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First published online May 20, 2009
doi: 10.1242/10.1242/jcs.043257

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β8 integrin regulates neurogenesis and neurovascular homeostasis in the adult brain

Department of Cancer Biology, University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA

View larger version (32K): [in this window] [in a new window]   Fig. 1. Genetic background influences post-natal survival of β8–/– mice. (A) Kaplan-Meier survival plot using wild-type (n=40) and β8–/– (n=39) mice. Note that approximately 60% of β8–/– mice die by post-natal day 30 (P30) whereas the remaining animals survive for up to 5 months. (B) Weight analysis using P30 and P60 control and mutant male mice (n=7-10 males per genotype, although similar results were observed with female mice). Note that β8–/– mice weigh significantly less than wild-type littermates. *P<0.0001 compared with wild-type group. (C,D) Images of 4-month-old (P120) wild-type (C) and β8–/– (D) littermates. β8–/– mice have a hunched posture and limb paresis; these phenotypes develop with 100% penetrance in mutant mice that survive to adulthood. (E) Footprint analysis performed with 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 brain regions of wild-type mice, but is not detected in β8–/– animals. SVZ, subventricular zone of lateral ventricle; OB, olfactory bulbs; DG, hippocampal dentate gyrus; CB, cerebellum.

View larger version (81K): [in this window] [in a new window]   Fig. 2. Adult β8–/– mice have widespread neurovascular pathologies. (A,B) Coronal sections through cerebral cortices of wild-type (A) and β8–/– (B) mice were immunostained with anti-laminin to reveal cerebral blood vessels. (C) Quantification of cerebral blood vessels based on anti-laminin immunoreactivity of P60 wild-type (n=5) and β8–/– (n=5) mice, *P=0.002 as compared with 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) Quantification of GFAP-expressing astrocytes in sections from wild-type (n=4) and β8–/– (n=4) mice, *P<0.001 as compared with wild-type group. (G,H) Transmission electron microscopy reveals 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) Quantification 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 labeled with both anti-lamina and anti-GFAP. The percentages of lamina-expressing blood vessels contacting more than one GFAP+ astrocytes were quantified per x400 field, *P=0.002 as compared with the wild-type group.

View larger version (42K): [in this window] [in a new window]   Fig. 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) cortices, biotin labeled cerebral endothelial cells (arrows), but did not cross the blood-brain barrier and label the brain parenchyma. By 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 anti-occludin, anti-claudin 5 or anti-actin antibodies. Note that both occludin and claudin 5 are expressed in β8–/– brains.

View larger version (61K): [in this window] [in a new window]   Fig. 4. Size-reduced olfactory bulbs in adult β8–/– mice. (A,B) P90 brains from wild-type (A) and β8–/– (B) littermates that were killed and cardiac-perfused with Bouin's fixative. In comparison to the wild-type mouse (arrow in A), note the smaller olfactory bulbs in the β8–/– animal (arrow in B). (C) 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 cytoarchitecture. 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 than in 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). RMS, rostral migratory stream; DCX, doublecortin; GFAP, glial fibrillary acidic protein.

View larger version (93K): [in this window] [in a new window]   Fig. 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). By 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) and β8–/– (n=3) sagittal brain sections that were immunofluorescently labeled with anti-doublecortin antibody to visualize migrating neuroblasts, *P=0.004 compared with 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. 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 with wild-type samples. v, ventricle; SCJ, striatocortical junction; DCX, doublecortin; GFAP, glial fibrillary acidic protein.

View larger version (47K): [in this window] [in a new window]   Fig. 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 killed 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–/– (n=11) mice 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 with wild-type samples. (D,E) Ultrastructural analyses of SVZ regions dissected from P90 wild-type (D) and β8–/– (E) mice. 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 higher numbers of apoptotic cells in the SVZ of β8–/– mice. *P<0.001 compared with wild-type samples. V, ventricle; BrdU, bromodeoxyuridine; a, SVZ type a neuroblast; b, SVZ neural stem cell; c, SVZ transit amplifying cell.

View larger version (37K): [in this window] [in a new window]   Fig. 7. β8 integrin promotes NPC proliferation and self-renewal in vitro. (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 medium 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 with wild-type neurospheres. (D) Detergent-soluble protein lysates from wild-type or β8–/– neurospheres were immunoblotted with anti-v and -β8 integrin polyclonal antibodies, or anti-actin to control for protein loading. (E) A proliferation and self-renewal assay in which 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 seven compared with wild-type neurospheres.

View larger version (28K): [in this window] [in a new window]   Fig. 8. Autocrine TGFβ signaling does not promote NPC proliferation and self-renewal. (A) Quantification 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–/– (n=3 per experiment) mice. Neurosphere conditioned medium (+/– exogenous LAP-TGFβ1) was transferred to transiently transfected HEK293T cells, followed by quantification of luciferase activity. Wild-type neurospheres induce a robust TGFβ-mediated luciferase activity; however, note the significant decrease in luciferase activity using conditioned medium from β8–/– neurospheres, *P=0.002 compared with non-conditioned media; **P<0.0001 compared with wild-type cells; ***P<0.0001 compared with wild-type cells. (B) Exogenous TGFβ1 does not rescue β8–/– neurosphere proliferation and self-renewal defects. Ten SVZ neurospheres of similar sizes (75 µm diameter) 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 because of the absence of new neurospheres generated in the β8–/– samples treated with TGFβ1. *P<0.0001 at all passages compared with untreated wild-type samples; **P<0.001 for P2 and **P=0.001 for P3 and P4 compared with 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 with untreated P2 samples; P<0.0001 compared with untreated P3 samples; *P=0.0004 compared with untreated P1 samples; **P<0.001 compared with untreated P2 samples.

View larger version (30K): [in this window] [in a new window]   Fig. 9. A model for vβ8 integrin adhesion and signaling at the neural-vascular interface. vβ8 integrin is expressed in perivascular neural cells where it mediates interactions with multiple ECM ligands, including latent TGFβs (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.

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