Overexpression of Nogo-A changes nerve growth factor signaling dynamics in PC12 cells

Abstract

The nerve growth factor (NGF) receptor TrkA is a tightly regulated receptor tyrosine kinase that activates neuronal signaling pathways promoting cell survival in addition to axonal and dendritic outgrowth. Previously, we showed that NGF and TrkA signaling is altered in neuron-like PC12 cells that overexpress Nogo-A, a protein known to influence axonal outgrowth and dendritic arborization associated with neuronal plasticity. In the present report, we provide evidence for changes in NGF-mediated receptor-level and downstream signaling that occur in cells overexpressing Nogo-A. NGF stimulation increased the association of Nogo-A with TrkA, which corresponded to a decrease in sustained phosphorylation of TrkA and its downstream effectors Erk1/2, indicating that Nogo-A plays a role in the temporal regulation of this pathway. Furthermore, co-immunoprecipitation of the p75 neurotrophin receptor (p75^NTR) with TrkA was significantly reduced in cells overexpressing Nogo-A, suggesting that Nogo-A blocked this interaction. Analysis of calcium and calmodulin involvement in NGF-induced activation of Erk1/2 revealed a calcium and calmodulin-dependent inhibition of sustained phosphorylation in Nogo-A-overexpressing cells but not in wild type cells, suggesting that Nogo-A facilitated the activation of calcium/calmodulin to alter NGF signaling. Taken together, these results provide evidence for Nogo-A regulation of NGF signaling, in part by modifying calcium and calmodulin-dependent mechanisms.

1. Introduction

The control of neuronal growth and survival is orchestrated by the combined actions of factors that either support or inhibit axonal growth, pathfinding, and target innervation [1, 2]. When properly regulated, these opposing mechanisms contribute to neuronal homeostasis and serve to accurately guide the growth and extension of axons and dendrites throughout development and maintain their survival and function in adulthood. However, following injury or disease, these homeostatic mechanisms can be disrupted, resulting in the inhibition of recovery. Understanding the underlying mechanisms involved in these homeostatic processes will provide a major step toward the development of interventions that reverse this inhibition so that recovery can proceed in a properly regulated manner.

The neurite inhibitory protein Nogo-A, originally characterized as a myelin-associated neurite outgrowth inhibitor, is expressed in neurons during development [3, 4], after injury [5, 6] and throughout adulthood in various regions of the brain, such as the cerebral cortex [6], hippocampus [3, 7] and neural stem cells of the hippocampus [8] and subventricular zone [9]. Unlike the inhibitory role of myelin-associated Nogo-A [10, 11], neuronal Nogo-A has a growth-supporting function, which was shown in studies comparing the regenerative response to optic nerve injury between oligodendrocyte-specific and neuron-specific Nogo-A knockout mice [12]. Although the function of neuronally-expressed Nogo-A as a positive influence on neuronal growth is now recognized, particularly in the context of recovery following injury [12–14] and on the stability of dendritic spines [15], its regulatory role in neuronal homeostasis and the signal transduction mechanisms responsible are not fully understood.

In a recent report [16] we showed that Nogo-A interacts with the nerve growth factor (NGF) receptor TrkA to alter NGF signaling outcomes from growth promoting to the activation of a ceramide-dependent cell death pathway, which implicated Nogo-A as a regulator of NGF signaling. Our goal in the present study was to explore additional NGF signaling pathways affected by Nogo-A that may contribute to this regulation. Our present results show that Nogo-A overexpression was associated with changes in both TrkA/p75NTR receptor dynamics and temporal regulation of Erk1/2 phosphorylation by calcium (Ca²⁺) and calmodulin (CaM)-mediated mechanisms following NGF stimulation. These findings implicate neuronal Nogo-A as a potential mediator of neuronal homeostasis by influencing NGF-mediated signaling mechanisms.

2. Materials and methods

2.1. Reagents

The sources of reagents and antibodies were as follows: NGF (rmβ-NGF) was from R&D Systems (1156-NG); BAPTA-AM was from Calbiochem (196419); W5 (CA315) and W7 (CA320) were from Enzo/Biomol; anti-Trk (B-3) (sc-7268) was from Santa Cruz; anti-TrkA (2505), anti-phospho-TrkA (Y490) (4619) and anti-phospho-Erk1/2 (9106) were from Cell Signaling; anti Erk1 was from Santa Cruz (sc-94-G); anti-p75^NTR was from Millipore (07-476); anti-GAPDH was from Trevigen (2275-PC). The Nogo-A-specific monoclonal antibody 11C7, raised against rat Nogo-A amino acid sequence 623 – 640 [17], was generously provided by Prof. Martin Schwab.

2.2. Cell culture

PC12 cells were obtained from American Type Culture Collection (ATCC). This cell line is not listed among the commonly misidentified cell line by the International Cell Line Authentication Committee (ICLAC). The generation of the stably transfected PC12 cell line overexpressing Nogo-A (PC12/Nogo-A) was described previously [16]. In general, cells not exceeding passage 20 were grown on poly-D-lysine-coated culture dishes in RPMI plus GlutaMAX medium supplemented with 5% fetal bovine serum at 37 °C in a humid atmosphere with 5% CO₂. For experiments, cells were used at approximately 80% confluency and were placed in serum-free RPMI medium for 3-4 hours before treatments to reduce serum interference. The calcium chelator BAPTA-AM (25 μM final concentration) and calmodulin inhibitors W5 and W7 (each at and 10 μM final concentration) were added 30 minutes before NGF (10 ng/ml) treatment.

2.3. Co-Immunoprecipitation

PC12 cells (1.5 – 2 x 10⁶ cells) were solubilized in one ml lysis/immunoprecipitation buffer (150 mM NaCl, 10 mM Tris, 1 mM EDTA, 1% Triton X-100, 0.5% NP-40, protease inhibitor cocktail, 50 mM NaF, 1 mM Na₃VO₄), centrifuged to remove insoluble material and incubated with 1 μg (5 μl) anti-Trk antibody (Santa Cruz, cat.# sc-414) for one hour at 4 °C. Twenty-five μl of anti-mouse magnetic beads (Dynabeads, pre-washed as described in manufacturer’s instructions) were added and the mixture was incubated overnight at 4 °C on a rocking platform. The beads were isolated and washed twice with lysis/immunoprecipitation buffer. After the last wash, proteins were eluted from the beads with 20 μl reducing sample buffer. The eluted proteins were separated on 4-12% NuPage gels and transferred to PVDF membranes for western blot analysis. For uniformity, all samples within each experiment were blotted to a single membrane to insure equal treatment and exposure times.

2.4. Cell lysis and western blot analysis

At the designated termination times, cells were placed on ice, washed with cold phosphate-buffered saline and immediately solubilized in 100 μl 2X sample buffer. Cellular proteins were separated on 4-12% NuPage bis-tris gels (Invitrogen) and then transferred to PVDF membranes. After blocking in tris-buffered saline/0.05% Tween-20 (TBS-T) containing 5% non-fat dry milk, the membranes were incubated overnight at 4 °C with primary antibody (1:1000 dilution) in TBS-T containing 5% non-fat dry milk. For primary antibodies against phosphotyrosine epitopes, the membranes were washed with TBS-T after blocking and the primary antibody was diluted in TBS-T/%5 bovine serum albumin. (It should be noted that in blots showing phospho-Erk1/2 results we used anti-Erk1 antibody staining to represent total Erk for normalization because its reactivity was not affected by the phosphorylation state of Erk1. In our hands, two different primary antibodies we tested against total Erk1/2 showed substantially reduced reactivity towards phosphorylated Erk1/2 than unphosphorylated Erk1/2, which precluded their use for normalizing total Erk1/2). The membranes were washed four times with TBS-T and then incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (Pierce or Jackson ImmunoResearch). Immunoreactivity was detected by enhanced chemiluminescence (Pierce) using an Azure Biosystems 600 imager. For uniformity, all samples within each experiment were blotted to a single membrane to insure equal treatment and exposure times

2.5. Data analysis

Data were analyzed by two-tailed paired Student’s t-test using GraphPad Prism software version 10.2.3 (GraphPad Software, USA). Results are expressed as mean ± SD from at least three independent experiments. p values ≤ 0.05 were considered significant.

3. Results

3.1. Nogo-A interaction with TrkA increases with NGF stimulation

To determine if there was an activation-dependent association between TrkA and Nogo-A, we stimulated PC12 cells stably overexpressing Nogo-A (PC12/Nogo-A) for increasing times with 10 ng/ml NGF followed by immunoprecipitation of TrkA with a C-terminus-directed Trk antibody and western blotting for Nogo-A. Both phosphorylated (Figure 1A panel a) and total (Figure 1A panel b) immunoprecipitated TrkA are shown, which corroborate our previous finding that, in cells overexpressing Nogo-A, sustained (15 – 60 minutes) NGF-induced phosphorylation of TrkA is significantly reduced [16]. Figure 1A, panel c shows that in PC12/Nogo-A cells there was a significant increase over basal levels in the amount of Nogo-A co-immunoprecipitated with TrkA following NGF treatment. This increase was consistently detected (N=4) 15–30 minutes following peak TrkA phosphorylation (5 minutes). Wild type cells (PC12/wt), which express substantially less Nogo-A, showed no detectable Nogo-A co-immunoprecipitating with TrkA. These results suggest that Nogo-A and TrkA can form a complex that increases following NGF stimulation and may be linked to decreased TrkA phosphorylation.

Fig. 1.

(A) Effects of Nogo-A overexpression on TrkA phosphorylation and co-immunoprecipitated proteins following NGF activation. PC12/wt or PC12/Nogo-A cells were serum starved for 3-4 hours then stimulated with 10 ng/ml NGF for the indicated times. Cell lysates were subjected to immunoprecipitation with anti-Trk antibody followed by western blotting of the immunoprecipitated proteins for phospho-TrkA (Y674/675) (panel a), TrkA (panel b), Nogo-A detected with antibody 11C7 (panel c), p75^NTR (panel d). For uniform comparison, all samples for this figure were run on a single blot. Similar results were obtained in four separate immunoprecipitation experiments. (B) Comparison of p75^NTR expression between PC12/wt and PC12/Nogo-A cells. Equivalent amounts of total cell lysates from unstimulated PC12/wt and PC12/Nogo-A cells were subjected to western blotting with anti-p75^NTR and anti-GAPDH antibodies. Similar results were obtained in four separate western blotting experiments.

3.2. p75^NTR association with TrkA is blocked in PC12/Nogo-A cells.

To address the question of whether Nogo-A disrupts the association of p75^NTR with TrkA, we first confirmed that PC12/wt and PC12/Nogo-A cells express comparable levels of p75^NTR. Figure 1B shows that western blotting of total cellular protein from the two cell types produced similar immunoreactivity for p75^NTR. Next, we immunoprecipitated TrkA from PC12/wt and PC12/Nogo-A cells treated at different times with 10 ng/ml NGF, followed by western blotting with an anti-C-terminus p75^NTR antibody to determine the relative amounts of co-immunoprecipitated p75^NTR. Figure 1 (panel d) shows that p75^NTR co-precipitated with TrkA in the wild type cells, but the levels of p75^NTR co-precipitated from PC12/Nogo-A cells were barely detectible, suggesting that Nogo-A prevented p75^NTR from associating with TrkA. Interestingly, in wild type cells, p75^NTR co-precipitated with TrkA even under unstimulated conditions (0 time), suggesting that p75^NTR is constitutively associated with TrkA. These results indicate that increased Nogo-A reduces the interaction between TrkA and p75^NTR.

3.3. Nogo-A overexpression changes the temporal dynamics of Erk1,2 phosphorylation following NGF stimulation.

The activation and temporal regulation of the mitogen-activated protein kinase (MAPK) pathway are critical for determining cell fate decisions guiding cell growth and survival [18, 19]. To examine the effect of Nogo-A overexpression on NGF-activated signaling pathways downstream from TrkA, we treated PC12/wt and PC12/NogoA cells with 10 ng/ml NGF and measured the phosphorylation of Erk1/2 by western blots probed with phospho-specific antibodies. Figure 2A shows that NGF-induced phosphorylation of combined Erk1,2 in PC12/Nogo-A cells was transient, peaking at five minutes, while PC12/wt cells showed phosphorylation that was sustained for over 60 minutes. Additionally, the 5-minute (transient) phosphorylation in the PC12/Nogo-A cells showed a significant increase over that for PC12/wt cells (Figure 2B), suggesting an inverse regulation between transient and sustained Erk1/2 phosphorylation. These results suggest that Nogo-A influences the temporal regulation of components of the MAPK pathway involved in Erk1/2 phosphorylation.

Fig. 2.

Nogo-A overexpressing cells show inhibition of sustained but increased transient NGF-induced phosphorylation of Erk1/2. A, Equivalent aliquots from PC12/wt or PC12/Nogo-A cell lysates used in Fig. 1 were subjected to western blotting for phospho-Erk1/2, total Erk1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). For uniform comparison, all samples for this figure were run on a single blot. Similar results were obtained in four separate immunoprecipitation experiments. B, Phospho-Erk1/2-reactive bands at the five-minute time point were quantified by densitometry (Image J) to determine differences between PC12/wt and PC12/Nogo-A cells. Data represent means ± SD from four separate experiments (N=4). Data were analyzed by two-tailed paired Student’s t-test (p = .0249). p < 0.05 was considered significant.

3.4. Calcium and calmodulin mediate suppression of sustained Erk1/2 phosphorylation following NGF treatment in PC12/Nogo-A cells.

Calcium (Ca²⁺) and Calmodulin (CaM) have been implicated in the temporal regulation of NGF-activated Erk1/2 signaling [20, 21], which prompted us to compare the effects of Ca²⁺ and CaM inhibitors on NGF-induced Erk1/2 phosphorylation between PC12/wt and PC12/Nogo-A cells. We used the Ca²⁺ chelator BAPTA-AM (25 μM) and the CaM inhibitors W5 (control) and W7 (each at 10 μM to minimize non-specific effects). To evaluate temporal changes in phosphorylation, we normalized Erk1 and Erk2 phosphorylation at each time point following NGF treatment to the maximum phosphorylation (five-minute control for BAPTA-AM experiments and five-minute W5-treated for W7 experiments). We analyzed Erk1 and Erk2 separately because past studies have demonstrated that, although often functionally redundant, differences do exist between these two kinases [22, 23]. Figure 3, A & B show representative western blots for PC12/wt cells pretreated for 30 minutes before NGF stimulation with BAPTA-AM (Fig. 3 A), W5 (control) or W7 (Fig. 3 B). These blots were then analyzed quantitatively for inhibitor-induced changes in Erk1 and Erk2 phosphorylation (Fig. 3, C – F). Both the calcium chelator BAPTA-AM (Figure 3, C & D) and the CaM inhibitor W7 (Fig. 3, E & F) produced a partial but significant decrease in transient (five-minute) phosphorylation for both Erk1 and Erk2, while sustained phosphorylation (15 and 30 minutes) was not significantly different from controls. These results confirmed that Ca²⁺ and CaM play a role in NGF-mediated transient activation of Erk1/2 in wild type cells [20, 21] and provided us with a basis to compare the effects of Nogo-A overexpression on Ca²⁺ and CaM regulation of NGF-induced Erk1/2 phosphorylation.

Fig. 3.

Inhibitors calcium and calmodulin decrease transient NGF-induced Erk1/2 phosphorylation in PC12/wt cells. PC12/wt cells were serum starved for 3-4 hours then incubated with vehicle (control), BAPTA-AM (25 μM), W5 (control) (10 μM) or W7 (10 μM) for 30 minutes. Cells then were stimulated with 10 ng/ml NGF for the indicated time points followed by analysis of Erk1/2 phosphorylation by western blotting with a phospho-specific antibody. For uniform comparison, all samples for this figure were run on a single blot. Similar results were obtained in four separate immunoprecipitation experiments. A and B, Representative immunoblots showing the effects of BAPTA-AM treatment (A) or W7 treatment (B) on NGF-induced Erk1/2 phosphorylation over time (30 minutes). C and D, Densitometry quantification of NGF-induced Erk1 (C) and Erk2 (D) phosphorylation for control and BAPTA-treated cells. E and F, Similar quantification of Erk1 (E) and Erk2 (F) phosphorylation for W5 and W7-treated cells. Data represent mean ± SD from three separate experiments (N=3). Data were analyzed by two-tailed paired Student’s t-test (p values are indicated above the bars). p < 0.05 was considered significant.

As shown above (Figure 2), sustained phosphorylation of Erk1/2 was considerably reduced in PC12/Nogo-A relative to PC12/wt cells following NGF stimulation. Pretreatment of PC12/Nogo-A cells with the Ca²⁺ chelator BAPTA-AM (Figure 4, A, C & D) or the CaM inhibitor W7 (Figure 4, B, E & F) prior to NGF treatment produced a significant increase over control in NGF-induced sustained Erk1/2 phosphorylation (Figure 4) with no apparent change in transient (5 minute) phosphorylation. Considering only the sustained phosphorylation, these results indicate that increased levels of cellular Nogo-A produced a Ca²⁺ and CaM-dependent suppression of NGF-induced sustained Erk1/2 phosphorylation. Taken together, the data from Figures 3 and 4 reveal that transient and sustained phosphorylation of Erk1/2 can be differentiated by their inverse sensitivities to inhibitors of Ca²⁺/CaM and that Nogo-A can influence this Ca²⁺/CaM-mediated regulation.

Fig. 4.

Calcium and calmodulin inhibitors increase transient NGF-induced phosphorylation of Erk1/2 in PC12/Nogo-A cells. PC12/Nogo-A cells were serum starved for 3-4 hours then incubated with vehicle (control), BAPTA-AM (25 μM), W5 (control) (10 μM) or W7 (10 μM) for 30 minutes. Cells then were stimulated with 10 ng/ml NGF for the indicated time points followed by analysis of Erk1/2 phosphorylation by western blotting with a phospho-specific antibody. For uniform comparison, all samples for this figure were run on a single blot. Similar results were obtained in four separate immunoprecipitation experiments. A and B, Representative immunoblots showing the effects of BAPTA-AM treatment (A) or W7 treatment (B) on NGF-induced Erk1/2 phosphorylation over time (30 minutes). C and D, Densitometry quantification of NGF-induced Erk1 (C) and Erk2 (D) phosphorylation for control and BAPTA-treated cells. E and F, Similar quantification of Erk1 (E) and Erk2 (F) phosphorylation for W5 and W7-treated cells. Data represent mean ± SD from five separate experiments (N=5). Data were analyzed by two-tailed paired Student’s t-test (p values are indicated above the bars). p < 0.05 was considered significant, ns = not significant.

4. DISCUSSION

The present study is an extension of our previous work that revealed the effect of Nogo-A on NGF-mediated survival signaling by showing that Nogo-A overexpression resulted in a ceramide-mediated delayed cell death of PC12 cells following NGF treatment [16]. In this previous study we proposed that overexpressed Nogo-A interfered with TrkA/p75^NTR signaling, which led to activation of the NGF → p75^NTR → neutral sphingomyelinase → ceramide pathway and eventual cell death. This result suggested to us that Nogo-A, when expressed at normal levels, may act as a modulator of NGF signaling without activating an apoptotic pathway. In support of this hypothesis, we now report that Nogo-A is associated with changes in the temporal signaling dynamics of NGF-mediated Erk1/2 activation. Using our PC12 cell line that overexpresses the Nogo-A protein, we show that Nogo-A associates with TrkA in an activation-dependent manner, prevents p75^NTR association with TrkA, and elicits Ca²⁺/CaM-dependent changes in transient and sustained Erk1/2 phosphorylation following NGF stimulation. Although it is uncertain whether the overexpression of Nogo-A recapitulates its function at the physiologic level, our results suggest a potential mechanism by which Nogo-A influences critical NGF signaling pathways associated with regulating axonal outgrowth and neuronal survival.

Several proteins have been identified as TrkA binding proteins, many of which modify TrkA function. The best known and most studied of these is p75^NTR, whose cooperation with TrkA is necessary for NGF to stimulate neuronal differentiation and survival [24–26]. Interestingly, p75^NTR, by itself or partnered with Nogo66 receptor/Lingo1 or sortilin can contribute negatively to neurite outgrowth [27] and survival [28]. These types of signaling mechanisms in which TrkA does not participate, may occur more frequently due to the availability of p75^NTR following disruption of TrkA/p75^NTR interaction by Nogo-A. Many other proteins associate with TrkA to either positively or negatively influence TrkA signaling and function; these include the sorting receptor sortilin [29], Mint2 [30], members of the LIG (leucine-rich repeat and immunoglobulin) family proteins [31, 32], suppressor of cytokine signaling-2 (SOCS2) [33], and amyloid precursor protein (APP) [34]. Our previous study [16] showed that TrkA and Nogo-A associated with each other in unstimulated cells, suggesting that Nogo-A is a member of the TrkA binding-protein family that serves to regulate neuronal survival. Our current findings lend additional support for the functional significance of interaction between these two proteins by demonstrating an increase Nogo-A/TrkA association following treatment with NGF. Although detailed molecular mechanisms responsible for Nogo-A association with NGF-activated TrkA and the subsequent temporal alterations in phosphorylation of TrkA and the downstream effectors Erk1/2 have yet to be established, the concurrent suppression of p75^NTR association with TrkA, even in unstimulated conditions, is likely an important contributing factor to Nogo-A-mediated downstream changes in NGF signaling.

MAPK has been shown to be an essential pathway involved in the formation of corticospinal axonal projections in vivo and in vitro [35, 36] and is a major signaling pathway stimulated by the activation of TrkA. For these reasons we chose Erk1/2 phosphorylation as a representative indicator to evaluate the effect of Nogo-A overexpression on signaling dynamics downstream from TrkA. By comparing the activation of Erk1/2 between PC12/wt and PC12/Nogo-A cells, we found that Nogo-A overexpression changed Erk1/2 phosphorylation from sustained to transient following NGF activation. This difference is reminiscent of studies contrasting NGF-induced sustained Erk1/2 phosphorylation with epidermal growth factor (EGF)-induced transient phosphorylation of Erk1/2 [37–39]. While both NGF and EGF activate the same signaling pathway, the temporal differences in Erk1/2 phosphorylation change the signaling outcomes; NGF promotes differentiation whereas EGF stimulates proliferation. While the full consequences of Nogo-A-mediated effects on NGF signaling remain to be established, the dramatic changes we observed in the temporal dynamics of Erk1/2 phosphorylation following NGF stimulation between PC12/wt and PC12/Nogo-A cells not only demonstrate distinct mechanistic divisions between transient and sustained phosphorylation of Erk1/2, but also suggest that Nogo-A may change or shift the outcomes of NGF signaling due to this transition.

What, then, are the mechanisms by which Nogo-A alters NGF-mediated Erk1/2 signaling? As discussed above, we present evidence that 1) Nogo-A associates with TrkA in an activation-dependent manner, and 2) in cells overexpressing Nogo-A, TrkA interactions with p75^NTR are diminished. These events likely play a role, directly or indirectly, in shifting NGF-mediated TrkA phosphorylation from sustained to transient, which subsequently defines, to some extent, the temporal change we observed in Erk1/2 phosphorylation [39]. We addressed the above question further by examining the role of Ca²⁺ and CaM in Nogo-A-mediated changes in Erk1/2 phosphorylation following NGF stimulation. Previous studies have shown that Ca²⁺ and CaM-associated mechanisms are involved in the regulation of transient [20, 21] and sustained [40] Erk1/2 activation. Our results indicate that Nogo-A influences a Ca²⁺/CaM-dependent mechanism to alter NGF-mediated Erk1/2 signaling, although the precise details of how Nogo-A regulates Ca2+/CaM to affect signaling pathways downstream from TrkA remain to be investigated.

A noteworthy observation concerning the effect of inhibitors on transient (5-minute) Erk1/2 phosphorylation is that the small but mostly non-significant inhibitory effect in PC12/Nogo-A cells (Figure 4, C – F) does not reflect the significant amount of inhibition we observed for PC12/wt (Figure 3, C – F). A possible explanation for this difference is that this early 5-minute time point is a combination of the inhibitor-induced decreased transient response, as seen for the PC12/wt cells, with the initial portion of the sustained response, which increases in the presence of inhibitors for PC12/Nogo-A cells. As a result, any change may be difficult to detect in PC12/Nogo-A cells because these simultaneous opposing changes offset each other. In PC12/wt cells there was a measurable and significant decrease in the 5-minute transient response in the presence of the inhibitors because there was no opposing increase in sustained phosphorylation to offset the inhibitor-induced decrease, suggesting that sustained phosphorylation was already at its maximum.

There are other important TrkA-related signaling mechanisms that Nogo-A-mediated activation of Ca2+ and CaM may influence. Interestingly, CaM also binds to TrkA in a calcium-dependent manner and is involved in the intracellular processing of TrkA, as revealed by studies in which CaM inhibition led to the proteolytic cleavage of 41 kDa C-terminal fragment of TrkA [41, 42]. Increased CaM activity by Nogo-A also may inhibit TrkA proteolytic processing and the release of cytosolic bioactive peptides from TrkA, such as the 41 kDa C-terminal fragment. Furthermore, Nogo-A, through its influence on Ca2+ and CaM-mediated mechanisms, may reduce TrkA recycling from endosomal compartments to the plasma membrane [43] and therefore decrease sustained activation of the receptor as well as cell survival [44]. Our results are consistent with this hypothesis since an inhibition of internalized receptor recycling back to the plasma membrane may explain, at least partially, the decrease in sustained TrkA phosphorylation and cell survival [16].

5. Conclusion

Our current findings provide insight into how Nogo-A contributes to neuronal homeostasis by influencing NGF signaling pathways. Our results support a model, summarized schematically in Figure 5, in which Nogo-A changes the signaling dynamics and specificity of TrkA-induced Erk1,2 activation by influencing Ca²⁺/CaM-dependent regulatory mechanisms that govern transient and sustained phosphorylation of Erk1/2 following NGF activation. We propose that Nogo-A is involved in regulating the balance between negative and positive signaling by modifying both TrkA/p75^NTR receptor-level events and the strength and duration of NGF-activated downstream signaling pathways. This work provides a foundation for further investigations into the influence of Nogo-A on signaling mediated by neurotrophins and possibly other growth factors.

Fig. 5.

Summary schematic depicting proposed changes in NGF-mediated Erk1,2 signaling resulting from Nogo-A overexpression in PC12 cells. (A) In wild type cells NGF activates the TrkA/p75^NTR complex to induce both a transient and sustained phosphorylation of Erk1,2. The transient response is at least partially dependent on Ca2+/CaM. (B) In Nogo-A-overexpressing cells, Nogo-A blocks the interaction between TrkA and p75NTR resulting in transient Erk1,2 phosphorylation but a Ca2+/CaM-dependent inhibition of sustained phosphorylation.

*Evidence for the absence of p75^NTR intracellular domain (ICD) cleavage and activation of the neutral sphingomyelinase → ceramide pathway leading to apoptosis in PC12/Nogo-A cells was provided previously (16).

Highlights.

• Nogo-A is a TrkA binding protein

• Nogo-A interferes with the association of p75^NTR with TrkA

• Nerve growth factor-mediated TrkA signaling is altered by Nogo-A

• Calcium and calmodulin mediate Nogo-A-induced changes to TrkA signaling

Abbreviations

APP

amyloid precursor protein

BAPTA-AM

1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester)

Ca²⁺

calcium ion

CaM

calmodulin

Erk1,2

extracellular signal-regulated kinase 1/2

GAPDH

glyceraldehyde 3-phosphate dehydrogenase

LIG

leucine-rich repeat and immunoglobulin

NGF

nerve growth factor

nSMase

neutral sphingomyelinase

P75^NTR

p75 neurotrophin receptor

P75^NTR ICD

p75 neurotrophin receptor intracellular domain

SOCS2

suppressor of cytokine signaling-2

TrkA

tropomyosin receptor kinase A

Data availability

All data generated during this study are contained within the article.