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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 GSK3 β phosphorylation of CRMP2 without changing GSK3 β activity or expression. (A) Top: Timeline of experimental procedures. Bottom: Schematic of GSK3β and Cdk5 phosphorylation sites within the rat CRMP2 sequence. Numbers represent amino acid residues within the CRMP2 sequence. Representative immunoblots of GSK3β-phosphorylated CRMP2 (CRMP2 pThr509/pThr514), total CRMP2, and β III-tubulin (B), inactivated Ser9-phosphorylated GSK3β and β III-tubulin (E), and total GSK3β and β III-tubulin (G) from naïve cortical neurons compared to those exposed to KCl for 30 min or 96 h. Summary of the relative levels of the indicated proteins (C,D,F,H). Expression of GSK3β-phosphorylated CRMP2 (CRMP2 pThr509/pThr514) is decreased following 30 min or 96 h exposure to KCl (C) whereas total CRMP2 expression was not affected by KCl exposures (D) (*p < 0.05 compared to control; One-Way ANOVA, Tukey's post-hoc analysis) (n = 4). KCl treatment did not alter phosphorylation of GSK3β (F) nor total GSK3β expression (H) did not change following 30 min or 96 h KCl treatment (One-Way ANOVA, Tukey's post-hoc analysis) (n = 5).
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Figure 1: KCl-induced activity decreases GSK3 β phosphorylation of CRMP2 without changing GSK3 β activity or expression. (A) Top: Timeline of experimental procedures. Bottom: Schematic of GSK3β and Cdk5 phosphorylation sites within the rat CRMP2 sequence. Numbers represent amino acid residues within the CRMP2 sequence. Representative immunoblots of GSK3β-phosphorylated CRMP2 (CRMP2 pThr509/pThr514), total CRMP2, and β III-tubulin (B), inactivated Ser9-phosphorylated GSK3β and β III-tubulin (E), and total GSK3β and β III-tubulin (G) from naïve cortical neurons compared to those exposed to KCl for 30 min or 96 h. Summary of the relative levels of the indicated proteins (C,D,F,H). Expression of GSK3β-phosphorylated CRMP2 (CRMP2 pThr509/pThr514) is decreased following 30 min or 96 h exposure to KCl (C) whereas total CRMP2 expression was not affected by KCl exposures (D) (*p < 0.05 compared to control; One-Way ANOVA, Tukey's post-hoc analysis) (n = 4). KCl treatment did not alter phosphorylation of GSK3β (F) nor total GSK3β expression (H) did not change following 30 min or 96 h KCl treatment (One-Way ANOVA, Tukey's post-hoc analysis) (n = 5).

Mentions: CRMP2 has recently been suggested as a potential mediator of activity-dependent neurite outgrowth in cerebellar granule neurons (Tan et al., 2013). Unlike other central neurons, cerebellar granule cells require slightly depolarizing conditions for survival in vitro. Therefore, it is difficult to generalize this finding to other neuronal populations within the central nervous system. As such, it is not known if CRMP2 is involved in outgrowth induced by depolarization in neurons where it is not necessary for survival. As the ability of CRMP2 to enhance neurite outgrowth is highly dependent upon its phosphorylation state (for review see Khanna et al., 2012), Western blot analysis was used to determine the level of GSK3β-phosphorylated CRMP2 following acute (30 min) and chronic exposure to KCl (96 h) treatment (Figure 1A). The 96 h KCl treatment was chosen as it reproducibly increased neurite outgrowth. Additionally, as it was unknown how long any biochemical changes, such as phosphorylation, would be sustained, a more acute (30 min) treatment was also used. Treatment with 25 mM KCl reduced the level of GSK3β-phosphorylated CRMP2 by ~60.8% (acute) and ~54.8% (chronic) compared to control (p < 0.05), while total CRMP2 expression did not change (Figures 1B–D). Therefore, exposure to KCl leads to increased levels of active, unphosphorylated 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 GSK3 β phosphorylation of CRMP2 without changing GSK3 β activity or expression. (A) Top: Timeline of experimental procedures. Bottom: Schematic of GSK3β and Cdk5 phosphorylation sites within the rat CRMP2 sequence. Numbers represent amino acid residues within the CRMP2 sequence. Representative immunoblots of GSK3β-phosphorylated CRMP2 (CRMP2 pThr509/pThr514), total CRMP2, and β III-tubulin (B), inactivated Ser9-phosphorylated GSK3β and β III-tubulin (E), and total GSK3β and β III-tubulin (G) from naïve cortical neurons compared to those exposed to KCl for 30 min or 96 h. Summary of the relative levels of the indicated proteins (C,D,F,H). Expression of GSK3β-phosphorylated CRMP2 (CRMP2 pThr509/pThr514) is decreased following 30 min or 96 h exposure to KCl (C) whereas total CRMP2 expression was not affected by KCl exposures (D) (*p < 0.05 compared to control; One-Way ANOVA, Tukey's post-hoc analysis) (n = 4). KCl treatment did not alter phosphorylation of GSK3β (F) nor total GSK3β expression (H) did not change following 30 min or 96 h KCl treatment (One-Way ANOVA, Tukey's post-hoc analysis) (n = 5).
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Related In: Results  -  Collection

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Figure 1: KCl-induced activity decreases GSK3 β phosphorylation of CRMP2 without changing GSK3 β activity or expression. (A) Top: Timeline of experimental procedures. Bottom: Schematic of GSK3β and Cdk5 phosphorylation sites within the rat CRMP2 sequence. Numbers represent amino acid residues within the CRMP2 sequence. Representative immunoblots of GSK3β-phosphorylated CRMP2 (CRMP2 pThr509/pThr514), total CRMP2, and β III-tubulin (B), inactivated Ser9-phosphorylated GSK3β and β III-tubulin (E), and total GSK3β and β III-tubulin (G) from naïve cortical neurons compared to those exposed to KCl for 30 min or 96 h. Summary of the relative levels of the indicated proteins (C,D,F,H). Expression of GSK3β-phosphorylated CRMP2 (CRMP2 pThr509/pThr514) is decreased following 30 min or 96 h exposure to KCl (C) whereas total CRMP2 expression was not affected by KCl exposures (D) (*p < 0.05 compared to control; One-Way ANOVA, Tukey's post-hoc analysis) (n = 4). KCl treatment did not alter phosphorylation of GSK3β (F) nor total GSK3β expression (H) did not change following 30 min or 96 h KCl treatment (One-Way ANOVA, Tukey's post-hoc analysis) (n = 5).
Mentions: CRMP2 has recently been suggested as a potential mediator of activity-dependent neurite outgrowth in cerebellar granule neurons (Tan et al., 2013). Unlike other central neurons, cerebellar granule cells require slightly depolarizing conditions for survival in vitro. Therefore, it is difficult to generalize this finding to other neuronal populations within the central nervous system. As such, it is not known if CRMP2 is involved in outgrowth induced by depolarization in neurons where it is not necessary for survival. As the ability of CRMP2 to enhance neurite outgrowth is highly dependent upon its phosphorylation state (for review see Khanna et al., 2012), Western blot analysis was used to determine the level of GSK3β-phosphorylated CRMP2 following acute (30 min) and chronic exposure to KCl (96 h) treatment (Figure 1A). The 96 h KCl treatment was chosen as it reproducibly increased neurite outgrowth. Additionally, as it was unknown how long any biochemical changes, such as phosphorylation, would be sustained, a more acute (30 min) treatment was also used. Treatment with 25 mM KCl reduced the level of GSK3β-phosphorylated CRMP2 by ~60.8% (acute) and ~54.8% (chronic) compared to control (p < 0.05), while total CRMP2 expression did not change (Figures 1B–D). Therefore, exposure to KCl leads to increased levels of active, unphosphorylated 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