Treatment of inflammatory and neuropathic pain by uncoupling Src from the NMDA receptor complex

Abstract

Chronic pain hypersensitivity depends on N-methyl-D-aspartate receptors (NMDARs). However, clinical use of NMDAR blockers is limited by side effects resulting from suppression of the physiological functions of these receptors. Here we report a means to suppress pain hypersensitivity without blocking NMDARs, but rather by inhibiting the binding of a key enhancer of NMDAR function, the protein tyrosine kinase Src. We show that a peptide consisting of amino acids 40-49 of Src fused to the protein transduction domain of the HIV Tat protein (Src40-49Tat) prevented pain behaviors induced by intraplantar formalin and reversed pain hypersensitivity produced by intraplantar injection of complete Freund's adjuvant or by peripheral nerve injury. Src40-49Tat had no effect on basal sensory thresholds, acute nociceptive responses or cardiovascular, respiratory, locomotor or cognitive functions. Thus, through targeting of Src-mediated enhancement of NMDARs, inflammatory and neuropathic pain are suppressed without the deleterious consequences of directly blocking NMDARs, an approach that may be of broad relevance to managing chronic pain.

Fig. 1

Src40-49Tat suppresses the Src-NMDAR interaction in vitro and in vivo. (a) Cartoon illustrating the main hypothesis. Domain structure of Src shows Src-homology (SH) domains 1,2,3 and the unique domain (UD). (b) Dot blot of ND2.1-GST fusion protein probed with biotinylated Src unique domain peptide with overlapping sequence of 40-58, followed by streptavidin-HRP (SA-HRP). (c) Effect of Src 40-49 on mEPSCs. Left panel, average mEPSCs in the first two min after breaking in to whole-cell configuration was superimposed with averages obtained after 8–14 min. Right panel: the ratio of measurements during the first two min and from 8–14 min (Src40-49 n = 11; sSrc40-49 n =5). (d) Dot blot of ND2.1-GST fusion protein probed with biotinylated Src40-49Tat or scrambled Src40-49Tat followed by SA-HRP. (e) In vitro binding assay of assays with ND2.1-GST and Src unique domain, with no peptide, Src40-49Tat or sSrc40-49Tat (30μM). Src unique domain bound to ND2.1-GST was probed with antibody against Src, stripped and reprobed with antibody against GST. (f–h) Immunoblots of coimmunoprecipitates obtained with antibody against NR2B (anti-NR2B, f) or antibody against Src (anti-Src, g–h) from brain crude synaptosomes (f–g) in vitro incubated with Src40-49Tat or sSrc40-49Tat (10μM) or (h) from animals with or without Src40-49Tat (100pmol g⁻¹) intravenous injection (45 min before sample collection). Blots were probed with respective antibodies as labeled.

Fig. 2

Src40-49Tat suppresses formalin-induced behaviors in rats with intravenous or intrathecal administration. (a) Time course of flinches induced by formalin (2.5%), with Src40-49Tat (1pmol g⁻¹, i.v.) or sSrc40-49Tat (1pmol g⁻¹, i.v.) 45 min before formalin ( n=10). (b–c) The effect of Src40-49Tat (0.001–10pmol g⁻¹, i.v. n=10) on formalin-induced phase 2 (9–60min, b) or phase1 (0–8 min, c) flinches. Scrambled Src40-49Tat (1pmol g⁻¹, n=10), Tat-protein transduction domain alone (Tat-PTD) (1pmol g⁻¹ n=10) or saline vehicle (n=28) were used as controls (*p<0.05 vs. saline controls). (d) The effect of Src40-49Tat, Scrambled Src40-49Tat or Tat-PTD (i.v., 1pmol g⁻¹ n=8) on formalin-induced paw edema. (e–f) The effect of intrathecal Src40-49Tat (5×10⁻⁴ pmol g⁻¹, 30 min pretreatment) on formalin-induced flinches. (e) time course, (f) total number of flinches (n=8, **p<0.01 vs. saline treated controls). (mean±s.e.m. for all groups)

Fig. 3

Src40-49Tat disrupts Src association with ND2 and with NMDAR, and prevents formalin-induced increase in tyrosine phosphorylation of the NR2B in spinal cord dorsal horn. (a) Representative immunoblots of lysate (left) or co-immunoprecipitates obtained with antibody against Src from spinal cord crude synaptosomes in naïve animals, or in animals with Src40-49Tat and sSrc40-49Tat (10pmol g⁻¹, i.v., 45 before formalin). Samples were collected 30 min following formalin. The blots were probed with antibody against NR1, and stripped and reprobed with antibodies against ND2 and against Src. (b-c) Representative immunoblots (left panel) of immunoprecipitates obtained with antibody against NR2B from ipsilateral dorsal lumbar spinal cord following formalin injection, probed with antibodies against phosphotyrosine (anti-pTyr ) and against NR2B. Right panel shows a histogram of relative tyrosine phosphorylation of NR2B (Relative p-NR2B density; see Methods), normalized to naïve. (b) Samples were collected in naïve rats or at 10, 30, and 60 min following formalin injection, (c) Rats were treated intrathecally with Src40-49Tat or sSrc40-49Tat (5×10⁻⁴ pmol g⁻¹), and samples were collected 60 min following formalin injection. (mean±s.e.m.; n=6–10, each analyzed individually *p<0.05 vs. naïve control).

Fig. 4

Src40-49Tat reverses CFA-induced thermal and mechanical hypersensitivity. (a) CFA injection reduces ipsilateral paw withdrawal latency (PWL) to radiant heat 24 h following CFA. (b) The effect of Src40-49Tat (10 pmol g⁻¹, i.v.) on CFA-induced thermal hypersensitivity in terms of percentage Maximal Possible Effect (MPE; see Methods) of PWL. Inset: immunoblot and histogram showing tyrosine phosphorylation of NR2B 24 h after CFA and effects of Src40-49Tat or sSrc40-49Tat on normalized p-NR2B band intensity. (c) Histogram illustrating effects Src40-49Tat (0.1, 1 or 10 pmol g⁻¹, i.v.) or of sSrc40-49Tat (10 pmol g⁻¹, i.v.) or saline (0) on CFA-induced changes in PWL, expressed as percentage MPE of PWL. (d) The effect of Src40-49Tat and sSrc40-49Tat (10pmol g⁻¹ i.v.) on CFA-induced reduction of paw withdrawal threshold (PWT) to Von-Frey stimuli in the ipsilateral paw, 6 h following CFA. (*p<0.05, **p<0.01 vs. control; mean±s.e.m.; n=5–9).

Fig. 5

The effect of Src40-49Tat on formalin-induced behavior is occluded in Src null mutant mice. Time course (a) and total phase 2 flinches (b) induced by 1.5% formalin in Src^−/− mice and littermate wild-type mice (mean±s.e.m, n=5 mice, ***p<0.005 vs. wild-type littermate control, Src^+/+). (c) Representative immunoblots (left panel) of immunoprecipitates obtained with NR2B antibody from spinal cord of Src^−/− or Src^+/+ mice, 60 min after formalin injection. The immunoprecipitates were probed with antibodies against phosphotyrosine and against NR2B. Right panel: a histogram of relative p-NR2B band density of spinal dorsal horn from Src^+/+ and Src^−/− mice treated with formalin, normalized to Src^+/+ mice (mean±s.e.m.; n=3–7 mice,. *p<0.05 vs. wild-type littermates). (d–f) The effect of Src40-49Tat (0.5pmol g⁻¹ i.v.) on formalin-induced flinches in (d–e)wild-type control mice and (f) Src null mutant mice (mean±s.e.m., n=6–8 mice, ***p<0.005 vs. non-treated control mice).

Fig. 6

The effect of Src40-49Tat on peripheral nerve injury-induced tactile and cold hypersensitivity. (a–b) The effect of Src40-49Tat on PNI-induced decrease of paw withdrawal thresholds (PWT) in rats with (a) intrathecal Src40-49Tat, sSrc40-49Tat (5×10⁻⁴ pmol g⁻¹) or (b) intravenous Src40-49Tat (10pmol g⁻¹) administration. BI, before injury, PNI, peripheral nerve injury (n=10–16, *p<0.05, vs. saline control). (c) The effect of Src40-49Tat on acetone-induced flinches in rats following PNI. Acetone was applied before or 30 and 60 min after Src40-49Tat or sSrc40-49Tat (10 pmol g⁻¹ i.v.) (*p<0.05 vs. pre-peptide control. n=4). (d) The effect of intrathecal Src40-49Tat (5×10⁻⁴ pmol g⁻¹) on PNI-induced increase in relative p-Tyr-NR2B density in rat spinal cord. Left panel: a histogram of the relative p-NR2B density normalized to sham, and right panel: representative immunoblots of immunoprecipitates obtained with antibody against NR2B and probed with antibodies against phosphotyrosine and against NR2B. (sample were collected 5 h following Src40-49Tat; n=5, *p<0.05 vs. sham; #p<0.05 vs. PNI ). (e) PWT in Src^−/− and Src^+/+ mice, day 1–22 after PNI. Inset: representative immunoblots of immunoprecipitates obtained with antibody against NR2B from spinal cord of Src^−/− and Src^+/+ mice, 22 days following PNI, probed with antibodies against phosphotyrosine and against NR2B (f) The effect of Src40-49Tat (5 pmol g⁻¹, i.v.) on PWT of Src^−/− and Src^+/+ mice, day 14 after PNI (e–f: n=8–11, *p<0.05, **p<0.01, ***p<0.001 vs. controls). (mean±s.e.m for all groups).