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The functionalized amino acid (S)-Lacosamide subverts CRMP2-mediated tubulin polymerization to prevent constitutive and activity-dependent increase in neurite outgrowth.

Wilson SM, Moutal A, Melemedjian OK, Wang Y, Ju W, François-Moutal L, Khanna M, Khanna R - Front Cell Neurosci (2014)

Bottom Line: Whereas (S)-LCM was ineffective in targeting VGSCs, the presumptive pharmacological targets of (R)-LCM, (S)-LCM was more efficient than (R)-LCM in subverting neurite outgrowth.Knockdown of CRMP2 by siRNA in cortical neurons resulted in reduced CRMP2-dependent neurite outgrowth; incubation with (S)-LCM phenocopied this effect.Taken together, these results suggest that changes in the phosphorylation state of CRMP2 are a major contributing factor in activity-dependent regulation of neurite outgrowth.

View Article: PubMed Central - PubMed

Affiliation: Paul and Carole Stark Neurosciences Research Institute, Indiana University School of Medicine Indianapolis, IN, USA.

ABSTRACT
Activity-dependent neurite outgrowth is a highly complex, regulated process with important implications for neuronal circuit remodeling in development as well as in seizure-induced sprouting in epilepsy. Recent work has linked outgrowth to collapsin response mediator protein 2 (CRMP2), an intracellular phosphoprotein originally identified as axon guidance and growth cone collapse protein. The neurite outgrowth promoting function of CRMP2 is regulated by its phosphorylation state. In this study, depolarization (potassium chloride)-driven activity increased the level of active CRMP2 by decreasing its phosphorylation by GSK3β via a reduction in priming by Cdk5. To determine the contribution of CRMP2 in activity-driven neurite outgrowth, we screened a limited set of compounds for their ability to reduce neurite outgrowth but not modify voltage-gated sodium channel (VGSC) biophysical properties. This led to the identification of (S)-lacosamide ((S)-LCM), a stereoisomer of the clinically used antiepileptic drug (R)-LCM (Vimpat®), as a novel tool for preferentially targeting CRMP2-mediated neurite outgrowth. Whereas (S)-LCM was ineffective in targeting VGSCs, the presumptive pharmacological targets of (R)-LCM, (S)-LCM was more efficient than (R)-LCM in subverting neurite outgrowth. Biomolecular interaction analyses revealed that (S)-LCM bound to wildtype CRMP2 with low micromolar affinity, similar to (R)-LCM. Through the use of this novel tool, the activity-dependent increase in neurite outgrowth observed following depolarization was characterized to be reliant on CRMP2 function. Knockdown of CRMP2 by siRNA in cortical neurons resulted in reduced CRMP2-dependent neurite outgrowth; incubation with (S)-LCM phenocopied this effect. Other CRMP2-mediated processes were unaffected. (S)-LCM subverted neurite outgrowth not by affecting the canonical CRMP2-tubulin association but rather by impairing the ability of CRMP2 to promote tubulin polymerization, events that are perfunctory for neurite outgrowth. Taken together, these results suggest that changes in the phosphorylation state of CRMP2 are a major contributing factor in activity-dependent regulation of neurite outgrowth.

No MeSH data available.


Related in: MedlinePlus

Chronic KCl depolarization increases the association of CRMP2 and tubulin. (A) Timeline of experimental procedures. (B) Summary of CRMP2-binding to tubulin from cortical cell lysates as determined by ELISA. The values obtained from these experiments are arbitrary optical densities. Thus, we have normalized the values of treated groups to the control group, which was set to 100%. This allows us to compare values across experiments. KCl exposure increased CRMP2 binding by ~43.5% compared to control. As (S)-LCM was previously shown not to impact CRMP2/tubulin binding, its co-application did not alter the effect of KCl. (*p < 0.05 compared to control; One-Way ANOVA, Tukey's post-hoc analysis) (n = 4).
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Figure 9: Chronic KCl depolarization increases the association of CRMP2 and tubulin. (A) Timeline of experimental procedures. (B) Summary of CRMP2-binding to tubulin from cortical cell lysates as determined by ELISA. The values obtained from these experiments are arbitrary optical densities. Thus, we have normalized the values of treated groups to the control group, which was set to 100%. This allows us to compare values across experiments. KCl exposure increased CRMP2 binding by ~43.5% compared to control. As (S)-LCM was previously shown not to impact CRMP2/tubulin binding, its co-application did not alter the effect of KCl. (*p < 0.05 compared to control; One-Way ANOVA, Tukey's post-hoc analysis) (n = 4).

Mentions: To determine the potential mechanism underlying CRMP2's role in KCl-facilitated neurite outgrowth, we examined the ability of CRMP2 to bind tubulin following chronic exposure to KCl (Figure 9A). Lysates from cortical neurons that had been exposed to KCl for 96 h were incubated on tubulin-coated plates. An ELISA was then used to determine the amount of bound CRMP2. Consistent with the notion that a loss of phosphorylation should translate into an increase in CRMP2 activity, KCl exposure led to a ~43.5% increase in the binding of CRMP2 to tubulin compared to control (p < 0.05) (Figure 9B). As (S)-LCM was previously demonstrated to alter CRMP2 function by inhibiting its ability to enhance the intrinsic GTPase activity of tubulin, the addition of (S)-LCM did not alter the effect of KCl on CRMP2/tubulin binding [(143.5 ± 11.9) compared to KCl alone (133.5 ± 4.6)] (p > 0.05) (Figure 9).


The functionalized amino acid (S)-Lacosamide subverts CRMP2-mediated tubulin polymerization to prevent constitutive and activity-dependent increase in neurite outgrowth.

Wilson SM, Moutal A, Melemedjian OK, Wang Y, Ju W, François-Moutal L, Khanna M, Khanna R - Front Cell Neurosci (2014)

Chronic KCl depolarization increases the association of CRMP2 and tubulin. (A) Timeline of experimental procedures. (B) Summary of CRMP2-binding to tubulin from cortical cell lysates as determined by ELISA. The values obtained from these experiments are arbitrary optical densities. Thus, we have normalized the values of treated groups to the control group, which was set to 100%. This allows us to compare values across experiments. KCl exposure increased CRMP2 binding by ~43.5% compared to control. As (S)-LCM was previously shown not to impact CRMP2/tubulin binding, its co-application did not alter the effect of KCl. (*p < 0.05 compared to control; One-Way ANOVA, Tukey's post-hoc analysis) (n = 4).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4109617&req=5

Figure 9: Chronic KCl depolarization increases the association of CRMP2 and tubulin. (A) Timeline of experimental procedures. (B) Summary of CRMP2-binding to tubulin from cortical cell lysates as determined by ELISA. The values obtained from these experiments are arbitrary optical densities. Thus, we have normalized the values of treated groups to the control group, which was set to 100%. This allows us to compare values across experiments. KCl exposure increased CRMP2 binding by ~43.5% compared to control. As (S)-LCM was previously shown not to impact CRMP2/tubulin binding, its co-application did not alter the effect of KCl. (*p < 0.05 compared to control; One-Way ANOVA, Tukey's post-hoc analysis) (n = 4).
Mentions: To determine the potential mechanism underlying CRMP2's role in KCl-facilitated neurite outgrowth, we examined the ability of CRMP2 to bind tubulin following chronic exposure to KCl (Figure 9A). Lysates from cortical neurons that had been exposed to KCl for 96 h were incubated on tubulin-coated plates. An ELISA was then used to determine the amount of bound CRMP2. Consistent with the notion that a loss of phosphorylation should translate into an increase in CRMP2 activity, KCl exposure led to a ~43.5% increase in the binding of CRMP2 to tubulin compared to control (p < 0.05) (Figure 9B). As (S)-LCM was previously demonstrated to alter CRMP2 function by inhibiting its ability to enhance the intrinsic GTPase activity of tubulin, the addition of (S)-LCM did not alter the effect of KCl on CRMP2/tubulin binding [(143.5 ± 11.9) compared to KCl alone (133.5 ± 4.6)] (p > 0.05) (Figure 9).

Bottom Line: Whereas (S)-LCM was ineffective in targeting VGSCs, the presumptive pharmacological targets of (R)-LCM, (S)-LCM was more efficient than (R)-LCM in subverting neurite outgrowth.Knockdown of CRMP2 by siRNA in cortical neurons resulted in reduced CRMP2-dependent neurite outgrowth; incubation with (S)-LCM phenocopied this effect.Taken together, these results suggest that changes in the phosphorylation state of CRMP2 are a major contributing factor in activity-dependent regulation of neurite outgrowth.

View Article: PubMed Central - PubMed

Affiliation: Paul and Carole Stark Neurosciences Research Institute, Indiana University School of Medicine Indianapolis, IN, USA.

ABSTRACT
Activity-dependent neurite outgrowth is a highly complex, regulated process with important implications for neuronal circuit remodeling in development as well as in seizure-induced sprouting in epilepsy. Recent work has linked outgrowth to collapsin response mediator protein 2 (CRMP2), an intracellular phosphoprotein originally identified as axon guidance and growth cone collapse protein. The neurite outgrowth promoting function of CRMP2 is regulated by its phosphorylation state. In this study, depolarization (potassium chloride)-driven activity increased the level of active CRMP2 by decreasing its phosphorylation by GSK3β via a reduction in priming by Cdk5. To determine the contribution of CRMP2 in activity-driven neurite outgrowth, we screened a limited set of compounds for their ability to reduce neurite outgrowth but not modify voltage-gated sodium channel (VGSC) biophysical properties. This led to the identification of (S)-lacosamide ((S)-LCM), a stereoisomer of the clinically used antiepileptic drug (R)-LCM (Vimpat®), as a novel tool for preferentially targeting CRMP2-mediated neurite outgrowth. Whereas (S)-LCM was ineffective in targeting VGSCs, the presumptive pharmacological targets of (R)-LCM, (S)-LCM was more efficient than (R)-LCM in subverting neurite outgrowth. Biomolecular interaction analyses revealed that (S)-LCM bound to wildtype CRMP2 with low micromolar affinity, similar to (R)-LCM. Through the use of this novel tool, the activity-dependent increase in neurite outgrowth observed following depolarization was characterized to be reliant on CRMP2 function. Knockdown of CRMP2 by siRNA in cortical neurons resulted in reduced CRMP2-dependent neurite outgrowth; incubation with (S)-LCM phenocopied this effect. Other CRMP2-mediated processes were unaffected. (S)-LCM subverted neurite outgrowth not by affecting the canonical CRMP2-tubulin association but rather by impairing the ability of CRMP2 to promote tubulin polymerization, events that are perfunctory for neurite outgrowth. Taken together, these results suggest that changes in the phosphorylation state of CRMP2 are a major contributing factor in activity-dependent regulation of neurite outgrowth.

No MeSH data available.


Related in: MedlinePlus