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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

Targeting CRMP2 prevents activity-dependent increase in neurite outgrowth. (A) Timeline of experimental procedures. (B–E) Representative tracings of cortical neurons expressing EGFP and incubated for 96 h in vehicle, 25 mM KCl, 200 μM (S)-LCM, or 25 mM KCl + 200 μM (S)-LCM. (F) Total outgrowth of cortical neurons exposed to 25 mM KCl in the presence or absence of 200 μM (S)-LCM. (G) In naïve neurons, KCl exposure increased outgrowth by ~40% compared to vehicle. Co-application of (S)-LCM blunted the KCl-induced increase to ~10% (*p < 0.05 compared to control; Student's t-test) (n = 110–379 cells, across 8 separate culture wells) (scale bar = 50 μm).
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Figure 8: Targeting CRMP2 prevents activity-dependent increase in neurite outgrowth. (A) Timeline of experimental procedures. (B–E) Representative tracings of cortical neurons expressing EGFP and incubated for 96 h in vehicle, 25 mM KCl, 200 μM (S)-LCM, or 25 mM KCl + 200 μM (S)-LCM. (F) Total outgrowth of cortical neurons exposed to 25 mM KCl in the presence or absence of 200 μM (S)-LCM. (G) In naïve neurons, KCl exposure increased outgrowth by ~40% compared to vehicle. Co-application of (S)-LCM blunted the KCl-induced increase to ~10% (*p < 0.05 compared to control; Student's t-test) (n = 110–379 cells, across 8 separate culture wells) (scale bar = 50 μm).

Mentions: Previously, CRMP2 was identified to be involved in activity-dependent neurite outgrowth of cerebellar granule cells (Tan et al., 2013). Unlike other central neurons, cerebellar granule cells require slightly depolarizing conditions for survival in vitro. Therefore, it is difficult to generalize this finding to other neuron populations within the central nervous system. As such, it is not known if CRMP2 is involved in outgrowth induced by depolarization in neurons where it is not necessary for survival. To determine the involvement of CRMP2 in activity-driven neurite outgrowth, cortical neurons overexpressing EGFP were exposed to 25 mM KCl and maintained for 96 h to ascertain the extent of activity dependent neurite outgrowth (Figure 8A). Consistent with previous findings (Tan et al., 2013), chronic depolarization with 25 mM KCl led to a ~43% increase in total neurite outgrowth (143.1 ± 11.5) compared to control (100 ± 6.6) (Figure 8B–G). Notably, blockade of CRMP2-mediated neurite outgrowth by (S)-LCM was sufficient to prevent activity dependent growth induced by KCl (68.4 ± 3.8 vs. 61.7 ± 3.5) (p > 0.05). As our earlier data demonstrated that (S)-LCM is not affecting VGSC function in these neurons, these data suggest that KCl-facilitated neurite outgrowth is dependent on CRMP2.


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)

Targeting CRMP2 prevents activity-dependent increase in neurite outgrowth. (A) Timeline of experimental procedures. (B–E) Representative tracings of cortical neurons expressing EGFP and incubated for 96 h in vehicle, 25 mM KCl, 200 μM (S)-LCM, or 25 mM KCl + 200 μM (S)-LCM. (F) Total outgrowth of cortical neurons exposed to 25 mM KCl in the presence or absence of 200 μM (S)-LCM. (G) In naïve neurons, KCl exposure increased outgrowth by ~40% compared to vehicle. Co-application of (S)-LCM blunted the KCl-induced increase to ~10% (*p < 0.05 compared to control; Student's t-test) (n = 110–379 cells, across 8 separate culture wells) (scale bar = 50 μm).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Targeting CRMP2 prevents activity-dependent increase in neurite outgrowth. (A) Timeline of experimental procedures. (B–E) Representative tracings of cortical neurons expressing EGFP and incubated for 96 h in vehicle, 25 mM KCl, 200 μM (S)-LCM, or 25 mM KCl + 200 μM (S)-LCM. (F) Total outgrowth of cortical neurons exposed to 25 mM KCl in the presence or absence of 200 μM (S)-LCM. (G) In naïve neurons, KCl exposure increased outgrowth by ~40% compared to vehicle. Co-application of (S)-LCM blunted the KCl-induced increase to ~10% (*p < 0.05 compared to control; Student's t-test) (n = 110–379 cells, across 8 separate culture wells) (scale bar = 50 μm).
Mentions: Previously, CRMP2 was identified to be involved in activity-dependent neurite outgrowth of cerebellar granule cells (Tan et al., 2013). Unlike other central neurons, cerebellar granule cells require slightly depolarizing conditions for survival in vitro. Therefore, it is difficult to generalize this finding to other neuron populations within the central nervous system. As such, it is not known if CRMP2 is involved in outgrowth induced by depolarization in neurons where it is not necessary for survival. To determine the involvement of CRMP2 in activity-driven neurite outgrowth, cortical neurons overexpressing EGFP were exposed to 25 mM KCl and maintained for 96 h to ascertain the extent of activity dependent neurite outgrowth (Figure 8A). Consistent with previous findings (Tan et al., 2013), chronic depolarization with 25 mM KCl led to a ~43% increase in total neurite outgrowth (143.1 ± 11.5) compared to control (100 ± 6.6) (Figure 8B–G). Notably, blockade of CRMP2-mediated neurite outgrowth by (S)-LCM was sufficient to prevent activity dependent growth induced by KCl (68.4 ± 3.8 vs. 61.7 ± 3.5) (p > 0.05). As our earlier data demonstrated that (S)-LCM is not affecting VGSC function in these neurons, these data suggest that KCl-facilitated neurite outgrowth is dependent on CRMP2.

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