Abstract WP141: Cyclic-AMP Induces Nogo-A Receptor NgR1 Internalization and Inhibits Nogo-A-Mediated Collapse of Growth Cone

Recovery of stroke and neuronal injuries requires the promotion of axonal regeneration from the remaining neurons. However, axonal regeneration is inhibited by diverse axonal growth inhibitors, such as Nogo-A. C-terminal domain of Nogo-A, Nogo-66 binds to the Nogo-A receptor 1 (NgR1) and induces the collapse of growth cones and inhibition of neurite outgrowth. NgR1 is also a receptor for additional axonal growth inhibitors. In this study, by using indirect immunofluorescence and biotinylation method, we have found that a cell-permeable cAMP analog (dibutyryl-cAMP) and other intracellular cAMP-elevating agents, such as forskolin, which directly activates adenylyl cyclase, and rolipram, which inhibits cyclic nucleotide phosphodiesterase, all induced rapid internalization of the cell surface NgR1 in Neuroscreen-1 (NS-1) cells. This endocytosis of NgR1 is lipid raft mediated. These cAMP-elevating agents induced a reversible distribution of NgR1 between the cell surface and intracellular compartment; NgR1 distributed to the cell surface at low levels of cAMP and distributed to an intracellular compartment at high levels of cAMP. Using pharmacological activators and inhibitors of protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac), we found that NgR1 internalization is independent of PKA but dependent on Epac. There is a correlation between the decrease in cell surface expression of NgR1 decreased sensitivity of NS-1 cells to Nogo-66-induced growth cone collapse. Therefore, besides axonal growth inhibitors affecting neurons, neurons by themselves self-regulate their own sensitivity to extracellular cues such as axonal growth inhibitors. This normal cellular regulatory mechanism may be therapeutically applied to overcome axonal growth inhibitors and enhance functional recovery after stroke and neuronal injuries.

Download Full-text

Abstract P803: Pituitary Adenylate Cyclase-Activating Polypeptide via Cyclic-AMP Inhibits Nogo-A by Internalizing Its Receptor NgR1

Stroke ◽

10.1161/str.52.suppl_1.p803 ◽

2021 ◽

Vol 52 (Suppl_1) ◽

Author(s):

Rayudu GOPALAKRISHNA ◽

Charlotte Y Lin ◽

Andrew Oh ◽

William J Mack

Keyword(s):

Cyclic Amp ◽

Cell Surface ◽

Adenylate Cyclase ◽

Specific Inhibitor ◽

Axonal Growth ◽

Surface Expression ◽

Cell Surface Expression ◽

Growth Inhibitors ◽

Immunofluorescence Method ◽

Pituitary Adenylate Cyclase

Introduction: After a stroke, axonal regeneration is inhibited by diverse axonal growth inhibitors, such as Nogo-A. They bind to the Nogo-A receptor 1 (NgR1) and induce the collapse of growth cones and inhibit neurite outgrowth. Since NgR1 is the receptor for a variety of axonal growth inhibitors, it is a crucial target for the prevention of axonal growth inhibition. Pituitary adenylate cyclase-activating polypeptide (PACAP) has neuroprotective and neurotrophic activities and increases neuritogenesis and synaptic plasticity. It enhances functional recovery after stroke in various animal models. Methods: Neuroscreen-1 (NS-1) cells were selected for this study as they produce rapid and robust neurite outgrowth with NGF. Cell surface NgR1 was detected using the indirect immunofluorescence method. The internalization of NgR1 was quantitated using the biotinylation method and Western immunoblotting. Results: Using the indirect immunofluorescence method, we found that PACAP (PACAP-38) induced a rapid decrease in the cell surface expression of NgR1 in NS-1 cells. The biotinylation method revealed that PACAP induced the internalization of NgR1. This internalization of NgR1 was blocked by pretreatment of NS-1 cells with SQ 22536, an inhibitor for adenylate cyclase, suggesting that cAMP plays a crucial role in the internalization of NgR1. The protein kinase A (PKA)-specific inhibitor KT5720 did not block PACAP-induced NgR1 internalization, whereas the exchange protein directly activated by cAMP (Epac)-specific inhibitor ESI-09 blocked this internalization. Collectively, this data suggests that PACAP-induced NgR1 internalization is independent of PKA but is dependent on Epac. The PACAP-induced decrease in cell surface expression of NgR1 and its internalization desensitized NS-1 cells to Nogo-66-induced growth cone collapse and enhanced neuritogenesis. Conclusion: Cyclic-AMP and Epac are involved in the PACAP-induced desensitization of neuronal cells to Nogo-A and increase in neuritogenesis. Since PACAP crosses the blood-brain barrier, it may be a useful therapeutic agent to overcome axonal growth inhibitors and enhance functional recovery after stroke.

Download Full-text

GLUT8 Subcellular Localization and Absence of Translocation to the Plasma Membrane in PC12 Cells and Hippocampal Neurons

Endocrinology ◽

10.1210/en.2005-0668 ◽

2005 ◽

Vol 146 (11) ◽

pp. 4727-4736 ◽

Cited By ~ 20

Author(s):

Mathieu Widmer ◽

Marc Uldry ◽

Bernard Thorens

Keyword(s):

Plasma Membrane ◽

Protein Kinase ◽

Cell Surface ◽

Pc12 Cells ◽

Hippocampal Neurons ◽

Osmotic Shock ◽

Surface Expression ◽

Cell Surface Expression ◽

Intracellular Compartment ◽

Early Endosomes

GLUT8 is a high-affinity glucose transporter present mostly in testes and a subset of brain neurons. At the cellular level, it is found in a poorly defined intracellular compartment in which it is retained by an N-terminal dileucine motif. Here we assessed GLUT8 colocalization with markers for different cellular compartments and searched for signals, which could trigger its cell surface expression. We showed that when expressed in PC12 cells, GLUT8 was located in a perinuclear compartment in which it showed partial colocalization with markers for the endoplasmic reticulum but not with markers for the trans-Golgi network, early endosomes, lysosomes, and synaptic-like vesicles. To evaluate its presence at the plasma membrane, we generated a recombinant adenovirus for the expression of GLUT8 containing an extracellular myc epitope. Cell surface expression was evaluated by immunofluorescence microscopy of transduced PC12 cells or primary hippocampal neurons exposed to different stimuli. Those included substances inducing depolarization, activation of protein kinase A and C, activation or inhibition of tyrosine kinase-linked signaling pathways, glucose deprivation, AMP-activated protein kinase stimulation, and osmotic shock. None of these stimuli-induced GLUT8 cell surface translocation. Furthermore, when GLUT8myc was cotransduced with a dominant-negative form of dynamin or GLUT8myc-expressing PC-12 cells or neurons were incubated with an anti-myc antibody, no evidence for constitutive recycling of the transporter through the cell surface could be obtained. Thus, in cells normally expressing it, GLUT8 was associated with a specific intracellular compartment in which it may play an as-yet-uncharacterized role.

Download Full-text

Efficiency of acetylcholine receptor subunit assembly and its regulation by cAMP.

The Journal of Cell Biology ◽

10.1083/jcb.113.3.623 ◽

1991 ◽

Vol 113 (3) ◽

pp. 623-636 ◽

Cited By ~ 48

Author(s):

A F Ross ◽

W N Green ◽

D S Hartman ◽

T Claudio

Keyword(s):

Cell Surface ◽

Acetylcholine Receptor ◽

Cell Lines ◽

Fibroblast Cell ◽

Surface Expression ◽

Mouse Fibroblast ◽

Cell Surface Expression ◽

Intracellular Camp ◽

Temperature Sensitive ◽

Subunit Assembly

Assembly of nicotinic acetylcholine receptor (AChR) subunits was investigated using mouse fibroblast cell lines stably expressing either Torpedo (All-11) or mouse (AM-4) alpha, beta, gamma, and delta AChR subunits. Both cell lines produce fully functional cell surface AChRs. We find that two independent treatments, lower temperature and increased intracellular cAMP can increase AChR expression by increasing the efficiency of subunit assembly. Previously, we showed that the rate of degradation of individual subunits was decreased as the temperature was lowered and that Torpedo AChR expression was acutely temperature sensitive, requiring temperatures lower than 37 degrees C. We find that Torpedo AChR assembly efficiency increases 56-fold as the temperature is decreased from 37 to 20 degrees C. To determine how much of this is a temperature effect on degradation, mouse AChR assembly efficiencies were determined and found to be only approximately fourfold more efficient at 20 than at 37 degrees C. With reduced temperatures, we can achieve assembly efficiencies of Torpedo AChR in fibroblasts of 20-35%. Mouse AChR in muscle cells is also approximately 30% and we obtain approximately 30% assembly efficiency of mouse AChR in fibroblasts (with reduced temperatures, this value approaches 100%). Forskolin, an agent which increases intracellular cAMP levels, increased subunit assembly efficiencies twofold with a corresponding increase in cell surface AChR. Pulse-chase experiments and immunofluorescence microscopy indicate that oligomer assembly occurs in the ER and that AChR oligomers remain in the ER until released to the cell surface. Once released, AChRs move rapidly through the Golgi membrane to the plasma membrane. Forskolin does not alter the intracellular distribution of AChR. Our results indicate that cell surface expression of AChR can be regulated at the level of subunit assembly and suggest a mechanism for the cAMP-induced increase in AChR expression.

Download Full-text

Faculty Opinions recommendation of Assembly and cell surface expression of TAP-independent, chloroquine-sensitive and interferon-gamma-inducible class I MHC complexes in transformed fibroblast cell lines are regulated by tapasin.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1009214.121258 ◽

2002 ◽

Author(s):

Tim Elliott

Keyword(s):

Cell Surface ◽

Cell Lines ◽

Interferon Gamma ◽

Fibroblast Cell ◽

Surface Expression ◽

Class I ◽

Cell Surface Expression ◽

Class I Mhc ◽

Fibroblast Cell Lines

Download Full-text

Faculty Opinions recommendation of Mechanism of cell surface expression of the Streptococcus mitis platelet binding proteins PblA and PblB.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1082761.535676 ◽

2007 ◽

Author(s):

Tim Mitchell

Keyword(s):

Cell Surface ◽

Binding Proteins ◽

Surface Expression ◽

Cell Surface Expression ◽

Streptococcus Mitis ◽

Platelet Binding

Download Full-text

Persistent HIV Transcription Induces Sialyl-Lewis-X Glyco-Antigen Cell-Surface Expression

SSRN Electronic Journal ◽

10.2139/ssrn.3565035 ◽

2020 ◽

Author(s):

Florent Colomb ◽

Leila B. Giron ◽

Leticia Kuri Cervantes ◽

Tongcui Ma ◽

Samson Adeniji ◽

...

Keyword(s):

Cell Surface ◽

Surface Expression ◽

Sialyl Lewis X ◽

Cell Surface Expression ◽

Lewis X ◽

Hiv Transcription ◽

Sialyl Lewis

Download Full-text

Influence of β-D-mannuronic acid, as a new member of non-steroidal anti-inflammatory drugs family, on expression pattern of chemokines and their receptors in rheumatoid arthritis

Current Drug Discovery Technologies ◽

10.2174/1570163816666191023103118 ◽

2019 ◽

Vol 16 ◽

Author(s):

Mona Aslani ◽

Arman Ahmadzadeh ◽

Zahra Aghazadeh ◽

Majid Zaki-Dizaji ◽

Laleh Sharifi ◽

...

Keyword(s):

Cell Surface ◽

Mrna Expression ◽

Surface Expression ◽

Phase Iii ◽

Cell Surface Expression ◽

Optimum Dose ◽

High Dose ◽

Mannuronic Acid ◽

Anti Inflammatory ◽

High Doses

Background: : Based on the encouraging results of phase III clinical trial of β-D-mannuronic acid (M2000) (as a new anti-inflammatory drug) in patients with RA, in this study, we aimed to evaluate the effects of this drug on the expression of chemokines and their receptors in PBMCs of RA patients. Methods:: PBMCs of RA patients and healthy controls were separated and the patients' cells were treated with low, moderate and high doses (5, 25 and 50 μg/mL) of M2000 and optimum dose (1 μg/mL) of diclofenac, as a control in RPMI-1640 medium. Real-time PCR was used for evaluating the mRNA expression of CXCR3, CXCR4, CCR2, CCR5 and CCL2/MCP-1. Cell surface expression of CCR2 was investigated using flow cytometry. Results:: CCR5 mRNA expression reduced significantly, after treatment of the patients' cells with all three doses of M2000 and optimum dose of diclofenac. CXCR3 mRNA expression down-regulated significantly followed by treatment of these cells with moderate and high doses of M2000 and optimum dose of diclofenac. CXCR4 mRNA expression declined significantly after treatment of these cells with moderate and high doses of M2000. CCL2 mRNA expression significantly reduced only followed by treatment of these cells with high dose of M2000, whereas, mRNA and cell surface expressions of CCR2 diminished significantly followed by treatment of these cells with high dose of M2000 and optimum dose of diclofenac. Conclusion:: According to our results, M2000 through the down-regulation of chemokines and their receptors may restrict the infiltration of immune cells into the synovium.

Download Full-text