VCAM1 Is Essential to Maintain the Structure of the SVZ Niche and Acts as an Environmental Sensor to Regulate SVZ Lineage Progression

Cell Stem Cell

Article VCAM1 Is Essential to Maintain the Structure of the SVZ Niche and Acts as an Environmental Sensor to Regulate SVZ Lineage Progression Erzsebet Kokovay,1 Yue Wang,2 Gretchen Kusek,2 Rachel Wurster,2 Patty Lederman,2 Natalia Lowry,2 Qin Shen,3,4,* and Sally Temple2,4,* 1Department

of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA Stem Cell Institute, Regenerative Research Foundation, Rensselaer, NY 12144, USA 3Center for Stem Cell Biology and Regenerative Medicine, School of Medicine, Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing, 100084, China 4These authors contributed equally to this work *Correspondence: [email protected] (Q.S.), [email protected] (S.T.) http://dx.doi.org/10.1016/j.stem.2012.06.016 2Neural

SUMMARY

Neurons arise in the adult forebrain subventricular zone (SVZ) from Type B neural stem cells (NSCs), raising considerable interest in the molecules that maintain this life-long neurogenic niche. Type B cells are anchored by specialized apical endfeet in the center of a pinwheel of ependymal cells. Here we show that the apical endfeet express high levels of the adhesion and signaling molecule vascular cell adhesion molecule-1 (VCAM1). Disruption of VCAM1 in vivo causes loss of the pinwheels, disrupted SVZ cytoarchitecture, proliferation and depletion of the normally quiescent apical Type B cells, and increased neurogenesis in the olfactory bulb, demonstrating a key role in niche structure and function. We show that VCAM1 signals via NOX2 production of reactive oxygen species (ROS) to maintain NSCs. VCAM1 on Type B cells is increased by IL-1b, demonstrating that it can act as an environmental sensor, responding to chemokines involved in tissue repair. INTRODUCTION The SVZ of the lateral ventricle in the adult brain harbors NSCs that continue to generate new neurons throughout life, and it is the largest neurogenic niche in the adult brain (Doetsch and Alvarez-Buylla, 1996). Prior work has shown that the SVZ lineage is initiated from slowly dividing Type B NSCs, which produce the transit-amplifying Type C cells that in turn produce Type A neuroblasts, and these migrate anteriorly along the rostral migratory stream (RMS), eventually differentiating into neurons that incorporate into olfactory bulb circuits (AlvarezBuylla et al., 2001). Recent studies have begun to elucidate the 3D structure of this adult stem cell niche. The germinal zone, which is only approximately 50 mm thick, is sandwiched between two layered structures: on the parenchymal side, the SVZ is bounded by an exten220 Cell Stem Cell 11, 220–230, August 3, 2012 ª2012 Elsevier Inc.

sive planar network of blood vessels, the SVZ plexus, and on the ventricular side, by the layer of ciliated ependymal cells (Kazanis et al., 2010; Luo et al., 2008; Mirzadeh et al., 2008; Shen et al., 2008; Tavazoie et al., 2008). A subpopulation of Type B cells have apical processes that penetrate the ependymal layer, and some of these also have a basal process projecting onto the SVZ plexus blood vessels, thus spanning these two important humoral compartments (Mirzadeh et al., 2008; Shen et al., 2008). The apical processes are typically in groups, arising from a cluster of Type B cells, and the adjacent ependymal cells form a pinwheel around this center, like petals around the central stigma in an inflorescence; these pinwheels are specific to the striatal germinal zone (Mirzadeh et al., 2008) (Figure 1). Thus, by interacting with the ependymal layer, the apical processes of Type B cells could serve as anchors to provide structural integrity to the niche, and they are positioned perfectly to sense changes in signals in the adjacent humoral compartment, the cerebrospinal fluid (CSF). In prior studies, we demonstrated that NSCs can home to the SVZ vascular plexus using molecules that also are important in hematopoietic stem cell (HSC) homing: stromal differentiation factor-1 (SDF1) and its receptor, chemokine (C-X-C motif) receptor 4 (CXCR4) (Kokovay et al., 2010). However, as yet, the molecules responsible for positioning and holding the apical endfeet of Type B cells within the ependymal layer are not known. In the bone marrow, once HSCs home to the bone marrow niche attracted by SDF1/CXCR4, they are held in place by binding to VCAM1, expressed on the surface of bone marrow stromal cells (Lapidot et al., 2005; Papayannopoulou et al., 1995). VCAM1 expression is upregulated in response to cytokines, increasing adhesion of HSCs in the bone marrow niche (Simmons et al., 1992). Given that the importance of SDF1/ CXCR4 for stem cell homing is conserved between blood and brain, we investigated whether VCAM1 function is similarly conserved in the brain niche. Strikingly, we found that bright anti-VCAM1 staining is located on the endfeet of Type B cells in the center of pinwheels. Blocking VCAM1 function severely disrupted the niche structure, with loss of the pinwheels, massive activation of the quiescent Type B cells, and consequent depletion of the NSC population. Hence, we identify VCAM1 as a key molecule responsible for

Cell Stem Cell VCAM1 Is Essential for the Neural Stem Cell Niche

Figure 1. VCAM1 Is Expressed on the Apical Processes of Type B Cells (A) VCAM1 (red) is strongly expressed on the surface of the striatal side of the lateral ventricle and the dorsal wedge (arrow), but not on the medial or septal side or in the overlying corpus callosum, as shown in this low-magnification image of a coronal section. (B) High-magnification image of a coronal section showing bright punctate VCAM1 expression (green) on GFAP+ (red) processes. The GFAP+ cells have thin radial processes (arrows in lower panel). (C) En face view of the SVZ wholemount showing VCAM1+ (red) staining in the center of ependymal pinwheels revealed by b-catenin (blue). (D) Immunocytochemistry of VCAM1 (green) and GFAP (red) showing polarized membrane expression on cultured SVZ cells. Blue, DAPI nuclear staining. (E) High-magnification confocal projection image angled 45
showing VCAM1 (red) cupping GFAP processes. (F) High-magnification confocal image of the most superficial layer of the SVZ wholemount showing VCAM1 (red) on GFAP (green) cells in the center of pinwheel structures revealed by anti-b-catenin (white). LV, lateral ventricle; stm, striatum; cc, corpus callosum; sept, septum. Scale bars: 20 mm. See also Figure S1.

maintaining the integrity of the neurogenic niche and the NSC state. RESULTS VCAM1 Is Concentrated on the Apical Processes of Type B Cells in the Adult Mouse SVZ VCAM1, also known as CD106, is a member of the immunoglobin (Ig) superfamily. It is a cell surface sialoglycoprotein first identified on cytokine-activated endothelium (Osborn et al., 1989). By immunohistochemistry of coronal sections, confirmed by in situ hybridizations (Figure S1 available online), we observed strong punctate staining of VCAM1 along the striatal side of the ventricular wall and the dorso-lateral edge of the SVZ, which contains many aligned neuroblast chains flowing into the RMS, but not on the septal side of the ventricular wall (Figure 1A), indicating specific expression in the neurogenic niche. Double immunolabeling showed that all of the VCAM1+ punctate structures near the ventricular surface were associated with GFAP+ cells (n = 134 cells), and high magnification revealed that these structures were the apical endfeet of GFAP+ Type B cells, which penetrate into the ependymal layer and protrude (to varying extents) into the lateral ventricle (Figure 1B). These VCAM1+ GFAP+ cells typically extended radial processes

basally into the SVZ toward the SVZ plexus layer (Figure 1B, arrows). In order to understand better the VCAM1+ endfeet structures in relation to the SVZ cytoarchitecture, we immunostained SVZ wholemounts, an organotypic preparation of the SVZ in which the normal 3D cell-cell relationships are preserved (Doetsch et al., 1999a). When viewed en face from the ventricular side, a distinctive pattern of staining emerged that was visible with different VCAM1 antibodies. Using anti-b-catenin to reveal the ependymal junctions in the SVZ wholemounts, we saw that the VCAM1+ endfeet were intermingled in the ependymal layer, singly or in groups, sometimes of more than 10 (Figure 1C). Interestingly, polarized VCAM1+ staining was visible on the membrane of cultured, elongated GFAP+ adult SVZ cells, (Figure 1D), indicating that asymmetric localization of VCAM1 can be preserved in vitro. In wholemounts immunostained for GFAP, the VCAM1+ endfeet typically overlapped or abutted GFAP+ elements in the center of the pinwheels (Figures 1E and 1F). Advancing from this surface layer down into the SVZ tissue, most of the VCAM1 staining disappeared rapidly, and the vast majority of Type C cells, revealed by Tomato fluorescence in a Mash1-Cre-Tomato mouse (Ascl1tm1.1(cre/ERT2); B6.CgGt(ROSA)26Sortm9(CAG-tdTomato)) (Kim et al., 2011) (Figure S1), or staining for the oligodendrocyte transcription factor 2 Cell Stem Cell 11, 220–230, August 3, 2012 ª2012 Elsevier Inc. 221

Cell Stem Cell VCAM1 Is Essential for the Neural Stem Cell Niche

Figure 2. Blocking VCAM1 Disrupts the Type B and Ependymal Cytoarchitecture (A and B) Confocal images of the SVZ wholemount stained for b-catenin (green) to reveal the ependymal layer, and stained for GFAP (red) to reveal the Type B cells, comparing the effect of control antibody infusion (A) with that of infusion of VCAM1 blocking antibody (B) for 6 days in vivo. (C) Images illustrating the dramatic changes in GFAP+ processes using wholemounts from a GFAP-GFP (green) transgenic mouse also immunostained for GFAP (red) after VCAM1 blocking or control antibody treatment. (D) Increased GFAP-GFP+ (green) Ki67+ (red) cells following VCAM1 block. (E) Quantification of (D). (F) The number of GFAP-GFP+ apical Type B cells is significantly reduced by VCAM1 block. (G) Quantification of EdU+ cells in the olfactory bulb, 3 weeks after pump removal and EdU injection. (H) Quantification of EdU+ cells in the SVZ. Scale bars: 20 mm. Error bars = SEM. See also Figure S2.

(OLIG2), were negative, as were doublecortin (DCX)+ Type A cells (not shown). Thus, VCAM1 expression is rapidly downregulated with lineage progression. Blocking VCAM1 Disrupts the Position and Function of Cells in the SVZ Niche The specific expression pattern of VCAM1, which has known adhesion properties, suggested it could be important in maintaining Type B cell position, with their apical processes intercalated in the ependymal layer. To test whether inhibiting VCAM1 function would disrupt the niche architecture, we used an 222 Cell Stem Cell 11, 220–230, August 3, 2012 ª2012 Elsevier Inc.

osmotic minipump to continuously deliver VCAM1 blocking antibody or isotype control antibody into the lateral ventricle of hGFAP-GFP mice for 6 days, then examined the resulting SVZ wholemounts. Inspection of the ependymal layer showed a dramatic effect of blocking VCAM1. In the SVZs from animals treated with isotype control antibodies, b-catenin staining revealed the normal honeycomb-like pattern and a recognizable pinwheel arrangement of ependymal cells around concentrations of GFAP+ processes (Figure 2A). In contrast, the SVZs from animals treated with VCAM1 blocking antibodies showed pockets of severe

Cell Stem Cell VCAM1 Is Essential for the Neural Stem Cell Niche

disruption in the ependymal layer, revealed by abnormal knots of b-catenin staining and bundles of GFAP+ processes, with loss of many of the basic pinwheel structures (Figure 2B). Interestingly, in addition to an increase in b-catenin, which could indicate an increase in adherens junctions, we observed upregulation of the tight junction protein ZO-1 in VCAM1-blocking-antibodytreated mice (Figure S2). This suggests that perturbations in SVZ cell-cell interactions could contribute to the observed increase in Type B cell division. Disruption was also seen in the GFAP+ apical Type B cell population located just beneath the ependymal layer (Figures 2B and 2C). The number of GFAP-GFP+ cell bodies was dramatically reduced, to 44.8% of control (control, 1,915 ± 292.6 versus 858.2 ± 237.2 after VCAM1 block, p < 0.03) (Figures 2B and 2F), and their processes, normally beautifully aligned, especially in the dorsal aspect of the SVZ, were disarrayed and shorter (Figure 2C). Blocking VCAM1 did not increase apoptotic cell death, so that is unlikely to be the cause of the GFAP+ cell loss (caspase 3 staining, not shown). Type B cells situated at the apical surface rarely divide (Doetsch et al., 1997, 1999a, 1999b; Mirzadeh et al., 2008; Morshead et al., 1994; Shen et al., 2008), supporting the concept that the ependymal layer forms a quiescent niche for these cells. Notably, VCAM1 inhibition stimulated apical GFAP+ Type B cell proliferation, so that there were significantly more GFAP/Ki67-double positive cells within 9 mm of the ependymal layer where the majority of Type B cells reside (Doetsch et al., 1997; Mirzadeh et al., 2008; Shen et al., 2008) (7-fold increase from 3.46% ± 1.49% in control to 22.85% ± 5.76% after anti-VCAM1 treatment, p < 0.02, Figures 2D and 2E). The number of MASH1+ Type C cells in the SVZ after 6 days of blocking VCAM1 was reduced, although not significantly (44.51 ± 12.60 for control versus 28.65 ± 5.34 for anti-VCAM1, average cells per stack, p = 0.28) (Figure S2). We labeled cells in S phase by injecting mice with EdU on days 4, 5, and 6 after insertion of pumps with VCAM1 or control antibody, and we let the mice survive for 3 more weeks as a chase period. The EdU label is retained by newborn neurons that migrate to the olfactory bulb and by slowly dividing progenitor cells that remain in the SVZ, which are thought to represent dormant stem cells (Johansson et al., 1999). Blocking VCAM1 resulted in more cells leaving the SVZ and migrating to the olfactory bulb (VCAM1 block, 146.8 ± 25.16 EdU+ cells/field in the olfactory bulb; control, 82.48 ± 5.55, p < 0.05). In contrast there were significantly fewer label-retaining cells left in the SVZ (VCAM1 block, 5.05 ± 0.87 EdU+ cells in the SVZ; VCAM1 block, 12.85 ± 0.78, p < 0.0006 Figures 2G and 2H). These results show that VCAM1 is critical for the organization of Type B cells and ependymal cells at the ventricular surface and suggest that blocking VCAM1 results in loss of the more quiescent ependymal niche, with activation of Type B cells to a proliferative state. The trend toward fewer transit amplifying Type C cells after 1 week and more olfactory neurons indicates rapid depletion of the early stages of the lineage and increased migration out of the SVZ. Inhibiting VCAM1 Function Disrupts Neuroblast Chain Migration In several wholemount preparations we found that neuroblast chains in the SVZ were severely disrupted after VCAM1 function

was blocked, with abnormal clumps of DCX+ neuroblasts visible even at low magnification, and denuded areas where chains were severely reduced (Figure 3A). Confocal imaging revealed that DCX+ cells were bunched together, wrapped by disorganized Type B cells (Figures 3B and 3C). Interestingly, in sagittal sections we also observed several patches of neuroblast chains outside the SVZ in the striatal parenchyma (Figure 3D, arrow). As we did not see expression of VCAM1 on neuroblasts in vivo (Figure S1), disruption of neuroblast chains is most likely indirect. Neuroblast disruption could be due to disorganization of the associated GFAP+ processes, or to disruption of the ependymal cilia that normally direct neuroblast chain migration (Sawamoto et al., 2006); indeed, after treatment with VCAM1-blocking antibody, there were patches in the SVZ where cilia were disorganized or absent (Figure S3). VCAM1 Is Required for NSC Maintenance Given that Type B NSCs rather specifically express VCAM1, and given its ability to act as a signaling molecule in other systems, we investigated whether VCAM1 function impacted cardinal features of NSC behavior, including self-renewal and the ability to produce differentiated progeny. We generated a lentiviral vector expressing Vcam1 shRNA that gave over 90% transduction and reduced the Vcam1 level in adult SVZ cells to 59.61% ± .02% of empty vector (EV) control (Figure 4A). We tested the impact of reducing Vcam1 levels on neurosphere formation under conditions that enable a measure of NSC self-renewal (Pastrana et al., 2011), and on cells plated in adherent conditions to assess an impact on differentiation. As in vivo, the vast majority of VCAM1+ cells in vitro, in neurosphere, or in adherent cultures are GFAP+ (>95%), and we rarely observed (<1%) VCAM1 expression on neuroblasts. shRNA knockdown of Vcam1 had no effect on primary neurosphere formation, but it dramatically reduced secondary neurosphere formation to 44.70% ± 4.47% of control and tertiary neurosphere formation to 29.84% ± 2.39% of control (***p < 0.001) (Figures 4B and 4C). Similarly, blocking with VCAM1 antibody did not significantly affect primary neurosphere generation, but reduced secondary neurosphere formation to just 38.07% ± 6.61% of control (p < 0.01) (Figure 4D). These findings are consistent with Vcam1 being important for maintaining the NSC state, and the rapid loss of Type B and Type C cells seen after blocking VCAM1 in vivo would be expected to result in fewer neurosphere-generating cells, as observed here. Dissociated cultures of SVZ cells treated with shRNA Vcam1 lentivirus or EV control lentivirus for 4 DIV showed reduced Nestin+ progenitor cells and fewer cells expressing the proliferation marker Ki67, but increased numbers of EGFR+ cells, a marker of activated Type B and Type C cells, and DCX+ Type A neuroblasts. These observations are consistent with in vivo results showing that blocking VCAM1 stimulates lineage progression (Figure 4E). Conversely, addition of exogenous VCAM1 increased expression of NSC markers and decreased expression of lineage progression markers (Figure S4). Consistent with this, knockdown of Vcam1 reduced expression of genes associated with stem cell maintenance (Aguirre et al., 2010; Fasano et al., 2007, 2009; Imayoshi et al., 2010; Nam and Benezra, 2009) and upregulated genes associated with lineage progression and differentiation in this system, which produces Cell Stem Cell 11, 220–230, August 3, 2012 ª2012 Elsevier Inc. 223

Cell Stem Cell VCAM1 Is Essential for the Neural Stem Cell Niche

Figure 3. Neuroblast Chain Migration Is Disordered after Blocking VCAM1 (A) Low-magnification images of SVZ wholemounts stained for DCX (magenta). (B and C) High-magnification images show disrupted neuroblast chains (magenta) and the disrupted GFAP-GFP+ Type B cell layer (green) after VCAM1 block (C) versus control antibody (B). (D) High-magnification coronal section shows neuroblast chains (red) outside of the SVZ after VCAM1 block. Dali, blue nuclei. Scale bars: 20 mm. See also Figure S3.

largely new GABAergic olfactory neurons (Hack et al., 2005) (Figures 4F and 4G). These data show that VCAM1 function is critical for SVZ NSC maintenance, and without it, the NSC population rapidly depletes and differentiates. Activating VCAM1 Increases ROS in SVZ NSCs VCAM1 can signal through activation of the NADPH oxidase enzyme NOX2 and production of ROS in endothelial cells, upregulating molecules that alter cell-cell and cell-matrix adhesion (Cook-Mills, 2002, 2006; Cook-Mills et al., 2011). Interestingly, it has recently been shown that proliferative NSCs in the SVZ have high levels of ROS generated by the NOX2 enzyme (Le Belle et al., 2011), and that this oxidative status is important for maintenance of the stem cell state. By immunostaining, we found that NOX2 and VCAM1 were expressed with a high degree of overlap, suggesting that apical processes rich in VCAM1 are also rich in this enzyme (Figure 5A). To examine a functional association, we knocked down Vcam1 in neurosphere-expanded adult SVZ cells and saw significantly reduced Nox2 expression by qPCR (Figure 5B). If VCAM1 functions via NOX2, then we would expect that activation of VCAM1 would result in increased ROS. To test this, we generated beads coated with the anti-VCAM1 antibody, a configuration that has been shown to bind, dimerize, and activate VCAM1 (Deem et al., 2007; Matheny et al., 2000) (in contrast to the use of the soluble antibody, as described above, which binds VCAM1 and blocks its interaction with its coreceptor; Baron et al., 1994). These VCAM1-antibody-activating beads, or beads 224 Cell Stem Cell 11, 220–230, August 3, 2012 ª2012 Elsevier Inc.

coated with IgG antibodies as control, were overlaid on 4 DIV adherent adult SVZ cultures in the presence of a ROS sensor dihydroethidine, which when oxidized binds to DNA and produces a fluorescent signal (Figure 5D) (Cook-Mills, 2002, 2006). Treatment of NSCs with VCAM1-activating beads resulted in a 9.1% increase in ROS sensor fluorescence compared to control beads (Figures 5C and 5E), indicating that this signaling pathway is downstream of VCAM1. Thus signaling through VCAM1 increases ROS activity, which is consistent with VCAM1 helping to maintain the appropriate ROS status beneficial for NSC maintenance and function (Le Belle et al., 2011). VCAM1 Increases in Response to IL-1b, Acting as an Environmental Sensor on Type B Cells In endothelial cells and the bone marrow stem cell niche, VCAM1 is upregulated by the cytokines interleukin 1b (IL-1b) and SDF1, an effect that plays an important role in the immune response (Cook-Mills et al., 2011; Oostendorp and Do¨rmer, 1997). Thus, upregulation of VCAM1 in endothelium activates signaling cascades that open up the vasculature, allowing immune cells access to sites of inflammation. IL1 receptor 1 (IL1R1) is found on the GFAP+ processes of Type B cells in the SVZ (Figure 6A). Furthermore, IL-1b is expressed by the choroid plexus within the lateral ventricle near the SVZ (Figure 6B), and it is present in CSF and upregulates upon injury (Bonneh-Barkay et al., 2010; Chiaretti et al., 2005; Gourin and Shackford, 1997; SzmydyngerChodobska et al., 2009), suggesting that IL-1b in the environment could modulate VCAM1 function.

Cell Stem Cell VCAM1 Is Essential for the Neural Stem Cell Niche

Figure 4. NSC Self-Renewal Depends on VCAM1 Function (A) qPCR of Vcam1 expression in adult SVZ cells treated with lentiviral shRNA against Vcam1. (B) Reduced secondary neurosphere formation after Vcam1 shRNA treatment. (C) Primary, secondary, and tertiary neurosphere formation of the above. ***p < 0.001. (D) Reduction in secondary sphere formation by SVZ cells treated with VCAM1 blocking antibody versus isotype control antibody. **p < 0.01. (E) Quantification of immunostained adult SVZ cells in adherent culture shows that Vcam1 knockdown for 7 days reduced Ki67+ proliferating cells and Nestin+ cells and increased EGFR+ and DCX+ cells. (F and G) qPCR analysis of adult SVZ cells treated with EV or Vcam1 shRNA virus and then grown in neurosphere culture for 14 DIV. (F) Genes implicated in SVZ NSC maintenance and (G) in NSC differentiation. ***p < 0.001. Error bars = SEM. See also Figure S4.

To investigate the role of SDF1 and IL-1b in NSC VCAM1 expression, we treated neurosphere-expanded cells from hGFAP-GFP mice (Zhuo et al., 1997) with 10 ng/ml of SDF1 or IL-1b and used qPCR and flow cytometry to analyze the effect on VCAM1 expression. Although SDF1 increased Vcam1 message it did not result in an increase in VCAM1 protein under these conditions (not shown). However, treatment of neurosphere cells with IL-1b gave a more than 3-fold increase in Vcam1 message measured by qPCR (Figure 6C) and a significant increase in VCAM1 protein expression in the GFAP-GFP+ population but not in the GFAP-GFP population (1216 ± 47.48 versus 847 ± 29.13 control, p < 0.01, Figure 6D) when we measured using flow cytometry. The proportion of cells that express VCAM1 did not change with IL-1b treatment, but the signal increased, suggesting that IL-1b treatment increased VCAM1 expression in cells that were already VCAM1+ (Figure 6E). Given that reduced VCAM1 resulted in reduced NSC maintenance and increased Type B cell proliferation and lineage progression, we tested whether IL-1b would have an opposing effect. Indeed, daily treatment with 10 ng/ml IL-1b on cultured adult SVZ cells increased the number of cells expressing CD133, a marker associated with SVZ NSCs (Becker-

vordersandforth et al., 2010; Mirzadeh et al., 2008), and significantly reduced the number of MASH1+ Type C cells and cell proliferation, revealed by lowered Ki67 expression (Figure 6F). Furthermore, injecting IL-1b directly into the lateral ventricles of adult mice in vivo resulted in significantly fewer EdU+ proliferating cells in the SVZ (Figure 6G). These data support the conclusion that upregulation of VCAM1 via IL-1b treatment favors maintenance of the NSC state and inhibits lineage progression. DISCUSSION Here we examined the molecular underpinnings responsible for the highly organized cytoarchitecture of the adult SVZ neurogenic zone. Our results indicate that VCAM1 is essential for maintaining this adult stem cell niche, playing a key role in both its structure and function by regulating the positioning of Type B cells and their state of quiescence versus activation. In addition, VCAM1, which has important roles in inflammation, can act as an environmental sensor for factors in the CSF or other humoral compartments, responding to cytokines such as IL-1b to regulate NSC activity. Cell Stem Cell 11, 220–230, August 3, 2012 ª2012 Elsevier Inc. 225

Cell Stem Cell VCAM1 Is Essential for the Neural Stem Cell Niche

Figure 5. VCAM1 Activation Results in ROS Signaling (A) Immunostaining shows that NOX2 is expressed in GFAP+ cells closely overlapping with VCAM1+ staining. (B) qPCR analysis shows that Nox2 expression is reduced after lentiviral Vcam1 knockdown versus EV control. ***p < 0.001. (C) Phase contrast and fluorescent images of 4 DIV adult SVZ cells treated with a ROS sensor in the presence of anti-VCAM1-activating beads or control IgG beads. (D) Diagram of the experimental design. (E) Analysis of fluorescence intensity shows that VCAM1 activation increases ROS production. Scale bars: 20 mm. Error bars = SEM.

VCAM1 is expressed rather specifically by the apical Type B cells in the SVZ, displaying one of the cleanest expression patterns discovered to date for these cells. The small fraction of cells that express VCAM1 but are GFAP in tissue culture remains to be identified and may be a consequence of in vitro manipulation as these were not seen in vivo. The fact that VCAM1 is expressed by radial glia in the embryo (Sheppard et al., 1995) and enriched in the most primitive cells in the VZ during development (Kawaguchi et al., 2008) indicates a continuity of function in CNS progenitor cells throughout life, fitting with their lineal relationship (Alvarez-Buylla et al., 2001). Interestingly, after blocking VCAM1 in the SVZ, we saw patches of small neuroblast chains in the striatal parenchyma. Thus, by helping to maintain the cytoarchitecture, VCAM1 may be important for restricting germinal cells to the SVZ locale. Alternatively, the blocking antibody could activate normally dormant cells outside of the niche, which is worthy of further investigation. Our data show that VCAM1 is critical for anchoring Type B cells within the ependymal niche. Type B cells that are closely apposed to ependymal cells are largely quiescent (Doetsch et al., 1997, 1999b), and ependymal cells likely secrete specific 226 Cell Stem Cell 11, 220–230, August 3, 2012 ª2012 Elsevier Inc.

factors that maintain this population in a relatively dormant state. Other stem cell types such as HSCs and Drosophila germ cells are similarly dependent on contact with adjacent support cells to retain stem cell function (Nystul and Spradling, 2007; Xie and Spradling, 2000). We propose that VCAM1 is a key contributing factor to adult NSC maintenance by anchoring them close to factors located at the ependymal surface and controlling their detachment toward blood vessels that release factors that stimulate NSC lineage progression (Kokovay et al., 2010; Louissaint et al., 2002). VCAM1 might also help regulate cell-cell junction formation, and it is interesting that after blocking VCAM1 we see a marked upregulation of b-catenin and ZO-1, markers of adherens and tight junctions, respectively. This in turn could impact the mode of division, as junctions can regulate symmetric versus asymmetric division in embryonic NSCs (Go¨tz and Huttner, 2005), which will be a point for future studies. In other cell types, VCAM1 signals by interacting with alpha4beta1 integrin (VLA4; Cook-Mills et al., 2011). However, we did not detect VLA4 expression by qPCR or immunohistochemistry in the SVZ (not shown), so the counter-ligand for VCAM1 in the SVZ remains to be elucidated.

Cell Stem Cell VCAM1 Is Essential for the Neural Stem Cell Niche

Figure 6. IL-1b Upregulates VCAM1 Expression and Inhibits Lineage Progression (A) A confocal image of an SVZ wholemount taken just below the ependymal layer where the majority of Type B cells reside, showing that a subset of GFAP-GFP+ cells with radial morphology express IL-1R1. (B) Confocal image of choroid plexus stained for IL-1b (left) or negative control IgG (right). (C) qPCR analysis shows increased Vcam1 expression in SVZ cells treated with IL-1b versus vehicle control. (D) VCAM1 fluorescence intensity measured using flow cytometry. SVZ cells treated with 10 ng/ml IL-1b recombinant protein show increased VCAM1 immunostaining compared to vehicle. (E) Flow analysis of neurosphere-expanded cells from GFAP-GFP mice treated with IL-1b or vehicle shows no significant differences in subpopulation composition. (F) Quantification of positive cells in adherent SVZ cell culture treated with recombinant IL-1b protein or PBS vehicle for 4 DIV. (G) Quantification of EdU+ cells in the SVZ 18 hr after IL-1b or vehicle injection into the lateral ventricle.*p < 0.05, **p < 0.01, ***p < 0.0001. Scale bars: 20 mm. Error bars = SEM.

In endothelial cells, VCAM1 signals through NOX2 and ROS production (Cook-Mills et al., 2011). Although the role of ROS in inflammation, DNA damage, and cell death is well recognized, their role as second messengers in activating multiple signaling cascades is becoming increasingly appreciated (Jiang et al., 2011; Petry et al., 2010; Vieira et al., 2011). Induction of ROS generation by cell surface receptors such as FGFR1–4, PDGFRa/b, and VEGFR2, which have been implicated in neural stem and progenitor cell proliferation, occurs via the NOX enzymes (Petry et al., 2010). In a prior study, it was shown that ROS stimulation increased neurogenesis and self-renewal in cultured SVZ NSCs that are in an activated state, and treatment with antioxidants interfered with self-renewal by reducing ROS production and subsequently reducing PI3K/AKT pathway activity (Le Belle et al., 2011). It was suggested that quiescent NSCs in vivo might have low ROS levels, but that once activated, high ROS levels ensured sustained self-renewal while generating differentiated progeny. Our findings are consistent with a role for ROS in NSC maintenance and quiescence, downstream of VCAM1. It is possible that VCAM1 helps maintain a level of ROS compatible with NSC quiescence, and that abnormally low or high levels are detrimental to NSC function in the niche. The fact that the NSCs respond to factors that also potently act on endothelial cells is interesting given the importance of the vascular niche to stem cell function (Louissaint et al., 2002; Palmer et al., 2000; Shen et al., 2004). Although we did not observe any marked changes in the SVZ vascular plexus after VCAM1 block in vivo (not shown), it is possible that this manipulation, or application of IL-1b, alters endothelial permeability and thereby the release of factors that impact NSC activity.

The proximity of VCAM1-rich apical endings to the CSF in the lateral ventricle allows them to serve as detectors of CSF changes. Rather little is known about how the CSF regulates NSCs, but the fact that it contains multiple growth factors, chemokines, and cytokines, and that its composition varies with age and disease (Emerich et al., 2005; Johanson et al., 2008, 2011; Veening and Barendregt, 2010), implies that it is an important regulatory compartment for NSC homeostasis. For example, IL-1b is secreted into the CSF and increases with inflammation, age, and Alzheimer’s disease (Cacabelos et al., 1991; Marques et al., 2007; Sun et al., 2009). In our study, we found that increased IL-1b stimulates VCAM1 levels on NSCs and reduces proliferation and prevents lineage progression, which is somewhat counterintuitive because damage-associated cytokines might be expected to stimulate stem cell differentiation to aid tissue repair. However, it is also possible that this effect helps preserve NSCs, preventing their large-scale activation and depletion. In addition, upregulation of VCAM1 could play a protective role in maintaining the integrity of the ependymal zone during inflammation. It will be interesting to examine whether VCAM1 in the adult NSC niche can promote leukocyte adhesion, thus attracting them from the CSF to areas of CNS damage, because while the white blood cell count in the CSF is normally low, it can increase in CNS infections and be protective (Ransohoff et al., 2003). In the other major adult neurogenic zone, the hippocampal dentate gyrus, increased IL-1b reduces proliferation and neurogenesis in young and old mice, and contributes to reduced neurogenesis after stress (Koo and Duman, 2008; Koo et al., 2010; Kuzumaki et al., 2010; Luo et al., 2008). Reducing IL-1b can Cell Stem Cell 11, 220–230, August 3, 2012 ª2012 Elsevier Inc. 227

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increase hippocampal neurogenesis in aging (Gemma et al., 2007) but decrease injury-induced increased neurogenesis (Spulber et al., 2008). Thus it appears that IL-1b has an important role in regulating hippocampal neurogenesis and can have opposing effects depending on the context. It will be worthwhile to examine whether VCAM1 is also an important mediator of NSC function in the hippocampus in vivo during disease and aging, given that it interacts with IL-1b to regulate neurogenesis in hippocampal NSC cultures (Barkho et al., 2006). Understanding the impact and mechanism of action of neuroinflammatory molecules on adult stem cell niches is important to determine the consequences on adult neural cell genesis. For example, our data suggest that chronic impairment of VCAM1 function would lead to NSC depletion, and it is tempting to suggest that such a mechanism could help explain why prolonged inflammation eventually leads to memory deficits (Bettcher et al., 2011). In addition to regulation via cytokines, VCAM1 function is modulated by molecules such as bilirubin, vitamin E, and metalloproteinases (Cook-Mills et al., 2011), and VCAM1 can be cleaved, releasing the ectodomain, which binds and activates its receptor (Garton et al., 2006). Hence there are a number of ways in which VCAM1 function could be manipulated to augment NSC maintenance to offset deficits due to aging or disease. EXPERIMENTAL PROCEDURES SVZ Wholemount Dissection Adult 2- to 3-month-old Swiss Webster mice (Taconic Farms), hGFAP-GFP transgenic mice (FVB/N-Tg(GFAPGFP)14Mes/J, Jackson Laboratory), or Mash1Cre-Tomato mice (Ascl1tm1.1(cre/ERT2); B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato); Kim et al., 2011) were sacrificed and SVZ wholemounts were dissected, as described (Doetsch et al., 1999a; Lois and Alvarez-Buylla, 1993). Briefly, a 2–4 mm long strip of SVZ tissue of the striatal wall was dissected, from under the corpus callosum to the ventral tip of the lateral ventricle. The SVZ wholemounts were fixed in cold 4% paraformaldehyde in 0.1 M PBS for 30 min and washed with PBS before staining. Adult SVZ Cell Culture SVZs were dissected from Swiss Webster or hGFAP-GFP mice, incubated in 100 U papain in DMEM at 37
C on a shaker for 45 min, and gently triturated. Dissociated cells were washed 33 in PBS with centrifugation at 400 3 g at 4
C. For adherent cultures, cells were seeded in poly-l-ornithine (Sigma) coated 24-well plates (Corning) or Terasaki plates (Nunc) at 40,000, 2,000, or 200 cells per well in serum-free culture medium as described (Kokovay et al., 2010). The neurosphere assays to test self-renewal were conducted as described (Pastrana et al., 2011). Briefly, cells were cultured in ultra-low attachment 6-well plates (Corning) at three cells/ml for primary spheres and one cell/ml for secondary spheres in serum-free culture medium with 20 ng/ml of FGF2 and 20 ng/ml EGF (Invitrogen). For treatment with recombinant proteins, neurospheres were dissociated with 50U papain in 5 mls DMEM for 30 min at 37
C on a shaker, washed 33 with PBS, and treated with 1 mg/ml VCAM1 protein, 10 ng/ml IL-1b, or 10 ng/ml SDF1 (Peprotech) overnight.

FACS Analysis Neurospheres from GFAP-GFP or Swiss Webster (for negative control of GFP signal) mice were dissociated and treated with 10 ng/ml mouse recombinant IL-1b, SDF1, or vehicle (PBS) overnight. On ice, cells (60,000 cells per condition, n = 3) were blocked with heat-inactivated FBS for 15 min and incubated with 0.2 mg/ml Alexa Fluor 647 conjugated anti-mouse VCAM1 or isotype control (eBioscience). Gates were set manually using the negative controls. The fluorescence intensity and mean number of positive cells were measured on BD Biosciences FACS Aria II and the percentage of positive cells was determined for each phenotype. VCAM1 Activating Beads and ROS Detection Beads were coated with IgG or anti-VCAM1 as described (Deem et al., 2007; Matheny et al., 2000). Briefly, 10 mm diameter streptavidin-coated microspheres (80 ml/ sample, Bangs Laboratories) were labeled with 24 mg biotinconjugated goat anti-rat IgG, washed 33 in PBS by centrifugation, and then incubated with 16 mg rat anti-VCAM1 or isotype control antibody at 4
C for 1 hr. The beads were washed 33 in PBS and resuspended in PBS. Adult SVZ cells were grown in adherent culture for 4 DIV then incubated in 2 mM of the ROS sensor dihydroethidium in culture medium with no anti-oxidants (n = 3 per condition). Antibody coated beads were added to each well. After 30 min, five fields of view were captured using a 203 objective. The pixel strength of each cell nucleus was measured using ImageJ software (Ferreira and Rasband, 2011). EdU Labeling To label proliferating cells, EdU (Invitrogen) (10 mg/ml solution) was injected intraperitoneally (50 mg/kg) 2 hr before mice were sacrificed. To detect label-retaining cells and newborn olfactory bulb cells, EdU was injected once daily for 3 days and mice were sacrificed 3 weeks after the final injection. EdU was detected using the Click-it Kit (Invitrogen), prior to the performance of additional immunohistochemistry. VCAM1 Blocking Experiments For in vivo VCAM1 blocking, miniosmotic pumps (model 1007D Alzet) were preloaded with rat anti-mouse VCAM1 antibody (BD Biosciences) or rat IgG2ak control antibody at 0.5 mg/ml in artificial CSF hGFAP-GFP or GFAP-GFP-BAC mice (FVB/N-SwissWebster-Tg(GFAPEGFP)CV94Gsat/ Mmmh). Mice were anesthetized with 3% isoflurane and a 3 mm cannula (Alzet) connected to the pumps was inserted stereotactically into the lateral ventricle (0.85 mm lateral of Bregma). After 5–7 days of infusion, mice were sacrificed and the SVZ wholemounts were dissected and fixed for immunostaining. For each group, three to five random fields from each wholemount were imaged using a two-photon Zeiss 510 Meta confocal microscope or a Leica TCS SP5 II confocal microscope, and data were pooled from two or three wholemounts. The number of GFAP-GFP+ cell bodies in the ependymal layer was counted in a 1 mm z-stack and divided by the field area. Brain sections for analysis were generated as described in the Supplemental Information. For in vitro VCAM1 inhibition, neurospheres were treated with 10 mg/ml of VCAM1 blocking or control antibody for 7 days, and the numbers of neurospheres per well were counted (n = 3–5 per condition). Neurospheres were dissociated and then incubated for 7 more days in the appropriate antibody and were quantified. Statistics All data are expressed as mean ± SEM. Student’s t test and ANOVA were performed using Graphpad Prism software. SUPPLEMENTAL INFORMATION

Vcam1 Lentiviral shRNA Preparation An shRNA was designed (Dharmacon) and plasmids were generated by insertion of the hairpin oligonucleotides into the FUGW-H1 lentiviral construct (Fasano et al., 2007). To package, the lentiviral constructs were cotransfected using Superfect (QIAGEN) with pCMV-VSVG and pCMV-DVPR into 293FT cells grown under selective conditions. Supernatant was harvested 2 and 3 days later and was concentrated by ultracentrifugation. Lentiviruses were used at 10 multiplicity of infection (MOI) for cell transduction. The oligonucleotide sequence targeting Vcam1 was 50 GACTGACTGTTGTAACTTA 30 .

228 Cell Stem Cell 11, 220–230, August 3, 2012 ª2012 Elsevier Inc.

Supplemental Information for this article includes four figures and Supplemental Experimental Procedures and can be found with this article online at http://dx.doi.org/10.1016/j.stem.2012.06.016. ACKNOWLEDGMENTS This research was funded by NIH grant NS051531 from the National Institutes of Neurological Disorders and Stroke. The authors would like to thank

Cell Stem Cell VCAM1 Is Essential for the Neural Stem Cell Niche

Christopher Fasano and John Dimos for help with Vcam1 shRNA design and production.

Doetsch, F., Garcı´a-Verdugo, J.M., and Alvarez-Buylla, A. (1999b). Regeneration of a germinal layer in the adult mammalian brain. Proc. Natl. Acad. Sci. USA 96, 11619–11624.

Received: November 2, 2011 Revised: May 23, 2012 Accepted: June 26, 2012 Published: August 2, 2012

Emerich, D.F., Skinner, S.J., Borlongan, C.V., Vasconcellos, A.V., and Thanos, C.G. (2005). The choroid plexus in the rise, fall and repair of the brain. Bioessays 27, 262–274.

REFERENCES Aguirre, A., Rubio, M.E., and Gallo, V. (2010). Notch and EGFR pathway interaction regulates neural stem cell number and self-renewal. Nature 467, 323–327. Alvarez-Buylla, A., Garcia-Verdugo, J.M., and Tramontin, A.D. (2001). A unified hypothesis on the lineage of neural stem cells. Nat. Rev. 2, 287–293. Barkho, B.Z., Song, H., Aimone, J.B., Smrt, R.D., Kuwabara, T., Nakashima, K., Gage, F.H., and Zhao, X. (2006). Identification of astrocyte-expressed factors that modulate neural stem/progenitor cell differentiation. Stem Cells Dev. 15, 407–421. Baron, J.L., Reich, E.P., Visintin, I., and Janeway, C.A., Jr. (1994). The pathogenesis of adoptive murine autoimmune diabetes requires an interaction between alpha 4-integrins and vascular cell adhesion molecule-1. J. Clin. Invest. 93, 1700–1708. Beckervordersandforth, R., Tripathi, P., Ninkovic, J., Bayam, E., Lepier, A., Stempfhuber, B., Kirchhoff, F., Hirrlinger, J., Haslinger, A., Lie, D.C., et al. (2010). In vivo fate mapping and expression analysis reveals molecular hallmarks of prospectively isolated adult neural stem cells. Cell Stem Cell 7, 744–758. Bettcher, B.M., Wilheim, R., Rigby, T., Green, R., Miller, J.W., Racine, C.A., Yaffe, K., Miller, B.L., and Kramer, J.H. (2011). C-reactive protein is related to memory and medial temporal brain volume in older adults. Brain, Behav., and Immun. 26, 103–108. Bonneh-Barkay, D., Zagadailov, P., Zou, H., Niyonkuru, C., Figley, M., Starkey, A., Wang, G., Bissel, S.J., Wiley, C.A., and Wagner, A.K. (2010). YKL-40 expression in traumatic brain injury: an initial analysis. J. Neurotrauma 27, 1215–1223.

Fasano, C.A., Dimos, J.T., Ivanova, N.B., Lowry, N., Lemischka, I.R., and Temple, S. (2007). shRNA knockdown of Bmi-1 reveals a critical role for p21-Rb pathway in NSC self-renewal during development. Cell Stem Cell 1, 87–99. Fasano, C.A., Phoenix, T.N., Kokovay, E., Lowry, N., Elkabetz, Y., Dimos, J.T., Lemischka, I.R., Studer, L., and Temple, S. (2009). Bmi-1 cooperates with Foxg1 to maintain neural stem cell self-renewal in the forebrain. Genes Dev. 23, 561–574. Ferreira, T., and Rasband, W. (2011). The Image J User Guide-IJ1.45. Garton, K.J., Gough, P.J., and Raines, E.W. (2006). Emerging roles for ectodomain shedding in the regulation of inflammatory responses. J. Leukoc. Biol. 79, 1105–1116. Gemma, C., Bachstetter, A.D., Cole, M.J., Fister, M., Hudson, C., and Bickford, P.C. (2007). Blockade of caspase-1 increases neurogenesis in the aged hippocampus. Eur. J. Neurosci. 26, 2795–2803. Go¨tz, M., and Huttner, W.B. (2005). The cell biology of neurogenesis. Nat. Rev. Mol. Cell Biol. 6, 777–788. Gourin, C.G., and Shackford, S.R. (1997). Production of tumor necrosis factoralpha and interleukin-1beta by human cerebral microvascular endothelium after percussive trauma. J. Trauma 42, 1101–1107. Hack, M.A., Saghatelyan, A., de Chevigny, A., Pfeifer, A., Ashery-Padan, R., Lledo, P.M., and Go¨tz, M. (2005). Neuronal fate determinants of adult olfactory bulb neurogenesis. Nat. Neurosci. 8, 865–872. Imayoshi, I., Sakamoto, M., Yamaguchi, M., Mori, K., and Kageyama, R. (2010). Essential roles of Notch signaling in maintenance of neural stem cells in developing and adult brains. J. Neurosci. 30, 3489–3498. Jiang, F., Zhang, Y., and Dusting, G.J. (2011). NADPH oxidase-mediated redox signaling: roles in cellular stress response, stress tolerance, and tissue repair. Pharmacol. Rev. 63, 218–242.

Cacabelos, R., Barquero, M., Garcı´a, P., Alvarez, X.A., and Varela de Seijas, E. (1991). Cerebrospinal fluid interleukin-1 beta (IL-1 beta) in Alzheimer’s disease and neurological disorders. Methods Find. Exp. Clin. Pharmacol. 13, 455–458.

Johanson, C.E., Duncan, J.A., 3rd, Klinge, P.M., Brinker, T., Stopa, E.G., and Silverberg, G.D. (2008). Multiplicity of cerebrospinal fluid functions: New challenges in health and disease. Cerebrospinal Fluid Res. 5, 10.

Chiaretti, A., Genovese, O., Aloe, L., Antonelli, A., Piastra, M., Polidori, G., and Di Rocco, C. (2005). Interleukin 1beta and interleukin 6 relationship with paediatric head trauma severity and outcome. Childs Nerv. Syst. 21, 185–193, discussion 194.

Johanson, C., Stopa, E., Baird, A., and Sharma, H. (2011). Traumatic brain injury and recovery mechanisms: peptide modulation of periventricular neurogenic regions by the choroid plexus-CSF nexus. J. Neural Transm. 118, 115–133.

Cook-Mills, J.M. (2002). VCAM-1 signals during lymphocyte migration: role of reactive oxygen species. Mol. Immunol. 39, 499–508.

Johansson, C.B., Momma, S., Clarke, D.L., Risling, M., Lendahl, U., and Frise´n, J. (1999). Identification of a neural stem cell in the adult mammalian central nervous system. Cell 96, 25–34.

Cook-Mills, J.M. (2006). Hydrogen peroxide activation of endothelial cellassociated MMPs during VCAM-1-dependent leukocyte migration. Cell Mol. Biol. (Noisy-le-grand) 52, 8–16. Cook-Mills, J.M., Marchese, M.E., and Abdala-Valencia, H. (2011). Vascular Cell Adhesion Molecule-1 Expression and Signaling During Disease: Regulation by Reactive Oxygen Species and Antioxidants. Antioxid. Redox. Signal. 15, 1607–1638. Deem, T.L., Abdala-Valencia, H., and Cook-Mills, J.M. (2007). VCAM-1 activation of endothelial cell protein tyrosine phosphatase 1B. J. Immunol. 178, 3865–3873. Doetsch, F., and Alvarez-Buylla, A. (1996). Network of tangential pathways for neuronal migration in adult mammalian brain. Proc. Natl. Acad. Sci. USA 93, 14895–14900.

Kawaguchi, A., Ikawa, T., Kasukawa, T., Ueda, H.R., Kurimoto, K., Saitou, M., and Matsuzaki, F. (2008). Single-cell gene profiling defines differential progenitor subclasses in mammalian neurogenesis. Development 135, 3113–3124. Kazanis, I., Lathia, J.D., Vadakkan, T.J., Raborn, E., Wan, R., Mughal, M.R., Eckley, D.M., Sasaki, T., Patton, B., Mattson, M.P., et al. (2010). Quiescence and activation of stem and precursor cell populations in the subependymal zone of the mammalian brain are associated with distinct cellular and extracellular matrix signals. J. Neurosci. 30, 9771–9781. Kim, E.J., Ables, J.L., Dickel, L.K., Eisch, A.J., and Johnson, J.E. (2011). Ascl1 (Mash1) defines cells with long-term neurogenic potential in subgranular and subventricular zones in adult mouse brain. PLoS ONE 6, e18472.

Doetsch, F., Garcı´a-Verdugo, J.M., and Alvarez-Buylla, A. (1997). Cellular composition and three-dimensional organization of the subventricular germinal zone in the adult mammalian brain. J. Neurosci. 17, 5046–5061.

Kokovay, E., Goderie, S., Wang, Y., Lotz, S., Lin, G., Sun, Y., Roysam, B., Shen, Q., and Temple, S. (2010). Adult SVZ lineage cells home to and leave the vascular niche via differential responses to SDF1/CXCR4 signaling. Cell Stem Cell 7, 163–173.

Doetsch, F., Caille´, I., Lim, D.A., Garcı´a-Verdugo, J.M., and Alvarez-Buylla, A. (1999a). Subventricular zone astrocytes are neural stem cells in the adult mammalian brain. Cell 97, 703–716.

Koo, J.W., and Duman, R.S. (2008). IL-1beta is an essential mediator of the antineurogenic and anhedonic effects of stress. Proc. Natl. Acad. Sci. USA 105, 751–756.

Cell Stem Cell 11, 220–230, August 3, 2012 ª2012 Elsevier Inc. 229

Cell Stem Cell VCAM1 Is Essential for the Neural Stem Cell Niche

Koo, J.W., Russo, S.J., Ferguson, D., Nestler, E.J., and Duman, R.S. (2010). Nuclear factor-kappaB is a critical mediator of stress-impaired neurogenesis and depressive behavior. Proc. Natl. Acad. Sci. USA 107, 2669–2674. Kuzumaki, N., Ikegami, D., Imai, S., Narita, M., Tamura, R., Yajima, M., Suzuki, A., Miyashita, K., Niikura, K., Takeshima, H., et al. (2010). Enhanced IL-1beta production in response to the activation of hippocampal glial cells impairs neurogenesis in aged mice. Synapse 64, 721–728. Lapidot, T., Dar, A., and Kollet, O. (2005). How do stem cells find their way home? Blood 106, 1901–1910. Le Belle, J.E., Orozco, N.M., Paucar, A.A., Saxe, J.P., Mottahedeh, J., Pyle, A.D., Wu, H., and Kornblum, H.I. (2011). Proliferative neural stem cells have high endogenous ROS levels that regulate self-renewal and neurogenesis in a PI3K/Akt-dependant manner. Cell Stem Cell 8, 59–71. Lois, C., and Alvarez-Buylla, A. (1993). Proliferating subventricular zone cells in the adult mammalian forebrain can differentiate into neurons and glia. Proc. Natl. Acad. Sci. USA 90, 2074–2077. Louissaint, A., Jr., Rao, S., Leventhal, C., and Goldman, S.A. (2002). Coordinated interaction of neurogenesis and angiogenesis in the adult songbird brain. Neuron 34, 945–960. Luo, J., Shook, B.A., Daniels, S.B., and Conover, J.C. (2008). Subventricular zone-mediated ependyma repair in the adult mammalian brain. J. Neurosci. 28, 3804–3813. Marques, F., Sousa, J.C., Correia-Neves, M., Oliveira, P., Sousa, N., and Palha, J.A. (2007). The choroid plexus response to peripheral inflammatory stimulus. Neuroscience 144, 424–430. Matheny, H.E., Deem, T.L., and Cook-Mills, J.M. (2000). Lymphocyte migration through monolayers of endothelial cell lines involves VCAM-1 signaling via endothelial cell NADPH oxidase. J. Immunol. 164, 6550–6559. Mirzadeh, Z., Merkle, F.T., Soriano-Navarro, M., Garcia-Verdugo, J.M., and Alvarez-Buylla, A. (2008). Neural stem cells confer unique pinwheel architecture to the ventricular surface in neurogenic regions of the adult brain. Cell Stem Cell 3, 265–278. Morshead, C.M., Reynolds, B.A., Craig, C.G., McBurney, M.W., Staines, W.A., Morassutti, D., Weiss, S., and van der Kooy, D. (1994). Neural stem cells in the adult mammalian forebrain: a relatively quiescent subpopulation of subependymal cells. Neuron 13, 1071–1082. Nam, H.S., and Benezra, R. (2009). High levels of Id1 expression define B1 type adult neural stem cells. Cell Stem Cell 5, 515–526.

anisms of lodgement of transplanted murine hemopoietic progenitors between bone marrow and spleen. Proc. Natl. Acad. Sci. USA 92, 9647–9651. Pastrana, E., Silva-Vargas, V., and Doetsch, F. (2011). Eyes wide open: a critical review of sphere-formation as an assay for stem cells. Cell Stem Cell 8, 486–498. Petry, A., Weitnauer, M., and Gorlach, A. (2010). Receptor activation of NADPH oxidases. Antioxid. Redox. Signal. 13, 467–487. Ransohoff, R.M., Kivisa¨kk, P., and Kidd, G. (2003). Three or more routes for leukocyte migration into the central nervous system. Nat. Rev. Immunol. 3, 569–581. Sawamoto, K., Wichterle, H., Gonzalez-Perez, O., Cholfin, J.A., Yamada, M., Spassky, N., Murcia, N.S., Garcia-Verdugo, J.M., Marin, O., Rubenstein, J.L., et al. (2006). New neurons follow the flow of cerebrospinal fluid in the adult brain. Science 311, 629–632. Shen, Q., Goderie, S.K., Jin, L., Karanth, N., Sun, Y., Abramova, N., Vincent, P., Pumiglia, K., and Temple, S. (2004). Endothelial cells stimulate self-renewal and expand neurogenesis of neural stem cells. Science 304, 1338–1340. Shen, Q., Wang, Y., Kokovay, E., Lin, G., Chuang, S.M., Goderie, S.K., Roysam, B., and Temple, S. (2008). Adult SVZ stem cells lie in a vascular niche: a quantitative analysis of niche cell-cell interactions. Cell Stem Cell 3, 289–300. Sheppard, A.M., McQuillan, J.J., Iademarco, M.F., and Dean, D.C. (1995). Control of vascular cell adhesion molecule-1 gene promoter activity during neural differentiation. J. Biol. Chem. 270, 3710–3719. Simmons, P.J., Masinovsky, B., Longenecker, B.M., Berenson, R., TorokStorb, B., and Gallatin, W.M. (1992). Vascular cell adhesion molecule-1 expressed by bone marrow stromal cells mediates the binding of hematopoietic progenitor cells. Blood 80, 388–395. Spulber, S., Oprica, M., Bartfai, T., Winblad, B., and Schultzberg, M. (2008). Blunted neurogenesis and gliosis due to transgenic overexpression of human soluble IL-1ra in the mouse. Eur. J. Neurosci. 27, 549–558. Sun, Y., Lu, C.J., Lin, C.H., and Wen, L.L. (2009). Interleukin-1beta is increased in the cerebrospinal fluid of patients with small infarcts. Eur. J. Neurol. 16, 858–863. Szmydynger-Chodobska, J., Strazielle, N., Zink, B.J., Ghersi-Egea, J.F., and Chodobski, A. (2009). The role of the choroid plexus in neutrophil invasion after traumatic brain injury. J. Cereb. Blood Flow Metab. 29, 1503–1516.

Nystul, T., and Spradling, A. (2007). An epithelial niche in the Drosophila ovary undergoes long-range stem cell replacement. Cell Stem Cell 1, 277–285.

Tavazoie, M., Van der Veken, L., Silva-Vargas, V., Louissaint, M., Colonna, L., Zaidi, B., Garcia-Verdugo, J.M., and Doetsch, F. (2008). A specialized vascular niche for adult neural stem cells. Cell Stem Cell 3, 279–288.

Oostendorp, R.A., and Do¨rmer, P. (1997). VLA-4-mediated interactions between normal human hematopoietic progenitors and stromal cells. Leuk. Lymphoma 24, 423–435.

Veening, J.G., and Barendregt, H.P. (2010). The regulation of brain states by neuroactive substances distributed via the cerebrospinal fluid; a review. Cerebrospinal Fluid Res. 7, 1.

Osborn, L., Hession, C., Tizard, R., Vassallo, C., Luhowskyj, S., Chi-Rosso, G., and Lobb, R. (1989). Direct expression cloning of vascular cell adhesion molecule 1, a cytokine-induced endothelial protein that binds to lymphocytes. Cell 59, 1203–1211.

Vieira, H.L., Alves, P.M., and Vercelli, A. (2011). Modulation of neuronal stem cell differentiation by hypoxia and reactive oxygen species. Prog. Neurobiol. 93, 444–455.

Palmer, T.D., Willhoite, A.R., and Gage, F.H. (2000). Vascular niche for adult hippocampal neurogenesis. J. Comp. Neurol. 425, 479–494. Papayannopoulou, T., Craddock, C., Nakamoto, B., Priestley, G.V., and Wolf, N.S. (1995). The VLA4/VCAM-1 adhesion pathway defines contrasting mech-

230 Cell Stem Cell 11, 220–230, August 3, 2012 ª2012 Elsevier Inc.

Xie, T., and Spradling, A.C. (2000). A niche maintaining germ line stem cells in the Drosophila ovary. Science 290, 328–330. Zhuo, L., Sun, B., Zhang, C.L., Fine, A., Chiu, S.Y., and Messing, A. (1997). Live astrocytes visualized by green fluorescent protein in transgenic mice. Dev. Biol. 187, 36–42.