The functionalized amino acid (S)-Lacosamide subverts CRMP2-mediated tubulin polymerization to prevent constitutive and activity-dependent increase in neurite outgrowth

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.

A membrane-delimited N-myristoylated CRMP2 peptide aptamer inhibits CaV2.2 trafficking and reverses inflammatory and postoperative pain behaviors

“…These were performed exactly as described by Wilson et al 86 Briefly, 96-well plates (Nunc MaxiSorp, Thermo Scientific) were coated with tubulin (200 ng per well, Cytoskeleton Inc) and incubated at room temperature overnight. The next day, the plates were washed and blocked with 3% BSA to minimize nonspecific adsorptive binding to the plates.…”

“…CRMP2 bound to tubulin with half-saturation concentration of ~607 nM. 86 Binding of heat-denatured CRMP2 demonstrated nonsaturable background binding to tubulin. A control peptide, tat-CBD3 rev , did not affect CRMP2-tubulin binding.…”

Chemical shift perturbation mapping of the Ubc9-CRMP2 interface identifies a pocket in CRMP2 amenable for allosteric modulation of Nav1.7 channels

“…MST, a method that monitors the thermophoretic movement of molecules in optically generated microscopic temperature gradients thereby permitting analysis of biomolecular interaction was performed as described previously [34,35]. In a typical MST-experiment the concentration of the labeled molecule is kept constant while the concentration of the unlabeled interaction partner is varied.…”

CRMPs Function in Neurons and Glial Cells: Potential Therapeutic Targets for Neurodegenerative Diseases and CNS Injury

“…CRMP2 binds to the N-type voltage-gated calcium channels to evoke channel function, promote neuropathic pain in vivo [96][97][98], and induce the release of calcitonin gene-related peptide (CGRP) [99] in a Cdk5-phosphorylation-dependent manner [97]. Thus, the inhibition of CRMP2 phosphorylation may also be beneficial in decreasing the abnormal calcium influx and the neuropathic pain that follows.…”

“…However, the Ser-522 residue is highly resistant to phosphatase treatments [124]. Alternatively, (S)-LCM treatment in vitro and in vivo were shown to reduce the expression of Cdk5-and GSK3β-phosphorylated CRMP2 [97,98,125], which has been confirmed to be effective for the prevention of postoperative and neuropathic pain [100].…”