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(GE Healthcare Bio-Sciences AB, Uppsala, Sweden, http:// ...... genesis and inhibits glial differentiation by independent mechanisms. Cell 2001;104:365–376.
TISSUE-SPECIFIC STEM CELLS Nitric Oxide Exposure Diverts Neural Stem Cell Fate from Neurogenesis Towards Astrogliogenesis RUXANDRA COVACU,a ALEXANDRE I. DANILOV,a BO SONNICH RASMUSSEN,a KATARINA HALLE´ N,b MORTEN C. MOE,c ANNA LOBELL,a CLAS B. JOHANSSON,d MIKAEL A. SVENSSON,c TOMAS OLSSON,a LOU BRUNDINa a

Department of Clinical Neuroscience, Division of Neurology, Neuroimmunology Unit, bDepartment of Molecular Medicine and Surgery, Division of Urology, cDepartment of Neurosurgery, Karolinska Institutet, Stockholm, Sweden; dBaxter Laboratory in Genetic Pharmacology, Stanford University School of Medicine, Stanford, California, USA Key Words. Autoimmune disease • Nitric oxide • Neural stem cell • Neural differentiation • Glial differentiation

ABSTRACT Regeneration of cells in the central nervous system is a process that might be affected during neurological disease and trauma. Because nitric oxide (NO) and its derivatives are powerful mediators in the inflammatory cascade, we have investigated the effects of pathophysiological concentrations of NO on neurogenesis, gliogenesis, and the expression of proneural genes in primary adult neural stem cell cultures. After exposure to NO, neurogenesis was downregulated, and this corresponded to decreased expression of the proneural gene neurogenin-2 and ␤-III-tubulin. The de-

creased ability to generate neurons was also found to be transmitted to the progeny of the cells. NO exposure was instead beneficial for astroglial differentiation, which was confirmed by increased activation of the Janus tyrosine kinase/signal transducer and activator of transcription transduction pathway. Our findings reveal a new role for NO during neuroinflammatory conditions, whereby its proastroglial fate-determining effect on neural stem cells might directly influence the neuroregenerative process. STEM CELLS 2006;24:2792–2800

INTRODUCTION

It has been demonstrated that stem cells are activated during neuroinflammatory conditions during which they migrate to and differentiate at the site of injury [18, 19]. In experimental autoimmune encephalomyelitis, an animal model for MS, stem cells can differentiate into oligodendrocytes in demyelinated lesions typical for this disease [19]. Conversely, neurogenesis has been reported to be impaired during inflammation [20, 21], and this effect is especially evident when microglial cells are activated [22]. Since microglial activation and subsequent excessive NO production are characteristics of neuroinflammation, this implies a role for NO in downregulation of neurogenesis. This has never been demonstrated at a cellular level to date. In this study, we have addressed whether NO affects neural stem cell differentiation. We used primary neural stem/progenitor cell cultures from adult rats. In determining the exposure levels, we have been guided by our own and other investigators’ determinations of NO levels in cerebrospinal fluid and brain tissue [13–17, 23]. When exposed to 5–10 times the normal

Nitric oxide (NO) is a gaseous second messenger involved in numerous physiological processes such as blood pressure regulation, neurotransmission, and inflammation. Studies in Drosophila have shown its involvement in development, since NO inhibits proliferation during embryogenesis and in the imaginal discs in metamorphosis [1, 2]. The cytostatic role of NO has also been described in amphibians [3] and in the mammalian central nervous system (CNS) [4 – 6]. When excessively produced, NO becomes detrimental to the tissue in which it is produced. It induces cell death in neural progenitor cells [7], in mature oligodendrocytes [8], and in neurons [9]. NO production leads to nitrosylation and peroxynitrosylation of proteins, can also cause DNA damage [10], or may interfere with DNA repair [11]. The level of NO production correlates with disease activity in various neuroinflammatory conditions such as bacterial meningitis [12], systemic lupus erythematosus [13], traumatic brain injury [14], and in multiple sclerosis (MS) [15–17].

Correspondence: Lou Brundin, M.D., Ph.D., Department of Clinical Neuroscience, Division of Neurology, Karolinska Institutet, 171 76 Stockholm, Sweden. Telephone: ⫹46-8-51775412; Fax: ⫹46-8-51773757; e-mail: [email protected] Received December 20, 2005; accepted for publication August 8, 2006; first published online in STEM CELLS EXPRESS August 17, 2006; available online without subscription through the open access option. ©AlphaMed Press 1066-5099/2006/$20.00/0 doi: 10.1634/stemcells.2005-0640

STEM CELLS 2006;24:2792–2800 www.StemCells.com

Covacu, Danilov, Rasmussen et al. levels of NO (corresponding to 0.1 mM (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl) amino]diazen-1-ium-1,2-diolate [DETANONO:ate]), the NSCs differentiate into astrocytes and oligodendrocytes but generate strikingly fewer neurons. Furthermore, our results reveal that the expression of the proneuronal gene neurogenin-2 was downregulated in cultures exposed to pathological concentrations of NO, which might explain the increased formation of astrocytes in these cultures. We thus demonstrate that at pathological concentrations NO has a skewing effect on NSC differentiation, diverting them from a proneuronal to a proastroglial fate.

MATERIALS

AND

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(DAKO, DakoCytomation Norden AB, Stockholm, Sweden, http://www.dakoab.se/), guinea pig anti-doublecortin (Chemicon), goat anti-mouse IgG Alexa 594, goat anti-mouse IgM Alexa 488 and goat anti-rabbit IgG Alexa 350 (Molecular Probes, Invitrogen, Stockholm, Sweden, http://www.invitrogen. com), rabbit anti-Sox2 (Chemicon), and rat anti-nestin (Chemicon). All antibodies were used at 1:100 dilutions. For Western blotting, the anti-Tuj antibody was diluted 1:200, and the antimouse immunoglobulin horseradish peroxidase (HRP)-conjugate (DAKO) was diluted 1:1,000. pSTAT-1 (1:1,000) and STAT-1 (1:100) were obtained from Cell Signaling (In Vitro; Stockholm, Sweden, http://www.cellsignal.com).

METHODS Immunocytochemistry

Neural Stem Cell Culture Neural stem cells were isolated from adult female Dark Agouti rats (Scanbur B&K, Sollentuna, Sweden, http://www.scanbur. eu/), 7– 8 weeks old, according to a protocol previously described by Johansson et al. [24]. All experiments were performed with cells that had undergone two passages. For propagation, the cells were cultured in Dulbecco’s modified Eagle’s medium/F-12 containing B27 supplement, penicillin (100 U/ml), streptomycin (100 ␮g/ml) (Life Technologies, Invitrogen AB, Stockholm, Sweden, http://www.invitrogen.com), and epidermal growth factor (EGF; Sigma-Aldrich, Stockholm, Sweden, http://www.sigmaaldrich.com) at 20 ng/ml. For differentiation, spheres were split into single cell suspensions, seeded on poly(D-lysine)-coated plates (Sigma-Aldrich), and cultured in medium lacking EGF but supplemented with 1% fetal calf serum (FCS) (Life Technologies). As a source of NO, we used DETA-NONO:ate (Alexis Biochemicals, Goteborg, Switzerland, http://www.axxora.com/), and NO release was measured using an NO-selective electrode (see next section). Cells were exposed to DETA-NONO:ate at concentrations of 1, 0.1, 0.05, and 0.01 mM for 1 hour, 8 hours, or 24 hours. The cells were incubated in a humidified chamber containing 5% CO2/5% O2/90% nitrogen at 37°C. Oxygen levels were measured using an oxygen meter (TED 60; Teledyne, City of Industry, CA, http://www.teledyne-ai.com/). Two different exposure procedures were used. In the “immediate differentiation” experiments, cells were differentiated directly following a 24-hour exposure. In the “delayed differentiation” experiments, cells were allowed to proliferate in the presence of EGF for 4 days (or 1–2 days for the 5-bromo-2⬘deoxyuridine [BrdU]/␤-III-tubulin double staining) after NO exposure before being differentiated. All animal experiments were approved by the local ethics committee.

Detection of NO Using an NO-Selective Electrode NO concentrations were measured using a Clark-type electrode (Iso-NO; World Precision Instruments, Stevenage, U.K., http:// www.wpi-europe.com/). DETA-NONO:ate was added to the medium with constant stirring at 37°C, and the concentration of NO was measured for 24 hours.

Antibodies For immunocytochemistry, the following antibodies were used: mouse anti-␤-III-tubulin (Tuj1), (Chemicon Europe, Ltd., Hampshire, U.K., http://www/chemicon.com), mouse anti-O4 (Chemicon), rabbit anti-glial fibrillary acidic protein (GFAP) www.StemCells.com

Cells differentiated on coated coverslips and fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) were blocked in PBS/0.1% saponin/5% goat serum and incubated with primary antibody overnight (o.n.). For the BrdU immunostaining, cells were permeabilized with 4 M HCl for 10 minutes before incubation with the primary antibody. After washing, the secondary antibody was applied for 1 hour at 37°C. Labeled cells were visualized using a fluorescence microscope (Leica Microsystems AB, Sollentuna, Sweden, http://www.leica.com) and counted manually. Quantification of the total number of cells, visualized by 4,6-diamidino-2phenylindole staining, was performed using Leica QWinV3 analysis software. The whole area of the coverslips was analyzed. Percentages of neurons/oligodendrocytes in exposed cultures were compared with unexposed cultures, and the result was expressed as percentage of control.

Proliferation Assay Cells were seeded into 96-well U-bottomed plates in octaplicates or hexaplicates at 2 ⫻ 105, 1 ⫻ 105, or 5 ⫻ 104 cells per well in propagation medium. Half of the replicates were exposed to 0.1 mM DETA-NONO:ate for 24 hours. Cells were then washed and cultured for an additional 48 hours before pulsing with [3H]thymidine (1 ␮Ci/well) for 48 hours. Cells were harvested using a Tomtec cell harvester (PerkinElmer Wallac, Turku, Finland, http:// http://www.perkinelmer.com). The incorporated radioactivity was measured using a ␤-liquid scintillation counter, 1450 Microbeta Plus (PerkinElmer Wallac). To mark proliferating cells, the cultures were pulsed with 10 ␮M BrdU (Sigma-Aldrich) for 1–2 days.

Western Blotting Cells were seeded into six-well plates at a concentration of 2 million cells per well in propagation medium. After exposure to 0.1 mM DETA-NONO:ate for 1 hour, 8 hours, or 24 hours, the cells were cultured for 5 days in differentiation medium. Cell homogenates were separated by SDS-polyacrylamide gel electrophoresis (PAGE), and transfer to a nitrocellulose membrane was performed. After blocking in PBS/Tween (0.01%) with 5% nonfat milk, the membrane was incubated with primary antibody at 4°C o.n. After washing, the secondary HRP-conjugated antibody was applied and incubated for 1 hour at room temperature. Bands were detected using an enhanced chemiluminescence Western Blotting Detection kit (GE Healthcare Bio-Sciences AB, Uppsala, Sweden, http:// www1.amershambiosciences.com/). The band net intensity was measured using an Electrophoresis Documentation anal-

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Figure 1. Measurement of NO release from (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl) amino]diazen-1-ium-1,2-diolate (DETA-NONO:ate) and characterization of NSC cultures. (A): NO release from DETA-NONO:ate in culture medium measured using an NO-sensitive Clark-type electrode (n ⫽ 5). (B, C): NSC cultures passaged twice were attached on poly-D-lysine-coated glasses and labeled for Sox-2 (red) and nestin (green) (B), and the nuclei were visualized with 4,6-diamidino-2-phenylindole (blue) (C). Scale bars ⫽ 25.4 ␮m. Abbreviations: DETA-NO, DETA-NONO:ate; NO, nitric oxide.

ysis system 290 from Eastman Kodak (Rochester, NY, http:// www.kodak.com).

Total mRNA Isolation and Reverse Transcription Cells were exposed to DETA-NONO:ate at 0.1 or 0.01 mM for 24 hours. Total mRNA was isolated at different time-points during the experiment: after passage of neural spheres, after exposure, and after 4 hours, 24 hours, or 120 hours of differentiation. An RNeasy mini kit (Qiagen, West Sussex, U.K., http:// www1.qiagen.com/) was used for mRNA isolation. Reverse transcription was performed with 10 ␮l of total RNA, random hexamer primers (0.1 ␮g/ml; Gibco BRL, Invitrogen AB, Stockholm, Sweden, http://www.gibcobrl.com), and Superscript Reverse Transcriptase (200 U; Gibco BRL).

Real-Time Polymerase Chain Reaction Primers were designed using Primer Express software (Applied BioSystems, Foster City, CA, http://www.appliedbiosystems. com): ␤-actin, 5⬘-CGTGAAAAGATGACCCAGATCA-3⬘ and 5⬘-AGAGGCATACAGGGACAACACA-3⬘; for Notch-1, 5⬘GGGCAAACCATGCAGGAAT-3⬘ and 5⬘-AGCGCAAACTGCCACAAGT-3⬘; for Hes-1, 5⬘-CAGAAAGTCATCAAAGCCTATCATG-3⬘ and 5⬘-TCAGTGTTTTCAGTTGGCTCAAAC-3⬘; for Mash-1, 5⬘-AGCGCAACCGGGTCAA-3⬘ and 5⬘CGCGCGGATGTATTCGA-3⬘; for Neurogenin-2, 5⬘-CCAACTCCACGTCCCATAC-3⬘ and 5⬘-GAGGTGCATAACGGTGCTTCTC-3⬘; for Math-3, 5⬘-ATCTGGGCCTTGTCTGAAGTCTT-3⬘ and 5⬘-GCTGAGAGAGACCTTT-ACATAGCATTT3⬘; for ␤-III-tubulin, 5⬘-GGGCCTTTG-GACACCTATTCA-3⬘ and 5⬘-GCCCTCTGTATAGTGC CCTTTG-3⬘; and for GFAP, 5⬘-ACAGACTTTCTCCAACCTCCAGAT-3⬘ and 5⬘-TCTTTACCACGATGTTCCTCTTGA-3⬘. For quantification of mRNA levels, the QuantiTect SYBR (Qiagen) Green methodology was used according to the manufacturer’s instructions. Amplification was performed using an ABI prism 7700 Sequence Detection System (PerkinElmer Life and Analytical Sciences, Boston, http://www.perkinelmer.com). Relative mRNA quantities of the target genes and housekeeping genes were calculated using standard curves made for each primer pair. The standard curve was constructed of a fourfold dilution series of pooled samples. The relative expression of the target genes was expressed as the quantity ratio of the respective target gene and housekeeping gene for each individual sample. The housekeeping gene used throughout was ␤-actin, although results were confirmed using 18S.

Cell Death Evaluation by Propidium Iodide-Labeling After dissociating the neural spheres, the single cells were cultured at 100,000 cells per well in 24-well plates with propagation medium. The cells were exposed to 0.1 and 1 mM DETA-NONO:ate for 1 hour, 8 hours, and 24 hours. Directly after exposure, the cells were washed in PBS and stained with 5 ␮g/ml propidium iodide (PI). The analysis was done using flow cytometry (FACS Calibur; BD Biosciences, San Jose, CA, http://www.bdbiosciences.com) and CellQuest Software (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com).

Terminal Deoxynucleotidyl Transferase dUTP NickEnd Labeling Assay Cells were seeded on coated glasses at 30,000 cells/well, exposed to 0.1 mM DETA-NONO:ate for 24 hours. After exposure the cells were differentiated for 24 and 120 hours. The terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) labeling was performed using an ApoAlert DNA Fragmentation Assay kit (Becton, Dickinson and Company) according to the manufacturer’s instructions.

Statistics The percent values from at least six independent experiments in each group were used to calculate the statistics using the Wilcoxon signed rank test. The level of significance was p ⬍ .05. The statistics for the data presented in Figure 4D were calculated using the Mann-Whitney test. The level of significance was p ⬍ .01.

RESULTS Primary Neural Stem Cell Cultures Exposed to Nitric Oxide Are Less Prone to Generate Neurons To study the effect of NO on neural stem cell differentiation, primary cell cultures were exposed to different concentrations of the NO donor DETA-NONO:ate. This donor has a half-life of approximately 22 hours [25], which is suitable for our studies in which the purpose has been to mimic neuroinflammatory conditions with prolonged NO production. The physiological concentrations of NO in the normal brain have been estimated to range between 10 and 100 nM [26]. The NO release from 1 mM DETANONO:ate in our experiments peaked at 2 hours to reach 3,324 ⫾ 626 nM NO, whereas approximately 500 nM was released from 0.1 mM (Fig. 1A). To characterize the stem cell population in cultures after the second passage, cells were stained for Sox-2 and nestin. According to the flow cytometry analysis (supplemental online

Covacu, Danilov, Rasmussen et al.

Figure 2. Representative images of neurons in exposed and unexposed cultures. (A–D): Neurons immunolabeled for ␤-III-tubulin (red) in unexposed (A, B) and 0.1 mM (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl) amino]diazen-1-ium-1,2-diolate-exposed cultures (C, D). Scale bars: A, C ⫽ 190 ␮m; B, D ⫽ 50 ␮m. Cell nuclei are 4,6-diamidino2-phenylindole-labeled (blue).

data) 75%–90% of the cells were double-labeled for these markers. Using imaging and manual counting the number of double labeled cells could be estimated to higher than 90%. This suggests that the majority of cells were in an undifferentiated state when exposed to NO. In the immediate differentiation studies (Figs. 2, 3A). the cells were exposed to DETA-NONO:ate for 24 hours followed directly by differentiation for 5 days and immunolabeling for neurons (␤-III-tubulin) and oligodendrocytes (O4). Figure 2A–2D depicts the ␤-III-tubulin-immunoreactive neurons in unexposed and 0.1 mM diethylenetriaamine (DETA-NONO:ate)-exposed cultures. Substantially fewer neurons were formed in the 0.1 mM- and 1 mM-exposed cultures, where the number of neurons was reduced by 77 ⫾ 10% and 100%, respectively, compared to controls (Fig. 3B). This effect was also evident when cells were cultured with FCS instead of EGF during exposure. The percentage of neurons at concentrations lower than 0.1 mM tended to increase compared to unexposed controls (not statistically significant). NO-depleted DETA had a minor effect on neurogenesis and only at the highest concentrations used. In parallel to reduced neurogenesis, no reduction in the number of oligodendrocytes was observed. There was a trend of an increasing number of oligodendrocytes when higher concentrations of DETA-NONO:ate were used (Fig. 3C), although this was not statistically significant. We performed a kinetic assay in which cells were exposed to 0.1 mM DETA-NONO:ate for 1 hour, 8 hours, or 24 hours. Neurogenesis was quantified by Western blotting for ␤-III-tubulin (Fig. 3D). The tubulin bands detected in the 8 and 24 hours lanes had 85% and 99% reduced intensities, respectively, compared with the control (n ⫽ 3; Fig. 3E). These results indicate that the effect of NO on neurogenesis occurs within 8 hours of exposure to 0.1 mM DETA-NONO:ate. This concentration of DETA-NONO:ate corresponds to 500 nM NO, which is 5 times the NO concentration found in healthy brain. These experiments demonstrate that neural stem cell cultures exposed to relatively low, but pathophysiological, concentrations of NO have an impaired ability to differentiate into neurons but not into oligodendrocytes. www.StemCells.com

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Figure 3. Fewer neurons generated in stem cell cultures exposed to NO. (A): Schematic picture outlining the design of the immediate differentiation experiments. (B): Percentage of neurons (␤-III-tubulin-immunoreactive) in cultures exposed to 0.01–1 mM (Z)-1-[2-(2aminoethyl)-N-(2-ammonioethyl) amino]diazen-1-ium-1,2-diolate (DETA-NONO:ate) presented as percentage of unexposed control (0), p ⬍ .05 for 0.1 or 1 mM compared with unexposed control (n ⫽ 6). (C): Percentage of oligodendrocytes in cultures exposed to 0.01–1 mM DETA-NONO:ate is presented as percentage of control (n ⫽ 6). (D): Western blot analysis of ␤-III-tubulin expression in cultures exposed to 0.1 mM DETA-NONO:ate for 1 hour, 8 hours, or 24 hours and differentiated for 120 hours. (E): Net intensities of ␤-III-tubulin bands were normalized to ␤-actin band intensities. Data from three independent experiments were pooled and presented as percentage of control (0 h). Bars ⫽ means ⫾ SEM; 0, unexposed control; ⴱ, p ⬍ .05; n ⫽ number of experiments. Abbreviations: EGF, epidermal growth factor; FCS, fetal calf serum; h, hour(s); int., intensity; NO, nitric oxide.

The Progeny of NO-Exposed Neural Stem Cell Cultures Fail to Differentiate into Neurons To test whether neurogenesis could be recovered in exposed cultures, we performed delayed differentiation experiments (Fig. 4A). Cells were allowed to proliferate for 96 hours following NO exposure and before differentiation. Neurogenesis was however still significantly lower in 0.1 mM- and 1 mM-exposed cultures compared with controls (Fig. 4B). As equal proliferation levels were measured in both control and exposed cultures post-exposure (Fig. 4C and supplemental online data), the observed effect could not be attributed to impaired proliferation. We next addressed the question of whether the cells that had been proliferating were still committed to the same fate as cells in control cultures. Thus, cells were exposed to NO, pulsed with BrdU, differentiated, and double-labeled for BrdU and ␤-III-tubulin or GFAP. BrdUimmunoreactive cells occurred at equal levels in exposed and unexposed cultures (supplemental online data). Neurons were rarely detected following 0.1 mM DETA-NONO:ate (500 nM NO) exposure, and there were significantly fewer progenyderived BrdU/␤-III-tubulin double-positive neurons in these cultures (Fig. 4D, 4F) compared with the unexposed ones (Fig. 4E). The formation of astrocytes was extensive in both unexposed and exposed cultures (Fig. 4G).

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Figure 4. The progeny of NO-exposed stem cell cultures fail to differentiate into neurons. (A): Schematic illustration of delayed differentiation experiments. Cells were allowed to proliferate for 96 hours after exposure and prior to differentiation for 120 hours. (B): Percentage of neurons (␤-III-tubulin-immunoreactive) shown as percentage of control (0) in cultures exposed to 0.01–1 mM (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl) amino]diazen-1-ium-1,2-diolate (DETA-NONO:ate); significant reduction, p ⬍ .05 for 0.1 or 1 mM compared with unexposed control; n ⫽ 6. (C): Proliferation levels of control (white bars) and 0.1 mM DETA-NONO:ate-exposed (black bars) cultures; n ⫽ 2 (mean ⫾ SEM). (D): Percentage of BrdU-labeled progeny cells that differentiated into neurons, labeled for ␤-III-tubulin; data show the mean percentage of BrdU/␤-III-tubulin double positive cells of five different glasses for each group (mean ⫾ SD); n ⫽ 3 different experiments. (E–G): Unexposed (E) and 0.1 mM-exposed (F) cultures that have been immunolabeled for BrdU (green) and ␤-III-tubulin (red). In spite of equal proliferation after exposure, neurogenesis was severely affected in exposed cultures (F). Scale bars ⫽ 40 ␮m. (G): Astrocyte formation in 0.1 mM-exposed cultures in which glial fibrillary acidic protein (red) and BrdU (green) double-positive cells can be detected. Scale bars ⫽ 125 ␮m. The image was obtained from the culture periphery, which accounts for lower cell density. Bars ⫽ means ⫾ SEM; 0 ⫽ unexposed control; ⴱ, p ⬍ .05; ⴱⴱ, p ⬍ .01; n ⫽ number of experiments. Abbreviations: BrdU, 5-bromo-2⬘-deoxyuridine; c.p.m., counts per minute; EGF, epidermal growth factor; FCS, fetal calf serum; h, hour(s); NO, nitric oxide; unexp., unexposed.

NO Downregulates Neuronal Gene Expression In the experiments described thus far, the effect of NO on neurogenesis was assayed by the measurement of ␤-III-tubulin production in fully differentiated neurons. To investigate whether NO affects early stages of neurogenesis, we used realtime reverse transcription-polymerase chain reaction (PCR) to quantify the transcription of genes known to be active at these stages. These genes included Notch-1 and Hes-1, which are expressed in undifferentiated cells [27, 28]; Neurogenin-2 (Ngn-2) and Mash-1, which are expressed at the proneuronal stage [29, 30]; Math-3 for committed progenitor cells [31]; and ␤-III-tubulin for differentiated neurons (see [32] for review). We used two experimental set-ups. In the first set-up, the cells were attached to poly(D-lysine)-coated plates before exposure. Because coating might induce differentiation and to ensure that the cells were in an undifferentiated condition when exposed, a second type of experiment was performed. The results are presented in Figure 5A–5F. Here, the cells were in suspension during NO exposure and were attached to coated plates following exposure. Figure 5A–5F depicts the relative mRNA expression of the genes mentioned above in unexposed cultures and cultures exposed to 0.01 or 0.1 mM DETA-NONO:ate. A twofold change in expression was considered to be relevant. Exposure to 0.1 mM DETA-NONO:ate led to decreased mRNA levels of three of the genes studied (Notch-1, Ngn-2, and ␤-III-tubulin).

The expression of Notch-1 was low in all cultures regardless of NO exposure. After 24 hours of differentiation, Notch expression was approximately twofold lower in the 0.1 mMexposed cultures compared with controls (Fig. 5A). We also detected a twofold decrease in the expression of Ngn-2 in these cultures, which was apparent directly after exposure and after 24 hours of differentiation but was reduced at the later time point (Fig. 5B). The expression of ␤-III-tubulin reached detectable levels after 24 hours of differentiation and was twofold lower in the 0.1 mM-exposed cultures compared to unexposed cultures (Fig. 5C). The expression of Hes-1 and Mash-1 remained unchanged by exposure (Fig. 5D, 5E). The expression of Math-3 was downregulated directly after exposure, but expression resumed after 4 hours of differentiation (Fig. 5F). To test whether the downregulatory effects of NO on gene expression were limited to Notch-1 and the neuronal genes Ngn-2 and ␤-III-tubulin, mRNA expression of the astrocytic intermediate filament Gfap was also quantified. As apparent in Figure 5G, Gfap mRNA levels increased prominently after 24 hours of differentiation in all cultures and were not downregulated after exposure. In summary, we demonstrate that NO downregulates the expression of Notch-1 and the neuronal genes Ngn-2 and ␤-III-tubulin but not that of the astrocytic gene Gfap.

Levels of GFAP Protein Increase in Cultures Exposed to 0.1 mM DETA-NONO:ate Because NO did not effect the GFAP mRNA expression levels, we investigated its effect on the GFAP protein levels, which

Covacu, Danilov, Rasmussen et al.

Figure 5. Nitric oxide (NO) downregulates the expression of neuronal genes Ngn-2 and ␤-III-tubulin and promotes astroglial differentiation. Real-time reverse transcription-polymerase chain reaction was used to quantify the expression of genes at different stages of neuronal differentiation in control (black bars), 0.01 mM-exposed (empty bars), and 0.1 mM-exposed (gray bars) cultures. Cells were cultured nonadherently and exposed to NO for 24 hours. After washing, the cells were put on coated glasses and differentiated. Genes analyzed were (A) Notch-1 (B), Ngn-2 (C), ␤-III-tubulin (D), Hes-1 (E), Mash-1, and (F) Math-3. (G): The figure depicts the expression of the astrocytic gene Gfap. The housekeeping gene used throughout was ␤-actin. Time points were 0 (after passage of neural spheres; n ⫽ 3); a.e. (n ⫽ 5); and 4 hours, 24 hours, and 120 hours, showing the duration of differentiation; n ⫽ 2, n ⫽ 5, and n ⫽ 2, respectively. For illustrative purposes, mean ⫾ SEM of pooled experiments are presented. (H1): GFAP protein expression assessed by Western blotting in cells exposed to 0.1 mM (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl) amino]diazen-1-ium-1,2-diolate for 1 hour, 8 hours, or 24 hours; 0 h represents unexposed control. (H2): Activation of the Janus tyrosine kinase/STAT-1 pathway in these cultures was assessed by Western blotting for phosphorylated STAT-1. (H3): Expression of whole STAT-1. (H4): ␤-Actin expression was used as loading control. (I): Net intensities of GFAP bands normalized to ␤-actin expression are given as percentage of control. Data are shown as mean ⫾ SEM of two experiments. Abbreviations: a.e., after exposure; GFAP, glial fibrillary acidic protein; h, hour(s); int., intensity; pSTAT-1, phosphorylated signal transducer and activator of transcription 1; STAT-1, signal transducer and activator of transcription 1.

reflect the level of astrogliogenesis in our cultures. The cultures were exposed to 0.1 mM DETA-NONO:ate for 0 hours, 1 hour, 8 hours, or 24 hours and differentiated for 5 days before Western blotting (Fig. 5H1). Figure 5I depicts a 200% increase of the GFAP band intensity in exposed cultures compared with that in unexposed controls, suggesting that the percentage of cells differentiating into astrocytes increased in cultures exposed to pathological NO concentrations. The effect of exposure on the Janus tyrosine kinase (JAK)/signal transducer and activator of transcription (STAT)-1/3 signal transduction pathway, which directs astroglial differentiation, was also investigated. Increased levels of phosphorylated STAT-1 were already evident after 1 hour of exposure to 0.1 mM DETA-NONO:ate (Fig. 5H2). www.StemCells.com

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Figure 6. Low neurogenesis in 0.1 mM-exposed cultures is not related to cell death or oxygen levels. (A): Representative histograms from fluorescence-activated cell sorting analysis of PI-stained stem cells 1 hour, 8 hours, or 24 hours after exposure to 0.1 mM (Z)-1-[N-(2aminoethyl)-N-(2-ammonioethyl) amino]diazen-1-ium-1,2-diolate; 0 h is unexposed control. (B): Cell death assessed in control and 0.1 mMexposed cultures after nitric oxide (NO) removal and differentiation for 24 hours and 120 hours; n ⫽ 2. (C): Neurogenesis assessed by ␤-IIItubulin-immunoreactive in cultures exposed to NO at 5% oxygen (white bars) and 20% oxygen levels (dotted bars). The data are given as percentage of control; n ⫽ 2. Bars show means ⫾ SEM. Abbreviations: aft, after; h, hour(s); PI, propidium iodide; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; unexp., unexposed.

The Effect of NO on Neurogenesis Is Not Related to Cell Death or Oxygen Availability Because the detrimental effect on neurogenesis was evident at pathological concentrations of NO, it was relevant to assess cell mortality in our experiments. Cell death was assessed by PI staining 1 hour, 8 hours, or 24 hours directly after exposure to 0.1 mM DETA-NONO:ate. The level of PI-positive cells in cultures exposed for 1 hour or 8 hours did not vary from controls. However, in the 24-hour-exposed cultures there was an approximately 15% increase in the percentage of PI-positive cells (Fig. 6A). To ensure that decreased neurogenesis in these cultures was not related to ongoing cell death after initiation of differentiation, TUNEL staining was performed after 24 hours and 120 hours of differentiation. No significant differences from unexposed controls were detected (Fig. 6B).

Oxygen Availability All experiments in this study were performed using 5% oxygen to mimic the in vivo conditions in which the oxygen levels in the brain range from 0.1% to 5% [33–35]. Since oxygen availability can affect the outcome of NO exposure [36], we compared the differentiation of cultures exposed at normal oxygen levels with cultures that had been exposed at low oxygen conditions. These experiments revealed comparable numbers of neurons in cultures exposed at 21% and 5% oxygen (Fig. 6C).

DISCUSSION The main aim of this study was to elucidate the effects of neuropathological NO concentrations on primary adult NSC differentiation. We report that NSC cultures exposed to neuropathological concentrations of NO have a reduced ability to generate neurons. The reverse was true for gliogenesis, for which differentiation of NSC into oligodendrocytes but most prominently into astrocytes was favored by exposure to NO. The fate-diverting effect was transmitted to the progeny of the exposed cells.

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At the time of exposure, the cells were maintained in undifferentiated conditions with EGF to avoid any effect on the results due to the established cytotoxicity of pathological NO on differentiated neurons [9, 37–39]. The undifferentiated state of the cultures was also confirmed by immunolabeling for Sox-2 and nestin, where more than 90% of the cells were found to be double-positive. Furthermore, the cells were pulsed with NO for a limited period of time, which corresponds to the natural disease course occurring in meningitis, MS exacerbation, or trauma. Plausible explanations for the NO-induced decrease in neurogenesis are that either the differentiation of the stem cells is diverted from a neuronal fate towards an astroglial fate or that early neuronal progenitor cells are selectively depleted during NO exposure. The kinetic assay revealed that there was already a significant decrease in neurogenesis but no increase in cell death after 8 hours. During the differentiation period there was also an equivalent amount of cell death in unexposed and exposed cultures. Moreover, the cultures used had undergone two passages and were maintained with EGF, which has been shown to revert progenitor cells into a more undifferentiated state [40]. Even though the presence of progenitor cells cannot be excluded after the short propagation period [41], the high percentage of Sox-2 and nestin double-positive cells and the low expression of Ngn-2 before exposure confirms the immature state of the culture. After exposure, Ngn-2 expression levels were high in control and 0.01 mM-exposed cultures but twofold lower in the 0.1 mM-exposed cultures. Cell death of an Ngn2expressing progenitor cell pool can be excluded since there was very low expression of this gene prior to exposure. In addition, the flow cytometry measurements of the Sox-2/nestin doublepositive cells before and after exposure do not show major changes in the distribution of the different populations of cells. To study whether the percentage of neurons could be recuperated in the exposed cultures, delayed differentiation experiments were performed. We allowed the cells to proliferate after exposure to see whether the newly generated daughter cells could differentiate into neurons. However, the difference in percentage of neurons remained significantly lower in exposed cultures. Because NO has been shown to be cytostatic, we performed proliferation assays to ensure that the exposed cells were able to generate daughter cells. In addition, the cultures were fed with BrdU, and the newly generated cells could be visualized with immunofluorescence. Equal proliferation levels, measured with [3H]thymidine incorporation and BrdU immunolabeling, were detected in exposed and unexposed cultures. Even though the density of BrdU-labeled cells were similar in the different cultures, the fate of the BrdU-labeled progeny was affected since significantly fewer of these cells differentiated into neurons compared with the progeny of unexposed cells. These results suggest that the downregulative effects of NO on neurogenesis could be irreversible. Because neither effects on proliferation or cell death could account for the reduced neurogenesis, the effects on genetic determinants of stem cell fate were investigated. It has now been established in several systems that NO can regulate gene expression [42, 43]. We investigated a panel of genes expressed at different stages of neurogenesis with the greatest effect recorded on the expression of Ngn-2 and ␤-III-tubulin. In the developing mammalian cerebral cortex, neurogenins are expressed in the ventricular zone and only during the period of neurogenesis [29]

A New Role for NO in Neuroinflammatory Conditions when they are involved in determining the neural fate of progenitor cells [44]. The neuronal gene ␤-III-tubulin is a terminal differentiation gene expressed downstream of Ngn-2 [32]. Considering the hierarchic order of these genes, it is not surprising that downregulation of Ngn-2 in 0.1 mM-exposed culture leads to a similar effect on ␤-III-tubulin expression. The expression of Notch-1 was also downregulated in 0.1 mM-exposed cultures. Notch has mainly been related to maintenance of the undifferentiated phenotype. However, recent findings have demonstrated its involvement in astrocytic differentiation [45– 48], and Notch is also expressed in the postmitotic neurons of the adult rat retina [49]. Since Notch activation is regarded as one of the strongest signals of gliogenesis [45, 46], the finding of a simultaneous downregulation of Notch-1 contrasts with the finding of increased glial differentiation following NO exposure. The expression of the Notch target gene Hes-1 was not affected, however, implying either that the downregulation of Notch might not be reflected in downstream signaling events or that other signaling pathways maintain the Hes expression. The present study thus supports previous findings that NO affects gene expression, and the observed effect is not a consequence of cell death or low proliferation. The quantification of the Gfap expression also implies that the downregulatory effect of NO was specific mainly to neuronal genes. On the contrary, analysis of the expression of GFAP protein levels revealed an increase in cultures that had been exposed to NO. To further prove the role of NO in cell fate determination, we assayed the activation of the JAK-STAT-1 pathway. Recent published studies [50] have demonstrated that during development, when neurogenesis precedes astrogliogenesis, neurogenins actively downregulate the astroglial differentiation pathway, which also involves blockage of JAK-STAT1/3 signaling. In our study, we noted an increased level of phosphorylated STAT-1 in cultures exposed to NO, indicating that this pathway may be pathophysiologically important.

CONCLUSION Our results show that NO has an instructive role in the determination of stem cell differentiation, directing the cells from a neuronal fate to an astroglial fate. This effect occurs at NO levels (0.1 mM DETA-NONO:ate) that are lower than those required for initiating necrosis or apoptosis and is transmitted to the progeny of the exposed cells. The effect is mediated through downregulation of Ngn-2, which might explain the increased activation of the JAKSTAT pathway and astroglial differentiation. An increasing number of studies contribute to understanding the role of NO in the maintenance and regulation of the regenerative pool in the CNS. It has now been confirmed by several groups that at physiological concentrations NO is a negative regulator of stem/progenitor cell pool proliferation and is needed for initiation of differentiation [1– 6, 51–54]. However, we here demonstrate for the first time that NO at concentrations occurring in neuropathological conditions can affect the differentiation of NSC. Although these results are based on in vitro experiments, they have clinical implications for disease states such as MS and trauma. In MS, there is a continuous, unexplained loss of neurons, resulting in disability and loss of cognitive functions [55]. Furthermore, the problem of excessive glial scar formation after trauma has been a limiting factor in many experiments of neural

Covacu, Danilov, Rasmussen et al. repair. The diversion of stem cell fate caused by NO may give new insights into these basic and clinical problems.

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Williams Foundation, The Swedish Society for Neurologically Disabled, and Karolinska Institutet.

ACKNOWLEDGEMENTS We thank Dr. R.A. Harris for linguistic advice. This study was supported by Torsten and Ragnar Soderberg Foundation, Montel

DISCLOSURES

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