Potential conflict of interest: Nothing to report.
Paradoxical dysregulation of the neural stem cell pathway sonic hedgehog-gli1 in autoimmune encephalomyelitis and multiple sclerosis†
Article first published online: 7 NOV 2008
Copyright © 2008 American Neurological Association
Annals of Neurology
Volume 64, Issue 4, pages 417–427, October 2008
How to Cite
Wang, Y., Imitola, J., Rasmussen, S., O'Connor, K. C. and Khoury, S. J. (2008), Paradoxical dysregulation of the neural stem cell pathway sonic hedgehog-gli1 in autoimmune encephalomyelitis and multiple sclerosis. Ann Neurol., 64: 417–427. doi: 10.1002/ana.21457
- Issue published online: 7 NOV 2008
- Article first published online: 7 NOV 2008
- Manuscript Accepted: 4 JUN 2008
- Manuscript Revised: 30 MAY 2008
- Manuscript Received: 7 SEP 2007
- NIAID. Grant Numbers: AI043496, AI071448
- National Multiple Sclerosis Society. Grant Number: RG3945
- Fidelity Foundation
- Cereer Transition Fellow of the NMSS
- Top of page
- Materials and Methods
- Supporting Information
Neurovascular niches have been proposed as critical components of the neural stem cell (NSC) response to acute central nervous system injury; however, it is unclear whether these potential reparative niches remain functional during chronic injury. Here, we asked how central nervous system inflammatory injury regulates the intrinsic properties of NSCs and their niches.
We investigated the sonic hedgehog (Shh)-Gli1 pathway, an important signaling pathway for NSCs, in experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis (MS), and its regulation by inflammatory cytokines.
We show that Shh is markedly upregulated by reactive and perivascular astroglia in areas of injury in MS lesions and during EAE. Astroglia outside the subventricular zone niche can support NSC differentiation toward neurons and oligodendrocytes, and Shh is a critical mediator of this effect. Shh induces differential upregulation of the transcription factor Gli1, which mediates Shh-induced NSC differentiation. However, despite the increase in Shh and the fact that Gli1 was initially increased during early inflammation of EAE and active lesions of MS, Gli1 was significantly decreased in spinal cord oligodendrocyte precursor cells after onset of EAE, and in chronic active and inactive lesions from MS brain. The Th1 cytokine interferon-γ was unique in inducing Shh expression in astroglia and NSCs, while paradoxically suppressing Gli1 expression in NSCs and inhibiting Shh-mediated NSC differentiation.
Our data suggest that endogenous repair potential during chronic injury appears to be limited by inflammation-induced alterations in intrinsic NSC molecular pathways such as Gli1. Ann Neurol 2008;64:417–427
Multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) are inflammatory and demyelinating diseases of the central nervous system (CNS) characterized by robust perivascular immune cell infiltration, demyelination, astroglia activation, and axonal injury followed by astroglial plaques.1 Although spontaneous remyelination occurs in MS, it is greatly limited.2 Premyelinating oligodendrocytes have been described in MS and EAE lesions.1, 3 However, oligodendrocyte precursor cell (OPC) differentiation and functional remyelination is lacking in MS or EAE lesions, unlike what happens in neurotoxin-induced demyelination.4 In addition, the activation of the subventricular zone (SVZ) niche in EAE and in MS is limited,5, 6 compared with stroke or other models of focal neurodegeneration, where the SVZ stem cells migrate to areas of injury and differentiate to replace the missing cells.7 These results suggest that the microenvironment may be permissive for the neural stem/precursor cells in some diseases and not permissive in others.8
Astroglia are the most abundant cells in the CNS and are part of the typical neural stem cell (NSC) niches in adult forebrain, namely, the SVZ and the dentate gyrus. Astroglia act as multipotent stem cells, as well as niche cells, supporting the NSCs by cell-cell contact and by producing niche molecules.9, 10 It is unclear whether astroglia outside the typical niche can function as niche cells under certain conditions. It is hypothesized that the injury itself could reactivate programs in astroglia and endothelial cells. We have shown that the SVZ niche responds to the inflammatory microenvironment11 in EAE, and those areas of injury create atypical injury-induced niches formed by perivascular as well as parenchymal astroglia and endothelial cells. One of the mediators is stromal cell-derived factor (SDF-1α) that acts through CXCR4 receptor on NSCs, facilitating the migration and differentiation of exogenous stem cells.8, 12 Thus, the area of injury can re-create a transient permissive microenvironment or ectopic niches to facilitate stem cell migration, differentiation, and integration, raising the possibility that other stem cell regulators may be reexpressed in these areas, but confirmation of their role is still lacking.13
Sonic hedgehog is one member of the hedgehog family morphogens that play important roles in development of many tissues and organs. Shh is crucial in regulating stem cell niches and NSC proliferation in postnatal telecephalon,14 adult hippocampus,15 and SVZ.16 Shh is also required for oligodendrogenesis14, 17 and plays a role in OPC migration.18 Shh signaling is tightly regulated; when Shh binds to its receptor Patched1, it releases the inhibition of the associated signaling receptor Smoothened (Smo), resulting in upregulation and nuclear translocation of Gli transcription factors, Gli1, Gli2, and Gli3.19 Increased Shh reactivity has been reported after neural injury,20, 21 and Shh may play a role in neuronal repair and oligodendrocyte maturation.22, 23 However, it is unclear how Shh is regulated during inflammation outside the classic NSC niche in adult CNS.
Here, we demonstrate that inflammation in EAE and MS induces reactivation of the developmental molecule Shh but paradoxically inhibits its downstream transcription factor Gli1. We show that nonniche astroglia regulate the NSCs differentiation by inducing the expression of Shh. Inflammatory cytokines, specifically interferon (IFN)-γ, can increase Shh in astroglia but paradoxically downregulate Gli1 expression in NSCs, thus disrupting the program of Shh-induced NSC differentiation. These data suggest a novel mechanism underlying the negative effects of inflammation on repair during chronic injury.
Materials and Methods
- Top of page
- Materials and Methods
- Supporting Information
C57BL/6 and ubiquitan C promotor controlled green fluorescent protein (GFP) transgenic C57BL/6 mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Myelin oligodendrocyte (MOG) specific T cell receptor (TCR) transgenic 2D2 mice were provided by Dr Vijay K. Kuchroo of Harvard Medical School. All mice were housed according to National Institutes of Health guidelines, and the Animal Care Committee of Harvard University approved all experiments.
Human Brain Specimen
Human brain samples were obtained from the Rocky Mountain MS Center Tissue Bank (Englewood, CO) and the Human Brain and Spinal Fluid Resource Center, University of California (Los Angeles, CA). MS samples from 25 cases are listed in Supplemental Table 1 (see supplementary method for details).
Primary Neural Stem Cell and Astroglia Isolation and Culture
Embryonic day 14.5 (E14.5) or E12.5 NSCs were isolated from cerebral cortices of timed C57BL/6 or UBC-GFP mouse embryos. Adult NSCs and astroglia were isolated from the SVZ or the spinal cords of adult C57BL/6 mice, respectively.11 For the cytokine treatment experiment, astroglia were isolated from the neonatal mice hemispheres on postnatal day 0 (see supplemental method for details).
Neural Stem Cell Differentiation
Neurospheres initially cultured in fibroblast growth factor/epidermal growth factor (FGF/EGF)–containing media were plated on poly-D-lysine–coated coverslips or on top of adult astroglia monolayer without FGF/EGF for 5 days in presence of 5μg/ml Shh neutralizing antibodies (Sigma-Aldrich, St. Louis, MO; or 5E1 from Developmental Studies Hybridoma Bank, Iowa City, IA) or the same concentration of control IgG.
Gli1 Gene Silencing
NSCs were treated with recombinant mouse Shh, amino-terminal peptide (1μg/ml) for 24 hours, then transfected with 2μM SMARTpool Gli1 small, interfering RNA (siRNA) (Dharmacon, Lafayette, CO) with mouse NSC nucleofector device (Amaxa, Gaithersburg, MD). Suppression of RNA expression was evaluated after 24 hours and 4 days.
The comparisons for percentage of cells in this article were presented as mean ± standard deviation. We used unpaired t test with Welch's correction for statistical analysis of percentage data and expression profiles, and Mann–Whitney U test for fold difference data using Prism 4.0.
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- Materials and Methods
- Supporting Information
Stem Cell Regulator Shh Is Upregulated by Astroglia in Multiple Sclerosis and Experimental Autoimmune Encephalomyelitis
We found that Shh is expressed in the SVZ (see Supplemental Fig 1A) and constitutively expressed at low levels in the spinal cord of adult naive mice, where it colocalizes with glial fibrillary acidic protein (GFAP) (see Supplemental Fig 1B). We then examined Shh expression in normal human brain and chronic active MS (Figs 1A, E). We found significant upregulation of Shh in hypertrophic astroglia (see Fig 1F) associated with enhanced GFAP expression (see Figs 1G, H) in the MS lesions compared with normal brain (see Figs 1B–D). Shh expression was most intense at the border of the lesions and the adjacent normal-appearing white matter, and less so at the center of the lesions (see Fig 1K). Shh expression profile of astroglia from MS was much greater than in normal brain (see Figs 1I, J).
We next examined the effect of the CNS inflammation on Shh expression during EAE. Shh expression was profoundly increased in hypertrophic astroglia in the spinal cord during the course of EAE, associated with the increased expression of GFAP. The upregulation of Shh was observed as early as day 10 after immunization, before the onset of clinical signs, peaking between days 20 and 30 (see Supplemental Figs 1B, B′). Furthermore, Shh was also upregulated in perivascular astroglia (see Supplemental Fig 1C) that have been proposed as atypical niches in stroke and EAE.8, 12, 13 Shh was not upregulated in the SVZ niche but was significantly increased in the corpus callosum astroglia of diseased animals (see Supplemental Figs 2A–D). The upregulation of Shh was confirmed by Western blot of EAE spinal cord from day 20 after immunization (see Supplemental Fig 1D) and its colocalization with GFAP confirmed by fluorescence-activated cell sorting (FACS) analysis (see Supplemental Fig 1E). Slight upregulation of Shh was also detected in neurons in the EAE spinal cord. No Shh expression was detected on oligodendrocyte lineage cells (data not shown). No change in the stem cell regulators bone morphogenetic proteins (BMP) 2 and 4 were observed in EAE (see Supplemental Fig 2E).
Next, we investigated the specific inflammatory mediators that may upregulate Shh in astroglia. Astroglia from postnatal day 0 were cultured for 7 days with a panel of cytokines including IFN-γ, transforming growth factor-β (TGF), interleukin-4 (IL), -10, and -17 or were cocultured with splenocytes from MOG-TCR transgenic mice that had been activated with MOG in vitro. Western blot analysis showed that most of the cytokines tested were able to upregulate Shh on astroglia, but IFN-γ was the most potent inducer of Shh compared with untreated cells (p < 0.05) (see Supplemental Fig 3A). The astroglial upregulation of Shh by IFN-γ was confirmed by immunofluorescence staining (see Supplemental Fig 3B).
Adult Astroglia Regulate Neural Stem Cell Neuronal and Glial Differentiation by Shh
We hypothesized that adult spinal cord astroglia may play a role in maintaining endogenous progenitor population by providing a favorable microenvironment similar to a typical stem cell niche. To test this hypothesis, we cultured GFP-tagged E12.5 NSCs on a monolayer of adult spinal cord astroglia in serum-free and FGF/EGF-free conditions for 5 days. Differentiation of NSCs in medium alone was low (see Supplemental Fig 4C), but when cocultured with astroglia, there was an increase in the number of differentiated progeny. Immunofluorescence staining for neurons microtubule-associated protein 2 (MAP2), oligodendrocyte precursors (NG2), and astroglia (GFAP) among the GFP+ cell population (Fig 2A) showed that coculture with astroglia induced differentiation of a substantial number of neurons (13.7 ± 7.1%; see Fig 2B), as well as oligodendrocyte precursors (17.4 ± 3.7%; see Fig 2C) and astroglia (71.8 ± 5.0%; this number may not represent total differentiated astroglia because the GFAP can be expressed in a portion of undifferentiated NSCs as well; see Fig 2D). Addition of anti-Shh neutralizing antibody to the coculture inhibited differentiation compared with rat IgG control (see Fig 2A). Blocking Shh reduced neuronal differentiation by 70% (5.2 ± 4.9%; p = 0.0070) and oligodendrocyte precursors by 50% (8.8 ± 3.7%; p < 0.0001), which is comparable with FGF/EGF withdrawal–induced differentiation. Interestingly, the GFAP+ astroglia differentiation was totally abrogated by anti-Shh antibody (p < 0.0001). We also found that the maturation of the differentiated neurons was greatly delayed by blocking the Shh pathway (see Fig 2A, insets), as shown by a significant reduction of neurites and branch-point complexity (see Figs 2E, F) (p < 0.0001). These data suggest that adult spinal cord astroglia can induce NSC neuronal, oligodendroglial, and astroglia differentiation, and that this process is at least partially mediated by Shh.
Unique Role of Gli1 during Shh-Induced Neural Stem Cell Differentiation
To confirm the role of Shh-induced differentiation and investigate the downstream mediators, we pretreated NSCs with Shh, followed by FGF/EGF withdrawal–induced differentiation in the presence of Shh. Under conditions of FGF/EGF withdrawal, NSCs stop proliferating and start to downregulate the expression of the immature marker nestin. Notably, on day 4 after FGF/EGF withdrawal, 77.4% of the NSCs remained nestin-positive, whereas only 26% of the cells pretreated with Shh were nestin-positive (see Supplemental Fig 4A). Furthermore, Shh-treated NSCs generated significantly more MAP2+ neurons (22.6 ± 6.0%) than those under FGF/EGF withdrawal (17.0 ± 2.3%; p = 0.0019) (see Supplemental Fig 4D). Similarly, Shh promoted more NG2+ OPCs (p = 0.0003) (see Supplemental Fig 4F) but significantly less GFAP+ astroglia (p < 0.0001) (see Supplemental Fig 4E) than control. Interestingly, although control cultures showed no O4+ cells on day 5, we observed a substantial number of O4+ cells in the Shh-treated culture (see Supplemental Fig 4C). Differentiation marker and O4-positive cells started to emerge as early as day 3 in Shh-treated conditions (see Supplemental Fig 4B), suggesting that Shh accelerates oligodendrocyte differentiation. These data confirm the critical role of Shh on oligodendrocyte differentiation.
Shh signaling is mediated by the transcription factors Gli1, Gli2, and Gli3. Gli1 was found to be important in the Shh-responding NSCs,24 but less is known about whether it plays the major role in NSC differentiation during development and in disease. To identify the Shh downstream signals in NSCs differentiation, we examined Gli1, Gli2, and Gli3 messenger RNA (mRNA) expression in Shh-treated E14.5 NSCs. We found 10-fold induction of Gli1 by Shh but no significant induction of Gli2 and Gli3, suggesting that Gli1 is the predominant signaling pathway of Shh in NSCs (Fig 3A). We confirmed the induction of Gli1 at the protein level by intracellular staining of Gli1 in Shh-stimulated NSCs. Shh-treated cells showed about threefold Gli1 induction (mean fluorescence intensity [MFI], 35.1) compared with Shh-untreated cells (MFI, 12.9) and secondary antibody control (MFI, 1.73) (see Fig 3B).
We confirmed the role of Gli1 in NSC differentiation by performing transient knockdown of Gli1. We evaluated the effects of Gli1 siRNA on the differentiation of Shh-treated E14.5 NSCs. Gli1 mRNA level was knocked down by 71.3% 24 hours after siRNA transfection compared with the cells with mock transfection (see Fig 3C). Reduction of Gli1 protein persisted for at least 4 days after the transfection as detected by FACS analysis, as shown by MFI reduction from 147 to 38.2 (secondary control, 2.44) (see Fig 3D). We observed a significant reduction of MAP2+ neuronal differentiation in siRNA-transfected cells (12.5 ± 4.5%), compared with 19.8 ± 3.7% in mock-transfected cells (p = 0.0009), and an increase in GFAP+ astroglial differentiation in siRNA-transfected cells (58.4 ± 8.8%), compared with 23.6 ± 6.9% in control cells (p < 0.0001) (see Figs 3E–G). The effect of siRNA transfection on oligodendrocyte differentiation could not be evaluated because of failure of OPC differentiation even after mock transfection, because OPCs are more sensitive to environmental damage.25 These data confirm the critical role of Gli1 in mediating Shh-induced NSC differentiation.
Interestingly, we found that neurons preserve Gli1 immunoreactivity and aggregation in the nuclei of MAP2+ cells (see Supplemental Figs 5A, B), but GFAP+ astroglia had low expression without nuclear localization and aggregation (see Supplemental Figs 5C, D). We confirmed these findings by FACS analysis showing that MAP2+ neurons express greater levels of Gli1 (MFI, 149) compared with GFAP+ astroglia (MFI, 75) (see Supplemental Fig 5E), suggesting a role of Gli1 in mature neuron function.
In Vivo Inflammation Suppresses Gli1 Expression in Experimental Autoimmune Encephalomyelitis and Multiple Sclerosis Lesions
Because we found increased Shh expression in the CNS after injury, we examined the expression of Shh receptor and downstream signaling molecules in EAE and MS. The Shh receptor smoothened (Smo) expression was upregulated in the spinal cord in EAE with kinetics similar to the upregulation of Shh (see Supplemental Fig 6A). Interestingly, despite a slight increase in Gli1 mRNA before the onset of EAE (day 10), we found Gli1 expression markedly decreased after the onset of the EAE (day 16) and throughout the course of the disease (Fig 4A). By immunofluorescence staining, we found a significant amount of Gli1 immunoreactivity localized in the nuclei of OPCs and neurons as expected (see Fig 4B; see Supplemental Figs 6B, C), whereas astroglia show very low Gli1 immunoreactivity in both EAE and naive CNS (see Supplemental Fig 6D). However, we observed a reduction of Gli1 expression in the spinal cord OPCs during the peak and chronic phases of EAE (see Fig 4B). The MFI of Gli1 expression in the naive OPCs was 177.01 ± 31.94 compared with 70.55 ± 34.4 and 104.95 ± 40.17 on days 18 and 30 after immunization, respectively, in EAE mice (see Fig 4C). Compared with the naive mice, the OPCs in EAE mice exhibited significant downregulation of Gli1 (p < 0.001). Although Gli1 was expressed by neurons, there is no significant difference in Gli1 expression between naive and EAE animals (see Supplemental Fig 6C), thus confirming that the reduction of Gli1 expression in EAE spinal cords was due to the downregulation of Gli1 in the OPCs.
Olig1 and Olig2 genes are important for remyelination,26 and Olig genes were shown to be regulated by Shh.27 We observed a similar regulation in EAE; Olig2-expressing cells are increased in all stages of EAE (see Supplementary Fig 7A), but we did not detect significant changes in Olig1 and Olig2 expression levels during EAE progression (see Supplementary Fig 7B).
We further examined Gli1 expression in MS lesions of different stages and in normal controls (see Figs 4D, E). Consistent with our findings in EAE, Gli1 mRNA was upregulated in active lesions but was significantly decreased in chronic active lesions (p = 0.0025) and inactive lesions (p = 0.0031) from MS brain compared with normal brain, suggesting that long-term inflammation impairs Gli1 signal in chronic MS lesions. Thus, despite the notable increase of Shh in the lesions, Gli1, the critical downstream mediator for NSC differentiation, is decreased in MS and EAE. Taken together, our data suggest that downregulation of Gli1 signal in the OPCs may contribute to the impaired oligodendrocyte maturation in EAE and MS.
Interferon-γ Suppresses Gli1 Expression and Perturbs the Shh-Induced Neural Stem Cell Differentiation
To identify the mechanism of Gli1 downregulation by inflammation, we analyzed Gli gene expression in E14.5 NSCs in the presence of inflammatory cytokines. As shown in Figure 5A, Gli1 expression was reduced threefold by IFN-γ. We focused on Gli1 because it is the major transcription factor of Shh in NSCs. There were less than twofold change in Gli2 and Gli3 regulation by the cytokines, which is considered not significant for real-time quantitative RNA assay (see Supplemental Figs 8A, B). IFN-γ suppressed Shh-induced upregulation of Gli1 in embryonic NSCs (see Fig 5B) and adult SVZ NSCs (see Supplemental Fig 8C) in a dose-dependent manner, whereas it paradoxically increased Shh mRNA expression by more than 100-fold (see Fig 5C) and Shh protein levels by more than 2-fold (see Fig 5D). Thus, IFN-γ inhibits Shh-Gli1 signaling in the Shh responder cells despite upregulating an autocrine increase in Shh in NSCs.
We then examined the consequences of IFN-γ–mediated Shh-Gli1 dysregulation on NSC differentiation. IFN-γ was added to Shh-induced NSC differentiation before FGF/EGF withdrawal and after Gli1 had already been induced by Shh. Shh-treated neurospheres attached to the poly-D-lysine–coated surface, and NSCs migrated out of the neurospheres and differentiated into neurons, oligodendrocytes, and astroglia. In contrast, the IFN-γ–treated neurospheres remained unattached with few cells migrating out and remained in an undifferentiated state (see Fig 5E). This was not due to IFN-γ toxicity on NSCs because we found no difference in the percentage of apoptotic or necrotic cells by Annexin V and 7-AAD staining (see Supplemental Fig 8D). Taken together, our data suggest that inflammatory cytokines such as IFN-γ paradoxically inhibit Gli1 that mediates NSC differentiation even in the presence of exogenous Shh or autocrine increase in Shh by the NSCs.
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- Supporting Information
Adult stem cells are viewed as repositories for repair potential. But it is becoming clear that repair potential requires both stem cell competence and a permissive microenvironment. The mobilization of endogenous precursor cells has been proposed as a strategy to increase the regeneration in neurological diseases. In MS, progenitor cells are seen around the lesions, but there is a lack of mature oligodendrocytes.1, 3 The lack of repair in MS has been attributed to increased Jagged 1 or hyaluronan in lesions that impairs the maturation of OPCs.26, 28 Here, we show that astroglia contribute to the creation of ectopic niches during EAE and produce Shh that should lead to differentiation of NSCs. However, the inflammatory microenvironment, specifically IFN-γ, perturb the Shh-induced differentiation by downregulating Gli1 in vivo and in vitro. Our data suggest that inflammation may initiate the establishment of ectopic niches in the CNS, but chronic inflammation impairs effective repair.
Glial cells have well-established roles that include maintaining the ionic milieu of neurons, modulating synaptic action by controlling the uptake of neurotransmitters, and providing a scaffold during neural development.29 Furthermore, astroglia in the hippocampus and SVZ are multipotent stem cells, and also function as niche cells supporting stem cell proliferation and maturation.9, 30 A large number of neural progenitor cells, such as OPCs and doublecortin (Dcx)+ migrating neuronal progenitors, are maintained in the adult CNS and mobilized during CNS injury.31, 32 The local microenvironment, especially adult astroglia, may contribute to the progenitor response, raising the question whether astroglia outside the typical niche areas could become niche-like cells under circumstances such as injury or inflammation. Our data show that Shh is expressed in adult spinal cord astroglia at a low level under normal conditions, and that astroglia support neurogenesis and oligodendrogenesis. Thus, we demonstrate that astroglia outside the niche areas can sustain the differentiation of NSCs. We also report that this effect is mediated in part by Shh, because Shh blockade inhibits neuronal and oligodendrocyte differentiation of NSCs cocultured with astroglia.
We further observed that Shh itself is a potent mediator of neuronal and oligodendroglial differentiation, and it signals through the transcription factor Gli1. Interestingly, astroglia and maturing oligodendrocytes did not have significant expression of Gli1, whereas postmitotic neurons preserve high levels of Gli1 in the nuclei, suggesting that Gli1 can control neuronal function, because motor neurons can upregulate Shh and its receptor Smo after injury in adult rats20 and in mouse EAE (our observation).
Hedgehog signaling has been postulated to play a pivotal role in healing and repair processes,33 and inappropriate activation of this pathway has been implicated in several types of cancer.34 In vertebrates, the Gli genes, Gli1, Gli2, and Gli3, mediate the Shh morphogenetic signal by regulating expression of Shh target genes. Although the three Gli molecules function combinatorially in a context-dependent manner, the response to Shh signaling largely relies on regulation of Gli1 transcripts.35 Our observations highlight the fact that the Shh-Gli1 signal is critical for NSC differentiation, which mediates the astroglia-induced neuronal and oligodendroglial differentiation.
Olig1 and Olig2 genes are important in remyelination in models of toxin-induced demyelination and are also expressed in MS lesions.36 Shh is believed to be critical for Olig gene expression during development as demonstrated by gain- or loss-of-function studies.27 We have observed dysregulation of Shh-Gli1 signaling with upregulated Shh but downregulated Gli1, despite an increase in Olig1+ and Olig2+ cells in chronic EAE. These apparently contradictory findings suggest the complexity of Shh-Olig pathway. For instance, little Shh is expressed in adult CNS even when Olig2 is increased in toxin-induced demyelination model, which also suggests independence of Olig gene activation from Shh.37 Furthermore, there is no evidence of direct correlation between Olig gene expression and the Shh downstream signaling molecule Gli1. Our data suggest that Olig1 and Olig2 genes may be upstream of Gli1, or more likely, use different signal transduction pathways than Gli1 and have different responses to inflammatory signals. On the other hand, our observations show that Shh is involved in differentiation of neural progenitors through Gli1, whereas previous reports have associated Shh and Gli1 with proliferation of NSCs and progenitors.38 These findings suggest a dual function for Gli1 that is context dependent: During conditions of self-renewal, Gli1 helps maintain the NSC proliferation, and during differentiation, it contributes to oligodendrogenesis. This is in keeping with other oligodendrocyte genes such as Olig1/2 genes that have dual roles in NSC proliferation and differentiation.39
During neuronal injury or inflammation, activated astroglia can upregulate a series of molecules including GFAP, major histocompatibility complex class II, but also neural regulatory factors.26 However, the local inflammatory response involves multiple cytokines, and the outcome for the exposed activated astroglia may be supportive or inhibitory for neural precursor cell proliferation and differentiation. Although Shh is a developmental molecule, upregulation of Shh is observed with neuronal injury.20 Shh can be regulated by IFN-γ in cerebellar neurons,40 and we found that activated astroglia in the spinal cord of EAE and in MS lesions strongly upregulate the niche molecule Shh. We further demonstrate in vitro that Shh can be regulated by multiple cytokines, and most potently by IFN-γ. Conversely, it has been reported that Shh is produced by CD4+ T cells, and promotes proliferation and survival of activated T cells.41 We did not detect significant expression of Shh in infiltrating cells in EAE; however, the Shh secreted by reactive astroglia may play a role in local T-cell survival and proliferation, which may further contribute to astroglia activation in a positive feedback loop. Our data suggest that inflammation can redirect mature astroglia from outside the typical adult stem cell niches to form a nichelike microenvironment that can support precursor cell proliferation and differentiation.
In contrast with the favorable changes to the stem cell microenvironment, we demonstrate that the stem cell response to Shh is attenuated by inflammation both in vivo and in vitro. The same proinflammatory cytokine, namely, IFN-γ, also downregulates Gli1 signal in the Shh responder cells, thus directly abrogating the NSC/progenitor cell differentiation induced by Shh. This paradoxical effect of IFN-γ on Shh-producing astroglia and Shh-responder NSCs may explain the fact that neural progenitor cells or OPCs proliferate in MS and EAE but do not undergo full maturation because of impaired Shh-Gli1 signal. Our findings confirm the hypothesis that inflammation initiates the neural repair process, but chronic inflammation impairs repair. This may also suggest that any potential therapy to upregulate Shh signal has to be combined with modulation of inflammation.
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This work was supported by the NIAID (AI043496, AI071448, S.J.K.) National Multiple Sclerosis Society (RG3945, S.J.K.) and Fidelity Foundation to S.J.K. K.C.O. is a Cereer Transition Fellow of the NMSS.
We thank Mr. K. Dole for providing the human brain samples and Dr A. Vaknin-Dembinsky for help in siRNA transfection.
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Additional Supporting Information may be found in the online version of this article.
|ANA_21457_sm_Supfig1.tif||17427K||Supplemental Figure 1. Shh is up-regulated in reactive astroglia in EAE. (A) Immunofluorescence staining for Shh and GFAP in SVZ and corpus callosum (CC) of normal adult mice. Shh is co-localized with GFAP+ astroglia in the SVZ. Scale bar, 50 μm. (B) Immunofluorescence staining for Shh (green) and GFAP (red) in spinal cord white matter from naïve and MOG-induced EAE mice on day 10, 20, 30 post-immunization (n=2 at each time point with three repeats). Scale bar, 50 μm. (B′) Profile analysis on representative astroglial cells shows the expression level of Shh (green) and GFAP (red) in spinal cord white matter from naïve and MOG-induced EAE mice on day 10, 20, 30 post-immunization (n=2 at each time point with three repeats). (C) Shh-expressing reactive astroglia are forming niche-like structures around the proliferating cells in the EAE spinal cord white matter. BrdU was injected to EAE mice daily for 6 consecutive days from 14 dpi. The proliferating cells (majority were infiltrating cells) in day 20 EAE spinal cords were labeled with anti-BrdU antibody (blue) and astroglia were labeled with anti-GFAP (red) and anti-Shh (green). Scale bar, 20 μm. (D) Western blot analysis on the protein lysate from lumbosacral spinal cords shows increased Shh expression on day 20. (E) FACS analysis of astroglia from brain and spinal cord on day 20. There is an increase of both Shh and GFAP in EAE CNS. And there is significant increase of Shh in gated GFAP+ population. Dotted line, secondary antibody control. Blue line, astroglia from naïve mice. Shaded red area, EAE 20 dpi.|
|ANA_21457_sm_Supfig2.tif||14975K||Supplemental Figure 2. BMP and Shh expression in SVZ during EAE. (A, B) (A) Immunofluorescence staining for BMP2 (green) and BMP4 (red) in spinal cord white matter during EAE showing no change compared to naive. Scale bar, 20 μm. for Shh (green) and GFAP (red) staining of SVZ and adjacent corpus callosum (CC) from naïve and MOG-induced EAE mice on 20 days dpi. Scale bar, 50 μm and 20 μm, respectively. (DC) Magnified image of two representative astroglia from the SVZ from naïve and 20 dpi EAE mice and the profile analysis of the Shh (green) and GFAP (red) intensity in the astroglia. The areas of the profile analysis are indicated by the arrow line. Scale bar, 20 μm. (ED) Profile analysis of Shh and GFAP intensity of representative astroglia from the CC of naïve and EAE mice. The chart indicates the intensity of Shh and GFAP alone the arrow line represented areas in the images. The expression of Shh was significantly up-regulated in the astroglia in the CC of EAE (**p< 0.01, average of 40 cells each, unpaired t-test with Welch's correction). Scale bar, 20 μm. The images were acquired using the same parameters. (E) Immunofluorescence staining for BMP2 (green) and BMP4 (red) in spinal cord white matter during EAE shows no change compared to naive. Scale bar, 20 μm.|
|ANA_21457_sm_Supfig3.tif||19944K||Supplemental Figure 3. Upregulation of Shh in astroglia in vitro. (A) Western blot of Shh expression on P0 astroglia treated with IFN-γ, TGF-β, IL-4, IL-10, IL-17 or 7 days of co-culture with MOG-activated MOG-TCR transgenic splenocytes. Shown is the average of three replicates under the same conditions and graphed as a ratio of Shh to β-Actin. *p < 0.05 compared to control by unpaired t-test with Welch's correction. (B) Immunofluorescence staining of Shh on IFN-γ-treated astroglia. Scale bar, 50 μm.|
|ANA_21457_sm_Supfig4.tif||16361K||Supplemental Figure 4. Shh promotes neural stem cell differentiation associated with increased expression of Gli1. (A) Shh treatment accelerates NSC differentiation compared to FGF/EGF withdrawal. Percentage of differentiating NSCs 2 days after FGF/EGF withdrawal as indicated by loss of intracellular nestin expression by FACS. (B) O4+ (Committed oligodendrocyte linage marker) cells emerged as early as three days of differentiation induced by Shh. (C) Immunofluorescence staining for neurons (MAP2+), astroglia (GFAP+), OPCs (NG2+) and committed oligodendrocytes (O4+) by Shh-driven NSC differentiation compared with FGF/EGF withdrawal as control. The percentage of differentiation marker positive cells was averaged from ten 200x fields. Shh treatment significantly increased neuronal (p=0.0019) (D) and oligodendroglial differentiation (p=0.0003) (F), but decreased astroglial differentiation (p<0.0001) (E). The numbers represent the percentage of specific marker positive cells. **p<0.01, ***p<0.001 by unpaired t-test with Welch's correction. Scale bar, 20 μm.|
|ANA_21457_sm_Supfig5.tif||15693K||Supplemental Figure 5. Intracellular localization of Gli1. (A) Neural stem cells were differentiated under FGF/EGF withdrawal plus Shh on PDL-coated coverslips for 5 days, followed by immunofluorescence staining and confocal microscopy analysis for Gli1 (green) and MAP2 (red) showing nuclear localization of Gli1 in neurons. Scale bar, 10 μm (B) 2.5-D profile analysis of Gli1 expression by LSM510 software. (C) Immunofluorescence staining for Gli1 (green) and astroglia (GFAP+, red) and confocal microscopy analysis showing low Gli1 expression without nuclear aggregation. (D) 2.5-D profile analysis of Gli1 expression by LSM510 software. (E) Flow cytometric analysis for Gli1 expression intensity in MAP+ neurons (shaded), GFAP+ astroglia (heavy line) and O4+ oligodendrocytes (dashed line). Thin line, secondary antibody control.|
|ANA_21457_sm_Supfig6.tif||17376K||Supplemental Figure 6. Expression of Shh downstream signals in EAE. (A) Immunofluorescence staining for Shh receptor Smo on spinal cord white matter from MOG-induced EAE mice on days 10, 20, 30 after immunization shows increased expression of Smo. (B) 3-D view of Gli1 localization in the nuclei of NG2+ OPCs in naïve and 30 dpi EAE. (C) Immunofluorescence staining and profile analysis for Gli1 (green) in grey matter neurons (NeuN+, red) from naïve and EAE on days 18 (peak) and 30 (chronic phase) after immunization. (D) Immunofluorescence staining shows Gli1 (green) expression is absent in spinal cord white matter astroglia during EAE (GFAP+, red). Nuclei were counterstained with TO-PRO-3 (blue). Scale bar, 20μm.|
|ANA_21457_sm_Supfig7.tif||35196K||Supplemental Figure 7. Olig1 and Olig2 gene expression in EAE. (A) The number of Olig2+ OPCs was increased during the course of EAE as indicated by the average number of cells per 630x power field. (B) Olig1 and Olig2 immunofluorescence staining of naïve and EAE spinal cord white matter on 10 (before onset), 18-20 (peak) and 30 (chronic) dpi. Scale bar, 50 μm.|
|ANA_21457_sm_Supfig8.tif||35006K||Supplemental Figure 8. Effect of IFN-γ on NSC survival. (A, B) NSCs were treated with Shh together with IFN-γ, TGF-β, IL-4, IL-10 or IL-17 for 48 hours and real-time PCR analysis for Gli2 and Gli3 expression was performed. (C) Dose response of IFN-γ inhibition of Shh-Gli1 signaling in adult NSCs. (D) E14.5 NSCs were treated with IFN-γ for 5 days. Flow cytometry analysis shows no increase of apoptotic (Annexin V+) and necrotic (7-AAD+) death.|
|ANA_21457_sm_SupTab1.doc||25K||Supplemental Table 1. Clinical MS lesion and control patient information used in Gli1 expression study|
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