alpha1-Adrenergic receptors regulate neurogenesis and gliogenesis

Abstract

The understanding of the function of alpha(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 alpha(1)-adrenergic receptor subtypes in tissues in which they are normally expressed. We identified intriguing alpha(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 alpha(1A)-adrenergic receptor transgenic mice or normal mice given the alpha(1A)-adrenergic receptor-selective agonist, cirazoline. Neonatal neurospheres isolated from normal mice expressed a mixture of alpha(1)-adrenergic receptor subtypes, and stimulation of these receptors resulted in increased expression of the alpha(1B)-adrenergic receptor subtype, proneural basic helix-loop-helix transcription factors, and the differentiation and migration of neuronal progenitors for catecholaminergic neurons and interneurons. alpha(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, alpha(1)-adrenergic receptor stimulation increased the expression of glial markers at the expense of neuronal differentiation. In vivo, S100-positive glial and betaIII tubulin neuronal progenitors colocalized with either alpha(1)-adrenergic receptor subtype in the olfactory bulb. Our results indicate that alpha(1)-adrenergic receptors can regulate both neurogenesis and gliogenesis that may be developmentally dependent. Our findings may lead to new therapies to treat neurodegenerative diseases.

Fig. 1.

Constructs used for systemic overexpression in transgenic mice. A, CAM α_1A-AR; B, CAM α_1B-AR; C, α_1A-AR promoter-driving EGFP only; D, α_1B-AR EGFP-tagged receptor.

Fig. 2.

α_1A-AR promoter-EGFP cells localize in the SVZ in vivo. A, α_1A-AR promoter-EGFP-expressing cells (green) are found in the SVZ and RMS of adult mice. B, α_1A-AR promoter-EGFP cells are abundant in the SVZ, where nestin (in red) is located. C, magnification of the boxed area in B. α_1A-AR promoter-EGFP cells colocalize with nestin. D, α_1A-AR promoter-EGFP cells are abundant in the SVZ, where Notch-1 (in red) is located. Some α_1A-AR promoter-EGFP cells colocalize with Notch-1 (E) or vimentin (F) near the lateral ventricle (LV) border. G, some α_1A-AR promoter-EGFP cells express Dlx2 in the nucleus (white arrows) in the SVZ and RMS, whereas other α_1A-AR promoter-EGFP cells do not (yellow arrows). H, α_1A-AR promoter-EGFP cells line the fourth ventricle. I, α_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 α_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 μm.

Fig. 3.

AR expression in neonatal neurospheres and pluripotency. A, [¹²⁵I]Iodo-2-[β-(4-hydroxyphenyl)-ethyl-aminomethyl]tetralone saturation binding of normal neonatal neurospheres. B, competition binding with 5-methylurapidil indicates that normal neonatal neurospheres expressed 33% of the α_1A-AR subtype and 67% of the α_1B-AR (red circles). Upon incubation with 10 μM phenylephrine (Phe) for 24 h, the subtype composition became 14% of the α_1A-AR and 86% of the α_1B-AR subtype (blue circles). C, competition binding with ¹²⁵I-cyanopindolol and ICI-118,551, a β₂-AR selective antagonist indicates that normal neonatal neurospheres also express a majority (86%) of the low-affinity β₁-AR subtype. D, neurosphere self-renewal assay. CAM α_1A-AR has a lower efficiency to regenerate neurospheres than normal or KOs. E, normal neonatal neurospheres differentiate into all three cell-types (red, GFAP; magenta, MAP2; green, NG2 upon incubation with serum, 2% FBS). F, neurospheres isolated from CAM α_1A-AR mice differentiated into all three cell types but were mostly neurons. G, neurospheres isolated from the α_1A-AR KO mice were mostly astrocytes with a reduced number of neurons. H, quantitation of cell-type differentiation by 2% FBS for neurospheres isolated from all mouse models. At least three different coverslips (each containing 40-300 cells) were analyzed from three separate experiments. White bar, 10 μm. #, P < 0.05; ^*, P < 0.01.

Fig. 4.

α_1A-AR induces progenitor proliferation and differentiation. A, in vivo BrdU incorporation for 2 h indicates that 2 to 3 months CAM α_1A-AR mice have increased the number of proliferating progenitor cells in the SVZ (A) and SGZ (B) compared with CAM α_1B-AR or either KO mice. C, α_1A-promoter EGFP cells in the SVZ express BrdU (arrows). Normal neonatal neurospheres and phenylephrine stimulation for 0 (D, control), 3 (E), or 20 days (F) differentiates into neurons (Map2 in magenta), astrocytes (GFAP in red), and oligodendrocyte progenitors (NG2 in green). G, CAM α_1A-AR neurospheres at 0 days basally expressed more neurons than normal controls. KO of the α_1A -AR increased expression of astrocytes (H, red). I, quantified cell types for D to H. At least three different coverslips (each containing 40-300 cells) were analyzed from three separate experiments. White bar, 10 μm. ^*, P < 0.05.

Fig. 5.

α_1A-AR agonist increases BrdU incorporation in vivo. In vivo BrdU incorporation for 2 weeks was followed by 2 (A), 7 (B), or 14 days (C) of chase in normal mice (control) or mice that were treated with the α_1A-AR selective agonist, cirazoline (Ciraz), for 3 months. A, α_1A-AR stimulation increases the number of nestin and/or BrdU⁺ cells at 2 days of chase. At 7 (B) and 14 days (C), α_1A-AR stimulation increased BrdU+ cells compared with control. ^*, P < 0.05 compared with matched control. B + N, BrdU⁺/Nestin⁺-colabeled cells.

Fig. 6.

α₁-ARs regulate key transcription factor mRNA involved in neonatal neuronal differentiation. RNA was isolated from neurospheres and subjected to real-time PCR. When differentiated by 1% serum, normal neonatal neurospheres significantly increased the RNA of the α_1B-AR subtype, Dlx2, Mash1, Math1, Ngn1, and NeuroD, consistent with neuronal differentiation (gray triangles). When phenylephrine-stimulated, CAM α_1A-AR neurospheres increased the RNA of Dlx2, Mash1, and NeuroD (green circles), whereas CAM α_1B-AR neurospheres increased the RNA of Math-1 and Ngn-1 (red circles). KO of the α_1A-AR subtype increased the RNA of nestin (magenta triangles), suggesting a reversion to a more undifferentiated state. Other factors tested (Notch-1, Zic-1, Sox-2) were not regulated by α₁-ARs. α_1B-AR KO (blue triangles). Samples are obtained from three independent experiments, performed in duplicate, and were normalized to α-tubulin gene expression as internal controls. ^*, P < 0.05; ^**, P < 0.001.

Fig. 7.

α₁-ARs differentiate interneurons and catecholaminergic neurons through PI3K in neonatal neurospheres. A, Western blot analysis of normal neonatal neurospheres stimulated with phenylephrine (Phe 10 μM) increased whereas α_1A-AR KO decreased the expression of tyrosine hydroxylase, GAD-65/67, and dopamine β-hydroxylase. β₁-Integrin decreases expression with phenylephrine stimulation, whereas no R1 subunit of the NMDA receptor could be detected. Br, brain extract control. B, neuronal differentiation as assessed by MAP2 expression levels via Western analysis is stimulated by phenylephrine but decreased when coincubated with the PI3K inhibitor LY294002 (20 μM). C, decreased astrocyte expression as assessed by GFAP Western analysis was reduced with phenylephrine but increased when coincubated with LY294002. Other inhibitors used were 10 μM PD98059 (mitogen-activated protein kinase kinase inhibitor, PD), 10 μM SB203580 (p38 inhibitor, SB), 10 μM SP600125 (c-Jun NH₂-terminal kinase inhibitor, SP), and 0.5 μM Go6983 (PKC inhibitor, Go). ^*, P < 0.05; ^**, P < 0.01.

Fig. 8.

α₁-ARs regulate apoptosis and migration of neonatal progenitors. A, normal neonatal neurospheres treated with 10 μM phenylephrine for 0 (control), 2, or 3 days increase the number of TUNEL-positive cells. B, TUNEL-positive cells expressed GFAP but not MAP2. C, dissociated neonatal normal neurospheres treated with 10 μM phenylephrine or CAM α_1A-AR-derived neurospheres increased migration comparable with 2% serum. α_1A-AR KO-derived neurospheres displayed decreased migration. Total cells counted ranged from 40 to 240 per coverslip using at least three different cell preparations tested. ^*, P < 0.05; ^**, P < 0.01.

Fig. 9.

α₁-ARs differentiate normal adult neurospheres into glial cells. A, Western blot analysis of normal adult neurospheres stimulated with phenylephrine (Phe 10 μM) decreased the expression of markers associated with undifferentiated stem cells, such as notch-1, nestin, and vimentin, but increased the expression of GFAP and CC1, markers for astrocytes and oligodendrocytes, respectively. The NMDA receptor neuronal marker NR1 decreased in expression upon α₁-AR stimulation. Similar results repeated in another normal adult neurosphere cell line. Both adult neurosphere cell lines were negative for the neuronal markers tyrosine hydroxylase, GAD-65/67, and dopamine β-hydroxylase. α_1A-AR promoter-EGFP (B and D) or α_1B-AR-EGFP tagged (C and E) cells (green) colocalized (arrows) with S100-positive or βIII tubulin-positive cells in the adult olfactory. Mice were aged 2 to 3 months. White bar, 10 μm.