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TISSUE-SPECIFIC STEM CELLS

Neuropeptide Y Promotes Neurogenesis in Murine Subventricular Zone

^aNeuroprotection and Neurogenesis in Brain Repair Group, Center for Neuroscience and Cell Biology, Faculty of Medicine, University of Coimbra, Coimbra, Portugal;
^bLaboratory of Neuropsychiatry, Department of Neuroscience and Pharmacology, University of Copenhagen and Rigshospitalet University Hospital, Copenhagen, Denmark

Key Words. Subventricular zone • Calcium imaging • Neuronal differentiation • Functional binding • Neurogenesis • Neuropeptide Y

Correspondence: Correspondence: João O. Malva, Ph.D., Center for Neuroscience and Cell Biology, Institute of Biochemistry, Faculty of Medicine, University of Coimbra, 3004-504 Coimbra, Portugal. Telephone: 351-239-112254; Fax: 351-239-822776; e-mail: jomalva{at}fmed.uc.pt

Received on January 17, 2008; accepted for publication on March 24, 2008.

First published online in STEM CELLS EXPRESS  April 3, 2008.

	ABSTRACT

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
Stem cells of the subventricular zone (SVZ) represent a reliable source of neurons for cell replacement. Neuropeptide Y (NPY) promotes neurogenesis in the hippocampal subgranular layer and the olfactory epithelium and may be useful for the stimulation of SVZ dynamic in brain repair purposes. We describe that NPY promotes SVZ neurogenesis. NPY (1 µM) treatments increased proliferation at 48 hours and neuronal differentiation at 7 days in SVZ cell cultures. NPY proneurogenic properties are mediated via the Y1 receptor. Accordingly, Y1 receptor is a major active NPY receptor in the mouse SVZ, as shown by functional autoradiography. Moreover, short exposure to NPY increased immunoreactivity for the phosphorylated form of extracellular signal-regulated kinase 1/2 in the nucleus, compatible with a trigger for proliferation, whereas 6 hours of treatment amplified the phosphorylated form of c-Jun-NH₂-terminal kinase signal in growing axons, consistent with axonogenesis. NPY, as a promoter of SVZ neurogenesis, is a crucial factor for future development of cell-based brain therapy.

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

	INTRODUCTION

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
Constitutive neurogenesis takes place in the rodent adult mammalian brain, and particularly in the subventricular zone (SVZ). SVZ harbors a population of stem cells that proliferate and give rise to neurons and glial cells [1, 2]. SVZ-derived neuroblasts migrate long distances along the rostral migratory stream toward the olfactory bulb, where they functionally differentiate into GABA and dopaminergic interneurons, involved in odor discrimination [3, 4]. Interestingly, following cerebral injuries such as ischemia, epileptogenesis, or focal neuronal degeneration, SVZ neurogenesis increases, and SVZ-derived neurons are able to repopulate damaged areas [5–8]. Therefore, SVZ cells represent a source of repairing cells with the potential to be recruited through endogenous stimulation of neurogenesis or grafted following in vitro manipulation. Therefore, it is critical to identify proneurogenic factors able to promote neuronal progenitor expansion and neuronal differentiation.

Neuropeptide Y (NPY), a 36-amino acid peptide abundantly and widely distributed in the brain, regulates a wide range of physiological processes, including food intake, sexual behavior, and mood [9–12]. At the cellular level, NPY displays neuroprotective properties in the striatum and the hippocampus [13–15] and modulates neuronal activity [16, 17], inhibiting hippocampal hyperactivity in epilepsy; it has therefore has been proposed as an endogenous anticonvulsant [18–21].

Recently, NPY has been shown to promote neurogenesis in the dentate gyrus (DG) of the hippocampus and the olfactory epithelium (OE) of the rodent brain [22–26]. Indeed, in the DG, NPY, secreted by interneurons, promotes the proliferation of neuronal progenitor cells and, thereby, the production of new granule neurons [26]. In the OE, olfactory receptor neurons are constantly regenerated from a population of basal cells that retain stem cell characteristics [22, 27]. In this system, NPY locally produced by sustentacular cells, another cell type composing the OE, has been shown to stimulate proliferation of the basal cells [24]. In both systems, the proneurogenic effects have been shown to be mediated through Y1 receptor activation [24, 26]. Together, these recent data convincingly demonstrate that NPY supports neurogenesis in the DG and the OE. However, very little is known about the action of NPY on SVZ neurogenesis.

In the present work we investigated the biological effects of NPY on neurogenesis in SVZ. Our data clearly show that NPY is an inducer of neurogenesis in SVZ cells, providing new perspectives and molecular targets for the development of new treatments for neurodegenerative diseases and brain injuries.

	MATERIALS AND METHODS

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
All experiments were performed in accordance with NIH and European (86/609/EEC) guidelines for the care and use of laboratory animals.

SVZ Cultures
SVZ cells were cultured from 1–3-day-old C57Bl/6 donor mice. Fragments of SVZ were dissected out from 450-µm-thick coronal brain sections, digested in 0.025% trypsin and 0.265 mM EDTA (Gibco, Rockville, MD, http://www.invitrogen.com) in Hanks' balanced saline solution (Gibco), and dissociated by gentle trituration with a P1000 pipette. The cell suspension was diluted in serum-free culture medium (SFM) composed of Dulbecco's modified Eagle's medium/Ham's F-12 medium with GlutaMAX-I (Gibco) supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin (Gibco), 1% B27 (Gibco), 10 ng/ml epidermal growth factor (Gibco), and 10 ng/ml basic fibroblast growth factor (Gibco). Single cells were then plated on uncoated Petri dishes at a density of 3,000 cells per cm². The neurospheres were allowed to develop in a 95% air-5% CO₂ humidified atmosphere at 37°C.

Six- to 8-day-old neurospheres were adhered for 48 hours onto poly-D-lysine-coated glass coverslips in SFM devoid of growth factors. Then, the neurospheres were allowed to develop for 48 hours or 7 days at 37°C in the absence or in the presence of either 1 µM NPY, 1 µM Y1 receptor agonist ([Leu³¹, Pro³⁴]-NPY), 300 nM Y2 receptor agonist (NPY13–36), 1 µM Y5 receptor agonist (NPY [19–23]-[Gly¹, Ser³, Gln⁴, Thr⁶, Ala³¹, Aib³², Gln³⁴]-PP), or 1 µM NPY with 1 µM Y1 receptor antagonist (BIBP3226) (all from Bachem AG, Bubendorf, Switzerland, http://www.bachem.com).

Single-Cell Calcium Imaging
To functionally characterize neuronal differentiation in SVZ cells, variations of intracellular calcium concentration ([Ca²⁺]_i) following stimulation with 50 mM KCl and 100 µM histamine (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) were analyzed. KCl depolarization causes the increase of [Ca²⁺]_i in neurons, whereas stimulation with histamine increases [Ca²⁺]_i in stem/progenitor cells [28].

SVZ cultures were loaded for 40 minutes at 37°C with 5 µM Fura-2 AM (Molecular Probes, Eugene, OR, http://probes.invitrogen.com), 0.1% fatty acid-free bovine serum albumin (BSA), and 0.02% pluronic acid F-127, in Krebs buffer (132 mM NaCl, 1 mM KCl, 1 mM MgCl₂, 2.5 mM CaCl₂, 10 mM glucose, 10 mM HEPES, pH 7.4). After a 10-minute postloading period at room temperature, the coverslip was mounted on RC-20 chamber in a PH3 platform (Warner Instruments, Hamden, CT, http://www.warneronline.com) on the stage of an inverted Axiovert 200 fluorescence microscope (Carl Zeiss, Göttingen, Germany, http://www.zeiss.com). Cells (approximately 100 cells per field) were continuously perfused with Krebs and stimulated by applying 100 µM histamine or high-potassium Krebs solution (containing 50 mM KCl, isosmotic substitution with NaCl) by the mean of a fast-pressurized (95% air, 5% CO₂ atmosphere) system (AutoMate Scientific Inc., Berkeley, CA, http://www.autom8.com). [Ca²⁺]_i was evaluated by quantifying the ratio of the fluorescence emitted at 510 nm following alternate excitation (750 milliseconds) at 340 and 380 nm, using a Lambda DG4 apparatus (Sutter Instrument, Novato, CA, http://www.sutter.com) and a 510-nm band-pass filter (Carl Zeiss) before fluorescence acquisition with a x40 objective and a CoolSNAP digital camera (Roper Scientific, Tucson, AZ, http://www.roperscientific.com). Acquired values were processed using the MetaFluor software (Universal Imaging Corporation, Marlow, U.K., http://www.universal-imaging.co.uk). Histamine/KCl values for Fura-2 ratio were calculated to determine the extent of neuronal maturation in cultures. The percentage of cells displaying a neuronal-like profile was calculated on the basis of the histamine (Hist)/KCl ratio.

Immunocytochemistry
Cells were fixed for 30 minutes in 4% paraformaldehyde in phosphate-buffered saline (PBS); permeabilized in 0.25% Triton X-100 (Sigma-Aldrich); incubated overnight with the following primary antibodies: mouse monoclonal anti-microtubule-associated protein-2 (anti-MAP-2) antibody (1:200; Sigma-Aldrich), mouse monoclonal anti-neuronal nuclear protein (anti-NeuN) antibody (1:100; Chemicon, Temecula, CA, http://www.chemicon.com), rabbit polyclonal anti-phosphorylated form of stress-activated protein kinase (anti-P-SAPK)/c-Jun-NH₂-terminal kinase (JNK) (1:100), rabbit monoclonal anti-phosphorylated form of extracellular signal-regulated kinase 1/2 (anti-P-ERK1/2) (1:50), or mouse monoclonal anti-tau (1:500) (all from Cell Signaling Technology, Danvers, MA, http://www.cellsignal.com); and incubated for 1 hour at room temperature with the appropriate secondary antibodies: anti-rabbit IgG labeled with Alexa Fluor 488 or Alexa Fluor 594 (1:200) or anti-mouse IgG labeled with Alexa Fluor 594 (1:200) (all from Molecular Probes). Nuclei were counterstained with Hoechst 33342 (2 µg/ml in PBS containing 0.25% BSA; Molecular Probes). Preparations were mounted in DakoCytomation fluorescent medium (DakoCytomation, Carpinteria, CA, http://www.dakocytomation.com).

Proliferation Assay
5-Bromo-2'-deoxyuridine (BrdU; 10 µM; Sigma-Aldrich) was added in the last 4 hours of the culture session. BrdU was unmasked and labeled following successive passages in 1% Triton X-100, ice-cold 0.1 M HCl, and 2 M HCl at 37°C and in borate buffer (0.1 M Na₂B₄O₇·10H₂O, pH 8.5) and incubation with the primary rat monoclonal anti-BrdU antibody (1:50; Oxford Biotechnology, Raleigh, NC, http://www.immunologicalsdirect.com/index) overnight at 4°C and with the secondary anti-rat IgG labeled with Alexa Fluor 594 (1:200; Molecular Probes) for 1 hour at room temperature (RT). Nuclei counterstaining and mounting were performed as described previously.

Apoptosis Assay
The terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) method was used to detect apoptotic nuclei. Briefly, permeabilized cells were subsequently incubated in terminal deoxynucleotidyl transferase buffer (0.25 U/µl terminal transferase, 6 µM biotinylated dUTP, pH 7.5; Boehringer Mannheim, Mannheim, Germany, http://www.boehringer.com) for 1 hour 30 minutes at 37°C, in 300 mM NaCl and 30 mM sodium citrate buffer for 15 minutes, in avidin-biotin-peroxidase complex for 30 minutes (1:100; Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com), and in 30 mM Tris-HCl (pH 7.6) buffer containing diaminobenzidine chromogen (0.025%; Sigma-Aldrich), 0.08% NiCl₂, and 0.003% H₂O₂. The preparations were mounted in Depex (BDH, Poole, U.K., http://pt.vwr.com/app/Home). Transmission images were recorded using a digital camera coupled to an Axioskop microscope (Carl Zeiss).

Quantitative Polymerase Chain Reaction Analysis
Total cellular RNA from SVZ explants and SVZ neurospheres was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) according to the manufacturer's instructions. Purified RNA was treated with DNA-Free (Ambion, Huntingdon, U.K., http://www.ambion.com). Total RNA (1 µg) was reverse transcribed using TaqMan RT master mix (Applied Biosystems, Naerum, Denmark, http://www.appliedbiosystems.com) and random hexamers in a 100-µl reaction on a PTC-200 DNA engine Thermal Cycler (VWR International, Albertslund, Denmark). Relative quantification of the cDNA was performed in 96-well plates on the iCycler (Bio-Rad, Grenaa, Denmark, http://www.bio-rad.com) programmed for 2 minutes at 50°C, 3 minutes at 95°C, and 40 cycles of 15 seconds at 95°C and 1 minute at 60°C. Each well contained 2 µl of cDNA mixed with 10 µl of SYBR Green I master mix (Bio-Rad), 4.8 µl of water, 0.2 µl of uracil-N-glycosylase (0.1 U/µl; Epicentre Biotechnologies, Madison, WI, http://www.epibio.com), and 750 nM forward and reverse primers (MWG Biotech, Ebersberg, Germany, http://www.mwg-biotech.com). All samples were run in duplicate. Collected data were analyzed using the iCycler software, and a cycle threshold (Ct) for each sample was determined. Relative quantification was achieved by subtracting each Ct sample with the in-plate Ct of the reference control gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Using the - method, we also determined fold mRNA expression of Y2, Y5, and NPY compared with Y1 mRNA. Regulations were verified using RNA polymerase II as a reference gene. A group of reference genes was tested, and GAPDH and RNA polymerase II were found to be the least regulated genes. Water controls and genomic DNA controls were run simultaneously with the samples on all plates and did not differ significantly from background. The following primer sequences were used: Y1 (forward, GCT TAT GGG GCG GTG ATT ATT CT; reverse, ACC GCA ACG AGC AAG TCT GAG), Y2 (forward, AAG TGG CCT GGG GAA GAG AAG AGT; reverse, GAG GCA AAA CGT ACA GGA TGA GCA), Y5 (forward, CAA CCT GGC CTT CTC CGA CAT; reverse, GCT TTG CCG AAC ATC CAC TGA), NPY (forward, CTC TGC GAC ACT ACA TCA ATC TCA TCA; reverse, GGG CGT TTT CTG TGC TTT CCT T), GAPDH (forward, TGC ACC ACC AAC TGC TTA G; reverse, GGA TGC AGG GAT GAT GTT C), and RNA polymerase II (forward, TGC GCA CCA CGT CCA ATG ATA; reverse, GGA GCG CCA AAT GCC GAT AA). The efficiency of all primers was between 90% and 110%.

Y1 Agonist-Stimulated [³⁵S]-GTPS Functional Binding
The functional binding method was described previously [19, 29]. Brains of six naïve adult C57Bl/6 mice (25–35 g; Taconic, Lille Skensved, Denmark, http://www.taconic.com) were quickly removed following decapitation, frozen in cold isopentane, and stored at –80°C until use. The brains were coronally sliced, using a cryostat (Thermo Shandon Inc., Pittsburgh, http://www.thermo.com), at 15 µm at the level of the SVZ: +0.98 to +0.02 mm relative to bregma [30]. Sections were incubated in assay buffer A (50 mM Tris-HCl, 3 mM MgCl₂, 0.2 mM EGTA, 100 mM NaCl, pH 7.4) for 10 minutes at RT and preincubated in assay buffer B (assay buffer A + 0.2 mM dithiothreitol, 1 µM 1,3-dipropyl-8-cyclopentyl-xanthine [no. C-101, PerkinElmer Life and Analytical Sciences, Waltham, MA, http://www.perkinelmer.com], 0.5% wt/vol BSA, and 2 mM GDP [no. G7127; Sigma-Aldrich]) for 20 minutes at RT to shift all G-proteins into the inactivated state. Subsequently, incubation was performed in assay buffer B + 40 pM [³⁵S]-GTPS (1,250 Ci/mmol; NEG030H250UC; PerkinElmer) for 2 hours at 25°C with 10^–5 M human [Leu³¹, Pro³⁴]-NPY (Bachem), a Y1 receptor agonist. Basal and nonspecific binding were determined by incubation in assay buffer B + 40 pM [³⁵S]-GTPS and in buffer B + 40 pM [³⁵S]-GTPS + 10 µM nonlabeled GTPS (no. 89378; Sigma-Aldrich), respectively. Specificity of the Y1 binding was confirmed by adding 10^–6 M BIBP3226 (no. E3620; Bachem) during the preincubation and incubation. Incubation was terminated by washing twice for 5 minutes in ice-cold 50 mM Tris-HCl buffer (pH 7.4). After a brief rinse in deionized H₂O, sections were dried under a cooled steam of air and exposed to Kodak Biomax MR films (Kodak, Rochester, NY, http://www.kodak.com) together with ¹⁴C-microscales (GE Healthcare, Hillerød, Denmark, http://www.gehealthcare.com) for 5 days at –20°C. The films were developed in Kodak GBX developer. Finally, the films were digitalized using a COHU, high-performance charge-coupled device camera, and quantifications were performed measuring the optical density using computer-assisted image analysis (Scion Image, NIH) calibrated to the ¹⁴C-microscale (nCi/g equivalent tissue). Means of absolute values in nCi/g minus basal binding values were determined.

Data Analysis
Fluorescence images were recorded using an LSM 510 Meta confocal microscope or an Axioskop 2 Plus fluorescence microscope (both from Carl Zeiss). In all the in vitro experiments, measurements were performed at the border of the neurospheres, where migrating cells emerged, forming a dense cell monolayer. In all in vitro experiments, each experimental condition was assayed in three different wells. Except where otherwise specified, the experiments were replicated three times. Percentages of BrdU-, TUNEL-, P-ERK1/2- (at 1 hour), and NeuN-immunoreactive cells were derived from cells counted in five independent microscopy fields in each coverslip with a x40 objective (approximately 200 cells per field). Quantifications of P-ERK1/2- and P-SAPK/JNK-positive nuclei at both 5 and 15 minutes were done in two independent cultures in at least 20 nonoverlapping fields (magnification, x40). Measurements of total length (micrometers) of the ramifications and quantification of the number of ramifications per neurosphere were done in approximately 20 nonoverlapping fields in each coverslip using digital images (magnification, x20) (n= 3 coverslips from three different cultures). In single-cell calcium imaging experiments, the percentage of neuron-like cells was calculated in a microscopy field containing approximately 100 cells in each coverslip and using a x40 objective. Statistical significance was determined using two-tailed Student's t test or one-way analysis of variance followed by Bonferroni-corrected post hoc t test for multiple comparisons. All data are presented as means ± SEM. Statistical significance level was set for p values <.05.

	RESULTS

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
NPY Promotes SVZ Cell Proliferation and Exerts No Effect on Cell Death
To investigate the effect of NPY on proliferation, SVZ neurospheres derived from newborn mice were treated for 48 hours and 7 days in the absence (control condition) or presence of 1 µM NPY. The thymidine analogue BrdU that incorporates in DNA in S-phase of the cell cycle was added for the last 4 hours of both culture sessions. Nuclei were then immunostained for BrdU, as shown in Figure 1A. NPY induced a significant increase in the percentage of BrdU-positive cells compared with control after 48 hours (control 48 hours, 6.0% ± 0.7%; NPY 48 hours, 9.1% ± 0.7%; p < .01; Fig. 1B). The effect was not maintained after 7 days (control 7 days, 4.8% ± 0.6%; NPY 7 days, 3.9% ± 0.3%), probably because of a shift from proliferation to neuronal differentiation (described below).

View larger version (39K): [in this window] [in a new window]   Figure 1. NPY promotes proliferation in subventricular zone (SVZ) culture but exerts no effect on cell death. (A): Representative confocal photos of SVZ cell nuclei in SVZ cell cultures maintained for 48 hours in the absence (control) or in the presence of 1 µM NPY and immunolabeled for BrdU (red nuclei). (B): Percentage of BrdU-immunostained nuclei. (C): Representative transmission photography of SVZ cell nuclei in a control SVZ culture stained using the Tunel method (dark nuclei). (D): Percentages of Tunel-stained nuclei in cultures maintained for 48 hours in the absence (control) or the presence of 1 µM NPY. (A, C): Mean percentages ± SEM of two independent experiments are represented. **, p < .01, using analysis of variance with Bonferroni's correction for comparison with SVZ control cultures exposed to normal medium for 48 hours. Abbreviations: BrdU, 5-bromo-2'-deoxyuridine; NPY, neuropeptide Y; Tunel, terminal deoxynucleotidyl transferase dUTP nick-end labeling.

Y1, Y2, and Y5 Receptors and NPY mRNAs Are Detected in the SVZ
To determine the expression of Y1, Y2, and Y5 receptors in the SVZ, quantitative polymerase chain reactions (q-PCRs) were performed in cDNAs from both neonatal SVZ neurospheres and adult SVZ explants. The Ct (i.e., the number of cycles necessary for a gene to be linearly expanded) was determined for each gene and normalized by subtracting each Ct sample from in-plate Ct of the housekeeping reference gene GAPDH. The smaller the Ct value the more the gene is expressed compared with in-plate tested genes.

In SVZ neonatal cultures and adult SVZ explants, the Y1 gene was found to be expressed at higher levels than Y5 and Y2, respectively (in neonatal cultures, normalized Ct_Y1, 13.7 ± 0.2; normalized Ct_Y5, 17.5 ± 0.6; normalized Ct_Y2, 17.8 ± 0.4; in adult SVZ explants, normalized Ct_Y1, 7.2 ± 0.7; normalized Ct_Y5, 8.5 ± 0.6; normalized Ct_Y2, 9.3 ± 0.5; Fig. 2A, 2B). NPY mRNA was also detected in both SVZ cultures and explants (normalized Ct_NPY, 15.0 ± 0.5 and 3.1 ± 0.6, respectively; Fig. 2A, 2B).

View larger version (22K): [in this window] [in a new window]   Figure 2. Y1R, Y2R, and Y5R cDNA relative quantification in subventricular zone (SVZ). (A): Y1R, Y2R, and Y5R cDNA relative quantification in SVZ neonatal cultures. Ct values are means ± SEM of quantitative polymerase chain reactions (q-PCRs) in four different 8–10-day-old SVZ cultures. Fold expression compared with Y1 is shown as the inset. (B): Y1R, Y2R, and Y5R cDNA relative quantification in SVZ explants from adult mice. Ct values are means ± SEM of q-PCRs in at least eight mice for each receptor. All samples were run in duplicate. Fold expression compared with Y1 is shown as the inset. Reference gene cDNA: glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Normalization was achieved by subtracting Ct of GAPDH from Ct of target gene. Abbreviations: Ct, cycle threshold; NPY, neuropeptide Y; R, receptor.

View larger version (68K): [in this window] [in a new window]   Figure 3. Neuronal differentiation in control subventricular zone (SVZ) cultures and in cultures exposed to 1 µM NPY. (A–D): Immunodetection of microtubule-associated protein-2-positive (A, B) and neuronal nuclear protein (NeuN)-positive (C, D) neurons. (E): Percentage of NeuN-immunostained neurons. Shown are means ± SEM of two independent experiments. ***, p < .0001 using two-tailed Student's t test for comparison with SVZ control cultures. Abbreviation: NPY, neuropeptide Y.

SVZ cell cultures were loaded with the Fura-2AM calcium probe, perfused continuously for 15 minutes with Krebs solution, and subsequently stimulated for 2 minutes with 50 mM KCl and with 100 µM histamine, as shown in Figure 4A. Figure 4B–4D represents characteristic profiles of fluorescence records displayed by at least 20 cells of control, NPY-treated, and NPY/BIBP3226-treated cultures (Fig. 4B–4D, respectively). In control cultures, most of the cells responded to histamine but not to KCl, whereas in NPY-treated cultures, the scenario was completely different (Fig. 4B, 4C, respectively). Indeed, following 7 days of treatment, the percentage of neuronal-like cells increased significantly compared with control cultures, suggesting a proneurogenic effect of NPY (control, 6.8% ± 2.8%; NPY, 27.8% ± 6.4%; p < .01; Fig. 4E).

View larger version (24K): [in this window] [in a new window]   Figure 4. NPY induces neuronal differentiation in SVZ cultures through Y1 receptors. (A): Subventricular zone (SVZ) cultures were perfused continuously with Krebs solution for 15 minutes and stimulated for 2 minutes (from minute 5 to minute 7) with 50 mM KCl and for 2 minutes (from minute 10 to minute 12) with 100 µM Hist. (B–D): Representative single-cell calcium imaging profiles of the response of at least 20 cells in a nontreated culture (control) (B), a culture maintained 7 days with 1 µM of NPY alone (C), and a culture maintained concomitantly with 1 µM NPY and with 1 µM Y1 receptor antagonist (BIBP3226) (D). (E): Percentage of neuron-like responding cells in SVZ cultures maintained for 7 days in the absence (control) or in the presence of either 1 µM NPY, 1 µM Y1 receptor agonist ([Leu31 Pro34]-NPY), 300 nM Y2 receptor agonist (NPY13–36), 1 µM Y5 receptor agonist (NPY[19–23]-[Gly1, Ser3, Gln4, Thr6, Ala31, Aib32, Gln34]-PP), or 1 µM NPY with 1 µM Y1 receptor antagonist (BIBP3226). Means ± SEM of four independent experiments are represented. **, p < .01, using one-way analysis of variance with Bonferroni's correction for comparison with SVZ control cultures. Abbreviations: Hist, histamine; NPY, neuropeptide Y; sec, seconds.

Evidence for Functional In Vivo Y1 Activity in the SVZ
Detection of Y1 receptor mRNA in adult SVZ explants (Fig. 2) together with the results obtained in vitro indicated that, in vivo, this receptor is present and functionally active in the SVZ. Evaluation of Y1 receptor function in the SVZ was performed in vivo, in adult mice brain slices, using [Leu³¹, Pro³⁴]-NPY-stimulated [³⁵S]-GTPS binding, a method that allows the detection of G-protein-coupled NPY receptor activation [19, 29].

Figure 5 shows pseudocolored autoradiograms of unstimulated (basal binding; Fig. 5A) and [Leu³¹, Pro³⁴]-NPY-stimulated (Fig. 5B) mouse brain slices, nonspecific binding (Fig. 5C), and after specific blocking of [Leu³¹, Pro³⁴]-NPY-stimulated [³⁵S]-GTPS binding by the Y1 antagonist BIBP3226 (Fig. 5D). A significant increase in [Leu³¹, Pro³⁴]-NPY-stimulated [³⁵S]-GTPS binding compared with basal binding was found in the SVZ ([Leu³¹, Pro³⁴]-NPY-stimulated, 222.6 ± 5.0 nCi/g; basal, 194.7 ± 2.8 nCi/g; p= .0005). For comparison, high levels of [Leu³¹, Pro³⁴]-NPY-stimulated [³⁵S]-GTPS binding were observed in the same slices in the neocortex (motor cortex, M1, layers I–III), consistent with a previous study [34] ([Leu³¹, Pro³⁴]-NPY-stimulated, 317.6 ± 18.9 nCi/g; basal, 188.7 ± 7.3 nCi/g; p= .0027). To test the specificity of the [Leu³¹, Pro³⁴]-NPY-stimulated [³⁵S]-GTPS binding, slices were also coincubated with the Y1 antagonist BIBP3226. Means of absolute values of radioactivity minus basal binding were calculated. BIBP3226 incubation completely abolished [Leu³¹, Pro³⁴]-NPY-stimulated [³⁵S]-GTPS binding ([Leu³¹, Pro³⁴]-NPY-stimulated, 27.9 ± 3.5 nCi/g; [Leu³¹, Pro³⁴]-NPY-stimulated + BIBP3226, 2.0 ± 2.6 nCi/g; p= .0018; Figure 5). Likewise, in the neocortex, an area known to contain high levels of functional Y1 receptors, BIBP3226 almost completely blocked [Leu³¹, Pro³⁴]-NPY-stimulated [³⁵S]-GTPS binding in the same slices ([Leu³¹, Pro³⁴]-NPY-stimulated, 128.9 ± 23.3 nCi/g; [Leu³¹, Pro³⁴]-NPY-stimulated + BIBP3226, 26.3 ± 13.4 nCi/g; p= .0029). Taken together, these results show that the SVZ neurogenic niche contains functional Y1 receptors.

View larger version (109K): [in this window] [in a new window]   Figure 5. Y1 receptor functional binding in the subventricular zone (SVZ). Pseudocolored autoradiograms show Y1 receptor functional binding in the SVZ of mice using [Leu31, Pro34]-NPY-stimulated [35S]-GTPS binding. (A): Basal binding. (B): Y1 agonist: [Leu31, Pro34]-NPY (10–5 M). (C): Nonspecific binding. (D): [Leu31, Pro34]-NPY (10–5 M) + BIBP3226 (10–6 M). (E): Magnification of dashed section in (A), corresponding to the SVZ. (F): Magnification of dashed section in (B). (G): Magnification of dashed section in (D). Scale bar= 1 mm. (H): Levels of [Leu31, Pro34]-NPY-stimulated [35S]-GTPS binding and specificity of Y1 binding in the SVZ of mice. Means ± SEM of absolute values minus basal binding values obtained in a total of six naïve mice. ***, p < .0005, using paired t test for comparison with [Leu31, Pro34]-NPY-stimulated [35S]-GTPS binding.

View larger version (39K): [in this window] [in a new window]   Figure 6. NPY binding activates the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. (A): Representative fluorescence confocal photos of the P-ERK1/2 (red and green) immunocytochemistry and Hoechst staining (blue nuclei), in control cultures and in cultures maintained for 5 minutes and 1 hour in the presence of 1 µM NPY. Arrows depict P-ERK1/2-positive nuclei (red) and cell cytoplasm (green). (B): Number of P-ERK1/2-positive nuclei per neurosphere. Measurements were done in at least five neurospheres in each coverslip. Means ± SEM of two independent cultures per condition are represented. **, p < .005 using two-tailed Student's t test for comparison with subventricular zone (SVZ) control cultures exposed to normal medium for 5 minutes. (C): Number of P-ERK1/2-positive cells, expressed as percentage of total number of cells, in control cultures and in cultures maintained for 1 hour in the presence of 1 µM NPY. Means ± SEM of three independent experiments are represented. *, p < .05 using two-tailed Student's t test for comparison with SVZ control cultures exposed to normal medium for 1 hour. Abbreviations: NPY, neuropeptide Y; P-ERK, phosphorylated form of extracellular signal-regulated kinase 1/2.

View larger version (42K): [in this window] [in a new window]   Figure 7. NPY binding activates the stress-activated protein kinase (SAPK)/c-Jun-NH2-terminal kinase (JNK) signaling pathway. (A–D): Representative fluorescence confocal photos of the phosphorylated form of stress-activated protein kinase (P-SAPK)/JNK (green) and Hoechst staining (blue nuclei) in cultures maintained for 6 hours in control medium (A) or in the presence of 1 µM NPY (B–D). (D): High-magnification fluorescence confocal photos of a growth cone-like structure of a P-SAPK/JNK-positive cell (C) in a subventricular zone (SVZ) culture treated with 1 µM NPY for 6 hours. (E, F): Total length (µm) of the ramifications per neurosphere (E) and number of ramifications per neurosphere (F). Means ± SEM are shown. Measurements were done in approximately 20 nonoverlapping fields in each coverslip using digital images (magnification, x20) (from three different cultures; total number of neurospheres counted was at least 20 neurospheres in each culture). *, p < .05; **, p < .01, using two-tailed Student's t test for comparison with SVZ control cultures. (G, H): Representative fluorescence confocal photographs of P-SAPK/JNK (green), tau (red), and Hoechst staining (blue nuclei) in control cultures (G) and in cultures maintained for 6 hours in the presence of 1 µM NPY (H). Abbreviation: NPY, neuropeptide Y.

	DISCUSSION

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

We also quantified functional neuronal differentiation using a method recently developed by our group, based on the measurements of [Ca²⁺]_i amplitude responses following stimulation with KCl or histamine [28]. Briefly, high KCl solution in neurons leads to an increase in [Ca²⁺]_I, whereas in immature SVZ cells or glial cells the [Ca²⁺]_i remained unchanged. On the other hand, perfusion with 100 µM histamine increases [Ca²⁺]_i in SVZ immature cells but neither in neurons nor in glial cells [28, 31–33]. The percentage of cells displaying a neuronal-like profile (i.e., with a Hist/KCl ratio below 0.8) was calculated [28]. Using this method, we showed that NPY increases the number of functional SVZ neurons. Moreover, this effect is mediated by Y1 receptors, since the increase in neuronal differentiation was reproduced using the Y1 receptor agonist [Leu³¹, Pro³⁴]-NPY but abolished in the presence of NPY and the Y1 antagonist BIBP3226.

Y1-mediated neurogenesis has been shown to occur in the OE and DG. Indeed, in DG and primary olfactory cell cultures, increase in neuronal proliferation is mimicked by incubation with Y1 selective agonists and inhibited in the presence of BIBP3226 [24, 26]. In addition, proliferation is decreased in the DG of Y1 receptor KO mice [26]. Moreover, when functional binding assays were performed in adult naïve mice, functional Y1 receptors were shown to be present in the SVZ, consistent with the concept that endogenous NPY may promote neurogenesis in vivo via Y1 receptors.

NPY triggered the phosphorylation of both the MAPKs, ERK1/2 and SAPK/JNK. Activation of the ERK pathway by NPY has already been shown in other neurogenic systems and has been correlated with NPY-induced neuroproliferation. In NPY-treated DG and primary olfactory cell cultures, inhibition of ERK1/2 phosphorylation or upstream kinase activation abolished the neuroproliferative effect of NPY [24, 26]. In our experimental model, increase in SVZ proliferation by NPY may, in part, be mediated through ERK1/2 phosphorylation, as ERK1/2 activation is required for SVZ cell proliferation [35]. Moreover, nuclear localization of P-ERK1/2 is correlated to proliferation, as P-ERK1/2 substrates are mainly transcription factors involved in the activation of gene expression related to cell-cycle progression [36–38]. After 1 hour of treatment with NPY, P-ERK1/2 is found in the cytoplasm, where it can phosphorylate neurofilament heavy chain proteins and is associated with microtubules regulating cytoskeleton organization and neuronal function [38–40].

NPY was found to increase P-SAPK/JNK immunoreactivity first in the nucleus and then in the cytoplasm. This is consistent with a previous study where NPY induced phosphorylation of SAPK/JNK in synaptosomes prepared from dentate gyrus [41]. SAPK/JNKs appear to play an important role in neural differentiation and maturation. Indeed, in the nucleus, P-SAPK/JNK phosphorylates transcription factors, such as c-jun, involved in expression of genes related to neuronal differentiation [38, 42]. The JNK pathway is involved in C17.2 neural progenitor cell differentiation induced by interferon- [43], and JNK1 is required for neurogenesis in mouse embryonic bodies [44]. In the present study, after 6 hours of NPY treatment, P-SAPK/JNK was found in the cytoplasm, in neurite-like structures, colocalizing with tau, a microtubule-associated protein mainly present in axons. Moreover, NPY treatment increased the total length and number of P-SAPK/JNK-positive ramifications. Indeed, the SAPK/JNK pathway is involved in neurite outgrowth and axonogenesis [42, 45]. JNK3 expression, for instance, significantly increases the number and length of neurites in NGF-induced neuronal differentiation of PC12 cells [46]. JNK1 is responsible for MAP-2 phosphorylation and regulates microtubule assembly [47]. Consistent with our results, P-SAPK/JNK immunoreactivity is associated with tau-positive axons and growth cones in rat embryonic day 18 hippocampal neurons [45]. Together, these data suggest that NPY promotes neuronal differentiation and axonal sprouting through activation of the SAPK/JNK pathway.

Previous studies on the OE and the DG, together with the results shown in the present work in the SVZ, highlight the proneurogenic properties of NPY [24–26]. NPY is secreted locally in the DG by interneurons and in the OE by sustentacular cells. In this study, NPY mRNA was detected in both SVZ neurospheres and brain explants. In vivo, NPY may be secreted by SVZ cells acting locally in an autocrine/paracrine manner to stimulate both proliferation and neuronal differentiation. Indeed, the SVZ microenvironment, where stem and progenitor cells reside, is endowed with factors regulating neurogenesis, referred to as the neurogenic niche [48]. For instance, explants of adult SVZ secrete soluble factors that enhance proliferation, cell survival, and neuronal differentiation in SVZ cell cultures [49], and NPY may be part of the SVZ soluble factor repertoire. To further support this hypothesis, it has recently been shown that proliferation is reduced in the caudal part of the SVZ of Y1 knockout mice, suggesting that endogenous secretion of NPY promotes proliferation in normal mice [50]. Although neighboring striatum contains NPYergic neurons [51], SVZ endogenous production of NPY may contribute to basal neurogenesis. Moreover, in single-cell calcium imaging studies, the number of neurons in cultures incubated with both NPY and BIBP3226 was found to be slightly lower than in control conditions, probably because of the blocking of the endogenous and exogenous effect of NPY by BIBP3226.

	CONCLUSION

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
The present work highlights for the first time the proneurogenic properties of NPY in the SVZ, the major source of repairing cells in the mammalian brain. The use of NPY, as a neurogenesis activator, opens new perspectives for the development of SVZ cell-based therapies for brain repair.

	DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
We thank Dr. Luisa Cortes for help and expertise in confocal microscopy. This work was supported by FCT Portugal and by FEDER, POCI/NSE/58492/2004, PTDC/SAU-NEU/68465/2006, FRH/PBD/26462/2006, SFRH/BD/12731/2003, Danish National Research Council Grant 64750, the Lundbeck Foundation, Friis Foundation, and Elsass Foundation.

	FOOTNOTES

 
Author contributions: F.A.: conception and design, provision of study material, collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript; L.B.: provision of study material, collection and/or assembly of data, data analysis and interpretation; H.K. and S.H.C.: collection and/or assembly of data, data analysis and interpretation; R.F., B.S., and S.G.: collection and/or assembly of data; D.P.D.W.: conception and design, financial support, data analysis and interpretation, manuscript writing, final approval of manuscript; J.O.M.: conception and design, financial support, data analysis and interpretation, administrative support, manuscript writing, final approval of manuscript.

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Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 

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