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JBC Papers in Press. Published on March 26, 2008 as Manuscript M800053200 The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M800053200 LEPTIN PROMOTES ADULT HIPPOCAMPAL NEUROGENESIS IN VIVO AND IN VITRO Jacob C. Garza, Ming Guo, Wei Zhang and Xin-Yun Lu Department of Pharmacology, University of Texas Health Science Center, San Antonio, TX 78229 Running Title: Leptin and neurogenesis Address correspondence to: Xin-Yun Lu, M.D., Ph.D., Department of Pharmacology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229, Phone: 210-5670803, E-mail: lux3@uthscsa.edu Leptin, an adipose-derived hormone, has been implicated in several physiological processes involving the hippocampus. However, the role of leptin in adult hippocampal neurogenesis remains unknown. Here we show that leptin promotes neurogenesis in the dentate gyrus of adult mice as well as in cultured adult hippocampal progenitor cells. Chronic administration of leptin to adult mice increased cell proliferation, but had no significant effect on differentiation and survival of newly proliferated cells in the dentate gyrus. The expression of the leptin receptor, LepRb, was detected in hippocampal progenitor cells by RT-PCR and immunohistochemistry. Leptin treatment also promoted proliferation of cultured adult hippocampal progenitor cells. Analysis of signal transduction pathways revealed that leptin stimulated phosphorylation of Akt and STAT3, but not Erk1/2 . Furthermore, pretreating the cells with specific inhibitors of Akt or STAT3 attenuated leptin-induced cell proliferation in vitro in a dose-dependent manner. Taken together, our results provide evidence that leptin regulates adult hippocampal neurogenesis and suggest that activation of Akt and STAT3 signaling pathways may underlie the mechanism of action of leptin in neurogenesis. INTRODUCTION The dentate gyrus of the hippocampus is one of the two brain regions where adult neurogenesis persists throughout life. Adult neurogenesis is regulated by physiological and pathological events and modulated by pharmacological manipulations at any of three primary stages: cell proliferation, differentiation and survival. Neurogenesis in the dentate gyrus has been found to be negatively influenced by stress and is suppressed in various animal models of depression (1-3). Conversely, new neuron generation in the dentate gyrus is stimulated by treatment with antidepressants (4,5). Also, neurotrophins, growth factors, and cytokines have been shown to be capable of modulating neurogenesis of the adult dentate gyrus (6-8). Leptin is an adipocyte-derived cytokine encoded by the obese (ob) gene. It circulates as a 16-KDa peptide and is transported into the brain via a saturable transport system (9-11). Leptin signals in the brain by binding to the long form leptin receptor (LepRb), a Type I cytokine receptor, which results in Janus kinase 2 (Jak2)mediated phosphorylation of two tyrosine residues (Tyr985 and Tyr1138) in the cytoplasmic tail of the receptor (12). Phosphorylated Tyr985 of LepRb recruits SH2-containing proteintyrosine phosphatase-2, leading to activation of the the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway (13,14). Phosphorylated Tyr1138 of LepRb activates signal transducer and activator of transcription 3 (STAT3), which dimerizes and is translocated to the nucleus where it acts as a transcription factor (12). Jak2 also phosphorylates insulin receptor substrate-1 and 2 (IRS1/2), resulting in activation of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway (15,16). Leptin is well known for its role in the control of food intake and body weight, which is believed to be mediated via interaction with LepRb in the hypothalamus (17). Recent findings demonstrate that leptin facilitates spatial learning and memory (18-20) and produces antidepressant-like effects (21,22). Adult neurogenesis has been proposed to mediate hippocampal-dependent learning and therapeutic actions of antidepressants (23-26). However, whether leptin regulates adult Downloaded from www.jbc.org by on May 18, 2008 1 Copyright 2008 by The American Society for Biochemistry and Molecular Biology, Inc. hippocampal neurogenesis remains unknown. In the present study, we investigated the impact of leptin on cell proliferation, differentiation and survival in the dentate gyrus of adult mice. We also characterized the possible signaling mechanisms by which leptin exerts its effects on neurogenesis in cultured adult hippocampal stem/progenitor cells. METHODS AND MATERIALS Animals- Male C57BL/6J mice, weighing 20-25 g, were purchased from Jackson Laboratories (Bar Harbor, ME). Animals were housed in groups of five, and maintained on a 12-h light/dark cycle (lights on at 07:00 h) with ad libtum access to food and water. Animals were habituated to the housing conditions for 710 days prior to the beginning of the experimental procedures. All procedures were carried out in accordance with the National Institutes of Health Guide. Drugs- Recombinant rat leptin (R&D Systems, Minneapolis, MN) was dissolved in sterile saline (for in vivo experiments) or in cell culture medium (for in vitro experiments). Akt inhibitor VIII (1,3-Dihydro-1-(1-((4-(6-phenyl1H-imidazo[4,5-g]quinoxalin-7yl)phenyl)methyl)-4-piperidinyl)-2Hbenzimidazol-2-one), an isozyme-selective Akt inhibitor specific for Akt-1/2, and STAT3 inhibitor Cucurbitacin I I (27,28) were dissolved in dimethyl sulfoxide (DMSO) in a stock solution of 5 mg/mL (Calbiochem, La Jolla, Ca). Effect of leptin on cell proliferation and differentiation in the dentate gyrus of adult mice- Leptin at a dose of 1 mg/kg was used to study its effect on cell proliferation in the adult dentate gyrus of the hippocampus. This dose of leptin was chosen based upon its effectiveness in inducing antidepressant-like effects in rats (21) and mice (29). To study the time-course effects of leptin, 36 mice were used. Animals received i.p. injection of saline (n = 18) or leptin (1 mg/kg, n = 18), twice daily at the beginning and end of the light cycle. Body weight was measured daily during the period of treatment. Saline-treated and leptin-treated animals were assigned to three groups for 1, 5 or 14 days of treatment (n = 6/group). Subsequently, mice were injected with bromodeoxyuridine (BrdU), a thymidine analog that can be incorporated into the DNA during the S-phase of cell division, to label proliferating cells and track down the fate of newly proliferated cells. Animals received three i.p. injections of BrdU in total (50 mg/kg body weight in 0.1M PBS buffer, pH 7.2), spacing at 8-hour intervals over 24 hours. The time interval corresponds with the length of S phase of cell division reported in mice (approximately 6-9 h) (30,31). Animals were transcardially perfused 2 hours after the last BrdU injection for immunohistochemical evaluation of BrdU-labeled cells. To evaluate the effect of leptin on cell differentiation, ten mice received i.p. injection with saline (n = 5) or leptin (1 mg/kg, n = 5) for 14 days followed by BrdU labeling as described in Experiment 1. To track the fate of newly proliferated cells, animals were sacrificed 28 days after the last BrdU injection. Effect of leptin on cell survival in the adult dentate gyrus- In order to determine the effect of leptin on survival of newly proliferated cells, two groups of mice (n = 6/group) were administered with BrdU as described above (3 X 50 mg/kg, 8 h intervals) before treatment. The two groups of mice were treated with either saline or leptin (1 mg/kg, i.p.) for 14 days before trascardiac perfusion. Immunohistochemistry tissue preparation- Animals were anesthetized with an intramuscular (i.m.) injection of rabbit cocktail (43 mg/kg ketamine, 9 mg/kg xylazine and 1.4 mg/kg acepromazine in saline) and perfused through the ascending aorta using 0.1M PBS followed by 4% paraformaldehyde (PFA) in PBS. The brain was removed and fixed overnight in 4% PFA, and then transferred to 30% sucrose in PBS. Brains were cut into 40 M coronal sections on a cryostat and stored in cryoprotectant (30% sucrose, 30% ethylene glycol, 1% polyvinyl pyrrolidone, 0.05M sodium phosphate buffer) until processing for immunohistochemistry. BrdU immunostaining- The brain sections from the same experiment were processed for BrdU immunostaining simultaneously in order to minimize staining variations within and between groups. Since BrdU is incorporated into the DNA, the sections were first pretreated to denature DNA in order to Downloaded from www.jbc.org by on May 18, 2008 2 make it assessable to BrdU antibody. Brain sections were incubated in a solution containing 50% formamide and 50% 2X SSC for 2 hours at 65C. Then, the sections were briefly rinsed in PBS buffer, treated with 1% hydrogen peroxide for 10 min, and incubated in 2N HCl solution with 0.3% Triton X-100 for 30 min followed by incubation with 0.1M boric acid for 10 min. Subsequently, the sections were blocked in a solution containing 0.3% Triton X-100 and 3% normal goat serum in PBS for 1 h, and then incubated with mouse monoclonal anti-BrdU antibody (1:200, Roche, Indianapolis, IN) in the blocking solution overnight at 4 C. After rinsing in PBS buffer, the sections were then incubated with biotinylated goat anti-mouse antibody in blocking solution (1:400) for 1 h at room temperature, followed by incubation with avidin-biotin complex (1:100, Vector Laboratories, Burlingame, CA) for 1 h. BrdU immunoreactivity was revealed using 3��iaminobenzidine (DAB; Vector Laboratories, Burlingame, CA). Double fluorescence immunolabelingTo determine the phenotypes of BrdU-labeled cells, brain sections were processed for BrdU, neuronal nuclei (NeuN) or anti-glial fibrillary acidic protein (GFAP) fluorescent double labeling. Briefly, sections were first pretreated for DNA denaturation as described above, and then incubated with rat-anti-BrdU antibody (1:200, Accurate Chemical, Westburg, NY) and mouse anti-NeuN (1:400, Chemicon, Temecula, CA) or rabbit anti-GFAP (1:1000, Chemicon, Temecula, CA). After washing in PBS (0.1 M) buffer, sections were incubated for 4 h with fluorescent secondary antibodies: Alexa Fluor 488 goat anti-rat IgG to reveal immunoreactivity of BrdU and Alexa Fluor 546 goat anti-mouse IgG or Alexa Fluor 546 goat anti-rabbit IgG to reveal immunoreactivity of NeuN or GFAP, respectively (1:400 for all three antibodies, Molecular Probes, Eugene, ON). Then, the sections were washed in PBS and mounted onto coated glass slides and coverslipped with fluorescent mounting medium. Cell quantification and analyses- The coronal tissue sections were mounted on polylysine coated glass slides and were numerically coded, and stereological counts were performed by experimenters blinded to the experimental condition of each sample. The brain was sectioned at 40 m and every sixth section spaced 240 m apart throughout the entire rostral/caudal extent of the hippocampus (from Bregma -1.06 to Bregma -3.80) was used to assess the number of BrdU-labeled cells within the sub-granular zone (SGZ) along the superior and inferior blades of the dentate gyrus. This strategy ensures that the same BrdU-labeled cell will not be counted twice on adjacent sections and also guarantees that the area of the dentate gyrus counted for each animal remains consistent. BrdU-labeled cells were counted in sub granular zone which is defined as the border between the granule cell layer and the hilus. Counting was accomplished at 400��magnification using an Olympus BX51 microscope equipped with a 40��objective lens. This allowed clear visualization of each cell and enabled us to distinguish single cells from clusters. Cells were counted throughout each section by focusing through each focal plane of the section to ensure that all BrdU-labeled cells were visible. The number of BrdU-labeled cells was multiplied by 6 to obtain the total number of cells per dentate gyrus. Double-labeled cells for BrdU and NeuN or GFAP were visualized and counted with a 60x oil immersion objective using a Zeiss 510 confocal and multiphoton microscope. The number of BrdU-labeled cells that expressed NeuN or GFAP was determined by counting a minimum of 35 BrdU-labeled cells on the sections spanning the entire dentate gyrus as described previously. Images were captured with a 60��oil immersion lens on an Olympus FV500 confocal microscope using Fluoview Imaging Software (Olympus, Tokyo, Japan). The extent of co-localization was validated by viewing cells on three planes (X, Y, and Z) using Z-plane sectioning. Cells single-labeled for BrdU or double-labeled for BrdU/NeuN or BrdU/GFAP were counted. The percentage of BrdU cells double-labeled for NeuN or GFAP was calculated by dividing the number of doublelabeled cells by the total number of BrdU cells and multiplying 100. Cell culture- Adult rat hippocampal stem/progenitor cells (Chemicon, Temecula, Ca) were grown in neural expansion media containing Dulbecco�� modified Eagle�� Downloaded from www.jbc.org by on May 18, 2008 3 medium (DMEM), and F-12 Nutrient Mixture, 1��B-27 supplement, 2 mM L-glutamine, 1��solution of penicillin and streptomycin, and FGF-2 (20 ng/mL) in 100 mm polystyrene tissue culture dishes or 8 well chamber slides coated with poly-L-ornithine (10mg/ml) and mouse laminin (5 g/ml). The cultured cells were maintained in a 37C humidified incubator with 5% CO2 and complete change of media containing fresh FGF-2 every other day and passaged once every 5-6 days. Expression of LepRb in adult rat hippocampal stem/progenitor cellsTo determine whether LepRb transcript is expressed in adult rat hippocampal stem/progenitor cells, reverse transcription PCR (RT-PCR) was performed. Briefly, adult hippocampal stem cells were collected and total RNA was extracted. The extracted RNA was subsequently used to generate cDNA using SuperScript First Strand Synthesis System for RT��CR. A 572 bp fragment corresponding to the c-terminal region of LepRb was amplified using the primers LepRb Forward (5��TGACCAGTGTAACAGTGCTAAC-3�� and LepRb Reverse (5��GGGCATGTAAGGTACAAAG-3��. The RTPCR reaction conditions consisted of an initial denaturing stage at 94��for 4 minutes followed by 35 cycles at 94��for 30 seconds, 50��for 45 seconds, and 72��for 1 minute. A final elongation step at 72��for 10 minutes was added to the end of the program. The PCR product was visualized by electrophoresis on a 1% agarose gel. Immunocytochemistry was employed to detect protein expression of LepRb in adult hippocampal stem/progenitor cells. Cells were plated on glass coverslips in 24 well plates and grown in neural expansion media containing FGF-2. After 24 h the cells were fixed in 4% PFA, rinsed in PBS, and processed for immunocytochemistry for LepRb. Blocking solution (0.3% Triton X-100, 1% bovine serum albumin, and 3% normal goat serum in PBS) was added to each well for 1 h after which rabbit anti-LepRb (1:100, Linco, St. Charles, MO) and mouse anti-Nestin (1:1000, Chemicon, Temecula, CA) in blocking solution were added and incubated overnight at 4 C. After rinsing in PBS buffer, the cells were then incubated with Alexa Fluor 546 goat anti-rabbit and Alexa Fluor 488 goat anti-mouse (1:400 for both antibodies, Molecular Probes, Eugene, OR) secondary antibody for 4 h. The coverslips were washed and mounted on glass slides with ProLong Gold antifade reagent containing 4��,6-diamidino-2-phenylindole (DAPI) (Invitrogen, Carlsbad, CA). Effect of leptin on cell proliferation and differentiation in adult hippocampal stem/progenitor cell culture- Cell proliferation and differentiation experiments were carried out in 8 well chamber slides. For cell proliferation studies, each well of the chamber slide was seeded with 10,000 cells in 500 l of neural expansion media containing FGF-2. After 24 h, the media was replaced with fresh media with or without leptin for 48 h. Fresh media containing BrdU (10 M) was added for 4 h after which the cells were immediately fixed in 4% PFA. Cell proliferation was determined by performing immunocytochemistry for BrdU and counting the number of BrdU labeled cells in each well. For each well, eight random fields were chosen under the 20X objective and the total number of BrdU-labeled cells was counted. Cell differentiation experiments were carried out similar to cell proliferation experiments with some modifications. Cells were seeded in 8 well culture slides with neural expansion media containing FGF-2. The media was replaced 24 h later with either fresh neural expansion media without FGF-2 or fresh neural expansion media without FGF-2 containing leptin for 48 h after which media containing BrdU (10 M) was added for 4 h. FGF-2 was removed from the media in order to allow differentiation of neural precursors. Cells were maintained with fresh changes of media every other day for 8 additional days and then fixed with 4% PFA. Neural differentiation was determined by performing double-labeling immunocytochemistry for BrdU and the neuronal marker TuJ1 or the glial marker GFAP. For BrdU detection, the fixed cells were incubated in 2N HCl with 0.3% Triton X-100 for 30 minutes followed by 0.1M boric acid (pH 8.0) for 10 minutes and three rinses with PBS prior to incubation blocking solution (0.3% Triton X-100, 1% bovine serum albumin, and 3% normal goat serum in PBS) for 1 h. Next, the Downloaded from www.jbc.org by on May 18, 2008 4 cells were incubated with a mouse monoclonal anti-BrdU antibody (1:400) in the blocking solution overnight at 4 C. After three PBS rinses, the Alexa Fluor 488 goat anti-mouse secondary antibody (1:400) was applied in blocking solution for 4 h at room temperature. The cells were rinsed in PBS three times and visualized using a fluorescent microscope. For double immunocytochemical detection with antibodies against BrdU, TuJ1 or GFAP, the procedure was performed as described for BrdU detection except two primary antibodies were added simultaneously for incubation overnight. Effect of leptin on phosphorylation of Akt, STAT3 and ERK1/2 in adult hippocampal stem/progenitor cells- Cells were plated on polyornithine and laminin coated 100 mm polystyrene plates at a density of 1 ��106 cells per plate and grown in neural expansion media for 24 h. The media was replaced with neural expansion media without FGF-2 for 24 h after which fresh media containing 0, 0.3 nM, or 3.0 nM leptin was added. The cells were collected after 15 minutes and total protein was extracted using lysis buffer (50 mM Hepes, pH 7.6, 1% Triton X-100, 150 mM NaCl, 20 mM Napyrophosphate, 20 mM ��glycerophosphate, 10 mM NaF) containing a mixture of phosphatase inhibitors (Leupeptin, Aprotinin, Naorthovanadate, PMSF, Ser/Thr phosphatase inhibitor cocktail, Tyr phosphatase inhibitor cocktail). The total protein concentration was assessed by the Bradford assay and 40 g of total protein was run on an SDS-PAGE gel. The proteins were then transferred to a nitrocellulose membrane and blocked in a solution of 1% dry milk and 0.1% Tween-20 in 1��tris buffered saline (TBS) and subsequently incubated in primary antibody (anti-Akt, anti-phosphorylated Akt on Thr308, anti-STAT3, anti-phosphorylated STAT3 on Tyr705 and anti-ERK1/2 and antiphosphorylated ERK1/2 on Thr202/Tyr204) (all antibodies from Cell Signaling, 1:1000) in a solution of 1% BSA and 0.1% Tween-20 in TBS overnight at 4�� After washing the membrane was incubated in secondary antibody conjugated to horseradish peroxidase (1:10,000) in blocking solution for 1 h. Next, the membrane was washed and the electrogenerated chemiluminescence (ECL) reaction solutions were added (solution 1: 0.1M Tris-HCl, luminol, p-coumaric acid; solution 2: 0.1M Tris-HCl, hydrogen peroxide) Effect of signal transduction inhibition on leptin�� actions on adult hippocampal stem/progenitor cell proliferation- Adult hippocampal stem/progenitor cells were seeded in 8 well chamber slides as described for proliferation studies. Twenty-four hours after plating, the cells were incubated with fresh media containing various concentrations of Akt inhibitor VIII or STAT3 inhibitor Cucurbitacin I for 4 h before the treatment with leptin or vehicle. Cells were grown for 48 h and then BrdU was added and incubated for 4 h. Statistical analyses- The results were expressed as mean ��SEM. Statistical analyses were performed with one-factor ANOVA for the time-course effect of leptin on cell proliferation, two-tailed student�� t-test for cell differentiation and survival studies, one-factor ANOVA with repeated measures for body weight, and twofactor ANOVA for the in vitro studies. StudentNewman-Keuls post hoc comparisons followed ANOVAs. P < 0.05 was considered statistically significant. RESULTS Effect of leptin on new cell proliferation in the dentate gyrus of adult mice- The timecourse effect of leptin on adult hippocampal cell proliferation was determined in mice at a dose (1 mg/kg body weight) that has been shown to be effective in inducing antidepressant-like effects (21,29). Cell proliferation was assessed by administration of BrdU at a relatively low dose (3 x 50 mg/kg over 24 h) in order to minimize toxic effects. This dose of BrdU was selected based on several previous studies that have demonstrated that repeated i.p. injections of 50 mg/kg is sufficient to label proliferating cells in mice without having toxic effects on proliferating cells in the dentate gyrus (30,3537). BrdU-labeled cells, often found in clusters, were distributed in the inner layer of the granular cell layer and the hilus of the dentate gyrus (Figure 1). The BrdU-labeled cells were counted in both subgranular cell layer and hilus of the dentate gyrus. The total number of BrdUlabeled cells in the dentate gyrus was not significantly affected by leptin treatment for 1 or Downloaded from www.jbc.org by on May 18, 2008 5 5 days as compared to their respective vehicletreated control groups (Figure 1). After 14 days of treatment, however, leptin significantly increased the number of BrdU-labeled cells in the dentate gyrus (Figure 1, P < 0.01), indicating enhanced cell proliferation. No BrdU-positive cells were observed in the paraventricular thalamus, a brain area adjacent to the dentate gyrus, where neurogenesis has not been reported to occur (Figure 1). Effect of leptin on cell differentiation and survival in the dentate gyrus of adult miceTo determine if leptin treatment could affect the fate of new-born cells, BrdU-labeled cells were analyzed 28 days after BrdU administration in mice treated with leptin or vehicle. This length of time is known to be sufficient for newly proliferated cells to differentiate into their mature phenotypes (38). The total number of BrdU-labeled cells remained significantly higher in the leptin-treated group than the vehicletreated group 28 days after BrdU administration (Figure 2A, P < 0.05). The extent of differentiation of survived BrdU-labeled cells was determined by double-labeling immunohistochemistry with antibodies against BrdU and the neuronal marker, NeuN, or the glial marker, GFAP (Figure 2B-D). Analysis of colocalization of BrdU with NeuN or GFAP using confocal microscopy indicated that the majority of BrdU-labeled cells possessed the neuronal phenotype, a low percentage of BrdUlabeled cells were labeled for GFAP (Figure 2B). The percentages of BrdU-labeled cells that were double-labeled for either NeuN or GFAP were not significantly altered by leptin treatment (P = 0.49 for NeuN, and P = 0.86 for GFAP). To determine the effect of leptin on survival of newly proliferated cells, BrdU was administered to mice to label proliferating cells before leptin treatment began. The survival of newly proliferated cells was evaluated by quantifying BrdU-labeled cells after 14 days of leptin or vehicle treatment. There was no significant difference in the number of survived BrdU-labeled cells between vehicle- (100 ��18.9%) and leptin- (131 ��28%) treated groups (P = 0.89). Effect of leptin on body weight- Body weight of the mice was measured throughout the course of leptin treatment. Body weight gain was significantly decreased by leptin treatment from day 3 after the initiation of leptin treatment [F(1,130) = 8.742, P = 0.01], and this effect persisted until the end of the experiment (Figure 3), suggesting that chronic intermittent administration of leptin did not develop tachyphylaxis to the actions of leptin. Expression of LepRb in adult hippocampal stem/progenitor cellsTo determine whether adult hippocampal progenitor cells express LepRb, we examined LepRb gene and protein expression in cultured adult hippocampal stem/progenitor cells by RT-PCR and immunocytochemistry. A 572 bp band corresponding to the C-terminal region of the LepRb was detected by RT-PCR (Figure 4A), indicating the presence of LepRb transcripts in adult hippocampal progenitor cells. The presence of LepRb in cultured hippocampal stem/progenitor cells was further confirmed by immunocytochemical staining with antibodies against LepRb and Nestin, an intermediate filament family member that has been used as a marker for progenitor cells (32-34) (Figure 4B). The immunoreactivity of LepRb was observed in the cytoplasma. Nucleus showed punctuate LepRb immunoreactivity. The LepRb-positive cells were also positive for Nestin (Figure 4B). No immunostaining was seen when primary antibody was omitted. Taken together, these results indicate the presence of both mRNA and protein of LepRb in adult hippocampal progenitor cells. Effect of leptin on cell proliferation and differentiation in adult hippocampal stem/progenitor cultureTo determine whether leptin has a direct role in cell proliferation, cultured adult hippocampal stem/progenitor cells were incubated with different concentrations of leptin (1-30 nM) prior to BrdU labeling. Analyses of the number of BrdU-labeled cells revealed an inverted-U shape of the dose-response relationship between leptin and cell proliferation (Figure 5A). The maximal effect of leptin was observed with the dose of 1 nM (148.3 ��5.4% relative to vehicle control). ANOVA indicated a significant effect of the treatment [F(4,18) = 4.5, P < 0.01]. Post hoc analyses showed that the number of BrdUlabeled cells was significantly increased following 1 nM and 3 nM of leptin, but higher Downloaded from www.jbc.org by on May 18, 2008 6 concentrations of leptin at 10 nM or 30 nM elicited no significant effects. It is possible that leptin at high concentrations is cytotoxic or binds to other receptors nonspecifically, which may consequently counteract leptin�� actions mediated through LepRb. To determine whether leptin affects cell differentiation in adult hippocampal stem/progenitor cells, cells were grown for 8 days following BrdU labeling without FGF-2 in the media to allow differentiation. Leptin treatment showed no significant effect on the percentages of BrdU-labeled cells that were positive for neuronal marker TuJ1 or the glial marker GFAP (Figure 6). These in vitro findings are consistent with the results from in vivo studies showing the effects of leptin on cell proliferation and differentiation in the dentate gyrus of mice (as shown in Figures 1 and 2). Activation of intracellular signal transduction pathways by leptin in adult hippocampal stem/progenitor culture- Leptin has been demonstrated to activate multiple signal transduction pathways, which requires phosphorylation. To determine the signaling pathways that can be activated by leptin in adult hippocampal stem/progenitors, phosphorylation of Akt on Thr308, STAT3 on Tyr705, and ERK1/2 on Thr202/Tyr204 was evaluated by Western blot following leptin treatment at different doses. Basal phosphorylation of Akt in cultured hippocampal progenitor cells was low. Leptin treatment for 15 min dose-dependently elevated the level of phosphorylated Akt (Figure 7A and 7B). STAT3 showed a moderate level of basal phosphorylation in cultured hippocampal progenitor cells, and leptin treatment for 15 min could further increased STAT3 phosphorylation (Figure 7C and 7D). In contrast, phosphorylated ERK1/2 was not significantly altered after leptin treatment at either dose (Figure 7E and 7F). These results suggest the PI3K/Akt and Jak2/ STAT3 signaling pathways are recruited by leptin in adult hippcampal progenitor cells. Effect of blockade of PI3K/Akt and Jak2/STAT3 signaling pathways on leptinstimulated adult hippocampal cell proliferationTo further determine whether activation of the PI3K/Akt signaling pathway mediates the action of leptin on cell proliferation, adult hippocampal stem/progenitor cells were exposed to different concentrations of the Akt inhibitor VIII followed by leptin treatment. Treating the adult hippocampal stem/progenitor cells with the Akt inhibitor VIII significantly reduced the number of BrdU-labeled cells in a dose-dependent manner when compared to the leptin-treated group [F(4,15) = 6.001; P < 0.01] (Figure 8). Treating the cells with different concentrations of STAT3 inhibitor Cucurbitacin I also reduced the number of BrdU-labeled cells when compared to leptin-treated cells as well as the control group [F(4,13) = 25.203; P < 0.0001] (Figure 9). These results suggest that both the PI3K/Akt and the JAK/STAT3 pathways are involved in the stimulatory effect of leptin on proliferation of neural progenitor cells. DISCUSSION Downloaded from www.jbc.org by on May 18, 2008 In the present study, we have demonstrated that leptin promotes adult hippocampal neurogenesis both in vitro and in vivo. The leptin-stimulated neurogenesis mainly resulted from increased cell proliferation since leptin showed no significant effect on cell differentiation and survival. Leptin signaling recruited the PI3K/Akt and Jak2/ STAT3 pathways in adult hippocampal stem/progenitor cells and inhibition of these signal transduction pathways led to the attenuation of leptin�� actions on proliferation of progenitor cells, suggesting that a mechanism dependent of Akt and STAT3 activation mediates leptin�� actions. Cell proliferation in the dentate gyrus was increased by chronic but not short-term or acute administration of leptin, as indicated by the changes in total number of BrdU-labeled cells after 1d, 5d or 14d of leptin treatment. This temporal profile of leptin�� actions on cell proliferation is similar to the findings with chronic treatment using different classes of existing antidepressants (26,39,40). Interestingly, we observed a tendency toward a decrease in the number of BrdU-positive cells in the dentate gyrus over treatment time in control animals. A similar finding was also shown in the study by Nakagawa et al. (40), in which the number of BrdU-positive cells in the dentate gyrus in control animals appeared to be reduced after chronic (14d) vehicle injection in comparison with acute (1d) injection. It is possible that the 7 repeated injection procedure is stressful to the animals, which could cause suppression of cell proliferation. In support of this, stress hormones and stress have been demonstrated to decrease cell proliferation in the dentate gyrus (41-44). While many newly proliferated cells in the dentate gyrus die shortly after birth, some of them survive and differentiate (38). Confocal microscopic analysis demonstrated that the majority of newly proliferated cells in the dentate gyrus differentiate into neurons, and a small percentage of new cells differentiate into glia 28 days after BrdU labeling. However, the percentage of neurons and glial cells out of the total BrdU-labeled cells did not differ between leptin-treated and vehicle-treated groups, indicating that differentiation of new cells in the dentate gyrus was not influenced by leptin treatment. Another stage of neurogenesis is survival of new-born cells. Recently, it has been reported that chronic intracerebroventricular infusion of leptin protects hippocampal neurons in the CA1 and CA3 from cell death induced by neuronal insults (45). To determine whether leptin affects the survival of newly proliferated cells in the dentate gyrus, BrdU was administered to animals prior to the onset of 2 weeks of leptin injections. Leptin exhibited no significant effect on survival of newly proliferated cells in the dentate gyrus. The differential effects of leptin on survival of mature neurons in CA1 and CA3 versus newborn neurons in the dentate gyrus of the hippocampus may imply that leptin�� actions on cell survival may be dependent on the developmental status of the neuron. Alternatively, a constant higher concentration of leptin in the brain may be required for inducing significant effects on the survival of new born cells in the dentate gyrus. Leptin is known to suppress appetite, increase energy expenditure, and reduce body weight gain (46-48). Given that dietary restriction and physical activity have been shown to enhance hippocampal neurogenesis (49-52), one possibility is that leptin�� effects on neurogenesis could be secondary to the changes in energy homeostasis. If this is the case, a temporal correlation between weight loss and neurogenic activity would be predicted. However, body weight gain was seen to be decreased without significant effect on hippocampal neurogenesis after 5 days of leptin treatment. Also, dietary restriction promotes neurogenesis by increasing survival of newly generated neuronal cells rather than increasing the proliferation (49,50), which is in contrast to the stimulatory effect of leptin on cell proliferation without obvious effect on differentiation. Moreover, it has been demonstrated that locomotor activity was not altered by leptin following either acute or chronic administration (21,53). These findings support that leptin�� effect on neurogenesis is dissociated from its impact in energy homeostasis. Nonetheless, the possibility of a contribution of leptin-induced negative energy balance following chronic administration (14 days) to its neurogenic effect in vivo can not be ruled out completely. Our in vivo results from cell proliferation, differentiation and survival studies in mice suggest that leptin-stimulated hippocampal neurogenesis is likely to be the consequence of increasing cell proliferation rather than modifying the fate of cells after they are generated. A similar neuroproliferative effect without changing cell differentiation was observed in cultured adult hippocampal progenitor cells. The detection of mRNA and protein of LepRb in these cells suggest that leptin regulates neurogenesis possibly via direct interaction with LepRb on hippocampal progenitor cells. LepRb has the full signaling capability and is able to stimulate multiple intracellular signal transduction pathways including PI3K-Akt, Jak2-STAT3 and ERK1/2 signaling pathways (54). To determine the signaling mechanisms responsible for the neuroproliferative effect of leptin, activation of the PI3K-Akt, Jak2-STAT3 and ERK1/2 pathways were examined by assessing phosphorylation of Akt, STAT3 and ERK1/2 in hippocampal stem/progenitor cell culture. We found that leptin dose-dependently increased phosphorylation of Akt and STAT3 but not ERK1/2. Based upon these results, we hypothesized that the PI3K-Akt and Jak2STAT3 signaling pathways may mediate leptininduced neural progenitor cell proliferation. Consistent with this, inhibition of Akt activity attenuated leptin-induced cell proliferation. Our Downloaded from www.jbc.org by on May 18, 2008 8 results are in agreement with the observation that PI3K-Akt transduces intracellular signals that control adult hippocampal neural progenitor cell proliferation (55,56). Overexpression of Akt increases cell proliferation, while expression of a dominant negative Akt inhibits proliferation (55,56). A question that remains to be addressed is how Akt mediates leptin�� actions on neurogenesis. It has been shown that Akt inhibits glycogen synthase kinase GSK-3b activation by inducing its phosphorylation (57). GSK-3b is known to negatively regulate Wnt/catenin signaling via phosphorylating -catenin, which mediates ��catenin degradation (58). Both in vitro and in vivo studies have recently identified Wnt/��catenin signaling as a important pathway in adult hippocampal neurogenesis involved in the control of proliferation of progenitor cells and neuronal differentiation (59,60). Based upon these findings, we speculate that Akt may mediate leptin�� effects on hippocampal progenitor cell proliferation through promoting the Wnt-��catenin signaling. The observation of inhibition of STAT3 by Cucurbitacin reversed leptin-induced increase in proliferation of hippocampal progenitor cells suggests a potential role of Jak2-STAT3 signaling. Studies have demonstrated that STAT3 is important for the maintenance of neural progenitors. Expression of a constitutively active form of STAT3 in neural progenitors derived from the embryonic mouse neocortex increases the number of neurospheres formed by progenitor cells (61). Conversely, deletion of STAT3 results in a decrease in the number of primary neurospheres (61). These findings support that STAT3 promotes proliferation of neural progenitor cells. However, the effect of STAT3 on differentiation fate of neural progenitor cells remains controversial. On one hand, it has been shown that neuronal differentiation is promoted by suppression of STAT3 (62) and inhibited by overexpression of this signaling molecule (61). On the other hand, activation of STAT3 has been reported to be associated with granulocyte colony-stimulating factor-stimulated neurogenesis (63). Also, STAT3 has been demonstrated to be required for the neurotrophic effects of ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) on developing sensory neurons (64). Therefore, further investigation is needed in order to clarify the role of STAT3 in neurogenesis. The ERK1/2 pathway has also been shown to play an important role in regulating proliferation of neural progenitors (65,66). However, ERK1/2 phosphorylation was not increased in response to leptin treatment. This is somewhat unexpected because ERK1/2 has been shown to be phosphorylated by the synthetic cannabinoid HU210, a stimulator of cell proliferation (67). The lack of leptin-induced activation of ERK1/2 could be due to a dephosphorylation mechanism that negatively regulates ERK1/2 phosphorylation. Alternatively, our results may suggest that the ERK1/2 pathway is not recruited by leptin signaling in mediating hippocampal neurogenesis. In summary, our results indicate that leptin increases the production of new neurons in the adult dentate gyrus. Analysis of signaling pathways in vitro suggests that leptin may promote proliferation of neural progenitor cells through a mechanism which is dependent on Akt and STAT3 activation. These results support a novel role of leptin in the processes of adult hippocampal neurogenesis, providing new insights into the mechanisms of neurogenic regulation. Future work will be needed to determine whether the same signaling mechanisms operate in vivo and whether hippocampal neurogenesis is required for leptin�� actions on hippocampal-related processing such as learning, memory and emotional responses. ACKNOWLEDGEMENT We thank Margaret Wey for technical assistance in tissue preparation. This work was supported by Scientist Development Award AHA0530345N (X.Y.L.) and NIH grants MH073844 and MH076929 (X.Y.L.). 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(2005) J Clin Invest 115(11), 3104-3116 Downloaded from www.jbc.org by on May 18, 2008 12 FIGURE LEGENDS Figure 1. Effect of leptin administration on cell proliferation in the dentate gyrus of adult mice. BrdU labeling was used to assess cell proliferation. Mice were injected i.p. with leptin (1 mg/kg) or vehicle twice daily for 1, 5, or 14 days followed by BrdU labeling. The number of BrdU-labeled cells was counted in the subgranular zone and hilus of the dentate gyrus. A. Acute treatment (1d) with vehicle or leptin. B. Short-term treatment (5d) with vehicle or leptin. C. Chronic treatment (14d) with vehicle or leptin. Images in D and E showing BrdU-labeled cells in the adult dentate gyrus of mice treated with vehicle (left panel) or leptin (right panel) for 14 days. Images in D��and E��showing BrdU-labeled cells in the paraventricular thalamus treated with vehicle (left panel) or leptin (right panel) for 14 days. Image in F indicating high magnification view of proliferating cells. D3V, dorsal 3rd ventricle; DG, dentate gyrus; GCL, granule cell layer; PVT, paraventricular thalamus, SGZ, subgranular zone. Scale bar = 100 m in D and E, 50 in D��and E�� and 10 in F. Data are expressed as the mean number of BrdU-labeled cells per dentate gyrus ��SEM (n = 6 per group). **P < 0.01. Figure 2. Effect of leptin administration on cell differentiation. Mice were injected with leptin (1 mg/kg) or vehicle twice daily for 14 days followed by BrdU labeling and were perfused 28 days later. A. A significantly higher number of BrdU-postive cells remained in the leptin-treated group compared to the control group 28 days after BrdU labeling. B. Quantitative analysis showing the effect of leptin on the percentage of BrdU-labeled cells double-labeled for NeuN or GFAP. Results are expressed as mean ��SEM (n = 5 per group). *P < 0.05, compared to the vehicle-treated control group. C. Confocal microscopic images showing co-localization of BrdU with NeuN. D. Confocal microscopic images showing co-localization of BrdU with GFAP. White arrows indicating double labeled cells. Scale bars in C and D = 50 m for the images showing BrdU, NeuN/GFAP and, 10 m for the 3D images. Figure 3. Effect of leptin administration on body weight. Body weight was measured for 14 days during the treatment with leptin (1 mg/kg, i.p.). Data are expressed as mean ��SEM. **P<0.01. n = 6 per group. Figure 4. Expression of the long form leptin receptor (LepRb) in adult hippocampal stem/progenitor cells. A. RT-PCR using cDNA extracted from adult stem/hippocampal stem cells produced a 572 bp band corresponding to the C-terminal region of LepRb. B. Confocal microscopic images showing that adult hippocampal stem/progenitor cells stained with Nestin (green) are positive for immunoreactivity of the leptin receptor (red). DAPI staining (blue) revealing nuclei. Scale bar = 10 m Figure 5. Effects of leptin treatment on proliferation of adult hippocampal stem/progenitor cells. Cells were treated with various concentrations of leptin (1~30 nM) for 48 h and labeled with BrdU (10 M) in the last 4 h of incubation. A. Quantitative analysis revealed that the number of BrdU-labeled cells was increased by leptin treatment at concentrations of 1 nM and 3 nM when compared to control. Data are expressed as mean ��SEM. **P < 0.01. B. Representative microscopic images showing BrdU-labeled adult hippocampal progenitor cells in control (left panel) and leptin-treated groups (right panel). Scale bar = 100 m. Figure 6. Effect of leptin treatment on differentiation of cultured adult hippocampal stem/progenitor cells. Cells that were treated with leptin (1 nM) for 48 h and labeled with BrdU (10 M) in the last 4 h of incubation were allowed to differentiate for 8 days before fixation for immunohistochemical processing. A. Representative microscopic images showing that BrdU-labeled cells differentiated into neuronal (TuJ1-positive in red) or glial (GFAP-positive in green) cells. B. Quantitative analysis indicating that the percentage of BrdU-labeled cells that were positive for TuJ1 or GFAP was not altered by leptin treatment. Data are expressed as mean ��SEM. Scale bar = 10 m. Figure 7. Effect of leptin on phosphorylation of Akt, STAT3 and ERK1/2 in cultured adult hippocampal stem/progenitor cells. Cells were stimulated with leptin (0.3 nM and 3 nM) or vehicle for 15 min. The phosphorylation of Akt, STAT3 and ERK1/2 was detected using phospho-specific antibodies. A. Representative Downloaded from www.jbc.org by on May 18, 2008 13 immunoblot showing phosphorylated Akt and total Akt. B. The ratio of densitomitric measurements of phosphoAkt and total Akt expressed as percent of control C. Representative immunoblot showing phosphorylated STAT3 and total STAT3. D. The ratio of densitomitric measurements of phospho-STAT3 and total STAT3 expressed as percent of control. E. Representative immunoblot showing phosphorylated ERK1/2 and total ERK1/2. F. The ratio of densitomitric measurements of phospho-ERK1/2 and total ERK1/2 expressed as percent of control **P < 0.01 as compared with control. The data are representative of two individual experiments. Figure 8. Effect of inhibition of Akt on leptin-induced cell proliferation. Cultured adult hippocampal stem/progenitor cells were incubated with various concentrations of Akt inhibitor VIII (0.01~1.0 M) for 4 h followed by treatment with leptin (1 nM) for 48 h and labeling with BrdU (10 M) during the last 4 h of incubation. A. Quantitative analysis revealed that pretreatment with Akt inhibitor VIII attenuates the leptininduced increase in the number of BrdU-labeled cells in a dose-dependent manner. B. Representative microscopic images of BrdU-labeled cells from three treatment conditions: control-control (left), controlleptin (center), and Akt inhibitor VIII-leptin (right). *P < 0.05. Scale Bar = 100 m. Figure 9. Effect of inhibition of STAT3 on leptin-induced cell proliferation. Cultured adult hippocampal stem/progenitor cells were incubated with various concentrations of cucurbitacin I (0.1~10 nM) for 4 h followed by treatment with leptin (1 nM) for 48 h and labeling with BrdU (10 M) during the last 4 h of incubation. A. Quantitative analysis revealed that pretreatment with cucurbitacin I attenuated the leptin-induced increase in the number of BrdU-labeled cells. B. Representative microscopic images of BrdU-labeled cells from three treatment conditions: control-control (left), control-leptin (center), and cucurbitacin I - leptin (right) *P < 0.05. Scale Bar = 100 m. 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