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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

Working model for CRMP2-mediated, activity-dependent increase in neurite outgrowth and preferential inhibition by (S)-LCM. In control neurons (black arrows), CRMP2-mediated neurite outgrowth is driven by the binding of CRMP2 and tubulin, as well as, the enhancement of tubulin polymerization. Phosphorylation of CRMP2 by GSK3β and/or Cdk5 impairs its binding to tubulin, leading to neurite outgrowth arrest. Neuronal activity driven by KCl depolarization (blue arrows) leads to reduced priming by Cdk5 (residue S522), which prevents subsequent phosphorylation by GSK3β (residues T509/T514/S518; latter residue not shown). This transition to the unphosphorylated form results in increased binding of CRMP2/tubulin and CRMP2-dependent tubulin polymerization, thereby increasing neurite outgrowth. (S)-Lacosamide (green arrows) interacts with CRMP2 to directly impair its GAP activity, thus preventing the enhancement of tubulin polymerization and, therefore, neurite outgrowth (arrow size denotes the effect of treatment, i.e., increases in size imply a positive effect and decreases in size imply a negative effect).
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Figure 10: Working model for CRMP2-mediated, activity-dependent increase in neurite outgrowth and preferential inhibition by (S)-LCM. In control neurons (black arrows), CRMP2-mediated neurite outgrowth is driven by the binding of CRMP2 and tubulin, as well as, the enhancement of tubulin polymerization. Phosphorylation of CRMP2 by GSK3β and/or Cdk5 impairs its binding to tubulin, leading to neurite outgrowth arrest. Neuronal activity driven by KCl depolarization (blue arrows) leads to reduced priming by Cdk5 (residue S522), which prevents subsequent phosphorylation by GSK3β (residues T509/T514/S518; latter residue not shown). This transition to the unphosphorylated form results in increased binding of CRMP2/tubulin and CRMP2-dependent tubulin polymerization, thereby increasing neurite outgrowth. (S)-Lacosamide (green arrows) interacts with CRMP2 to directly impair its GAP activity, thus preventing the enhancement of tubulin polymerization and, therefore, neurite outgrowth (arrow size denotes the effect of treatment, i.e., increases in size imply a positive effect and decreases in size imply a negative effect).

Mentions: Our findings demonstrate that KCl-facilitated neurite outgrowth in cultured cortical neurons is a CRMP2-dependent process. Specifically, neuronal activity led to changes in CRMP2 activity through regulation of its phosphorylation state (Figure 10). Interestingly, decreased levels of GSK3β-phosphorylated CRMP2 were observed following activity that were secondary, not to changes in GSK3β expression or activity, but rather decreased CRMP2 priming by Cdk5. CRMP2 has been suggested to be involved in many processes with potential activity-dependent components such as epilepsy, pain, and schizophrenia (Johnston-Wilson et al., 2000; Nakata et al., 2003; Czech et al., 2004; Fallin et al., 2005; Ryu et al., 2008; Brittain et al., 2011a; Wilson et al., 2012a,b). These findings may provide key insight into the importance of CRMP2 within these neuropathologies. Previous work in our lab suggested the potential anti-epileptogenic benefit of targeting CRMP2-mediated neurite outgrowth following CNS insult (Wilson et al., 2012a). The findings presented here further validate CRMP2 as a potential therapeutic target in processes involving maladaptive activity-dependent neurite outgrowth.


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)

Working model for CRMP2-mediated, activity-dependent increase in neurite outgrowth and preferential inhibition by (S)-LCM. In control neurons (black arrows), CRMP2-mediated neurite outgrowth is driven by the binding of CRMP2 and tubulin, as well as, the enhancement of tubulin polymerization. Phosphorylation of CRMP2 by GSK3β and/or Cdk5 impairs its binding to tubulin, leading to neurite outgrowth arrest. Neuronal activity driven by KCl depolarization (blue arrows) leads to reduced priming by Cdk5 (residue S522), which prevents subsequent phosphorylation by GSK3β (residues T509/T514/S518; latter residue not shown). This transition to the unphosphorylated form results in increased binding of CRMP2/tubulin and CRMP2-dependent tubulin polymerization, thereby increasing neurite outgrowth. (S)-Lacosamide (green arrows) interacts with CRMP2 to directly impair its GAP activity, thus preventing the enhancement of tubulin polymerization and, therefore, neurite outgrowth (arrow size denotes the effect of treatment, i.e., increases in size imply a positive effect and decreases in size imply a negative effect).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 10: Working model for CRMP2-mediated, activity-dependent increase in neurite outgrowth and preferential inhibition by (S)-LCM. In control neurons (black arrows), CRMP2-mediated neurite outgrowth is driven by the binding of CRMP2 and tubulin, as well as, the enhancement of tubulin polymerization. Phosphorylation of CRMP2 by GSK3β and/or Cdk5 impairs its binding to tubulin, leading to neurite outgrowth arrest. Neuronal activity driven by KCl depolarization (blue arrows) leads to reduced priming by Cdk5 (residue S522), which prevents subsequent phosphorylation by GSK3β (residues T509/T514/S518; latter residue not shown). This transition to the unphosphorylated form results in increased binding of CRMP2/tubulin and CRMP2-dependent tubulin polymerization, thereby increasing neurite outgrowth. (S)-Lacosamide (green arrows) interacts with CRMP2 to directly impair its GAP activity, thus preventing the enhancement of tubulin polymerization and, therefore, neurite outgrowth (arrow size denotes the effect of treatment, i.e., increases in size imply a positive effect and decreases in size imply a negative effect).
Mentions: Our findings demonstrate that KCl-facilitated neurite outgrowth in cultured cortical neurons is a CRMP2-dependent process. Specifically, neuronal activity led to changes in CRMP2 activity through regulation of its phosphorylation state (Figure 10). Interestingly, decreased levels of GSK3β-phosphorylated CRMP2 were observed following activity that were secondary, not to changes in GSK3β expression or activity, but rather decreased CRMP2 priming by Cdk5. CRMP2 has been suggested to be involved in many processes with potential activity-dependent components such as epilepsy, pain, and schizophrenia (Johnston-Wilson et al., 2000; Nakata et al., 2003; Czech et al., 2004; Fallin et al., 2005; Ryu et al., 2008; Brittain et al., 2011a; Wilson et al., 2012a,b). These findings may provide key insight into the importance of CRMP2 within these neuropathologies. Previous work in our lab suggested the potential anti-epileptogenic benefit of targeting CRMP2-mediated neurite outgrowth following CNS insult (Wilson et al., 2012a). The findings presented here further validate CRMP2 as a potential therapeutic target in processes involving maladaptive activity-dependent neurite outgrowth.

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