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Address correspondence to Toru Kondo, Centre for Brain Repair, University of Cambridge, E.D. Adrian Building, Forvie site, Robinson Way, Cambridge CB2 2PY, UK. Tel.: 44-1223-334121. Fax: 44-1223-334121. email: tk294{at}cam.ac.uk
Abstract
Neural stem cell (NSC) differentiation is precisely controlled by a network of transcription factors, which themselves are regulated by extracellular signals (Bertrand et al., 2002; Shirasakiand and Pfaff, 2002). One way that the activity of such transcription factors is controlled is by the regulation of their movement between the cytosol and nucleus (Vandromme et al., 1996. Lei and Silver, 2002). Here we show that the basic helixloophelix transcription factor OLIG2, which has been shown to be required for motor neuron and oligodendrocyte development, is found in the cytoplasm, but not the nucleus, of astrocytes in culture and of a subset of astrocytes in the subventricular zone. We demonstrate that the accumulation of OLIG2 in the nucleus of NSCs blocks the CNTF-induced astrocyte differentiation and that the translocation of OLIG2 to the cytoplasm is promoted by activated AKT. We propose that the AKT-stimulated export of OLIG2 from the nucleus of NSCs is essential for the astrocyte differentiation.
Key Words: OLIG2; astrocyte differentiation; nuclear export; CRM1; AKT
Abbreviations used in this paper: bFGF, basic FGF; bHLH, basic helix-loop-helix; CNTF, ciliary neurotrophic factor; GFAP, glial fibrillary acidic protein; LMB, leptomycin B; NES, nuclear export signal; NF, neurofilament; NSC, neural stem cell; PI, propidium iodide; SVZ, subventricular zone; VZ, ventricular zone.
Introduction
OLIG2 is a basic helixloophelix (bHLH) transcription factor required for oligodendrocyte development (Lu et al., 2000; Takebayashi et al., 2000; Zhou et al., 2000, 2001). OLIG2 also blocks astrocyte differentiation, by inhibiting complex formation between STAT3 and the transcription coactivator p300 (Lu et al., 2002; Takebayashi et al., 2002; Zhou and Anderson 2002; Gabay et al., 2003; Fukuda et al., 2004). Despite the crucial role of OLIG2 in glial fate decision, little is known about how OLIG2 itself is regulated during glial development. Previous findings have suggested that when neural stem cells (NSCs) begin to differentiate into glialfibrillaryacidicprotein (GFAP)-positive astrocytes in culture, OLIG2 disappears from the nucleus and is sometimes detected in the cytoplasm (Fukuda et al., 2004), raising the possibility that the translocation of OLIG2 from the nucleus to cytoplasm might be crucial for astrocyte differentiation.
Here we show that OLIG2 is detected in the cytoplasm of GFAP-positive astrocytes in the subventricular zone (SVZ) of adult mouse brain, which have been shown to act as NSCs that give rise to both neurons and glia. We demonstrate that Leptomycin B (LMB), which is a specific inhibitor of CRM1-dependent nuclear export (Fornerod et al., 1997; Yoshida and Horinouchi, 1999), blocks both the export of OLIG2 from the nucleus and astrocyte differentiation induced by ciliary neurotrophic factor (CNTF). Moreover, we show that an OLIG2 mutant lacking a putative CRM1 binding site is deficient in both CNTF-induced nuclear export and astrocyte differentiation. Finally, we show that AKT, which is activated by CNTF stimulation, phosphorylates OLIG2 in vitro and that an OLIG2 mutant lacking an AKT phosphorylation site is also deficient in both CNTF-induced nuclear export and astrocyte differentiation. These findings suggest that AKT-dependent nuclear export of OLIG2 plays a crucial part in astrocyte differentiation.
Results and discussion
Export of OLIG2 from the nucleus is essential for astrocyte differentiation
We first investigated whether OLIG2 localized in the cytoplasm of astrocyte in vivo. We immunolabeled sections of either newborn or adult mouse brain for both OLIG2 and a neural markerSSEA1 for NSCs, neurofilament (NF) for neurons, or GFAP for astrocytes. In the SVZ of the adult brain, 44% of the cells with OLIG2 in the nucleus stained for SSEA1, and 38% stained for NF (Fig. 1, a and b). None of GFAP-positive cells had OLIG2 in the nucleus, although 20% of them had OLIG2 in the cytoplasm (Fig. 1 c). In the ventricular zone (VZ) of the newborn brain, we also found that 57% of the cells with OLIG2 in the nucleus stained for SSEA1, and 41% stained for NF; none of GFAP-positive cells had OLIG2 in the nucleus, although 4% of them had OLIG2 in the cytoplasm (unpublished data). It seems, therefore, that, in the VZ and SVZ, OLIG2 is mainly in the nucleus of both NSCs and neurons, by contrast, the few astrocytes contained OLIG2 exclusively in their cytoplasm.
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CNTF-dependent OLIG2 translocation from the nucleus depends on CRM1
Because it seemed likely that OLIG2 export from the nucleus would depend on the nuclear export signal receptor CRM1, we looked for CRM1-binding sites in OLIG2. We found one putative nuclear export signal sequence (NES; 146-LSKIATLLL-154). To examine whether this sequence was a functional NES, we replaced the leucines at 152 and 154 to alanines and examined the localization of the mutant protein in Cos7 cells. When FLAG-tagged wild-type OLIG2 (OLIG2-w) was overexpressed in Cos7 cells, it was diffusely distributed (Fig. 3 a), although in the presence of LMB it accumulated in the nucleus (Fig. 3 b), suggesting that OLIG2 is normally exported from the nucleus by CRM1 in Cos7 cells. By contrast, when FLAG-tagged mutant OLIG2 (OLIG2NES) was expressed in Cos7 cells, it accumulated in the nucleus even in the absence of LMB (Fig. 3 c), confirming that 146-LSKIATLLL-154 is the relevant CRM1-dependent export signal in OLIG2.
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It has been shown that the nuclear corepressor N-CoR, which inhibits astrocyte differentiation, is also exported from the nucleus, following CNTF stimulation (Hermanson et al., 2002), raising the possibility that OLIG2 and N-CoR may be physically associated. When we immunolabeled cultured NSCs with anti N-CoR antibodies, 10% of the cells showed nuclear staining (Fig. 4 a). CNTF stimulation caused the N-CoR to translocate to the cytoplasm (Fig. 4 b), as reported previously (Hermanson et al., 2002). However, we detected OLIG2 in the nucleus of >90% of the cultured NSCs (Fukuda et al., 2004), making it unlikely that OLIG2 is associated with N-CoR in the cells. To test this conclusion further, we transfected either a control vector expressing GFP or a vector expressing OLIG2NES, cultured them in CNTF for 2 d, and then immunolabeled them for N-CoR and either GFP or FLAG. In the cells transfected with either the control vector or the OLIG2NES vector, N-CoR was detected only in the cytoplasm (Fig. 4, c and d), whereas OLIG2NES remained in the nucleus (Fig. 4 d). It seems, therefore, that OLIG2 and N-CoR independently regulate astrocyte differentiation.
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To investigate the relationship between AKT-dependent OLIG2 phosphorylation and OLIG2 localization, we overexpressed the OLIG2-w or OLIG2-S30A in Cos7 cells and stained them for FLAG. Whereas OLIG2-w was detected mainly in the cytoplasm (Fig. 5 e), OLIG2-S30A was detected mainly in the nucleus (Fig. 5 f), suggesting that the translocation of OLIG2 from the nucleus depends on AKT-dependent phosphorylation of serine 30. We then examined whether OLIG2-S30A can block astrocyte differentiation. We overexpressed OLIG2-S30A in NSCs, cultured them in the presence of CNTF for 2 d, and then labeled them for GFAP. Whereas >50% of the cells transfected with the control vector differentiated into GFAP-positive astrocytes (Fig. 5, g and i), <5% of the cells expressing OLIG2-S30A did so (Fig. 5, h and i), suggesting that AKT-dependent translocation of OLIG2 from the nucleus is essential for CNTF-induced astrocyte differentiation. Interestingly, BMPs also promote astrocyte differentiation, and they can activate both STAT3 and AKT (D'Alessandro et al., 1994; Gross et al., 1996; Ghosh-Choudhury et al., 2002; Rajan et al., 2003).
In the developing spinal cord, OLIG2 is expressed only in the ventral VZ, where motor neurons and oligodendrocytes, but not astrocytes, are generated (Lu et al., 2000; Takebayashi et al., 2000; Zhou et al., 2000). It therefore seems unlikely that the mechanism of nuclear-to-cytoplasm translocation of OLIG2 that we describe here is relevant for astrocyte differentiation in the spinal cord. However, we found that OLIG2 localized in the cytoplasm of GFAP-positive astrocytes in both the VZ of newborn mouse brain and SVZ of adult mouse brain, suggesting that the translocation of OLIG2 from the nucleus might operate in astrocyte differentiation in the brain. Moreover, once VZ/SVZ cells are dissociated and expanded in culture, independently of their origins, they start to express OLIG2, proliferate indefinitely, and generate neurons, astrocytes and oligodendrocytes (Gabay et al., 2003; Fukuda et al., 2004). Recent evidence has also shown that the inhibition of OLIG2 activity in cultured NSCs blocks their proliferation and induces astrocyte differentiation (Hack et al., 2004). Since nuclear accumulation of OLIG2 is crucial to block astrocyte differentiation in cultured NSCs, these findings suggest that translocation of OLIG2 from the nucleus on its own might be sufficient to induce astrocyte differentiation in cultured NSCs.
Such NSCs are an attractive potential source of neural cells for cell therapy, as it has been shown that they can promote functional recovery in various types of CNS damage (Cao et al., 2002; Okano, 2002; Hallbergson et al., 2003). Hence, it will be important to understand the regulation of OLIG2 in NSCs, especially as there is another evidence that oligodendrocyte precursor cells in the adult brain can behave as NSCs (Nunes et al., 2003).
Materials and methods
Mice and cell culture
All animal procedures were performed according to the guidelines of the University of Cambridge Centre for Brain Repair. Neuroepithelial cells were isolated from telencephalons of E14.5 CD1 mice and expanded for up to 4 wk in N2-supplemented DME/F-12 containing 10 ng/ml bFGF (Peprotech) and 10 ng/ml EGF (Peprotech) as described previously (Nakashima et al., 1999). Recombinant CNTF (Peprotech) was used for the induction of astrocytes at a concentration of 20 ng/ml. Cos7 cells were maintained in DME supplemented with 10% FCS. In some experiments, LMB (25 ng/ml; Sigma-Aldrich) and LY294002 (50 µM; Calbiochem) were used to inhibit CRM1-dependent nuclear export and PI3 kinase activity, respectively.
Immunochemistry
Newborn and adult CD1 mice (P35w) were killed under deep anesthesia and 10-µm frozen sections of the brain were prepared. Sections were treated with acetone at 20°C for 20 min and 70% ethanol at 20°C for 10 min, blocked with 10% FCS at room temperature for 1 h. Cultured cells were labeled as described previously (Fukuda et al., 2004). The following antibodies were used to detect antigens: mouse anti-NF (1:200; Chemicon), mouse anti-GFAP (1:200; Sigma-Aldrich), rabbit anti-GFAP antibodies (1:500; DakoCytomation), mouse anti-S100ß antibody (1:500; Sigma-Aldrich), mouse anti-SSEA1 (1:20; Santa Cruz Biotechnology Corp.), and rabbit anti-OLIG2 antibodies (1:2,000; gift from H. Takebayashi, National Institute for Physiological Sciences, Okazaki, Japan), rabbit anti-STAT3 antibodies (1:200; Santa Cruz Biotechnology Corp.), rabbit anti-N-CoR antibodies (1:200; Santa Cruz Biotechnology Corp.), mouse anti-GFP antibody (1:200; Molecular Probe), mouse anti-FLAG M2 antibody (1:200; Sigma-Aldrich), or their combinations, followed by Alexa-dyeconjugated secondary antibodies (1:200; Molecular Probe) or Texas redconjugated antimouse IgM (1:200; Jackson ImmunoResearch Laboratories), and finally stained with propidium iodide (PI; 1 µg/ml) or Hoechst dye 33342 (1 µg/ml; Molecular Probe) to visualize all nuclei. The stained sections or cells were mounted in Citifluor mounting medium (CitiFluor). The fluorescence images were acquired using an upright fluorescence microscope (model LeiTZDMRD; Leica), a CCD camera (model C4742-95; Hamamatsu), 40x and 60x objectives, and an OpenLab software (Improvision). Images were combined for figures using Adobe Photoshop 5.5.
Transfection
Transfection into NSCs was performed using the Nucleofector according to the supplier's instruction (amaxa). On the following day, the cells were harvested and recultured on poly-L-ornithine (15 µg/ml; Sigma-Aldrich) and fibronectin (1 µg/ml; Invitrogen) -coated eight-well chamber slide (Nunc) as described before (Nakashima et al., 1999). The cells were then induced to differentiate into astrocytes. Transfection into Cos7 cells was performed using the Superfect Transfection Reagent according to the supplier's instruction (QIAGEN). On the following day, the cells were harvested and recultured on coverslips for another 2 d before immunolabeling.
Vector constructs
The expression vector of OLIG2-w (FLAGOLIG2pcDNA3) was described before (Fukuda et al., 2004). Mutant form of OLIG2 with the deletion of either NES or AKT phosphorylation site was made using AccuTaq LA DNA polymerase (Sigma-Aldrich) and sets of oligonucleotides containing mutant sequence. The following oligonucleotide DNA primers were synthesized: for the full-length mouse olig2 cDNA, the 5' primer was 5'-TTGAATTCATGGACTCGGACGCCAGCCT-3', and the 3' primer was 5'- AACTCGAGTCACTTGGCGTCGGAGGTGAG-3'. For a mutant in the NES, the sense primer was 5'-ACGGCGCTGGCGGCGCGAAACTACATC-3', and the anti-sense primer was 5'-CGCCGCCAGCGCCGTGGCGATCTTGGA-3'. For a mutant in the AKT phosphorylation site, the sense primer was 5'-AGCAAGGGGGGAGCCAGCAGCGGCTT-3', and the anti-sense primer was 5'-AAGCCGCTGCTGGCTCCCCCCTTGCT-3'. The mutant cDNAs were fused with FLAG in the pcDNA3 expression vector (Invitrogen) as described previously (Fukuda et al., 2004), resulting in FLAGOLIG2NESpcDNA3 (OLIG2NES) and FLAGOLIG2S30ApcDNA3 (OLIG2S30A). pIRES-EGFP (Becton Dickinson) was used as a control vector.
In vitro kinase assay
The in vitro kinase assay was performed following the instructions of the supplier (Cell Signaling Technology). In brief, we transfected 10 µg of either OLIG2-w or OLIG2-S30A to 5 x 106 NSCs using Nucleofector (amaxa). 2 d later, the transfected NSCs were harvested and suspended in cell lysis buffer (Cell Signaling Technology). The cells were sonicated briefly and cell debris was removed by centrifugation. The proteins were immunoprecipitated using anti-FLAG antibody and protein ASepharose (Amersham Bioscience), washed twice in the lysis buffer and three times in the AKT kinase buffer, and were subjected for AKT kinase assay following supplier's instructions (Cell Signaling Technology). The reactions were quenched by addition of SDS-PAGE buffer and analyzed using Western blotting and anti-phospho-AKT substrate antibody (Cell Signaling Technology).
Online supplemental material
Figure S1 shows that PI3K inhibitor LY294002 blocks both S100ß expression and nuclear export of OLIG2 induced by CNTF. Online supplemental materials are available at http://www.jcb.org/cgi/content/full/jcb.200404104/DC1.
Acknowledgments
We are grateful to M. Raff for a critical reading of the manuscript. H. Takebayashi for anti-OLIG2 antibodies, and Y. Hooks for mouse brain sections.
T. Setoguchi was supported by the Yamada Science Foundation. T. Kondo was supported by Merck, Sharp, and Dohme.
Submitted: 19 April 2004
Accepted: 13 August 2004
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