Dissecting the role of the CRMP2–neurofibromin complex on pain behaviors

Abstract

Neurofibromatosis type 1 (NF1), a genetic disorder linked to inactivating mutations or a homozygous deletion of the Nf1 gene, is characterized by tumorigenesis, cognitive dysfunction, seizures, migraine, and pain. Omic studies on human NF1 tissues identified an increase in the expression of collapsin response mediator protein 2 (CRMP2), a cytosolic protein reported to regulate the trafficking and activity of presynaptic N-type voltage-gated calcium (Cav2.2) channels. Because neurofibromin, the protein product of the Nf1 gene, binds to and inhibits CRMP2, the neurofibromin-CRMP2 signaling cascade will likely affect Ca channel activity and regulate nociceptive neurotransmission and in vivo responses to noxious stimulation. Here, we investigated the function of neurofibromin-CRMP2 interaction on Cav2.2. Mapping of >275 peptides between neurofibromin and CRMP2 identified a 15-amino acid CRMP2-derived peptide that, when fused to the tat transduction domain of HIV-1, inhibited Ca influx in dorsal root ganglion neurons. This peptide mimics the negative regulation of CRMP2 activity by neurofibromin. Neurons treated with tat-CRMP2/neurofibromin regulating peptide 1 (t-CNRP1) exhibited a decreased Cav2.2 membrane localization, and uncoupling of neurofibromin-CRMP2 and CRMP2-Cav2.2 interactions. Proteomic analysis of a nanodisc-solubilized membrane protein library identified syntaxin 1A as a novel CRMP2-binding protein whose interaction with CRMP2 was strengthened in neurofibromin-depleted cells and reduced by t-CNRP1. Stimulus-evoked release of calcitonin gene-related peptide from lumbar spinal cord slices was inhibited by t-CNRP1. Intrathecal administration of t-CNRP1 was antinociceptive in experimental models of inflammatory, postsurgical, and neuropathic pain. Our results demonstrate the utility of t-CNRP1 to inhibit CRMP2 protein-protein interactions for the potential treatment of pain.

CRMP2 and voltage-gated ion channels: potential roles in neuropathic pain

“…Efforts to more fully understand neurofibromin-mediated regulation of CRMP2, and its consequences for ion channel activity, led to the synthesis of complementary peptide arrays on which CRMP2 and neurofibromin’s C-terminus were tiled respectively [117]. Multiple binding domains were identified, and corresponding peptides were synthesized—including a unique CRMP2-neurofibromin regulating peptide (CNRP1) that, when conjugated to tat (t-), disrupts the CRMP2-neurofibromin interaction while also inhibiting membrane localization of Cav2.2, depolarization-evoked Ca 2+ influx, and Ca 2+ currents in rat DRGs [117].…”

“…Multiple binding domains were identified, and corresponding peptides were synthesized—including a unique CRMP2-neurofibromin regulating peptide (CNRP1) that, when conjugated to tat (t-), disrupts the CRMP2-neurofibromin interaction while also inhibiting membrane localization of Cav2.2, depolarization-evoked Ca 2+ influx, and Ca 2+ currents in rat DRGs [117]. To test the hypothesis that neurofibromin prevents CRMP2’s association with additional protein partners engaged in Cav2.2 trafficking, CRMP2 binding interactions within a nanodisc-embedded synaptic membrane library (which preserved membrane protein integrity in a virtually native environment without use of detergents) were assessed [117]. Mass spectrometry of coimmunoprecipitated CRMP2-bound proteins, in the presence and absence of t-CNRP1, identified syntaxin 1A as a novel CRMP2 binding partner (at residues 456-480) [117].…”

“…To test the hypothesis that neurofibromin prevents CRMP2’s association with additional protein partners engaged in Cav2.2 trafficking, CRMP2 binding interactions within a nanodisc-embedded synaptic membrane library (which preserved membrane protein integrity in a virtually native environment without use of detergents) were assessed [117]. Mass spectrometry of coimmunoprecipitated CRMP2-bound proteins, in the presence and absence of t-CNRP1, identified syntaxin 1A as a novel CRMP2 binding partner (at residues 456-480) [117]. …”

“…Notably, syntaxin 1A binds CRMP2 in the same region as the C-terminal domain of neurofibromin [117]. Disrupting the CRMP2-syntaxin 1A interaction via t-CNRP1 inhibited evoked CGRP release in spinal cord, and intrathecal administration of t-CNRP1 alleviated nociceptive behaviors in carrageenan-induced inflammatory pain, post-surgical pain, and HIV-induced (gp120-evoked) sensory neuropathic pain [117].…”

“…Disrupting the CRMP2-syntaxin 1A interaction via t-CNRP1 inhibited evoked CGRP release in spinal cord, and intrathecal administration of t-CNRP1 alleviated nociceptive behaviors in carrageenan-induced inflammatory pain, post-surgical pain, and HIV-induced (gp120-evoked) sensory neuropathic pain [117]. This evidence suggests that neurofibromin plays a physiological role in sequestration of CRMP2 from syntaxin 1A to disrupt Cav2.2 trafficking, limit synaptic vesicle docking, mitigate CGRP release, and ultimately, control pain signaling.…”