Robert S. Papay

Localization of the mouse α1A‐adrenergic receptor (AR) in the brain: α1AAR is expressed in neurons, GABAergic interneurons, and NG2 oligodendrocyte progenitors

α1‐Adrenergic receptors (ARs) are not well defined in the central nervous system. The particular cell types and areas that express these receptors are uncertain because of the lack of high avidity antibodies and selective ligands. We have developed transgenic mice that either systemically overexpress the human α1A‐AR subtype fused with the enhanced green fluorescent protein (EGFP) or express the EGFP protein alone under the control of the mouse α1A‐AR promoter. We confirm our transgenic model against the α1A‐AR knockout mouse, which expresses the LacZ gene in place of the coding region for the α1A‐AR. By using these models, we have now determined cellular localization of the α1A‐AR in the brain, at the protein level. The α1A‐AR or the EGFP protein is expressed prominently in neuronal cells in the cerebral cortex, hippocampus, hypothalamus, midbrain, pontine olivary nuclei, trigeminal nuclei, cerebellum, and spinal cord. The types of neurons were diverse, and the α1A‐AR colocalized with markers for glutamic acid decarboxylase (GAD), gamma‐aminobutyric acid (GABA), and N‐methyl‐D‐aspartate (NMDA) receptors. Recordings from α1A‐AR EGFP‐expressing cells in the stratum oriens of the hippocampal CA1 region confirmed that these cells were interneurons. We could not detect expression of the α1A‐AR in mature astrocytes, oligodendrocytes, or cerebral blood vessels, but we could detect the α1A‐AR in oligodendrocyte progenitors. We conclude that the α1A‐AR is abundant in the brain, expressed in various types of neurons, and may regulate the function of oligodendrocyte progenitors, interneurons, GABA, and NMDA receptor containing neurons. J. Comp. Neurol. 497:209–222, 2006.

α1-Adrenergic Receptor Stimulates Interleukin-6 Expression and Secretion through Both mRNA Stability and Transcriptional Regulation: Involvement of p38 Mitogen-Activated Protein Kinase and Nuclear Factor-κB

Our previous studies have demonstrated that activation of α1-adrenergic receptors (ARs) increased interleukin-6 (IL-6) mRNA expression and protein secretion, which is probably an important yet unknown mechanism contributing to the regulation of cardiac function. Using Rat-1 fibroblasts stably transfected with the α1A-AR subtype and primary mouse neonatal cardiomyocytes, we elucidated the basic molecular mechanisms responsible for the α1-AR modulation of IL-6 expression. IL-6 mRNA production mediated by α1-AR peaked at 1 to 2 h. Studies of the mRNA decay rate indicated that α1-AR activation enhanced IL-6 mRNA stability. Analysis of IL-6 promoter activity using a series of deletion constructs indicated that α1-ARs enhanced IL-6 transcription through several transcriptional elements, including nuclear factor κB (NF-κB). Inhibition of α1-AR mediated IL-6 production and secretion by actinomycin D and the increase of intracellular IL-6 levels by α1-AR activation suggest that α1-AR mediated IL-6 secretion through de novo synthesis. Both IL-6 transcription and protein secretion mediated by α1-ARs were significantly reduced by chemical inhibitors for p38 mitogen-activated protein kinase (MAPK) and NF-κB and by a dominant-negative construct of p38 MAPK. Serum level of IL-6 was elevated in transgenic mice expressing a constitutively active mutant of the α1A-AR subtype but not in a similar mouse model expressing the α1B-AR subtype. Our results indicate that α1-ARs stimulated IL-6 expression and secretion through regulating both mRNA transcription and stability, involving p38 MAPK and NF-κB pathways.

Mouse α1B‐adrenergic receptor is expressed in neurons and NG2 oligodendrocytes

α1‐Adrenergic receptors (ARs) are well‐known mediators of the sympathetic nervous system, are highly abundant in the brain, but are the least understood in the central nervous system. The particular cell types in the brain that contain these receptors or their functions are not known because of the lack of high avidity antibodies and selective ligands. We developed transgenic mice that endogenously overexpress the α1B‐AR subtype fused with the enhanced green fluorescent protein (EGFP). Endogenous expression was obtained by using a 3.4 kb fragment of the mouse α1B‐AR promoter. Using this model, we determined cellular localization of the α1B‐AR throughout the brain. The α1B‐AR‐EGFP fusion protein is expressed in neurons throughout the brain and in the Purkinje cells of the cerebellum. The α1B‐AR is also expressed in NG2 oligodendrocyte precursor cells in both neonatal cell cultures and in the adult cerebral cortex, but is weakly expressed in mature oligodendrocytes. The α1B‐AR was not observed in astrocytes or in cerebral vascular smooth muscle, cell types previously suggested to contain α1‐ARs. We conclude that the α1B‐AR is highly abundant throughout the brain, predominately in neurons, and may be involved in the development of the oligodendrocyte. In adult NG2 cells, implicated in stem cell‐like functions, the α1B‐AR may also play a role. This is the first report of a transgenic tagged‐GPCR approach to determine in vivo localization of a receptor. J. Comp. Neurol. 478:1–10, 2004.

Mice expressing the α1B‐adrenergic receptor induces a synucleinopathy with excessive tyrosine nitration but decreased phosphorylation

We had previously reported that systemic overexpression of the α1B‐adrenergic receptor (AR) in a transgenic mouse induced a neurodegenerative disease that resembled the parkinsonian‐like syndrome called multiple system atrophy (MSA). We now report that our mouse model has cytoplasmic inclusion bodies that colocalize with oligodendrocytes and neurons, are positive for α‐synuclein and ubiquitin, and therefore may be classified as a synucleinopathy. α‐Synuclein monomers as well as multimers were present in brain extracts from both normal and transgenic mice. However, similar to human MSA and other synucleinopathies, transgenic mice showed an increase in abnormal aggregated forms of α‐synuclein, which also increased its nitrated content with age. However, the same extracts displayed decreased phosphorylation of α‐synuclein. Other traits particular to MSA such as Purkinje cell loss in the cerebellum and degeneration of the intermediolateral cell columns of the spinal cord also exist in our mouse model but differences still exist between them. Interestingly, long‐term therapy with the α1‐AR antagonist, terazosin, resulted in protection against the symptomatic as well as the neurodegeneration and α‐synuclein inclusion body formation, suggesting that signaling of the α1B‐AR is the cause of the pathology. We conclude that overexpression of the α1B‐AR can cause a synucleinopathy similar to other parkinsonian syndromes.

Long-term α1A-adrenergic receptor stimulation improves synaptic plasticity, cognitive function, mood, and longevity.

The role of α1-adrenergic receptors (α1ARs) in cognition and mood is controversial, probably as a result of past use of nonselective agents. α1AAR activation was recently shown to increase neurogenesis, which is linked to cognition and mood. We studied the effects of long-term α1AAR stimulation using transgenic mice engineered to express a constitutively active mutant (CAM) form of the α1AAR. CAM-α1AAR mice showed enhancements in several behavioral models of learning and memory. In contrast, mice that have the α1AAR gene knocked out displayed poor cognitive function. Hippocampal brain slices from CAM-α1AAR mice demonstrated increased basal synaptic transmission, paired-pulse facilitation, and long-term potentiation compared with wild-type (WT) mice. WT mice treated with the α1AAR-selective agonist cirazoline also showed enhanced cognitive functions. In addition, CAM-α1AAR mice exhibited antidepressant and less anxious phenotypes in several behavioral tests compared with WT mice. Furthermore, the lifespan of CAM-α1AAR mice was 10% longer than that of WT mice. Our results suggest that long-term α1AAR stimulation improves synaptic plasticity, cognitive function, mood, and longevity. This may afford a potential therapeutic target for counteracting the decline in cognitive function and mood associated with aging and neurological disorders.

α1-Adrenergic Receptors Regulate Neurogenesis and Gliogenesis

The understanding of the function of α1-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 α1-adrenergic receptor subtypes in tissues in which they are normally expressed. We identified intriguing α1A-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 α1A-adrenergic receptor transgenic mice or normal mice given the α1A-adrenergic receptor-selective agonist, cirazoline. Neonatal neurospheres isolated from normal mice expressed a mixture of α1-adrenergic receptor subtypes, and stimulation of these receptors resulted in increased expression of the α1B-adrenergic receptor subtype, proneural basic helix-loop-helix transcription factors, and the differentiation and migration of neuronal progenitors for catecholaminergic neurons and interneurons. α1-Adrenergic receptor stimulation increased the apoptosis of astrocytes and regulated survival of neonatal neurons through phosphatidylinositol 3-kinase signaling. However, in adult normal neurospheres, α1-adrenergic receptor stimulation increased the expression of glial markers at the expense of neuronal differentiation. In vivo, S100-positive glial and βIII tubulin neuronal progenitors colocalized with either α1-adrenergic receptor subtype in the olfactory bulb. Our results indicate that α1-adrenergic receptors can regulate both neurogenesis and gliogenesis that may be developmentally dependent. Our findings may lead to new therapies to treat neurodegenerative diseases.

Novel α1-Adrenergic Receptor Signaling Pathways: Secreted Factors and Interactions with the Extracellular Matrix

α1-Adrenergic receptor (α1-ARs) subtypes (α1A, α1B, and α1D) regulate multiple signal pathways, such as phospholipase C, protein kinase C (PKC), and mitogen-activated protein kinases. We employed oligonucleotide microarray technology to explore the effects of both short- (1 h) and long-term (18 h) activation of the α1A-AR to enable RNA changes to occur downstream of earlier well characterized signaling pathways, promoting novel couplings. Polymerase chain reaction (PCR) studies confirmed that PKC was a critical regulator of α1A-AR-mediated gene expression, and secreted interleukin (IL)-6 also contributed to gene expression alterations. We next focused on two novel signaling pathways that might be mediated through α1A-AR stimulation because of the clustering of gene expression changes for cell adhesion/motility (syndecan-4 and tenascin-C) and hyaluronan (HA) signaling. We confirmed that α1-ARs induced adhesion in three cell types to vitronectin, an interaction that was also integrin-, FGF7-, and PKC-dependent. α1-AR activation also inhibited cell migration, which was integrin- and PKC-independent but still required secretion of FGF7. α1-AR activation also increased the expression and deposition of HA, a glycosaminoglycan, which displayed two distinct structures: pericellular coats and long cable structures, as well as increasing expression of the HA receptor, CD44. Long cable structures of HA can bind leukocytes, which this suggests that α1-ARs may be involved in proinflammatory responses. Our results indicate α1-ARs induce the secretion of factors that interact with the extracellular matrix to regulate cell adhesion, motility and proinflammatory responses through novel signaling pathways.

α1A-Adrenergic receptor prevents cardiac ischemic damage through PKCδ/GLUT1/4-mediated glucose uptake.

Abstract While α1-adrenergic receptors (ARs) have been previously shown to limit ischemic cardiac damage, the mechanisms remain unclear. Most previous studies utilized low oxygen conditions in addition to ischemic buffers with glucose deficiencies, but we discovered profound differences if the two conditions are separated. We assessed both mouse neonatal and adult myocytes and HL-1 cells in a series of assays assessing ischemic damage under hypoxic or low glucose conditions. We found that α1-AR stimulation protected against increased lactate dehydrogenase release or Annexin V+ apoptosis under conditions that were due to low glucose concentration not to hypoxia. The α1-AR antagonist prazosin or nonselective protein kinase C (PKC) inhibitors blocked the protective effect. α1-AR stimulation increased 3H-deoxyglucose uptake that was blocked with either an inhibitor to glucose transporter 1 or 4 (GLUT1 or GLUT4) or small interfering RNA (siRNA) against PKCδ. GLUT1/4 inhibition also blocked α1-AR-mediated protection from apoptosis. The PKC inhibitor rottlerin or siRNA against PKCδ blocked α1-AR stimulated GLUT1 or GLUT4 plasma membrane translocation. α1-AR stimulation increased plasma membrane concentration of either GLUT1 or GLUT4 in a time-dependent fashion. Transgenic mice overexpressing the α1A-AR but not α1B-AR mice displayed increased glucose uptake and increased GLUT1 and GLUT4 plasma membrane translocation in the adult heart while α1A-AR but not α1B-AR knockout mice displayed lowered glucose uptake and GLUT translocation. Our results suggest that α1-AR activation is anti-apoptotic and protective during cardiac ischemia due to glucose deprivation and not hypoxia by enhancing glucose uptake into the heart via PKCδ-mediated GLUT translocation that may be specific to the α1A-AR subtype.

Long-term α1B-adrenergic receptor activation shortens lifespan, while α1A-adrenergic receptor stimulation prolongs lifespan in association with decreased cancer incidence

The α1-adrenergic receptor (α1AR) subtypes, α1AAR and α1BAR, have differential effects in the heart and central nervous system. Long-term stimulation of the α1AAR subtype prolongs lifespan and provides cardio- and neuro-protective effects. We examined the lifespan of constitutively active mutant (CAM)-α1BAR mice and the incidence of cancer in mice expressing the CAM form of either the α1AAR (CAM-α1AAR mice) or α1BAR. CAM-α1BAR mice have a significantly shortened lifespan when compared with wild-type (WT) animals; however, the effect was sex dependent. Female CAM-α1BAR mice lived significantly shorter lives, while the median lifespan of male CAM-α1BAR mice was not different when compared with that of WT animals. There was no difference in the incidence of cancer in either sex of CAM-α1BAR mice. The incidence of cancer was significantly decreased in CAM-α1AAR mice when compared with that in WT, and no sex-dependent effects were observed. Further study is warranted on cancer incidence after activation of each α1AR subtype and the effect of sex on lifespan following activation of the α1BAR. The implications of a decrease in cancer incidence following long-term α1AAR stimulation could lead to improved treatments for cancer.

α1A-Adrenergic Receptors Regulate Cardiac Hypertrophy In Vivo Through Interleukin-6 Secretion

The role of α1-adrenergic receptors (ARs) in the regulation of cardiac hypertrophy is still unclear, because transgenic mice demonstrated hypertrophy or the lack of it despite high receptor overexpression. To further address the role of the α1-ARs in cardiac hypertrophy, we analyzed unique transgenic mice that overexpress constitutively active mutation (CAM) α1A-ARs or CAM α1B-ARs under the regulation of large fragments of their native promoters. These constitutively active receptors are expressed in all tissues that endogenously express their wild-type counterparts as opposed to only myocyte-targeted transgenic mice. In this study, we discovered that CAM α1A-AR mice in vivo have cardiac hypertrophy independent of changes in blood pressure, corroborating earlier studies, but in contrast to myocyte-targeted α1A-AR mice. We also found cardiac hypertrophy in CAM α1B-AR mice, in agreement with previous studies, but hypertrophy only developed in older mice. We also discovered unique α1-AR–mediated hypertrophic signaling that was AR subtype-specific with CAM α1A-AR mice secreting atrial naturietic factor and interleukin-6 (IL-6), whereas CAM α1B-AR mice expressed activated nuclear factor-κB (NF-κB). These particular hypertrophic signals were blocked when the other AR subtype was coactivated. We also discovered that crossbreeding the two CAM models (double CAM α1A/B-AR) inhibited the development of hypertrophy and was reversible with single receptor activation, suggesting that coactivation of the receptors can lead to novel antagonistic signal transduction. This was confirmed by demonstrating antagonistic signals that were even lower than normal controls in the double CAM α1A/B-AR mice for p38, NF-κB, and the IL-6/glycoprotein 130/signal transducer and activator of transcription 3 pathway. Because α1A/B double knockout mice fail to develop hypertrophy in response to IL-6, our results suggest that IL-6 is a major mediator of α1A-AR cardiac hypertrophy.