TISSUE-SPECIFIC STEM CELLS

Bidirectional Regulation of Neurogenesis by Neuronal Nitric Oxide

Synthase Derived from Neurons and Neural Stem Cells

CHUN-XIA LUO,a,b

XING JIN,aCHANG-CHUN CAO,

aMING-MEI ZHU,

aBIN WANG,

a

LEI CHANG,a QI-GANG ZHOU,a,b HAI-YIN WU,a,b DONG-YA ZHUa,b

aDepartment of Pharmacology, School of Pharmacy, Nanjing Medical University, Nanjing, People’s Republic of

China; bLaboratory of Cerebrovascular Disease, Nanjing Medical University, Nanjing, People’s Republic of China

Key Words. Neuronal nitric oxide synthase • Proliferation • Differentiation • Telomerase • CREB

ABSTRACT

It has been demonstrated that neuronal nitric oxide syn-thase (nNOS) negatively regulates adult neurogenesis.However, the cellular and molecular mechanisms underly-

ing are poorly understood. Here, we show that nNOSfrom neural stem cells (NSCs) and from neurons play op-

posite role in regulating neurogenesis. The NSCs treatedwith nNOS inhibitor N5-(1-imino-3-butenyl)-L- ornithine(L-VNIO) or nNOS gene deletion exhibited significantly

decreased proliferation and neuronal differentiation, indi-cating that NSCs-derived nNOS is essential for neurogene-

sis. The NSCs cocultured with neurons displayed asignificantly decreased proliferation, and deleting nNOSgene in neurons or scavenging extracellular nitric oxide

(NO) abolished the effects of coculture, suggesting thatneurons-derived nNOS, a source of exogenous NO for

NSCs, exerts a negative control on neurogenesis. Indeed,the NSCs exposed to NO donor DETA/NONOate displayeddecreased proliferation and neuronal differentiation. The

bidirectional regulation of neurogenesis by NSCs- and

neurons-derived nNOS is probably related to their distinctsubcellular localizations, mainly in nuclei for NSCs and incytoplasm for neurons. Both L-VNIO and DETA/NONOate

inhibited telomerase activity and proliferation in wild-type(WT) but not in nNOS2/2 NSCs, suggesting a nNOS-telo-

merase signaling in neurogenesis. The NSCs exposed toDETA/NONOate exhibited reduced cAMP response ele-ment binding protein (CREB) phosphorylation, nNOS

expression, and proliferation. The effects of DETA/NON-Oate were reversed by forskolin, an activator of CREB

signaling. Moreover, disrupting CREB phosphorylation byH-89 or LV-CREB133-GFP simulated the effects ofDETA/NONOate, and inhibited telomerase activity. Thus,

we conclude that NSCs-derived nNOS stimulates neuro-genesis via activating telomerase, whereas neurons-derived

nNOS represses neurogenesis by supplying exogenous NOthat hinders CREB activation, in turn, reduces nNOSexpression in NSCs. STEM CELLS 2010;28:2041–2052

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

INTRODUCTION

It is now widely accepted that neurogenesis is persistent inmammalian brain until adult [1, 2]. Proliferation of neuralstem cells (NSCs) in adult brain can be induced by diversephysiological and pathological stimuli [3–5]. Newly generatedneurons in the adult neocortex can replace dying neurons andform long-distance connections [6, 7], which raises optimismfor the reparative potential of endogenous progenitors to theneurodegenerative disorders. Developing effective regenera-tive strategies for these disorders may require a better under-standing of the cellular and molecular mechanisms underly-ing. Although the adult neurogenesis under physiological andpathological conditions is regulated by many endogenous neu-rotransmitters, such as glutamate, serotonin, and nitric oxide

(NO) [8, 9], the details of neurogenesis regulation are notcompletely discovered.

Three genetically different isoforms of nitric oxide syn-thase (NOS), including neuronal NOS (nNOS), endothelialNOS (eNOS), and inducible NOS, account for NO production.Of the three isoforms of NOS described, nNOS is mainlyexpressed in neurons [10]. Recently, nNOS is known to bemuch more widely distributed in other cell types of brain,including NSCs [11]. Although we and others have estab-lished that nNOS-derived NO exerts a negative control on theadult neurogenesis in normal and ischemic brains in vivo[12–15], the cellular and molecular mechanisms underlyingthe role of nNOS in neurogenesis remains unclear.

Telomere length is progressively shortened with each celldivision and the telomere maintenance appears to be essentialfor the prolonged persistence of proliferation and self-renewal

Author contributions: C.-X.L.: conception and design, data analysis and interpretation, manuscript writing; X.J.: cell culture, cellproliferation assay, collection and assembly of data; C.-C.C.: immunofluorescence, collection and assembly of data.; M.-M.Z.: NOassay in living cells, lentivirus production and infection of cultures; B.W.: RT-PCR, telomerase activity assay; L.C.: Western blotanalysis; Q.-G.Z.: Western blot analysis; H.-Y.W.: technical support; D.-Y.Z.: conception and design, data analysis and interpretation,manuscript writing.

Correspondence: Dong-Ya Zhu, M.D., Ph.D., Department of Pharmacology, School of Pharmacy, Nanjing Medical University, Nanjing210029, People’s Republic of China. Telephone: 86-25-86862818; Fax: 86-25-86862818; e-mail: dyzhu@njmu.edu.cn Received May6, 2010; accepted for publication September 2, 2010; first published online in STEM CELLS EXPRESS September 15, 2010. VC AlphaMedPress 1066-5099/2010/$30.00/0 doi: 10.1002/stem.522

STEM CELLS 2010;28:2041–2052 www.StemCells.com

of stem cells [16, 17]. Telomerase reverse transcriptase(TERT), the catalytic subunit of telomerase, extends telomeresequences after cell divisions [16]. Proliferative NSCs exhibithigh levels of telomerase activity [18], and NSCs from telo-merase-deficient mice exhibit reduced proliferation in vivoand in vitro [19]. Many studies have discovered that theeNOS in endothelial cells stimulates telomerase activity [20,21]. Moreover, proliferation and telomerase activity of thecultured subventricular zone (SVZ) neurospheres fromeNOS�/� mice were lower than that from wild-type mice[22]. Thus, it could be possible that nNOS in NSCs promotesproliferation and self-renewal by targeting telomerase.

NO donor has been proved to inhibit proliferation of cul-tured neurospheres [23, 24], raise a possibility that exogenousNO exerts a negative control on proliferation of NSCs. It hasbeen indicated that activated cAMP response element bindingprotein (CREB) is highly expressed in immature dividing cellsin adult mouse and zebrafish brains and that CREB regulatesneural stem/progenitor cells (NSPCs) proliferation in embry-onic zebrafish brain [25]. In our previous studies, we foundthat nNOS repressed CREB phosphorylation in ischemic hip-pocampus [15] and exogenous NO downregulates CREBphosphorylation in neurons [26]. Furthermore, nNOS genepromoter contains two cAMP response elements [27] andstimulation of cAMP pathway activates nNOS expression incultured cerebellar granule neurons [28] and human A673neuroepithelial cells [29]. Thus, exogenous NO may downreg-ulate nNOS expression by inhibiting CREB activation in theNSCs, thereby repressing proliferation.

Here, we report that NSCs-derived nNOS and neurons-derived nNOS regulate NSCs proliferation and differentiationbidirectionally. NSCs-derived nNOS is positive, whereas neu-rons-derived nNOS is negative to neurogenesis. Furthermore,we explore the possible mechanisms underlying the bidirec-tional regulation of neurogenesis.

MATERIALS AND METHODS

Animals

Homozygous nNOS-deficient mice (B6;129S4-Nos1tm1Plh, nNOS�/�;The Jackson Laboratory, Bar Harbor, ME, http://jaxmice.jax.org)were mated to generate nNOS�/� embryos, and wild-type (WT)embryos with similar genetic background (B6129SF2, nNOSþ/þ)were obtained by mating B6129SF1 female with B6129SF1 male.These mice were maintained in Model Animal Research Center ofNanjing University. Adult female and embryonic C57/BL/6 micefrom Laboratory Animal Center of Nanjing Medical University werealso used in this study. All experimental protocols using animalswere approved by the Institutional Animal Care and Use Committeeof Nanjing Medical University.

Cell Cultures

Embryonic NSCs were isolated from embryonic day 14 (E14)mouse cortex as described [24, 30]. Cells were floating culturedin proliferation medium containing 20 ng/ml basic fibroblastgrowth factor (bFGF; Sigma-Aldrich, St. Louis, MO, http://www.sigmaaldrich.com), 20 ng/ml epidermal growth factor (EGF;Sigma-Aldrich), and 2% B27 supplement (Grand Island, NY,http://www.invitrogen.com), and passaged every 4–6 days. Theseembryonic NSCs still possessed proliferation and self-renewalcapacity and were able to generate differentiated progeny until10th passage (supporting information Fig. 1A–1F). EmbryonicNSCs of the 2nd to 10th passage were used in this study.

Adult NSCs were isolated and cultured as previouslydescribed [31, 32], with some modifications. In brief, the dentategyri of 2-month-old female mice (10 mice were used in each pri-

mary isolation experiment) were dissected and digested with0.125% trypsin (Gibco) and 250 U/ml DNase I (Sigma-Aldrich)at 37�C for 20 minutes and undifferentiated progenitors were

Figure 1. Subcellular localizations of nNOS in NSCs and in neuronsare distinct. (A): Immunofluorescence of nNOS in the embryonic NSCsand neurons. Monolayer-cultured NSCs were stained with antibodiesagainst nNOS (red) and nestin (green) and cultured neurons werestained with antibodies against nNOS (red) and b-III-tubulin (green).Arrows mark nNOSþ neurons. Nuclei were counterstained with Hoechst33258 (blue). In the NSCs, immunofluorescence of nNOS was predomi-nantly located in the nuclei, whereas in the neurons, it was predomi-nantly located in the cytoplasms. (B): Nitric oxide (NO) production inthe embryonic NSCs and neurons. Monolayer-cultured NSCs and neu-rons were incubated with a NO-sensitive fluorescent dye DAF-FM DA.For neurons, fluorescent signals were distributed in the cytoplasms at 30and 60 minutes after incubation. For NSCs, fluorescent signals weremainly found in the nuclei at 30 minutes after incubation, and spreadall over the cytoplasms at 60 minutes. Arrows mark nNOSþ neurons.Nuclei were counterstained with Hoechst 33264 (blue). (C): Immuno-blots showing nNOS levels in nuclear and cytoplasmic fractions in theembryonic NSCs and neurons. Scale bars ¼ 20 lm (A), 25 lm (B).Abbreviations: DAF: 4-amino-5-methylamino-2,7-difluorofluoresceindiacetate; nNOS, neuronal nitric oxide synthase; NSC, neural stem cell.

2042 Bidirectional Regulation of Neurogenesis

enriched by centrifugation with Percoll (GE Healthcare Bio-Sci-ences AB, Bjorkgatan, Sweden, http://www.biocompare.com).Adult NSCs were floating cultured in proliferation medium simi-larly to embryonic NSCs. The proliferation, self-renewal capacity,and multiple differentiating potential of adult NSCs were wellidentified as the embryonic NSCs identification. Adult NSCs ofthe second to fourth passage were used in this study.

For bromodeoxyuridine (BrdU) incorporation and cocultureexperiments, embryonic or adult NSCs were planted on the cov-erslips (2 cm � 2 cm) coated with polyornithine (10 lg/ml;Sigma-Aldrich) and laminin (5 lg/ml; Invitrogen, Carlsbad, CA,http://www.invitrogen.com) at 1 � 104 cells/cm2, cultured as amonolayer.

Primary hippocampal neurons were isolated from embryonicday 18 (E18) mouse and cultured in neurobasal medium (Gibco)containing 2% B27 supplement as reported [24, 33]. Culturedneurons were identified at 10 days in vitro, and the proportion ofb-III-tubulinþ cells was about 92% (supporting information Fig.1G).

The details for embryonic and adult NSCs and primary neu-rons cultures are provided in supporting information.

Coculture of NSCs and Neurons

Before coculture, NSCs were planted on polyornithine/laminin-coated coverslips and cultured for 24 hours. Then the coverslipswith NSCs were turned over and put slightly on the substrativehippocampal neurons grown in dishes for 10 days to undergo co-culture for 24 hours. Neurobasal medium containing 20 ng/mlbFGF, 20 ng/ml EGF, and 2% B27 was used as cocultured me-dium. After coculture, the upper coverslip with NSCs wasremoved from the dish with neurons to perform the immunocyto-chemistry of NSCs.

All cultures including NSCs, neurons, and cocultures weremaintained in an incubator (HERAcell 150, Thermo Fisher Scien-tific, Waltham, MA, http://www.thermofisher.com) with humidi-fied atmosphere of 95% air and 5% CO2 at 37

�C.

Cell Proliferation Assays

Cell proliferation was assessed by cell counting for the neuro-sphere-cultured NSCs [34] and by BrdU incorporation for themonolayer-cultured NSCs [35]. Neurospheres were centrifugedand triturated to single-cell suspension, and then the cells wereseeded in 24-well plates at 2 � 104 cells/ml (0.5 ml/well). N5-(1-imino-3-butenyl)-L-ornithine (L-VNIO; Alexis Biochemicals, Post-fach, Switzerland, http://www.enzolifesciences.com), 3,3-bis(ami-noethyl)-1-hydroxy-2-oxo-1-triazene (DETA/NONOate; Sigma-Aldrich), carboxy-PTIO potassium salt (C-PTIO; Sigma-Aldrich),forskolin (Sigma-Aldrich), or H-89 dihydrochloride hydrate (H-89; Sigma-Aldrich) were added at the desired concentrationswhen seeding. Seventy-two hours later, the neurospheres gener-ated in each well were dissociated to single-cell suspension andthe number of cells was counted on a hemocytometer. Data werenormalized to the percentage of control.

For BrdU incorporation, NSCs were plated on polyornithine/laminin-coated coverslips and cultured as a monolayer for 24hours, and then were treated with drugs (L-VNIO, DETA/NON-Oate, and C-PTIO) or cocultured with neurons. During the last 2hours of coculture, 10 lM BrdU (Sigma-Aldrich) was added tolabel the dividing cells. NSCs were fixed in phosphate-bufferedsolution (PBS) containing 4% paraformaldehyde for 15 minutesat room temperature and BrdUþ cells were visualized asdescribed previously [15]. Briefly, cells were denatured in 2 MHCl (37�C for 30 minutes), rinsed in 0.1 M boric acid (pH 8.5)for 10 minutes, and blocked in PBS containing 3% normal goatserum, 0.3% (w/v) Triton X-100, and 0.1% bovine serum albumin(BSA) at room temperature for 30 minutes, followed by incuba-tion with mouse monoclonal anti-BrdU (1:1,000; Sigma-Aldrich)at 4�C overnight. Subsequently, cells were developed with theABC kit (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com) and detected with diaminobenzidine (Vector Lab-

oratories). The number of BrdUþ cells was counted in 10high-power fields systematically across the coverslip under aDIC microscope (Olympus IX71, Tokyo, Japan, http://www.olympus-global.com).

NSCs Differentiation

Monolayer-cultured NSCs were allowed to differentiate in growthfactor-free DMEM/F12 (1:1) medium containing 2% B27 and0.5% fetal bovine serum. During differentiation, the cultures weretreated with L-VNIO (100 lM), or DETA/NONOate (50 lM).Five days later, the cultures were fixed and stained with b-III-tubulin and glial fibrillary acidic protein (GFAP) antibodies tomark neurons and astrocytes, respectively. The percentages ofneurons and astrocytes were calculated in 10 high-power fieldssystematically across the coverslip.

Immunofluorescence

Fixed cultures were blocked in PBS containing 3% normal goatserum, 0.3% (w/v) Triton X-100, and 0.1% BSA at room temper-ature for 1 hour, and incubated with primary antibody at 4�Covernight. The primary antibodies used were as follows: mouseanti-nestin (1:100; Santa Cruz Biotechnology, Santa Cruz, CA,http://www.scbt.com), mouse anti-b-III-tubulin (1:150, Chemicon,Temecula, CA, http://www.millipore.com), mouse anti-GFAP(1:1,000, Chemicon), rabbit anti-nNOS (1:1,000, Chemicon), rab-bit anti-phosphorylated CREB (pCREB; Ser133; 1:500, Cell Sig-naling Technology, Beverly, MA, http://www.cellsignal.com), andrabbit anti-caspase-3 (1:300, Cell Signaling Technology). Subse-quently, cells were incubated with secondary antibodies goat anti-rabbit Cy3 (1:200; Chemicon) and goat anti-mouse FITC (1:100;Chemicon) for 2 hours at room temperature. Finally, cells werecounterstained with Hoechst 33258 (Sigma-Aldrich) to label thenuclei, imaged with a fluorescence microscope (Axio Imager,Zeiss, Oberkochen, Germany, http://www.zeiss.com), and ana-lyzed with Image-Pro Plus software (Media Cybernetics, SilverSpring, MD, http://www.mediacy.cm).

NO Production in Living Cells

The production of NO in living cells was examined using theNO-sensitive fluorescent dye [36], which reacts with NO in thepresence of oxygen to form highly fluorescent triazolofluorescein.We used a membrane-permeable form of the dye, 4-amino-5-methylamino-2,7-difluorofluorescein diacetate (DAF-FM DA),which can be taken up by cells and hydrolyzed by cellular ester-ases to form membrane-impermeable compound DAF-FM. TheNSCs grown on coverslips for 24 hours or neurons cultured invitro for 11 days were treated with a selective eNOS inhibitorN5-(1-iminoethyl)-L-ornithine (L-NIO; 10 lM; Biomol ResearchLaboratories, Plymouth Meeting, PA, http://www.bioscreening.com) for 1 hour, and loaded with 10 lM DAF-FM DA (Calbio-chem, San Diego, CA, http://www.emdbiosciences.com) in theabsence or presence of 10 lM C-PTIO in the dark incubator. At30 and 60 minutes after incubation of DAF-FM DA, fluorescentsignals of NO produced by cells were captured with a confocallaser-scanning microscope (LSM510, Zeiss).

Fluoro-Jade Staining

Fluoro-Jade staining was performed using a solution containing0.001% Fluoro-Jade and 0.1% acetic acid as previously described[37]. The details are provided in supporting information.

Lentivirus Production and Infection of NSCs

A recombinant lentivirus, LV-CREB133-GFP, was generatedwith the plasmid pCMV-CREB133 (Clontech Laboratories,Mountain View, CA, http://www.clontech.cm) as described insupporting information. LV-CREB133-GFP expresses a mutantvariant of the human CREB protein (CREB133) that contains aserine to alanine mutation corresponding to amino acid 133 in themutant mouse CREB protein. This mutation blocks phosphoryla-tion of CREB, thus preventing transcription.

Luo, Jin, Cao et al. 2043

www.StemCells.com

LV-CREB133-GFP or LV-GFP (control LV) were added intocultured NSCs at 5 ll/dish (diameter 3.5 cm) when passaged.Twenty-four hours later, the medium was fully changed withfresh medium without lentrivirus. After infected with therecombinant lentivirus for 5 days, NSCs were passaged forexperiments. The procedures concerning recombinant lentiviruswere performed following National Institutes of Healthguidelines.

Telomerase Activity Assay

Telomerase activity of cultured NSCs was detected using TRA-PEZE XL telomerase detection kit (Millipore, Billerica, MA,http://www.millipore.com). The single-cell suspension of NSCswas seeded in uncoated dishes, cultured for 72 hours to formmoderate neurospheres, and then treated with L-VNIO (100 lM)or DETA/NONOate (50 lM). Twenty-four hours after the treat-ment, the neurospheres were collected and lysed with CHAPSLysis Buffer (Millipore). The telomerase activity was detectedwith telomeric repeat amplification protocol (TRAP) [16] withsome modifications. The fluorescence energy transfer primerswere used to generate fluorescently labeled TRAP products,which were quantitatively measured with fluorescence platereader (SpectraMax M2e, Molecular Devices, Sunnyvale, CA,http://www.moleculardevices.com) or visualized following poly-acrylamide gel electrophoresis on a 10% nondenaturing gel andSYBR Green I (Invitrogen) staining.

Western Blot Analysis

Western bolt analysis was performed as described previously[15]. The primary antibodies were as follows: mouse anti-nNOS(1:600, BD company, Franklin Lakes, NJ, http://www.bdbiosciences.com), rabbit anti-pCREB(Ser133; 1:1,000, Cell Signaling Tech-nology) or rabbit anti-CREB (1:1,000, Cell Signaling Technol-ogy). Appropriate horseradish peroxidase-linked secondary anti-bodies were used for detection by enhanced chemiluminescence(Pierce, Rocdford, IL, http://www.piercenet.com). The details areprovided in supporting information.

Statistical Analysis

Comparisons among multiple groups were made with one-wayANOVA (one factor) or two-way ANOVA (two factors) followedby Scheffe’s post hoc test. Comparisons between two groupswere made with two-tail student’s t test. Data were presented asmean 6 SEM, p < .05 was considered statistically significant.

RESULTS

Subcellular Localizations of nNOS inNSCs and in Neurons

To determine the subcellular localizations of nNOS in differ-ent nerve cell types, we measured nNOS immunofluorescencein the cultured embryonic NSCs and neurons. Interestingly,we found that in NSCs, nNOS was confined to the nucleus,whereas in neurons, it was predominantly located in cyto-plasm (Fig. 1A). To further confirm this finding, we examinedthe production of NO in the living cell cultures using NO-sen-sitive fluorescent dye DAF-FM DA. Because the cultureswere pretreated with a selective eNOS inhibitor L-NIO, NOwas principally produced by nNOS. Identically, the fluores-cent signal of NO appeared largely in the nuclei of embryonicNSCs, whereas it was equably distributed in cytoplasm ofneurons, when incubated with DAF-FM DA for 30 minutes.After incubation with DAF-FM DA for 60 minutes, however,both NSCs and neurons exhibited a similar NO distribution(Fig. 1B). The dynamic change of NO distribution in NSCsmight result from the diffusion of NO from nucleus to cyto-

plasm, because NO is diffusible gaseous molecule [12].Importantly, Western blot analysis also confirmed the nuclearlocalization of nNOS in embryonic NSCs and cytoplasmiclocalization of nNOS in neurons (Fig. 1C).

NSCs-Derived nNOS Is Necessaryfor NSCs Proliferation

To reveal the biological significance of distinct subcellularlocalizations of nNOS in NSCs and in neurons, we investi-gated the role of nNOS in NSCs in regulating their own pro-liferation. We used two independent approaches to treatnNOS in the cultured embryonic NSCs. In a pharmacologicalapproach, we treated embryonic NSCs with L-VNIO, a potentand highly selective inhibitor of nNOS [38], to inhibit nNOSenzymatic activity. L-VNIO (10, 50, or 100 lM) was added tothe cultured NSCs at the time of seeding. After 72 hours offloating culture, L-VNIO decreased neurospheres formationconcentration-dependently compared with control, quantifiedby cell counting (Fig. 2A, 2B). Similar results were obtainedwith BrdU incorporation experiments using monolayer-cul-tured NSCs. Treatment with 100 lM L-VNIO for 24 hours(10 lM BrdU was introduced at 22 hours after L-VNIO treat-ment) reduced cell proliferation significantly. The ratio ofBrdU-positive cells in L-VNIO-treated NSCs was 36.4% 63.9%, compared with 51.2% 6 1.7% in control (Fig. 2C, 2D,p < .05). Moreover, 100 lM L-VNIO did not affect the apo-ptosis of the cultured NSCs (supporting information Fig. 2).In a genetic approach, we examined the proliferation of theembryonic NSCs from null mutant mice lacking nNOS gene(nNOS�/�). Consistent with pharmacological manipulation,the deletion of nNOS resulted in a marked decrease in bothneurospheres formation (Fig. 2E, 2F, p < .05) and BrdU-posi-tive cells (Fig. 2G, 2H, p < .01), compared with WT control.Taken together, these findings indicate that the autochthonousnNOS is necessary for NSCs proliferation.

Moreover, the role of NSCs-derived nNOS in adult neuro-genesis was also investegated by both neurosphere-formationand BrdU-incorporation experiments. L-VNIO (100 lM) sig-nificantly slowed down the neurosphere-formation of floatingcultured adult NSCs (p < .01; Fig. 2I, supporting informationFig. 3A) and decreased the ratio of BrdUþ cells of mono-layer-cultured adult NSCs (p < .01; Fig. 2J, supporting infor-mation Fig. 3B).

Neurons-Derived nNOS Inhibits NSCs Proliferation

It is well known that NO derived from nNOS negatively regu-lates the adult neurogenesis in vivo [13–15]. The role ofNSCs-derived nNOS seems to conflict with the reports above.Actually, neurogenesis in the adult brain may be regulatednot only by autochthonous nNOS in NSCs but also by nNOSexpressed in other cell types, especially in neurons. It is pos-sible that NO derived from other cell types inhibits NSCs pro-liferation. Accordingly, we exposed the cultured embryonicNSCs to a long half-life NO donor DETA/NONOate to mimicNO from other cell types. After exposure for 72 hours,DETA/NONOate decreased neurospheres formation substan-tially and concentration-dependently (Fig. 3A, 3B). To deter-mine whether the effect of DETA/NONOate on embryonicNSCs proliferation was due to NO release, a cell-impermeableNO scavenger C-PTIO (10 lM, an effective and nontoxicconcentration, see supporting information Fig. 4) was addedalong with 30 lM DETA/NONOate. As expected, C-PTIOabolished the inhibitory effect of DETA/NONOate on neuro-sphere-formation completely (Fig. 3A, 3C, p < .01). More-over, DETA/NONOate also inhibited neurosphere-formationin the adult NSCs (p < .01; Fig. 3D, supporting informationFig. 5A). Consistent with neurosphere-formation experiments,

2044 Bidirectional Regulation of Neurogenesis

BrdU incorporation experiments revealed that treatment withDETA/NONOate (50, 100, 200 lM) for 24 hours inhibitedthe proliferation of monolayer-cultured embryonic NSCs con-centration-dependently (Fig. 3E, 3F), and the effect was abol-ished by C-PTIO (Fig. 3E, 3G, p < .01). Similarly, exposureof adult NSCs to DETA/NONOate reduced ratio of BrdUþ

cells (p < .01; Fig. 3H, supporting information Fig. 5B).Additionally, 100 lM DETA/NONOate did not induce apo-ptosis of the monolayer-cultured NSCs (supporting informa-tion Fig. 2). Together, our results suggest that exogenous NOinhibits NSCs proliferation.

Next, we investigated the role of nNOS expressed in neu-rons in regulating NSCs proliferation. To establish the interac-tion between neurons and NSCs, the embryonic NSCs mono-layer cultured for 24 hours were cocultured with neuronswhich had been grown for 10 days in vitro. At 22 hours aftercoculture, 10 lM BrdU was incorporated. Two hours later,the NSCs were fixed for BrdU staining. The embryonic NSCscocultured with neurons exhibited a markedly reduced ratio ofBrdU-positive cells (25.6% 6 3.4% vs. 42.3% 6 4.5%; Fig.3I, 3J, p < .05), suggesting a negative regulation of NSCs

proliferation by the cocultured neurons. To examine whethernNOS expressed in neurons is responsible for the inhibitoryeffect of neurons on proliferation, we treated the cocultureswith C-PTIO to remove NO outside NSCs. As shown inFigure 3I and 3J, the decline in embryonic NSCs proliferationwas rescued by C-PTIO (36.8% 6 1.9%, p < .05). To furtherconfirm the contribution of neurons-derived nNOS, wecocultured the embryonic NSCs from WT mice with theneurons from nNOS�/� mice. As expected, the embryonicNSCs cocultured with nNOS�/� neurons exhibited signifi-cantly faster proliferation than that cocultured with WT neu-rons did (ratio of BrdU-positive cells, 35.4% 6 2.3% vs.20.5% 6 0.7%, p < .01; Fig. 3K, 3L), indicating requirementof nNOS for the negative regulation of NSCs proliferation byneurons.

Telomerase Is Involved in the Effectsof Autochthonous and Exogenous NO

Proliferative NSCs exhibit high levels of telomerase activity[18], and NSCs from telomerase-deficient mice exhibit

Figure 2. Neural stem cells (NSCs)-derived nNOS is necessary for prolifera-tion. (A): Newly formed embryonicneurospheres after treatment with L-VNIO (a potent and highly selectivenNOS inhibitor) or vehicle for 72 hours.(B): Statistical graph showing the num-ber of cells of the embryonic neuro-spheres treated with 10, 50, or 100 lML-VNIO for 72 hours. (C): Representa-tives of BrdU-labeled cells of the mono-layer-cultured embryonic NSCs treatedwith 100 lM L-VNIO or vehicle for 24hours. (D): Statistical graph from datain (C). (E): Representatives of newlyformed nNOS�/� and WT embryonicneurospheres after 72 hours floating-cul-ture. (F): Statistical graph from data in(E). (G): Representatives of BrdU-la-beled cells of the monolayer-culturednNOS�/� and WT embryonic NSCs af-ter culture for 48 hours. (H): Statisticalgraph from data in (G). (I): Statisticalgraph showing the number of cells ofthe adult neurospheres treated with 100lM L-VNIO for 72 hours. (J): Statisticalgraph showing the number of BrdU-la-beled cells of the monolayer-culturedadult NSCs treated with 100 lM L-VNIO or vehicle for 24 hours. Data aremeans 6 SEM; *, p < .05, **, p < .01versus control or WT. All results wereobtained from five to six independentexperiments performed in triplicate.Scale bars ¼ 500 lm (A, E), 100 lm(C, G). Abbreviations: BrdU, bromo-deoxyuridine; L-VNIO, N5-(1-imino-3-butenyl)-L-ornithine; nNOS, neuronal ni-tric oxide synthase; WT, wild-type.

Luo, Jin, Cao et al. 2045

www.StemCells.com

Figure 3. Exogenous nitric oxide (NO) derived from NO donor or from neurons inhibits proliferation of neural stem cells (NSCs). (A–H): NOdonor inhibits proliferation of embryonic and adult NSCs. (A): Representatives of embryonic neurospheres treated with 30 lM DETA/NONOateor 10 lM C-PTIO alone or two drugs in combination for 72 hours. (B): Concentration-response relationship of DETA/NONOate in inhibiting em-bryonic neurosphere-formation. (C): The effect of C-PTIO on DETA/NONOate pharmacological action. (D): The effect of DETA/NONOate inthe adult neurosphere-formation. (E): Representatives of BrdU-labeled cells of monolayer-cultured embryonic NSCs treated with 100 lM DETA/NONOate or 10 lM C-PTIO alone or two drugs in combination for 24 hours. (F): Concentration-response relationship of DETA/NONOate inreducing BrdU-labeled cells. (G): The effect of C-PTIO on DETA/NONOate pharmacological action. (H): Reduction in BrdU-labeled cells ofthe adult NSCs by DETA/NONOate. (I, J): NO derived from neurons-nNOS inhibits proliferation of cocultured NSCs. (I): Representatives ofBrdU-labeled cells of the embryonic NSCs cocultured with neurons in the presence or absence of 10 lM C-PTIO. (J): Statistical graph showingthe number of BrdUþ cells in different groups. (K, L): Requirement of nNOS for the inhibitory effect of neurons on NSCs proliferation. (K):

Representatives of BrdU-labeled cells of the embryonic NSCs cocultured with nNOS�/� neurons or WT neurons. (L): Statistical graph showingthe number of BrdUþ cells in different groups. Data are means 6 SEM; *, p < .05, **, p < .01, versus control in (B–D), (F–H), and (J) or ver-sus coculture with WT neurons in (L); #, p < .05, ##, p < .01, versus DETA/NONOate alone in (C) and (G), or versus coculture with neurons in(J). All results were obtained from three to four independent experiments performed in triplicate. Scale bars ¼ 500 lm (A), 100 lm (E, I, K).Abbreviations: BrdU, bromodeoxyuridine; C-PTIO, carboxy-PTIO potassium salt; nNOS, neuronal nitric oxide synthase; WT, wild-type.

2046 Bidirectional Regulation of Neurogenesis

reduced proliferation in vivo and in vitro [19]. Consistentwith these reports, we observed that RNA interference silenc-ing TERT expression in cultured embryonic neurospheres sig-nificantly decreased proliferation and induced adhesion tosubstrate (supporting information Fig. 6). To determinewhether the bidirectional regulation of NSCs proliferation byautochthonous and exogenous NO depends on bidirectionalregulation of telomerase activity, we incubated the culturedembryonic neurospheres from nNOS�/� and WT mice with100 lM L-VNIO or 50 lM DETA/NONOate for 24 hours.Interestingly, the telomerase activity in WT neurospheres washigher than that in nNOS�/� neurospheres (223.1 6 11.6 vs.121.2 6 6.4, p < .01; Fig. 4A, 4B), implying that nNOS maybe implicated in the regulation of telomerase activity. In WTneurospheres, both drugs significantly decreased telomeraseactivity (p < .05) but ineffective in nNOS�/� neurospheres (p> .05; Fig. 4A, 4B), suggesting requirement of nNOS fortelomerase activity regulation by both autochthonous and ex-ogenous NO, and opposite effects of autochthonous and exog-enous NO on telomerase activity as well. Furthermore,reduced telomerase activity of the adult NSCs treated with ei-ther 100 lM L-VNIO or 50 lM DETA/NONOate (p < .05,Fig. 4C) was also observed.

Exogenous NO Represses Proliferationby Inhibiting CREB Activation

To explore the molecular mechanisms underlying the prolifer-ation inhibition by exogenous NO, we incubated the culturedembryonic NSCs with 50 lM DETA/NONOate in the absenceor presence of 15 lM forskolin, an activator of cAMP-de-pendent protein kinase (PKA)-CREB signaling, and foundthat DETA/NONOate significantly decreased pCREB level (p< .05), nNOS expression (p < .01) (Fig. 5A, 5B), and prolif-eration (Fig. 5C, p < .01). Forskolin completely reversedDETA/NONOate-induced pCREB and nNOS level reductions(Fig. 5A, 5B, p < .05), and partially restored NSCs prolifera-tion (Fig. 5C, p < .01). Similarly, the immunofluorescence ofpCREB duplicated the effect of these drugs on CREB phos-phorylation (Fig. 5D).

To further confirm the role of CREB activation in nNOSexpression and proliferation, we incubated the cultured em-bryonic NSCs with 10 lM H-89, an inhibitor of PKA. TheNSCs treated with H-89 exhibited a significantly decreased

nNOS level (Fig. 5E, 5F, p < .05) and proliferation (30.7%of control, p < .01; Fig. 5G). To prevent CREB activationspecifically, we prepared a recombinant lentivirus LV-CREB133-GFP expressing a mutant variant of CREB proteinwhich could not be phosphorylated at Ser133. The embryonicNSCs infected with LV-CREB133-GFP displayed markedlyreduced pCREB (50.6% of LV-GFP, p < .01; Fig. 5H, 5I).As expected, LV-CREB133-GFP infection inhibited nNOSexpression (Fig. 5H, 5I, p < .05) and proliferation of NSCs(Fig. 5J, 5K, p < .05). These findings collectively indicatethat the disruption of CREB phosphorylation and subsequentnNOS reduction may account for the effect of exogenous NOon NSCs proliferation.

In addition, NO regulates the upstream effectors of mito-gen-activiated protein kinase signaling, such as extracellularsignal-regulated kinase and c-Jun N-terminal kinase (JNK)[39, 40], we thus examined whether they were involved indownregulation of NSCs-derived nNOS expression by exoge-nous NO. However, neither MEK inhibitor U0126 nor JNKinhibitor SP600125 influenced nNOS expression of embryonicNSCs (supporting information Fig. 7).

Different Roles of Autochthonous andExogenous NO in Differentiation

Newborn neural cells have to undergo differentiation beforethey become mature and functional. We thus investigatedthe roles of nNOS from NSCs and neurons in differentiation.We treated embryonic NSCs with L-VNIO (100 lM) to in-hibit the autochthonous nNOS or DETA/NONOate (50 lM)to mimic neurons-derived nNOS during differentiation. Inter-estingly, although L-VNIO and DETA/NONOate had muchweak effect on the percentage of astrocytes (GFAPþ; Fig.6A, 6B), both drugs substantially reduced the percentage ofneurons (b-III-tubulinþ) at day 5 after differentiation (3.7%6 1.2% vs. 21.0% 6 2.1% for L-VNIO, p < .01; 9.0% 62.0% vs. 21.0% 6 2.1% for DETA/NONOate, p < .05; Fig.6A, 6B), indicating opposite roles of autochthonous and ex-ogenous NO in the differentiation of NSCs into neurons.More importantly, two drugs hindered neuronal neuritogene-sis during differentiation, especially L-VNIO (Fig. 6A). Twodrugs may affect differentiation via different mechanisms,because L-VNIO significantly increased the apoptotic ratio ofcells (12.4% 6 1.5% vs. 5.1% 6 0.8%, p < .05 for Fluoro-

Figure 4. Regulation of telomerase activity by nitric oxide (NO) in the embryonic and adult NSCs. (A): The representative of SYBR Green I-stained gel visualizing TRAP products from embryonic NSCs samples. (B): The statistical graph showing telomerase activities of embryonicnNOS�/� or WT NSCs treated with L-VNIO or DETA/NONOate. (C): The statistical graph showing telomerase activities of the adult NSCstreated with L-VNIO or DETA/NONOate. Data are means 6 SEM; n ¼ 4, *, p < .05, **, p < .01 versus control of WT in (B), or versus controlin (C). Abbreviations: IC, internal control; L-VNIO, N5-(1-imino-3-butenyl)-L-ornithine; NC, negative control; nNOS, neuronal nitric oxide syn-thase; TPG, total product generated; TRAP, telomeric repeat amplification protocol; WT, wild-type.

Luo, Jin, Cao et al. 2047

www.StemCells.com

Jade staining; 2.3% 6 0.5% vs. 1.3% 6 0.2%, p < .05 forcaspase-3 staining) but DETA/NONOate did not (p > .05;Fig. 6C–6F). Together, these data suggest that NSCs-derivednNOS is required, but exogenous NO is unfavorable for neu-ronal differentiation.

DISCUSSION

We report here that NSCs- and neurons-derived nNOS playopposite roles in regulating NSCs proliferation and

Figure 5. CREB activation and the effects of exogenous nitric oxide (NO) on nNOS expression and proliferation in the neural stem cells(NSCs). (A–D): DETA/NONOate inhibits nNOS expression and proliferation in the NSCs by reducing CREB phosphorylation. (A): Immunoblotsshowing CREB, pCREB, and nNOS levels in floating-cultured embryonic neurospheres treated with 50 lM DETA/NONOate in the absence orpresence of 15 lM forskolin for 18 hours. (B): Statistical graph from data in (A), n ¼ 3. (C): The number of NSCs from floating-cultured embry-onic neurospheres treated with DETA/NONOate (50 lM) alone or with forsklin (15 lM) for 72 hours. (D): Immunofluorescent images showingpCREB levels in monolayer-cultured embryonic NSCs treated with DETA/NONOate (50 lM) alone or with forskolin (15 lM) for 18 hours. Cellswere stained with antibodies against pCREB (red) and nestin (green). Nuclei were counterstained with Hoechst 33258 (blue). (E–K): CREB acti-vation is required for nNOS expression and proliferation in the NSCs. (E): Immunoblots showing CREB, pCREB, and nNOS levels in floating-cultured embryonic neurospheres treated with 10 lM H-89 (a PKA inhibitor) or vehicle for 18 hours. (F): Statistical graph from data in (E), n ¼3. (G): The number of NSCs from floating-cultured embryonic neurospheres treated with 10 lM H-89 or vehicle for 72 hours. (H): Immunoblotsshowing CREB, pCREB, and nNOS levels in floating-cultured embryonic neurospheres infected with LV-CREB133-GFP or LV-GFP. (I): Statisti-cal graph from data in (H), n ¼ 3. (J): The number of NSCs from floating-cultured embryonic neurospheres infected with LV-CREB133-GFP orLV-GFP for 72 hours (n ¼ 3). (K): Representatives of images showing the neurospheres infected with LV-CREB133-GFP or LV-GFP. Data aremeans 6 SEM; *, p < .05, **, p < .01, versus control; #, p < .05, ##, p < .01, versus DETA/NONOate alone in (B, C), versus LV-GFP in (I,

J). Scale bars ¼ 50 lm (D), 100 lm (J). Abbreviations: CREB, cAMP response element binding protein; GAPDH, glyceraldehyde phosphate de-hydrogenase; GFP, green fluorescent protein; pCREB, phosphorylated CREB; Ph, phase contrast.

2048 Bidirectional Regulation of Neurogenesis

differentiation. The bidirectional regulations of neurogenesisby nNOS might result from different subcellular localizationsof nNOS in NSCs and neurons, mainly in nuclei for NSCsand in cytoplasm for neurons. We also explored the possiblemolecular mechanisms underlying the bidirectional regula-tions of neurogenesis by nNOS. CREB activation and telo-merase activity changes may be critical.

Several lines of research in vivo, including ours, have pro-vided strong evidence that nNOS-derived NO is a negativeregulator of cell proliferation in neurogenic regions of theadult mammalian brain [13–15]. The effects of nNOS could

be a composite consequence of cell interactions, because it isexpressed not only in neurons [41] but also in astrocytes [42]and NSCs [43, 44]. Our in vitro findings that NSCs-derivednNOS promoted but neurons-derived nNOS inhibited neuro-genesis suggest that different sources of nNOS play differentroles. Thus, the negative role of nNOS in neurogenesis invivo may be explained by the inhibitory effect of neurons-derived nNOS overwhelmingly surpasses the facilitative effectof NSCs-derived nNOS because of predominant expression ofnNOS in neurons in the brains. An important circumstantialevidence is that nNOS�/� mice exhibited a transient decrease

Figure 6. Different effects of autochthonous and exogenous NO on NSCs differentiation and survival. (A, B): Inhibition of NSCs-derivednNOS or exposure to exogenous NO impairs neuronal differentiation. Monolayer-cultured embryonic NSCs were treated with 100 lM L-VNIO or50 lM DETA/NONOate for 5 days during differentiation. (A): Representatives of GFAPþ astrocytes and b-III-tubulinþ neurons. (B): Statisticalgraphs showing the ratio of GFAPþ and b-III-tubulinþ cells. (C–F): Inhibition of NSCs-derived nNOS leads to cell apoptosis. Monolayer-cul-tured embryonic NSCs were treated with 100 lM L-VNIO or 50 lM DETA/NONOate for 5 days during differentiation. (C): Representatives ofFluoro-Jadeþ nuclei (arrows). Fluoro-Jade, green; Hoechst 33258, blue. (D): Statistical graph showing the ratio of Fluoro-Jadeþ nuclei. (E): Sta-tistical graph showing the ratio of caspase-3þ nuclei. (F): Representatives of caspase-3þ nuclei (arrows). Caspase-3, red; Hoechst 33258, blue.Data are means 6 SEM; n ¼ 3–5, *, p < .05; **, p < .01, versus control. Scale bars ¼ 50 lm (A), 100 lm (C, F). Abbreviation: GFAP, glialfibrillary acidic protein; L-VNIO, N5-(1-imino-3-butenyl)-L-ornithine.

Luo, Jin, Cao et al. 2049

www.StemCells.com

in neural precursors proliferation at early postnatal ages [45],which greatly supports our findings that NSCs-derived nNOSis necessary for their own proliferation.

Meanwhile, we have to confront some discordant reports invitro that nitric oxide synthesis inhibition increases proliferationof neural precursors [46–48]. First, the NOS inhibitor theyused, Nx-nitro-larginine methylester (L-NAME), is nonspecific,which may influence all the three isoforms of NOS. Given thatNSCs express both nNOS and eNOS [43–44], we chose ahighly selective nNOS inhibitor L-VNIO [38] in this study.Although selective eNOS inhibitor L-NIO (10 lM) had littleeffect on NSCs proliferation (110.8% of control, supporting in-formation Fig. 8), eNOS may complicate the effect of L-NAME on proliferation. Second, the cultures they grew wereSVZ explants [46], cortical neural clusters [47], or cerebellargranule cells [48]. All these neural precursors were differentiat-ing and most of them could be labeled with NSE [46], microtu-bule-associated protein-2 (MAP-2) [47], or b-III-tubulin [48].The treatments with L-NAME reduced NO produced not onlyby undifferentiated but also by differentiated cells. As we indi-cated here, NO from differentiated neurons inhibits prolifera-tion. Thus, the proliferation increase by L-NAME could beexplained by neurons-derived nNOS dysfunction.

Telomere maintenance is essential for the prolonged per-sistence of proliferation and self-renewal of stem cells andtelomerase activity is responsible for proliferation capabilityof cells [16, 17]. Both nNOS pharmacological inhibition andgene knockout inhibited telomerase activity in the culturedNSCs (Fig. 4), indicating a role of autochthonous nNOS sig-naling in activating telomerase and promoting proliferation ofNSCs. The observations that eNOS signaling in endothelialcells stimulates telomerase [20, 21] support our findings indi-rectly. Surprisingly, exogenous NO that mimics neurons-derived nNOS signaling inhibited telomerase activity in NSCsalso (Fig. 4). In fact, the inhibitory effect of exogenous NOon telomerase appears only in WT NSCs but not in nNOS�/�

NSCs (Fig. 4), which further addresses that NSCs-derivednNOS is implicated in telomerase activation.

What are the molecular mechanisms underlying the effectsof exogenous NO on telomerase activity and proliferation inNSCs? CREB activation is important for NSPCs proliferationin embryonic zebrafish brain [25]. Our current findings alsoindicated that prohibiting CREB activation repressed prolifer-ation in the cultured NSCs (Fig. 5). Consistent with our previ-ous observation in neurons [26], the present data showed thatexogenous NO inhibited CREB phosphorylation in NSCs, andthat the inhibition of CREB phosphorylation reduced telo-merase activity (supporting information Fig. 9) and nNOSexpression (Fig. 5E–5K). Moreover, nNOS gene promotercontains two cAMP response elements [27]. Together, wesuppose a signaling pathway mediating the negative effect ofexogenous NO on NSCs proliferation: NO downregulatesnNOS expression in NSCs through reducing CREB phospho-rylation, and in turn, inhibits telomerase activity (Fig. 7).

Clearly, NO is the only product of nNOS, whereverexpressed in NSCs or neurons. Why does NSCs-derived NO andneurons-derived NO play completely different roles? The differ-ence of functions could be attributed to that nNOS in NSCs andneurons have distinct subcellular localizations, the former innuclei and the later in cytoplasms (Fig. 1A, 1B). Moreover,nNOS levels in neurons were much higher than that in NSCs(data not shown). The cytoplasms distribution and rich expres-sion of neurons-derived nNOS may be facilitative for the diffu-sion of NO to outside of cells. Indeed, cell-impermeable NOscavenger could dramatically remove the NO produced by cul-tured neurons (supporting information Fig. 10A) and partiallyabolish the inhibitory effect of neurons on the proliferation of

cocultured NSCs (Fig. 3J). On the other hand, NO derived fromNSCs to diffuse to the outside of NSCs is negligible, as cell-impermeable NO scavenger was unable to weaken NO signal ofthe cultured NSCs (supporting information Fig. 10B) or to in-hibit the proliferation of cultured NSCs (Fig. 3C, 3G). The weakdiffusion of NSCs-derived NO may attribute to its nuclei local-ization and lower expression. Moreover, the nuclei localizationmay play an important role in regulating nuclear signaling mole-cule, such as telomerase. We thus suppose that NSCs-derivedNO may act in an autocrine manner via nuclear signaling mole-cule, whereas neurons-derived NO may act in a paracrine man-ner via membrane or cytoplasmic receptors (Fig. 7).

The direction that NO regulates NSCs differentiationcould vary with the doses. It has been shown that 0.1 and 0.4lM DETA/NONOate promotes neuronal differentiation andneurite outgrowth [23], but 100 and 1,000 lM DETA/NON-Oate diverts NSCs fate from neurogenesis toward astroglio-genesis [49]. In the current study, our data showed that 50lM DETA/NONOate inhibited neuronal differentiation andimpaired neurite outgrowth (Fig. 6). Because the physiologi-cal concentrations of NO in the normal brain have been esti-mated to range between 10 and 100 nM [50], and the concen-tration of NO released from 50 lM DETA/NONOate isapproximately equivalent to physiological level [49], we thusdeduce that neurons-derived nNOS could have adverse effecton NSCs differentiation to neurons. In contrast, our findingssuggest that NSCs-derived nNOS is required for neuronal dif-ferentiation (Fig. 6). Moreover, NSCs-derived nNOS may beinvolved in the survival of newborn neurons, because theNSCs treated with L-VNIO exhibited decreased percentage ofb-III-tubulinþ cells (Fig. 6B) and increased the number ofdead cells with neuronal morphology (supporting informationFig. 11) and apoptotic ratio after differentiation for 5 days(Fig. 6E, 6F). Yet this presumption remains to be demon-strated by further research. Additionally, the regulation ofNSCs proliferation by NO may also vary with the concentra-tions. It was reported that NO donor NOC-18 promoted NSCsproliferation at 10 lM, whereas it inhibited proliferation at

Figure 7. A model of signal pathway whereby nNOS derived fromneurons and NSCs regulate neurogenesis oppositely. NO produced byNSCs-derived nNOS stimulates TERT, leads to proliferation, whereasNO produced by neurons-derived nNOS inhibits nNOS expression inthe nucleus of NSCs through a negative control on CREB phosphoryl-ation, thus reducing proliferation. Abbreviations: CBP, CREB bindingprotein; CREB, cAMP response element binding protein; nNOS, neu-ronal nitric oxide synthase; NO, nitric oxide; NSCs, neural stem cells;p-CREB, phosphorylated CREB; PKA, cAMP-dependent protein ki-nase; TERT, telomerase reverse transcriptase.

2050 Bidirectional Regulation of Neurogenesis

100 lM [51]. So, different concentrations of NO may affectproliferation through different signaling pathway.

CONCLUSION

Our results suggest that the nNOS expressed in NSCs pro-motes their own proliferation and is essential for neuronal dif-ferentiation, whereas the nNOS expressed in neurons inhibitsNSCs proliferation and is unfavorable to neuronal differentia-tion. Telomerase activation accounts for the promotive effectof NSCs-derived nNOS on neurogenesis. Neurons-derivednNOS may negatively regulate neurogenesis through disrupt-ing CREB activation and downregulating nNOS expression,and thereby inhibiting telomerase activity.

ACKNOWLEDGMENTS

This work was supported by grants from National Natural Sci-ence Foundation of China (30971021 to D.-Y.Z., 30901550to C.-X.L.), National Basic Research Program of China (973Program; 2011CB504404 to D.-Y.Z.), and Natural ScienceFoundation of Jiangsu Province (09KJB310004 to C.-X.L.).

DISCLOSURE OF POTENTIAL

CONFLICTS OF INTEREST

The authors indicate no potential conflicts of interest.

REFERENCES

1 Altman J. Are new neurons formed in the brains of adult mammals?Science 1962;135:1127–1128.

2 Kaplan MS, Hinds JW. Neurogenesis in the adult rat: Electron micro-scopic analysis of light radioautographs. Science 1977;197:1092–1094.

3 van Praag H, Kempermann G, Gage FH. Running increases cell prolif-eration and neurogenesis in the adult mouse dentate gyrus. Nat Neuro-sci 1999;2:266–270.

4 Parent JM, Valentin VV, Lowenstein DH. Prolonged seizures increaseproliferating neuroblasts in the adult rat subventricular zone-olfactorybulb pathway. J Neurosci 2002;22:3174–3188.

5 Liu J, Solway K, Messing RO et al. Increased neurogenesis in thedentate gyrus after transient global ischemia in gerbils. J Neurosci1998;18:7768–7778.

6 Magavi SS, Leavitt BR, Macklis JD. Induction of neurogenesis in theneocortex of adult mice. Nature 2000;405:951–955.

7 Chen J, Magavi SS, Macklis JD. Neurogenesis of corticospinal motorneurons extending spinal projections in adult mice. Proc Natl AcadSci USA 2004;101:16357–16362.

8 Lichtenwalner RJ, Parent JM. Adult neurogensis and the ischemicforebrain. J Cereb Blood Flow Metab 2006;26:1–20.

9 Abrous DN, Koehl M, Le Moal M. Adult neurogenesis: From precur-sors to network and physiology. Physiol Rev 2005;85:523–569.

10 Thomas M, Feron O. Nitric oxide synthase: Which, where, and why?J Clin Invest 1997;100:2146–2152.

11 Zhou L, Zhu DY. Neuronal nitric oxide synthase: Structure, subcellu-lar localization, regulation, and clinical implications. Nitric Oxide2009;20:223–230.

12 Zhu XJ, Hua Y, Jiang J et al. Neuronal nitric oxide synthase-derivednitric oxide inhibits neurogenesis in the adult dentate gyrus by down-regulating cyclic AMP response element binding protein phosphoryla-tion. Neuroscience 2006;141:827–836.

13 Packer MA, Stasiv Y, Benraiss A et al. Nitric oxide negatively regu-lates mammalian adult neurogenesis. Proc Natl Acad Sci USA 2003;100:9566–9571.

14 Moreno-Lopez B, Romero-Grimaldi C, Noval JA et al. Nitric oxide isa physiological inhibitor of neurogenesis in the adult mouse subven-tricular zone and olfactory bulb. J Neurosci 2004;24:85–95.

15 Luo CX, Zhu XJ, Zhou QG et al. Reduced neuronal nitric oxide syn-thase is involved in ischemia-induced hippocampal neurogenesis byup-regulating inducible nitric oxide synthase expression. J Neurochem2007;103:1872–1882.

16 Kim NW, Piatyszek MA, Prowse KR et al. Specific association ofhuman telomerase activity with immortal cells and cancer. Science1994;266:2011–2015.

17 Harley CB, Futcher AB, Greider CW. Telomeres shorten during agingof human fibroblasts. Nature 1990;345:458–460.

18 Caporaso GL, Lim DA, Alvarez-Buylla A et al. Telomerase activityin the subventricular zone of adult mice. Mol Cell Neurosci 2003;23:693–702.

19 Ferron S, Mira H, Franco S et al. Telomere shortening and chromo-somal instability abrogates proliferation of adult but not embryonicneural stem cells. Development 2004;131:4059–4070.

20 Vasa M, Breitschopf K, Zeiher AM et al. Nitric oxide activates telomer-ase and delays endothelial cell senescence. Circ Res 2000;87:540–542.

21 Matsushita H, Chang E, Glassford AJ et al. eNOS activity is reducedin senescent human endothelial cells: Preservation by hTERT immor-talization. Circ Res 2001;89:793–798.

22 Chen J, Zacharek A, Zhang C et al. Endothelial nitric oxide synthaseregulates brain-derived neurotrophic factor expression and neurogene-sis after stroke in mice. J Neurosci 2005;25:2366–2375.

23 Chen J, Zacharek A, Li Y et al. N-cadherin mediates nitric oxide-induced neurogenesis in young and retired breeder neurospheres. Neu-roscience 2006;140:377–388.

24 Hu M, Sun YJ, Zhou QG et al. Negative regulation of neurogenesisand spatial memory by NR2B-containing NMDA receptors. J Neuro-chem 2008;106:1900–1913.

25 Dworkin S, Malaterre J, Hollande F et al. cAMP response elementbinding protein is required for mouse neural progenitor cell survivaland expansion. Stem Cells 2009;27:1347–1357.

26 Zhang J, Huang XY, Ye ML et al. Neuronal nitric oxide synthasealteration accounts for the role of 5-HT1A receptor in modulatinganxiety-related behaviors. J Neurosci 2010;30:2433–2441.

27 Sasaki M, Gonzalez-Zulueta M, Huang H et al. Dynamic regulation ofneuronal NO synthase transcription by calcium influx through aCREB family transcription factor-dependent mechanism. Proc NatlAcad Sci USA 2000;97:8617–8622.

28 Karacay B, Li G, Pantazis NJ et al. Stimulation of the cAMP pathwayprotects cultured cerebellar granule neurons against alcohol-inducedcell death by activating the neuronal nitric oxide synthase (nNOS)gene. Brain Res 2007;1143:34–45.

29 Biossel JP, Bros M, Schrock A et al. Cyclic AMP-mediated upregula-tion of the expression of neuronal NO synthase in human A673 neuro-epithelioma cells results in a decrease in the level of bioactive NOproduction: Analysis of the signaling mechanisms that are involved.Biochemistry 2004;43:7197–7206.

30 Gritti A, Galli R, Vescovi AL. Cultures of stem cells of the centralnervous system. In:Fedoroff S, Richardson A, eds. Protocols for Neu-ral Cell Culture. 3rd ed. Totowa: Humana Press, 2001:173–197.

31 Palmer TD, Markakis EA, Willhoite AR et al. Fibroblast growthfactor-2 activates a latent neurogenic program in neural stem cellsfrom diverse regions of the adult CNS. J Neurosci 1999;19:8487–8497.

32 Babu H, Cheung G, Kettenmann H et al. Enriched monolayer precur-sor cell cultures from micro-dissected adult mouse dentate gyrus yieldfunctional granule cell-like neurons. Plos One 2007;2:e388.

33 Price PJ, Brewer GJ. Serum-free media for neural cell cultures. In:Fe-doroff S, Richardson A, eds. Protocols for Neural Cell Culture. 3rded. Totowa: Humana Press, 2001:255–264.

34 Dasgupta B, Gutmann DH. Neurofibromin regulates neural stem cellproliferation, survival, and astroglial differentiation in vitro and invivo. J Neurosci 2005;25:5584–5594.

35 Song H, Stevens CF, Gage FH. Astroglia induce neurogenesis fromadult neural stem cells. Nature 2002;417:39–44.

36 Nagano T. Practical methods for detection of nitric oxide. Lumines-cence 1999;14:283–290.

37 Luo CX, Zhu XJ, Zhang AX et al. Blockade of L-type voltage-gatedCa2þ channel inhibits ischemia-induced neurogenesis by down-regulat-ing iNOS expression in adult mouse. J Neurochem 2005;94:1077–1086.

38 Babu BR, Griffith OW. N5-(1-Imino-3-butenyl)-L-ornithine: A neuro-nal isoform selective mechanism-based inactivator of nitric oxide syn-thase. J Biol Chem 1998;273:8882–8889.

39 Freeman SE, Patil VV, Durham PL. Nitric oxide-proton stimulation oftrigeminal ganglion neurons increases mitogen-activated protein kinaseand phosphatase expression in neurons and satellite glial cells. Neuro-science 2008;157:542–555.

40 Pei DS, Song YJ, Yu HM et al. Exogenous nitric oxide negativelyregulates c-Jun N-terminal kinase activation via inhibiting endogenous

Luo, Jin, Cao et al. 2051

www.StemCells.com

NO-induced S-nitrosylation during cerebral ischemia and reperfusionin rat hippocampus. J Neurochem 2008;106:1952–1963.

41 Bredt DS, Hwang PM, Snyder SH. Localization of nitric oxidesynthase indicating a neural role for nitric oxide. Nature 1990;347:768–770.

42 Arbones ML, Ribera J, Agullo L et al. Characteristics of nitricoxide synthase type I of rat cerebellar astrocytes. Glia 1996;18:224–232.

43 Wang T, FitzGerald TJ, Haregewoin A. Differential expression of ni-tric oxide synthase in EGF-responsive mouse neural precursor cells.Cell Tissue Res 1999;296:489–497.

44 Torroglosa A, Murillo-Carretero M, Romero-Grimaldi C et al. Nitricoxide decreases subventricular zone stem cell proliferation by inhibi-tion of epidermal growth factor receptor and phosphoinostitide-3-ki-nase/Akt pathway. Stem Cells 2007;25:88–97.

45 Chen J, Tu Y, Moon C et al. The localization of neuronal nitric oxidesynthase may influence its role in neuronal precursor proliferation andsynaptic maintenance. Dev Biol 2004;269:165–182.

46 Matarredona ER, Murillo-Carretero M, Moreno-Lopez B et al. Nitricoxide synthesis inhibition increases proliferation of neural precursorsisolated from the postnatal mouse subventricular zone. Brain Res2004;995:274–284.

47 Cheng A, Wang S, Cai J et al. Nitric oxide acts in a positive feedback loopwith BDNF to regulate neural progenitor cell proliferation and differentia-tion in the mammalian brain. Dev Biol 2003;258:319–333.

48 Ciani E, Calvanese V, Crochemore C et al. Proliferation of cerebellarprecursor cells is negatively regulated by nitric oxide in newborn rat.J Cell Sci 2006;119:3161–3170.

49 Covacu R, Danilov AI, Rasmussen BS et al. Nitric oxide exposurediverts stem cell fate from neurogenesis towards astrogliogenesis.Stem Cells 2006;24:2792–2800.

50 Shibuke K. An electrochemical microprobe for detecting nitric oxiderelease in brain tissue. Neurosci Res 1990;9:69–76.

51 Carreira BP, Morte MI, Inacio A et al. Nitric oxide stimulates the pro-liferation of neural stem cells bypassing the epidermal growth factorreceptor. Stem Cells 2010;28:1219–1230.

See www.StemCells.com for supporting information available online.

2052 Bidirectional Regulation of Neurogenesis