CRISPR/Cas9 editing of Nf1 gene identifies CRMP2 as a therapeutic target in neurofibromatosis type 1-related pain that is reversed by (S)-Lacosamide

Abstract

Neurofibromatosis type 1 (NF1) is a rare autosomal dominant disease linked to mutations of the Nf1 gene. Patients with NF1 commonly experience severe pain. Studies on mice with Nf1 haploinsufficiency have been instructive in identifying sensitization of ion channels as a possible cause underlying the heightened pain suffered by patients with NF1. However, behavioral assessments of Nf1 mice have led to uncertain conclusions about the potential causal role of Nf1 in pain. We used the clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (CRISPR/Cas9) genome editing system to create and mechanistically characterize a novel rat model of NF1-related pain. Targeted intrathecal delivery of guide RNA/Cas9 nuclease plasmid in combination with a cationic polymer was used to generate allele-specific C-terminal truncation of neurofibromin, the protein encoded by the Nf1 gene. Rats with truncation of neurofibromin, showed increases in voltage-gated calcium (specifically N-type or CaV2.2) and voltage-gated sodium (particularly tetrodotoxin-sensitive) currents in dorsal root ganglion neurons. These gains-of-function resulted in increased nociceptor excitability and behavioral hyperalgesia. The cytosolic regulatory protein collapsin response mediator protein 2 (CRMP2) regulates activity of these channels, and also binds to the targeted C-terminus of neurofibromin in a tripartite complex, suggesting a possible mechanism underlying NF1 pain. Prevention of CRMP2 phosphorylation with (S)-lacosamide resulted in normalization of channel current densities, excitability, as well as of hyperalgesia following CRISPR/Cas9 truncation of neurofibromin. These studies reveal the protein partners that drive NF1 pain and suggest that CRMP2 is a key target for therapeutic intervention.

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

“…To investigate the physiological role of the CRMP2-neurofibromin interaction, a CRISPR/Cas9 strategy truncating the C-terminal domain of neurofibromin was adopted and then DRGs from these CRISPR/CAs9-edited rats were electrophysiologically characterized [115]. These recordings demonstrated that CRISPR/Cas9-editing of Nf1 and neurofibromin truncation in rat DRGs resulted in ion channels with biophysical properties essentially parallel to those of Nf1 +/− DRGs; augmented Na + and Ca 2+ currents were observed in addition to hyperexcitability (determined by increased action potential frequency and decreased threshold), while K + currents were not significantly different [112].…”

“…These recordings demonstrated that CRISPR/Cas9-editing of Nf1 and neurofibromin truncation in rat DRGs resulted in ion channels with biophysical properties essentially parallel to those of Nf1 +/− DRGs; augmented Na + and Ca 2+ currents were observed in addition to hyperexcitability (determined by increased action potential frequency and decreased threshold), while K + currents were not significantly different [112]. In both male and female rats, neurofibromin truncation led to the development of thermal hyperalgesia [115]. …”

“…Biochemical examination of DRGs showed that neurofibromin truncation permitted an upregulation in phosphorylation of CRMP2 by Cdk5 [115]. Given the previous identification of ( S )-LCM as a selective inhibitor of Cdk5-mediated phosphorylation of CRMP2, DRGs with neurofibromin truncation were treated with ( S )-LCM; this inhibited membrane localization of Cav2.2 and normalized the previously detected changes in both Na + and Ca 2+ currents as well as action potential frequency, threshold, and overall hyperexcitability [115].…”

“…Given the previous identification of ( S )-LCM as a selective inhibitor of Cdk5-mediated phosphorylation of CRMP2, DRGs with neurofibromin truncation were treated with ( S )-LCM; this inhibited membrane localization of Cav2.2 and normalized the previously detected changes in both Na + and Ca 2+ currents as well as action potential frequency, threshold, and overall hyperexcitability [115]. Oral administration of ( S )-LCM to rats previously subjected to CRISPR/Cas9-editing of Nf1 reduced thermal hyperalgesia in both male and females [115]. A subsequent study demonstrated that lentiviral knock-down of CRMP2 expression in rodents subjected to neurofibromin truncation was sufficient to attenuate thermal hyperalgesia and normalize Na + current density [116].…”

Evaluation of edonerpic maleate as a CRMP2 inhibitor for pain relief

“…Actions of CRMP2 are tightly regulated by post-translational modifications [1]. In chronic neuropathic pain, we found that CRMP2 phosphorylation by cyclin-dependent kinase 5 (Cdk5) on Serine 522 (S522) was sufficient for mechanical allodynia [2] through the facilitation of CaV2.2 and NaV1.7 function [4][5][6][7][8]. By contrast, CRMP2 phosphorylation by the src tyrosineprotein kinase Fyn on tyrosine 32 (Y32) inhibits NaV1.7 [4].…”