Yongquan Luo

D2 Dopamine Receptors Stimulate Mitogenesis Through Pertussis Toxin-Sensitive G Proteins and Ras-Involved ERK and SAP/JNK Pathways in Rat C6-D2L Glioma Cells

Abstract: Dopamine D2 receptors are members of the G protein‐coupled receptor superfamily and are expressed on both neurons and astrocytes. Using rat C6 glioma cells stably expressing the rat D2L receptor, we show here that dopamine (DA) can activate both the extracellular signal‐regulated kinase (ERK) and c‐Jun NH2‐terminal kinase (JNK) pathways through a mechanism involving D2 receptor‐G protein complexes and the Ras GTP‐binding protein. Agonist binding to D2 receptors rapidly activated both kinases within 5 min, reached a maximum between 10 and 15 min, and then gradually decreased by 60 min. Maximal activation of both kinases occurred with 100 nM DA, which produced a ninefold enhancement of ERK activity and a threefold enhancement of JNK activity. DA‐induced kinase activation was prevented by either (+)‐butaclamol, a selective D2 receptor antagonist, or pertussis toxin, an uncoupler of G proteins from receptors, but not by (−)‐butaclamol, the inactive isomer of (+)‐butaclamol. Cotransfection of RasN17, a dominant negative Ras mutant, prevented DA‐induced activation of both ERK and JNK. PD098059, a specific MEK1 inhibitor, also blocked ERK activation by DA. Transfection of SEK1(K → R) vector, a dominant negative SEK1 mutant, specifically prevented DA‐induced JNK activation and subsequent c‐Jun phosphorylation without effect on ERK activation. Furthermore, stimulation of D2 receptors promoted [3H]thymidine incorporation with a pattern similar to that for kinase activation. DA mitogenesis was tightly linked to Ras‐dependent mitogen‐activated protein kinase (MAPK) and JNK pathways. Transfection with RasN17 and application of PD098059 blocked DA‐induced DNA synthesis. Transfection with FlagΔ169, a dominant negative c‐Jun mutant, also prevented stimulation of [3H]thymidine incorporation by DA. The demonstration of D2 receptor‐stimulated MAPK pathways may help to understand dopaminergic physiological functions in the CNS.

The Roles of Dopamine Oxidative Stress and Dopamine Receptor Signaling in Aging and Age-Related Neurodegeneration

Aging is accompanied by a decline of functions controlled by the central dopaminergic system, such as reduced locomotor activity, motivation, impairment of memory formation, and learning deficits. The molecular mechanisms underlying age-related impairment of dopaminergic functions are unknown. Current literature and our own recent work, which are reviewed and summarized in the present paper, suggest that dopamine oxidative stress and its subsequent signaling may contribute to the aging of dopaminergic system.

Intrastriatal injection of dopamine results in DNA damage and apoptosis in rats

OVERFLOW of the neurotransmitter dopamine (DA) in striatum is implicated in the neurodegenerative processes in ischemia, hypoxia and local exposure to high concentrations of excitatory amino acids. However, how DA causes neurotoxicity is not understood. We report that intrastriatal injection of DA (0.5–1 μmol/μl) in Wistar rats produces a robust increase in apoptotic cell death as determined by both a terminal deoxynucleotidyl transferase catalyzed dUTP-biotin nick labeling (TUNEL) and Klenow polymerase catalyzed [32P]dCTP labeled DNA ladder. Cells in which apoptosis was induced by DA are characterized by condensed chromatin, DNA fragmentation, shrinkage and irregular shapes. The apoptotic cell death induced by DA is not due to the effect of hyperosmolar solution since intrastriatal injection of identical concentrations of NaCl on opposite sides of the same rat brains shows little TUNEL-positive labeling. The number of apoptotic cells is proportional to the amount of DA and length of exposure period. With DA concentrations from 0 to 1 μmol/μl, the maximal toxic effect appears at a concentration of 1 μmol/μl after 24 h exposure. Demonstration of DA-induced apoptosis in vivo may provide a potential molecular mechanism for DA neurotoxicity.

SDF1α/CXCR4 Signaling, via ERKs and the Transcription Factor Egr1, Induces Expression of a 67-kDa Form of Glutamic Acid Decarboxylase in Embryonic Hippocampal Neurons

Stromal cell-derived factor α (SDF1α) and its cognate receptor CXCR4 play an important role in neuronal development in the hippocampus, but the genes directly regulated by SDF1α/CXCR4 signaling are unknown. To study the role of CXCR4 targeted genes in neuronal development, we used neuronal cultures established from embryonic day 18 rats. Hippocampal neurons express CXCR4 receptor proteins and are stimulated by SDF1α resulting in activation of extracellular signal-regulated kinase (ERK)1/2 and the transcription factor cAMP-response element-binding protein. SDF1α rapidly induces the expression of the early growth response gene Egr1, a transcription factor involved in activity-dependent neuronal responses, in a concentration-dependent manner. Gel-shift analysis showed that SDF1α enhances DNA binding activity to the Egr1-containing promoter for GAD67. Chromatin immunoprecipitation analysis using an Egr1 antibody indicated that SDF1α stimulation increases binding of Egr1 to a GAD67 promoter DNA sequence. SDF1α stimulation increases the expression of GAD67 at both the mRNA and protein levels, and increases the amount and neurite localization of γ-aminobutyric acid (GABA) in neurons already expressing GABA. SDF1α-induced Egr1/GAD67 expression is mediated by the G protein-coupled CXCR4 receptor and activation of the ERK pathway. Reduction of Egr1 gene expression using small interfering RNA technology lowers the level of GAD67 transcripts and inhibits SDF1α-induced GABA production. Inhibition of CXCR4 activation in the developing mouse brain in utero greatly reduced Egr1 and GAD67 mRNA levels and GAD67 protein levels, suggesting a pivotal role for CXCR4 signaling in the development of GABAergic neurons in vivo. Our data suggest that SDF1α/CXCR4/G protein/ERK signaling induces the expression of the GAD67 system via Egr1 activation, a mechanism that may promote the maturation of GABAergic neurons during development.

Physiological levels of β-amyloid increase tyrosine phosphorylation and cytosolic calcium

The a beta peptide is a neurotoxic peptide that accumulates in the brains of Alzheimer patients, but is also present in body fluids at subnanomolar levels. The potential effects of these low levels of a beta are unclear. We now show that one such action is to increase tyrosine phosphorylation in PC12 cells and olfactory neuroblasts. Application of a beta 25-35 or a beta 1-40 induces a dose-dependent increase in the tyrosine phosphorylation in both whole cells and in vitro. The increase in tyrosine phosphorylation is both rapid and sensitive, being stimulated by picomolar doses of a beta and occurring within 1 min of application. Calcium imaging experiments provide further support for the role of tyrosine phosphorylation in the action of a beta. While a beta does not alter calcium metabolism under basal conditions, the addition of a beta induces a rapid increase in cytoplasmic calcium in olfactory neuroblasts that have been treated with the tyrosine phosphatase inhibitor, sodium orthovanadate or in PC12 cells treated with nerve growth factor. These responses could be blocked by the tyrosine kinase inhibitor, herbimycin. These calcium responses displayed an obligate requirement for the presence of matrix proteins. The identification of a rapid, sensitive assay for the action of a beta may facilitate investigations of its mechanism of action.

Physiological levels of β-amyloid peptide stimulate protein kinase C in PC12 cells

Alzheimers beta-amyloid peptide (A beta) is normally present at nanomolar concentrations in body fluids and in the medium of cultured cells. In vitro experiments have shown that A beta has neurotrophic effects and can promote neuronal adhesion and elongation of axon-like processes. In an attempt to understand the molecular mechanisms underlying such effects, we have recently reported that nanomolar doses of A beta can stimulate protein tyrosine phosphorylation and activate phosphatidylinositol-3-kinase in neuronal cells. Here we show evidence that A beta can also activate protein kinase C, a serine/threonine kinase, in PC12 cells. First, using a serine-containing S6 peptide as an exogenous substrate, we found that nanomolar levels of A beta peptides 1-40 or 1-42 significantly stimulated an S6 phosphorylating kinase activity, whereas the A beta40-1 reverse sequence peptide had no effect. Down-regulation of PKC by prolonged (18 h) treatment with 1 microM PMA prevented the A beta-induced S6 phosphorylation. Using a more specific PKC substrate, N-terminal acetylated peptide (4-14) from myelin basic protein, we then demonstrated that A beta indeed increased PKC activity and that this activity could be blocked by the PKC inhibitor, staurosporine. Finally, immunoblotting experiments showed that A beta induced translocation of PKCgamma from cytosol to membrane and also significantly reduced cytosolic PKCalpha levels. Taken together, these data suggest that physiological levels of A beta can regulate PKC activity.

Plumbagin Promotes the Generation of Astrocytes from Rat Spinal Cord Neural Progenitors Via Activation of the Transcription Factor Stat3

J. Neurochem. (2010) 115, 1337–1349.

Dopamine stimulates redox-tyrosine kinase signaling and p38 MAPK in activation of astrocytic C6-D2l cells

An increase in dopamine (DA) availability in rat brain has been suggested to participate in certain neurodegenerative processes. However, the regulatory effects of DA on glial cells have not been extensively studied. Using a rat C6 glioma cell line stably expressing recombinant D2L receptors, we have found that micromolar levels of DA stimulate mitogenesis and glial fibrillary acidic protein (GFAP) expression, both serving as parameters of reactive gliosis. This mitogenesis occurs about 29 h after exposure to DA and requires D2-receptor-mediated intracellular redox-tyrosine kinase activation. Either DA or quinpirole, a D2 receptor agonist, stimulates protein tyrosine phosphorylation. Application of either DPI, a potent inhibitor of NADPH-dependent oxidase, or NAC, an anti-oxidant, effectively prevented DA-induced tyrosine phosphorylation and DNA synthesis. Preincubation of (+)-butaclamol, a D2 receptor antagonist, inhibits both DA-stimulated tyrosine phosphorylation and mitogenesis. DA at micromolar levels also stimulates GFAP expression. This DA-regulated GFAP expression can be completely inhibited by SB203580, a selective p38 MAPK inhibitor, but not influenced by (+)-butaclamol and genistein, a protein tyrosine kinase inhibitor. Thus, our data suggest that regulation of DNA synthesis and GFAP expression induced by DA is mediated by independent signaling pathways. The mitogenesis requires a D2-receptor-mediated protein tyrosine kinase cascade, while GFAP expression needs a D2-receptor-independent p38 MAPK activation. This observation may help to understand the processes of reactive gliosis in some dopaminergic-related neurodegenerative diseases.

Transforming growth factor β induces aβ-responsive calcium fluxes in neurons

The beta-amyloid (a beta) peptide is a neurotoxic peptide that accumulates in the brains of Alzheimer patients, but is also present in body fluids at subnanomolar levels. The potential effects of these low levels of a beta are unclear. We have recently shown that physiologic levels of a beta increase tyrosine phosphorylation and induce increases in cytosolic calcium. The basement membrane mixture, Matrigel, is required for observation of the a beta-induced calcium response. We now show that transforming growth factor beta (TGF beta) is the active component in Matrigel eliciting the a beta/calcium response. The response to the type of TGF beta varies depending on the cell type with TGF beta 1 eliciting a beta responsiveness in olfactory neuroblasts, and TGF beta 2 eliciting a beta responsiveness in PC12 cells.

Dopamine Stimulates Astrocytic C6‐D2L Cells via Tyrosine Kinase and p38 MAPK Activation

Dopamine (DA), a neurotransmitter of the central nervous system, plays a fundamental role in the control of a variety of physiological functions including locomotor activity, learning, reward behavior, and hormone synthesis and release. 1 However, overflow of DA in the brain has been suggested to participate in certain neurodegenerative processes, which include ischemia, 2 hypoxia, 3 and neurotoxicities induced by excitatory amino acids 4 and methamphetamine. 5 For example, the striatal DA concentration rapidly reaches concentrations as high as 0.2 mM after ligation of the cervical artery in the gerbil ischemic model. 6 Decrease in the endogenous DA concentration by chemical lesion of the nigrostriatal dopaminergic pathway attenuates ischemic insult to the striatum. 7 Moreover, direct administration of DA into striata results in apoptosis and neurodegeneration. 8,9,32 Using in vitro neonatal striatal cell cultures, we have demonstrated that DA induces apoptosis through an oxidationJNK-c-Jun activation pathway. 10