β8 Integrin Regulates Neurogenesis and Neurovascular Homeostasis in the Adult Brain

Abstract

Central nervous system (CNS) neurovascular units are multicellular complexes consisting of neural cells, blood vessels and a milieu of growth factors and extracellular matrix (ECM) proteins. ECM-mediated adhesion and signaling events within neurovascular units contribute to proper CNS development and physiology; however, the molecular mechanisms that control these events remain largely undetermined. Previous studies from our group and others showed that ablation of the ECM receptor, β8 integrin, in neural progenitor cells (NPCs) of the embryonic mouse brain results in severe developmental neurovascular pathologies and premature death. Here, we have interrogated integrin functions in the adult brain by studying mice harboring a homozygous null β8 gene mutation generated on an outbred background that permits survival for several post-natal months. We show that adult β8-/- mice display widespread defects in neurovascular unit homeostasis, including increased numbers of intracerebral blood vessels with pronounced perivascular astrogliosis. Furthermore, in neurogenic regions of the adult brain, where NPCs cluster around blood vessels in neurovascular niches, β8 integrin is essential for normal control of NPC proliferation and survival. Analysis of NPCs cultured ex vivo reveals that the growth and survival defects correlate, in part, with diminished integrin-mediated activation of latent transforming growth factor β1 (TGFβ1), which is an ECM protein ligand for β8 integrin. Collectively, these data reveal necessary functions for β8 integrin in modulating neurogenesis and neurovascular unit homeostasis in the adult mouse brain.

Introduction

The vertebrate CNS contains an elaborate network of blood vessels that intimately associate with neuronal and glial elements to form integrated complexes, or neurovascular units. Neural and vascular cells within such units interact mainly via basement membranes that contain an assortment of growth factors and ECM proteins. Precise regulation of cell-ECM communication within neurovascular units is essential for proper formation and function of the vascular system within the CNS. For example, glial- and neuronal-derived cues regulate blood vessel morphogenesis and blood-brain barrier development (Abbott et al., 2006; Iadecola, 2004). Vascular-derived cues also influence neural cell functions; for example, neural progenitor cells (NPCs) in specific regions of the adult brain preferentially reside near cerebral blood vessels within `neurovascular niches'. Cell-cell contacts as well as niche-derived growth factors and ECM proteins dictate NPC growth, survival, and differentiation (Palmer et al., 2000; Wurmser et al., 2004). How perivascular astrocytes and NPCs decipher vascular basement membrane-derived ECM cues, and how these events influence neural cell functions remains enigmatic.

Integrins are heterodimeric cell surface receptors for many ECM proteins. In both mice and humans there are 26 integrin genes: 18 genes encoding α subunits, and 8 genes that encode β subunits (Hynes, 2002). Many integrins are expressed in post-natal astrocytes and NPCs (Hall et al., 2006; McCarty et al., 2005), although functional roles for these integrins in adult neurovascular physiology remain obscure. Our group and others have reported that αvβ8 integrin is necessary for normal regulation of neurovascular development in the brain, since deletion of the αv or β8 integrin genes in developing neural progenitors and astrocytes, but not endothelial cells, leads to embryonic and neonatal blood vessel patterning defects and intracerebral hemorrhage (McCarty et al., 2004; Proctor et al., 2005). αvβ8 integrin-mediated regulation of neurovascular development depends on activation of latent forms of TGFβ, which are secreted from cells as ECM-bound complexes that have minimal bioactivity (Annes et al., 2003). Integrin adhesion to the latent forms of TGFβ1 and TGFβ3 mediates their bioactivation and induces canonical TGFβ receptor signaling (Cambier et al., 2005; Mu et al., 2002; Sheppard, 2004). The importance of integrin-mediated TGFβ activation has been demonstrated in vivo by mutating the integrin-binding motif within the TGFβ1 gene. Mice harboring this mutation die prematurely and display phenotypes that are identical to those observed in TGFβ1 gene knockouts (Yang et al., 2007). Furthermore, mouse embryos harboring this mutated TGFβ1 gene in combination with a TGFβ3-/- gene develop brain-specific neurovascular pathologies that appear identical to the phenotypes in αv and β8 integrin knockouts (Mu et al., 2008).

Here, we have analyzed mice genetically null for β8 integrin to determine functions for this integrin in neural-vascular cell adhesion and communication, mainly within neurogenic regions of the post-natal brain. We show that adult β8-/- mice develop severe deficits in neurogenesis and neurovascular homeostasis, and that these pathologies are associated with diminished integrin-mediated activation of TGFβ's. These data identify β8 integrin as a key regulator of neurovascular physiology in the adult brain, and indicate that integrin-mediated TGFβ activation is an important component in this process.

Results

Genetic Background Influences Post-Natal Viability of β8-/- Mice

Previous reports have demonstrated that most αv-/- and β8-/- embryos on a C57BL6/129S4 mixed genetic background die by E11 due to placental defects (Bader et al., 1998; McCarty et al., 2002; Zhu et al., 2002). αv-/- mice that survive beyond E11 develop progressive intracerebral hemorrhage, a cleft palate, and die in the early neonatal period (Bader et al., 1998). Similar results have been described for β8-/- mice, although the incidence of cleft palate formation was reported to be significantly lower (Zhu et al., 2002). We reasoned that strain-specific genetic modifiers might influence embryonic and post-natal viability in β8-/- mice. Therefore, heterozygous mutant mice (β8+/-) on a C57BL6/129S4 genetic background were backcrossed for two generations with the ICR/CD-1 outbred strain, and F2 progeny were subsequently interbred to generate wild type control (β8+/+) and homozygous mutant (β8-/-) progeny. Nearly all β8-/- progeny from β8+/- intercrosses were born alive and in nearly the predicted Mendelian distributions: twenty-six β8-/- mice of 123 total F2 neonates (21%) were identified at birth, as compared to the expected 25%, suggesting a low percentage of in utero lethality. All β8-/- neonates displayed grossly obvious intracerebral hemorrhage (Supplemental Figure 1A) that was very similar to that reported for C57BL6/129S4 mutants (Zhu et al., 2002). Although β8-/- pups were initially identified based on the intracerebral hemorrhage phenotype, all genotypes were confirmed at the time of weaning using PCR-based methods to amplify β8 gene sequences using genomic DNA isolated from ear snips (Supplemental Figure 1B).

Nearly 90% of β8-/- neonates (23 of 26 β8-/- mutants identified) survived beyond the first post-natal week (Figure 1A), although most β8-/- mice were distinguishable from wild type littermates based on phenotype. Mutant animals were generally smaller than control littermates (Figures 1B) and displayed an abnormal posture (Figure 1D). Approximately 50% of β8-/- mice that survived beyond the first week died between post-natal days 21 and 30 (Figure 1A). The remaining mutants survived for up to five months but developed progressively more severe neurological phenotypes, including ataxia and paresis that eventually led to an abnormal gait (Figure 1E). β8 integrin protein expression was analyzed by immunoblotting tissue homogenates prepared from various adult brain regions including the subventricular zone (SVZ) of the lateral ventricle, the olfactory bulbs, as well as the hippocampal dentate gyrus and cerebellum. As shown in Figure 1F, β8 integrin protein was expressed at similar levels in all brain regions analyzed from wild type mice; in contrast, β8 integrin protein was not detected in brain lysates from adult β8-/- mice.

Figure 1. Genetic Background Influences Post-Natal Survival of β8-/- Mice.

(A); Kaplan-Meier survival plot using wild type (n=40) and β8-/- mice (n=39). Note that approximately 60% of β8-/- mice die by P30 whereas the remaining animals survive for up to five post-natal months. (B); Weight analysis using P30 and P60 control and mutant male mice (n=7 to 10 males per genotype, although similar results were observed with female mice). Note that β8-/- mice weigh significantly less compared to wild type littermates, *p < 0.0001 compared to wild type group (C, D); Images of four month-old (P120) wild type (C) and β8-/- (D) littermates. β8-/- mice display a hunched posture and limb paresis; these phenotypes develop with 100% penetrance in mutant mice that survive to adulthood. (E); Footprint analysis performed with post-natal day 90 (P90) wild type (upper panel) and β8-/- (lower panel) animals with hind paws painted blue and fore paws painted red. Note that the β8-/- mouse (like all other β8-/- mutants examined) displays an abnormal gait characterized by pronounced dragging of the limbs. Arrow indicates direction of movement. (F); Brain tissue homogenates were prepared from P60 wild type and β8-/- mice. Detergent-soluble protein lysates were immunoblotted with an anti-β8 integrin polyclonal antibody. Note that β8 integrin protein is expressed in the various brains regions of wild type mice, but is not detected in β8-/- animals. Abbreviations: SVZ, subventricular zone of lateral ventricle; OB, olfactory bulbs; DG, hippocampal dentate gyrus; CB, cerebellum.

Defects in Neurovascular Unit Homeostasis in Adult β8-/- Mice

Analysis of serial brain sections from β8-/- mice older than four weeks of age showed the absence of obvious intracerebral hemorrhage (Supplemental Figure 1C). This appears to be due to resolution of hemorrhage that was present at birth (Supplemental Figure 1A), similar to what our group and others have shown in conditional αv and β8 integrin knockout mice (McCarty et al., 2004; Proctor et al., 2005). The diminished neurovascular pathologies do not reflect reduced expression of β8 integrin in the post-natal brain, since in wild type mice β8 integrin protein expression is maintained into adulthood (data not shown), suggesting continued functions for this integrin in the post-natal brain.

Next, an anti-laminin antibody, which highlights vascular basement membranes, was used to quantify numbers of blood vessels within the cerebral cortices of adult wild type and β8-/- animals. In comparison to wild type controls (Figure 2A) approximately two-fold more lamininpositive blood vessels were detected within cerebral cortices of β8-/- adult mice (Figure 2B). Many of the mutant vessels showed more intense laminin immunoreactivity (Figures 2B), suggesting higher levels of endogenous laminin protein expression. In order to test if the increased blood vessel densities in adult β8-/- brains were due to ongoing angiogenesis, we intraperitoneally injected the thymidine analog bromodeoxyuridine (BrdU) into adult wild type and β8-/- mice. BrdU incorporation in vascular endothelial cells was analyzed by double fluorescence staining with anti-BrdU and anti-laminin antibodies. However, we did not detect obvious differences in BrdU incorporation in control versus β8-/- endothelial cells (data not shown). These data suggest that the increased cerebral blood vessel densities in adult β8-/- mice are not due to pathological angiogenesis, but may be secondary to vascular damage in the embryonic and neonatal periods (Supplemental Figure 1).

Figure 2. Adult β8-/- Mice Display Widespread Neurovascular Pathologies.

(A, B); Coronal sections through cerebral cortices of wild type (A) and β8-/- (B) were immunostained with anti-laminin to reveal cerebral blood vessels. (C); Quantitation of cerebral blood vessels based on anti-laminin immunoreactivity for P60 wild type (n=5) and β8-/- mice (n=5), *p = 0.002 as compared to wild type group. (D, E); Double immunofluorescence analysis using anti-CD34 (green) and anti-GFAP (red) reveals increased numbers of astrocytes in β8-/- brains. (F); Quantitation of GFAP-expressing astrocytes in sections from wild type (n=4) versus β8-/- (n=4) mice, *p<0.001 as compared to wild type group. (G, H); Transmission electron microscopy reveals ultrastructural neurovascular unit pathologies in β8-/- mice. Note the increased vessel coverage by perivascular astrocyte endfeet in the mutant (H) versus control (G) samples (arrows). (I); Quantitation of cerebral blood vessel coverage by astrocytes within cerebral cortices of wild type and β8-/- mice. Coronal sections through the cerebral cortices of P60 wild type (n=5) and β8-/- (n=5) brains were immunofluorescently labeled with both anti-laminin and anti-GFAP. The percentages of laminin-expressing blood vessels contacting more than one GFAP⁺ astrocyte were quantified per 400X field, *p = 0.002 as compared to the wild type group.

Analysis of control (Figure 2D) and mutant (Figure 2E) cerebral cortices with an anti-GFAP antibody revealed nearly five times more GFAP-expressing astrocytes in adult β8-/- mice (Figure 2F), and similar patterns were observed in most other brain regions (data not shown). To determine if the elevated numbers of GFAP-expressing cells were the result of increased astrocyte proliferation, we analyzed GFAP-expressing cells following intraperitoneal injection of BrdU via double immunofluorescence labeling. Cells that were positive for both GFAP and BrdU were not detected (data not shown), suggesting that the increased numbers of GFAP cells were not the result of astrocyte hyperplasia. Many of these GFAP-expressing cells in the β8-/- brains were closely associated with cerebral blood vessels (Figure 2E). Ultrastructural analyses also revealed increased astrocyte coverage of cerebral blood vessels in adult β8-/- mice (Figure 2H), as opposed to wild type controls (Figure 2G). Astrocyte coverage of blood vessels was also quantified using anti-GFAP and anti-LM double immunofluorescence staining, which revealed nearly three-fold more cerebral blood vessels coverage by astrocyte endfeet in β8-/- mice (Figure 2I).

The increased numbers of perivascular astrocytes suggested that cerebral blood vessels in adult β8-/- mice might have compromised blood-brain barrier properties. Hence, blood-brain barrier permeability was analyzed using a reactive derivative of biotin (EZLink-NHS-Biotin). This small molecule (approximately 500 Daltons) is water-soluble, membrane impermeable, and covalently couples to free amine groups commonly present on many protein and macromolecules. Upon pathological disruption of the blood-brain barrier this tracer labels components of the brain parenchyma (Nitta et al., 2003). As shown in Figure 3A and 3C, in both wild type and β8-/- brains passage of reactive biotin into parenchymal regions surrounding cortical cerebral blood vessels was not detected. This is in contrast to periventricular regions of wild type and β8-/- mouse brains, which displayed robust passage of amine-reactive biotin into the brain parenchyma (Figure 3B, D). These data are consistent with a recent report showing that blood vessels within the SVZ and other periventricular regions of the adult mouse brain display enhanced blood-brain barrier permeability (Tavazoie et al., 2008). To further analyze the blood-brain barrier in wild type and adult β8-/- mice, we immunofluorescently stained brain sections with an anti-zona occludens protein 1 (ZO1) antibody to label endothelial cell tight junctions that comprise the blood-brain barrier (Wolburg and Lippoldt, 2002). However, we do not detect obvious differences in the expression patterns of ZO1 in cerebral blood vessels in adult wild type or β8-/- brains (Figure 3E, F). We also immunoblotted brain protein lysates using antibodies directed against occludin and claudin 5, proteins expressed in tight junctions that form the blood-brain barrier (Nitta et al., 2003; Wolburg and Lippoldt, 2002). As shown in Figure 3G, similar amounts of occludin and claudin 5 proteins were detected in homogenates from wild type and β8-/- cerebral cortices.

Figure 3. Lack of Obvious Blood-Brain Barrier Disruption in Adult β8-/- Mice.

(A-D); P60 wild-type (n=3) or β8-/- (n=5) mice were perfused with amine-reactive biotin and sagittal brain sections were labeled with fluorescently conjugated streptavidin. In both wild type (A) and mutant (C) cerebral cortices, biotin labeled cerebral endothelial cells (arrows), but did not cross the blood-brain barrier and label the brain parenchyma. In contrast, vessels within the choroid plexus (CP) and nearby periventricular (PV) regions of wild type (B) and mutant (D) mice are highly permeable to the amine-reactive biotin, although β8 integrin-dependent differences were not apparent. (E, F); Frozen coronal brain sections from adult wild type (E) and mutant (F) mice were immunofluorescently labeled with anti-zona occludin 1 (ZO1), a marker for endothelial cell tight junctions that form the blood-brain barrier. No apparent difference in ZO1 protein expression was detected in wild type and β8-/- cerebral blood vessels (arrows in E, F). (G); Detergent soluble brain lysates from the cerebral cortices of P60 wild type and β8-/- mice were immunoblotted with an anti-occludin, anti-claudin 5, or anti-actin antibodies. Note that both occludin and claudin 5 are expressed in β8-/- brains.

Olfactory Bulb Size Reduction in Adult β8-/- Mice

Gross and microscopic examination of wild type (n=3) and mutant (n=3) brains (Figure 4A, B) revealed approximately 15% reduction in brain size in all β8-/- mice [Brain weight (mgs): wild type, 537.85 +/- 16.43; β8-/-, 444.59 +/- 18.25]. Microscopic analyses of adult wild type and β8-/- brains revealed that most mutant animals displayed varying degrees of hydrocephalus (Supplemental Figure 1C). Additionally, in comparison to wild type littermates, olfactory bulbs in all adult β8-/- mice examined were nearly 50% smaller [Olfactory Bulb Weight (mgs): wild type, 25.16 +/- 1.60; β8-/-, 13.76 +/- 1.89]. Microscopic analysis of β8-/- olfactory bulbs revealed an apparently normal cytoarchitecture (Figure 4C); however, in comparison to control mice (Figure 4D) β8-/- olfactory bulbs (Figure 4E) showed reduced numbers of doublecortin-expressing (DCX⁺) neuroblasts within the rostral migratory stream (RMS). After migrating tangentially along the RMS to the olfactory bulbs, DCX⁺ neuroblasts migrate radially and form functional interneurons within the olfactory bulb granule cell layers (Sawamoto et al., 2006). Analysis of granule cell layers, however, did not reveal obvious differences in numbers of DCX⁺ interneurons in wild type or β8-/- olfactory bulbs (arrows in Figure 4D, E).

Figure 4. Size-Reduced Olfactory Bulbs in Adult β8-/- Mice.

(A, B); Gross images of P90 brains from wild type (A) and β8-/- (B) littermates that were sacrificed and cardiac-perfused with Bouin's fixative. In comparison to the wild type mouse (arrow in A), note the obvious size-reduced olfactory bulbs in the β8-/- animal (arrow in B). (C); Microscopic images of H&E-stained coronal sections through olfactory bulbs of P90 wild type and β8-/- mice. Note that β8-/- olfactory bulbs are significantly smaller, but have an apparently normal olfactory bulb cytoarchitecture. Abbreviations: GLO, glomerular layer; EPL, external plexiform layer; MCL, mitral cell layer; IPL, internal plexiform layer; GCL, granule cell layer; RMS, rostral migratory stream. (D, E); Coronal sections through olfactory bulbs taken from P90 wild type (D) and β8-/- (E) mice were immunofluorescently labeled with anti-DCX (green) to visualize neuroblasts and differentiated neurons and anti-GFAP (red) to identify astrocytes. Note that fewer DCX⁺ neuroblasts (green) are present in the RMS of β8-/- olfactory bulb versus the wild type control (arrowheads in D, E). However, similar numbers of GFAP⁺ astrocytes (red) and differentiated neurons (green) are present in the olfactory bulbs of β8-/- mice in comparison to wild type control mice (arrows in D, E). Abbreviations: RMS, rostral migratory stream; DCX, doublecortin; GFAP, glial fibrillary acidic protein.

Adult β8-/- Mice Display Pathologies in the SVZ and RMS

Various mouse mutants with abnormalities in SVZ neurogenesis and/or RMS migration develop size-reduced olfactory bulbs (Belvindrah et al., 2007; Chazal et al., 2000). Therefore, sections from the SVZ and RMS of wild type and β8-/- mice were analyzed using H&E histology and immunofluorescence to identify DCX⁺ neuroblasts and GFAP⁺ astrocytes. The RMS cytoarchitecture in wild type mice consisted of DCX⁺ neuroblasts forming closely bundled cell chains (Figure 5A), with GFAP⁺ astrocytes forming distinct `glial tubes' that ensheath the RMS (Figure 5A). In contrast, the RMS cytoarchitectures in β8-/- mice were abnormally organized (Figure 5B). GFAP⁺ astrocytes that normally make up glial tubes were intermingled with DCX⁺ cells (Figure 5B), and β8-/- neuroblasts did not form closely bundled chains, resulting in an increased RMS diameters as determined by measuring the boundaries defined by the migrating DCX⁺ cells (Figure 5C). Analysis of H&E-stained brain sections from the SVZ of wild type (Figure 5D) and β8-/- mice (Figure 5E) showed an abnormal cytoarchitecture in the SVZ of mutant animals. Immunofluorescence quantitation revealed nearly three-fold more DCX⁺ cells in the β8-/- SVZ, in comparison to wild type control mice (Figure 5F). β8-/- DCX⁺ cells were often clustered in the SVZ, further supporting integrin-dependent abnormalities in neuroblast migration.

Figure 5. Rostral Migratory Stream and Subventricular Zone Abnormalities in Adult β8-/- Mice.

(A, B); Sagittal brain sections from P90 wild type (A) and β8-/- (B) mice that were H&E stained to reveal the RMS cytoarchitecture. Sections were also immunofluorescently labeled with an anti-DCX antibody (green) to label migrating neuroblasts and an anti-GFAP antibody (red) to label astrocytes in glial tubes that ensheath the RMS. Note the apparently normal DCX⁺ neuroblast cell chains and the GFAP⁺ glial tubes in wild type brain sections (arrows in A). In contrast, the β8-/- RMS is wider with displaced DCX⁺ cells and intermingled GFAP⁺ astrocytes (arrows in B). (C); Rostral migratory stream widths were quantified in wild type (n=3) or β8-/- (n=3) sagittal brain sections that were immunofluorescently labeled with anti-doublecortin antibody to visualize migrating neuroblasts, *p = 0.004 compared to wild type samples. (D, E); Sagittal brain sections from P90 wild type (D) and β8-/- (E) mice were H&E stained to reveal the cytoarchitecture of the SVZ. Sections were also immunofluorescently labeled with an anti-DCX antibody (green) to identify neuroblasts and anti-GFAP (red) to identify NPCs and astrocytes. Note the abnormal cytoarchitecture of the SVZ, as revealed by H&E staining in β8-/- samples (arrows in E). Additionally, more DCX⁺ neuroblasts (arrows in E), often found in clusters, were present in the SVZ of β8-/-mice. (F); DCX⁺ cells were quantified within the SVZ of control (n=6) and mutant mice (n=6) by analyzing sagittal brain sections immunofluorescently labeled with anti-DCX, *p = 0.0001 compared to wild type samples. Abbreviations: v, ventricle; SCJ, striatocortical junction; DCX, doublecortin; GFAP, glial fibrillary acidic protein.

To determine possible functions for β8 integrin in NPC proliferation in vivo, we intraperitoneally injected BrdU into post-natal day 60 (P60) wild type (n=5) and β8-/- mutant (n=5) mice once per day for two consecutive days. Twenty-four hours after the second injection animals were sacrificed and brains were fixed by intracardiac perfusion, sagitally sliced, and embedded in freezing medium. Mitotic cells were quantified after immunostaining sections with an anti-BrdU monoclonal antibody. As shown in Figure 6A-C, in comparison to wild type mice, there was a two-fold reduction in numbers of BrdU⁺ cells in the SVZ of β8-/- mutants. Some BrdU⁺ cells were detected in the RMS and olfactory bulbs of mutant animals, suggesting that neuroblast migration from the SVZ is not completely defective in the absence of β8 gene expression (data not shown).

Figure 6. Neural Cell Proliferation and Survival Defects in the β8-/- Subventricular Zone.

(A, B); Sagittal brain sections from P60 wild type (A) and β8-/- (B) mice that were sacrificed after receiving intraperitoneal injections of BrdU were immunofluorescently labeled with anti-BrdU antibody. Dashed white lines denote ventricle boundaries. (C); Brain sections from wild type (n=6) and β8-/- mice (n=11) were immunofluorescently labeled with anti-BrdU antibody and BrdU⁺ cells were quantified by analyzing serial sections. Note the significant reduction in the numbers of proliferating cells in the SVZ of β8-/- mutant mice, *p < 0.001 compared to wild type samples. (D, E); Ultrastructural analyses of SVZ regions dissected from P90 wild type (D) and β8-/- mice (E). Unlike the wild type SVZ (D), which contains distinct units of a, b, and c cells, the SVZ in β8-/- mice has an abnormal cytoarchitecture with increased numbers of apoptotic cells (arrows in E). (F); Serial coronal sections through the SVZ of P60 wild type (n=7) and β8-/- (n=9) mice were immunofluorescently labeled to identify TUNEL⁺ cells and these cells were quantified. Note the increased numbers of apoptotic cells in the SVZ of β8-/- mice (F), *p < 0.001 compared to wild type samples. Abbreviations: V, ventricle; BrdU, bromodeoxyuridine; a, SVZ type a neuroblast; b; SVZ neural stem cell; c, SVZ transit amplifying cell.

Next, the ultrastructure of the adult β8-/- SVZ was analyzed using transmission electron microscopy. As shown in Figure 6D, the SVZ in wild type mice consisted of distinct units of type A, B, and C cells arranged near the ependymal cell lining of the brain ventricles. In contrast, β8-/- mice displayed cytoarchitectural abnormalities in the SVZ, with obvious disorganization of the A, B, and C cell units (Figure 6E). Electron microscopy also identified increased numbers of apoptotic cells in subventricular regions of β8-/- mice (Figure 6E). Apoptosis in the SVZ of wild type (n=4) and β8-/- adult mice (n=6) was quantified by measuring DNA fragmentation with fluorescent terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL). As shown in Figure 6F, in comparison to wild type littermates there was nearly a 10-fold increase in apoptotic cells numbers in the SVZ of β8-/- mice. Increased numbers of TUNEL⁺ cells were not detected in the corpus putamen or in the cerebral cortices of adult β8-/- mice (data not shown), indicating that apoptosis in the β8-/- brain occurs more selectively in the SVZ.

β8 Integrin is Necessary for Neurosphere Proliferation and Survival In Vitro

We next cultured neurospheres from SVZ regions of P60 wild type and β8-/- mice (n=3 per genotype). In comparison to wild type neurospheres (Figure 7A), β8-/- neurospheres (Figure 7B) were noticeably smaller and this was confirmed by measuring sphere diameters (Figure 7C). Antibodies directed against the αv and β8 integrin subunits were also used to determine integrin protein expression in neurospheres cultured from wild type and β8-/- mice (Figure 7D). αv and β8 integrin proteins were detected in wild type mouse neurospheres (Figure 7D). There was no detectable β8 integrin protein in adult neurospheres isolated from the β8-/- SVZ (Figure 7D) or by PCR-based amplification of β8 genomic sequences using specific DNA primers (data not shown). αv integrin protein expression was not significantly reduced in β8-/- neurospheres (Figure 7D). β8 integrin only heterodimerizes with the αv subunit, but αv integrin can also heterodimerize with β1, β3, β5, and β6 subunits (Hynes, 2002). We did not detect differences in expression of the β1, β3, β5, integrin subunits in β8-/- neurospheres. β6 integrin was not detected in wild type or β8-/- neurospheres (data not shown). Our results demonstrating αv and β8 integrin protein expression in adult mouse NPCs are consistent with previous reports showing αv and β8 integrin mRNA expression in human fetal SVZ NPCs (Hall et al., 2006) and αv, β5, and β8 integrin protein expression in neonatal rat brain neurospheres (Jacques et al., 1998). Lastly, it is likely that the CNS defects in β8-/- mice are due to loss of αvβ8 integrin expression and function, since αv is the only integrin subunit that is reported to heterodimerize with β8 integrin (Hynes et al., 2002; Nishimura et al., 1998). αvβ5 integrin is also expressed in adult NPCs (Figure 7D); however, ablation of the mouse β5 integrin gene does not influence NPC growth and survival in the SVZ or neuroblast migration in the RMS (Belvindrah et al., 2007).

Figure 7. β8 Integrin Regulates Cell Proliferation and Self-Renewal in Neurospheres.

(A, B); Neural progenitors were cultured from the SVZs of P60 wild type (A) and β8-/- (B) adult mice. NPCs proliferate to form free-floating neurospheres in serum-free media containing EGF and bFGF. (C); Diameters of 300 randomly selected neurospheres from P60 wild type mice (n=3) or β8-/- mice (n=3) were measured. Note the reduced diameters of β8-/- neurospheres, *p = 0.0006 compared to wild type neurospheres. (D); Detergent soluble protein lysates from wild type or β8-/- neurospheres were immunoblotted with anti-αv and -β8 integrin polyclonal antibodies, or anti-actin to control for protein loading. (E); A proliferation and self-renewal assay in which six individual neurospheres from each of two wild type and two β8-/- mice (n=12 neurospheres per genotype) were physically dissociated and the total numbers of newly formed spheres were quantified over seven sequential passages. Note the progressive reduction in new sphere formation in cells genetically null for β8 integrin, *p < 0.001 for passage two to passage 7 compared to wild type neurospheres.

We further analyzed possible β8 integrin-dependent proliferation/self-renewal functions in primary neurospheres cultured from the SVZ of P60 adult wild type and β8-/- mice (n=3 per genotype). Selected neurospheres with similar diameters (~75 μm) were physically dissociated and their ability to regenerate new neurospheres was assayed over multiple passages. In comparison to wild type samples (Figure 7E), there was a reduction in the numbers of neurospheres generated from individual β8-/- neurospheres. These defects were more pronounced after sequential passaging, suggesting progressive depletion of cells within β8-/- neurospheres with self-renewing and/or long-term proliferative capacities. Short-term cell proliferation was also quantified by adhering individual NPCs (from dissociated neurospheres) onto laminin-coated dishes and then pulsing cells with BrdU-containing growth media for one hour. The numbers of mitotic cells were then quantified by anti-BrdU immunofluorescence. As shown in Supplemental Figure 2B, a significant reduction in adult cell proliferation was detected in β8-/- versus wild type control NPCs. These findings suggest that β8 integrin plays a critical role in regulating NPC proliferation and/or self-renewal in vitro. These data also support the in vivo results showing reduced cell proliferation in the SVZ of adult β8-/- mice (Figures 6).

Diminished TGFβ1 Activation in β8-/- Neurospheres

Neurospheres cultured from SVZ regions of P60 wild type and β8-/- adult mice (n=3 per genotype) were used to analyze β8 integrin-mediated TGFβ activation. Conditioned media from wild type or β8-/- neurospheres was transferred to 293T cells transfected with a plasmid containing the plasminogen activator inhibitor promoter fused to luciferase cDNA (PAI1-luciferase) (Abe et al., 1994) and luciferase activity was assayed using a luminometer. Alternatively, purified latent TGFβ1 (LAP-TGFβ1) was added to wild type or β8-/- neurospheres and then transferred to control or transfected 293T cells. The use of 293T cells in this assay allows for quantitation of TGFβ-induced signaling events that are minimally influenced by endogenous β8 integrin expression (Supplemental Figure 3).

As shown in Figure 8A we detected a statistically significant increase in luciferase activity using conditioned media from both wild type and β8-/- neurospheres, suggesting that one or more TGFβ's produced endopgenously by neurospheres are activated via integrin-independent mechanisms. This may be due to activation of LAP-TGFβ2, which does not contain an RGD binding motif, but is expressed in brain neurospheres (Roussa et al., 2006). Nonetheless, conditioned media from wild type neurospheres that had been pre-incubated with exogenous LAP-TGFβ1 induced robust luciferase activity (Figure 8A). Conditioned media from β8-/- neurospheres treated with exogenous LAP-TGFβ1 induced statistically significantly lower levels of luciferase activity (Figure 8A). Adult rodent neurospheres also express αvβ1, αvβ3, and αvβ5 integrins (Jacques et al., 1998), and activation of exogenous LAP-TGFβ1 in β8-/- neurospheres may occur via one or more of these integrins.

Figure 8. Autocrine TGFβ Signaling Does Not Promote Neurosphere Proliferation and Self-Renewal.

(A); Quantitation of β8 integrin-mediated TGFβ activation using PAI1-lucferase reporter assays. The results are from two independent experiments using conditioned media from neurospheres cultured from P60 wild type (n=3 per experiment) or β8-/- mice (n=3 per experiment). Neurosphere conditioned media (+/- exogenous LAP-TGFβ1) was transferred to transiently transfected 293T cells, followed by quantitation of luciferase activity. Wild type neurospheres induce a robust TGFβ-mediated luciferase activity; however, note the significant decrease in luciferase activity using conditioned media from β8-/- neurospheres, *p = 0.002 compared to non-conditioned media; **p<0.0001 compared to wild type cells; ***p<0.0001 compared to wild type cells. (B); Exogenous TGFβ1 does not rescue β8-/- neurosphere proliferation and self-renewal defects. Ten SVZ neurospheres of similar sizes (~75 μm diameters) from wild type mice (n=2) or β8-/- mice (n=2) were dissociated, and new neurosphere formation was quantified over four passages. Addition of active TGFβ1 (10 ng/ml) significantly inhibits neurosphere formation in both wild type and β8-/- samples in each passage. The experiment was stopped after P4 due to the absence of new neurospheres generated in the β8-/- samples treated with TGFβ1. *p<0.0001 at all passages compared to untreated wild type samples; **p<0.001 for P2 and **p=0.001 for P2 and P3 compared to untreated β8-/- samples. (C); Antibody-mediated inhibition of TGFβ does not inhibit wild type neurosphere self-renewal and proliferation. Five individual SVZ neurospheres cultured from each of two wild type mice (n=10 total neurospheres) were dissociated and allowed to reform new spheres over three sequential passages, either in the presence of 5 μM isotype control IgG, 5 μM anti-TGFβ, and/or active TGFβ1 (10 ng/ml). Note that inhibition of TGFβ alone does not influence neurosphere self-renewal, but does largely reverse the growth inhibitory effects of bioactive TGFβ1, ^†p=0.002 compared to untreated P2 samples; ^††p<0.0001 compared to untreated P3 samples; *p=0.0004 compared to untreated P1 samples; **p<0.001 compared to untreated P2 samples.

To determine if integrin-activated TGFβ's function in an autocrine manner to promote NPC proliferation and survival, we added bioactive TGFβ1 to wild type neurospheres. Exogenous TGFβ1 was also added to β8-/- neurospheres in an attempt to `rescue' the self-renewal and proliferation defects. As shown in Figure 8B, addition of active TGFβ1 (10 ng/ml) resulted in robust inhibition of self-renewal in both wild type and β8-/- neurospheres over multiple passages. We also observed similar neurosphere growth inhibition at lower TGFβ1 concentrations, ranging from 0.01 to 1.0 ng/ml (data not shown). These data are consistent with other in vitro and in vivo results showing that exogenous TGFβ's inhibit NPC self-renewal and proliferation (Battista et al., 2006; Falk et al., 2008; Wachs et al., 2006). Lastly, addition of an antibody that neutralized TGFβ1, TGFβ2, and TGFβ3 (Dasch et al., 1989) did not impair wild type neurosphere self-renewal (Figure 8C), suggesting that TGFβ autocrine signaling pathways are not essential for NPC proliferation and self-renewal.

DISCUSSION

Does β8 Integrin Differentially Regulate Blood Vessel Functions in the Embryonic versus Adult Brain?

Our group and others have used various conditional gene ablation strategies to demonstrate that αvβ8 integrin in perivascular NPCs regulates blood vessel morphogenesis in the embryonic mouse brain; yet, the severe brain-specific pathologies in αv and β8 integrin mutant mice resolve by adulthood (McCarty et al., 2004; Proctor et al., 2005). We initially attributed resolution of the vascular pathologies to mosaic Cre transgene expression and heterogeneous integrin gene deletion in neural progenitor cells. However, in the present study we also observe resolution of hemorrhage in adult mice that are genetically null for β8 integrin in all cells. Also, while this manuscript was in the advanced stages of revision Munger and colleagues reported neurological phenotypes and premature death in the absence of ongoing hemorrhage in adult β8-/- mice (Aluwihare et al., 2009). These results significantly negate our original hypothesis that neural cells expressing αvβ8 integrin, which had `escaped' Cre-mediated gene deletion, contribute to repair of the hemorrhagic phenotypes. It remains possible that other pathways are activated in integrin mutant mice, and these may compensate for loss of normal β8 integrin functions. However, an alternative mechanism that we now favor is that β8 integrin in perivascular neural cells regulates active phases of blood vessel growth, like those that occur during embryonic and neonatal brain development. Angiogenesis in most regions of the rodent brain largely terminates by post-natal day 30 (P30) (Plate, 1999), which coincides with the reduction of neurovascular pathologies in adult αv and β8 integrin mutant mice (Figure 4, Supplemental Figure 1). In contrast, BrdU labeling experiments in rodents have shown that endothelial cells in neurogenic regions of the brain (also termed neurovascular niches) retain angiogenic characteristics (Kerever et al., 2007; Mercier et al., 2002; Palmer et al., 2000). Hence, it is enticing to speculate that similar to it's function in embryonic brain NPC's, β8 integrin in adult NPC's continues to mediate interactions with blood vessels in neurovascular niches and these events promote NPC growth, survival and migration.

It is possible that the neurovascular unit defects we report herein are related, in part, to prior embryonic neurovascular pathologies, or to the post-hemorrhagic hydrocephalus that develops in the adult brain. Indeed, similar to what we observe in β8-/- mice, intraventricular hemorrhage in the human neonatal brain can lead to long-term neurological deficits associated with hydrocephalus (Roland and Hill, 2003). Previous studies in rats with experimentally induced hydrocephalus have shown an increase in reactive astrocytes throughout the brain (Del Bigio and Zhang, 1998). Hence, the β8-/- mice may serve as potentially useful models for studying progressive neurodegeneration related to post-hemorrhagic hydrocephalus. Based on the following experimental data, however, we also argue that β8 integrin also provides essential NPC-intrinsic growth and survival functions in the SVZ: (i) β8 integrin protein is expressed in the SVZ in vivo and in SVZ NPCs cultured ex vivo (Figures 1 and 7); (ii) β8-/- NPCs isolated from the adult brain and cultured in media that partly mimics the SVZ microenvironment display abnormal growth and survival behaviors (Figure 7); (iii) adult mice displaying mild or severe hydrocephalus show similar NPC defects in the SVZ in vivo and in ex vivo cultures (data not shown); and (iv) hydrocephalus can develop as a result of cortical atrophy due to impaired developmental and adult neurogenesis (Tomita et al., 2003) and we detect impaired neuronal differentiation in embryonic β8-/- NPCs (Supplemental Figure 2E).

Is There a β8 Integrin-TGFβ Paracrine Signaling Axis in Adult Neuovascular Niches?

αvβ8 integrin is a receptor for latent forms of TGFβ1 and TGFβ3, and some pathologies in αv and β8 integrin mutant mice have been linked to reduced integrin-mediated activation of TGFβ's (Lacy-Hulbert et al., 2007; Travis et al., 2007). In this report we have shown that β8-/- NPCs display a modest, yet statistically significant reduction in activation of latent TGFβ1 (Figure 8A), suggesting that the brain pathologies we detect in β8-/- mice may be caused by diminished TGFβ activation and signaling. However, much of our experimental data argue against an important role for integrin-activated TGFβ's acting in an autocrine manner to promote NPC growth and survival. First, we have not been able to `rescue' NPC proliferation and self-renewal defects by exogenous addition of bioactive TGFβ1 to β8-/- neurosphere cultures (Figure 8B), suggesting other pathways are involved. Second, a blocking antibody directed against bioactive TGFβ's does not induce growth defects in wild type NPC's similar to those that develop in β8-/- NPCs (Figure 8C), suggesting that TGFβ's play limited physiological roles in neurosphere growth. Lastly, genetic ablation of TGFβ receptor II signaling in embryonic neural cells using a Nestin-Cre transgene (J. Shin and J.H. McCarty, unpublished data), or in adult SVZ NPCs using a GLAST-CreERT2 transgene (Colak et al., 2008), does not impair brain development or post-natal neurogenesis. Hence, we propose a model in which β8 integrin-activated TGFβ's may act in a paracrine manner to regulate endothelial cell or pericyte functions, in turn leading to the production of vascular-derived factors that promote NPC growth and survival (Figure 9). Indeed, other reports have suggested similar TGFβ-dependent paracrine mechanisms for astrocyte-mediated regulation of endothelial cell functions (Cambier et al., 2005; Garcia et al., 2004). It also remains possible, and indeed likely, that β8 integrin is a receptor for other growth-promoting ECM ligands within adult neurovascular niches.

Figure 9. A Model for αvβ8 Integrin Adhesion and Signaling at the Neural-Vascular Interface.

αvβ8 integrin is expressed in perivascular neural cells where it mediates interactions with multiple ECM ligands, including latent TGFβ's (LAP-TGFβ), present within vascular basement membranes. Integrin-mediated adhesion induces signaling pathways in cerebral blood vessels via canonical TGFβ receptors (RI/RII). Activation of these pathways in endothelial cells leads to the production of growth factors, and probably other vessel-derived survival cues, which subsequently modulate NPC behaviors. Genetic ablation of β8 integrin causes loss of integrin-mediated adhesion to ECM ligands, which uncouples the neural-vascular cell connections and leads to imbalances in NPC proliferation and survival.

Does β8 Integrin Have Functions Outside of Adult Neurovascular Niches?

While these studies demonstrate necessary roles for β8 integrin in adult neurovascular units and SVZ neurogenesis, this integrin may also be important in other populations of neural cellsthat contribute to brain physiology. Indeed, we detect abnormal development of the hippocampus and dentate gyrus in adult β8-/- mice (Supplemental Figure 4), suggesting more general functions for β8 integrin within other neurogenic brain regions. NPCs within the SVZ also generate oligodendroglial progenitors that myelinate axons in various regions of the adult brain (Menn et al., 2006). Conditional αv integrin knockout mice develop progressive neurodegenerative defects due to cerebellar white matter demyelination (McCarty et al., 2004); however, these cerebellar pathologies are not apparent in adult β8-/- mice (Supplemental Figure 5), suggesting that multiple αv-containing integrins regulate oligodendroglial progenitor cell functions. Interestingly, αvβ6 integrin is also a receptor for latent TGFβ's (Munger et al., 1999) and is expressed in the adult mouse brain (Chan et al., 2003). Neurodegeneration has not been reported in β6-/- mice, however, indicating that defective adhesion and signaling in αv conditional knockouts may be due to combined loss of β6 and β8 functions. Hence, it is likely that β8 integrin has important roles in multiple neural cell types in the adult brain.

Materials and Methods

Mice, Antibodies, and Immunofluorescence

ICR/CD-1 mice were purchased from Jackson Laboratories. β8+/- mice (Zhu et al., 2002) were obtained from the Mutant Mouse Regional Resource Center. Adult mice were anesthetized and brains were fixed by cardiac perfusion with ice-cold 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS). Brains were removed and post-fixed overnight in 4% PFA/PBS and then processed for standard paraffin embedding. Paraffin-embedded brain sections (7 μm/section) were generated, and sections were analyzed using a Zeiss Axio Imager.Z1 microscope. The following antibodies were purchased from commercial sources and used for immunofluorescence on tissue section and/or cells: rabbit anti-laminin (1:100, Sigma), mouse anti-GFAP mAb (2.5 μg/ml, Chemicon), rabbit anti-GFAP pAb (10 μg/ml, DAKO), rat anti- BrdU (Accurate Chemical), rabbit anti-DCX (Chemicon), anti-tubulin-III/TUJ1 (1 μg/ml GeneTex), anti-ZO1 and rat anti-mouse HSPG (antibody 10E4, 10 μg/ml Seikagaku). Prior to immunofluorescent staining with anti-DCX and anti-GFAP, paraffin sections were processed for heat-based antigen retrieval at pH 9 according to manufacturer's protocol (DAKO). For anti-ZO1 immunofluorescence, brains were removed without prior fixation and embedded in OCT for frozen section preparation. TUNEL fluorescence was performed on paraformaldehyde-fixed and paraffin-embedded brain sections using the manufacturer protocols (Promega). Secondary antibodies used for immunofluorescence were goat anti-rabbit and goat anti-mouse conjugated to Alexa488 or ALexa594 (Molecular Probes). The following antibodies used for immunoblotting were purchased from commercial sources: rabbit anti-β-actin antibody (Sigma-Aldrich), rabbit anti-occludin and rabbit anti-claudin 5 antibodies (Zymed). The anti-αv antibody was generated against a synthetic peptide (CKRVRPPQEEQEREQLQPHENGEGTSEA) corresponding to a region of the chicken αv cytoplasmic tail. The anti-β8 integrin antibodies used for immunoblotting have been described previously (McCarty et al., 2005; McCarty et al., 2004).

Neurosphere Cultures

NPCs were cultured from the SVZ of individual adult mice using standard protocols (Rietze and Reynolds, 2006). A detailed description of the procedure is available in the Supplementary Materials and Methods. For secondary sphere formation assays, single spheres with similar diameters (~75 μm) were mechanically dissociated and plated in one well of a 24 well plate. After intervals of 7 days (for multiple passages) the number of newly formed spheres was counted. Alternatively, neurosphere self-renewal was quantified in the presence of exogenously added TGFβ1 (10 ng/ml) and/or anti-TGFβ antibody (5 μM) (both from R&D Systems). To analyze short-term proliferation, neurospheres were allowed to adhere overnight to glass coverslips coated with 5 μg/ml laminin (Sigma-Aldrich). Cells were then pulsed for 1 h with 1 μM BrdU and then fixed, permeabilized, and immunostained with a rat anti-BrdU monoclonal antibody. For neurosphere immunoblotting, cell lysates were prepared in RIPA Buffer (10mM Tris, pH7.4, 1% NP40, 0.5% Deoxycholate, 0.1% SDS, 150mM NaCl and 1mM EDTA) and protein concentrations were determined (BCA Assay, Thermo Scientific). Protein lysates were mixed with an equal volume of 2X sample buffer, heated at 100° C, resolved by SDS-PAGE, and then immunoblotted with the various antibodies. For immunofluorescence analyses, neurospheres were fixed in 4% PFA, embedded in paraffin, and then sectioned at 7 μm intervals. Spheres were then immunofluorescently labeled with primary and secondary antibodies. To quantify differentiation, neurospheres adhering to laminin-coated coverslips were cultured in 1% serum-containing growth medium. Seven days later cells were fixed, immunofluorescently labeled, and GFAP-expressing astrocytes and Tuj1-expressing neurons were quantified.

BrdU Incorporation Assays

BrdU (BD Pharmingen) was injected at 65 μg/g body weight into mice once daily for two consecutive days. 24 hours after the last injection mice were cardiac-perfused with 4% paraformaldehyde and the brains were removed and post-fixed overnight. Brains were cryopreserved with 10% and 20% sucrose in PBS at 4°C, then embedded in Tissue Tek OCT compound (Sakura, Inc). Frozen sections (7 μm thickness per section) were generated and at least eight serial sections per brain were analyzed to quantify BrdU incorporation. Briefly, genomic DNA was denatured in 2N HCl for 1 hour at 37°C and then neutralized with 0.1M sodium borate buffer for 30 minutes at room temperature. Slides were then blocked with 10% goat serum (Vector Labs) in PBS for 30 minutes and incubated in primary antibody overnight at 4°C, followed by incubation with Alexa488-conjugated goat anti-rat secondary antibodies.

Luciferase Reporter Assays

293T cells were plated into poly-lysine-coated six well plates at 4×10⁵ cells per well and allowed to attach overnight. Cells were then transfected with a PAI1-Luciferase plasmid (Abe et al., 1994) using Effectene reagents (Qiagen). After 24 hours the transfection media was removed and serum free media was added for an additional 24 hours. Meanwhile, one hundred wild type or β8-/- adult neurospheres of similar diameters (~75 μm) were added to six well dishes containing 2 mls of neurosphere growth media. LAP-TGFβ1 (SLC, R&D Systems) at 10 ng/ml (or PBS as a negative control) was added to neurospheres. After 16 hours conditioned media was collected, passed through a sterile 0.2 μm filter, and added to transfected 293T cells (see above) for 16 hours. As a control to confirm PAI1-Luciferase plasmid transfection, bioactive TGFβ1 (R&D Systems) was added at 5 ng/ml to transfected 293T cells and incubated for 4 hr. Cell lysates were prepared and luciferase substrates were added according to the Enhanced Luciferase Assay kit (BD Biosciences). Lysates were plated in duplicate wells in a 96 well plate and luciferase activities were determined using a Lumistar Galaxy Luminometer (BMG Lab Technologies). The murine VEGF-A ELISA reagents were purchased from R&D Systems.

Biotin Perfusions

P60 control and mutant animals were deeply anesthetized with Avertin (250 mg/kg) and a 20G needle was inserted into the left ventricle of the heart. Animals were first perfused with 20 mls of PBS to flush the circulation so that no residual cells or serum proteins were labeled with reactive biotin. Animals were then perfused for five continuous minutes with 1 mg/ml EZLink-NHS-Biotin (Pierce Chemicals, Inc.) dissolved in PBS (Approximately 7.5 total mls per animal). Animals were then perfused with 4% paraformaldehyde/PBS fixative to preserve brain morphology. Brains were removed and coronally sliced into 1 mm segments followed by cryopreservation in 30% Sucrose/PBS, embedded in OCT, and cut in 7 μm sections. Frozen brain sections were rehydrated in PBS and blocked with 10% goat serum in PBS and visualized using streptavidin conjugated to Alexa 488 fluorophore (Molecular Probes).