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Previous Article | Next Article
The Journal of Neuroscience, June 15, 2000, 20(12):4635-4645
Axonal Regulation of Schwann Cell Proliferation and Survival and the Initial Events of Myelination Requires PI 3-Kinase Activity
Patrice Maurel1 and James L. Salzer1, 2, 3 Departments of 1 Cell Biology, 2 Neurology, and the 3 Kaplan Cancer Center, New York University Medical Center, New York, New York 10016
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
In this report, we have investigated the signaling pathways that are activated by, and mediate the effects of, the neuregulins and axonal contact in Schwann cells. Phosphatidylinositol 3-kinase (PI 3-kinase) and mitogen-activated protein kinase kinase (MAPK kinase) are strongly activated in Schwann cells by glial growth factor (GGF), a soluble neuregulin, and by contact with neurite membranes; both kinase activities are also detected in Schwann cell-DRG neuron cocultures. Inhibition of the PI 3-kinase, but not the MAP kinase, pathway reversibly inhibited Schwann cell proliferation induced by GGF and neurites. Cultured Schwann cells undergo apoptosis after serum deprivation and can be rescued by GGF or contact with neurites; these survival effects were also blocked by inhibition of PI 3-kinase. Finally, we have examined the role of these signaling pathways in Schwann cell differentiation in cocultures. At early stages of coculture, inhibition of PI 3-kinase, but not MAPK kinase, blocked Schwann cell elongation and subsequent myelination but did not affect laminin deposition. Later, after Schwann cells established a one-to-one relationship with axons, inhibition of PI 3-kinase did not block myelin formation, but the myelin sheaths that formed were shorter, and the rate of myelin protein accumulation was markedly decreased. PI 3-kinase inhibition had no observable effect on the maintenance of myelin sheaths in mature myelinated cocultures. These results indicate that activation of PI 3-kinase by axonal factors, including the neuregulins, promotes Schwann cell proliferation and survival and implicate PI 3-kinase in the early events of myelination.
Key words: Schwann cell; PI 3-kinase; MAP kinase; proliferation; survival; myelination; signaling pathways
INTRODUCTION
The development of myelinated nerve fibers in the peripheral nervous system depends on complex interactions between Schwann cells and axons. Axon-associated factors promote the generation of Schwann cells during development via mitogenic (Wood and Bunge, 1975
; Salzer et al., 1980a
The neuregulin family of growth factors plays a crucial role during the early stages of Schwann cell development (Lemke, 1996
Little is known about the signaling pathways by which the neuregulins mediate these diverse biological effects on Schwann cells. Neuregulins bind to receptor tyrosine kinases of the erbB family. After ligand binding, the erbBs undergo dimerization and phosphorylation, thereby recruiting Src homology 2 domain-containing signal-transducing proteins (Burden and Yarden, 1997
We now report that GGF and axonal contact result in a robust activation of PI 3-kinase and that this activation is required for the mitogenic and survival activities of GGF and axons on Schwann cells. MAP kinase kinase (MAPK kinase) is also robustly activated and contributes to the survival mediated by GGF, but it is not required for axon-mediated survival. In a Schwann cell-DRG neuron coculture system, inhibition of PI 3-kinase, but not that of MAPK kinase, also blocks myelination, possibly by blocking initial Schwann cell-axon associations. These results indicate that activation of PI 3-kinase is crucial for the contact-dependent regulation of Schwann cell development by axons.
Parts of this paper have previously appeared in abstract form (Maurel and Salzer, 1999
MATERIALS AND METHODS
Inhibitors, antibodies, and cell culture materials. The PI 3-kinase inhibitor 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one (LY294002; Sigma,St. Louis, MO) and the MAPK kinase inhibitor U0126 (Promega, Madison, WI ) were prepared in DMSO (Sigma) at stock concentrations of 20 and 10 mM, respectively. Dilutions were made directly in the appropriate culture media, keeping DMSO at a constant final concentration of 0.2% (final LY294002 concentration of 2.5-20 µM; final U0126 concentration of 1-10 µM). Monoclonal antibodies included the fluorescein-conjugated anti-bromodeoxyuridine (BrdU) antibody (Boehringer Mannheim, Indianapolis, IN), the anti-myelin basic protein (MBP) antibody SMI 94, and the anti-neurofilament antibodies SMI 31 and SMI 32 (Sternberger Monoclonals, Lutherville, MD). Rabbit polyclonal antibodies included the anti-Akt, anti-phospho-Akt, anti-MAPK, and anti-phospho-MAPK from New England Biolabs (Beverly, MA), the anti-MBP antibody 644 and an anti-protein zero (P0) antibody (gifts of D. Colman, Mount Sinai Medical Center, New York, NY), and the anti-laminin antibody L-9393 (Sigma). Secondary antibodies were rhodamine-conjugated donkey anti-rabbit IgG, fluorescein-conjugated donkey anti-mouse IgG (Chemicon, Temecula, CA), coumarin-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA), 125I-anti-rabbit IgG (ICN Biomedicals, Costa Mesa, CA), and horseradish peroxidase-conjugated goat anti-rabbit IgG (Pharmacia, Piscataway, NJ). Materials used for cell cultures included DMEM (BioWhittaker, Walkersville, MD), MEM, DME/Ham's F12, L15, and L-glutamine (Life Technologies, Gaithersburg, MD), FBS (HyClone, Logan, UT), collagen (Biomedical Technologies, Stoughton, MA), poly-L-lysine, glucose, 5-fluorodeoxyuridine, uridine, insulin, progesterone, putrescine, selenium, ascorbic acid (Sigma), transferrin (Jackson ImmunoResearch), nerve growth factor (NGF; Harlan Bioproducts for Science, Indianapolis, IN), recombinant human GGF (rhGGF2; gift of Cambridge NeuroScience, Cambridge, MA), and FGF-2 (gift of D. Rifkin, New York University Medical Center, New York, NY).
Cell cultures. Cultures of primary rat Schwann cells and DRG neurons were established as described previously (Einheber et al., 1993
Neurite membrane preparation. Procedures for isolating neurite membranes were modified from previously described methods (Salzer et al., 1980b
Proliferation assays. To investigate the effect of PI 3-kinase and MAPK kinase inhibitors on the proliferation of Schwann cells mediated by soluble growth factors, we plated 50,000 cells in D media onto poly-L-lysine-coated glass coverslips. After 24 hr, forskolin (4 µM) was added to the cultures. The next day, the cultures were treated for 24 hr with either LY294002 or U0126 in fresh D media supplemented with forskolin and GGF (5 ng/ml) or FGF-2 (10 ng/ml). Control cultures were maintained in D media with or without forskolin.
Two approaches were used to investigate the effect of LY294002 and U0126 on the proliferation of Schwann cells induced by contact with neurites. We first examined Schwann cells proliferating in cocultures with DRG neurons. For these studies, 10,000 Schwann cells in C media were seeded onto dissociated DRG neurons. After 24 hr, cultures were fed fresh C media with (treated) or without (control) kinase inhibitors for an additional 24 hr. In other studies, two aliquots of neurite membranes, each equivalent to one-third of a 35 mm DRG neuron culture dish, were centrifuged (200 × g; 10 min; 4°C) at a 24 hr interval on 50,000 Schwann cells cultured in D media alone or supplemented either with LY294002 or U0126. Cells were processed for BrdU immunostaining after an additional 24 hr in culture.
Schwann cell proliferation was assessed using a BrdU nuclear-labeling assay. In all conditions, BrdU was added during the last 4 hr of culture at a final concentration of 20 µM; immunostaining was performed according to the manufacturer's instructions (Boehringer Mannheim). All cultures were mounted on glass slides in Citifluor (Ted Pella, Redding, CA) containing Hoechst at 2 µg/ml and examined by epifluorescence microscopy (Axiophot microscope; Carl Zeiss, Thornwood, NY). BrdU- and Hoechst-labeled nuclei from five to seven random high-power fields per culture were counted. Four to six cultures per condition in two to three separate experiments were analyzed in each study. Typically over 3000 cells were counted per condition. The rate of proliferation of the Schwann cells was determined as the ratio of BrdU- to Hoechst-labeled nuclei (BrdU index).
Survival assays. To investigate the effect of PI 3-kinase and MAPK kinase inhibitors on the survival of Schwann cells mediated by GGF and the neurite membrane preparation, we cultured 30,000 cells in D media for 24 hr. Cells were then washed five times with DMEM and maintained in serum-free media (DMEM and 2 mM L-glutamine) alone or supplemented with forskolin (4 µM) and GGF (50 ng/ml) with or without LY294002 or U0126. In other studies neurite membranes were added as described in Proliferation assays, with or without the inhibitors. Cells were treated with fresh media every day, for 3 d (GGF) or 2 d (membrane fraction).
Using the Schwann cell-DRG neuron coculture system, we seeded dissociated DRG neurons with 10,000 Schwann cells in serum-containing C media. After 24 hr, some cultures were fed serum-free media (control), whereas others were switched to serum-free media supplemented with LY294002 or U0126 for 3 d. The serum-free media consisted of DME/Ham's F12, 2 mM L-glutamine, 10 µg/ml transferrin, 5 µg/ml insulin, 20 nM progesterone, 100 µM putrescine, 30 nM selenium, and 50 ng/ml NGF.
Schwann cell survival was determined by the TUNEL assay, which was performed with the Apoptosis Detection System (Promega, Madison, WI) according to the manufacturer's instructions. TUNEL and Hoechst-labeled nuclei from 7 to 10 random high-power fields were counted per culture, with four to six cultures per condition in two to three separate experiments, using an Axiophot microscope. The level of apoptosis was determined as the ratio of TUNEL to Hoechst-labeled nuclei (TUNEL index).
Myelination assays. Myelinating Schwann cell-neuron cocultures were prepared by seeding purified DRG neuron cultures with 200,000 Schwann cells in C media. The next day the cultures were switched to, and maintained in, serum-free media for 3 d to allow the Schwann cells to populate the neurites. Basal lamina formation and myelination were then initiated by feeding the cultures C media supplemented with 50 µg/ml ascorbic acid [day 0 (d0)] and maintained for 15 d. To determine the role of PI 3-kinase and MAPK kinase in the process of myelination, kinase inhibitors LY294002 and U0126 were added either at d0 or at d4. Cultures were analyzed for the expression of myelin proteins (MBP and P0) and for laminin, a major basal lamina component, by immunofluorescence and slot blot. Cultures for immunofluorescence microscopy were processed as described previously (Einheber et al., 1997
Western and slot blot analysis of kinase activities and myelin protein expression. In vivo, full activation of the serine/threonine protein kinase Akt results from the PI 3-kinase-dependent phosphorylation on Thr-308 and Ser-473 residues (Alessi et al., 1996
MBP and P0 levels from myelinating cocultures treated with or without LY294002 were determined by slot blot analysis. Five micrograms of total proteins were directly slot blotted onto nitrocellulose and probed with either anti-MBP 644 or anti-P0 polyclonal antibodies, which were detected with an 125I-anti-rabbit IgG antibody. Quantitation was performed using the PhosphorImager 425 and the ImageQuant software (Molecular Dynamics, Sunnyvale, CA).
Statistical analysis. One-way ANOVA, followed by a Bonferroni post-test, was performed with the Prism software package (GraphPad, San Diego, CA), with p < 0.05 considered to be statistically significant.
RESULTS
Mitogen activation of PI 3-kinase is required for the induction of Schwann cell proliferation
To elucidate whether the PI 3-kinase or the MAP kinase signaling pathways were involved in the induction of Schwann cell proliferation, we treated cells with the neuregulin-1 isoform GGF in the presence or absence of LY294002, a specific inhibitor of PI 3-kinase (Vlahos et al., 1994
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Figure 1. Activation of PI 3-kinase by GGF is required for the induction of Schwann cell proliferation. A, B, Schwann cells, maintained for 24 hr in serum-containing media supplemented with 4 µM forskolin, were incubated an additional 24 hr with 5 ng/ml GGF, without (GGF) or with either the PI 3-kinase inhibitor LY294002 at 2.5-20 µM (LY2.5-LY20) or the MAPK kinase inhibitor U0126 at 1-10 µM (U1-U10). Controls were cultures maintained in serum-containing media without (Ctrl) or with forskolin (Fk). LY294002, in contrast to U0126, inhibits the GGF-induced incorporation of BrdU into Schwann cell nuclei (A) in a concentration-dependent manner (B; mean ± SEM from 3 separate experiments). Scale bar, 100 µm. C, PI 3-kinase and MAPK kinase activities were assessed on Western blots with polyclonal antibodies recognizing phosphorylated Akt (p-Akt) and MAP kinase p44/p42 (p-p44, p-p42), downstream effectors of PI 3-kinase and MAPK kinase, respectively. GGF strongly activates both PI 3-kinase and MAPK kinase, which are specifically inhibited by their respective inhibitor. The inhibition of PI 3-kinase activity correlates with the inhibition in BrdU incorporation.
GGF induced a robust activation of both PI 3-kinase and MAPK kinase in the Schwann cells, as evidenced by the increased phosphorylation of Akt and MAP kinase p44/p42, respectively, detected by Western blot analysis (Fig. 1C). No measurable activity of either kinase was present in quiescent Schwann cells maintained in D media, and only low levels of phospho-Akt were detected in the presence of forskolin. In the presence of LY294002, GGF-induced phospho-Akt levels progressively decreased in a dose-dependent response, whereas the levels of phospho-MAPK p44/p42 were not affected, indicating the specificity of this inhibitor. This finding also indicates that in Schwann cells, activation of MAP kinase by GGF is direct and does not result from the protein kinase activity associated with PI 3-kinase itself (Bondeva et al., 1998
Because LY294002 is a reversible inhibitor of PI 3-kinase, we next examined whether the inhibition of Schwann cell proliferation is reversible. Schwann cells, cultured for 24 hr in the presence of LY294002 and GGF, were briefly rinsed to remove the inhibitor and then maintained on GGF-supplemented media for an additional 24 hr. Removal of the inhibitor completely reversed the inhibition of Schwann cell proliferation, resulting in a BrdU index comparable with that of Schwann cells that had not been treated with the PI 3-kinase inhibitor (Fig. 2). These results indicate that the inhibition of Schwann cell proliferation by LY294002 was not caused by a nonspecific, cytotoxic effect.
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Figure 2. The inhibition of BrdU incorporation by PI 3-kinase is reversible. Schwann cells, maintained for 24 hr in serum-containing media supplemented with 4 µM forskolin, were then incubated 24 hr with 5 ng/ml GGF, without (GGF) or with the PI 3-kinase inhibitor LY294002 at 20 µM (LY20). A set of cultures was kept in serum-containing media without forskolin (Ctrl). LY294002 was removed by washing the treated cultures five times with DMEM, and cultures were then maintained an additional 24 hr in GGF- and forskolin-supplemented serum-containing media (wash). All the other cultures were identically washed but subsequently maintained in the media they were in before washing. Cells incorporated BrdU comparably with cultures never treated with the PI 3-kinase inhibitor (mean ± SEM from 3 separate experiments).
Schwann cells proliferate in response to a number of other soluble growth factors. We examined whether FGF-2 (basic FGF) that, in combination with forskolin is a Schwann cell mitogen (Davis and Stroobant, 1990
Axons promote Schwann cell proliferation via PI 3-kinase
We next determined whether the mitogenic activity of neurites also depends on PI 3-kinase activity. Schwann cells maintained in serum-containing media proliferate slowly (Fig. 3). When cocultured with DRG neurons, the labeling index increased by almost 10-fold, consistent with the known mitogenic activity of DRG neurons. The addition of LY294002 at 20 µM to the culture media resulted in a striking decrease (~67%) in the incorporation of BrdU by the Schwann cells, whereas the MAPK kinase inhibitor (10 µM) had a negligible effect on proliferation that was not statistically significant.
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Figure 3. Neurons promote Schwann cell proliferation via PI 3-kinase. Ten thousand Schwann cells were seeded onto purified DRG neuron cultures in serum-containing media. Twenty-four hours later, cultures were fed fresh media without (CC) or with LY294002 at 20 µM (CC + LY20) or U0126 at 10 µM (CC + U10) for an additional 24 hr. Controls were Schwann cells maintained in serum-containing media for 48 hr (Sc). The cultures were then processed for BrdU immunostaining. Values represent the mean BrdU index ± SEM from three separate experiments.
These results suggest that the axonal induction of Schwann cell proliferation was dependent on PI 3-kinase activation. Consistent with this possibility, PI 3-kinase activity was detected in the coculture system (data not shown), although it was not clear whether this activity and its inhibition by LY294002 were primarily associated with Schwann cells, DRG neurons, or both. We therefore added neurite membranes to cultures of Schwann cells that, from previous studies (Salzer et al., 1980a
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Figure 4. Neurite membranes promote Schwann cell proliferation via PI 3-kinase. A, Two aliquots of a neurite membrane preparation were centrifuged onto Schwann cell cultures at a 24 hr interval in serum-containing media without kinase inhibitors (mb) or supplemented with LY294002 at 20 µM (mb + LY20) or U0126 at 10 µM (mb + U10). BrdU incorporation was assessed after an additional 24 hr in culture. Controls were Schwann cells maintained in serum-containing media (Ctrl). B, Schwann cells, in culture for 24 hr in serum-containing media, were preincubated for 30 min with either inhibitor before centrifuging the neurite membrane fraction onto the cells. Lysates were made after 30 min of contact with the membranes. Contact with the neurite membranes strongly activates PI 3-kinase, and MAPK kinase to a lesser extent, in the Schwann cells. The inhibition of PI 3-kinase, but not that of MAPK kinase (B), correlates with the inhibition of the neurite membrane-induced incorporation of BrdU in Schwann cell nuclei (A; mean ± SEM from 2 separate experiments). p-Akt, Phosphorylated Akt; p-p44, p-p42, phosphorylated MAP kinase p44/p42.
We next assayed the effect of neurite membranes on Schwann cell proliferation. We found a fivefold increase in the BrdU-labeling index of Schwann cells treated with membranes; this increase was dependent on the activity of PI 3-kinase but not that of MAPK kinase (Fig. 4A). Thus, LY294002 completely abolished the increase in BrdU incorporation, whereas the MAPK kinase inhibitor U0126 had no effect. Taken together, these results indicate that contact-dependent axonal mitogens, including the neuregulins, activate both PI 3-kinase and MAP kinase pathways and that Schwann cell proliferation specifically requires PI 3-kinase activation.
Schwann cell survival promoted by soluble GGF requires PI 3-kinase
Postnatal Schwann cells cultured in a serum-free media undergo programmed cell death that can be blocked by soluble neuregulins (Syroid et al., 1996
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Figure 5. Schwann cell survival promoted by GGF requires PI 3-kinase. Schwann cells were cultured for 3 d in either serum-containing media (Ctrl) or serum-free media (SFM). In some cultures, the serum-free media were supplemented with 4 µM forskolin and 50 ng/ml GGF, without (GGF) or with 20 µM LY294002 (LY20) or 10 µM U0126 (U10). Apoptotic cells were detected by the TUNEL assay. Values represent the mean BrdU index ± SEM from three separate experiments.
Axons promote Schwann cell survival via contact-dependent signals that require PI 3-kinase
Axon-dependent signals have been shown to promote the survival of Schwann cell precursors and early postnatal Schwann cells (Dong et al., 1995
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Figure 6. Neurons promote Schwann cell survival via PI 3-kinase. Schwann cells cocultured for 3 d with neurons in serum-free media without kinase inhibitors (A, D), with 20 µM LY294002 (B, E), or with 10 µM U0126 (C, F) were analyzed for apoptosis by Hoechst staining (A-C) and TUNEL assay (D-F). Schwann cells in contact with neurons exhibit very few apoptotic cells (A, D). When treated with the PI 3-kinase inhibitor, numerous Schwann cells undergo apoptosis, as indicated by pyknotic nuclear fragments that are labeled in the TUNEL assay (B, E). No nuclear fragmentation and TUNEL cells were observed in cocultures treated with the MAPK kinase inhibitor (C, F). Scale bar, 50 µm.
These studies do not distinguish whether Schwann cell survival was mediated by cell contact or the release of soluble factors by neurons. They also do not distinguish whether the primary effect of the inhibitor is on neurons, Schwann cells, or both. Although we did not notice an increase in the number of TUNEL neurons in cocultures treated with the PI 3-kinase inhibitor (data not shown), it remained possible that a decreased viability of the neurons indirectly affected the survival of the Schwann cells. To address these issues, we determined whether neurite membranes could prevent Schwann cells from apoptosis after serum withdrawal. The results are shown in Figure 7. The eightfold increase in the TUNEL index observed after serum withdrawal over a period of 2 d was completely abolished by the addition of neurite membranes. The PI 3-kinase inhibitor LY294002 (20 µM), but not the MAPK kinase inhibitor U0126 (10 µM), totally inhibited the survival promoted by the addition of neurite membranes. These results strongly suggest that neurons promote Schwann cell survival via contact-dependent signals that activate the PI 3-kinase pathway.
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Figure 7. Axons promote Schwann cell survival via contact-dependent signals that require PI 3-kinase. Schwann cells were cultured for 2 d in either serum-containing media (Ctrl) or serum-free media (SFM). In some cultures, the serum-free media were supplemented by centrifuging two aliquots of neurite membranes at 24 hr intervals, without (mb) or with 20 µM LY294002 (LY20) or 10 µM U0126 (U10). Apoptotic cells were detected by the TUNEL assay. Values represent the mean BrdU index ± SEM from two separate experiments.
PI 3-kinase is required for initial events of myelination
In addition to regulating Schwann cell proliferation and survival, axons promote their differentiation. To determine whether PI 3-kinase and/or MAPK kinase were involved in the axonal induction of Schwann cell differentiation, we analyzed the effects of PI 3-kinase and MAPK kinase inhibitors on myelination in DRG neuron-Schwann cell cocultures. Specifically, cocultures were treated with LY294002 (20 µM) or U0126 (10 µM) beginning on day 0 or day 4 after addition of ascorbate and maintained for up to 15 d. Myelination was then assessed by immunostaining for MBP; results are shown in Figure 8. Unlike control cultures, which myelinated extensively (Fig. 8A), inhibition of PI 3-kinase beginning at day 0 completely blocked myelination in treated cultures (Fig. 8B). In contrast, myelin sheaths were present in cultures when PI 3-kinase was inhibited beginning 4 d after the addition of ascorbic acid (Fig. 8C). Inhibition of MAPK kinase beginning on either day had no effect on the progress of myelination (data not shown). In separate studies, we confirmed the presence of both PI 3-kinase and MAPK kinase activities in the coculture system (Zanazzi, Teng, Kraemer, and Salzer, personal communication) and their specific inhibition by LY294002 and U0126, respectively (data not shown).
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Figure 8. Inhibition of PI 3-kinase perturbs myelination. Myelinating Schwann cell-DRG neuron cocultures were set up as described in Materials and Methods. A-C, Immunostaining of 11-d-old cocultures for MBP, not treated with the PI 3-kinase inhibitor (A) or incubated with 20 µM LY294002 from day 0 (B; onset of myelination) or from day 4 (C; 4 d after the onset of myelination). The inhibition of PI 3-kinase from day 0 to day 11 completely inhibits myelination (B). In contrast, inhibition of PI 3-kinase from day 4 to day 11 does not prevent the appearance of myelin sheaths. However, these sheaths seem shorter in length than those in untreated cultures of the same age. D-F, Corresponding fields counterstained with Hoechst. Scale bar, 50 µm.
Because Schwann cell proliferation requires PI 3-kinase, the absence of myelination might have resulted from insufficient numbers of Schwann cells to segregate bundles of axons efficiently and promote myelination. To address this possibility, we established cocultures with an excess of Schwann cells (2 × 106 instead of 2 × 105 cells). In the presence of LY294002, the cocultures still failed to myelinate, whereas untreated companion cultures myelinated normally (data not shown). These results suggest that PI 3-kinase inhibition affects axon-Schwann cell interactions required for myelination that are distinct from proliferation. We also did not detect an increase in Schwann cell apoptosis in the LY294002-treated cocultures (data not shown), presumably because of serum factors that are present in myelinating media.
Although MBP-positive segments were present in cocultures treated with the PI 3-kinase inhibitor beginning at d4, the segments that did form appeared to be of shorter length compared with those of untreated cultures of the same age (Fig. 8, compare A, C). We therefore quantitated MBP and P0 protein levels and counted the number and length of MBP-positive segments at various times during the course of myelination (Fig. 9). In control cultures, the amount of MBP and P0 dramatically increased during the first 15 d of culture in myelinating media (Fig. 9A,B). When the cultures were treated with LY294002 from day 0 to day 15, the accumulation of both MBP and P0 was completely inhibited with no increase over that of day 0 cultures (data not shown). In contrast, MBP and P0 continued to accumulate in cocultures treated with LY294002 from day 4, although the amount was reduced 50% compared with that of controls at day 15. In these cocultures, the number of MBP-positive segments was similar to that of untreated cultures of the same age (Fig. 9C). However their length was much shorter (Fig. 9D). In control cocultures, myelinated segments averaged 79 µm in length at day 4, 106 µm at day 7, and 120 µm at day 11 and did not increase significantly at later times. When the 4-d-old cocultures were maintained in media supplemented with the PI 3-kinase inhibitor, the myelinated segments that formed remained at 80 µm at 7 d and beyond.
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Figure 9. LY294002-treated cocultures have shorter myelin sheaths. Myelinating cocultures were maintained for up to 15 d, without LY294002 ( ) or with 20 µM LY294002 added from day 4 (
). Cultures were analyzed at different days (d0 to d15) for the presence of the myelin proteins MBP (A) and P0 (B) (expressed as the percent of the total amount present in 15-d-old controls) and for the number (C) and length (D) of the myelin sheaths. Values represent the mean ± SEM of five (A, B), three (C; 54 high-power fields), or two (D; >1000 segments measured for each time point) separate experiments.
Finally we examined the effects of LY294002 on well established, myelinated cultures. Interestingly, chronic inhibition of PI 3-kinase in 5-week-old myelinating cultures had no apparent adverse effects, such as demyelination, suggesting that PI 3-kinase activity is not required for the long-term maintenance of mature myelin sheaths (data not shown).
Effect of PI 3-kinase inhibition on laminin deposition
The addition of ascorbate to the culture media initiates the assembly of a basal lamina by the Schwann cell, which is required for these cells to ensheathe or myelinate axons (Eldridge et al., 1987
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Figure 10. PI 3-kinase does not affect laminin expression. Myelinating cocultures were maintained for up to 11 d, without LY294002 (A, D) or with 20 µM LY294002 added from day 0 (B, E) or from day 4 (C, F). Cultures were immunostained for laminin (A-C) and neurofilament (D-F). Schwann cell nuclei are counterstained with Hoechst. Scale bar, 50 µm.
DISCUSSION
The intracellular signaling pathways activated by axonal contact that regulate Schwann cell development and differentiation have been elusive. In this study, we have shown that both the PI 3-kinase and MAP kinase pathways are potently activated by GGF and by axonal contact. Using specific pharmacological inhibitors, we have also demonstrated that PI 3-kinase but not MAPK kinase activation is crucial for the generation and initial differentiation of Schwann cells. These results are considered further below.
PI 3-kinase is a key signaling pathway in the generation of Schwann cells
A major finding of this study is that activation of PI 3-kinase by axons and neuregulins is required for Schwann cell proliferation and survival. We demonstrated that PI 3-kinase is robustly activated in Schwann cells after treatment with GGF and neurite membranes, consistent with the presence of multiple binding sites for the p85 adaptor of PI 3-kinase on erbB3. To investigate the role of this activation, we used LY294002 as a specific inhibitor of PI 3-kinase (Vlahos et al., 1994
PI 3-kinase activity is also required for the trophic effects of GGF and neurite membranes on Schwann cells. Thus, inhibition of PI 3-kinase reversed the effects of GGF, DRG neurons, and neurite membranes on Schwann cell survival (Figs. 5-7). These results are consistent with the key role the PI 3-kinase pathway plays in the survival of many cells, including the trophic effects of other growth factors on Schwann cells (Campana et al., 1999
A number of downstream effectors of PI 3-kinase have been identified that are candidates to promote Schwann cell proliferation and survival. Of particular interest is the proto-oncogene Akt, which is activated by PI 3-kinase-dependent phosphorylation and by its phosphorylated lipid products. Akt prevents apoptosis in many cells (Franke et al., 1997
(GSK-3
MAP kinase has a dispensable role in Schwann cell proliferation and survival
In addition to PI 3-kinase, MAP kinase is also robustly activated by neurite membranes and, as reported previously (Kim et al., 1997
Role of the PI 3-kinase pathway in Schwann cell differentiation
These studies also indicate that the formation of the myelin sheath is dependent on PI 3-kinase activity. Myelination proceeds in a series of distinctive stages (Webster, 1992
We considered, as an alternative possibility, that PI 3-kinase might be necessary for the synthesis of the basal lamina. Basal lamina formation, which is dependent on axonal signals, is essential for the initial events of Schwann cell ensheathment and myelination (Bunge et al., 1986
At later stages of differentiation, PI 3-kinase seems to be required for the maturation but not the maintenance of myelin sheaths. Thus, cocultures in which PI 3-kinase was inhibited beginning on day 4, a time when Schwann cells have assembled a basal lamina and begun to segregate axons (Fernandez-Valle et al., 1998
In summary, our results strongly implicate PI 3-kinase in the contact-dependent regulation of Schwann cell development by axon signals, including the neuregulins. In the future, it will be of interest to determine whether PI 3-kinase activity is also required for the proliferation of Schwann cells during Wallerian degeneration, which may be mediated in part via erbB receptor activation (Kwon et al., 1997
FOOTNOTES Received Feb. 14, 2000; revised April 3, 2000; accepted April 4, 2000.
This work was supported by the National Institutes of Health Grant NS 26001 (J.L.S.) and the National Multiple Sclerosis Society Grant FA 1302-A-1 (P.M.). We thank G. Zanazzi and S. Einheber for helpful discussions and critical reviews of this manuscript and M. Marchionni and Cambridge Neuroscience for providing rhGGF2.
Correspondence should be addressed to Dr. Patrice Maurel, Department of Cell Biology, New York University Medical Center, 550 First Avenue, New York, NY 10016. E-mail: maurep01{at}endeavor.med.nyu.edu.
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