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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

KCl-induced activity decreases Cdk5-phosphorylated CRMP2. (A) Timeline of experimental procedures. (B) Schematic of GSK3β and Cdk5 phosphorylation sites within the rat CRMP2 sequence. Numbers represent amino acid residues within the CRMP2 sequence. (C) Representative immunoblots of Cdk5-phosphorylated CRMP2 (CRMP2 pSer522) and β III-tubulin from naïve cortical neurons compared to those exposed to KCl for 30 min or 96 h. (D) KCl exposure lead to a time-dependent decrease in the level of Cdk5-phosphorylated CRMP2 (CRMP2 pSer522) (*p < 0.05 compared to control and #p < 0.05 compared to 30 min KCl treatment; One-Way ANOVA, Tukey's post-hoc analysis; n = 5).
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Figure 2: KCl-induced activity decreases Cdk5-phosphorylated CRMP2. (A) Timeline of experimental procedures. (B) Schematic of GSK3β and Cdk5 phosphorylation sites within the rat CRMP2 sequence. Numbers represent amino acid residues within the CRMP2 sequence. (C) Representative immunoblots of Cdk5-phosphorylated CRMP2 (CRMP2 pSer522) and β III-tubulin from naïve cortical neurons compared to those exposed to KCl for 30 min or 96 h. (D) KCl exposure lead to a time-dependent decrease in the level of Cdk5-phosphorylated CRMP2 (CRMP2 pSer522) (*p < 0.05 compared to control and #p < 0.05 compared to 30 min KCl treatment; One-Way ANOVA, Tukey's post-hoc analysis; n = 5).

Mentions: In the case of CRMP2, substrate recognition by GSK3β first requires phosphorylation by Cdk5 at a downstream site (Ser522), which “primes” the protein for subsequent GSK3β phosphorylation (Cole et al., 2006) (see Figure 2B). Therefore, Western blot analysis of Cdk5-phosphorylated CRMP2 was used to determine if the KCl-induced decrease in GSKβ phosphorylation is due to a reduction in Cdk5 priming. Both acute and chronic exposure to KCl (Figure 2A) decreased the level of Cdk5-phosphorylated CRMP2 in a time dependent manner by ~44.8% (acute) and ~84.9% (chronic) compared to control (p < 0.05) (Figures 2C,D), suggesting that the activity-dependent decrease in GSK3β-phosphorylated CRMP2 can be attributed to decreased levels of Cdk5-primed CRMP2.


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)

KCl-induced activity decreases Cdk5-phosphorylated CRMP2. (A) Timeline of experimental procedures. (B) Schematic of GSK3β and Cdk5 phosphorylation sites within the rat CRMP2 sequence. Numbers represent amino acid residues within the CRMP2 sequence. (C) Representative immunoblots of Cdk5-phosphorylated CRMP2 (CRMP2 pSer522) and β III-tubulin from naïve cortical neurons compared to those exposed to KCl for 30 min or 96 h. (D) KCl exposure lead to a time-dependent decrease in the level of Cdk5-phosphorylated CRMP2 (CRMP2 pSer522) (*p < 0.05 compared to control and #p < 0.05 compared to 30 min KCl treatment; One-Way ANOVA, Tukey's post-hoc analysis; n = 5).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: KCl-induced activity decreases Cdk5-phosphorylated CRMP2. (A) Timeline of experimental procedures. (B) Schematic of GSK3β and Cdk5 phosphorylation sites within the rat CRMP2 sequence. Numbers represent amino acid residues within the CRMP2 sequence. (C) Representative immunoblots of Cdk5-phosphorylated CRMP2 (CRMP2 pSer522) and β III-tubulin from naïve cortical neurons compared to those exposed to KCl for 30 min or 96 h. (D) KCl exposure lead to a time-dependent decrease in the level of Cdk5-phosphorylated CRMP2 (CRMP2 pSer522) (*p < 0.05 compared to control and #p < 0.05 compared to 30 min KCl treatment; One-Way ANOVA, Tukey's post-hoc analysis; n = 5).
Mentions: In the case of CRMP2, substrate recognition by GSK3β first requires phosphorylation by Cdk5 at a downstream site (Ser522), which “primes” the protein for subsequent GSK3β phosphorylation (Cole et al., 2006) (see Figure 2B). Therefore, Western blot analysis of Cdk5-phosphorylated CRMP2 was used to determine if the KCl-induced decrease in GSKβ phosphorylation is due to a reduction in Cdk5 priming. Both acute and chronic exposure to KCl (Figure 2A) decreased the level of Cdk5-phosphorylated CRMP2 in a time dependent manner by ~44.8% (acute) and ~84.9% (chronic) compared to control (p < 0.05) (Figures 2C,D), suggesting that the activity-dependent decrease in GSK3β-phosphorylated CRMP2 can be attributed to decreased levels of Cdk5-primed CRMP2.

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