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. Author manuscript; available in PMC: 2014 Mar 1.
Published in final edited form as: J Neurochem. 2013 Jan 20;124(6):869–879. doi: 10.1111/jnc.12070

Suppression of pain-related behavior in two distinct rodent models of peripheral neuropathy by a homopolyarginine-conjugated CRMP2 peptide

Weina Ju †,∫, Qi Li ¥, Yohance M Allette ‡, Matthew S Ripsch ‡, Fletcher A White †,‡,§,, Rajesh Khanna †,‖,¶,§,⊥,*
PMCID: PMC3584205  NIHMSID: NIHMS417650  PMID: 23106100

Abstract

The N-type voltage-gated calcium channel (CaV2.2) is a clinically endorsed target in chronic pain treatments. As directly targeting the channel can lead to multiple adverse side effects, targeting modulators of CaV2.2 may prove better. We previously identified ST1-104, a short peptide from the collapsin response mediator protein 2 (CRMP2), which disrupted the CaV2.2–CRMP2 interaction and suppressed a model of HIV-related neuropathy induced by antiretroviral therapy but not traumatic neuropathy. Here, we report ST2-104 –a peptide wherein the cell-penetrating TAT motif has been supplanted with a homopolyarginine motif, which dose-dependently inhibits the CaV2.2–CRMP2 interaction and inhibits depolarization-evoked Ca2+ influx in sensory neurons. Ca2+ influx via activation of vanilloid receptors is not affected by either peptide. Systemic administration of ST2-104 does not affect thermal or tactile nociceptive behavioral changes. Importantly, ST2-104 transiently reduces persistent mechanical hypersensitivity induced by systemic administration of the antiretroviral drug 2′,3′-dideoxycytidine (ddC) and following tibial nerve injury (TNI). Possible mechanistic explanations for the broader efficacy of ST2-104 are discussed.

Keywords: CaV2.2, CRMP2, peptide inhibitor, polyarginine motif, calcium imaging, neuropathic pain

Introduction

Persistent pain prevalence has been reported to range from ~10 to 25% in Asia, Africa, Europe and the Americas resulting in a projected ~105 million people in the United States and ~600 million people globally suffering from chronic pain (Goldberg & McGee 2011), with a cost estimate of more than $100 billion per year in direct health-care expenditure and lost work time in the United States alone (Harstall 2003). Thus, developing novel therapies for chronic pain syndromes remains a large unmet medical need. The N-type voltage-gated calcium channel (CaV2.2) is a clinically endorsed target for treatment of chronic pain, with the synthetic peptide omega-conotoxin, Ziconotide (Prialt®) approved for treatment of severe, refractory pain (Doggrell 2004). The intrathecal route of administration and relatively poor tolerability, combined with major side effects being confusion, somnolence, orthostatic hypotension and nausea, severely limit the use of Ziconotide (Schmidtko et al. 2010, Bowersox et al. 1992). We have advanced a strategy focused on developing novel chemical entities to alleviate pain by taking advantage of a new approach to targeting CaV2.2: targeting modulators of channel trafficking. Interference of the protein-protein interaction between the target collapsin response mediator protein 2 (CRMP2) and CaV2.2 in sensory neurons by unique peptides effectively diminishes trafficking of the ion channel to the plasma membrane and serves to attenuate peripheral sensitization in rodent models of inflammation or neuropathic pain (Brittain et al. 2011b, Piekarz et al. 2012, Ripsch et al. 2012, Wilson et al. 2011, Wilson et al. 2012a). Fusing a Ca2+ channel binding domain (CBD3) to the HIV-1 transactivator of transcription domain (TAT) resulted in a cell-permeable protein, which reduced CaV2.2-mediated currents in vitro, suppressed neuropeptide release from sensory neurons and inhibited excitatory synaptic transmission in dorsal horn neurons of the spinal cord (Brittain et al. 2011b). Additionally, the TAT-conjugated CBD3 (ST1-104) administration in vivo reduced nociceptive behavior in a number of pain models, including models of peripheral neuropathy induced by nucleoside reverse transcriptase inhibitors (NRTIs) such as 2′3′-dideoxycytidine (ddC; Zalcitabine®) (Brittain et al. 2011b) or 2′,3′-didehydro-3′-deoxythymidine (d4T; Stavudine®) (Piekarz et al. 2012) or a chronic inflammatory pain model involving focal demyelination of the sciatic nerve (Wilson et al. 2011). ST1-104 was found to be mildly anxiolytic without affecting memory retrieval, sensorimotor function, or depression (Brittain et al. 2011b). Notably, sympathetic activity was not affected by ST1-104 (Brittain et al. 2011b, Wilson et al. 2011). ST1-104 was ineffective, however, in suppressing persistent mechanical hypersensitivity induced by a tibial nerve injury (TNI) (Wilson et al. 2011).

In this investigation, we tested the effects of a variant to the parental TAT-conjugated CBD3 (i.e. ST1-104) peptide – a nona-arginine (R9)-conjugated CBD3 (i.e. ST2-104) peptide; the cell penetrating peptide (CPP) R9 motif was chosen due to its superior cell penetrating abilities (Wender et al. 2000). We show that ST2-104 interferes with the CaV2.2–CRMP2 interaction and inhibits Ca2+ influx from sensory neurons. In addition to reversing mechanical hypersensitivity due to the NRTI ddC, we further document that R9-conjugation bestows upon CBD3 the ability to reverse TNI-induced pain-related behavior that was shown to be recalcitrant to the parental CBD3 peptide (Brittain et al. 2011b).

Methods

Animals

Pathogen-free, adult female Sprague-Dawley rats (150–200 g; Harlan Laboratories, Madison, WI) were housed in temperature (23 ± 3°C) and light (12-h light: 12-h dark cycle; lights on at 07:00 h) controlled rooms with standard rodent chow and water available ad libitum. The Institutional Animal Care and Use Committee of Indiana University/Purdue University in Indianapolis approved these experiments. All procedures were conducted in accordance with the Guide for Care and Use of Laboratory Animals published by the National Institutes of Health and the ethical guidelines of the International Association for the Study of Pain. Animals were randomly assigned to treatment or control groups.

Materials

Peptides (Table 1) were synthesized and HPLC-purified by Genscript USA Inc. (Piscataway, NJ). All chemicals, unless noted, were purchased from Sigma (St. Louis, MO). Fura-2AM was obtained from Invitrogen (Grand Island, NY). Antibodies were obtained as follows: anti-β-tubulin monoclonal (Promega, Madison, WI) and anti-CaV2.2 polyclonal (Origene Technologies Inc., Rockville, MD). Resiniferatoxin (RTX) was purchased from Tocris Bisoscience (Bristol, United Kingdom) and Capsaicin (Cap) was from Sigma.

Table 1.

Peptides used in this study.

Peptide Cell penetrating
motif		 Sequence
(N → C-termini)		 Molecular weight
(g/mol)
ST1-104 TAT: YGRKKRRQRRR ARSRLAELRGVPRGL 3192.8
ST9-104 TAT: YGRKKRRQRRR LGRPVGRLEALRSRA 3192.8
ST2-104 R9: RRRRRRRRR ARSRLAELRGVPRGL 3056.6
FITC-ST2-104 R9: RRRRRRRRR ARSRLAELRGVPRGL 3559.2
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Purification of recombinant CRMP2-GST

CRMP2-GST fusion protein was purified from BL21 (DE3) Escherichia coli bacterial lysates as described (Brittain et al. 2011a, Brittain et al. 2011b). Purified CRMP2 was dialyzed into phosphate buffered saline with 10% glycerol. The final dialyzed protein was quantified by SDS-PAGE by comparison to a bovine serum albumin protein standard curve. CRMP2-GST (~3 μM) aliquots were flash frozen on dry ice and stored at –80°C. That the purified protein was CRMP2 was verified by western blot analyses with two different CRMP2 antibodies (Sigma Aldrich and Immunobiological laboratories, Minneapolis, MN) as well as in a functional assay in which it was found to enhance tubulin polymerization (Wilson et al. 2012b) (data not shown).

Co-immunoprecipitation and Western blotting

Brain lysates prepared from embryonic day 19 rats were generated by homogenization and sonication in detergent-free RIPA buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, and 1 mM EDTA with protease inhibitors). Lysates were incubated overnight at 4°C with GST alone or CRMP2-GST (~0.5 μM) in the absence or presence of various concentrations of ST2-104 peptide with gentle rotation. Then, samples were incubated with glutathione sepharose beads (Amersham, Piscataway, NJ) for 1 h at 4°C to capture bound proteins, resolved by SDS-PAGE and processed for immunoblotting with antibodies against CRMP2 and CaV2.2.

Primary dorsal root ganglion (DRG) neuronal cultures

Sensory DRG neurons from adult mice were isolated as described (Brittain et al. 2011b). All animals were housed with free access to food and water in the Indiana University Laboratory Animal Research Center and used in procedures approved by the Animal Use and Care Committee of the Indiana University School of Medicine.

Calcium imaging

DRG neurons were loaded at 37°C with 3 μM Fura-2AM (Kd=25 μM, λex 340, 380 nm/λemi 512 nm) to follow changes in intracellular calcium ([Ca2+]c) in a standard bath solution containing 139 mM NaCl, 3 mM KCl, 0.8 mM MgCl2, 1.8 mM CaCl2, 10 mM NaHEPES, pH 7.4, 5 mM glucose exactly as previously described (Brittain et al. 2011b). Fluorescence imaging was performed with an inverted microscope, Nikon Eclipse TE2000-U, using objective Nikon Super Fluor 20× 0.75 NA and a Photometrics cooled CCD camera CoolSNAPHQ (Roper Scientific, Tucson, AZ) controlled by MetaFluor 6.3 software (Molecular Devices, Downingtown, PA). The excitation light was delivered by a Lambda-LS system (Sutter Instruments, Novato, CA). The excitation filters (340±5 and 380±7) were controlled by a Lambda 10-2 optical filter change (Sutter Instruments). Fluorescence was recorded through a 505 nm dichroic mirror at 535±25 nm. To minimize photobleaching and phototoxicity, the images were taken every ~2.4 seconds during the time-course of the experiment using the minimal exposure time that provided acceptable image quality. The changes in [Ca2+]c were monitored by following a ratio of F340/F380, calculated after subtracting the background from both channels.

ddC model of peripheral neuropathic pain

von Frey filaments were used to test mechanical sensitivity before and after peptide administration. Prior to initial von Frey tactile testing, all rodents were habituated to testing chambers for at least two days. Animals were tested for baseline responses at least two times before initiation of the injection paradigm using previously published methods (Bhangoo et al. 2007b). Mechanical hypersensitivity was established by a single injection (50 mg/kg) of the antiretroviral drug 2′,3′-dideoxycytidine (ddC, Sigma) given intraperitoneal (i.p.) in 150-200 g female Sprague Dawley rats. A single administration of ddC produced a significant bilateral decrease in paw withdrawal threshold (PWT) to von Frey hair stimulation from post-injection day 3 through 42, the last day of testing. The von Frey test was performed on six positions spaced across the glabrous side of the hind paw; two distinct locations for the distribution of each nerve branch (saphenous, tibial and sural) exactly as described previously to determine PWT to tactile stimuli (Brittain et al. 2011b, Bhangoo et al. 2007c). A threshold that exhibits ≥ –20 mN difference from the baseline threshold of testing in a given animal is representative of neuropathic pain (Brittain et al. 2011b, Joseph et al. 2004).

Tibial Nerve Injury (TNI) model of traumatic neuropathy

This injury was inflicted as previously described (Wang et al. 2011). Under isoflurane (2%) anesthesia the skin on the lateral surface of the thigh was incised and a section made directly through the biceps femoris muscle, exposing the sciatic nerve and its three terminal branches: the sural, common peroneal, and tibial nerves. The surgical procedure comprised an axotomy and ligation of the tibial nerve leaving the common peroneal and sural nerves intact (see Figure 5A). The tibial nerve was tightly ligated with 5.0 silk and sectioned distal to the ligation, removing 2-4 mm of the distal nerve stump. Effort was made to avoid contact with or stretching of the intact common peroneal and sural nerves. Muscle and skin were closed in two layers. Sham controls involved exposure of the sciatic nerve and its branches without any lesion.

Figure 5. ST2-104 peptide attenuates tibial nerve injury (TNI)-induced mechanical hypersensitivity.

Figure 5

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(A) Schematic of the sural, tibial, and common peroneal terminal nerve branches of the sciatic nerve and their dorsal root origins. Neuropathic pain was induced by ligation of the tibial nerve and 2–4 mm of the nerve distal to the ligation was removed. Paw-withdrawal thresholds (mN) were measured in TNI rodents before and after intraperitoneal administration of ST2-104 peptide (B) or gabapentin (GBP, C). PWT in uninjured rats (n=6 each) was not significantly altered by 10 mg/kg of ST2-104 at any of the times tested (p>0.5, one-way ANOVA with Dunnett’s post hoc test). PWT in TNI rodents was significantly reduced when compared to pre-TNI thresholds (n=6; white bars). Following intraperitoneal administration of ST2-104 (0.1, 1, or 10 mg/kg body weight; n=6 each), PWT increased above TNI levels for at least 2 h with 0.1 mg/kg ST2-104 and at least 4 h with the two highest ST2-104 doses (ANOVA with Dunnett’s post hoc test, *p<0.05). After a 200 mg/kg GBP injection, PWT increased above TNI levels for at least 1 h (ANOVA with Dunnett’s post hoc test, *p<0.05).

Thermal behavioral assessment

Thermal hyperalgesia was determined by measuring foot withdrawal latency of the response to heat stimulation (Bhangoo et al. 2007a). Each rat was placed in a box (22×12×12 cm) containing a smooth glass floor. A heat source (Ugo Basile Plantar™ Analgesia Instrument) was focused on a portion of the hind paw, which is flush against the glass, and a radiant thermal stimulus was delivered to that site. The stimulus shuts off automatically when the hind paw moved (or after 20 s to prevent tissue damage). The intensity of the heat stimulus was constant throughout all experiments. A thermal stimulus was delivered 4 times to each hind paw at five-minute intervals. The value for the response based on thermal latency was obtained by averaging the 4 measurements per animal. The baseline response for right and left hind paws was tested for two days prior to initiation of the injection paradigm.

Assessment of distribution of FITC-conjugated ST2-104 in nervous system of TNI-injured rats

Adult female Sprague-Dawley rats were euthanized with CO2 and transcardially perfused with saline followed by 4% paraformaldehyde 1 hour following i.p. injection of FITC-conjugated ST2-104. Lumbar ganglia 5 and 6 from behaviorally-tested TNI-treated rats (n = 2) were immediately removed and post fixed for 4 hours. Sagittal sections of the DRG were serially cut at 14 μm onto SuperFrost Plus microscope slides (Fisher Scientific, Pittsburgh, PA) and counterstained with Hoescht nuclear stain (1:1,000; Sigma Aldrich). At least 6 sections were obtained for analysis per DRG. Tissue was processed such that DRG sections on each slide were at intervals of 80 μm. Images of DRG were taken with an intensified CCD camera (Photometrics CoolSnap HQ2) coupled to a Nikon microscope (Nikon Eclipse Ti) using Nikon Elements software (Nikon Instruments Inc., Melville, NY, USA). Tissue sections were illuminated with a Lamda DG-4 175 W xenon lamp (Sutter Instruments, Novata, CA) and images captured using the same exposure for all tissue. Images are presented without any post-hoc manipulation.

Statistics

GraphPad Software (LaJolla, CA) was used to determine the statistical significance. The statistical significance of differences between means was determined by Student’s t-test or a one-way ANOVA followed by post hoc comparisons (Dunnett’s or Tukey’s test).

Results

We tested the hypothesis that replacement of the TAT motif with the presumably superior cell penetrating prowess of the homopolyarginine motif (Wender et al. 2000) would prevent CRMP2–CaV2.2 interaction, block Ca2+ influx in sensory neurons, and be effective in reducing pain-related behavior in various models of peripheral neuropathy.

We first tested if the nona-arginine (R9) motif conjugated to the CBD3 peptide of CRMP2, designated ST2-104 peptide, could interfere with the CaV2.2–CRMP2 interaction. Rat brain lysates were incubated with purified CRMP2-GST in the presence of DMSO (0.3%, control) or increasing concentrations (1–100 μM) of ST2-104 and then CRMP2-bound proteins recovered by incubation with glutathione sepharose beads. Probing of the CRMP2-enriched fraction with a CaV2.2 antibody demonstrated a robust interaction between CaV2.2 and CRMP2 (Figure 1; top blot, lanes 1, 2) whereas a glutathione beads only pull-down did not capture any CaV2.2 (Figure 1; top blot, lane 7). ST2-104, in a concentration-dependent manner, attenuated this interaction with greater than ~55% inhibition at 3 μM and >85% inhibition at 100 μM (Figure 1; top blot, lanes 2-6). The ST2-104 mediated disruption showed specificity in that the CRMP2-tubulin interaction (Fukata et al. 2002), was not affected by ST2-104 (Figure 1; middle blot, lanes 1-6).

Figure 1. ST2-104 inhibits the CaV2.2–CRMP2 interaction.

Figure 1

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Embryonic day 19 rat brain lysates (Lys) were incubated with recombinant CRMP2-GST protein (0.5 μM) in the absence (control, lane 1) or presence of increasing concentrations (as indicated, lanes 2-6) of ST2-104 peptide. The CRMP2-GST bound proteins were recovered by incubation with glutathione sepharose beads and immunoblotted with CaV2.2 (top) and β-tubulin (middle). Ponceau S stained blot (bottom) shows equal proteins across samples within each experiment. Lys + beads lane represents pull-down in which the CRMP2-GST protein was omitted. Representative blots from 2 separate experiments are shown.

We previously reported that uncoupling the CaV2.2–CRMP2 interaction with ST1-104 resulted in reduction of depolarization-induced Ca2+-influx in sensory neurons (Brittain et al. 2011b). Here, we asked if ST2-104, the R9 grafted analog of ST1-104, could work similarly. Calcium imaging experiments performed with Fura-2AM on adult mouse DRG neurons demonstrated that stimulation with high 90 mM KCl (a concentration where only CaV2 channels are recruited (Wheeler et al. 2012)) produced a transient rise in intracellular calcium ([Ca2+]c) (Figure 2A, B). Peak calcium influx was recorded within 20 sec of stimulation (Figure 2A-C). DRGs incubated for 20 min with ST1-104 or ST2-104 peptides showed a concentration-dependent decrease in K+-stimulated [Ca2+]c influx (Figure 2D). A concentration-response analysis indicated that the peptides inhibited Ca2+ influx in a concentration dependent manner with IC50 values of 12.1 ± 0.6 μM (n=4) for ST1-104 and 8.1 ± 0.5 μM for ST2-104 (n=4) (Figure 2D). While the concentration-response curves for inhibition of Ca2+ influx were not statistically different between the two peptides (p>0.05, one-way ANOVA with Tukey’s post hoc test) indicating that both were equipotent, ST2-104 was significantly better in its efficacy as maximum block by this peptide was ~50% greater than that by ST1-104 (Fig. 2D). 10 μM of a reverse CBD3 control peptide (ST9-104) had no effect on K+-stimulated [Ca2+]c influx (Figure 2C).

Figure 2. ST2-104 inhibits depolarization-evoked Ca2+ influx in dorsal root ganglion (DRG) neurons.

Figure 2

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(A) Differential interference contrast (DIC) and pseudocolored fluorescent images of a field of DRG neurons visualized for Fura-2, before (Basal) and after stimulation with KCl (90 mM K+). Arrowhead shows a typical responding sensory neuron. (B) Ca2+ imaging was performed on adult mouse DRG neurons using the ratiometric Ca2+-sensitive dye Fura-2. Following a 1 min baseline measurement, neurons were stimulated with 90 mM KCl for 10 sec (window denoted by arrows in B) arrows to induce Ca2+ influx. Representative traces and average ± S.E.M. (black error bars) for DRG neurons treated with vehicle control are shown with the change in fluorescence (peak – basal; ΔF) and time as indicated by the scale bars. (C) Bar graph shows the peak fluorescence response (adjusted for background) of DRGs incubated for 20 min with 1, 3, 10 or 30 μM ST2-104 peptide, 10 μM ST9-104 (reverse CBD3) peptide, or DMSO-treated (control) DRGs. Values represent the average ± SEM from two separate imaging experiments from 27-37 cells per condition. Asterisks indicate statistical significance compared with untreated cells (p < 0.05, one-way ANOVA with Dunnett’s post-hoc test). (D) Concentration-dependent effect of ST1-104 and ST2-104 peptides on depolarization-evoked Ca2+ influx in sensory neurons. Percent inhibition of peak Ca2+ influx normalized to the control is plotted. IC50 = 12.1 ± 0.6 μM (n=4) for ST1-104 and 8.1 ± 0.5 μM for ST2-104 (n=4).

A previous study had demonstrated that an arginine-rich hexapeptide (R4W2) acted in a stereo selective manner to antagonize vanilloid receptor 1 (VR1 or TRPV1) (Himmel et al. 2002). To test if ST2-104-mediated block of calcium imaging may be due to targeting of TRPV1 receptors, we performed additional imaging experiments using resiniferatoxin (RTX) or capsaicin (Cap) – selective agonists of vanilloid receptors in a subset of nociceptive DRGs (Caterina & Julius 2001, Greffrath et al. 2001, Winter et al. 1990) – as the stimuli for Ca2+ release. Stimulation with 20 nM RTX induced a robust increase in [Ca2+]c in ~18% (16 of 88) DRGs (Figure 3). The RTX-induced [Ca2+]c was unchanged in the presence of 10 μM of either ST1-104 or ST2-104 peptides (Figure 3B; p > 0.05; one-way ANOVA with Tukey’s post hoc test). Similar results were obtained following stimulation with 3 μM Cap (Figure 3C, D).

Figure 3. ST1-104 and ST2-104 do not affect Ca2+ influx evoked by vanilloid receptor activation in DRG neurons.

Figure 3

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Differential interference contrast (DIC) and pseudocolored fluorescent images of a field of DRG neurons visualized for Fura-2, before (Basal), after stimulation with vanilloid receptor activators resiniferatoxin (RTX, A) or Capsaicin (Cap, C) and after stimulation with KCl (90 mM K+). Arrowheads show typical responding sensory neurons. (B) Ca2+ imaging was performed on adult mouse DRG neurons using the ratiometric Ca2+-sensitive dye Fura-2. Following a 1 min baseline measurement, neurons were stimulated with 20 nM RTX (A) or 3 μM Cap (C) for 10 sec arrows to induce Ca2+ influx. (B, D) Bar graphs show the peak fluorescence response (adjusted for background) of DRGs incubated for 20 min with 10 μM ST1-104 or ST2-104 peptides, or vanilloid receptors activators RTX (B) or Cap (D) alone. Values represent the average ± SEM from two separate imaging experiments from 13-21 cells per condition. There were no differences in the Ca2+ influx stimulated by RTX and RTX + peptides (B) or Cap plus peptides (D) (p>0.5, one-way ANOVA with Dunnett’s post hoc test).

In our previous studies we demonstrated that three TAT-conjugated peptides with the CBD3 core sequence reduced chronic nociceptive behavior in an animal model of NRTI-induced painful neuropathy (Brittain et al. 2011b, Piekarz et al. 2012, Ripsch et al. 2012). ST1-104, however, was not effective in reversing tactile hypernociception in a TNI model (Wilson et al. 2011). Here, we tested the effects of ST2-104 on NRTI- and TNI-induced models of peripheral neuropathy.

To study changes in persistent mechanical hypersensitivity following a single injection of the NRTI 2′3′-dideoxycytidine (ddC), we investigated alterations in the threshold force of indentation (produced by von Frey filaments) required for eliciting a flexion hindpaw withdrawal reflex. A single administration of ddC (50 mg/kg) produced a significant bilateral decrease in paw withdrawal threshold to von Frey hair stimulation from post-injection day (PID) 3 through the last day of testing at PID 30 (Figure 4; p < 0.05; Student’s t-test). To determine if ST2-104 could reduce mechanical hypersensitivity elicited by ddC, we administered a single systemic dose of ST2-104 (0.1, 1 and 10 mg/kg) intraperitoneally to ddC-treated rats. Mechanical hypersensitivity was assessed 1, 2 or 4, and 24 hours following injection of peptide. Systemic injection of the 10 mg/kg dose of ST2-104 reversed paw withdrawal threshold (PWT) to pre-ddC levels for at least one hour (Figure 4; *, p<0.05, ANOVA with Dunnett’s post-hoc test) while the lower doses of ST2-104 were completely ineffective at affecting PWT at any times tested following the injection. A scramble control peptide (10 mg/kg) or saline controls did not elicit any change in PWT at any time tested following injection of ddC-injected rats (not shown).

Figure 4. ST2-104 peptide suppresses ddC-induced mechanical hypersensitivity.

Figure 4

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Paw-withdrawal thresholds (PWT in millinewtons, mN) were measured in ddC-treated rodents before and after intraperitoneal administration of ST2-104 peptide. PWT in rodents subjected to a single injection of ddC was significantly reduced when compared to pre-ddC thresholds (n=6; white bars). After the induction of neuropathic pain behavior using ddC, ST2-104 (0.1, 1, or 10 mg/kg body weight; n=6 each) was administered intraperitoneally and bilateral pain behavior was assessed using the Von-Frey filament test. Behavior was tested at 1, 2 or 4, and 24 h. Following administration of ST2-104, bilateral paw withdrawal thresholds increased above ddC levels (ANOVA with Dunnett’s post hoc test, *p<0.05, n=6).

The efficacy of ST2-104 was also tested in the TNI model of traumatic neuropathy at 0.5, 1, 2, and 4 hours following injection of peptide. At 1 hour post-injection, ST2-104 caused a dose-dependent reversal of PWT to pre-injury level with an almost 80% reversal observed at the highest dose of 10 mg/kg (Figure 5B; *, p<0.05, ANOVA with Dunnett’s post-hoc test). Significant accumulation of FITC-labeled ST2-104 was evident in sections of DRG and spinal cord at 1 h post-injection in the TNI-injured rats (see Figure 6). Partial reversal of PWT to pre-injury levels was observed with the 1 and 10 mg/kg doses of ST2-104 for 2 to 4 hours (Figure 5B; *, p<0.05, ANOVA with Dunnett’s post-hoc test). While both ST1-104 (Brittain et al. 2011b) and ST2-104 are effective at reversing ddC-induced mechanical hypersensitivity, in contrast to the failure of ST1-104 in reversing TNI-induced pain-related behavior (Wilson et al. 2011), ST2-104 was effective in this model even at a ten-fold lesser dose. Importantly, ST2-104, even at a 20-fold lesser dose, was almost as effective as the anti-epileptic, anti-neuropathic pain medication gabapentin (GBP; Neurontin®; Figure 5C), which targets the α2δ subunit of calcium channels (Gee et al. 1996, Maneuf et al. 2006) among other targets (Eroglu et al. 2009).

Figure 6. Distribution of fluorescein isothiocyanate (FITC)-conjugated ST2-104 peptide in tissues from TNI rats following intraperitoneal injection.

Figure 6

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Sections of lumbar DRGs and spinal cord from TNI-injured rodents, 60 min following injected with FITC-labeled ST2-104 (10 mg/kg i.p.). Nuclei of all cells stained with Hoechst (blue).

Because an arginine-rich hexapeptide (R4W2) was reported to block TRPV1 channels with analgesic activity (Planells-Cases et al. 2000), we tested if systemic administration of ST2-104 could alter noxious thermal and tactile stimulus-dependent behavioral outcomes in uninjured rats. ST2-104 (10 mg/kg, i.p.) failed to elicit changes in thermal latencies (control: 9.8 ± 0.1 sec (n=6) compared to ST2-104 at 30 min post-injection: 9.9 ± 0.1 sec (n=6), ST2-104 at 60 min post-injection: 9.7 ± 0.2 s (n=6), or ST2-104 at 120 min post-injection: 10.2 ± 0.2 s (n=6)) or PWT to tactile stimulus (Fig. 5C, leftmost bars). Collectively, these data demonstrate that ST2-104 is effective in reversing tactile hyperalgesia in two models of peripheral neuropathy and that this effect does not involve TRP channels.

Finally, confirmation that the distribution of ST2-104 after intraperitoneal injection reaches neuronal targets associated with noxious behavior, we collected DRG and spinal cord tissue samples from animals injected with FITC–R9CBD3 and detected the peptide in the DRG and spinal cord at least 1 hour later (Figure 6).

Discussion

In this study, we asked if the CBD3 peptide from CRMP2, conjugated to the cell penetrating peptide (CPP) motif nona-arginine (R9) instead of TAT, would be similar in its in vitro mechanism of action and efficacy in rodent neuropathic pain models. We chose to conjugate CBD3 to the cationic R9 peptide for several reasons. First, it is the most efficacious of currently known protein transduction domains with at least a 20–fold better penetrability into cells than TAT (Wender et al. 2000) or other homopolymeric amino acids (Mitchell et al. 2000). Second, R9 is well tolerated by cells, with low short- and long-term toxicological effects (Tunnemann et al. 2008). Third, the mechanism of R9 transduction into cells is well understood and involves binding to cell surface heparan sulfate proteoglycans, heparin sulfate-mediated endocytosis into vesicles, release of R9 from heparin sulfate upon cleavage by heparanases, culminating in release of unbound R9 into the cytosol due to vesicular leakage (Fuchs & Raines 2004). Three endocytic pathways – macropinocytosis, clathrin-mediated endocytosis and caveolae/lipid-raft-mediated endocytosis – are thought to be involved (Duchardt et al. 2007).

Similar to TAT-conjugated CBD3 (designated ST1-104), the R9-conjugated CBD3 (designated ST2-104) peptide interfered with the CaV2.2–CRMP2 interaction. The interference was specific, as the tubulin-CRMP2 interaction was not disrupted by ST2-104. The functional consequence of the disrupted CaV2.2– CRMP2 interaction was a significantly higher extent (i.e. efficacy) of inhibition of calcium influx in sensory neurons. This increased efficacy may possibly contribute to the mechanism of action of this peptide. That ST2-104 did not affect Ca2+ influx activated by vanilloid receptor activators resiniferatoxin and capsaicin rules out targeting of these receptors as a potential mechanism of action. These results are entirely consistent with our previous data that showed no effect of ST1-104 on TRPV1 current recordings following 30 nM to 3 μM of capsaicin (Brittain et al. 2011b) demonstrating that ST1-104 does not work through direct inhibition of TRPV1 channels. Our previous work has delineated that ST1-104 inhibits calcium entry via CaV2.2 (Brittain et al. 2011b) as well as CaV2.3 (R-type channels) and ST1-106 inhibits CaV2.3 and CaV3.1 (T-type channels) (Piekarz et al. 2012). Currently, we are exploring the precise channel target(s) of ST2-104.

Block of CaV2.2 has been shown to attenuate nociceptive behavior (Saegusa et al. 2001, Malmberg & Yaksh 1994). We have demonstrated that interfering with the CaV2.2–CRMP2 interaction with TAT-conjugated CBD3-scaffold peptides ST1-104, ST1-106, and ST1-107 mitigates inflammatory and neuropathic pain (Brittain et al. 2011b, Piekarz et al. 2012, Ripsch et al. 2012, Wilson et al. 2011). We recently also demonstrated that targeting the opposite interface of the CaV2.2–CRMP2 interaction with TAT-conjugated peptides derived from the first intracellular loop and the end of the carboxyl terminus of voltage-gated calcium channels phenocopies the suppression in pain behaviors (Wilson et al. 2012a), validating the importance of targeting channel modulating proteins such as CRMP2 as targets for chronic pain therapeutics. However, despite the anti-nociceptive promise of CBD3 peptides, ST1-104 was not unable to suppress tactile hypersensitivity in a nerve injury-induced pain scenario (Wilson et al. 2011). Several factors may account for the lack of effect of ST1-104 following nerve injury-induced pain including, but not limited to, mechanistic differences between injury- and drug-induced neuropathies; differential inflammatory responses; differential bioavailability of the peptide in the various injury models; and differential cell penetrating ability of the peptide. Reasoning that the reported superior cell penetrating ability of the nona-arginine CPP (Wender et al. 2000) may allow for greater uptake of the peptide which may result in superior biochemical and functional uncoupling of the CaV2.2–CRMP2 interaction and suppression of transmitter release from nociceptive neurons culminating in efficacy in chronic pain models, we tested the R9 grafted CBD3 (i.e. ST2-104) peptide in the TNI model of traumatic neuropathy. We found that ST2-104, like ST1-104 (Brittain et al. 2011b), was effective at reversing mechanical hypersensitivity induced by ddC-associated distal symmetrical polyneuropathy. Importantly, ST2-104 reversed mechanical hypersensitivity associated with nerve-injury possibly due to its superior cell transduction attributes (Wender et al. 2000). At the highest dose of ST2-104 (10 mg/kg administered systemically; n=6), paw withdrawal threshold values were within 20% of pre-injury values. At the same dose, ST1-104 was completely ineffective in this traumatic neuropathy model (Wilson et al. 2011). The effectiveness of ST2-104, but not ST1-104, in the TNI model could possibly be due to enhanced transduction of ST2-104 into sensory neurons and/or entry into other cells like microglia. It has been shown that microglia are indirectly involved in mechanical hypersensitivity following spared nerve injury (Suter et al. 2009). The lack of fluorescently labeled ST2-104 macrophages and microglia (Figure 6) is consistent with our previous findings with fluorescently labeled ST1-104 (Brittain et al. 2011b) and suggests that differential cellular recruitment by the peptides may not explain the observed differences in behavior.

Neurodegeneration has been suggested as an underlying etiology of neuropathic pain (Bordet & Pruss 2009). Proteomic data lends further support to the notion that common cellular and molecular changes may ensue during the course of neuroprotection leading to pain following traumatic neuropathic injuries. For example, Yue and colleagues noted marked chronic upregulation of proteins involved in inflammation and cytoprotection, in DRGs derived from rats subjected to chronic compression of the DRG (Zhang et al. 2008). Our previous studies demonstrated that ST1-104 attenuates Ca2+ influx via N-methyl-D-Aspartate receptors (NMDARs) and reduces neuronal death in a moderate controlled cortical impact model of traumatic brain injury (Brittain et al. 2011a) and reduces infarct volume in an animal model of focal cerebral ischemia (Brittain et al. 2012). Preliminary studies show that ST2-104 is also neuroprotective (R. Khanna and J. M. Brittain, unpublished observations) and suggest the testable hypothesis that another potential mechanism of action of ST2-104 in the TNI model may be neuropreservation and/or mitigation of inflammatory responses.

The reversal of mechanical hypersensitivity was transient and lasted at least 1 hour in the ddC model and 4 hours in the traumatic neuropathy model. These findings are entirely consistent with our previous results demonstrating longer periods of pain-related behavior reversal in the TNI versus the ddC model following systemic injection of a TAT-conjugated peptide from calcium channels that breaks the CaV2.2–CRMP2 interaction (Wilson et al. 2012a) as well as with the CRMP2-derived ST1-104 peptide (Brittain et al. 2011b). The ~2 hour half-life of R9 (Mitchell et al. 2000) combined with its rapid uptake that decreases over time (Sarko et al. 2010) could be possible factors that contribute to the transient nature of the pain reversing effects. Future studies in our laboratory will be aimed at exploring chemical modifications to increase peptide stability and/or use of alternative CPPs to overcome the drawback of short cytoplasmic half-life.

Collectively, these findings provide an instructive example of how tailoring different cell penetrating peptides to the parental CRMP2 CBD3 peptide can be used to achieve broader efficacy in a traumatic neuropathy model previously refractory to this novel therapeutic. These studies further underscore the importance of targeting the CaV2.2–CRMP2 modulatory axis in development of novel therapeutics to manage chronic neuropathic pain.

Acknowledgements

We thank Sarah Wilson, Joel M. Brittain and members of the Khanna laboratory for technical assistance and helpful discussions regarding this project. Purified CRMP2 protein was provided by Joel M. Brittain. This work was supported, in part, by grants from the Indiana Clinical and Translational Sciences Institute funded, in part by a Project Development Team Grant Number (RR025761) from the National Institutes of Health, National Center for Research Resources, Clinical and Translational Sciences Award, the Indiana State Department of Health – Spinal Cord and Brain Injury Fund (A70-9-079138 to R.K. and F.A.W.), NIH/NINDS (NS049136-06 to F.A.W.), NIDA (DA026040-04 to F.A.W.), a National Scientist Development from the American Heart Association (SDG5280023 to R.K.), a Neurofibromatosis New Investigator Award from the Department of Defense Congressionally Directed Military Medical Research and Development Program (NF1000099 to R.K.), a Ralph W. and Grace M. Showalter Foundation grant (to R.K.), the Indiana University Biomedical Committee – Research Support Funds (to R.K), and a Research Inventions and Scientific Commercialization grant (to R.K.) from the Indiana CTSI. R.K. is a co-founder of Sophia Therapeutics, LLC.

1Abbreviations

Cap

Capsaicin

CaV2.2

N-type voltage-gated Ca2+ channel

CBD

Ca2+ channel binding domain

CRMP2

collapsin response mediator protein 2

CPP

cell penetrating peptide

ddC

2′3′-dideoxycytidine

DIV

days in vitro

DRG

dorsal root ganglion

GBP

gabapentin

IC50

half maximal inhibitory concentration

NRTI

nucleoside reverse transcriptase inhibitors

PID

post-injection day

PWT

paw withdrawal threshold

R9

nona-arginine

RTX

resiniferatoxin

ST

Sophia Therapeutics

TAT

HIV-1 transactivator of transcription domain

TNI

tibial nerve injury

Footnotes

The authors declare no conflict of interests.

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