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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 binds to wildtype CRMP2 in solution measured by microscale thermophoresis (MST). (A) Illustration of hydration shell changes after small molecule [i.e., (S)-LCM] binding to a macromolecule. The changes in the hydration shell properties are detected in the MST instrument. (B) MST assay principle. The four stages in a thermophoresis experiment: (i) initial state (all molecules are randomly distributed); (ii) infrared (IR) laser is turned on, and thermophoresis commences; (iii) steady state flow while IR laser is turned on; and (iv) equilibration to initial state by back-diffusion with IR laser turned off. The green arrow represents the steady-state time point at which the MST measurements were analyzed for the graphs shown in (D,G). (C) MST time traces of concentrations of (S)-LCM ranging from 0.006 to 100 μM. Increasing concentrations altered thermodiffusion of NT-647 labeled CRMP2. (D) MST values were used to determine dissociation constant for binding of (S)-LCM to wildtype CRMP2, apparent Kd = 1.5 ± 0.01 μM; the curve was fit with an r2 value of 0.76. (E) Stereo view of the binding site for (S)-LCM within one monomer of the CRMP2 structure (Stenmark et al., 2007; Wang et al., 2010b). (S)-LCM is shown in capped-sticks representation. The amino acids mutated to alanines are indicated in green text. (F) MST time traces of concentrations of (S)-LCM ranging from 0.006 to 100 μM. MST experiments were repeated using NT-647 labeled CRMP25ALA harboring mutations in residues as indicated in (E). (G) No association could be detected between (S)-LCM and CRMP25ALA. The data could not be fitted with a curve (r2 = 0.09). A representative of range of data points obtained from at least 3 measurements is shown.
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Figure 3: (S)-LCM binds to wildtype CRMP2 in solution measured by microscale thermophoresis (MST). (A) Illustration of hydration shell changes after small molecule [i.e., (S)-LCM] binding to a macromolecule. The changes in the hydration shell properties are detected in the MST instrument. (B) MST assay principle. The four stages in a thermophoresis experiment: (i) initial state (all molecules are randomly distributed); (ii) infrared (IR) laser is turned on, and thermophoresis commences; (iii) steady state flow while IR laser is turned on; and (iv) equilibration to initial state by back-diffusion with IR laser turned off. The green arrow represents the steady-state time point at which the MST measurements were analyzed for the graphs shown in (D,G). (C) MST time traces of concentrations of (S)-LCM ranging from 0.006 to 100 μM. Increasing concentrations altered thermodiffusion of NT-647 labeled CRMP2. (D) MST values were used to determine dissociation constant for binding of (S)-LCM to wildtype CRMP2, apparent Kd = 1.5 ± 0.01 μM; the curve was fit with an r2 value of 0.76. (E) Stereo view of the binding site for (S)-LCM within one monomer of the CRMP2 structure (Stenmark et al., 2007; Wang et al., 2010b). (S)-LCM is shown in capped-sticks representation. The amino acids mutated to alanines are indicated in green text. (F) MST time traces of concentrations of (S)-LCM ranging from 0.006 to 100 μM. MST experiments were repeated using NT-647 labeled CRMP25ALA harboring mutations in residues as indicated in (E). (G) No association could be detected between (S)-LCM and CRMP25ALA. The data could not be fitted with a curve (r2 = 0.09). A representative of range of data points obtained from at least 3 measurements is shown.

Mentions: MST was used to determine if (S)-LCM could interact with purified CRMP2. Using an infrared laser, precise microscopic temperature gradients are generated within thin glass capillaries filled with a fluorescently labeled protein sample (i.e., CRMP2), and the atomistic movement of molecules along these temperature gradients is monitored in the presence of increasing concentrations of an unlabeled binding partner (S)-LCM (Figures 3A,B). As the concentration of (S)-LCM increased, it bound to CRMP2 thermodiffusing out of the heated infrared spot, resulting in a change in the MST signal and providing a readout of the binding between the CRMP2 and (S)-LCM. NT647-labeled CRMP2 protein was incubated with varying concentrations of (S)-LCM (0.006–100 μM) and apparent Kd values were obtained by fitting curves using the Hill method. MST experiments revealed that (S)-LCM bound to WT CRMP2 with an apparent Kd of 1.5 ± 0.01 μM (Figures 3C,D). Importantly, Kd of (S)-LCM is similar to what is observed for (R)-LCM in this assay (1.0 ± 0.04 μM; Figure 3D) (Wilson and Khanna, 2014). We had previously identified 5 key residues on CRMP2, essential for coordinating (R)-LCM binding and mutated them to alanines to create CRMP25ALA, whose function mimics that of wildtype CRMP2, yet is not impaired by the presence of (R)-LCM (Wang et al., 2010b; Wilson et al., 2012a) (Figure 3E). MST experiments revealed that (S)-LCM did not interact with NT647-labeled CRMP25ALA (Figures 3F,G), suggesting that the same binding pocket is necessary for coordinating both (R)- and (S)-LCM binding.


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 binds to wildtype CRMP2 in solution measured by microscale thermophoresis (MST). (A) Illustration of hydration shell changes after small molecule [i.e., (S)-LCM] binding to a macromolecule. The changes in the hydration shell properties are detected in the MST instrument. (B) MST assay principle. The four stages in a thermophoresis experiment: (i) initial state (all molecules are randomly distributed); (ii) infrared (IR) laser is turned on, and thermophoresis commences; (iii) steady state flow while IR laser is turned on; and (iv) equilibration to initial state by back-diffusion with IR laser turned off. The green arrow represents the steady-state time point at which the MST measurements were analyzed for the graphs shown in (D,G). (C) MST time traces of concentrations of (S)-LCM ranging from 0.006 to 100 μM. Increasing concentrations altered thermodiffusion of NT-647 labeled CRMP2. (D) MST values were used to determine dissociation constant for binding of (S)-LCM to wildtype CRMP2, apparent Kd = 1.5 ± 0.01 μM; the curve was fit with an r2 value of 0.76. (E) Stereo view of the binding site for (S)-LCM within one monomer of the CRMP2 structure (Stenmark et al., 2007; Wang et al., 2010b). (S)-LCM is shown in capped-sticks representation. The amino acids mutated to alanines are indicated in green text. (F) MST time traces of concentrations of (S)-LCM ranging from 0.006 to 100 μM. MST experiments were repeated using NT-647 labeled CRMP25ALA harboring mutations in residues as indicated in (E). (G) No association could be detected between (S)-LCM and CRMP25ALA. The data could not be fitted with a curve (r2 = 0.09). A representative of range of data points obtained from at least 3 measurements is shown.
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Related In: Results  -  Collection

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Figure 3: (S)-LCM binds to wildtype CRMP2 in solution measured by microscale thermophoresis (MST). (A) Illustration of hydration shell changes after small molecule [i.e., (S)-LCM] binding to a macromolecule. The changes in the hydration shell properties are detected in the MST instrument. (B) MST assay principle. The four stages in a thermophoresis experiment: (i) initial state (all molecules are randomly distributed); (ii) infrared (IR) laser is turned on, and thermophoresis commences; (iii) steady state flow while IR laser is turned on; and (iv) equilibration to initial state by back-diffusion with IR laser turned off. The green arrow represents the steady-state time point at which the MST measurements were analyzed for the graphs shown in (D,G). (C) MST time traces of concentrations of (S)-LCM ranging from 0.006 to 100 μM. Increasing concentrations altered thermodiffusion of NT-647 labeled CRMP2. (D) MST values were used to determine dissociation constant for binding of (S)-LCM to wildtype CRMP2, apparent Kd = 1.5 ± 0.01 μM; the curve was fit with an r2 value of 0.76. (E) Stereo view of the binding site for (S)-LCM within one monomer of the CRMP2 structure (Stenmark et al., 2007; Wang et al., 2010b). (S)-LCM is shown in capped-sticks representation. The amino acids mutated to alanines are indicated in green text. (F) MST time traces of concentrations of (S)-LCM ranging from 0.006 to 100 μM. MST experiments were repeated using NT-647 labeled CRMP25ALA harboring mutations in residues as indicated in (E). (G) No association could be detected between (S)-LCM and CRMP25ALA. The data could not be fitted with a curve (r2 = 0.09). A representative of range of data points obtained from at least 3 measurements is shown.
Mentions: MST was used to determine if (S)-LCM could interact with purified CRMP2. Using an infrared laser, precise microscopic temperature gradients are generated within thin glass capillaries filled with a fluorescently labeled protein sample (i.e., CRMP2), and the atomistic movement of molecules along these temperature gradients is monitored in the presence of increasing concentrations of an unlabeled binding partner (S)-LCM (Figures 3A,B). As the concentration of (S)-LCM increased, it bound to CRMP2 thermodiffusing out of the heated infrared spot, resulting in a change in the MST signal and providing a readout of the binding between the CRMP2 and (S)-LCM. NT647-labeled CRMP2 protein was incubated with varying concentrations of (S)-LCM (0.006–100 μM) and apparent Kd values were obtained by fitting curves using the Hill method. MST experiments revealed that (S)-LCM bound to WT CRMP2 with an apparent Kd of 1.5 ± 0.01 μM (Figures 3C,D). Importantly, Kd of (S)-LCM is similar to what is observed for (R)-LCM in this assay (1.0 ± 0.04 μM; Figure 3D) (Wilson and Khanna, 2014). We had previously identified 5 key residues on CRMP2, essential for coordinating (R)-LCM binding and mutated them to alanines to create CRMP25ALA, whose function mimics that of wildtype CRMP2, yet is not impaired by the presence of (R)-LCM (Wang et al., 2010b; Wilson et al., 2012a) (Figure 3E). MST experiments revealed that (S)-LCM did not interact with NT647-labeled CRMP25ALA (Figures 3F,G), suggesting that the same binding pocket is necessary for coordinating both (R)- and (S)-LCM binding.

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