Astrocytes Negatively Regulate Neurogenesis Through the Jagged1-Mediated Notch Pathway§

Authors

  • Ulrika Wilhelmsson,

    1. Center for Brain Repair and Rehabilitation, Department of Clinical Neuroscience and Rehabilitation, Institute of Neuroscience and Physiology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
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  • Maryam Faiz,

    1. Center for Brain Repair and Rehabilitation, Department of Clinical Neuroscience and Rehabilitation, Institute of Neuroscience and Physiology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
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  • Yolanda de Pablo,

    1. Center for Brain Repair and Rehabilitation, Department of Clinical Neuroscience and Rehabilitation, Institute of Neuroscience and Physiology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
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  • Marika Sjöqvist,

    1. Turku Centre for Biotechnology, University of Turku, Turku, Finland
    2. Department of Biosciences, Åbo Akademi University, Turku, Finland
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  • Daniel Andersson,

    1. Center for Brain Repair and Rehabilitation, Department of Clinical Neuroscience and Rehabilitation, Institute of Neuroscience and Physiology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
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  • Åsa Widestrand,

    1. Center for Brain Repair and Rehabilitation, Department of Clinical Neuroscience and Rehabilitation, Institute of Neuroscience and Physiology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
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  • Maja Potokar,

    1. Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia
    2. Celica Biomedical Center, Ljubljana, Slovenia
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  • Matjaž Stenovec,

    1. Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia
    2. Celica Biomedical Center, Ljubljana, Slovenia
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  • Peter L. P. Smith,

    1. Center for Brain Repair and Rehabilitation, Department of Clinical Neuroscience and Rehabilitation, Institute of Neuroscience and Physiology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
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  • Noriko Shinjyo,

    1. Department of Medical Chemistry and Cell Biology, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
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  • Tulen Pekny,

    1. Center for Brain Repair and Rehabilitation, Department of Clinical Neuroscience and Rehabilitation, Institute of Neuroscience and Physiology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
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  • Robert Zorec,

    1. Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia
    2. Celica Biomedical Center, Ljubljana, Slovenia
    3. IKERBASQUE, Basque Foundation for Science, Bilbao, Spain
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  • Anders Ståhlberg,

    1. Center for Brain Repair and Rehabilitation, Department of Clinical Neuroscience and Rehabilitation, Institute of Neuroscience and Physiology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
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  • Marcela Pekna,

    1. Center for Brain Repair and Rehabilitation, Department of Clinical Neuroscience and Rehabilitation, Institute of Neuroscience and Physiology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
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  • Cecilia Sahlgren,

    1. Turku Centre for Biotechnology, University of Turku, Turku, Finland
    2. Department of Biosciences, Åbo Akademi University, Turku, Finland
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  • Milos Pekny

    Corresponding author
    1. Center for Brain Repair and Rehabilitation, Department of Clinical Neuroscience and Rehabilitation, Institute of Neuroscience and Physiology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
    • Center for Brain Repair and Rehabilitation, Department of Clinical Neuroscience and Rehabilitation, Institute of Neuroscience and Physiology, Sahlgrenska Academy at the University of Gothenburg, Box 440, SE-405 30 Gothenburg, Sweden

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    • Telephone: +46 31 786 3269; Fax: +46 31 416 108


  • Author contributions: U.W. and M.F.: conception and design, collection and/or assembly of data, data analysis and interpretation, and manuscript writing; Y.d.P and D.A.: conception and design, collection and/or assembly of data, and data analysis and interpretation, M. Sjöqvist, Å.W., M. Potokar, M. Stenovec, P.L.P.S., and T.P.: collection and/or assembly of data and data analysis and interpretation; N.S.: provision of study material or patients; R.Z. and C.S.: conception and design, financial support, and data analysis and interpretation; A.S.: conception and design; M. Pekna: conception and design, data analysis and interpretation, and manuscript writing; M. Pekny: conception and design, financial support, data analysis and interpretation, and manuscript writing. U.W. and M.F. contributed equally to this article. Y.d.P. and M. Sjöqvist, contributed equally to this article.

  • Disclosure of potential conflicts of interest is found at the end of this article.

  • §

    First published online in STEM CELLSEXPRESS July 15, 2012.

Abstract

Adult neurogenesis is regulated by a number of cellular players within the neurogenic niche. Astrocytes participate actively in brain development, regulation of the mature central nervous system (CNS), and brain plasticity. They are important regulators of the local environment in adult neurogenic niches through the secretion of diffusible morphogenic factors, such as Wnts. Astrocytes control the neurogenic niche also through membrane-associated factors, however, the identity of these factors and the mechanisms involved are largely unknown. In this study, we sought to determine the mechanisms underlying our earlier finding of increased neuronal differentiation of neural progenitor cells when cocultured with astrocytes lacking glial fibrillary acidic protein (GFAP) and vimentin (GFAP−/−Vim−/−). We used primary astrocyte and neurosphere cocultures to demonstrate that astrocytes inhibit neuronal differentiation through a cell–cell contact. GFAP−/−Vim−/− astrocytes showed reduced endocytosis of Notch ligand Jagged1, reduced Notch signaling, and increased neuronal differentiation of neurosphere cultures. This effect of GFAP−/−Vim−/− astrocytes was abrogated in the presence of immobilized Jagged1 in a manner dependent on the activity of γ-secretase. Finally, we used GFAP−/−Vim−/− mice to show that in the absence of GFAP and vimentin, hippocampal neurogenesis under basal conditions as well as after injury is increased. We conclude that astrocytes negatively regulate neurogenesis through the Notch pathway, and endocytosis of Notch ligand Jagged1 in astrocytes and Notch signaling from astrocytes to neural stem/progenitor cells depends on the intermediate filament proteins GFAP and vimentin. STEM Cells2012;30:2320–2329

INTRODUCTION

Adult neurogenesis is restricted to two specific neurogenic niches: the subgranular zone (SGZ) of the hippocampus and the subventricular zone (SVZ) of the lateral ventricles. The cellular players within these niches are important for the regulation of neural stem/progenitor cell development and coordination of cell genesis. Increasing evidence suggests an important role for astrocytes in the neurogenic niche: astrocytes share certain properties with neural stem cells [1–3] and create an environment conducive to neurogenesis [4]. Astrocytes regulate neurogenesis by the secretion of factors such as Wnt3 [5], interleukin-1β, interleukin-6 [6], and thrombospondin-1 [7]. Astrocytes control the neurogenic niche also through membrane-associated factors [4], however, the identity of these factors and the mechanisms involved are largely unknown.

The Notch receptor family is involved in a large number of cell fate decisions during development and their activation can promote an undifferentiated, precursor cell state. Notch1 and its cognate ligand Jagged1 are expressed in the neurogenic niches in the adult mammalian brain [8]. Using transgenic Hes5-GFP mice, which enable the visualization of cells with active canonical Notch signaling, it was shown that canonical Notch signaling in the SGZ is restricted to type 1 neural stem cells with radial or nonradial morphology [9, 10]. Activated Notch in postnatal SVZ cells suppresses neuronal differentiation and decreases proliferation [11], and activation of Notch signaling in neural stem cells leads to increased astrogliogenesis in vitro [12]. Notch receptor activation promotes the survival of neural stem cells in vitro, and transient administration of Notch ligands to the brain of adult rats increased the numbers of newly generated precursor cells [13]. A series of recent publications supports the notion that Notch1 and Notch canonical signaling repressing pro-neuronal genes are required for the maintenance of a pool of proliferating undifferentiated cells in the adult SGZ and SVZ [9, 10, 14–16]. Thus, despite controversy regarding the actual mechanisms and effects on neuronal cell fate, Notch signaling plays an essential role in controlling adult neurogenesis. In SVZ, astrocytes were shown to express Jagged1 [12, 17], whereas in the adult hippocampus, Jagged1 mRNA expression was reported to be present in the SGZ [15] and in the granule cell layer (GCL) [8].

We and others have shown that ablation of the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin in mice [18, 19] creates an environment more permissive to transplantation of neural grafts or neural stem cells [20, 21] and increases axonal and synaptic regeneration [22–24]. Neuronal differentiation of neural progenitor cells was increased when cocultured with astrocytes lacking GFAP and vimentin (GFAP−/−Vim−/−) [21]. Although the altered cellular distribution of Wnt3 in the GFAP−/−Vim−/− astrocytes could be associated with changed secretion of this pro-neurogenic factor and thus explain this finding, a direct cell–cell signaling from astrocytes to neural stem/progenitor cells and its involvement in neurogenesis remains as an attractive alternative. In this study, we investigated the role of astrocyte membrane-associated factors in the regulation of neurogenesis.

MATERIALS AND METHODS

Mice

Mice carrying a null mutation in the GFAP and/or vimentin genes and wild-type controls [25, 26] were on a C57Bl6/129Sv/129Ola mixed genetic background. All mice were housed in a barrier facility, and experiments were conducted according to protocols approved by the Ethics Committee of the University of Gothenburg.

Neurosphere Culture

Postnatal day 4 (P4) mouse forebrain was dissected in Leibovitz medium (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) and enzymatically (0.1% trypsin, 0.5 mM EDTA in Hank's balanced salt solution [Invitrogen]) and mechanically dissociated into a single-cell suspension and plated in neurosphere medium (Neurobasal [Invitrogen] containing L-glutamine [2 mM, Invitrogen], penicillin/streptavidin [100 U/0.1 mg/ml (1×), Invitrogen], B27 [1:50, Invitrogen], basic fibroblast growth factor [20 ng/ml, Invitrogen], epidermal growth factor [20 ng/ml, Stem Cell Technologies, Vancouver, BC, Canada, http://www.stemcell.com], heparin [1 U/ml, Sigma-Aldrich, St. Louis, MS, http://www.sigmaaldrich.com], and fungizone [0.25 μg/ml, Bristol-Myers Squibb, New York City, NY, http://www.bms.com]). Neurospheres were dissociated with TrypLE (Invitrogen) into a single-cell suspension and replated under the same conditions as primary cultures. Ablation of GFAP, vimentin, or both did not alter the passaging capacity.

Neurosphere Differentiation

Seven-day-old P4 primary neurospheres were pipetted into 24-well plates (15 neurospheres per well) with glass coverslips coated with poly-L-ornithine (0.01 mg/ml, Sigma-Aldrich) and laminin (5 μg/ml, Invitrogen) and gently flooded with differentiation medium (neurosphere medium as described above without growth factors and heparin added). Alternatively, primary neurospheres were dissociated with TrypLE and plated at equal densities for differentiation. One day after plating, 1% fetal bovine serum (FBS, Invitrogen) was added. For experiments with conditioned medium, neurospheres were dissociated and plated in 90% neurosphere medium supplemented with 10% medium conditioned by astrocytes for 3 days. Higher concentrations of conditioned medium resulted in neurosphere cell death. After 5 days of differentiation, cells were fixed with 4% paraformaldehyde for immunocytochemistry.

Primary Astrocyte Culture

Primary astrocytes derived from forebrain of P2 mice were cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) with 10% FBS, as described [27]. For quantitative real-time polymerase chain reaction (qRT-PCR) analysis, astrocytes (Experiment 1) were prepared as described [28]. Alternatively, total RNA was extracted and purified with the RNeasy Micro Kit (Qiagen, Hilden, Germany, http://www.qiagen.com) (Experiments 2 and 3).

Cocultures

At confluence, primary astrocytes were trypsinized and plated in 24-well plates or on coated glass coverslips. Twenty-four hours later, the medium was changed to differentiation medium. After another 24 hours, primary neurospheres were enzymatically dissociated with TrypLE for 5 minutes at 37°C and plated on astrocytes. For the experiment in Figure 1D, neurospheres were stained with Hoechst 33342 dye (1:5,000, Invitrogen) for 10 minutes (to distinguish neurosphere cells from astrocytes) For experiments in Figure 1B and 1E, neurospheres were incubated with 5 μM 5-bromo-2-deoxyuridine (BrdU, Sigma-Aldrich) for 24 hours (to distinguish neurosphere cells from astrocytes) before dissociation and replating with astrocytes. For cultures with mixed GFAP−/−Vim−/− and wild-type astrocytes, equal numbers of cells of respective genotype were mixed and plated. One day after plating, 1% FBS was added. After 5 days of differentiation, cells were fixed with 4% paraformaldehyde for immunocytochemistry.

Figure 1.

Neuronal differentiation of stem/progenitor cells is determined by the environment and is cell–cell contact dependent. (A):GFAP−/−Vim−/− (G−/−V−/−; n = 8) neurospheres differentiate into more β-III-tubulin-expressing neurons (β-III-tubulinpos) than WT (n = 11), GFAP/ (G−/−; n = 6), or Vim/ (V/; n = 6) neurospheres. (B): Neuronal differentiation of P4 WT neurospheres cocultured with either WT, G/, V/, or G/V/− primary P2 astrocytes (n = 6, 3, 3, and 3, respectively). Images show 5-bromo-2-deoxyuridine (BrdU)-labeled dissociated WT neurospheres plated on top of WT and G/V/ astrocytes and allowed to differentiate for 5 days. Neurosphere cells are visualized by immunostaining with antibodies against BrdU (red), neurons are visualized by antibodies against β-III-tubulin (green), and nuclei are labeled with DAPI. Arrows point to neurosphere cells differentiated into neurons (β-III-tubulinposBrdUpos), arrowheads point to neurosphere cells not differentiated into neurons (β-III-tubulinnegBrdUpos). (C): Neuronal differentiation of dissociated WT neurosphere cells in astrocyte-conditioned medium. No difference in the percentage of β-III-tubulinpos neurons was observed between WT neurosphere cells cultured under control conditions (no conditioned medium, n = 4) and WT neurosphere cells cultured in the presence of medium conditioned by G−/−V−/− (n = 9) or WT (n = 7) astrocytes. (D): When neurosphere cells were exposed to conditioned medium from WT astrocytes while in direct contact with G−/−V−/− astrocytes or vice versa, neuronal differentiation was increased by direct contact with G−/−V−/− astrocytes (n = 4 in all groups). (E): The number of β-III-tubulinpos neurons from WT neurospheres was higher in the presence of G−/−V−/− astrocytes than WT astrocytes or mixed G−/−V−/− and WT astrocytes (n = 4 in all groups). N equals number of mice per experimental group. DAPI, nuclear staining; scale bars = 75 μm (A) and 30 μm (B); *, p <.05; **, p <.01; ***, p <.005; ****, p <.001 (A–C and E, ANOVA followed by Tukey honestly significant difference post hoc analysis; D, two-tailed t-test). Abbreviations: DAPI, 4′-6-diamidino-2-phenylindole; WT, wild type.

Immunocytochemistry and Cell Proliferation, Cell Death and Differentiation Assessment

To assess cell differentiation, cells were labeled with mouse anti-β-III-tubulin (1:2,000, Covance, Princeton, NJ, http://www.covance.com), rabbit anti-S100 (1:200, Dako, Glostrup, Denmark, http://www.dako.com), mouse anti-RIP (1:50, a kind gift from Dr. Hockfield, Yale University), and rat anti-BrdU (1:200, Nordic Biosite, Stockholm, Sweden, http://www.biosite.se) antibodies followed by Alexa fluorochrome-conjugated secondary antibodies (1:1,000, Invitrogen), and 4′-6-diamidino-2-phenylindole (DAPI, 1:10,000, Sigma-Aldrich). For detection of BrdU, cells were first treated with 2N HCl. Cells were counted using an inverted fluorescence microscope (Leica) and Volocity software (PerkinElmer, Waltham, MA, http://www.perkinelmer.com). Neuronal differentiation of neurosphere cells was quantified by counting the total number of β-III-tubulinposHoechtpos or β-III-tubulinposBrdUpos cells per well.

To quantify cell proliferation, cell death, and Notch activation under the differentiation conditions, neurospheres were dissociated and plated in differentiation media. To assess proliferation after 1 or 5 days, BrdU (5 μM) was added to the media for 6 hours, and the cells were then fixed and immunostained against BrdU. Alternatively, to assess cell death, propidium iodide (0.5 μg/ml) was added to the media for 15 minutes, and the cells were then washed and fixed. To assess activation of Notch, cells were incubated with goat anti-cleaved Notch1 (1:50, Santa Cruz Biotechnology, Santa Cruz, CA, http://www.scbt.com). All cells were counterstained with DAPI and the percentage of cells positive for BrdU, propidium iodide, or cleaved Notch1 was assessed using microscope and software as above.

Luciferase Assay to Measure Notch Signaling Activity

HEK-293 cells stably expressing the full-length Notch1 receptor (293HEK-FLN1) or neural stem/progenitor cell line derived from adult mouse forebrain [29] were transfected with 12XCSL-luciferase Notch reporter and a cytomegalovirus (CMV)-β-galactosidase construct by electroporation [30, 31]. Transfected cells were plated on monolayers of primary P2 astrocytes and cocultured for 24 hours and then lysed and analyzed with Luciferase Assay and β-galactosidase Assay Kits (Promega, Madison, WI, http://www.promega.com), according to the manufacturer's instructions. β-Galactosidase activity was used to assess transfection efficiency. Luciferase expression levels are given as light units relative to β-galactosidase expression.

Fluorescent Activated Cell Sorting Analyses of General Endocytosis, Ligand Internalization, and Membrane Bound Jagged1

For quantification of general endocytosis, P2 primary astrocyte cultures were incubated with fluorescence-tagged dextran-coated beads (Invitrogen) for 4 hours in 37°C. The cells were then detached, centrifuged (450g, 5 minutes), and extracellular fluorescence was quenched with trypan blue in phosphate-buffered saline (PBS) for 5 minutes in room temperature. The cells were centrifuged, excess trypan blue was removed, and the cells were resuspended in PBS and analyzed by fluorescent activated cell sorting (FACS) as described [32] using FACSCalibur (BD Pharmingen, San Diego, CA, http://www.bdbiosciences.com/). For ligand internalization, recombinant Notch1 extracellular domain fused to Fc fragment of human IgG1 (rN1ECD, 1 μg/ml, R&D Systems, Minneapolis, MN, http://www.rndsystems.com) were preincubated in PBS with Alexa 488 goat anti-human antibodies (1:200, Invitrogen) in +4°C for 1 hour. Primary astrocytes cultures were blocked in DMEM containing 10% goat serum and 1% bovine serum albumin (BSA) for 45 minutes at 37°C. The rN1ECD-Alexa 488 solution was diluted 1:5 in blocking solution and then added to the astrocytes for incubation at 37°C. The cells were then prepared for FACS analyses as described above.

For detection of surface levels of Jagged1, detached astrocytes were fixed in 4% paraformaldehyde, without further permeabilization, blocked with 3% BSA in PBS, and incubated with a fluorescent-conjugated antibody recognizing the extracellular domain of Jagged1 (R&D Systems) followed by FACS analyses as described above.

Quantification of Jagged1-Positive Vesicles

Live P2 primary astrocyte cultures were incubated with rabbit anti-Jagged1 antibodies (1:100, Abcam, Cambridge, U.K., http://www.abcam.com) and goat anti-rabbit Alexa Fluor 488 secondary antibodies (1:600, Invitrogen). The cells were fixed in 2% paraformaldehyde, and images of single astrocytes were acquired using a laser-scanning confocal microscope (LSM 510, Carl Zeiss, Oberkochen, Germany, http://www.zeiss.com) and were analyzed by ImageJ software (National Institute of Health, http://rsbweb.nih.gov/ij/) similarly as previously described [33].

Protein Extraction and Western Blot

For Western blot analysis, cells were lysed in RIPA buffer (0.15 M NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and 0.05 M Tris-HCl, pH 8) supplemented with protease inhibitors (Roche, Basel, Switzerland, http://www.roche-applied-science.com) and dithiothreitol (1 mM, Sigma-Aldrich). Lysates were centrifuged for 15 minutes at 15,000g and diluted with Laemmli sample buffer. Jagged1 was detected with goat anti-Jagged1 antibody (C-20, Santa Cruz Biotechnology).

Jagged1 Activation of Notch Signaling

Plates were coated with Protein G (50 μg/ml, Invitrogen) overnight at room temperature, washed in PBS, and blocked with 1% BSA in PBS. The plates were washed with PBS and incubated with recombinant Jagged1-Fc chimera (R&D Systems) or a ChromPure IgG Fc fragment (Jackson Immunoresearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com) at concentrations of 1, 2.5, or 5 μg/ml in 0.1% BSA in PBS for 4 hours at room temperature. Seven-day-old P4 primary neurospheres were dissociated with TrypLE into a single-cell suspension and immediately plated at equal densities on ligand-coated plates. One day after plating, 1% FBS was added. After 5 days of differentiation, cells were fixed with 4% paraformaldehyde for immunocytochemistry. No concentration-dependent differences were seen between 1 and 5 μg/ml. For γ-secretase inhibition, N-[N-(3,5-difluorophenylacetyl-l-alanyl)]-S-phenylglycine t-butylester (DAPT; 5 μM) was added to the media daily.

Reverse Transcription and qRT-PCR

Reverse transcription was performed with the iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA, http://www.bio-rad.com). qRT-PCR amplification analyses were carried out on a LightCycler 480 (Roche). Master mix was prepared with iQ SYBR Green Supermix (Bio-Rad Laboratories) and primer sequences used were 5′-ATCGCATCGTACTGCCTTTC-3′ and 5′-GGCAATCCCTGTGTTTTGTT-3′. PCR cycling condition was 3 minutes 95°C, followed by 45 cycles (20 seconds 95°C, 20 seconds 60°C, 20 seconds 72°C) and melt curve from 60°C to 95°C to determine correct PCR product formation. Among six reference genes measured, ACTB and GAPDH were the most stably expressed as shown by NormFinder [34] and were used to normalize expression levels. Data from three independent experiments were pooled using mean centering.

BrdU Injections and Entorhinal Cortex Lesions

Three-month-old male mice were used in all in vivo experiments. For basal cell proliferation, mice received a single intraperitoneal injection of BrdU (300 mg/kg) in sterile PBS and were killed 24 hours later. For cell fate determination experiments, 3-month-old males received BrdU twice daily (300 mg/kg) for 1 week and were killed 6 weeks after the first injection. Unilateral entorhinal cortex lesions were performed by transection of perforant pathway with a retractable wire knife as previously described [35]. For cell fate determination after lesion, mice received BrdU twice daily (200 mg/kg) during the first week after transection and were killed 2 weeks after the first injection.

Tissue Processing and Immunohistochemistry

Mice were anesthetized and perfused transcardially, and the dissected brains were postfixed with 4% paraformaldehyde overnight. After immersion in 30% sucrose in PBS, 40 μm horizontal sections were cut. For BrdU immunohistochemistry, sections were treated with 2N HCl. Following primary antibodies were used: rat anti-BrdU (1:100, Nordic Biosite), mouse anti-BrdU and rabbit anti-S100 (both 1:200, Dako), biotinylated mouse anti-NeuN (1:100, Chemicon, Temecula, CA, http://www.chemicon.com), rabbit anti-Sox2 (1:200, Chemicon), rabbit anti Tbr2 (1:200, Abcam), goat anti-Jagged or anti-Sox2 (both 1:50, Santa Cruz Biotechnology) antibodies. Secondary antibodies used were streptavidin-conjugated Cy3 (1:300, Sigma-Aldrich), or Alexa fluorochrome-conjugated secondary antibodies and nuclei marker TOPRO-3 (both Invitrogen), and for light microscopy, biotinylated donkey anti-rat antibodies (Jackson ImmunoResearch) and Vectastain ABC kit (BioNordika, Stockholm, Sweden, http://www.bionordika.se). Sections were mounted with Mowiol and coverslipped.

Hippocampal Cell Counts

Stereological counting of cells was performed using an epifluorescence microscope (Eclipse 80i, Nikon) and by using a modified optical dissector method as described previously [36]. For absolute counts of BrdUpos cells in the SGZ and GCL of the dentate gyrus at 24 hour time point and 2 weeks after lesion time point, every sixth horizontal section 40 μm thick covering a depth of 1,680 μm (seven sections) was used. For the 6 week time point, we used every fourth horizontal section 40 μm thick covering a depth of 960 μm (six sections). On each section, all cells positive for the respective marker or the combination thereof in the specified brain region were counted. The average cell number per section was multiplied by a factor to obtain the estimated total cell number for a depth of 1,680 μm through the hippocampus in the dorso-ventral direction. Colocalization of BrdU with NeuN, S100, Sox2, and Tbr2, respectively, was quantified in three to six sections per mouse by laser-scanning confocal microscopy (Leica Microsystems, Wetzlar, Germany, http://www.leica-microsystems.com/). Absolute numbers of double-positive cells were calculated by multiplying the ratio of double-positive cells by the total number of BrdUpos cells. For the assessment of absolute number of Sox2posS100neg neural stem/progenitor cells, stacks of optical sections of the dentate gyrus covering 6 μm (basal condition) and 12 μm (lesioned mice) on every sixth horizontal tissue section 40 μm thick (three sections) were acquired with a laser-scanning confocal microscope (Leica Microsystems).

To compare the area of GCL in GFAP−/−Vim−/− and wild-type mice, every fourth horizontal section 40 μm thick covering a depth of 960 μm (a total of six sections), with GCL visualized by NeuN antibodies, was photographed with an epifluorescence microscope (Nikon Instruments, Amstelveen, the Netherlands, http://www.nikoninstruments.com) and the GCL area was measured with ImageJ software (version 1.30, National Institutes of Health). The area of the GCL did not differ between GFAP−/−Vim−/− and wild-type mice (data not shown).

Statistical Analysis

Data were analyzed in SPSS (version 16.0, IBM, Armonk, NY, http://www.ibm.com) or Prism (version 5.0, GraphPad Software, La Jolla, CA, http://www.graphpad.com). One-factor or two-factor ANOVA was used followed by post hoc analysis (Bonferroni correction or Tukey honestly significant difference test). Two-tailed t-tests were used for comparisons between two groups. Differences were considered significant at p <.05. Values are presented as mean ± SEM.

RESULTS

Neuronal Differentiation of Stem/Progenitor Cells Is Determined by the Environment

To assess the mechanisms by which astrocytes regulate neuronal differentiation of neural stem/progenitor cells, we first determined the effects of GFAP and vimentin deficiency on differentiation of neurosphere cultures derived from P4 mice. There were no differences in neuronal differentiation between wild-type, GFAP/−, or Vim/− neurospheres (Fig. 1A). However, several fold more β-III-tubulinpos neurons were generated from GFAP/−Vim/− neurospheres than wild-type neurospheres (p <.001, Fig. 1A). This experiment was repeated with neurospheres from wild-type and GFAP−/−Vim−/− mice that were dissociated prior differentiation. Again, there were more β-III-tubulinpos neurons generated from GFAP/−Vim−/− than from wild-type neurospheres (1.9% ± 0.4% and 0.6% ± 0.1%, respectively, p <.001). Twofold more RIPpos oligodendrocytes were generated from GFAP/−Vim/− compared to wild-type neurospheres (0.4% ± 0.07% vs. 0.2% ± 0.02%, p <.0001). The neurosphere genotype had no effect on astrogenesis (95% ± 0.5% and 96% ± 0.4% S100pos cells for GFAP−/−Vim/− and wild-type neurospheres, respectively), cell proliferation (28% ± 2.5% and 29% ± 2.5% BrdUpos cells at 24 hours and 10% ± 0.8% and 10% ± 0.9% BrdUpos cells at 5 days of differentiation of GFAP−/−Vim−/− and wild-type neurospheres, respectively), or cell death (17% ± 1.0% and 15% ± 1.1% propidium iodidepos cells at 24 hours and 4% ± 0.6% and 4% ± 0.4% propidium iodidepos cells at 5 days of differentiation of GFAP−/−Vim−/− and wild-type neurospheres, respectively).

To assess the effects of the astrocyte environment on neuronal differentiation, we used an astrocyte-neurosphere coculture system in which P4 neurospheres were prelabeled with BrdU for 24 hours before coculturing with P2 astrocytes to exclude the contribution of neural stem/progenitor cells from the astrocyte cultures (Fig. 1B). Coculture of dissociated wild-type neurosphere cells on top of wild-type astrocytes increased neuronal differentiation 2.4-fold when compared to neurosphere cells plated directly on laminin-coated surface (3.2% ± 0.4%, n = 6 and 1.3% ± 0.4%, n = 11, respectively, p <.05). Also, the total number of neurosphere-derived cells was higher when neurospheres were cultured in the presence of astrocytes (data not shown). These results confirm that the presence of astrocytes is beneficial for the proliferation, survival, and neuronal differentiation of neurosphere cells as previously shown for adult hippocampus-derived neural progenitor cells [4, 37]. Comparable neuronal differentiation was seen when dissociated wild-type neurospheres were plated on top of wild-type, GFAP/−, or Vim/− astrocytes. However, when plated on top of GFAP−/−Vim−/− astrocytes, dissociated wild-type neurospheres generated 2.1-fold more neurons compared to plating on top of wild-type, GFAP/−, or Vim/− astrocytes (Fig. 1B; p <.01). These findings provide evidence for enhanced neuronal differentiation of neurosphere cells cocultured with GFAP−/−Vim−/− astrocytes. As the total number of neurosphere-derived cells did not differ between neurospheres cocultured with GFAP/−Vim/− and wild-type astrocytes (82,988 ± 4,005 and 88,424 ± 12,665 cells per coverslip), the absence of GFAP and vimentin in astrocytes does not appear to have any measurable effect on neurosphere cell survival and/or proliferation in this coculture system.

Effect of GFAP−/−Vim−/− Astrocytes Is Mediated by Cell–Cell Contact

Next, we determined whether the increase in neuronal differentiation was due to secretion of neurogenic factors by GFAP−/−Vim−/− astrocytes or due to signaling dependent on cell–cell contact. Neuronal differentiation of wild-type neurosphere cells was similar in medium conditioned by GFAP−/−Vim−/− or wild-type astrocytes (Fig. 1C). In contrast, when prelabeled neurosphere cells were exposed to conditioned medium from wild-type astrocytes while in direct contact with GFAP−/−Vim−/− astrocytes or vice versa, neuronal differentiation was increased by direct contact with GFAP/−Vim−/− astrocytes (p <.05, Fig. 1D). To further analyze the effect of cell–cell contact, we cocultured prelabeled dissociated neurosphere cells on wild-type, GFAP−/−Vim−/−, and mixed wild-type and GFAP−/−Vim−/− astrocytes. The presence of wild-type astrocytes abolished the increase in neuronal differentiation mediated by GFAP−/−Vim−/− astrocytes (p <.01, Fig. 1E), suggesting the presence of an inhibitory signaling from wild-type astrocytes to neurosphere cells.

GFAP−/−Vim−/− Astrocytes Show Reduced Notch Signaling

The increased neuronal differentiation of neurosphere cells cocultured with GFAP−/−Vim−/− astrocytes indicated a cell–cell contact-dependent signaling mechanism that is suppressed by contact with wild-type astrocytes. Since Notch signaling suppresses neuronal differentiation through cell–cell contact [38], we investigated the possible involvement of Notch signaling in GFAP−/−Vim−/− astrocyte-mediated neuronal differentiation by coculturing P2 wild-type and GFAP−/−Vim−/− astrocytes with Notch reporter cells [30, 31]. There was less Notch signaling between GFAP−/−Vim/− astrocytes and reporter cells than between wild-type astrocytes and reporter cells (p <.005; Fig. 2A).

Figure 2.

GFAP−/−Vim−/− astrocytes show reduced Notch signaling. (A): In coculture with Notch reporter cells, GFAP−/−Vim−/− (G−/−V−/−) astrocytes showed less Notch signaling than wild-type (WT) astrocytes (n = 4 in both groups). (B): Quantitative real-time polymerase chain reaction analysis of expression levels of Jagged1 in G−/−V−/− astrocytes relative to those in WT astrocytes showed downregulation of Jagged1 (data from three independent experiments with n = 3–5 mice per group in each experiment). (C): Western blot analysis showed less Jagged1 protein in G−/−V−/− astrocytes than in WT astrocytes (three independent experiments). (D): G−/−V−/− (n = 3) astrocytes showed reduced general endocytosis compared to WT (n = 5) astrocytes. (E): G−/−V−/− astrocytes showed a major reduction in Notch ligand-mediated endocytosis of the NEC, a prerequisite for Notch signaling, compared to WT (n = 4 in both groups). (F): Number of Jagged1pos vesicles was reduced in G−/−V−/− astrocytes compared to WT astrocytes (n = 35 astrocytes from two mice in each group). (G): Adult mouse neural stem cells transfected with a Notch reporter showed reduced Notch signaling activity when cocultured with G−/−V−/− compared to WT astrocytes (n = 8 per group). *, p <.05; **, p <.01; ***, p <.005; ****, p <.001 (two-tailed t-test). Abbreviations: MFI, mean fluorescence intensity; NEC, Notch extracellular domain; WT, wild type.

Next, we assessed the expression level of Jagged1, the principal Notch ligand, in GFAP−/−Vim−/− and wild-type astrocytes by qRT-PCR in three independent experiments. Expression of Jagged1 was downregulated by 40% in GFAP−/−Vim−/− astrocytes (p <.001; pooled data from three independent experiments, Fig. 2B). Western blot analysis confirmed that GFAP−/−Vim/− astrocytes contained less Jagged1 protein (p <.001, Fig. 2C). FACS analyses of Jagged1pos astrocytes showed comparable amount of cell membrane bound Jagged1 on wild-type and GFAP−/−Vim−/− astrocytes (46.9 ± 2.4 and 40.9 ± 4.1 mean fluorescence intensity, respectively). Thus, although the total amount of Jagged1 in GFAP−/−Vim−/− astrocytes is reduced, the membrane-associated fraction is not altered.

Notch ligand and receptor availability is known to be regulated by endocytosis and membrane trafficking [39], and we have previously shown that intermediate filaments are important for astrocyte vesicle trafficking dynamics and interferon-γ induced mobility of major histocompatibility complex (MHC) class II compartment [28, 40, 41]. Thus, we investigated both the general endocytosis as well as the endocytosis of Jagged1, which is important for eliciting a Notch signal [42]. In GFAP/−Vim−/− astrocytes, we observed a general reduction in endocytosis as shown by FACS analysis of the uptake of dextran-coated beads (p <.01, Fig. 2D) and a decrease in the Notch ligand-mediated internalization of the Notch extracellular domain (p <.001, Fig. 2E). Also, the number of Jagged1pos vesicles was reduced in GFAP−/−Vim−/− astrocytes (p <.05, Fig. 2F), suggesting that reduced endocytosis of Jagged1 in intermediate filament-deficient astrocytes might be the cause of decreased Notch signaling from GFAP−/−Vim−/− astrocytes to neural stem/progenitor cells.

To determine the efficiency of Notch signaling from GFAP−/−Vim−/− astrocytes specifically to neural stem cells, we transfected adult mouse neural stem cells [29] with a Notch reporter [30, 31]. We found that the Notch signaling activity in neural stem cells cocultured with GFAP/−Vim−/− compared to wild-type P2 astrocytes was reduced by 78% (p <.001, Fig. 2G). Next, we assessed Notch signaling in P4 neurospheres, which contain both astrocytes and stem/progenitor cells, by quantifying the expression of the Notch intracellular domain (NICD) using an antibody specific for cleaved/active Notch. After 5 days of differentiation, GFAP−/−Vim−/− neurospheres showed a 19% decrease in NICDpos cells compared to wild-type controls (62% ± 2.4% vs. 76% ± 0.1% NICDpos cells/well; p <.05).

Jagged1 Reverses the Increase in Neuronal Differentiation Mediated by GFAP−/−Vim−/− Astrocytes

To determine whether adding Jagged1 to the system would abrogate the effect of GFAP−/−Vim−/− astrocytes on neuronal differentiation, we allowed P4 neurosphere cells to differentiate in the presence of immobilized recombinant Jagged1-Fc or a control protein Fc [43]. Under control conditions, neuronal differentiation was greater in GFAP−/−Vim−/− neurosphere cells (p <.01, Fig. 3A). In the presence of Jagged1, however, neuronal differentiation of GFAP−/−Vim−/− neurospheres decreased to the level comparable to wild-type neurospheres (p <.05, Fig. 3A).

Figure 3.

Immobilized Jagged1 reverses the increase in neuronal differentiation mediated by GFAP−/−Vim/ astrocytes. (A): Differentiating WT (n = 4) neurospheres cultured in the presence of immobilized Jagged1 or a control protein (Fc) showed no differences in the percentage of β-III-tubulin-expressing (β-III-tubulinpos) neurons. In contrast, differentiating GFAP−/−Vim/ (G−/−V−/−; n = 4) neurospheres cultured in the presence of Fc showed greater neuronal differentiation than G−/−V−/− neurospheres cultured with Jagged1 or WT neurospheres cultured with Jagged1 or Fc. Thus, immobilized Jagged1 abrogated the proneurogenic effect of G−/−V−/− astrocytes on neurosphere cells. (B): Addition of DAPT, a γ-secretase inhibitor that prevents cleavage of the Notch receptor, to differentiating G−/−V−/− neurospheres, abrogated the Jagged1-mediated decrease in neuronal differentiation of G−/−V−/− (n = 4) neurospheres. N equals number of mice per experimental group. *, p <.05; **, p <.01 (ANOVA followed by Tukey honestly significant difference post hoc analysis). Abbreviations: DAPT, N-[N-(3,5-difluorophenylacetyl-l-alanyl)]-S-phenylglycine t-butylester; WT, wild type.

To investigate if this decrease was specific to Notch-mediated signaling, we added DAPT, a γ-secretase inhibitor that prevents cleavage and activation of the Notch receptor [44], to GFAP−/−Vim−/− neurosphere cells differentiating in the presence of Jagged1. The addition of DAPT abrogated the Jagged1-mediated decrease in neuronal differentiation of GFAP/−Vim−/− neurospheres (p <.05, Fig. 3B).

Increased Neurogenesis in the Hippocampal Dentate Gyrus of GFAP−/−Vim−/− Mice

Since our in vitro experiments were performed with astrocytes obtained from the whole brain and the neurogenic as well as other properties differ between astrocytes from different brain regions, we determined the effects of GFAP and vimentin ablation specifically on hippocampal neurogenesis. Astrocytes identified by S100 expression in the dentate gyrus of the hippocampus of wild-type and GFAP−/−Vim−/− mice were positive for Jagged1 (Fig. 4A). To assess endogenous stem/progenitor cell division in the hippocampal dentate gyrus, we administered a single pulse of BrdU to GFAP−/−Vim−/− and wild-type mice. No differences were seen in the number of proliferating cells (BrdUpos) in the SGZ and GCL between GFAP−/−Vim−/− and wild-type mice 24 hours after BrdU administration (Fig. 4B). To assess different subpopulations of neural stem/progenitor cells, we combined BrdU labeling with antibodies against transcription factors Sox2 and Tbr2. Sox2 is present in rarely dividing neural stem cells with radial glia-like morphology and active, horizontally aligned neural stem cells (both corresponding to type 1 cells), and early intermediate progenitor cells (type 2a cells) [9, 45]. As Sox2 is also present in non-neurogenic astrocytes, we combined Sox2 antibodies with astrocyte marker S100 to be able to exclude the Sox2-positive non-neurogenic astrocytes. Tbr2 expression is found in early to late intermediate stage progenitor cells (type 2a and 2b cells) [46]. Twenty four hours after a single BrdU injection, there was no difference in the fraction of Sox2posS100neg cells or Tbr2pos cells among BrdU labeled cells in the SGZ and GCL of GFAP−/−Vim−/− and wild-type mice (Fig. 4B). In addition, the total cell numbers of Sox2posS100neg cells and Tbr2pos cells were similar between GFAP−/−Vim−/− and wild-type mice (Fig. 4C). This suggests that reduced Jagged1-mediated Notch signaling from GFAP−/−Vim−/− astrocytes in the adult hippocampus does not affect neural stem cell pool maintenance, proliferation, and lineage progress into early intermediate progenitor cells.

Figure 4.

Increased neurogenesis in the hippocampal dentate gyrus of GFAP−/−Vim−/− mice under basal conditions and after injury. (A): S100pos astrocytes in the SGZ (arrows) and in the molecular cell layer (arrowheads) of the dentate gyrus show immunoreactivity for Jagged1. (B): After 24 hours of a single BrdU injection, GFAP−/−Vim−/− mice (G−/−V−/−; n = 4–7) and wild-type mice (WT; n = 4–6) had comparable number of dividing cells (BrdUpos) and subpopulations of dividing cells (BrdUposSox2posS100neg cells and BrdUposTbr2pos cells) in SGZ and GCL (SGZ+GCL). (C): The total numbers of Sox2pos and Tbr2pos cells were comparable between genotypes (n = 4–5 per group). (D): At 6 weeks, G/V/ mice had more BrdUpos cells in GCL and more BrdUposNeuNpos cells in SGZ+GCL and in the GCL alone than WT mice (n = 12 per group). There was no difference in the number of BrdUposS100pos cells. (E): SGZ and GCL in the dentate gyrus visualized with antibodies against BrdU, NeuN, and S100 (arrowhead depicts BrdUpos cell). (F): Images of BrdUposNeuNpos and BrdUposS100pos cells in the dentate gyrus of the hippocampus. (G): After 2 weeks of entorhinal cortex lesion, G/V/ mice showed lower number of BrdUpos cells compared to WT mice (n = 7 per group), but there was no difference between genotypes in the number of BrdUposNeuNpos cells (n = 4 per group) or BrdUposS100pos cells (n = 5 per group). (H): G/V/ mice showed a higher percentage of BrdUposNeuNpos cells in SGZ+GCL than WT mice (n = 4 per group). There was no difference in the percentage of BrdUposS100pos cells (n = 5 per group). Scale bars = 50 μm (A and E), 10 μm (F). *, p <.05; **, p <.01 (two-tailed t-test). Abbreviations: BrdU, 5-bromo-2-deoxyuridine; GCL, granule cell layer; SGZ, subgranular zone; WT, wild type.

To determine how the absence of GFAP and vimentin affects the differentiation and survival of newly formed astrocytes and neurons, we administered BrdU twice daily for a week and assessed the numbers of the newly formed cells 6 weeks after the first injection. We found that the two groups of mice had similar numbers of newly formed astrocytes (BrdUposS100pos). However, GFAP−/−Vim−/− mice had 40% more BrdUpos cells, which implies an enhanced survival of newly formed cells in the dentate gyrus and 74% more newly born neurons (BrdUposNeuNpos) in the GCL (p <.05, Fig. 4D–4F). Thus, in the absence of GFAP and vimentin, neurogenesis in the hippocampal dentate gyrus is increased.

Next, we asked whether the absence of GFAP and vimentin would affect the neurogenic response to injury. Entorhinal cortex lesion leads to hippocampal injury and promotes neurogenesis [47]. First, we assessed the total population of Sox2pos neural stem/progenitor cells by counting Sox2pos cells negative for the astrocyte marker S100. We found that 4 days after entorhinal cortex lesion, GFAP−/−Vim−/− (n = 3) and wild-type (n = 6) mice had comparable number of Sox2posS100neg neural stem/progenitor cells in the SGZ of the denervated hippocampus (69.6 ± 5.8 vs. 74.7 ± 4.0 cells per section, respectively). To examine the effect of entorhinal cortex lesion on cell proliferation, we administered BrdU twice daily during the first week after lesion. Two weeks later, GFAP−/−Vim−/− mice had lower number of BrdUpos cells in the SGZ and GCL on the lesioned side compared to wild-type mice (Fig. 4G; p <.05). To assess the fate of the dividing cells, we determined the percentage of astrocytes and neurons formed from BrdUpos cells. The total number of newly born neurons and astrocytes was comparable in GFAP−/−Vim−/−and wild-type mice (Fig. 4G), however, the percentage of newly born neurons (BrdUposNeuNpos) was higher in GFAP−/−Vim−/− than in wild-type mice (p <.05, Fig. 4H). Thus, while the lesion-triggered proliferative response in the hippocampus was lower, the cell fate was more directed toward neuronal lineage in GFAP−/−Vim−/− compared to wild-type mice.

DISCUSSION

Astrocytes Regulate Neurogenesis Through Cell–Cell Contact with Neural Stem/Progenitor Cells

Astrocytes play an active role in adult neurogenesis through the secretion of factors, of which several have been characterized [4–7], while the astrocyte membrane-associated factors involved in the regulation of neurogenesis have been far less studied [4]. Although primary astrocytes and especially hippocampus-derived primary astrocytes direct neural stem/progenitor cell differentiation toward a neuronal fate in vitro [4], astrocytes in the neurogenic niches may exert several distinct and possibly even opposing regulatory actions that affect neurogenesis. Thus, the net effect of astrocytes on neurogenesis should reflect the integration of all these effects. While factors secreted by astrocytes regulate neurogenesis positively [5–7], here we provide evidence that the negative control of neuronal differentiation of neural stem/progenitor cells by astrocytes is mediated through a cell–cell contact and that intermediate filament proteins GFAP and vimentin play an important role in this process. First, we showed that compared to wild-type neurospheres, neurogenesis from GFAP−/−Vim−/− neurospheres is highly increased. To discriminate between the effects of GFAP and vimentin ablation on the intrinsic properties of neural stem/progenitor cells and the effects on the niche, we cocultured neurosphere cells with prelabeled astrocytes. In this system, we found that simultaneous ablation of GFAP and vimentin in astrocytes increased neuronal differentiation of neurosphere cells. Furthermore, we showed that to exert this inhibitory effect on neurosphere differentiation, astrocytes need to be in direct contact with the neurosphere cells. These findings support the notion that wild-type astrocytes inhibit neuronal differentiation of stem/progenitor cells through cell–cell contact and the ablation of GFAP and vimentin lifts this inhibition. This conclusion is also supported by our previous report showing that GFAP−/−Vim−/− astrocytes support neuronal differentiation from adult rat hippocampus-derived neural stem/progenitor cells [21]. This does not exclude the possibility that the absence of GFAP and vimentin also directly affects the properties of neural stem/progenitor cells, which were shown to express vimentin and transiently also GFAP [48, 49]. This question merits further investigation.

Astrocytes Regulate Neurogenesis Through the Notch Pathway

Notch signaling is an evolutionarily conserved signaling pathway that controls many aspects of neuronal fate choice during development and plays an essential role in controlling adult neurogenesis [50]. Astrocytes in SVZ were previously shown to express Jagged1, however, reports concerning the effects of Notch signaling in SVZ on neurogenesis are controversial [12, 51]. Although cortical injury has been shown to be associated with increased Jagged1 expression in the ipsilateral SVZ [12], a direct link between altered Jagged1 expression or function in astrocytes and neurogenesis is lacking.

Our results show that in the absence of GFAP and vimentin, Jagged1 mRNA and protein levels as well as Notch signaling activity from astrocytes to reporter cells were decreased. Importantly, adult mouse neural stem cells [29] transfected with a Notch reporter showed reduced Notch signaling activity when cocultured with GFAP−/−Vim−/− astrocytes compared to wild type. Together, these findings point to the involvement of Notch pathway in the control of neural stem/progenitor cell differentiation. Additional support for this hypothesis comes from our data showing that compared to wild-type, Notch ligand endocytosis, a prerequisite for both Notch receptor and ligand activation [39, 42], was reduced by 71% in GFAP−/−Vim−/− astrocytes. Furthermore, the GFAP−/−Vim−/− astrocytes contained 36% fewer Jagged1pos vesicles than wild type, consistent with our previous reports that intermediate filaments have a role in astrocyte vesicle trafficking dynamics [28, 40]. Thus, it is conceivable that the activity of Notch ligands in GFAP−/−Vim−/− astrocytes is reduced, even though the amount of Jagged1 found at the cell membrane of astrocytes was not altered. Immobilized Jagged1, which is used to activate Notch signaling [52], abolished the increased neuronal differentiation mediated by GFAP−/−Vim−/− astrocytes. In contrast, the addition of a γ-secretase inhibitor DAPT, which prevents cleavage of the Notch receptor and inhibits Notch signaling, abrogated the effect of immobilized Jagged1 on neuronal differentiation of neurosphere cells. Together, these data imply that astrocytes exert their control of Notch signaling in the signal receiving cells through the endocytosis of Jagged1 and this process is dependent on the normal function of cytoplasmic intermediate filaments in astrocytes. Our results add support to the claims that Notch signaling inhibits neuronal differentiation of neural stem/progenitor cells.

Previously we showed that ablation of GFAP and vimentin attenuates reactive gliosis and creates a more favorable environment for the survival of neural grafts and neural stem cells as well as promotes axonal and synaptic regeneration [20, 21, 24, 53]. Here, we demonstrate that ablation of GFAP and vimentin increases hippocampal neurogenesis in unchallenged mice and neuronal cell fate determination after injury. However, we did not detect any changes in the population of type 1 and 2 neural stem/progenitor cells as determined by the number of cells expressing Tbr2 or Sox2 alone or in combination with BrdU incorporation. As judged from immunostaining with antibodies against Jagged1 (Fig. 4A), S100pos astrocytes are not the only cell type expressing Jagged1 in the SGZ. Jagged1 may also be present in neural stem cells or intermediate progenitor cells, and thus affect Notch signaling in the niche in an astrocyte-independent manner. Notch receptor activation promotes the survival of neural stem cells in vitro and transient administration of Notch ligands to the brain of adult rats increased the numbers of newly generated precursor cells [13]. After inducible conditional inactivation of Notch signaling in the brain of adult mice, neural stem cells differentiated into transit-amplifying precursors resulting in an increased number of immature neurons 1–3 weeks after Notch inactivation and followed by a dramatic loss of neurogenesis 2–3 months later [10, 16]. In contrast, in the GFAP−/−Vim−/− mice, hippocampal neurogenesis is still detectable and increased even in old age (18 months) [54]. In addition, GFAP−/−Vim−/− mice and wild-type mice used in this study showed comparable numbers of Sox2posS100neg neural stem/progenitor cells in the hippocampus under both basal conditions and after entorhinal cortex lesion, suggesting that the hippocampal neural stem cell pool is not depleted in GFAP−/−Vim−/− mice. Thus, the reduced inhibition of Notch signaling in the hippocampal neurogenic niche of the GFAP−/−Vim−/− mice has much milder effects than its complete inactivation. Notably, the choice of gene promoter (Gfap and Glast vs. Nestin) to induce the Notch gene inactivation in the neurogenic niche seems to affect the dynamics in neurogenesis differentially [10, 14, 15]. These distinct effects of Notch inactivation point to the complexity of the interplay between cell types and their role in Notch signaling in the hippocampus. In contrast to conditional Notch1 inactivation, which essentially abolishes long-term neurogenesis, the compensatory mechanisms in response to constitutive absence of GFAP and vimentin may account for the milder effects on astrocyte-mediated Notch signaling and neurogenesis observed in GFAP−/−Vim−/− mice. Notch signaling is also present in newly born neurons in GCL of the adult hippocampus where it regulates dendritic morphology [15, 55]. This may play a role in the integration of the new neurons into the neuronal networks and ultimately affect their survival. In addition, the ablation of GFAP and vimentin conceivably has multiple effects on the neurogenic niche, including the endogenous properties of neural stem cells and altered distribution or secretion of soluble neurogenic factors such as Wnt3 [21] that may promote neuronal differentiation or survival.

SUMMARY

We conclude that astrocytes inhibit neuronal differentiation of neural stem/progenitor cells through a cell–cell contact. Endocytosis of Notch ligand Jagged1 by astrocytes plays an essential role in this process and is dependent on normal function of the intermediate filament proteins GFAP and vimentin in these cells.

Acknowledgements

This work was supported by the Swedish Medical Research Council (project 11548), AFA Research Foundation, ALF Göteborg (project 11392), Sten A. Olsson Foundation for Research and Culture, Söderberg Foundations, Hjärnfonden, the Swedish Stroke Foundation, the Swedish Society for Medical Research, the Free Mason Foundation, Amlöv's Foundation, E. Jacobson's Donation Fund, NanoNet COST Action (BM1002), the EU FP 7 Program EduGlia (237956 to M.P. and R.Z.), the EU FP 7 Program TargetBraIn (279017 to M.P.), Trygg-Hansa, Academy of Finland (C.S.), Turku Graduate School for Biomedical Sciences and Academy of Finland (M. Sj.), and by the Research Agency of Slovenia (P30310, J30031, J30133, J34051, J34146, J33632 to R.Z).

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

The authors indicate no potential conflicts of interest.

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