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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 affect tubulin-CRMP2 binding. (A) 96-well plates coated with 200 ng tubulin were incubated with increasing concentrations of recombinant CRMP2 or heat-denatured CRMP2. The Y axis displays the OD450 absorbance of the ELISA using CRMP2-specific antibodies. CRMP2 bound to tubulin with half-saturation concentration of ~607 nM. Binding of heat-denatured CRMP2 demonstrated non-saturable background binding to tubulin. (B) Competitive binding assay revealed that (S)-LCM does not abrogate the binding of CRMP2 to tubulin. For (A,B), all measurements were performed in sextuplicate and error bars indicate standard error of the mean. Most of the error bars are smaller than the symbols.
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Figure 4: (S)-LCM does not affect tubulin-CRMP2 binding. (A) 96-well plates coated with 200 ng tubulin were incubated with increasing concentrations of recombinant CRMP2 or heat-denatured CRMP2. The Y axis displays the OD450 absorbance of the ELISA using CRMP2-specific antibodies. CRMP2 bound to tubulin with half-saturation concentration of ~607 nM. Binding of heat-denatured CRMP2 demonstrated non-saturable background binding to tubulin. (B) Competitive binding assay revealed that (S)-LCM does not abrogate the binding of CRMP2 to tubulin. For (A,B), all measurements were performed in sextuplicate and error bars indicate standard error of the mean. Most of the error bars are smaller than the symbols.

Mentions: It was previously determined that inhibition of CRMP2-mediated neurite outgrowth by (R)-LCM was mediated, not through attenuation of tubulin binding, but by impairing the ability of CRMP2 to enhance tubulin polymerization (Wilson et al., 2012a). To ensure that (S)-LCM functions in a similar manner, an ELISA-based competition assay was used to determine the ability of CRMP2 to bind tubulin in the presence of increasing concentrations of (S)-LCM. Similar to what was previously observed for (R)-LCM, binding of CRMP2 and tubulin was not impacted by as much as 1 mM (S)-LCM (Figure 4).


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 affect tubulin-CRMP2 binding. (A) 96-well plates coated with 200 ng tubulin were incubated with increasing concentrations of recombinant CRMP2 or heat-denatured CRMP2. The Y axis displays the OD450 absorbance of the ELISA using CRMP2-specific antibodies. CRMP2 bound to tubulin with half-saturation concentration of ~607 nM. Binding of heat-denatured CRMP2 demonstrated non-saturable background binding to tubulin. (B) Competitive binding assay revealed that (S)-LCM does not abrogate the binding of CRMP2 to tubulin. For (A,B), all measurements were performed in sextuplicate and error bars indicate standard error of the mean. Most of the error bars are smaller than the symbols.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: (S)-LCM does not affect tubulin-CRMP2 binding. (A) 96-well plates coated with 200 ng tubulin were incubated with increasing concentrations of recombinant CRMP2 or heat-denatured CRMP2. The Y axis displays the OD450 absorbance of the ELISA using CRMP2-specific antibodies. CRMP2 bound to tubulin with half-saturation concentration of ~607 nM. Binding of heat-denatured CRMP2 demonstrated non-saturable background binding to tubulin. (B) Competitive binding assay revealed that (S)-LCM does not abrogate the binding of CRMP2 to tubulin. For (A,B), all measurements were performed in sextuplicate and error bars indicate standard error of the mean. Most of the error bars are smaller than the symbols.
Mentions: It was previously determined that inhibition of CRMP2-mediated neurite outgrowth by (R)-LCM was mediated, not through attenuation of tubulin binding, but by impairing the ability of CRMP2 to enhance tubulin polymerization (Wilson et al., 2012a). To ensure that (S)-LCM functions in a similar manner, an ELISA-based competition assay was used to determine the ability of CRMP2 to bind tubulin in the presence of increasing concentrations of (S)-LCM. Similar to what was previously observed for (R)-LCM, binding of CRMP2 and tubulin was not impacted by as much as 1 mM (S)-LCM (Figure 4).

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