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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

(S)-LCM subverts CRMP2-mediated tubulin polymerization. (A) Effects of enantiomers of LCM on CRMP2-mediated microtubule assembly were measured by light scattering and absorbance at 412 nm. Also shown is the basal tubulin self-polymerization in the absence of any additional protein (no protein). Values represent measurements performed in triplicate. Background absorbance was subtracted from each measurement. Values from a representative experiment are illustrated. (B) Average area under the curve values ± s.e.m. calculated from the tubulin polymerization curves shown in (A). The addition of CRMP2 increased polymerization, while the inclusion of 3 μM (R)- or (S)-LCM led to a significant reduction in CRMP2-mediated enhancement of tubulin polymerization AUC compared to DMSO (*p < 0.05; One-Way ANOVA, Bonferroni post-hoc analysis).
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Figure 5: (S)-LCM subverts CRMP2-mediated tubulin polymerization. (A) Effects of enantiomers of LCM on CRMP2-mediated microtubule assembly were measured by light scattering and absorbance at 412 nm. Also shown is the basal tubulin self-polymerization in the absence of any additional protein (no protein). Values represent measurements performed in triplicate. Background absorbance was subtracted from each measurement. Values from a representative experiment are illustrated. (B) Average area under the curve values ± s.e.m. calculated from the tubulin polymerization curves shown in (A). The addition of CRMP2 increased polymerization, while the inclusion of 3 μM (R)- or (S)-LCM led to a significant reduction in CRMP2-mediated enhancement of tubulin polymerization AUC compared to DMSO (*p < 0.05; One-Way ANOVA, Bonferroni post-hoc analysis).

Mentions: Subsequently, the ability of CRMP2 to enhance tubulin polymerization in the presence of (S)-LCM was examined via turbidimetric assay. Based on the principle that light is scattered by microtubules to an extent that is proportional to the concentration of microtubule polymer, this assay determines the extent of tubulin polymerization by measuring changes in absorbance. Consistent with previous results (Wilson et al., 2012a), the addition of 200 nM recombinant CRMP2 protein dramatically enhanced tubulin polymerization by ~2.8-fold as demonstrated by area under the curve measurements compared to tubulin alone (p < 0.05) (Figures 5A,B). Similar to what is observed for (R)-LCM, the ability of CRMP2 to enhance tubulin polymerization was impaired by ~44.0% by as little as 3 μM (S)-LCM (p < 0.05) (Figure 5B). Additionally, FM4-64 labeling revealed that (S)-LCM does not impact synaptic bouton size—a phenomenon regulated by CRMP2 (Supplemental Figure 2).


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)

(S)-LCM subverts CRMP2-mediated tubulin polymerization. (A) Effects of enantiomers of LCM on CRMP2-mediated microtubule assembly were measured by light scattering and absorbance at 412 nm. Also shown is the basal tubulin self-polymerization in the absence of any additional protein (no protein). Values represent measurements performed in triplicate. Background absorbance was subtracted from each measurement. Values from a representative experiment are illustrated. (B) Average area under the curve values ± s.e.m. calculated from the tubulin polymerization curves shown in (A). The addition of CRMP2 increased polymerization, while the inclusion of 3 μM (R)- or (S)-LCM led to a significant reduction in CRMP2-mediated enhancement of tubulin polymerization AUC compared to DMSO (*p < 0.05; One-Way ANOVA, Bonferroni post-hoc analysis).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: (S)-LCM subverts CRMP2-mediated tubulin polymerization. (A) Effects of enantiomers of LCM on CRMP2-mediated microtubule assembly were measured by light scattering and absorbance at 412 nm. Also shown is the basal tubulin self-polymerization in the absence of any additional protein (no protein). Values represent measurements performed in triplicate. Background absorbance was subtracted from each measurement. Values from a representative experiment are illustrated. (B) Average area under the curve values ± s.e.m. calculated from the tubulin polymerization curves shown in (A). The addition of CRMP2 increased polymerization, while the inclusion of 3 μM (R)- or (S)-LCM led to a significant reduction in CRMP2-mediated enhancement of tubulin polymerization AUC compared to DMSO (*p < 0.05; One-Way ANOVA, Bonferroni post-hoc analysis).
Mentions: Subsequently, the ability of CRMP2 to enhance tubulin polymerization in the presence of (S)-LCM was examined via turbidimetric assay. Based on the principle that light is scattered by microtubules to an extent that is proportional to the concentration of microtubule polymer, this assay determines the extent of tubulin polymerization by measuring changes in absorbance. Consistent with previous results (Wilson et al., 2012a), the addition of 200 nM recombinant CRMP2 protein dramatically enhanced tubulin polymerization by ~2.8-fold as demonstrated by area under the curve measurements compared to tubulin alone (p < 0.05) (Figures 5A,B). Similar to what is observed for (R)-LCM, the ability of CRMP2 to enhance tubulin polymerization was impaired by ~44.0% by as little as 3 μM (S)-LCM (p < 0.05) (Figure 5B). Additionally, FM4-64 labeling revealed that (S)-LCM does not impact synaptic bouton size—a phenomenon regulated by CRMP2 (Supplemental Figure 2).

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