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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 does not alter slow-inactivation of voltage-gated sodium channels. (A) Voltage protocol for slow inactivation. Currents were evoked by 5 s prepulses between −100 and +20 mV and then fast-inactivated channels were allowed to recover for 5 s at a hyperpolarized pulse to −100 mV. The fraction of channels available was determined by a 15 ms test pulse at −10 mV. (B) Representative peak Na+ currents, in response to a step to −10 mV following a prepulse at −100 mV (black trace) and −50 mV (gray trace) in neurons in the absence (left) or presence (right) of 300 μM (S)-LCM. (C) Summary of steady-state slow inactivation curves from cortical neurons in the absence or presence of 300 μM (S)-LCM applied acutely or overnight. (D) For comparison, the fraction of current available following a −50 mV prepulse is depicted. (S)-LCM did not alter sodium channel steady-state slow inactivation in cortical neurons at either time (p > 0.05) (Student's t-test) (n = 6).
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Figure 6: (S)-LCM does not alter slow-inactivation of voltage-gated sodium channels. (A) Voltage protocol for slow inactivation. Currents were evoked by 5 s prepulses between −100 and +20 mV and then fast-inactivated channels were allowed to recover for 5 s at a hyperpolarized pulse to −100 mV. The fraction of channels available was determined by a 15 ms test pulse at −10 mV. (B) Representative peak Na+ currents, in response to a step to −10 mV following a prepulse at −100 mV (black trace) and −50 mV (gray trace) in neurons in the absence (left) or presence (right) of 300 μM (S)-LCM. (C) Summary of steady-state slow inactivation curves from cortical neurons in the absence or presence of 300 μM (S)-LCM applied acutely or overnight. (D) For comparison, the fraction of current available following a −50 mV prepulse is depicted. (S)-LCM did not alter sodium channel steady-state slow inactivation in cortical neurons at either time (p > 0.05) (Student's t-test) (n = 6).

Mentions: To ensure that (S)-LCM is unable to alter VGSC function at concentrations well above those required for CRMP2 binding, whole cell recordings were used to measure levels of slow inactivation (Figure 6A). Neither acute nor chronic (24 h) administration of 300 μM (S)-LCM altered the onset or extent of slow inactivation (Figures 6B,D). This concentration was chosen as it greatly surpasses those used for subsequent experiments.


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 does not alter slow-inactivation of voltage-gated sodium channels. (A) Voltage protocol for slow inactivation. Currents were evoked by 5 s prepulses between −100 and +20 mV and then fast-inactivated channels were allowed to recover for 5 s at a hyperpolarized pulse to −100 mV. The fraction of channels available was determined by a 15 ms test pulse at −10 mV. (B) Representative peak Na+ currents, in response to a step to −10 mV following a prepulse at −100 mV (black trace) and −50 mV (gray trace) in neurons in the absence (left) or presence (right) of 300 μM (S)-LCM. (C) Summary of steady-state slow inactivation curves from cortical neurons in the absence or presence of 300 μM (S)-LCM applied acutely or overnight. (D) For comparison, the fraction of current available following a −50 mV prepulse is depicted. (S)-LCM did not alter sodium channel steady-state slow inactivation in cortical neurons at either time (p > 0.05) (Student's t-test) (n = 6).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: (S)-LCM does not alter slow-inactivation of voltage-gated sodium channels. (A) Voltage protocol for slow inactivation. Currents were evoked by 5 s prepulses between −100 and +20 mV and then fast-inactivated channels were allowed to recover for 5 s at a hyperpolarized pulse to −100 mV. The fraction of channels available was determined by a 15 ms test pulse at −10 mV. (B) Representative peak Na+ currents, in response to a step to −10 mV following a prepulse at −100 mV (black trace) and −50 mV (gray trace) in neurons in the absence (left) or presence (right) of 300 μM (S)-LCM. (C) Summary of steady-state slow inactivation curves from cortical neurons in the absence or presence of 300 μM (S)-LCM applied acutely or overnight. (D) For comparison, the fraction of current available following a −50 mV prepulse is depicted. (S)-LCM did not alter sodium channel steady-state slow inactivation in cortical neurons at either time (p > 0.05) (Student's t-test) (n = 6).
Mentions: To ensure that (S)-LCM is unable to alter VGSC function at concentrations well above those required for CRMP2 binding, whole cell recordings were used to measure levels of slow inactivation (Figure 6A). Neither acute nor chronic (24 h) administration of 300 μM (S)-LCM altered the onset or extent of slow inactivation (Figures 6B,D). This concentration was chosen as it greatly surpasses those used for subsequent experiments.

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