Decoy peptides that bind dynorphin noncovalently prevent NMDA receptor-mediated neurotoxicity

Abstract

Prodynorphin-derived peptides elicit various pathological effects including neurological dysfunction and cell death. These actions are reduced by N-methyl-d-aspartate receptor (NMDAR) but not opioid receptor antagonists suggesting NMDAR-mediation. Here, we show that a conserved epitope (KVNSEEEEEDA) of the NR1 subunit of the NMDAR binds dynorphin peptides (DYNp) noncovalently. Synthetic peptides containing this epitope form stable complexes with DYNp and prevent the potentiation of NMDAR-gated currents produced by DYNp. They attenuate DYNp-evoked cell death in spinal cord and prevent, as well as reverse, DYNp-induced paralysis and allodynia. The data reveal a novel mechanism whereby prodynorphin-derived peptides facilitate NMDAR function and produce neurotoxicity. Furthermore, they suggest that synthetic peptides that bind DYNp, thus preventing their interaction with NMDAR, may be novel therapeutic agents for the treatment of spinal cord injury.

Fig. 1

Positive ion mode MALDI Mass spectrum of a solution containing an equimolar mixture of DYN (1pmole), DYN 2–17 (1 pmole), and KVNSE5DA (1 pmole). Complexes of DYN and DYN 2–17 with KVNSE₅DA are seen at amu 3426.8 and 3263.6. The insert shows a proposed model of the interaction of DYN and KVNSE₅DA.

Fig. 2

KVNSE₅DA prevents the potentiation of glutamate activated currents produced by DYN and DYN 2–17 in Xenopus oocytes expressing NR1 and NR2B subunits. Potentiation of glutamate induced currents by DYN is reversibly reduced by co-application of d-KVNSE₅DA (100μM) (A). Co-application of either d- (black symbols) or l-KVNSE₅DA (white symbols) significantly inhibits the potentiation of glutamate activated currents induced by DYN or DYN 2-17. [* p≤ 0.05, n = 7–11; Student’s t-test] (B). The inhibitory effect of l- and d-KVNSE₅DA upon DYN 2–17 is concentration-dependent (C).

Fig. 3

KVNSE₅DA attenuates the neurotoxic actions of DYN 2–17-in the spinal cord (A). DYN 2–17 (100 μM) induced significant cell death in cultured spinal cord neurons at 72 h (p< 0.01, F _(1,8)= 6.9, ANOVA; **p < 0.01 vs controls; Duncan test), which was significantly attenuated by co-administering an equimolar concentration of KVNSE₅DA(^*p < 0.01 vs. DYN 2–17; Duncan test).; KVNSE ₅DA alone had no effect. i.t. infusion of DYN 2–17 (5 nmol) produces long-lasting tactile allodynia which is prevented by co- infusion of l-KVNSE₅DA (p <0.01, F_(4,135) = 15.8, ANOVA; * p < 0.05 relative to Veh+DYN 2–17, Student-Newman-Keuls test: SNK). ○ Veh (n=7), □ Veh + DYN 2-17 (n=6), ▲ l-KVNSE₅DA 30 nmol + DYN 2–17 (n=5), ▼ l- KVNSE₅DA 100 nmol + DYN 2–17, ■ l- KVNSE₅DA 250 nmol + DYN 2–17 (n=8). (C) DYN 2–17 induced motor paralysis is reduced by co- or post-infusion of l- KVNSE₅DA (250 nmol). l-KVNSE₅DA (250 nmol) was infused with DYN 2–17 (75 nmol) or 5 min after DYN 2–17 (100 nmol) infusion (p≤ 0.01, *^ p< 0.05 relative to Veh + DYN 2–17; Kruskal-Wallis test). ○ Control (n=9), □ Veh + 75 nmol DYN 2–17 (n=21), ■ l- KVNSE₅DA 250 nmol + 75 nmol DYN 2–17 (n=20). ⋄ DYN 100 nmol, ◆ l- KVNSE₅DA 250 nmol + 100 nmol DYN (D) l-KVNSE5DA (250 nmol) also prevents the impairment of motor function (rotor-rod performance) that is apparent 21 days after DYN 2–17 (75 nmol) infusion (p< 0.01, F_{(1,89 )} = 9.7 , ANOVA; * p< 0.05, SNK), □ Vehicle + DYN 2–17 (n=5), ■ l-KVNSE₅DA 250 nmol + l-KVNSE₅DA (n=5).

Fig. 4

KVNSE₅DA reduces DYN 2–17-induced paralysis and motor neuron loss. (A) CGRP immunostaining of motor neurons in horizontal section of DYN 2–17-treated rat illustrates patchy loss of motor neurons (*). (B) Co-administration of l-KVNSE₅DA (250 nmol) significantly protected against motor neuron loss. (C) Cresyl Violet Nissl stain illustrates shows microglial invasion (*) into an area of DYN 2–17-treated tissue where there is profound motor neuron loss. This area is adjacent to intact ventral horn tissue, with normal complement of motor neurons (arrows). Scale bars a,b: 500μm; c:100μm. (D) Percentage of animals showing paralysis (score <3) or motor neuron loss, 72 h after infusion of vehicle(control; n=13), DYN 2–17 (DYN; n=12) or DYN + l-KVNSE₅DA (DYN/ l-KVNSE₅DAt; n=11) (†).