Cell-permeable peptide Tat-PSD-95 PDZ2 inhibits chronic inflammatory pain behaviors in mice

Abstract

Inflammatory conditions can lead to persistent debilitating pain, and the activation of N-methyl-D-aspartate receptors (NMDARs) has been shown to play an important role in the processing of inflammatory pain. Postsynaptic density protein-95 (PSD-95), a scaffolding protein, has been identified to interact with NMDARs at neuronal synapses of the central nervous system (CNS). However, the role of these interactions in the central sensitization of nociceptive processing has not been defined. In this study, we investigated the effect of disrupting NMDAR/PSD-95 interactions on chronic inflammatory pain behaviors. We constructed a fusion peptide, Tat-PSD-95 PDZ2, comprising the second PDZ domain of PSD-95, to disrupt specifically NMDARs/PSD-95 protein interactions. Western blot analysis showed that Tat-PSD-95 PDZ2 intraperitoneally injected into mice was delivered intracellularly into neurons in the CNS. By in vitro and in vivo binding assays, we found that the Tat-PSD-95 PDZ2 dose dependently inhibited the interactions between NMDARs and PSD-95. Furthermore, behavioral testing showed that mice given Tat-PSD-95 PDZ2 exhibited significantly reduced complete Freund's adjuvant (CFA)-induced chronic inflammatory pain behaviors compared to the vehicle-treated group. Our results indicate that by disrupting NMDAR/PSD-95 protein interactions, the cell-permeable fusion peptide Tat-PSD-95 PDZ2 provides a new target and approach for chronic inflammatory pain therapy.

Figure 1

In vitro and in vivo intracellular delivery of Tat-PSD-95 PDZ2 into mouse spinal cord neurons. (a) After incubation with His-tagged fusion peptides (Tat-PSD-95 PDZ2 or PSD-95 PDZ2 without Tat) for 30 min, Western blotting with anti-His antibody showed that the His-peptide was detected only in the neurons treated with Tat-PSD-95 PDZ2, but not in the neurons treated with PSD-95 PDZ2 or medium alone. Tubulin served as a loading control. (b) Western blot analysis demonstrated that after intraperitoneal injection, Tat-linked fusion peptides (Tat-PSD-95 PDZ2 and mutated Tat-PSD-95 PDZ2) were delivered into lumbar spinal cord of mice; PSD-95 PDZ2 without Tat was not detected in the spinal cord. Tat-PSD-95 PDZ2 was dose-dependently delivered into the spinal cord following systemic administration. 1: PSD-95 PDZ2 without Tat (8 mg/kg); 2: mutated Tat-PSD-95 PDZ2 (8 mg/kg); 3: Tat-PSD-95 PDZ2 (2 mg/kg); 4: Tat-PSD-95 PDZ2 (4 mg/kg); 5: Tat-PSD-95 PDZ2 (8 mg/kg). Tubulin served as a loading control.

Figure 2

Immunohistochemical staining demonstrated that only fusion peptide linked to Tat (Tat-PSD-95 PDZ2) was distributed in the spinal cord after intraperitoneal injection. (a, b) The Tat fusion peptide Tat-PSD-95 PDZ2 was accumulated in the cell bodies of the spinal cord. (c, d) PSD-95 PDZ2 without Tat was not detected in the spinal cord after systemic administration. b & d represent high magnification of the outlined areas in a & c, respectively. Scale bars: 50 μm in a for a & c; 10 μm in b for b & d. The data shown are representative of three independent experiments.

Figure 3

Disruption of NMDAR/PSD-95 protein interactions by Tat-PSD-95 PDZ2. (a) GST pull-down showed that Tat-PSD-95 PDZ2 dose-dependently inhibited the interactions between NMDA receptor NR2B and PSD-95 protein; mutated Tat-PSD-95 PDZ2 had no effect. (b) Co-immunoprecipitation showed that Tat-PSD-95 PDZ2 (8 mg/kg) markedly blocked the interaction between NR2A/2B and PSD-95 and that mutated Tat-PSD-95 PDZ2 (8 mg/kg) had no effect on this interaction compared to the effect of PSD-95 PDZ2 (8 mg/kg). The specificity of the NR2A/2B antibody was verified by preincubation with NR2 peptide. The amount of sample loaded for the input was 10% of that for the immunoprecipitation. IP: immunoprecipitation; IB: immunoblotting.

Figure 4

Intraperitoneal injection with Tat-PSD-95 PDZ2 significantly inhibited CFA-induced inflammatory pain behaviors in both development and maintenance phases. (a) Tat-PSD-95 PDZ2 dose-dependently inhibited inflammatory sensitization of the behavioral response induced by CFA injection on the ipsilateral side. However, mutated Tat-PSD-95 PDZ2 or PSD-95 PDZ2 had no effect. *P < 0.05 vs. the vehicle-treated group. (b) On the contralateral side, paw withdrawal thresholds in these peptides-treated groups were not significantly different from those of the vehicle-treated group. Data are expressed as mean ± SEM [n = 8 for Tat-PSD-95 PDZ2 (at different doses) and mutated Tat-PSD-95 PDZ2 groups; n = 6 for PSD-95 PDZ2 and the vehicle-treated groups].

Figure 5

Intrathecal injection with Tat-PSD-95 PDZ2 significantly inhibited CFA-induced inflammatory pain behaviors in both development and maintenance phases. (a) Tat-PSD-95 PDZ2 dose-dependently inhibited the CFA-induced decrease of paw withdrawal threshold on the ipsilateral side, but mutated Tat-PSD-95 PDZ2 or PSD-95 PDZ2 had no effect. *P < 0.05 vs. the vehicle-treated group. (b) On the contralateral side, these peptides did not significantly influence paw withdrawal threshold after intrathecal injections. Data are expressed as mean ± SEM [n = 8 for Tat-PSD-95 PDZ2 (at different doses), mutated Tat-PSD-95 PDZ2, and PSD-95 PDZ2 groups; n = 6 for the vehicle-treated group].

Figure 6

Intraperitoneal injection with Tat-PSD-95 PDZ2 had no effect on the baseline behavior and locomotor function of mice. (a) After intraperitoneal injection, these fusion peptides including Tat-PSD-95 PDZ2 had no significant effect on the baseline paw withdrawal threshold of the mice. (b) After intraperitoneal injection, these fusion peptides including Tat-PSD-95 PDZ2 did not show any effect on the tests of locomotor function. Data are expressed as mean ± SEM (n = 6 for each group).