Pharmacologically enabling the degradation of Na V 1.8 channels to reduce neuropathic pain

Abstract

In phase II clinical trials, Na V 1.8 channels were identified as viable targets to treat acute pain. Results were modest, however, and Na V 1.8 pore blockers must be given systemically, potentially leading to adverse effects, especially during prolonged use. A local, long-lasting approach is desirable, yet local anesthetics are neither specific nor long-lasting. In lieu of a pore blocker approach, we show a pharmacological method targeting the scaffolding and degradation of Na V 1.8 channels, which attenuated neuropathic pain behavior in mice. Na V 1.8 channels interact with the WW domain-containing scaffold protein called Magi-1. WW domains are typically found in ubiquitin ligases, and Na V 1.8 channels are susceptible to degradation by ubiquitin ligases. Here, we show Na V 1.8 and MAGI-1 colocalized in human tissues. We demonstrate that a lipidated peptide derived from the Na V 1.8 WW binding domain, at sub-micromolar concentrations, inhibited rodent dorsal root ganglion neuronal firing. The peptide reduced Na V 1.8 channel immunoreactivity and tetrodotoxin-resistant currents in human dorsal root ganglion neurons. We found that the lipidated peptide attenuated neuropathic pain behaviors in mice for multiple weeks after a single injection. Our results reveal that the Na V 1.8-targeted lipidated peptide provides local and sustained analgesia, serving as a viable alternative to Na V 1.8 pore blockers.

Keywords: Dorsal root ganglion neurons; Electrophysiology; Neuropathic pain; Peptides; Scaffold protein; Sodium channels; Trafficking; Ubiquitination.

Figure 1.

Magi-1 knockdown attenuated pain behaviors during entrapment injury. (A) Magi-1 is expressed in mouse peripheral terminals. (B) MAGI-1 and Na_V1.8 are colocalized in human skin. Colocalization assessed using ImageJ (NIH). Pearson coefficient was 0.61. (Ci) Thermal responses after injury and knockdown. Data represented as cumulative mean ± SEM. Pooled male and female behavior represented as cumulative mean ± SEM (n = 16). The genetic knockdown significantly increased withdraw threshold on day 15. Significance determined using repeated measures 2-way ANOVA with Bonferroni correction *P < 0.05; **P < 0.01; ***P < 0.001. (Cii) Total area under the curve (AUC) quantification for all mice under experimental condition presented in i (n = 16). (Di) von Frey mechanical sensitivity represented as cumulative mean ± SEM. Mechanical sensitivity data pooled male and female behavior (n = 16; day 20-25 n = 12-14). There was a significant increase in withdrawal threshold on day 15. Significance determined using repeated measures 2-way ANOVA with Bonferroni correction *P < 0.05; **P < 0.01; ***P < 0.001. (Dii) Total AUC quantification for all mice under experimental condition presented in i (n = 16). (E) Magi-1 knockdown modified percent weight borne on ipsilateral hind paw. There was a significant decrease in the percent weight borne by the scram group on the injured hind paw. The Magi-1 group was not statistically different. Significance determined using a paired Student t test **P < 0.01. ANOVA, analysis of variance; NIH, National Institute of Health.

Figure 2.

PY(A) peptide decreased Na_V1.8 expression. (A) PY(A) peptide sequence. (B) Proposed mechanism of lipidated peptides partitioning in the membrane and affecting Magi-1. (C) Cultured rat DRG neurons treated with 10 µM PY(A) peptide or 10 µM scrambled peptide control. Na_V1.8 immunofluorescence progressively decreases over 6 and 24 hours after incubation with PY(A) peptide. There is also an internalization of Na_V1.8 seen after 6 hours and continuing until 24 hours. (D) Quantification of Na_V1.8 immunofluorescence in cultured DRGs. Significance was determined using a one-way ANOVA with *P < 0.05; **P < 0.01; ***P < 0.001. (E) Cultured rat DRG neurons treated with PY(A) peptide + vehicle, PY(A) peptide + bafilomycin (100 nM), or scrambled peptide + vehicle. Na_V1.8 immunofluorescence is decreased at 6 and 24 hours; however, the addition of Bafilomycin prevents the loss of Na_V1.8 immunofluorescence. (F) Quantification of Na_V1.8 immunofluorescence in cultured DRG neurons. Significance was determined using a one-way ANOVA with *P < 0.05; **P < 0.01; ***P < 0.001. (G) Representative action potential firing from cultured neurons after 24 incubation with scrambled (above) PY(A) peptide (below). (H) Representative rheobase determination in cultured DRG neurons. PY(A) peptide increased threshold of firing. Red trace indicates selected trace to measure rheobase. For scrambled −140 pA, and for PY(A) peptide −440 pA. (I) Threshold of firing as a function of PY(A) peptide dose. The threshold for action potentials increased as the concentration of the peptide increased. Estimated IC₅₀ = 928 nM. ANOVA, analysis of variance; DRG, dorsal root ganglion.

Figure 3.

The PY(A) peptide preferentially targeted C fibers. (A) Sciatic nerve 24 hours after lipidated HA peptide injection. Sciatic nerve was immunostained using anti-HA and antiperipherin antibodies. Peripherin is a marker of unmyelinated fibers. Colocalization as assessed by Image J. Pearson coefficient was 0.72. (B) Schematic of ex vivo skin-nerve preparation. Naïve mice were first given an intradermal injection of 100 µM (20 µL) PY(A) peptide or scrambled peptide control unilaterally into one hind paw (n = 3 each). Twenty-four hours later, hind paws with intact sural nerve were isolated for recordings. Thermal, electrical, and mechanical stimulation were done at the same location during recordings. (C) von Frey sensitivity did not significantly differ between PY(A) peptide and scrambled peptide control. (D) Electrical thresholds were significantly higher in PY(A) peptide–treated mice compared to scrambled peptide–treated mice. Representative traces of fired action potentials after heat stimulation in (E) scrambled or (F) PY(A) peptide treatment. Insets show representative cumulative action potentials fired after heat stimulation. (G) The net number of action potentials fired after PY(A) peptide treatment was significantly decreased compared to scrambled peptide–treated animals. Significance determined using paired Student t test *P < 0.05; **P < 0.01.

Figure 4.

PY(A) peptide attenuated pain behaviors during entrapment injury. (Ai) Paw withdrawal latency was significantly increased after peptide injection form day 5 to day 25 represented as cumulative mean ± SEM (n = 12 male and female mice). Significance determined using repeated measures 2-way ANOVA with Bonferroni correction *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. (Aii) Total AUC quantification for all mice under experimental condition presented in i (n = 12). Significance determined using paired Student t test ****P < 0.0001. (Bi) von Frey mechanical sensitivity was significantly increased on days 10 and 15 represented as cumulative mean ± SEM. Significance determined using repeated measures 2-way ANOVA with Bonferroni correction *P < 0.05; **P < 0.01. (Bii) Total AUC quantification for all mice under experimental condition presented in i (n = 12). Significance determined using paired Student t test *P < 0.05. (Ci) PY(A) peptide–treated mice showed increased weight bearing on the ipsilateral paw. Scrambled-treated mice showed no difference after 25 days. Significance determined using paired Student t test. ***P < 0.001. (Cii) PY(A) peptide–treated mice showed a significant decrease in weight bearing on the uninjured (contralateral) paw. Scrambled-treated mice showed no difference after 20 days. Significance determined using paired Student t test. *P < 0.05. ANOVA, analysis of variance; AUC, area under the curve.

Figure 5.

PY(A)-H peptide decreased Na_V1.8 expression in human DRG neurons. (A) Human PY peptide (PY(A)-H) sequence. (B) Incubation of peptide alters Na_V1.8 expression in a first human donor. A similar Na_V1.8 immunofluorescence decrease is seen within 6 hours and continues to decrease at 24 hours of 10 µM PY(A)-H incubation. The scrambled peptide had no effect on Na_V1.8 expression. (C) Quantification of fluorescence in scrambled and PY(A)-H peptide–treated neurons over time. Significance was determined using one-way ANOVA **P < 0.01; ***P < 0.001. (D) Incubation to peptide alters Na_V1.8 expression in a second human donor. Na_V1.8 immunofluorescence decreased within 6 hours of 10 µM PY(A)-H incubation and continued to decrease at 24 hours. The scrambled peptide did not affect Na_V1.8 expression. (E) Quantification of fluorescence in scrambled and PY(A)-H peptide–treated neurons over time. Significance was determined using one-way ANOVA **P < 0.01; ***P < 0.001. (F) Immunofluorescence images of human DRG neurons taken from the third donor 24 hours after incubation with 10 µM scrambled peptide or PY(A)-H peptide. There was a reduction of Na_V1.8 immunofluorescence in PY(A)-H peptide–treated human DRG neurons compared to scrambled peptide. A marked reduction in immunofluorescence was noted particularly in the processes (indicated by white arrows). (G) Quantification of Na_V1.8 staining fluorescence in DRGs. Significance determined using unpaired Student t test *P < 0.05. (H) Representative I_Na at +20 mV of human DRG neurons after 24-hour incubation with 10 µM scrambled (top) or PY(A) peptide (bottom). (I) Quantification of I_Na current density at +20 mV pooled from donors 1, 2, and 3. Significance determined using unpaired Student t test ***P < 0.005 (n = 12). ANOVA, analysis of variance; DRG, dorsal root ganglion.