1-Adrenergic Receptors Regulate Neurogenesis and Gliogenesis

The understanding of the function of 1-adrenergic receptors in the brain has been limited due to a lack of specific ligands and antibodies. We circumvented this problem by using transgenic mice engineered to overexpress either wild-type receptor tagged with enhanced green fluorescent protein or constitutively active mutant 1-adrenergic receptor subtypes in tissues in which they are normally expressed. We identified intriguing 1A-adrenergic receptor subtype-expressing cells with a migratory morphology in the adult subventricular zone that coexpressed markers of neural stem cell and/or progenitors. Incorporation of 5-bromo-2-deoxyuridine in vivo increased in neurogenic areas in adult 1A-adrenergic receptor transgenic mice or normal mice given the 1A-adrenergic receptor-selective agonist, cirazoline. Neonatal neurospheres isolated from normal mice expressed a mixture of 1-adrenergic receptor subtypes, and stimulation of these receptors resulted in increased expression of the 1B-adrenergic receptor subtype, proneural basic helix-loop-helix transcription factors, and the differentiation and migration of neuronal progenitors for catecholaminergic neurons and interneurons. 1-Adrenergic receptor stimulation increased the apoptosis of astrocytes and regulated survival of neonatal neurons through phosphatidylinositol 3-kinase signaling. However, in adult normal neurospheres, 1-adrenergic receptor stimulation increased the expression of glial markers at the expense of neuronal differentiation. In vivo, S100-positive glial and III tubulin neuronal progenitors colocalized with either 1-adrenergic receptor subtype in the olfactory bulb. Our results indicate that 1adrenergic receptors can regulate both neurogenesis and gliogenesis that may be developmentally dependent. Our findings may lead to new therapies to treat neurodegenerative diseases. It is now recognized that neurogenesis continues in the mammalian brain after birth. The areas of the most active neurogenesis are the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the hippocampus (Lie et al., 2004). The SVZ contains neural stem cells (NSCs) whose progenitors migrate through defined pathways, such as the rostral migratory stream (RMS) that directs neuroblasts to the olfactory bulb where they mature into interneurons. In the hippocampus, new neurons are born in the SGZ and become functioning granule cells (Santarelli et al., 2003). The prevailing view is that NSCs are glial fibrillary acidic protein (GFAP)-positive cells of a radial glial lineage (Morshead and van der Kooy, 2004). NSCs are selfrenewing and multipotent cells that generate neurons, astroThis study was supported by the National Institutes of Health Heart, Lung, and Blood Institute [Grant 5-R01-HL61438]; National Institutes of Health National Center for Research Resources INBRE Program [Grant P20RR016741]; National Science Foundation, Faculty Early Career Development Award [Grant 0347259]; and National Science Foundation, Major Research Instrumentation Award [Grant 0619688]. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.109.057307. ABBREVIATIONS: SVZ, subventricular zone; AR, adrenergic receptor; bHLH, basic helix-loop-helix; BrdU, 5-bromo-2 -deoxyuridine; CAM, constitutively active mutant; EGF, epidermal growth factor; EGFP, enhanced green fluorescence protein; FBS, fetal bovine serum; FGF, fibroblast growth factor; NSC, neural stem cell; PBS, phosphate-buffered saline; RMS, rostral migratory stream; RT, room temperature; SGZ, subgranular zone; TAP, transient amplifying progenitor; TUNEL, terminal deoxynucleotidyl transferase; VEGF, vascular endothelial growth factor; GFAP, glial fibrillary acidic protein; KO, knockout; DMEM, Dulbecco’s modified Eagle’s medium; FACS, fluorescence-activated cell sorting; NMDA, N-methylD-aspartate; PKC, protein kinase C; PCR, polymerase chain reaction; MAP2, microtubule-associated protein-2; D-PBS, Dulbecco’s phosphatebuffered saline; PD98059, 2 -amino-3 -methoxyflavone; SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; SP600125, anthra[1–9-cd]pyrazol-6(2H)-one; LY294002, 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride; Go6983, 3-[1-[3(dimethylamino)propyl]-5-methoxy-1H-indol-3-yl]4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione; ICI-118,551, ( )-1-[2,3-(dihydro-7-methyl-1H-inden-4yl)oxy]-3-[(1-methylethyl)amino]-2-butanol. 0026-895X/09/7602-314–326$20.00 MOLECULAR PHARMACOLOGY Vol. 76, No. 2 Copyright © 2009 The American Society for Pharmacology and Experimental Therapeutics 57307/3500631 Mol Pharmacol 76:314–326, 2009 Printed in U.S.A. 314 at A PE T Jornals on A uust 7, 2017 m oharm .aspeurnals.org D ow nladed from cytes, and oligodendrocytes (Lie et al., 2004). Under normal conditions, neurogenesis in other central nervous system (CNS) regions is minimal, suggesting that specific mechanisms regulate where new neurons are produced. The 1-adrenergic receptor (AR) subtypes ( 1A, 1B, and 1D) 1 are G-protein-coupled receptors that regulate the sympathetic nervous system by binding and transducing the signals of norepinephrine and epinephrine. Within the peripheral nervous system, 1-AR activation is known to regulate the cardiovascular and other organ systems. Within the CNS, it has proven more difficult to ascribe 1-AR functions, let alone the subtype to a particular function, because of poorly selective ligands and weak antibodies (Jensen et al., 2009). However, evidence links central 1-ARs to the regulation of plasticity (Sirviö and MacDonald, 1999) and stimulation of GABAergic interneurons (Papay et al., 2006). Studies have also indirectly suggested a potential role of 1-ARs in neurogenesis. 1-AR activation increases vascular endothelial growth factor (VEGF) mRNA (Gonzalez-Cabrera et al., 2003), and VEGF has been suggested to increase the proliferation of neuronal precursors (Jin et al., 2002). VEGF localizes to the choroid plexus (Maharaj et al., 2006), which receives strong adrenergic innervation to regulate its secretory functions (Lindvall and Owman, 1981). The 1-ARs stimulate the shedding of epidermal growth factor (EGF) and fibroblast growth factor (FGF) (Chen et al., 2006), factors needed to maintain NSC niches. [H]Prazosin binding sites are found in SVZ neuroepithelia in rat embryonic day-13 embryos (Pabbathi et al., 1997) and in adult mice engineered to overexpress 1A-ARs tagged with enhanced green fluorescent protein (EGFP) to localize the receptor (Papay et al., 2006). Using 1-AR promoters expressing EGFP tags with or without the receptor (Papay et al., 2004, 2006), we identified a cell type in the SVZ in vivo that coexpressed markers of NSCs and/or their progenitors that can be labeled by 5-bromo-2deoxyuridine (BrdU). Subsequent studies on isolated neonatal neurospheres derived from normal mice and mice engineered to overexpress constitutively activate mutant (CAM) receptors or with their 1-ARs knocked out (KO) revealed that 1-ARs play an important role in the regulation of NSC/ progenitors and their differentiation into neurons. In contrast, 1-ARs expressed on adult neurospheres and isolated from normal mice regulated gliogenesis. However, 1-ARs colocalized with both glial and neuronal progenitors in the adult mouse olfactory bulb. Materials and Methods Animal Use. Mice were housed and provided veterinary care in an Association for Assessment and Accreditation of Laboratory Animal Care-accredited animal care facility. The experimental protocols used in this study conform to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health and was approved by the Animal Care and Use Committee at our institutions. Immunohistochemistry. For in vivo analysis, mice were cardiac-perfused, and brain sections wee made using a vibrotome as described previously (Papay et al., 2004, 2006). Primary antibodies used were rabbit notch1 at 1:50 (Santa Cruz Biotechnology, Santa Cruz, CA), mouse nestin at 1:100, rabbit Dlx2 at 1:200 (both from Chemicon, Temecula, CA), and rabbit vimentin at 1:5000 (Abcam Inc., Cambridge, MA). For in vitro analysis, cells were fixed in 4% paraformaldehyde for 30 min. Coverslips were blocked in 6% bovine serum albumin and 0.3% Trition X-100 in PBS for 1 h at room temperature (RT) and then incubated with the primary antibody chicken microtubule-associated protein-2 (MAP2) at 1:5000 (Novus Biologicals, Inc., Littleton, CO), rabbit NG2 at 1:7000 (a gift from Bill Stallcup), or mouse GFAP at 1:3000 (Chemicon) in blocking buffer for 24 h at 4°C. The coverslips were washed three times in PBS and then incubated with the corresponding secondary antibodies donkey antichicken cy5 at 1:500 (Jackson Immunoresearch Laboratories Inc., West Grove, PA), goat anti-rabbit 488 at 1:4000, and goat anti-mouse 568 at 1:4000 (both from Molecular Probes, Carlsbad, CA) for 1 h at RT. Coverslips were washed three times in PBS, transferred to a microscope slide with Vectashield containing 4 , 6 -diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA). Sections were analyzed using a confocal laser-scanning microscope (Aristoplan; Leica Micosystems, Inc., Deerfield, IL). Confocal images represent optical sections of 2 to 3 m axial resolution and an average of three to six line scans. Fluorescence in red, green, blue, and purple channels were collected simultaneously. Confocal images were reassembled, and the images were presented as a projection. Colocalization was confirmed in single confocal sections in which EGFP fluorescence is present in the same cell with each cell-type marker. Neonatal Neurosphere Isolation and Culture. Neurospheres were isolated from the periventricular regions of postnatal day 3 pups of neonatal normal, CAM 1A, CAM 1B, and their corresponding KO mice. Periventricular regions were digested for 20 min at 37°C in 3 ml of 0.05% trypsin, 0.53 mM EDTA, and 0.001% DNAase in D-PBS (10 mM HEPES, pH 7.6, and 0.5 g of glucose/500 ml). Six milliliters of B27 complete media (DMEM/Ham’s F-12, 1 B27 (Gibco), 20 ng/ml recombinant human EGF (Stem Cell Technologies, Vancouver, BC, Canada), 10 ng/ml recombinant human FGF (Abcam), 0.0002% heparin (Sigma), and 100 U/ml penicillin/streptomycin) was added, and the sample was centrifuged at 100g for 7 min. The supernatant was removed and the pellet titrated in 5 ml of D-PBS with a 5-ml pipette for 5 min. Cells were passed through a large 70m cell strainer into a 50-ml conical tube and centrifuged at 100g. Cells were resuspended in D-PBS, centrifuged, and the pellet was resuspended in D-PBS. Cells were passed through a 30m cell strainer (CellTrics; Partec, Swedesboro, NJ), centrifuged, and resuspended into 10 ml of B27 complete media and counted. Cells were split and fed every 2 to 3 days by centrifugation and replating. After 1 to 2 weeks, we picked 20 neurospheres from the culture of dissociated tissue. Of these isolated neurospheres, approximately 60% maintained good neurosphere growth when dissociated. We finally isolated six neurospheres for cell lines, which were all positive for nestin/notch and 1-AR expression, except for the KO cell lines. After reaching sufficient density, neurospheres were passaged by mechanical dissociation or with accutase (Sigma, St. Louis, MO). Neurospheres used for experimental purposes were all taken from third or later passages. Radioligand Binding. Membranes were prepared from neurospheres as described previously (Zuscik et al., 1998). Saturation or competition binding was performed using the 1-AR antagonist [I]iodo-2-[ -(4-hydroxyphenyl)-ethyl-aminomethyl]tetralone or the -AR antagonist I-cyanopindolol as the radioligand as described previously (Zuscik et al., 1998). Data were analyzed using Prism software (GraphPad Software Inc., San Diego, CA). Various AR antagonists used in radioligand binding were purchased from Sigma-Aldrich. Neurosphere Assay. Neurospheres were dissociated with accutase and mechanical titration and were diluted to single-cell level for replating in individual uncoated 24-well plates. The percentage of single cells (percentage of cloning efficiency) that regenerate neurospheres was determined. Differentiation Assay. Neurospheres were centrifuged at 100g for 7 min to remove growth factors and were resuspended in B27 media without EGF/FGF but were supplemented with 2% FBS. Cells 1 After the 1C-AR was reclassified as the 1A-AR, the 1C-AR designation is no longer used. 1-Adrenergic Receptors Regulate Neurogenesis 315 at A PE T Jornals on A uust 7, 2017 m oharm .aspeurnals.org D ow nladed from were then transferred to a 24-well plate containing a sterile precoated poly(D-lysine)/laminin coverslip (Biocoat; BD Biosciences, San Jose, CA). For phenylephrine-induced differentiation, 10 M phenylephrine was added daily to B27 complete media in the presence of 1 M propranolol and 0.1 M rauwolscine to block and 2-ARs, respectively. Coverslips were fixed with 4% paraformaldehyde in PBS for 30 min at RT and then washed twice with PBS for 5 min and used for immunohistochemistry or stored in PBS at 4°C. At least three different coverslips (each containing 40–300 cells) were analyzed from three separate experiments. In Vivo BrdU Incorporation. Mice were injected intraperitoneally with BrdU at 150 mg/kg body weight. Two hours after the injection, the mice were anesthetized and cardiac-perfused, and brains were sectioned as described previously (Papay et al., 2006). In chase experiments, normal mice or mice that received bottle water containing cirazoline at 10 mg/l for 12 weeks were injected twice daily intraperitoneally with BrdU at 50 mg/kg body weight for 2 weeks, then sacrificed at 2 days, 7 days, and 14 days after the last BrdU injection. Sections were first rinsed in 0.9% NaCl and then incubated in 1 N HCl in 0.9% NaCl at 37°C for 30 min. Sections were rinsed with 0.1 M borate buffer, pH 9.0, and then rinsed with Trisbuffered saline. Sections were then incubated with mouse anti-BrdU (Chemicon) at 1:5000 in blocking buffer for 2 days at 4°C and then incubated with Alexa Fluor 568 goat anti-mouse IgG (Molecular Probes) at 1:4000 in blocking buffer for 1 h at RT. Specimens were rinsed twice with distilled water before incubation with 2 ml of 10 mM copper sulfate in 50 mM ammonium acetate for 1 h at RT to remove autofluorescence. Samples were then rinsed twice with distilled water before being returned to PBS, transferred to a microscope slide containing Vectashield with 4 , 6 -diamidino-2-phenylindole, and BrdU nuclei were counted using confocal microscopy followed by stereology. Stereology. The optical dissector technique was used to estimate the density of BrdU and/or Nestin cells in the SGZ of the dentate gyrus of the hippocampus and the SVZ of the lateral ventricles. Random slices were selected, and the number of BrdU cells and volume of each structure were computed with Stereo Investigator software (MBF Bioscience; MicroBrightField Inc., Williston, VT). Cell density was expressed in cells per cubic millimeter, and the number of BrdUand Nestin-labeled cells per section was then averaged. Real-Time PCR. Neurospheres were treated with phenylephrine (10 M) or 1% FBS for 1, 3, or 7 days in the presence of rauwolscine (0.1 M) and propranolol (1 M). Total RNA was isolated using the RNeasy kit (QIAGEN, Valencia, CA), and 2 g of RNA was reversetranscribed using oligo(dT) primer with Superscript II Reverse Transcriptase (Invitrogen, Carlsbad, CA). Quantitative real-time PCR was performed with an iCycler (Bio-Rad, Hercules, CA) using iQ SYBR Green Supermix (Bio-Rad). The cDNA was amplified with primers for various neural transcriptional factors. The primers used included the following: Dlx2, 5 -aagggtgtctgtgcagatttc-3 and 5 cgtcgcagctttcacaact-3 ; Mash-1, 5 -catctcccccaactactcca-3 and 5 ccagcagctcttgttcctct-3 ; Math-1, 5 -acatctcccagatcccacag-3 and 5 gggcatttggttgtctcagt-3 ; Ngn-1, 5 -gagccggctgacaatacaat-3 and 5 -ctcaggttcttcctggagca-3 ; nestin, 5 -gaggggacctggaacatgaa-3 and 5 -gtccattctccatcctccca-3 ; NeuroD, 5 -gtgatgctggtactactggaattg-3 and 5 -gcaactgcatgggagttttc-3 ; 1A-AR, 5 -ggttcccaaaggaaacctgt-3 and 5 -ggtttcataccagggtggtg-3 ; 1B-AR, 5 -tctcagccaagtcctggttt-3 and 5 -gcgaacacctttacctgctc-3 ; and -tubulin, 5 -ggctgccctagagaaggatt-3 and 5 -aaacatccctgtggaagcag-3 . Samples were analyzed for relative gene expression using the 2 CT method (Livak and Schmittgen, 2001) and were normalized to -tubulin gene expression as an internal control. Data were obtained from three independent experiments performed in duplicate. Pathway Analysis. Normal neonatal neurospheres were pretreated for 1 h with individual inhibitors (either 20 M PD98059, 10 M SB203580, 10 M SP600125 (all from Calbiochem, San Diego, CA), 20 M LY294002 (Cell Signaling Technology, Danvers, MA), or 0.5 M Go6983 (Calbiochem) and then stimulated with phenylephrine (10 M) for 3 days. Neurospheres were then lysed and subjected to SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membrane, and probed with antibodies against mouse GFAP at 1:1000 or chicken MAP2 at 1:3000. Membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies. Chemiluminescence detection was performed by incubating membranes with SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL) followed by exposure onto X-ray film. Neuronal Cell Types. Normal neurospheres were stimulated with 10 M phenylephrine for 0, 3, 7, or 14 days, and lysates were subjected to Western blot analysis. Nonspecific binding was blocked by incubation with 5% milk and 0.1% Tween 20 in Tris-buffered saline for 2 h at RT. The membranes were then incubated with primary antibody in blocking buffer against goat anti-GAD-65/67 (1:100), rabbit anti1-integrin (1:500; Santa Cruz Biotechnology), sheep anti-tyrosine hydroxylase (1:200), rabbit anti-NMDAR1 (1: 100; Chemicon), sheep anti-dopamine -hydroxylase (1:3000; Novus Biologicals), or monoclonal anti-actin (Sigma) overnight at 4°C with gentle agitation. Membranes were washed three times with 0.1% Tween 20 in Tris-buffered saline and incubated with the appropriate horseradish peroxidase-coupled secondary antibodies for 1 h at RT followed by chemiluminescence detection. Apoptosis Assay. Neurospheres were grown and analyzed for apoptosis by using the FlowTACS Apoptosis Detection Kit (Trevigen, Gaithersburg, MD). Terminal deoxynucleotidyl transferase end-labeling of the free 3 -hydroxyl residues in the fragmented DNA was performed according to manufacturer’s instructions. In brief, normal neonatal neurospheres were treated with 10 M phenylephrine for 0 (control), 2, or 3 days, fixed in 3.7% formaldehyde, permeabilized, and labeled with primary antibodies against MAP2 and GFAP and the terminal deoxynucleotidyl transferase enzyme, followed by Strep-Fluorescein treatment and appropriate secondary antibodies. Cells were also treated with propidium iodide to label necrotic cells, which were not counted. Cells were analyzed under a fluorescence microscope. Total cells counted ranged from 40 to 240 per coverslip using at least three different cell preparations. Migration Assay. The effect of 1-AR stimulation on progenitor cell migration was determined by Boyden chamber assay as described previously (Sun et al., 2001). In brief, neurospheres derived from normal, CAM, and KO mice were dissociated in DMEM/Ham’s F-12 medium without growth factors at a density of 10 cells/transwell (Costar; Corning Life Sciences, Acton, MA) using a 0.4 M membrane. Phenylephrine (10 M) was added to the lower well, and Fig. 1. Constructs used for systemic overexpression in transgenic mice. A, CAM 1A-AR; B, CAM 1B-AR; C, 1A-AR promoter-driving EGFP only; D, 1B-AR EGFP-tagged receptor. 316 Gupta et al. at A PE T Jornals on A uust 7, 2017 m oharm .aspeurnals.org D ow nladed from after 48 h, the media from each of the upper and lower wells were aspirated, and cells were scraped and assayed for DNA content using the CyQuant Cell Proliferation assay kit (Invitrogen) and a CytoFluor II fluorescent plate reader. Isolation of Adult Normal Neurospheres. Adult normal neurospheres were isolated from the periventricular regions of 2 to 3 months normal mice. Periventricular regions were dissected in a Petri dish containing 10 ml of NeuroCult Tissue Collection Solution (StemCell Technologies). After dissection, the collection solution was discarded, and the tissue was divided into two piles, minced for 1 min in 1 ml of NeuroCult Dissociation Solution, and transferred to a 15-ml sterile conical tube being careful not to introduce air bubbles. The mincing was repeated twice. The pooled minced tissue was incubated for 7 min at 37°C in a beaker of prewarmed water. The suspension was gently mixed with 3 ml of NeuroCult Inhibition Solution, avoiding air bubbles. The mixture was centrifuged at 100g for 7 min, and the supernatant was removed. The pellet was resuspended in 1 ml of NeuroCult Resuspension Solution and triturated Fig. 2. 1A-AR promoter-EGFP cells localize in the SVZ in vivo. A, 1A-AR promoter-EGFP-expressing cells (green) are found in the SVZ and RMS of adult mice. B, 1A-AR promoter-EGFP cells are abundant in the SVZ, where nestin (in red) is located. C, magnification of the boxed area in B. 1A-AR promoter-EGFP cells colocalize with nestin. D, 1A-AR promoter-EGFP cells are abundant in the SVZ, where Notch-1 (in red) is located. Some 1A-AR promoter-EGFP cells colocalize with Notch-1 (E) or vimentin (F) near the lateral ventricle (LV) border. G, some 1A-AR promoter-EGFP cells express Dlx2 in the nucleus (white arrows) in the SVZ and RMS, whereas other 1A-AR promoter-EGFP cells do not (yellow arrows). H, 1A-AR promoterEGFP cells line the fourth ventricle. I, 1B-AR-EGFP-tagged cells are not localized near the ependymal border. J, FACS of dissociated periventricular cells from normal mice (control) or three different 1A-promoter EGFP mice (fluorescein isothiocyanate 1–3). Approximately 44.4% of periventricular cells express EGFP. Mice were aged 2 to 3 months. White bar, 10 m. 1-Adrenergic Receptors Regulate Neurogenesis 317 at A PE T Jornals on A uust 7, 2017 m oharm .aspeurnals.org D ow nladed from