Collapsin response mediator protein 2 (CRMP2) interacts with N-methyl-D-aspartate (NMDA) receptor and Na+/Ca2+ exchanger and regulates their functional activity

Abstract

Collapsin response mediator protein 2 (CRMP2) is traditionally viewed as an axonal growth protein involved in axon/dendrite specification. Here, we describe novel functions of CRMP2. A 15-amino acid peptide from CRMP2, fused to the TAT cell-penetrating motif of the HIV-1 protein, TAT-CBD3, but not CBD3 without TAT, attenuated N-methyl-d-aspartate receptor (NMDAR) activity and protected neurons against glutamate-induced Ca(2+) dysregulation, suggesting the key contribution of CRMP2 in these processes. In addition, TAT-CBD3, but not CBD3 without TAT or TAT-scramble peptide, inhibited increases in cytosolic Ca(2+) mediated by the plasmalemmal Na(+)/Ca(2+) exchanger (NCX) operating in the reverse mode. Co-immunoprecipitation experiments revealed an interaction between CRMP2 and NMDAR as well as NCX3 but not NCX1. TAT-CBD3 disrupted CRMP2-NMDAR interaction without change in NMDAR localization. In contrast, TAT-CBD3 augmented the CRMP2-NCX3 co-immunoprecipitation, indicating increased interaction or stabilization of a complex between these proteins. Immunostaining with an anti-NCX3 antibody revealed that TAT-CBD3 induced NCX3 internalization, suggesting that both reverse and forward modes of NCX might be affected. Indeed, the forward mode of NCX, evaluated in experiments with ionomycin-induced Ca(2+) influx into neurons, was strongly suppressed by TAT-CBD3. Knockdown of CRMP2 with short interfering RNA (siRNA) prevented NCX3 internalization in response to TAT-CBD3 exposure. Moreover, CRMP2 down-regulation strongly attenuated TAT-CBD3-induced inhibition of reverse NCX. Overall, our results demonstrate that CRMP2 interacts with NCX and NMDAR and that TAT-CBD3 protects against glutamate-induced Ca(2+) dysregulation most likely via suppression of both NMDAR and NCX activities. Our results further clarify the mechanism of action of TAT-CBD3 and identify a novel regulatory checkpoint for NMDAR and NCX function based on CRMP2 interaction with these proteins.

Keywords: CRMP2; Calcium Signaling; Glutamate; Glutamate Receptors; NMDA Receptor; Neurons; Sodium Calcium Exchange; TAT-CBD3.

FIGURE 1.

TAT-CBD3, but not CBD3 without TAT or TAT alone, strongly inhibits glutamate-induced Ca²⁺ dysregulation. Unless stated otherwise, in this and other similar figures, rat hippocampal neurons (12–14 days in vitro) were loaded with Ca²⁺-sensitive dye Fura-2FF-AM to monitor changes in [Ca²⁺]_c. Here and in other figures, the thin, gray traces show fluorescent signals from individual neurons, whereas thick, red traces represent mean ± S.E. (error bars) from individual experiments (n = 20–25 neurons/experiment). Here and in all other figures, where indicated, neurons were treated either with vehicle (A) (DMSO, 0.1%) or 10 μm TAT-CBD3 (B) for 10 min prior to glutamate exposure. C, neurons were treated with 10 μm CBD3. The peptide remained in the bath solution throughout the experiment. D, neurons were treated with 10 μm TAT for 10 min prior to glutamate exposure. Where indicated, 25 μm glutamate plus 10 μm glycine (Glu) were applied. E, the summary graph shows the average area under the curve (AUC) (a.u., arbitrary units), which represents a measure of [Ca²⁺]_c increase over time. A representative AUC is shown in A as a gray area under the mean ± S.E. trace. Under each experimental condition, the AUC was calculated for the same time (1080 s) following glutamate application. *, p < 0.01 comparing vehicle- and TAT-CBD3-treated neurons. n = 5 separate, individual experiments; the total number of analyzed neurons is 383.

FIGURE 2.

TAT-CBD3, but not CBD3 without TAT, strongly inhibits NMDA-induced increases in cytosolic Ca²⁺. Rat hippocampal neurons (12–14 days in vitro) were loaded with Ca²⁺-sensitive dye Fura-2AM to monitor changes in [Ca²⁺]_c. In these experiments, where indicated, short pulses of 30 μm NMDA (plus 10 μm glycine) were applied to neurons. Following recovery of [Ca²⁺]_c after the first NMDA pulse, neurons were treated with either vehicle (A) (DMSO, 0.1%), 10 μm TAT-CBD3 (C), or 10 μm CBD3 (D), as indicated. Then one or two more NMDA pulses were applied. B, before the second NMDA pulse, neurons were treated with 20 μm AP-5 as a positive control. E, the summary graph shows changes in average peak cytosolic Ca²⁺ concentration under different conditions. #, p < 0.01 compared with [Ca²⁺]_c before treatment. NT, non-treated cells. n = 5 separate, individual experiments; the total number of analyzed neurons is 217. *, p < 0.01 compared with changes in average peak [Ca²⁺]_c in NMDA-treated cells. n = 5 separate, individual experiments; the total number of analyzed neurons is 205. Error bars, S.E.

FIGURE 3.

TAT-CBD3 disrupts CRMP2 interaction with NR2B-NMDAR but does not cause NMDAR relocalization. A, immunoprecipitation (IP) was performed with IgG or anti-CRMP2 antibody (Sigma-Aldrich) followed by Western blotting (WB) with anti-NR2B antibody (BD Biosciences). Where indicated, prior to immunoprecipitation, cells were incubated with vehicle (0.1% DMSO) or 10 μm TAT-CBD3 for 10 min. B and D, confocal, inverted fluorescence images of representative neurons stained with anti-NR2B antibody (BD Biosciences). C and E, fluorescence intensity profiles for straight lines in B and D, respectively. n = 3 separate, individual experiments. The inset in B shows Western blot produced with cell lysate and anti-NR2B antibody (BD Biosciences) to illustrate antibody specificity. Scale bars, 15 μm.

FIGURE 4.

Na⁺/NMDG replacement in the bath solution induces reversal of Na⁺/Ca²⁺ exchanger. Neurons were co-loaded with a Na⁺-sensitive dye, SBFI-AM (A and C), and a Ca²⁺-sensitive dye, Fluo-4FF-AM (B and D). Measurements of cytosolic Ca²⁺ and Na⁺ were performed simultaneously. The thin, gray traces show fluorescent signals from individual neurons. The thick, red traces represent cytosolic Ca²⁺ mean ± S.E. (error bars), whereas thick, green traces represent cytosolic Na⁺ mean ± S.E. (error bars) from individual experiments (n = 20–25 neurons/experiment). The bath solution was supplemented with 5 μm nifedipine to block L-type voltage-gated Ca²⁺ channels, 1 μm tetrodotoxin to block Na⁺ channels, and 20 μm AP-5 to antagonize the NMDA receptor. To elevate cytosolic Na⁺, neurons were preincubated for 10 min with 1 mm ouabain (Ouab). Ouabain remained in the bath solution throughout the experiment. Where indicated, Na⁺ was replaced by equimolar NMDG to trigger NCX reversal. C and D, 10 μm MK801, an inhibitor of reverse NCX (12), was applied to link the Na⁺/NMDG-induced increase in cytosolic Ca²⁺ to NCX_rev activity. n = 5 separate, individual experiments; the total number of analyzed neurons is 121.

FIGURE 5.

Na⁺/Ca²⁺ exchanger reversal induced by Na⁺/NMDG replacement is inhibited by TAT-CBD3. Neurons were loaded with Fura-2FF-AM. Where indicated, neurons were treated either with vehicle (A), 10 μm TAT-CBD3 (B), 10 μm TAT-CBD3 A6K (C), or 10 μm TAT-scramble peptide (D) for 10 min prior to NCX reversal induced by Na⁺/NMDG replacement. E, neurons were treated with 10 μm CBD3, and the peptide remained in the bath solution throughout the experiment. The bath solution was supplemented with 5 μm nifedipine, 1 μm tetrodotoxin, and 20 μm AP-5. In addition, neurons were preincubated for 10 min with 1 mm ouabain (Ouab). Ouabain remained in the bath solution throughout the experiment. F, the summary graph shows the average AUC (a.u., arbitrary units), which represents a measure of [Ca²⁺]_c increase over time. Under each experimental condition, the AUC was calculated for the same time (1080 s) following Na⁺/NMDG replacement. Data are mean ± S.E. (error bars). *, p < 0.01 comparing TAT-CBD3-, TAT-CBD3 A6K-, and vehicle-treated neurons. n = 4 separate, individual experiments with each condition; the total number of analyzed neurons is 404.

FIGURE 6.

Ni²⁺ and TAT-CBD3, but not TAT-scramble peptide, suppress ion currents mediated by NCX_rev. In A, the ascending voltage ramp protocol employed in these experiments and current traces obtained in the absence (black and blue) and in the presence of 5 mm Ni²⁺ (green) applied to neurons 2 min prior to the voltage ramp. B and C, current traces obtained with untreated neurons (black and blue) and neurons treated with either 10 μm TAT-CBD3 (B, red trace) or 10 μm TAT-scramble (C, orange trace) applied 5 min prior to the voltage ramp. D, the summary graph shows changes in the peak ion current under different conditions. *, p < 0.05 compared with vehicle-treated cells (vehicle). n = 5. Error bars, S.E.

FIGURE 7.

CRMP2 co-immunoprecipitates with NCX3, but not with NCX1. TAT-CBD3 strengthens CRMP2-NCX3 interaction. A, immunoprecipitation (IP) was performed with IgG or anti-CRMP2 antibody followed by Western blotting (WB) with anti-NCX1 antibody. B, immunoprecipitation was performed with IgG or anti-NCX3 antibody followed by Western blotting with anti-CRMP2 antibody. C, prior to immunoprecipitation, where indicated, neurons were incubated either with 10 μm TAT-CBD3 or with 10 μm TAT-scramble peptide for 10 min. Each experiment was repeated three times.

FIGURE 8.

TAT-CBD3 triggers NCX3 internalization. A, C, and E, confocal, inverted fluorescence images of representative neurons stained with anti-NCX3 antibody. B, D, and F, fluorescence intensity profiles for straight lines in A, C, and E, respectively. n = 3 separate, individual experiments. The total numbers of analyzed neurons are 59 (vehicle-treated), 65 (TAT-CBD3-treated), and 51 (TAT-scramble-treated). Inset in A, Western blot produced with cell lysate and anti-NCX3 antibody (provided by Drs. Kenneth Philipson and Michela Ottolia, UCLA) to illustrate antibody specificity. Scale bars, 15 μm.

FIGURE 9.

CRMP2 down-regulation prevents TAT-CBD3-induced NCX3 internalization. Neurons were transfected during plating with a GFP construct and siRNA against CRMP2. A–C, a representative experiment (bright field (A), GFP fluorescence (B), and CRMP2 immunostaining (C)). In this experiment, neuron without GFP has robust CRMP2 expression, whereas neuron with GFP fluorescence demonstrates significantly decreased CRMP2 expression (marked by an arrow). D, a summary of CRMP2 expression measurements based on fluorescence intensity measurements. The fluorescence measurements were performed in somata of individual neurons, using a 3 × 3-μm region of interest. The fluorescence intensity is expressed in arbitrary units (a.u.). The data are mean ± S.E. (error bars), n = 21. E–G, a representative experiment without TAT-CBD3 pretreatment (bright field (E), GFP fluorescence (F), and NCX3 immunofluorescence (inverted) (G)). In H, fluorescence intensity profile for a straight line in G. In these experiments, 18 cells were analyzed; each cell had a similar pattern of NCX3 distribution. I–K, a representative experiment with 10 μm TAT-CBD3 pretreatment for 10 min (bright field (I), GFP fluorescence (J), and NCX3 immunofluorescence (inverted) (K)). L, fluorescence intensity profile for a straight line in K. In these experiments, 24 cells were analyzed; each cell had a similar pattern of NCX3 distribution. Scale bars, 15 μm.

FIGURE 10.

CRMP2 down-regulation strongly attenuates TAT-CBD3-induced inhibition of NCX_rev. Neurons, transfected during plating with a GFP construct and siRNA against CRMP2, were loaded with Fura-2FF-AM. In A–E, a representative experiment is shown (bright field (A), GFP fluorescence (B), pseudocolored image of Fura-2FF F₃₄₀/F₃₈₀ ratio taken prior to Na⁺/NMDG replacement at 300 s (C), pseudocolored image of Fura-2FF F₃₄₀/F₃₈₀ ratio taken after Na⁺/NMDG replacement at 1500 s (D), and changes in cytosolic Ca²⁺ concentration over time in the same experiment (E)). Where indicated, neurons were treated with 10 μm TAT-CBD3 for 10 min. The bath solution was supplemented with 5 μm nifedipine, 1 μm tetrodotoxin, and 20 μm AP-5. In addition, neurons were preincubated for 10 min with 1 mm ouabain prior to Na⁺/NMDG replacement. Ouabain remained in the bath solution throughout the experiment. A–D, arrows indicate a neuron that expresses GFP and has down-regulated CRMP2. E, the green trace shows fluorescent signal from this neuron, whereas black traces show fluorescent signals from other neurons in the field. F, the summary graph shows the average AUC (a.u., arbitrary units), which represents a measure of [Ca²⁺]_c increase over time. Under each experimental condition, the AUC was calculated for the same time (900 s) following Na⁺/NMDG replacement. Data are mean ± S.E. (error bars); *, p < 0.01 comparing siRNA-transfected (siRNA) and non-transfected (NT) neurons. n = 3 separate, individual experiments; the total number of analyzed neurons is 57. Scale bar in A, 30 μm, applicable to A–D.

FIGURE 11.

TAT-CBD3 inhibits the forward mode of NCX. Neurons were loaded with Fura-2FF. A, 5 μm ionomycin in combination with 20 μm AP-5 was applied to neurons. Where indicated, neurons were subjected to Na⁺/NMDG replacement (B) or treated with 10 μm TAT-CBD3 (C) for 10 min prior to the addition of ionomycin. D, the summary graph shows the time from the beginning of ionomycin application to the onset of Ca²⁺ dysregulation (t_dys), which reflects NCX_for activity. Data are mean ± S.E. (error bars). *, p < 0.01 comparing the effect of Na⁺/NMDG replacement and TAT-CBD3- and vehicle-treated neurons; n = 5 separate, individual experiments; the total number of analyzed neurons is 101.

FIGURE 12.

Proposed mechanisms of CRMP2- and TAT-CBD3-mediated regulation of Ca²⁺ signaling in neurons. CRMP2 binds to NMDAR, NCX, and CaV2.2 and, presumably, determines their localization and functional activity. We speculate that TAT-CBD3 binds to NMDAR and CaV2.2 and triggers CRMP2 dissociation from these proteins, leading to augmentation of CRMP2 binding to NCX. Disruption of the CRMP2-CaV2.2 complex leads to CaV2.2 internalization and a decrease in its activity (35). (Notably, voltage-gated Ca²⁺ channels do not significantly contribute to glutamate-induced Ca²⁺ dysregulation in our experiments (35).) Augmentation of CRMP2-NCX3 binding also leads to NCX internalization, and in both cases with CaV2.2 and NCX3, internalization leads to suppression of functional activity of these proteins. However, disruption of the CRMP2-NMDAR complex does not cause relocation of NMDAR and yet also results in suppression of NMDAR activity by an unknown mechanism. Inhibition of NMDAR in combination with suppression of NCX is responsible for a decrease in Ca²⁺ influx into neurons and protection against glutamate-induced Ca²⁺-dysregulation. The scheme illustrates neither structural peculiarities nor true stoichiometry of molecular components involved in Ca²⁺ signaling but serves the purpose of illustrating major CRMP2-associated events that alter Ca²⁺ signaling regardless of their localization within the presynaptic or postsynaptic neuron.