Studies on CRMP2 SUMOylation-deficient transgenic mice identify sex-specific NaV1.7 regulation in the pathogenesis of chronic neuropathic pain

Abstract

The sodium channel NaV1.7 is a master regulator of nociceptive neuronal firing. Mutations in this channel can result in painful conditions as well as produce insensitivity to pain. Despite being recognized as a "poster child" for nociceptive signaling and human pain, targeting NaV1.7 has not yet produced a clinical drug. Recent work has illuminated the NaV1.7 interactome, offering insights into the regulation of these channels and identifying potentially new druggable targets. Amongst the regulators of NaV1.7 is the cytosolic collapsin response mediator protein 2 (CRMP2). CRMP2, modified at Lysine 374 (K374) by addition of a small ubiquitin-like modifier (SUMO), bound NaV1.7 to regulate its membrane localization and function. Corollary to this, preventing CRMP2 SUMOylation was sufficient to reverse mechanical allodynia in rats with neuropathic pain. Notably, loss of CRMP2 SUMOylation did not compromise other innate functions of CRMP2. To further elucidate the in vivo role of CRMP2 SUMOylation in pain, we generated CRMP2 K374A knock-in (CRMP2 K374A/K374A ) mice in which Lys374 was replaced with Ala. CRMP2 K374A/K374A mice had reduced NaV1.7 membrane localization and function in female, but not male, sensory neurons. Behavioral appraisal of CRMP2 K374A/K374A mice demonstrated no changes in depressive or repetitive, compulsive-like behaviors, and a decrease in noxious thermal sensitivity. No changes were observed in CRMP2 K374A/K374A mice to inflammatory, acute, or visceral pain. In contrast, in a neuropathic model, CRMP2 K374A/K374A mice failed to develop persistent mechanical allodynia. Our study suggests that CRMP2 SUMOylation-dependent control of peripheral NaV1.7 is a hallmark of chronic, but not physiological, neuropathic pain. IntroductionThe voltage-gated sodium channel NaV1.7 is a "poster child" target in pain signaling: gain-of-function mutations in the human NaV1.7 gene SCN9A can produce sensory neuron hyperexcitability associated with severe pain as well as insensitivity to pain [16]. While other sodium channels regulate the propagation of action potentials along nerves, NaV1.7 is upstream and defines the threshold at which an action potential will be elicited [44].Alterations of NaV1.7 trafficking are important in the etiology of neuropathic pain. Mapping the NaV1.7 interactome has shed light on novel proteins involved in regulation of trafficking and degradation of NaV1.7 [10; 35]. In neuropathic pain, the expression of proteins regulating trafficking of voltage gated sodium channels (VGSCs) is dysregulated [4; 38]. In particular, upregulation of the VGSC β-subunits [4] and downregulation of Nedd4-2 (an E3 ubiquitin ligase)[38] following a spared nerve injury (SNI) [15], converge to functionally upregulate NaV1.7.We identified the collapsin response mediator protein 2 (CRMP2) as a regulator of NaV1.7 function [17-19; 22; 48]. Our laboratory uncovered the logical coding of CRMP2's cellular actions [11; 51]. The argument path underlying NaV1.7 regulation is defined by "IF CRMP2 is phosphorylated by cycli...

Studies on CRMP2 SUMOylation-deficient transgenic mice reveal novel insights into the trafficking of NaV1.7 channels

“…In contrast, none of these effects were observed in male mice, suggesting that the CRMP2 K374A/K374A mutation imposed a sexspecific regulation on NaV1.7. Of relevance to the role of NaV1.7 in pain, we found that CRMP2 K374A/K374A mice of both sexes failed to develop mechanical allodynia after a spinal nerve injury (SNI) (28), thus supporting the conclusion that CRMP2 SUMOylation-dependent regulation of NaV1.7 still holds in chronic neuropathic pain in male mice. Together, these studies underscore the critical role of CRMP2 SUMOylation as a regulatory mechanism underlying NaV1.7 functional expression.…”

“…Although CRMP2 SUMOylation was abolished in both male and female homozygous mice, we unexpectedly discovered that CRMP2 dependent NaV1.7 trafficking was sexually dimorphic (28). In female mice only, germline loss of CRMP2 SUMOylation reduced NaV1.7-CRMP2 binding, NaV1.7 membrane localization and NaV1.7 currents in DRG sensory neurons, compared to their wildtype (WT) littermates (28). Inhibiting clathrin assembly with Pitstop2 (29), rescued the decreased sodium currents back to the levels observed in DRG from WT female mice.…”

“…Our previous in vitro studies found that the CRMP2 K374A/K374A genotype leads to a reduction of sodium current in female, but not male, mice (28). Inhibiting clathrin assembly with the small molecule Pitstop2 (16), rescued the decrease in sodium currents observed in DRG neurons from female CRMP2 K374A/K374A mice (28).…”

“…Subsequent In vivo studies revealed that, following SNI, both CRMP2 K374A/K374A male and female mice become resistant to the development of mechanical allodynia compared to their WT littermates (28). From these data, one can surmise that although there is no impact of loss of CRMP2 SUMOylation on sodium currents in male mice, inflicting a neuropathic pain injury uncovers an effect in both genders (28). To resolve this discrepancy, we hypothesized that increased internalization of NaV1.7 in male CRMP2 K374A/K374A mice compromises its role in neuropathic pain.…”

“…To determine the role of CRMP2 SUMOylation in vivo, we generated CRMP2 K374A knock-in (CRMP2 K374A/K374A ) transgenic mice, in which the sole SUMOylation site on CRMP2 at Lysine 374 was replaced by an alanine (27). Although CRMP2 SUMOylation was abolished in both male and female homozygous mice, we unexpectedly discovered that CRMP2 dependent NaV1.7 trafficking was sexually dimorphic (28). In female mice only, germline loss of CRMP2 SUMOylation reduced NaV1.7-CRMP2 binding, NaV1.7 membrane localization and NaV1.7 currents in DRG sensory neurons, compared to their wildtype (WT) littermates (28).…”