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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

The effect of (S)-LCM on neurite outgrowth phenocopies siRNA knockdown of CRMP2. (A) Representative inverted black and white representative image of a cortical neuron 48 h following EGFP-transfection. (B–F) Representative tracings of neurons transfected with EGFP ± control siRNA, CRMP2 siRNA, 200 μM (S)-LCM, or CRMP2 siRNA + (S)-LCM. (G) Total outgrowth of neurons transfected with EGFP, control siRNA, or CRMPsiRNA combined with 24 h (S)-LCM treatment (200 μM). CRMP2 siRNA and (S)-LCM reduced outgrowth to a similar level. Combination of CRMP2 and (S)-LCM did not produce a further reduction. (H–K) Comparison of the effects of CRMP2 siRNA and (S)-LCM on # of branches, # of processes, mean process length, and maximum process length. (*p < 0.05 vs. EGFP alone, One-Way ANOVA, Tukey's post-hoc analysis) (values represent mean ± s.e.m.) (n = 86–320 cells, 8 separate culture wells) (scale bar = 50 μm).
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Figure 7: The effect of (S)-LCM on neurite outgrowth phenocopies siRNA knockdown of CRMP2. (A) Representative inverted black and white representative image of a cortical neuron 48 h following EGFP-transfection. (B–F) Representative tracings of neurons transfected with EGFP ± control siRNA, CRMP2 siRNA, 200 μM (S)-LCM, or CRMP2 siRNA + (S)-LCM. (G) Total outgrowth of neurons transfected with EGFP, control siRNA, or CRMPsiRNA combined with 24 h (S)-LCM treatment (200 μM). CRMP2 siRNA and (S)-LCM reduced outgrowth to a similar level. Combination of CRMP2 and (S)-LCM did not produce a further reduction. (H–K) Comparison of the effects of CRMP2 siRNA and (S)-LCM on # of branches, # of processes, mean process length, and maximum process length. (*p < 0.05 vs. EGFP alone, One-Way ANOVA, Tukey's post-hoc analysis) (values represent mean ± s.e.m.) (n = 86–320 cells, 8 separate culture wells) (scale bar = 50 μm).

Mentions: Having demonstrated that (S)-LCM interacts with CRMP2, we next investigated the possible effect of this interaction on CRMP2 function. If (S)-LCM acts to inhibit CRMP2-mediated neurite outgrowth, then its action should mimic the phenotype bestowed by CRMP2 siRNA without the off-target effects on other CRMP2-dependent signaling pathways. Consequently, neurite outgrowth was measured from EGFP-transfected primary cultured cortical neurons using the ImageXpress Micro and MetaXpress software systems (Molecular Devices). Consistent with previous reports (Wilson et al., 2012a), siRNA knockdown of CRMP2 led to a ~37% decrease in total outgrowth (62.6 ± 4.5) compared to control (100 ± 6.6) (see Figures 7A–G). Importantly, neurite outgrowth was not altered by control siRNA (85.1 ± 5.6) (p > 0.05). CRMP2 siRNA-mediated reduction in neurite outgrowth was recapitulated by overnight application of 200 μM (S)-LCM, which decreased total outgrowth by ~34% compared to control (66.2 ± 4.5) (p < 0.05) (Figures 7A–K). The effects of (S)-LCM and CRMP2 siRNA appeared to mutually occlude one another as (S)-LCM was not able to provide a further reduction following CRMP2 knockdown [(71.3 ± 3.3) vs. (62.6 ± 4.5)] (p > 0.05) (see Figures 7A–G). Total outgrowth is a composite summary of the following parameters: number of branches, number of processes, mean process length, and maximum process length (Figures 7H–K), all of which, aside from mean process length, were reduced by both (S)-LCM and CRMP2 siRNA, compared to both control siRNA and no treatment. Combined treatment of CRMP2 siRNA and (S)-LCM did not produce a further reduction in any parameter (data not shown).


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)

The effect of (S)-LCM on neurite outgrowth phenocopies siRNA knockdown of CRMP2. (A) Representative inverted black and white representative image of a cortical neuron 48 h following EGFP-transfection. (B–F) Representative tracings of neurons transfected with EGFP ± control siRNA, CRMP2 siRNA, 200 μM (S)-LCM, or CRMP2 siRNA + (S)-LCM. (G) Total outgrowth of neurons transfected with EGFP, control siRNA, or CRMPsiRNA combined with 24 h (S)-LCM treatment (200 μM). CRMP2 siRNA and (S)-LCM reduced outgrowth to a similar level. Combination of CRMP2 and (S)-LCM did not produce a further reduction. (H–K) Comparison of the effects of CRMP2 siRNA and (S)-LCM on # of branches, # of processes, mean process length, and maximum process length. (*p < 0.05 vs. EGFP alone, One-Way ANOVA, Tukey's post-hoc analysis) (values represent mean ± s.e.m.) (n = 86–320 cells, 8 separate culture wells) (scale bar = 50 μm).
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Related In: Results  -  Collection

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Figure 7: The effect of (S)-LCM on neurite outgrowth phenocopies siRNA knockdown of CRMP2. (A) Representative inverted black and white representative image of a cortical neuron 48 h following EGFP-transfection. (B–F) Representative tracings of neurons transfected with EGFP ± control siRNA, CRMP2 siRNA, 200 μM (S)-LCM, or CRMP2 siRNA + (S)-LCM. (G) Total outgrowth of neurons transfected with EGFP, control siRNA, or CRMPsiRNA combined with 24 h (S)-LCM treatment (200 μM). CRMP2 siRNA and (S)-LCM reduced outgrowth to a similar level. Combination of CRMP2 and (S)-LCM did not produce a further reduction. (H–K) Comparison of the effects of CRMP2 siRNA and (S)-LCM on # of branches, # of processes, mean process length, and maximum process length. (*p < 0.05 vs. EGFP alone, One-Way ANOVA, Tukey's post-hoc analysis) (values represent mean ± s.e.m.) (n = 86–320 cells, 8 separate culture wells) (scale bar = 50 μm).
Mentions: Having demonstrated that (S)-LCM interacts with CRMP2, we next investigated the possible effect of this interaction on CRMP2 function. If (S)-LCM acts to inhibit CRMP2-mediated neurite outgrowth, then its action should mimic the phenotype bestowed by CRMP2 siRNA without the off-target effects on other CRMP2-dependent signaling pathways. Consequently, neurite outgrowth was measured from EGFP-transfected primary cultured cortical neurons using the ImageXpress Micro and MetaXpress software systems (Molecular Devices). Consistent with previous reports (Wilson et al., 2012a), siRNA knockdown of CRMP2 led to a ~37% decrease in total outgrowth (62.6 ± 4.5) compared to control (100 ± 6.6) (see Figures 7A–G). Importantly, neurite outgrowth was not altered by control siRNA (85.1 ± 5.6) (p > 0.05). CRMP2 siRNA-mediated reduction in neurite outgrowth was recapitulated by overnight application of 200 μM (S)-LCM, which decreased total outgrowth by ~34% compared to control (66.2 ± 4.5) (p < 0.05) (Figures 7A–K). The effects of (S)-LCM and CRMP2 siRNA appeared to mutually occlude one another as (S)-LCM was not able to provide a further reduction following CRMP2 knockdown [(71.3 ± 3.3) vs. (62.6 ± 4.5)] (p > 0.05) (see Figures 7A–G). Total outgrowth is a composite summary of the following parameters: number of branches, number of processes, mean process length, and maximum process length (Figures 7H–K), all of which, aside from mean process length, were reduced by both (S)-LCM and CRMP2 siRNA, compared to both control siRNA and no treatment. Combined treatment of CRMP2 siRNA and (S)-LCM did not produce a further reduction in any parameter (data not shown).

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