Human Molecular Genetics, 2001, Vol. 10, No. 26 3009-3016
© 2001 Oxford University Press
Heterozygosity for the neurofibromatosis 1 (NF1) tumor suppressor results in abnormalities in cell attachment, spreading and motility in astrocytes
Department of Neurology, Washington University School of Medicine, Box 8111, 660 South Euclid Avenue, St Louis, MO 63110, USA, 1Center for Developmental Biology and The Kent Waldrep Foundation Center for Basic Research on Nerve Growth and Regeneration, University of Texas Southwestern Medical Center, Dallas, TX, USA and 2Department of Neurosurgery, University of Toronto, Toronto, Canada
Received August 17, 2001; Revised and Accepted October 19, 2001.
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ABSTRACT |
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Individuals with the neurofibromatosis 1 (NF1) tumor predisposition syndrome develop low-grade pilocytic astrocytomas at an increased frequency. Previously, we demonstrated that astrocytes from mice heterozygous for a targeted mutation in the Nf1 gene (Nf1+/– astrocytes) exhibit a cell autonomous growth advantage associated with increased RAS pathway activation. In this report, we extend our initial characterization of the effect of reduced Nf1 gene expression on astrocyte function by demonstrating that Nf1+/– astrocytes exhibit decreased cell attachment, actin cytoskeletal abnormalities during the initial phases of cell spreading, and increased cell motility. Whereas these cytoskeletal abnormalities were also observed in Nf1–/– astrocytes, astrocytes expressing a constitutively active RAS molecule showed increased cell motility and abnormal actin cytoskeleton organization during cell spreading, but exhibited normal cell attachment. Based on ongoing gene expression profiling experiments on human astrocytoma tumors, we demonstrate increased expression of two proteins implicated in cell attachment, spreading and motility (GAP43 and T-cadherin) in Nf1+/– and Nf1–/– astrocytes. These results support the emerging notion that tumor suppressor gene heterozygosity results in abnormalities in cell function that may contribute to the pathogenesis of non-tumor phenotypes in NF1.
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INTRODUCTION |
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Neurofibromatosis 1 (NF1) is an autosomal dominant tumor predisposition syndrome in which affected individuals develop both benign and malignant tumors at an increased frequency (1). One of the most frequently encountered tumors in individuals with NF1 is the World Health Organization grade I pilocytic astrocytoma (2). This tumor commonly occurs in the optic pathway and may be associated with visual loss. In addition, this astrocytic brain tumor may involve the hypothalamus or other brain regions, including the brainstem (3). The majority of these tumors behave in a benign fashion with slow patterns of growth and rare cases of malignant progression. Previous work from our laboratory has demonstrated that NF1-associated pilocytic astrocytomas demonstrate loss of heterozygosity for the NF1 gene and lack NF1 protein (neurofibromin) expression (4). This observation is consistent with the two hit hypothesis for tumor suppressor genes and suggests that NF1-associated astrocytomas require bi-allelic inactivation of the NF1 gene.
The NF1 gene was identified in 1990 by positional cloning (5–7) and its protein product, neurofibromin, was found to encode a 220–250 kDa cytoplasmic protein (8–10). Analysis of the predicted amino acid sequence of neurofibromin revealed a central 300 residue domain with significant homology to a family of proteins that accelerate the inactivation of RAS (11–14). RAS provides a mitogenic signal for many cell types, including astrocytes, raising the possibility that loss of neurofibromin would result in increased RAS activation and increased cell proliferation. Loss of neurofibromin has been demonstrated in NF1-associated pilocytic astrocytomas (4) and, in the one NF1-associated astrocytoma examined thus far, results in increased RAS pathway activation (15).
In addition to overt astrocytic tumor formation, individuals with NF1 also manifest with learning disabilities and regions of hyperintense signal on brain magnetic resonance imaging (MRI) studies (16,17). It is not known what accounts for these abnormalities, although the MRI hyperintense lesions are associated with increased astrogliosis (18). Moreover, examination of post-mortem brains from individuals with NF1 demonstrated increased astrogliosis (19). These observations suggest that reduced, but not absent, NF1 gene expression may be sufficient to confer a growth advantage on astrocytes in vivo and may underlie some of the non-tumor phenotypes in the brain in individuals affected with NF1 (20). To further explore this hypothesis, we have previously demonstrated that heterozygosity for the Nf1 tumor suppressor gene in mice with a targeted disruption of the mouse Nf1 gene resulted in increased astrocyte proliferation in vitro and in vivo (21). This increase in astrocyte number was specific to glial fibrillary acidic protein (GFAP)-immunoreactive cells (astrocytes) and was not observed in oligodendrocytes or microglia. Subsequent studies demonstrated that this Nf1 heterozygosity conferred a cell autonomous growth advantage for cultured astrocytes (22). Increased astrocyte proliferation was accompanied by increases in RAS pathway signaling and cooperated with N-RAS overexpression in transgenic mice. During the course of these experiments, we noted that Nf1 heterozygote astrocytes demonstrated reduced attachment to tissue culture plates, suggesting alterations in neurofibromin-associated cytoskeleton-mediated processes.
This initial observation prompted us to formally evaluate the consequence of reduced Nf1 gene expression on cytoskeleton-mediated processes. In this report, we demonstrate that Nf1 heterozygosity results in decreased astrocyte attachment, delayed actin cytoskeleton re-organization during cell spreading and increased cell motility. Based on ongoing gene expression profiling experiments on human astrocytoma tumors, we identified two proteins (GAP43 and T-cadherin) implicated in cell attachment, spreading and motility whose expression is upregulated in Nf1 heterozygote astrocytes. We further show that complete inactivation of the Nf1 gene in cultured astrocytes similarly affects cell attachment and motility as well as results in increased GAP43 and T-cadherin protein expression. These data extend our understanding of the consequence of NF1 haploinsufficiency and further the notion that NF1 heterozygote astrocytes may contribute to the pathology seen in the central nervous system in NF1 patients.
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RESULTS |
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Heterozygosity for Nf1 results in abnormal cytoskeleton-mediated processes
To evaluate the effect of reduced Nf1 gene expression on astrocyte processes dependent on the cytoskeleton, we measured cell attachment to fibronectin-coated plates as a function of time. Nf1 heterozygote and wild-type littermate cultures were established from postnatal day 2 pups, genotyped and pooled with at least two animals per genotype per group. As shown in Figure 1A, reduced cell attachment was observed in the Nf1 heterozygote (Nf1+/–) astrocytes at both 1 and 4 h after initial plating. This difference was maximal at 4 h, but was not detected after incubation overnight. This difference is also visually appreciated using phalloidin-BODIPY immunocytochemistry to highlight the actin cytoskeleton. As shown in Figure 1B, Nf1+/– astrocytes retain a tightly compacted actin cortical rim within 60–90 min of plating. This is in contrast to the more developed actin cytoskeleton observed in the wild-type astrocytes that have already begun to spread on the fibronectin matrix after 60–90 min. Collectively, these results suggest that heterozygosity for Nf1 results in abnormalities in the initial phases of cell spreading and attachment.
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To further explore potential defects in cytoskeletal-mediated processes, Nf1+/– and wild-type astrocytes were examined in a Boyden chamber assay for alterations in cell motility. After 6 h, a small, but significant increase (14–18%) in the number of migrating astrocytes was observed in the Nf1+/– compared to the wild-type cells (Fig. 1C). This subtle effect was highly reproducible and has been observed in five independently derived cultures.
RAS transgenic astrocytes demonstrate increased motility and abnormal actin cytoskeleton organization during cell spreading, but no abnormalities in cell attachment
Since the Nf1 tumor suppressor gene product, neurofibromin, functions as a GTPase-activating protein (GAP) for p21-RAS, we explored the possibility that the cytoskeletal abnormalities observed in the Nf1+/– astrocytes would be mirrored by increased RAS activity. B8 transgenic mice, in which an activated G12V-RAS molecule was targeted to astrocytes using the GFAP promoter, were employed as a model of astrocytes with constitutive RAS activation (23). These astrocytes failed to demonstrate significant differences in cell adhesion (Fig. 2A), but demonstrated abnormal cytoskeletal organization during cell spreading when compared to wild-type controls (Fig. 2B). Similar to what we observed with the Nf1+/– astrocytes, a small, but significant, increase in cell motility (15–18%) was observed using a Boyden chamber motility assay (Fig. 2C). Collectively, these data support the notion that some, but not all, of the Nf1+/– abnormalities are recapitulated by RAS activation. In this regard, it is possible that cell attachment represents one property that is uniquely affected by Nf1 heterozygosity and is not mirrored by oncogenic RAS pathway activation.
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Neurofibromin loss in astrocytes results in increased cell motility and decreased attachment
To provide further support for the notion that reduced Nf1 expression is directly responsible for the decrease in cell attachment and increase in cell motility, we completely inactivated Nf1 with Cre recombinase in vitro using adenovirus delivery. Conditional Nf1 mice (Nf1flox/flox) were generated by the insertion of lox P targeting sequences flanking exons 31 and 32 of the mouse Nf1 gene (24). These mice are phenotypically normal, but can be made neurofibromin-deficient by the addition of Cre recombinase. In these experiments, postnatal day 2 Nf1flox/flox mice in which both alleles contain LoxP insertions were cultured and exposed to adenovirus containing bacterial Cre recombinase or LacZ (control). Cells were collected and pooled prior to functional studies and analysis by western blot. A representative western blot (Fig. 3A) demonstrates loss of neurofibromin in the Cre recombinase-infected cultures, but not in untreated (data not shown) or LacZ control-infected cultures.
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Cell attachment experiments were performed on fibronectin-coated plates as previously performed using Nf1+/– astrocytes. In these experiments, we routinely observed a 32–33% reduction in cell attachment at both 1 and 4 h in neurofibromin-deficient cultures, but not in control or untreated parallel cultures (Fig. 3B). Similarly, increased astrocyte motility (24% increase) was observed in the Nf1-deficient astrocytes compared to control or untreated cells (Fig. 3C). We also observed similar changes in actin cytoskeleton organization during the initial phases of cell spreading in the Nf1–/– astrocytes as we described for the Nf1+/– astrocytes (data not shown). These results support the notion that reduced cell attachment relates specifically to Nf1 inactivation.
Nf1 heterozygosity or complete inactivation results in increased GAP43 and T-cadherin expression
Ongoing experiments in our laboratory are directed at identifying unique gene expression changes associated with human NF1-associated astrocytoma formation and progression. Several of the genes identified in these studies have been implicated in the regulation of cell attachment, adhesion and motility (D.H.Gutmann and M.Watson, manuscript in preparation). To determine whether some of the genes identified in human low grade tumors were associated with reduced Nf1 expression in astrocytes, we analyzed their expression by western blot in matched Nf1+/– and wild-type astrocytes. In these experiments, GAP43 and T-cadherin expression were both found to be increased in the Nf1+/– compared to the wild-type astrocyte cultures (Fig. 4A). Similar analysis on cultured B8 RAS transgenic astrocytes failed to reveal any significant change in either GAP43 or T-cadherin expression (data not shown), suggesting that the increased expression of these cytoskeleton-associated proteins was specifically related to reduced Nf1 gene expression and not oncogenic RAS activation. To provide corroborative evidence for an Nf1-specific effect, Nf1flox/flox astrocytes were generated and infected with adenovirus containing either Cre recombinase or LacZ. As shown in Figure 4, inactivation of Nf1 by Cre recombinase resulted in increased GAP43 and T-cadherin expression (Fig. 4B and C). No change in ezrin or ApoE expression was observed.
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DISCUSSION |
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Although the neurofibromatosis 1 gene is most commonly regarded as a tumor suppressor gene in which complete inactivation results in tumor formation, there is an accumulating body of data to support the notion that reduced, but not absent, NF1 gene expression is sufficient to confer a growth advantage and alter cell fate in vitro and in vivo. Mice heterozygous for Nf1 display abnormalities in fibroblast-mediated wound healing that is clearly related to reduced Nf1 gene expression and not complete inactivation (25). Similarly, Nf1 heterozygosity results in increased mast cell survival proliferation, colony formation and RAS pathway activation (26). Moreover, Nf1 heterozygosity is sufficient to rescue the coat color deficit in SteelW mice by modulating melanocyte cell fate (26). As previously reported for human NF1 brains, mice heterozygous for a targeted mutation in the Nf1 gene demonstrate a 50% increase in the number of GFAP immunoreactive astrocytes in the corpus callosum and hippocampal regions (21). This increase in glial cells is specific to astrocytes and was not observed when oligodendrocytes or microglia were counted. Moreover, this defect in cell growth control was shown to be cell autonomous using cultured astrocytes from postnatal day 2 Nf1+/– and wild-type littermates (22). The increase in cell proliferation was accompanied by increased RAS pathway activation, as previously reported in mast cells (26). Together, these results strongly argue for an Nf1 heterozygote effect that is cell autonomous and involves dysregulated RAS pathway activity.
The results presented here demonstrate that, in addition to increased astrocyte proliferation, Nf1+/– astrocytes have abnormalities attributable to the cytoskeleton, including reduced cell attachment, abnormal actin cytoskeleton organization during cell spreading and increased cell motility. Some, but not all, of these effects were mirrored by oncogenic RAS expression in astrocytes. Increased cell motility and abnormalities in actin cytoskeleton organization during cell spreading were observed in both the Nf1+/– and the B8 RAS transgenic astrocytes. In contrast, Nf1+/– astrocytes demonstrated decreased attachment in vitro whereas transgenic RAS astrocytes exhibit no significant differences in cell attachment. Preliminary experiments also have shown that Nf1+/– astrocytes cannot engraft in immunocompromised recipient mouse brains after stereotactic injection (M.L.Bajenaru and D.H. Gutmann, unpublished data). In contrast, wild-type astrocytes can engraft and are detectable for at least 4 weeks post-injection. This difference might reflect abnormal cell attachment properties inherent to Nf1+/– astrocytes.
To confirm that these effects were specifically related to reduced Nf1 expression, astrocytes from a conditional Nf1 knockout mouse were treated with adenovirus containing either the Cre recombinase or LacZ. Inactivation of neurofibromin by Cre recombinase resulted in decreased cell attachment similar to that observed with the Nf1+/– astrocytes. Neurofibromin-deficient astrocytes demonstrated a further increase in cell motility compared to the Nf1+/– astrocytes. These observations argue that reduced neurofibromin expression is sufficient to confer these changes. We further demonstrated Nf1-specific changes in the expression of two proteins implicated in cell spreading, attachment and motility, GAP43 and T-cadherin. The increases in GAP43 and T-cadherin were seen only in the Nf1+/– and Nf1 null astrocytes, but not in the B8 RAS transgenic astrocytes. These results support the hypothesis that the changes in GAP43 and T-cadherin expression are a direct result of reduced or absent neurofibromin expression and are not directly related to constitutive oncogenic RAS pathway activation. Preliminary results have demonstrated that RAS pathway inhibition using the MEK inhibitor PD98059 had no effect on the increased GAP43 expression in neurofibromin-deficient astrocytes (Y.L.Wu and D.H.Gutmann, unpublished data). These findings raise the possibility that neurofibromin dysfunction may not be equivalent to oncogenic RAS pathway activation (27).
There are several possibilities that may account for the observation that oncogenic RAS is not functionally identical to neurofibromin dysfunction (28). First, the level of RAS activation is higher in the B8 RAS transgenic astrocytes than in either Nf1+/– or Nf1–/– astrocytes. This higher level of RAS activity in the B8 transgenic astrocytes may result in the activation of RAS effector molecules that are not affected by neurofibromin dysfunction. For instance, the ability of RAS to activate Rho, Rac, Akt, JNK or Raf may differ depending on the level of RAS activity. It is also not clear whether all downstream RAS effectors are equally affected by neurofibromin dysfunction. Results from our laboratory have demonstrated greater elevations in AKT compared to MAPK activity in Nf1+/– astrocytes compared to wild-type cells (22). Secondly, the length of RAS activation is different in Nf1+/– or Nf1–/– cells, in that RAS in these astrocytes is regulatable by other GAPs (rasGAPs), whereas oncogenic RAS provides a long duration, constitutively active RAS signal, which is insensitive to normal rasGAP regulation. Thirdly, it is important to recognize that common oncogenic RAS mutations disable both intrinsic neurofibromin and p120-rasGAP-stimulated RAS hydrolysis. In contrast, RAS proteins in Nf1-deficient cells retain normal p120-rasGAP activity, which in many cells, may account for up to 90% of total GAP activity. Functionally, this has recently been shown to have tremendous biological impact at the cellular level. Whereas Nf1-deficient hematopoietic cells exhibit a profound growth advantage compared to wild-type cells (29) and consistently develop a profound myeloproliferative disorder (29,30), mice reconstituted with cells that overexpress N-RAS have defective long-term reconstitution, increased apoptosis and early death (31). Fourthly, it is not known whether neurofibromin dysfunction alone is sufficient to account for NF1-associated growth abnormalities or whether specific growth factors or cellular contexts are critical for uncovering the cellular effects of neurofibromin dysfunction. Support for a context dependency is underscored by the requirement for specific growth factors (GM-CSF) to reveal a significant growth advantage for Nf1-deficient myeloid cells (32). Fifthly, in B8 RAS transgenic astrocytes, we targeted Ha-RAS whereas Nf1+/– or Nf1–/– astrocytes have dysregulated N-RAS, Ha-RAS and K-RAS, each of which may result in distinct cellular consequences (33). Finally, it is possible that neurofibromin’s specific effect on the cytoskeleton is not solely dependent on its ability to regulate RAS. Non-RAS-GAP functions have been proposed for neurofibromin in both mammalian cells and Drosophila (34,35). Further work is clearly needed to determine the precise mechanisms by which neurofibromin regulates the RAS signaling pathway as well as to define the relationship between Nf1 expression, RAS regulation and cytoskeleton processes.
Neurofibromin has previously been linked to the actin and microtubule cytoskeleton. Neurofibromin was found to co-localize with microtubules in fibroblasts and other cell types (36) and to directly bind tubulin in affinity chromatography experiments (37). The ability of neurofibromin to interact with polymerized microtubules maps to the RAS-GAP domain and is differentially affected by selected non-conservative amino acid changes that destroy the ability of neurofibromin to accelerate RAS-GTP hydrolysis (38). In addition, neurofibromin subcellular localization and the interaction with RAS during B lymphocyte signaling are dependent on the cytoskeleton (39). Lastly, during neuron development in vitro and in vivo, neurofibromin co-localizes with actin during some stages of development and differentiation and with the microtubule cytoskeleton at other times (40). These observations suggest that neurofibromin might modulate cytoskeleton-mediated processes.
Although we did not perform an exhaustive survey of proteins associated with cell attachment, motility and spreading, the finding that T-cadherin and GAP43 were overexpressed in Nf1+/– and neurofibromin-null astrocytes raises the possibility that these secondary changes, as a result of reduced or absent neurofibromin function, may underlie some of the cytoskeletal abnormalities seen in Nf1+/– astrocytes. T-cadherin was originally cloned from the chicken embryo brain as a truncated cadherin-like molecule lacking both the transmembrane and cytoplasmic domains found in conventional cadherin molecules (41). T-cadherin is located in lipid rafts through a GPI-linked anchor and is thought to mediate calcium-dependent adhesion and provide a negative guidance cue for neurite outgrowth (42,43). Astrocytes have been shown to express T-cadherin in situ (44), yet little is known about the role of T-cadherin in astrocytoma formation or astrocyte growth control. Recent results from our laboratory have demonstrated that T-cadherin overexpression is a common feature of human high-grade astrocytomas and is associated with malignant transformation of astrocytes harboring oncogenic RAS (D.H.Gutmann and M.Watson, manuscript in preparation). Further work will be necessary to conclusively demonstrate that T-cadherin overexpression in astrocytes alters cell adhesion, motility or attachment. These experiments are in progress.
GAP43 is associated with the cell periphery, localizes to pseudopods (45), and also accumulates in lipid rafts (46). GAP43 is detected in astrocytes where its expression decreases as a function of differentiation in vitro and in vivo (47). Although functional studies have not been performed in astrocytes, the introduction of a dominant negative GAP43 molecule reduces peripheral nerve regeneration and stimulus-induced nerve sprouting (46). Additionally, reversion of RAS-transformed NIH-3T3 fibroblast cells correlates with decreased GAP43 expression (48), but is not related to the mitogenic responsiveness upon reintroduction of GAP43. These observations suggest that GAP43 is associated with malignant transformation, but is not the direct result of increased oncogenic RAS pathway activation. Constitutively active GAP43 remains at the membrane and is associated with membrane blebs, disorganized cell spreading and poor substrate adherence. We have recently observed increased GAP43 expression by gene expression profiling in human NF1-associated astrocytomas (D.H.Gutmann and M.Watson, manuscript in preparation), suggesting that some of the effects seen in the Nf1+/– astrocytes relate to GAP43 overactivation. Ongoing experiments in our laboratory aimed at establishing a more direct link between GAP43 overexpression and cytoskeleton-mediated processes in astrocytes have shown that overexpression of GAP43 in C6 rat astrocytomas lacking GAP43 expression is associated with dramatic alterations in actin cytoskeleton-associated processes (Z.-y.Huang and D.H.Gutmann, unpublished data). Further work on the direct link between Nf1 dysfunction, RAS pathway activation and GAP43 expression will be required.
Recent work from our laboratory has demonstrated that loss of neurofibromin in astrocytes both in vitro and in vivo may not be sufficient for astrocytoma formation (M.L.Bajenaru, N.M.Hedrick, Y.Zhu, J.Donahoe, L.F.Parada and D.H.Gutmann, manuscript in preparation). The observations reported herein suggest that additional gene expression changes may be required for astrocyte transformation. The identification of two new candidates, GAP43 and T-cadherin, raises the possibility that cooperating events are required for NF1-associated astrocytoma formation. The identification of these and other potential candidates will improve our understanding of the basic pathogenesis of NF1-associated brain tumors.
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MATERIALS AND METHODS |
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Antibodies
Neurofibromin antibodies were purchased from Santa Cruz Biotechnology (NF1GRP-D). The rabbit GAP43 polyclonal antibody was purchased from Chemicon International (AB5220) and the T-cadherin rabbit polyclonal antibody was purchased from Santa Cruz Biotechnology (H126). The ezrin mouse monoclonal antibody was purchased from BABCO (clone 4A5) and the ApoE antibody was purchased from Calbiochem (178479).
Mice
Nf1+/– mice were maintained by intercrossing Nf1+/– mice with wild-type mice. Genotyping was performed as previously described by Brannan et al. (49). The B8 RAS transgenic mouse line was maintained as a continuous colony by intercrossing B8 mice with wild-type ICR mice and genotyped using a LacZ primer set (23). Lastly, Nf1 conditional knockout mice were generated by Yuan Zhu and Luis Parada and were maintained as a continuous colony by intercrossing Nf1flox/flox mice (24). Genotyping was performed as previously described. All animals were maintained according to established animal studies protocols at Washington University.
Astrocyte cultures
Murine neocortical astrocyte cultures were established as previously described from postnatal day 2 pups (22). Genotypes were determined by PCR from tail DNA. Astrocytes are initially seated as separate cultures (passage 0; P0) and then pooled once the genotypes have been determined (P1). Experiments were performed on P2 cultures. The percent of astrocytes was determined by GFAP immunolabeling and previously determined to be >97% GFAP immunoreactive cells (50).
Primary astrocytes cultures were established from Nf1flox/flox newborn mice and cultured as described previously (50). Astrocytes were infected with 1010 plaque forming units per milliliter in 5 ml of culture media containing 10% horse serum for 1 h at 37°C. Titered adenovirus containing LacZ (Ad5-LacZ) or Cre recombinase (Ad5-Cre) was purchased from the University of Iowa Vector Core. Astrocytes were washed three times with PBS and cultured for an additional 6 days. Astrocyte cultures were pooled into untreated, adenoviral Cre-treated, and adenoviral LacZ-treated cultures and harvested for western blot or seeded for the motility and adhesion experiments. Only passage 1 cultures were used for these experiments.
Cell adhesion spreading and motility
Astrocyte adhesion was performed by pre-coating a 96-well plate with 10 µg/ml fibronectin (Sigma) in sterile PBS overnight at 4°C. The wells were washed twice with PBS and incubated with 2% heat inactivated bovine serum albumin (BSA) for 2 h at 37°C. Cells were resuspended in serum-free medium for 2 h at 37°C and seeded at a density of 5000 cells per well with six wells per condition. Cells were allowed to adhere to the well for 1 or 4 h, at which time they were washed three times with PBS, and stained with 0.5% crystal violet for 30 min at room temperature. The wells were then washed three times in water and solubilized in 1% SDS overnight at room temperature. The number of cells adherent was quantitated by absorbance at 540 nm. Each experiment was performed at least three times with identical results.
Cell spreading was analyzed by phalloidin cytochemistry on 1 x 105 astrocytes plated on 10 mg/ml of laminin. Cells were allowed to adhere for 60–90 min, after which time they were fixed in 3.7% paraformaldehyde for 10 min at room temperature and permeabilized with 0.1% Triton X-100. The actin cytoskeleton was visualized with BODIPY-conjugated phalloidin (0.2 U in 50 µl; Molecular Probes) for 20 min. Coverslips were then washed in PBS and mounted in one drop of Fluoromount G (EM Sciences) and examined on a Zeiss Axiophot microscope. Each experiment was repeated three times with identical results.
Cell motility was determined in Transwell Boyden assays containing 8 µm membranes. Briefly, the bottom surface of the gel was coated with Matrigel (Collaborative Research) and 10 000 cells were allowed to attach for 1 h. Cells were gently washed and incubated for 24 h to allow for migration. Cells were fixed in cold methanol for 30 min prior to staining with a Leukostat staining kit (Fisher Scientific) and counted visually. The number of migrating cells was counted in quadruplicate and the mean and SD determined for each condition. Each experiment was repeated three times with identical results.
Western blot
Western blotting was performed using established techniques in the laboratory by separating 100 µg of total protein for each sample by SDS–PAGE and analyzed using commercially available antibodies. The antibodies were used at the following dilutions: NF1-GRP (neurofibromin, 1:300), T-cadherin (1:1000), GAP43 (1:500), ApoE (1:2000) and ezrin (1:500). Western blots were developed by ECL chemiluminescence as described previously.
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ACKNOWLEDGEMENTS |
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We gratefully appreciate the expert advice of Dr Michael Chicoine in the Department of Neurosurgery, Dr Timothy Fleming in the Department of Ophthalmology, Dr Mark Watson in the Department of Pathology and Dr M.Livia Bajenaru in our laboratory as well as the technical assistance of Sean Brophy during the execution of these experiments. This work is funded by a grant from the American Cancer Society as well as the National Institutes of Health (NS36996 and NS41097).
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +1 314 362 7149; Fax: +1 314 362 2388; Email: gutmannd@neuro.wustl.edu
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REFERENCES |
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