored at the cell membranes of the small bipolar cells did not alter the effects of rCNTF on the mature astrocytes (⫹97 ⫾ 21.4% vs ⫹84 ⫾ 47% of control, ANOVA significant at 95%, t test 䡠䡠䡠P ⬍ 0.001). (b) Western blot demonstrating the presence of the three components of the trimeric CNTF receptor (LIFR, 200K mol wt; gp130, 130K mol wt; CNTFR␣, 80K mol wt) in mature astroglial cultures from wild-type mice. NL, nonlesioned; L, lesioned.
An Interglial Signaling System Which Is Potentially under Neuronal Control in the Adult Brain The biological significance of such a low-affinity receptor system for CNTF on mature astrocytes is but a matter of speculation. It is intriguing, however, that
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astrocytes are the only known source of CNTF in the adult brain (Sto¨ckli et al., 1991; Sendtner et al., 1994). Release of large amounts of CNTF in the extracellular space will therefore affect astrocytes via a paracrine/ autocrine system in the same time it affects neighboring neurons that express CNTFR␣ (Ip et al., 1993; MacLennan, 1996; Kordower et al., 1997). Following injury to the brain parenchyma, release of CNTF by dying astrocytes will thus provoke activation of surviving astrocytes in the close vicinity of the lesion, as well as exert a neuroprotective role as hypothesized by several authors (Thoenen, 1991; Sendtner et al., 1997). The strong potentiation of the CNTF effects on astrocytes by soluble CNTFR␣ suggests the possibility of an additional type of signaling system. CNTFR␣ is synthesized by neurons but it is linked to their membrane by a GPI anchor (Davis et al., 1991), which can be cleaved even in the intact brain since it is detected in a soluble form in the cerebrospinal fluid (Davis et al., 1993a). The presence of CNTFR␣ in a soluble form in the brain will transform the low-affinity astroglial receptor system into a high-affinity one and, as a consequence, will allow much smaller amounts of CNTF to provoke biochemical changes in astrocytes. Our data therefore are compatible with the hypothesis of a three-partner system in which astroglial release of CNTF would affect neighboring astrocytes in a manner which would strongly depend upon neuronal release of CNTFR␣ (Fig. 8). A CNTF-based interglial signal could thus be modulated by neurons in the absence of a lesion provoking the release of massive amounts of the cytokine. The physiological conditions under which such a system may operate are, however, not known and their very existence may even be questioned. On the one hand, and although release of GPI-anchored CNTFR␣ has been demonstrated for skeletal muscle following denervation, physiological conditions under which neurons may modulate a similar release are not known. On the other hand, the issue of a potential physiological release of CNTF by astrocytes (i.e., not following injury) remains unsolved. CNTF lacks a signal peptide, indicating that it is not released via conventional exocytotic mechanisms (Thoenen, 1991; Sendtner et al., 1994). It could, however, be released via a nonconventional energy-dependent pathway (Kamiguchi et al., 1995) reminiscent of those demonstrated for the 18-kDa isoform of bFGF and IL-1 (Rubartelli et al., 1990; Florkiewicz et al., 1995). This has not been observed, however, in astroglial cultures, the conditioned medium of which does not support the survival of ciliary ganglion cells (Rudge et al., 1992) and does not provoke phosphorylation of appropriate intracellular signaling proteins
CNTF May Act on Astrocytes via the LIF Receptor
381
FIG. 7. Effect of partial blockade of the LIFR on the effects of rCNTF on mature astrocytes. Addition of the partial LIFR antagonist hLIF05 to the culture medium provoked a 50% reduction of the increase of the intracellular CNTF levels provoked by the addition of 250 ng/ml of rCNTF in mature astrocytes (ANOVA significant at 95%, t test **P ⬍ 0.01).
(Rudge et al., 1995). The answer to that question, of a potential physiological release of CNTF by astrocytes in the mature brain, will determine whether the interglial signaling system defined in the present study is just another injury mechanism or whether it operates, under the control of neurons releasing CNTFR␣, under currently unknown physiological conditions.
EXPERIMENTAL METHODS Astroglial cultures were prepared from cerebral hemispheres of neonatal Swiss mice (Iffa Credo, France), CNTF knockout mice (BRL, Switzerland), and neonatal CNTFR␣ knockout mice (Regeneron Pharmaceutical Inc., U.S.A.). Cultures were either grown at confluence for 14 days, thus defining “mature” cultures, or maintained only for 7 days. In the latter case, identified as “immature” cultures, cells occupied less than 50% of the dish. Culture Conditions Astroglial cultures were prepared as previously described (Bardakdjian et al., 1979). After 7 or 14 days in vitro, the medium (minimal essential medium containing 2 mM glutamine, essential amino acids, 0.03% glucose, penicillin–streptomycin) was removed and replaced with the same medium but without fetal calf serum (FCS). Recombinant rat CNTF (Boehringer
Mannheim, Germany) (rCNTF) was then added to the medium at a concentration of 0, 10 (4.4 ⫻ 10 ⫺10 M), 30 (1.3 ⫻ 10 ⫺9 M), 50 (2.2 ⫻ 10 ⫺9 M), 100 (4.4 ⫻ 10 ⫺9 M), or 250 ng/ml (10 ⫺8 M). The cells were collected 3 days later. In preliminary studies, it was confirmed (Levison et al., 1996) using tritiated thymidine that CNTF does not provoke astroglial proliferation in in vitro cultures. Some of the mature astroglial cultures, prepared as described above, were mechanically lesioned using the fine tip of a plastic pipette 18 days after astroglial plating. After lesion, the medium was removed and cultures were treated with 250 ng/ml of rCNTF or left untreated. In other experiments, PIPLC (Boehringer) was added at the concentration of 1 U/ml and the medium was exchanged 24 h later. In half of the dishes, rCNTF was then applied as described above. PIPLC activity was checked by quantifying the acetylcholine esterase activity in the supernatant, according to the technique described by Ellman et al. (1964). In the experiments with soluble CNTFR␣, 200 ng/ml of mycsCNTFR␣ (kindly provided by Ralph Laufer, IRBM, Italy) was added to the medium without FCS after 14 days in vitro. Thirty minutes later, rCNTF was added at 0, 10, 30, 50, 100, and 250 ng/ml. The cells were collected 3 days later. To block a potential binding of CNTF on the dimeric LIFR, hLIF05 (kindly provided by Dr. Ann Vernallis, UK) was used. This compound is a specific antagonist of the LIF receptor that has been shown in other cell models to partially block the activation of the VIP gene
382
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FIG. 8. CNTF/LIF receptor as an interglial signaling system under neuronal control. This scheme presents a hypothesis in which the CNTF-based glial signaling system is open to a neuronal partner which can strongly amplify the binding efficiency of the ligand by providing the CNTFR␣ subunit and thus restore a high-affinity receptor system.
by CNTF (50% inhibition with 5 g/ml of hLIF05; Vernallis et al., 1997). hLIF05 was added 12 h before rCNTF (250 ng/ml), and the cells were collected 24 h after rCNTF treatment. In all cases, the medium was removed at the end of the experiments and each dish was rinsed three times with HBSS (Hanks’ balanced salt solution, Seromed, Germany). The cells were collected by scraping into 62.5 mM Tris–HCl (pH 6.8), 2 mM EDTA, 2 mM phenylmethylsulfonyl fluoride, 0.5% Triton X-100, and 2.3% sodium dodecyl sulfate. Biochemical Analysis Total protein content was determined by the method of Lowry et al. (1951) with bovine serum albumin as a standard. The proteins were analyzed by Western blotting under the conditions described by Laemmli (1970). Briefly, samples were boiled for 5 min after addition of 10% glycerol, 5% mercaptoethanol (or 5% dithiothreitol for the MAPK-P), and 5% bromophenol blue, and then
lysates were electrophoresed on 7.5% (LIFR, gp130, CNTFR␣, Stat3-P, and Stat1-P), 10% (GFAP and MAPKP), or 12% (CNTF) SDS–polyacrylamide gels. Gels were blotted on nitrocellulose, blocked for 1 h in 5% nonfat dry milk in TBS-T (20 mM Tris, pH 7.5/500 mM NaCl/ 0.1% Tween 20), and then probed overnight at 4°C with the following antibodies: polyclonal anti-CNTF (R&D Systems, UK, 1/500), polyclonal anti-CNTFR␣ (kindly provided by Dr. A. J. MacLennan, University of Florida, U.S.A., 1/1000), monoclonal anti-gp130 (Santa Cruz, U.S.A., 1/1000), polyclonal anti-LIFR (Santa Cruz, 1/500), monoclonal anti-p44/p42 MAP kinase (or antiphospho ERK1/ERK2, New England Biolabs, UK, 1/1000), polyclonal anti-ERK1 (Santa Cruz, 1/4000), polyclonal anti-phospho-specific Stat3 (Tyr705) (New England Biolabs, 1/1000), polyclonal anti-phospho-specific Stat1 (Tyr701) (New England Biolabs, 1/1000), monoclonal anti-Stat3 (Santa Cruz, 1/1000), and polyclonal anti-Stat1 (Santa Cruz, 1/1000). After washing with TBS-T, membranes were incubated with a horseradish peroxidase-conjugated donkey anti-rabbit sec-
CNTF May Act on Astrocytes via the LIF Receptor
ondary antibody (for CNTFR␣, LIFR, ERK1, phosphospecific Stat3, phospho-specific stat1, and stat1), a mouse anti-goat antibody (for CNTF), or a sheep antimouse antibody (for gp130, p44/p42 MAP kinase, and Stat3), followed by the enhanced chemiluminescent reaction (Amersham, Sweden), according to the manufacturer’s instructions. For GFAP, the sheets were incubated with a polyclonal anti-GFAP antibody (Dakopatts, Denmark, 1/100) and revealed with antirabbit IgG coupled to 125I, according to a previously described technique (Andres-Barquin et al., 1994). The sheets were placed in contact with a RPN6 film (Amersham). The levels of CNTF and GFAP were measured by densitometry and brought back to the total protein laid down. To limit variations in their processing, extracts from the control and all experimental conditions were treated in parallel on a single sheet for each specific experiment. In addition, to evaluate the variability between specific experiments, control extracts were subsequently reloaded on a single sheet and processed together. Statistical analysis used one-factor ANOVA and unpaired t test. Immunohistochemical Analysis The medium was removed and cells were rinsed two times with PBS. They were then fixed with cold methanol (⫺20°C) and probed 1 h at room temperature with the following antibodies: polyclonal anti-CNTF (1/100), polyclonal anti-CNTFR␣ (1/50), monoclonal anti-gp130 (1/100), and polyclonal anti-LIFR (1/100). After washing with PBS, cells were exposed to secondary antibodies linked to TRITC.
ACKNOWLEDGMENTS These studies were supported by INSERM and Association Franc¸aise contre les Myopathies. The authors gratefully acknowledge help from Ralph Laufer (IRBM P. Angeletti, Rome), John McLennan (University of Florida College of Medicine, Gainesville, FL), Austin Smith (Center for Genome Research, Edimburg, UK), Ann Vernallis (School of Health and Life Sciences, Aston, UK), Tom De Chiara and George Yancopoulos (Regeneron, Tarrytown, NY), Sharon L. Juliano (USUHS, Bethesda, MD), Philippe Hantraye (CNRS, Orsay, Italy), and Brigitte Onte´niente (INSERM, Cre´teil, Italy).
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