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Cyclic-AMP induces Nogo-A receptor NgR1 internalization and inhibits Nogo-A-mediated collapse of growth cone
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Cyclic-AMP induces Nogo-A receptor NgR1 internalization and inhibits Nogo-A-mediated collapse of growth cone Rayudu Gopalakrishna a, *, Aubree Mades a, Andrew Oh a, Angela Zhu a, Julie Nguyen a, Charlotte Lin a, Mark S. Kindy b, William J. Mack c a
Department of Integrative Anatomical Sciences, Keck School of Medicine, University of Southern California, Los Angeles, CA, 90089, USA Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, 33612, USA, James A. Haley VA Medical Center, Tampa, FL, 33612, USA c Department of Neurological Surgery, Keck School of Medicine, University of Southern California, Los Angeles, CA, 90089, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 December 2019 Accepted 3 January 2020 Available online xxx
The promotion of axonal regeneration is required for functional recovery from stroke and various neuronal injuries. However, axonal regeneration is inhibited by diverse axonal growth inhibitors, such as Nogo-A. Nogo-66, a C-terminal domain of Nogo-A, binds to the Nogo-A receptor 1 (NgR1) and induces the collapse of growth cones and inhibits neurite outgrowth. NgR1 is also a receptor for additional axonal growth inhibitors, suggesting it is an important target for the prevention of axonal growth inhibition. By using the indirect immunofluorescence method, we show for the first time that a cell-permeable cAMP analog (dibutyryl-cAMP) induced a rapid decrease in the cell surface expression of NgR1 in Neuroscreen1 (NS-1) cells. The biotinylation method revealed that cAMP indeed induced internalization of NgR1 within minutes. Other intracellular cAMP-elevating agents, such as forskolin, which directly activates adenylyl cyclase, and rolipram, which inhibits cyclic nucleotide phosphodiesterase, also induced this process. This internalization was found to be reversible and influenced by intracellular levels of cAMP. Using selective 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. The decrease in cell surface expression of NgR1 desensitized NS-1 cells to Nogo-66-induced growth cone collapse. Therefore, it is likely that besides axonal growth inhibitors affecting neurons, neurons themselves also self-regulate their sensitivity to axonal growth inhibitors, as influenced by intracellular cAMP/Epac. This normal cellular regulatory mechanism may be pharmacologically exploited to overcome axonal growth inhibitors, and enhance functional recovery after stroke and neuronal injuries. © 2020 Elsevier Inc. All rights reserved.
Keywords: Nogo-A cAMP NgR1 Axonal growth inhibitors Exchange protein directly activated by cAMP Growth cone
1. Introduction Functional recovery from stroke and other neuronal injuries requires the promotion of axonal regeneration from remaining neurons [1e5]. However, axonal regeneration is inhibited by diverse axonal growth inhibitors, such as Nogo-A, myelin-associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMgp), and chondroitin sulfate proteoglycans (CSPGs). Nogo-A, produced by oligodendrocytes and neurons, has emerged as a key
Abbreviations: NgR1, Nogo-A receptor 1; PKA, protein kinase A; Epac, exchange protein directly activated by cAMP; NS-1, Neuroscreen-1; PDE, cyclic nucleotide phosphodiesterase. * Corresponding author. Department of Integrative Anatomical Sciences, 1333 San Pablo Street, Keck School of Medicine, Los Angeles, CA, 90089, USA. E-mail address: [email protected] (R. Gopalakrishna).
axonal growth inhibitor [6]. Nogo-A has two distinct antineuritogenic domains: N-terminal amino-Nogo (Nogo-A-D20) and C-terminal Nogo-66 [7]. Nogo-66 binds to Nogo-A receptor 1 (NgR1) [8,9]. NgR1 can also serve as the receptor for other axonal growth inhibitors, such as MAG, OMgp, and CSPGs, and as such, it is an important mediator for axonal growth inhibition [10e13]. NgR1 signals through transmembrane coreceptors p75NTR (or TROY) and LINGO-1, leading to the activation of RhoA, which in turn activates its effector, RhoA-associated protein kinase (ROCK) [14]. This signaling cascade ultimately leads to actin cytoskeletal reorganization and the collapse of growth cones, inhibiting neurite outgrowth. Currently, therapies are targeting Nogo-A, MAG, NgR1, RhoA, and ROCK for drug development to enhance axonal sprouting and functional recovery after stroke and spinal cord injury [15,16]. Cyclic-AMP plays a crucial role in overcoming neurite outgrowth inhibition caused by myelin [17] and helping improve recovery
https://doi.org/10.1016/j.bbrc.2020.01.009 0006-291X/© 2020 Elsevier Inc. All rights reserved.
Please cite this article as: R. Gopalakrishna et al., Cyclic-AMP induces Nogo-A receptor NgR1 internalization and inhibits Nogo-A-mediated collapse of growth cone, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.009
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Fig. 1. Specificity of NgR1 staining and internalization of NgR1 by dibutyryl-cAMP, forskolin, and rolipram. (A) Confocal images and quantitative image densitometric analysis of NgR1 staining of “not permeabilized” NS-1 cells transfected with Accell NgR1 siRNA; cell-surface-associated NgR1 was detected by indirect immunofluorescence staining (red). Nuclei stained blue by DAPI. Image density was normalized to one cell (one nucleus) and expressed as percentage of the control. Where indicated, the values represent mean ± SE
Please cite this article as: R. Gopalakrishna et al., Cyclic-AMP induces Nogo-A receptor NgR1 internalization and inhibits Nogo-A-mediated collapse of growth cone, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.009
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from the neuronal injuries [18]. Embryonic neurons have higher cAMP levels than adult neurons, causing axonal growth inhibitors to promote rather than inhibit the growth of these neurons [19]. On the other hand, these axonal growth inhibitors inhibit adult neurons with lower cAMP levels. Cyclic-AMP may block the actions of axonal growth inhibitors by inducing a transcriptional activation of arginase-1 and subsequent elevation of polyamines [17]. In some cases, cAMP can still rapidly prevent the action of axonal growth inhibitors without involving transcriptional activation through an unknown mechanism [20]. Cyclic-AMP mediates its actions primarily through two effectors, protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac) [21,22]. While PKA is still an important player in mediating cAMP actions, some cAMP-mediated actions previously believed to be mediated by PKA may indeed be mediated instead by Epac [22]. There are two isoforms of Epac proteins: Epac 1, which is ubiquitously distributed in all tissues, and Epac 2, which is present at higher levels in neurons [21]. Epac is involved in mediating various cellular processes in neurons, such as apoptosis, neurotransmitter release, and axon growth [23]. The relative contribution of PKA and Epac in the cAMP-mediated actions to overcome axonal growth inhibitors is not known. Here we show for the first time that cell-permeable cAMP (dibutyryl-cAMP) and other agents that increase intracellular cAMP (forskolin and rolipram) rapidly induce the internalization of NgR1 and desensitize Neuroscreen-1 (NS-1) neuronal-like cells to Nogo66-induced growth cone collapse. This internalization is mediated by Epac. We further show that with a subsequent decrease in intracellular cAMP, NgR1 returns back to the cell surface. 2. Materials and methods
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were selected for this study because they produce rapid and robust neurite outgrowth with NGF. PKA-deficient PC12 cells (A132.7) that were originally cloned by Dr. John Wagner, and parent PC12 cells were kind gifts from Dr. Louis Hersh (University of Kentucky, Lexington). NS-1 cells, PKA-deficient PC12 cells, and parent PC12 cells were grown in flasks coated with poly-L-lysine in RPMI medium supplemented with 10% heat-inactivated horse serum, 5% fetal calf serum, 50 units/ml penicillin, and 0.05 mg/ml streptomycin. 2.3. Indirect immunofluorescence NS-1 cells were grown on 12-mm glass coverslips previously coated with poly-D-lysine to 50% confluency and treated with dibutyryl-cAMP, forskolin, or rolipram for 1 h. Cell surface NgR1 was detected in paraformaldehyde-fixed cells that were not permeabilized, while intracellular NgR1 was detected in cells permeabilized with 0.25% Triton-X-100. After blocking with 5% goat serum, cells were incubated with rabbit anti-NgR1 antibody (1:300 dilution) for 24 h at 4 C. Cells were then incubated with anti-rabbit goat secondary antibody conjugated with Alexa Fluor 594 for 1 h at room temperature. Nuclei were visualized with DAPI. Presented images were taken in a blinded fashion using an LSM 800 Zeiss confocal microscope and 63 1.4NA oil objective. Twelve images were collected for the stack, and the imaging intensity was quantitated using ImageJ software [24]. 2.3.1. Knockdown of NgR1 by siRNA transfection NS-1 cells were plated on glass coverslips. After 24 h, the cells were incubated with 1 mM Accell NgR1 siRNA in a delivery medium for 4 days, according to the manufacturer’s instructions. Then, NgR1 was detected using immunofluorescence staining. As a negative control, we used Accell non-targeting siRNA.
2.1. Materials Dibutyryl-cAMP, dibutyryl-cGMP, forskolin, rolipram, and 40 60 diamidino-2-phenylindole (DAPI) were obtained from SigmaAldrich. Mouse nerve growth factor (NGF), KT5720, and ESI-09 were from EMD Millipore. N6-benzoyl-cAMP and 8-(4chlorophenylthio)-20 -O-methyl-cAMP (8-pCPT) were from Cayman Chemical. Recombinant Nogo-A-Fc chimera, in which Nogo-A (aa1026-1090) is fused with mouse IgG2a (referred to as Nogo-66 throughout this study) was obtained from R&D Systems. Alexa Fluor 568-conjugated phalloidin was from Molecular Probes. NgR1 rabbit polyclonal antibody (H-120) which recognizes rat NgR1 was from Santa Cruz Biotechnology. The recombinant monoclonal rabbit antibody to NgR1 (clone M5) was from Absolute Antibody. An anti-rabbit secondary antibody conjugated with Alexa Fluor 594 and a goat anti-mouse IgG2a were from Jackson ImmunoResearch. Accell rat NgR1 siRNA, non-targeting siRNA control, and delivery medium were from Dharmacon. Phosphatidylinositolspecific phospholipase C from Bacillus cereus was from Invitrogen. 2.2. Cell culture Neuroscreen-1 (NS-1) cells (a clone derived from PC12 cells)
2.3.2. Removal of glycosylphosphatidylinositol(GPI)-anchored cellsurface associated proteins NS-1 cells, in serum-free medium, were incubated with phosphatidylinositol-specific phospholipase C (0.5 U/ml) for 2 h at 37 C. This treatment specifically removes GPI-anchored proteins from the cell surface. Then, the cells were washed and the cellsurface associated NgR1 was detected in “not permeabilized” cells. 2.4. Biotinylation assay for internalization NgR1 internalization was determined by biotinylation as described previously [25]. NS-1 cells were labeled with sulfoeNHSeSS-biotin (0.8 mg/ml) for 30 min at 4 C. Labeled cells were transferred to the incubator at 37 C and treated with dibutyryl-cAMP (200 mM), forskolin (5 mM), or rolipram (2 mM) for 15 min to induce internalization of NgR1. Then, cells were treated with glutathione to cleave the biotin-associated SeS bond to selectively strip it from the cell surface. Cell extracts were prepared by homogenization and the internalized biotinylated proteins were isolated with streptavidin-agarose beads. Proteins that eluted from these beads were subjected to Western immunoblotting using an anti-NgR1 antibody.
from three replicate estimations. Statistically different from control (*p < 0.05). (B) Immunofluorescence staining and quantitative image densitometric analysis of cell-surfaceassociated NgR1 of impermeable cells treated with phosphatidylinositol-specific phospholipase C to remove GPI-anchored cell surface proteins. (C) Confocal images and quantitative image densitometric analysis of cell surface NgR1 in cells treated with cAMP-elevating agents. NS-1 cells were treated with 200 mM dibutyryl-cAMP, or cAMP-generating agents 5 mM forskolin, and 2 mM rolipram for 1 h. Cell surface NgR1 was detected with “not permeabilized” cells. (D) Confocal images and quantitative image densitometric analysis of NgR1 in detergent-permeabilized cells. (E) Internalization of biotinylated cell-surface NgR1 by dibutyryl-cAMP, forskolin, and rolipram. NS-1 cell-surface proteins were biotinylated and treated with indicated agents for 15 min. By using streptavidin beads, the internalized biotinylated proteins were isolated and subjected to Western immunoblotting for NgR1. Total biotinylated protein that was not internalized and protein left after stripping with glutathione were shown. (F) Western immunoblotting of total (membrane and intracellular) NgR1 present in NS-1 cells treated with dibutyryl-cAMP, forskolin and rolipram for 1 h. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: R. Gopalakrishna et al., Cyclic-AMP induces Nogo-A receptor NgR1 internalization and inhibits Nogo-A-mediated collapse of growth cone, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.009
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2.5. Western immunoblotting Protein samples were subjected to SDS-polyacrylamide gel electrophoresis and the separated proteins were transferred to polyvinylidene fluoride membrane [26]. The blocked membranes were incubated with anti-NgR1 rabbit antibodies followed by goat anti-rabbit secondary antibodies conjugated with horseradish peroxidase. The immunoreactive bands were visualized by the SuperSignal West Femato Maximum Sensitivity Substrate kit (Pierce). These bands were analyzed by densitometric scanning using the Omega 12IC Molecular Imaging System.
was pre-aggregated with 125 ng/ml goat anti-mouse IgG2a. The cAMP-pretreated cells were then incubated with pre-aggregated Nogo-66 (12.5 nM) for 30 min. The treated cells were then fixed with glutaraldehyde, permeabilized with 0.1% Triton-X-100, and subsequently blocked with 1% bovine serum albumin. The cells were then stained with Alexa Fluor 586-conjugated phalloidin for 20 min. After washing the cells, the coverslips were mounted with Fluoromount medium, and images were taken in a blinded fashion using an LSM 800 Zeiss confocal microscope. The results are expressed as the percentage of collapsed growth cones to total counted growth cones [25].
2.6. Growth cone collapse assay 2.7. Statistical analysis We measured the collapse of actin-rich filopodia and lamellipodia-like structures, which are referred to as growth cones in this paper. NS-1 cells were grown on 12-mm glass coverslips and treated with NGF (50 ng/ml) for four days to induce neuronal differentiation. Then, the neuronal-like NS-1 cells were pretreated with dibutyryl-cAMP (200 mM) or forskolin (5 mM) for 1 h. Nogo-66
For NgR1 internalization and growth cone analysis, the data is expressed as the mean ± SE and analyzed using one-way analysis of variance, followed by post hoc Scheffe’s test. p < 0.05 was considered statistically significant. Statistical analysis was performed using StatView software.
Fig. 2. Role of PKA and Epac in cAMP-induced internalization of NgR1. (A) Confocal images showing cell surface NgR1 staining in NS-1 cells treated with activators and inhibitors of PKA and Epac. Where indicated, NS-1 cells were initially pretreated with 200 nM KT5720 or 10 mM ESI-09 for 1 h. Then, cells were treated with 0.5 mM N6-benzoyl-cAMP, 10 mM 8pCPT or 200 mM dibutyryl-cAMP for 1 h. (B) Quantitative image densitometric analysis of NgR1 staining from confocal images. The values represent mean ± SE from three replicate estimations. Statistical difference between means is indicated with (*p < 0.05).
Please cite this article as: R. Gopalakrishna et al., Cyclic-AMP induces Nogo-A receptor NgR1 internalization and inhibits Nogo-A-mediated collapse of growth cone, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.009
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3. Results
3.3. Reversal of NgR1 internalization
Previous studies have shown that the PC12 cell line, a parent cell line of NS-1, responded to Nogo-66-induced neurite outgrowth inhibition, thus suggesting the possible presence of NgR1 in this cell line [27]. Therefore, we first made sure that the immunoreactive protein observed on the cell surface of NS-1 cells was indeed NgR1. We initially induced knockdown of NgR1 in NS-1 cells utilizing Accell siRNA, which is efficient in transfecting cells. This resulted in a substantial decrease in cell-surface associated immunofluorescence staining of impermeable cells (not treated with detergent) incubated with anti-NgR1 antibody (Fig. 1A). On the other hand, the non-targeting control siRNA did not decrease this cell-surface NgR1 immunofluorescence staining. To further evaluate the specificity of the cell-surface-associated NgR1 staining in impermeable cells, we treated intact NS-1 cells with phosphatidylinositol-specific phospholipase C to remove specifically GPI-anchored cell-surface proteins, which includes NgR1. The phospholipase C treatment substantially decreased the immunofluorescence staining of impermeable NS-1 cells, suggesting that the observed immunofluorescence staining is caused by a cellsurface GPI-anchored protein, which is most likely NgR1 in this case (Fig. 1B). We used two different antibodies from two commercial sources, and both detected the NgR1 protein. Additionally, both antibodies detected the protein in the Western immunoblots corresponding to its apparent molecular weight of 65 kDa (glycosylated). We present the data utilizing anti-NgR1 antibody from Santa Cruz Biotechnology.
Since internalized proteins may be sorted out for degradation or recycled back to the plasma membrane, we have determined whether the dibutyryl-cAMP mediated internalized NgR1 returns back to the cell surface after removal of dibutyryl-cAMP. Initially, NS-1 cells were treated with dibutyryl-cAMP, and the cells were washed and kept in medium without added cAMP. Cycloheximide was included in the medium to prevent the synthesis of new proteins. Within 1 h after the removal of dibutyryl-cAMP, NgR1 had returned back to the cell surface, at the level seen prior to the dibutyryl-cAMP treatment (Fig. 3A and B). This suggests that the internalization of this receptor is a reversible process and influenced by intracellular levels of cAMP. However, we cannot exclude the possibility that NgR1 may be degraded with continued elevation of intracellular cAMP for several hours.
3.1. Internalization of NgR1 by cAMP When NS-1 cells were treated with cell-permeable dibutyrylcAMP, NgR1 that was present on the cell surface rapidly decreased within 1 h (Fig. 1C). Additionally, intracellular cAMP-elevating agents, such as forskolin, which directly activates adenylyl cyclase, and rolipram, which inhibits cyclic nucleotide phosphodiesterase (PDE), also decreased cell surface levels of NgR1 (Fig. 1C). We found an increase in the intracellular levels in NgR1 by cAMP-elevating agents, suggesting that they are inducing internalization of this receptor (Fig. 1D). However, dibutyryl-cGMP, a cell-permeable analog of cGMP, and phorbol 12-myristate 13-acetate, an activator for protein kinase C, did not induce the internalization of NgR1 (data not shown). The biotinylation assay revealed a rapid (15 min) and substantial internalization of NgR1 in NS-1 cells treated with dibutyryl-cAMP, forskolin, and rolipram (Fig. 1E). Western immunoblots showed no decrease in the total amount of NgR1 in the cells treated with these agents (Fig. 1F). This suggests that the internalized NgR1 is not appreciably degraded during this period. 3.2. Role of PKA and Epac in NgR1 internalization Since cAMP mediates its actions through at least two effectors, PKA and Epac, we determined the relative contribution of these two effectors to the cAMP-induced internalization of NgR1. Dibutyryl-cAMP, forskolin, and rolipram all induced the internalization of NgR1 in a PKA-deficient PC12 clone to the extent that is observed with wild type PC12 cells expressing PKA (data not shown). The PKA-specific activator N6-benzoyl-cAMP did not induce internalization of NgR1 (Fig. 2A and B). However, the Epacspecific activator 8-pCPT did induce this internalization. The PKAspecific inhibitor KT5720 did not block the dibutyryl-cAMPinduced NgR1 internalization, whereas the Epac-specific inhibitor ESI-09 blocked this internalization. Collectively, this data suggests that cAMP-induced NgR1 internalization is independent of PKA but is dependent on Epac.
Fig. 3. Reversal of cAMP-induced internalization of NgR1. (A) Confocal images showing cell surface NgR1 staining in NS-1 cells treated with dibutyryl-cAMP, which was subsequently removed through washing. NS-1 cells were treated with 200 mM of dibutyryl-cAMP for 30 min. In another set, cells were initially treated with dibutyrylcAMP for 30 min, then the cells were washed twice with a medium without dibutyrylcAMP. The cells were then kept in a new medium with cycloheximide (10 mg/ml) for 90 min. Another set of cells were continuously treated with dibutyryl-cAMP for 2 h. (B) Quantitative image densitometric analysis of NgR1 staining from confocal images. The values represent mean ± SE from three replicate estimations. Statistical difference between means is indicated with (*p < 0.05).
Please cite this article as: R. Gopalakrishna et al., Cyclic-AMP induces Nogo-A receptor NgR1 internalization and inhibits Nogo-A-mediated collapse of growth cone, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.009
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3.4. Internalization of NgR1 correlates with desensitization to Nogo-66 We determined whether the cAMP-induced decrease in the cell surface expression of NgR1 desensitizes NS-1 cells to the Nogo-66induced collapse of growth cones. Initially, we treated NS-1 cells with NGF for four days to induce neurite outgrowth with prominent growth cones. Nogo-66 rapidly induced a collapse of growth cones (Fig. 4 A and B). A pretreatment with dibutyryl-cAMP and forskolin for 1 h to induce the internalization of NgR1 desensitized NS-1 cells to Nogo-66-induced growth cone collapse. 4. Discussion NgR1 internalization may desensitize neurons to not only
Nogo-A but also to other axonal growth inhibitors. Since NgR1 is localized to lipid raft microdomains [28], it remains to be determined whether its coreceptors (p75NTR and LINGO-1) are internalized as well. The N-terminal domain of Nogo-A, Nogo-A-D20, along with its receptor, undergoes endocytosis and induces retrograde signaling in neurons [29]. However, NgR1 internalization, its cellular trafficking, and its degradation are not known. The internalization of NgR1 and the related desensitization of neurons to Nogo-66 are rapid events that occur in minutes by mechanism(s) not involving transcriptional activation. Thus, this is different from other reported mechanisms involving transcriptional activation occurring in several hours after the elevation of cAMP [17]. The observed internalization of NgR1 is reversibly regulated by intracellular cAMP. Epac may alter cytoskeletal organization and induce internalization of NgR1 (Fig. 4C). We postulate that NgR1 is
Fig. 4. Confocal images of growth cones induced by NGF. (A) Dibutyryl-cAMP and forskolin inhibited Nogo-66-induced growth cone collapse. Initially, NS-1 cells were treated with NGF to induce growth cones. Then, NGF-treated cells were incubated with Hank’s balanced salt solution (control), 200 mM dibutyryl-cAMP, or 5 mM forskolin for 1 h. This was followed by further incubation with 12.5 nM preaggregated Nogo-66 for 30 min. Then cells were fixed, permeabilized, and stained with Alexa Fluor 586-cojugated phalloidin. (B) Quantitative analysis of growth cone collapse induced by Nogo-66 and its prevention by pretreatment with dibutyryl-cAMP and forskolin. ‘Dibu-cAMP’ refers to ‘dibutyryl-cAMP’. The values represent mean ± SE from three replicate estimations. Statistical difference between means is indicated with asterisk (p < 0.05). (C) Schematic presentation describing the possible mechanism by which cAMP induces internalization of NgR1 and thereby desensitizes NS-1 cells to Nogo-66-induced growth cone collapse and axonal growth inhibition. Intracellular elevation of cAMP activates two effectors namely Epac and PKA. Epac signaling induces internalization of NgR1 as a rapid event. We hypothesize that NgR1 is taken into early endosomes, which act as signalosomes, and that the signaling they generate may restore axonal growth. With a decrease in cAMP, NgR1 may be inserted back into the membrane via recycling endosomes. As a later event (hours), PKA induces a transcriptional activation of a set of genes which ultimately overcome axonal growth inhibitors to enhance neurite outgrowth [17]. AC, adenylyl cyclase.
Please cite this article as: R. Gopalakrishna et al., Cyclic-AMP induces Nogo-A receptor NgR1 internalization and inhibits Nogo-A-mediated collapse of growth cone, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.009
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taken into early endosomes, which act as signalosomes, and the signaling they generate could potentially restore axonal growth. With a decrease in cAMP, NgR1 may be inserted back into the membrane via recycling endosomes. Although cAMP may elicit several cellular events, inducing the internalization of NgR1 is a very relevant mechanism in preventing Nogo-A-induced axonal growth inhibition. As a later event (hours), PKA induces a transcriptional activation of arginase-1 and subsequent increase in the synthesis of polyamines which further enhance neurite outgrowth [17]. Cyclic-AMP may be involved in the action of additional agents which are known to overcome the axonal growth inhibitors. For example, brain-derived neurotrophic factor, laminin, and the green tea polyphenol epigallocatechin-3-gallate overcome myelinderived axonal growth inhibitors [25,30,31]. All of these agents induce the elevation of intracellular cAMP [24,30,32]. Whether these agents can induce an internalization of NgR1 remains to be determined. In summary, this study suggests that cAMP and its effector Epac induce reversible internalization of NgR1 and thus decrease neuronal cell sensitivity to axonal growth inhibitors, such as NogoA. Therefore, besides axonal growth inhibitors affecting neurons, neurons themselves self-regulate their own sensitivity to extracellular cues such as axonal growth inhibitors. Additional in vitro studies with isolated neurons and in vivo models are certainly needed to understand the role of cAMP in NgR1 internalization. This normal cellular regulatory mechanism may be pharmacologically exploited to overcome axonal growth inhibitors and enhance functional recovery after stroke and various neuronal injuries.
[9]
[10]
[11]
[12]
[13]
[14]
[15] [16] [17]
[18]
[19]
[20]
Declaration of competing interest [21]
The authors declare that there is no conflict of interest in this study.
[22] [23]
Acknowledgments [24]
We thank Dr. Louis Hersh at the University of Kentucky for generously providing the PKA-deficient PC12 cell line. We thank Dr. Seth Ruffins and USC Stem Cell Microscopy Core for confocal microscopy. We also thank Calvin Le for excellent technical assistance. This work was supported by Keck School of Medicine, United States.
[25]
[26]
Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2020.01.009.
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Cyclic-AMP induces Nogo-A receptor NgR1 internalization and inhibits Nogo-A-mediated collapse of growth cone Rayudu Gopalakrishna a, *, Aubree Mades a, Andrew Oh a, Angela Zhu a, Julie Nguyen a, Charlotte Lin a, Mark S. Kindy b, William J. Mack c a
Department of Integrative Anatomical Sciences, Keck School of Medicine, University of Southern California, Los Angeles, CA, 90089, USA Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, 33612, USA, James A. Haley VA Medical Center, Tampa, FL, 33612, USA c Department of Neurological Surgery, Keck School of Medicine, University of Southern California, Los Angeles, CA, 90089, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 December 2019 Accepted 3 January 2020 Available online xxx
The promotion of axonal regeneration is required for functional recovery from stroke and various neuronal injuries. However, axonal regeneration is inhibited by diverse axonal growth inhibitors, such as Nogo-A. Nogo-66, a C-terminal domain of Nogo-A, binds to the Nogo-A receptor 1 (NgR1) and induces the collapse of growth cones and inhibits neurite outgrowth. NgR1 is also a receptor for additional axonal growth inhibitors, suggesting it is an important target for the prevention of axonal growth inhibition. By using the indirect immunofluorescence method, we show for the first time that a cell-permeable cAMP analog (dibutyryl-cAMP) induced a rapid decrease in the cell surface expression of NgR1 in Neuroscreen1 (NS-1) cells. The biotinylation method revealed that cAMP indeed induced internalization of NgR1 within minutes. Other intracellular cAMP-elevating agents, such as forskolin, which directly activates adenylyl cyclase, and rolipram, which inhibits cyclic nucleotide phosphodiesterase, also induced this process. This internalization was found to be reversible and influenced by intracellular levels of cAMP. Using selective 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. The decrease in cell surface expression of NgR1 desensitized NS-1 cells to Nogo-66-induced growth cone collapse. Therefore, it is likely that besides axonal growth inhibitors affecting neurons, neurons themselves also self-regulate their sensitivity to axonal growth inhibitors, as influenced by intracellular cAMP/Epac. This normal cellular regulatory mechanism may be pharmacologically exploited to overcome axonal growth inhibitors, and enhance functional recovery after stroke and neuronal injuries. © 2020 Elsevier Inc. All rights reserved.
Keywords: Nogo-A cAMP NgR1 Axonal growth inhibitors Exchange protein directly activated by cAMP Growth cone
1. Introduction Functional recovery from stroke and other neuronal injuries requires the promotion of axonal regeneration from remaining neurons [1e5]. However, axonal regeneration is inhibited by diverse axonal growth inhibitors, such as Nogo-A, myelin-associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMgp), and chondroitin sulfate proteoglycans (CSPGs). Nogo-A, produced by oligodendrocytes and neurons, has emerged as a key
Abbreviations: NgR1, Nogo-A receptor 1; PKA, protein kinase A; Epac, exchange protein directly activated by cAMP; NS-1, Neuroscreen-1; PDE, cyclic nucleotide phosphodiesterase. * Corresponding author. Department of Integrative Anatomical Sciences, 1333 San Pablo Street, Keck School of Medicine, Los Angeles, CA, 90089, USA. E-mail address: [email protected] (R. Gopalakrishna).
axonal growth inhibitor [6]. Nogo-A has two distinct antineuritogenic domains: N-terminal amino-Nogo (Nogo-A-D20) and C-terminal Nogo-66 [7]. Nogo-66 binds to Nogo-A receptor 1 (NgR1) [8,9]. NgR1 can also serve as the receptor for other axonal growth inhibitors, such as MAG, OMgp, and CSPGs, and as such, it is an important mediator for axonal growth inhibition [10e13]. NgR1 signals through transmembrane coreceptors p75NTR (or TROY) and LINGO-1, leading to the activation of RhoA, which in turn activates its effector, RhoA-associated protein kinase (ROCK) [14]. This signaling cascade ultimately leads to actin cytoskeletal reorganization and the collapse of growth cones, inhibiting neurite outgrowth. Currently, therapies are targeting Nogo-A, MAG, NgR1, RhoA, and ROCK for drug development to enhance axonal sprouting and functional recovery after stroke and spinal cord injury [15,16]. Cyclic-AMP plays a crucial role in overcoming neurite outgrowth inhibition caused by myelin [17] and helping improve recovery
https://doi.org/10.1016/j.bbrc.2020.01.009 0006-291X/© 2020 Elsevier Inc. All rights reserved.
Please cite this article as: R. Gopalakrishna et al., Cyclic-AMP induces Nogo-A receptor NgR1 internalization and inhibits Nogo-A-mediated collapse of growth cone, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.009
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Fig. 1. Specificity of NgR1 staining and internalization of NgR1 by dibutyryl-cAMP, forskolin, and rolipram. (A) Confocal images and quantitative image densitometric analysis of NgR1 staining of “not permeabilized” NS-1 cells transfected with Accell NgR1 siRNA; cell-surface-associated NgR1 was detected by indirect immunofluorescence staining (red). Nuclei stained blue by DAPI. Image density was normalized to one cell (one nucleus) and expressed as percentage of the control. Where indicated, the values represent mean ± SE
Please cite this article as: R. Gopalakrishna et al., Cyclic-AMP induces Nogo-A receptor NgR1 internalization and inhibits Nogo-A-mediated collapse of growth cone, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.009
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from the neuronal injuries [18]. Embryonic neurons have higher cAMP levels than adult neurons, causing axonal growth inhibitors to promote rather than inhibit the growth of these neurons [19]. On the other hand, these axonal growth inhibitors inhibit adult neurons with lower cAMP levels. Cyclic-AMP may block the actions of axonal growth inhibitors by inducing a transcriptional activation of arginase-1 and subsequent elevation of polyamines [17]. In some cases, cAMP can still rapidly prevent the action of axonal growth inhibitors without involving transcriptional activation through an unknown mechanism [20]. Cyclic-AMP mediates its actions primarily through two effectors, protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac) [21,22]. While PKA is still an important player in mediating cAMP actions, some cAMP-mediated actions previously believed to be mediated by PKA may indeed be mediated instead by Epac [22]. There are two isoforms of Epac proteins: Epac 1, which is ubiquitously distributed in all tissues, and Epac 2, which is present at higher levels in neurons [21]. Epac is involved in mediating various cellular processes in neurons, such as apoptosis, neurotransmitter release, and axon growth [23]. The relative contribution of PKA and Epac in the cAMP-mediated actions to overcome axonal growth inhibitors is not known. Here we show for the first time that cell-permeable cAMP (dibutyryl-cAMP) and other agents that increase intracellular cAMP (forskolin and rolipram) rapidly induce the internalization of NgR1 and desensitize Neuroscreen-1 (NS-1) neuronal-like cells to Nogo66-induced growth cone collapse. This internalization is mediated by Epac. We further show that with a subsequent decrease in intracellular cAMP, NgR1 returns back to the cell surface. 2. Materials and methods
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were selected for this study because they produce rapid and robust neurite outgrowth with NGF. PKA-deficient PC12 cells (A132.7) that were originally cloned by Dr. John Wagner, and parent PC12 cells were kind gifts from Dr. Louis Hersh (University of Kentucky, Lexington). NS-1 cells, PKA-deficient PC12 cells, and parent PC12 cells were grown in flasks coated with poly-L-lysine in RPMI medium supplemented with 10% heat-inactivated horse serum, 5% fetal calf serum, 50 units/ml penicillin, and 0.05 mg/ml streptomycin. 2.3. Indirect immunofluorescence NS-1 cells were grown on 12-mm glass coverslips previously coated with poly-D-lysine to 50% confluency and treated with dibutyryl-cAMP, forskolin, or rolipram for 1 h. Cell surface NgR1 was detected in paraformaldehyde-fixed cells that were not permeabilized, while intracellular NgR1 was detected in cells permeabilized with 0.25% Triton-X-100. After blocking with 5% goat serum, cells were incubated with rabbit anti-NgR1 antibody (1:300 dilution) for 24 h at 4 C. Cells were then incubated with anti-rabbit goat secondary antibody conjugated with Alexa Fluor 594 for 1 h at room temperature. Nuclei were visualized with DAPI. Presented images were taken in a blinded fashion using an LSM 800 Zeiss confocal microscope and 63 1.4NA oil objective. Twelve images were collected for the stack, and the imaging intensity was quantitated using ImageJ software [24]. 2.3.1. Knockdown of NgR1 by siRNA transfection NS-1 cells were plated on glass coverslips. After 24 h, the cells were incubated with 1 mM Accell NgR1 siRNA in a delivery medium for 4 days, according to the manufacturer’s instructions. Then, NgR1 was detected using immunofluorescence staining. As a negative control, we used Accell non-targeting siRNA.
2.1. Materials Dibutyryl-cAMP, dibutyryl-cGMP, forskolin, rolipram, and 40 60 diamidino-2-phenylindole (DAPI) were obtained from SigmaAldrich. Mouse nerve growth factor (NGF), KT5720, and ESI-09 were from EMD Millipore. N6-benzoyl-cAMP and 8-(4chlorophenylthio)-20 -O-methyl-cAMP (8-pCPT) were from Cayman Chemical. Recombinant Nogo-A-Fc chimera, in which Nogo-A (aa1026-1090) is fused with mouse IgG2a (referred to as Nogo-66 throughout this study) was obtained from R&D Systems. Alexa Fluor 568-conjugated phalloidin was from Molecular Probes. NgR1 rabbit polyclonal antibody (H-120) which recognizes rat NgR1 was from Santa Cruz Biotechnology. The recombinant monoclonal rabbit antibody to NgR1 (clone M5) was from Absolute Antibody. An anti-rabbit secondary antibody conjugated with Alexa Fluor 594 and a goat anti-mouse IgG2a were from Jackson ImmunoResearch. Accell rat NgR1 siRNA, non-targeting siRNA control, and delivery medium were from Dharmacon. Phosphatidylinositolspecific phospholipase C from Bacillus cereus was from Invitrogen. 2.2. Cell culture Neuroscreen-1 (NS-1) cells (a clone derived from PC12 cells)
2.3.2. Removal of glycosylphosphatidylinositol(GPI)-anchored cellsurface associated proteins NS-1 cells, in serum-free medium, were incubated with phosphatidylinositol-specific phospholipase C (0.5 U/ml) for 2 h at 37 C. This treatment specifically removes GPI-anchored proteins from the cell surface. Then, the cells were washed and the cellsurface associated NgR1 was detected in “not permeabilized” cells. 2.4. Biotinylation assay for internalization NgR1 internalization was determined by biotinylation as described previously [25]. NS-1 cells were labeled with sulfoeNHSeSS-biotin (0.8 mg/ml) for 30 min at 4 C. Labeled cells were transferred to the incubator at 37 C and treated with dibutyryl-cAMP (200 mM), forskolin (5 mM), or rolipram (2 mM) for 15 min to induce internalization of NgR1. Then, cells were treated with glutathione to cleave the biotin-associated SeS bond to selectively strip it from the cell surface. Cell extracts were prepared by homogenization and the internalized biotinylated proteins were isolated with streptavidin-agarose beads. Proteins that eluted from these beads were subjected to Western immunoblotting using an anti-NgR1 antibody.
from three replicate estimations. Statistically different from control (*p < 0.05). (B) Immunofluorescence staining and quantitative image densitometric analysis of cell-surfaceassociated NgR1 of impermeable cells treated with phosphatidylinositol-specific phospholipase C to remove GPI-anchored cell surface proteins. (C) Confocal images and quantitative image densitometric analysis of cell surface NgR1 in cells treated with cAMP-elevating agents. NS-1 cells were treated with 200 mM dibutyryl-cAMP, or cAMP-generating agents 5 mM forskolin, and 2 mM rolipram for 1 h. Cell surface NgR1 was detected with “not permeabilized” cells. (D) Confocal images and quantitative image densitometric analysis of NgR1 in detergent-permeabilized cells. (E) Internalization of biotinylated cell-surface NgR1 by dibutyryl-cAMP, forskolin, and rolipram. NS-1 cell-surface proteins were biotinylated and treated with indicated agents for 15 min. By using streptavidin beads, the internalized biotinylated proteins were isolated and subjected to Western immunoblotting for NgR1. Total biotinylated protein that was not internalized and protein left after stripping with glutathione were shown. (F) Western immunoblotting of total (membrane and intracellular) NgR1 present in NS-1 cells treated with dibutyryl-cAMP, forskolin and rolipram for 1 h. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
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2.5. Western immunoblotting Protein samples were subjected to SDS-polyacrylamide gel electrophoresis and the separated proteins were transferred to polyvinylidene fluoride membrane [26]. The blocked membranes were incubated with anti-NgR1 rabbit antibodies followed by goat anti-rabbit secondary antibodies conjugated with horseradish peroxidase. The immunoreactive bands were visualized by the SuperSignal West Femato Maximum Sensitivity Substrate kit (Pierce). These bands were analyzed by densitometric scanning using the Omega 12IC Molecular Imaging System.
was pre-aggregated with 125 ng/ml goat anti-mouse IgG2a. The cAMP-pretreated cells were then incubated with pre-aggregated Nogo-66 (12.5 nM) for 30 min. The treated cells were then fixed with glutaraldehyde, permeabilized with 0.1% Triton-X-100, and subsequently blocked with 1% bovine serum albumin. The cells were then stained with Alexa Fluor 586-conjugated phalloidin for 20 min. After washing the cells, the coverslips were mounted with Fluoromount medium, and images were taken in a blinded fashion using an LSM 800 Zeiss confocal microscope. The results are expressed as the percentage of collapsed growth cones to total counted growth cones [25].
2.6. Growth cone collapse assay 2.7. Statistical analysis We measured the collapse of actin-rich filopodia and lamellipodia-like structures, which are referred to as growth cones in this paper. NS-1 cells were grown on 12-mm glass coverslips and treated with NGF (50 ng/ml) for four days to induce neuronal differentiation. Then, the neuronal-like NS-1 cells were pretreated with dibutyryl-cAMP (200 mM) or forskolin (5 mM) for 1 h. Nogo-66
For NgR1 internalization and growth cone analysis, the data is expressed as the mean ± SE and analyzed using one-way analysis of variance, followed by post hoc Scheffe’s test. p < 0.05 was considered statistically significant. Statistical analysis was performed using StatView software.
Fig. 2. Role of PKA and Epac in cAMP-induced internalization of NgR1. (A) Confocal images showing cell surface NgR1 staining in NS-1 cells treated with activators and inhibitors of PKA and Epac. Where indicated, NS-1 cells were initially pretreated with 200 nM KT5720 or 10 mM ESI-09 for 1 h. Then, cells were treated with 0.5 mM N6-benzoyl-cAMP, 10 mM 8pCPT or 200 mM dibutyryl-cAMP for 1 h. (B) Quantitative image densitometric analysis of NgR1 staining from confocal images. The values represent mean ± SE from three replicate estimations. Statistical difference between means is indicated with (*p < 0.05).
Please cite this article as: R. Gopalakrishna et al., Cyclic-AMP induces Nogo-A receptor NgR1 internalization and inhibits Nogo-A-mediated collapse of growth cone, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.009
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3. Results
3.3. Reversal of NgR1 internalization
Previous studies have shown that the PC12 cell line, a parent cell line of NS-1, responded to Nogo-66-induced neurite outgrowth inhibition, thus suggesting the possible presence of NgR1 in this cell line [27]. Therefore, we first made sure that the immunoreactive protein observed on the cell surface of NS-1 cells was indeed NgR1. We initially induced knockdown of NgR1 in NS-1 cells utilizing Accell siRNA, which is efficient in transfecting cells. This resulted in a substantial decrease in cell-surface associated immunofluorescence staining of impermeable cells (not treated with detergent) incubated with anti-NgR1 antibody (Fig. 1A). On the other hand, the non-targeting control siRNA did not decrease this cell-surface NgR1 immunofluorescence staining. To further evaluate the specificity of the cell-surface-associated NgR1 staining in impermeable cells, we treated intact NS-1 cells with phosphatidylinositol-specific phospholipase C to remove specifically GPI-anchored cell-surface proteins, which includes NgR1. The phospholipase C treatment substantially decreased the immunofluorescence staining of impermeable NS-1 cells, suggesting that the observed immunofluorescence staining is caused by a cellsurface GPI-anchored protein, which is most likely NgR1 in this case (Fig. 1B). We used two different antibodies from two commercial sources, and both detected the NgR1 protein. Additionally, both antibodies detected the protein in the Western immunoblots corresponding to its apparent molecular weight of 65 kDa (glycosylated). We present the data utilizing anti-NgR1 antibody from Santa Cruz Biotechnology.
Since internalized proteins may be sorted out for degradation or recycled back to the plasma membrane, we have determined whether the dibutyryl-cAMP mediated internalized NgR1 returns back to the cell surface after removal of dibutyryl-cAMP. Initially, NS-1 cells were treated with dibutyryl-cAMP, and the cells were washed and kept in medium without added cAMP. Cycloheximide was included in the medium to prevent the synthesis of new proteins. Within 1 h after the removal of dibutyryl-cAMP, NgR1 had returned back to the cell surface, at the level seen prior to the dibutyryl-cAMP treatment (Fig. 3A and B). This suggests that the internalization of this receptor is a reversible process and influenced by intracellular levels of cAMP. However, we cannot exclude the possibility that NgR1 may be degraded with continued elevation of intracellular cAMP for several hours.
3.1. Internalization of NgR1 by cAMP When NS-1 cells were treated with cell-permeable dibutyrylcAMP, NgR1 that was present on the cell surface rapidly decreased within 1 h (Fig. 1C). Additionally, intracellular cAMP-elevating agents, such as forskolin, which directly activates adenylyl cyclase, and rolipram, which inhibits cyclic nucleotide phosphodiesterase (PDE), also decreased cell surface levels of NgR1 (Fig. 1C). We found an increase in the intracellular levels in NgR1 by cAMP-elevating agents, suggesting that they are inducing internalization of this receptor (Fig. 1D). However, dibutyryl-cGMP, a cell-permeable analog of cGMP, and phorbol 12-myristate 13-acetate, an activator for protein kinase C, did not induce the internalization of NgR1 (data not shown). The biotinylation assay revealed a rapid (15 min) and substantial internalization of NgR1 in NS-1 cells treated with dibutyryl-cAMP, forskolin, and rolipram (Fig. 1E). Western immunoblots showed no decrease in the total amount of NgR1 in the cells treated with these agents (Fig. 1F). This suggests that the internalized NgR1 is not appreciably degraded during this period. 3.2. Role of PKA and Epac in NgR1 internalization Since cAMP mediates its actions through at least two effectors, PKA and Epac, we determined the relative contribution of these two effectors to the cAMP-induced internalization of NgR1. Dibutyryl-cAMP, forskolin, and rolipram all induced the internalization of NgR1 in a PKA-deficient PC12 clone to the extent that is observed with wild type PC12 cells expressing PKA (data not shown). The PKA-specific activator N6-benzoyl-cAMP did not induce internalization of NgR1 (Fig. 2A and B). However, the Epacspecific activator 8-pCPT did induce this internalization. The PKAspecific inhibitor KT5720 did not block the dibutyryl-cAMPinduced NgR1 internalization, whereas the Epac-specific inhibitor ESI-09 blocked this internalization. Collectively, this data suggests that cAMP-induced NgR1 internalization is independent of PKA but is dependent on Epac.
Fig. 3. Reversal of cAMP-induced internalization of NgR1. (A) Confocal images showing cell surface NgR1 staining in NS-1 cells treated with dibutyryl-cAMP, which was subsequently removed through washing. NS-1 cells were treated with 200 mM of dibutyryl-cAMP for 30 min. In another set, cells were initially treated with dibutyrylcAMP for 30 min, then the cells were washed twice with a medium without dibutyrylcAMP. The cells were then kept in a new medium with cycloheximide (10 mg/ml) for 90 min. Another set of cells were continuously treated with dibutyryl-cAMP for 2 h. (B) Quantitative image densitometric analysis of NgR1 staining from confocal images. The values represent mean ± SE from three replicate estimations. Statistical difference between means is indicated with (*p < 0.05).
Please cite this article as: R. Gopalakrishna et al., Cyclic-AMP induces Nogo-A receptor NgR1 internalization and inhibits Nogo-A-mediated collapse of growth cone, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.009
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3.4. Internalization of NgR1 correlates with desensitization to Nogo-66 We determined whether the cAMP-induced decrease in the cell surface expression of NgR1 desensitizes NS-1 cells to the Nogo-66induced collapse of growth cones. Initially, we treated NS-1 cells with NGF for four days to induce neurite outgrowth with prominent growth cones. Nogo-66 rapidly induced a collapse of growth cones (Fig. 4 A and B). A pretreatment with dibutyryl-cAMP and forskolin for 1 h to induce the internalization of NgR1 desensitized NS-1 cells to Nogo-66-induced growth cone collapse. 4. Discussion NgR1 internalization may desensitize neurons to not only
Nogo-A but also to other axonal growth inhibitors. Since NgR1 is localized to lipid raft microdomains [28], it remains to be determined whether its coreceptors (p75NTR and LINGO-1) are internalized as well. The N-terminal domain of Nogo-A, Nogo-A-D20, along with its receptor, undergoes endocytosis and induces retrograde signaling in neurons [29]. However, NgR1 internalization, its cellular trafficking, and its degradation are not known. The internalization of NgR1 and the related desensitization of neurons to Nogo-66 are rapid events that occur in minutes by mechanism(s) not involving transcriptional activation. Thus, this is different from other reported mechanisms involving transcriptional activation occurring in several hours after the elevation of cAMP [17]. The observed internalization of NgR1 is reversibly regulated by intracellular cAMP. Epac may alter cytoskeletal organization and induce internalization of NgR1 (Fig. 4C). We postulate that NgR1 is
Fig. 4. Confocal images of growth cones induced by NGF. (A) Dibutyryl-cAMP and forskolin inhibited Nogo-66-induced growth cone collapse. Initially, NS-1 cells were treated with NGF to induce growth cones. Then, NGF-treated cells were incubated with Hank’s balanced salt solution (control), 200 mM dibutyryl-cAMP, or 5 mM forskolin for 1 h. This was followed by further incubation with 12.5 nM preaggregated Nogo-66 for 30 min. Then cells were fixed, permeabilized, and stained with Alexa Fluor 586-cojugated phalloidin. (B) Quantitative analysis of growth cone collapse induced by Nogo-66 and its prevention by pretreatment with dibutyryl-cAMP and forskolin. ‘Dibu-cAMP’ refers to ‘dibutyryl-cAMP’. The values represent mean ± SE from three replicate estimations. Statistical difference between means is indicated with asterisk (p < 0.05). (C) Schematic presentation describing the possible mechanism by which cAMP induces internalization of NgR1 and thereby desensitizes NS-1 cells to Nogo-66-induced growth cone collapse and axonal growth inhibition. Intracellular elevation of cAMP activates two effectors namely Epac and PKA. Epac signaling induces internalization of NgR1 as a rapid event. We hypothesize that NgR1 is taken into early endosomes, which act as signalosomes, and that the signaling they generate may restore axonal growth. With a decrease in cAMP, NgR1 may be inserted back into the membrane via recycling endosomes. As a later event (hours), PKA induces a transcriptional activation of a set of genes which ultimately overcome axonal growth inhibitors to enhance neurite outgrowth [17]. AC, adenylyl cyclase.
Please cite this article as: R. Gopalakrishna et al., Cyclic-AMP induces Nogo-A receptor NgR1 internalization and inhibits Nogo-A-mediated collapse of growth cone, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.009
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taken into early endosomes, which act as signalosomes, and the signaling they generate could potentially restore axonal growth. With a decrease in cAMP, NgR1 may be inserted back into the membrane via recycling endosomes. Although cAMP may elicit several cellular events, inducing the internalization of NgR1 is a very relevant mechanism in preventing Nogo-A-induced axonal growth inhibition. As a later event (hours), PKA induces a transcriptional activation of arginase-1 and subsequent increase in the synthesis of polyamines which further enhance neurite outgrowth [17]. Cyclic-AMP may be involved in the action of additional agents which are known to overcome the axonal growth inhibitors. For example, brain-derived neurotrophic factor, laminin, and the green tea polyphenol epigallocatechin-3-gallate overcome myelinderived axonal growth inhibitors [25,30,31]. All of these agents induce the elevation of intracellular cAMP [24,30,32]. Whether these agents can induce an internalization of NgR1 remains to be determined. In summary, this study suggests that cAMP and its effector Epac induce reversible internalization of NgR1 and thus decrease neuronal cell sensitivity to axonal growth inhibitors, such as NogoA. Therefore, besides axonal growth inhibitors affecting neurons, neurons themselves self-regulate their own sensitivity to extracellular cues such as axonal growth inhibitors. Additional in vitro studies with isolated neurons and in vivo models are certainly needed to understand the role of cAMP in NgR1 internalization. This normal cellular regulatory mechanism may be pharmacologically exploited to overcome axonal growth inhibitors and enhance functional recovery after stroke and various neuronal injuries.
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Declaration of competing interest [21]
The authors declare that there is no conflict of interest in this study.
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Acknowledgments [24]
We thank Dr. Louis Hersh at the University of Kentucky for generously providing the PKA-deficient PC12 cell line. We thank Dr. Seth Ruffins and USC Stem Cell Microscopy Core for confocal microscopy. We also thank Calvin Le for excellent technical assistance. This work was supported by Keck School of Medicine, United States.
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Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2020.01.009.
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Please cite this article as: R. Gopalakrishna et al., Cyclic-AMP induces Nogo-A receptor NgR1 internalization and inhibits Nogo-A-mediated collapse of growth cone, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.009