Spinal SIRPα1-SHP2 interaction regulates spinal nerve ligation-induced neuropathic pain via PSD-95-dependent NR2B activation in rats

Abstract

The fact that neuropathic pain mechanisms are not well understood is a major impediment in the development of effective clinical treatments. We examined whether the interaction between signal regulatory protein alpha 1 (SIRPα1) and Src homology-2 domain-containing protein tyrosine phosphatase 2 (SHP2), and the downstream spinal SHP2/postsynaptic density 95 (PSD-95)/N-methyl-d-aspartate receptor NR2B subunit signaling cascade play a role in neuropathic pain. Following spinal nerve ligation (L5), we assessed tactile allodynia using the von Frey filament test and analyzed dorsal horn samples (L4-5) by Western blotting, reverse transcription polymerase chain reaction, coimmunoprecipitation, and immunofluorescence. Nerve ligation induced allodynia, SIRPα1, SHP2, phosphorylated SHP2 (pSHP2), and phosphorylated NR2B (pNR2B) expression, and SHP2-PSD-95, pSHP2-PSD-95, PSD-95-NR2B, and PSD-95-pNR2B coimmunoprecipitation in the ipsilateral dorsal horn. In allodynic rats, injury-induced SHP2 immunoreactivity was localized in the ipsilateral dorsal horn neurons and coincident with PSD-95 and NR2B immunoreactivity. SIRPα1 silencing using small interfering RNA (siRNA; 1, 3, or 5μg/rat for 7days) prevented injury-induced allodynia and the associated changes in protein expression, phosphorylation, and coimmunoprecipitation. Intrathecal administration of NSC-87877 (an SHP2 antagonist; 1, 10, or 100μM/rat) and SIRPα1-neutralizing antibodies (1, 10, or 30μg/rat) suppressed spinal nerve ligation-induced allodynia, spinal SHP2 and NR2B phosphorylation, and SHP2/phosphorylated SHP2-PSD-95 and PSD-95-NR2B/phosphorylated NR2B coprecipitation. SHP2 siRNA led to similar effects as the NSC-87877 and SIRPα1 antibody treatments, except it prevented the allodynia-associated spinal SHP2 expression. In conclusion, our results suggest that a spinal SIRPα1-SHP2 interaction exists that subsequently triggers SHP2/PSD-95/NR2B signaling, thereby playing a role in neuropathic pain development.

BDNF contributes to the development of neuropathic pain by induction of spinal long-term potentiation via SHP2 associated GluN2B-containing NMDA receptors activation in rats with spinal nerve ligation

“…To clarify the underlying mechanisms by which how BDNF activates GluN2B-NMDA receptors in the spinal dorsal horn following spinal nerve injury, we focused on SHP2, a Src homology-2 (H2) domaincontaining protein tyrosine phosphatase-2, which has TrkB/TrkB and GluN2B-NMDA signaling pathway (Araki et al, 2000;Easton et al, 2006;Lin et al, 1999;Neel et al, 2003;Okada et al, 1996;Peng et al, 2012). We discovered that in intact rats, 7 days after intrathecal administration of BDNF (100 ng, twice per day for 2 days, see Fig.…”

“…To determine whether spinal SHP2 is involved in BDNF-mediated activation of GluN2B-NMDA receptors as well as in the induction of spinal LTP and post-injury pain hypersensitivity, we first examined effects of NSC-87877, a potent SHP2 protein tyrosine phosphatase inhibitor (Chen et al, 2006;Peng et al, 2012) on BDNF-induced GluN2B-NMDA receptors activation as well as spinal LTP induction and pain allodynia elicitation in intact rats. As shown in Fig.…”

“…Acting as a major regulator of RTK (Grossmann et al, 2010), SHP2 has been implicated in BDNF/TrkB (Araki et al, 2000;Easton et al, 2006) and GluN2B-NMDA receptors (Lin et al, 1999;Peng et al, 2012) signaling pathway. In our present study, we provide further evidence clarifying that spinal SHP2 is required for BDNF-mediated GluN2B-NMDA receptors activation as well as spinal LTP induction and pain hypersensitivity development in neuropathic rats.…”

“…Next, using a potent SHP2 protein tyrosine phosphatase inhibitor NSC-87877 (Chen et al, 2006;Peng et al, 2012) or RNA interference (RNAi) against SHP2, we disclosed that either blockade of SHP2 phosphorylation or knockdown of SHP2 in the spinal cord, could inhibit the BDNF-mediated GluN2B-NMDA receptors activation as well as spinal LTP induction and pain allodynia elicitation in intact rats, and reduce the nerve injury-evoked GluN2B-NMDA receptors up-regulation and spinal LTP occlusion, and also alleviate pain allodynia in neuropathic rats as well, implying that BDNF-induced SHP2 phosphorylation in the spinal dorsal horn is required for subsequent GluN2B-NMDA receptors activation and spinal LTP-like state induction, and ultimately for neuropathic pain development in rats following spinal nerve injury. It has been documented that BDNF enhances association of SHP2 with the NMDA receptor subunit GluN2B in the cortical postsynaptic density (PSD) (Lin et al, 1999), and phosphorylation of spinal SHP2 via signal regulatory protein alpha 1 (SIRPα1) induces SIRPα1-SHP2 interaction, which subsequently triggers SHP2/PSD-95/GluN2B signaling, and thereby playing a role in the development of neuropathic pain (Peng et al, 2012). Together these results with our present findings, we suggest that after nerve injury, increased BDNF causes phosphorylation of SHP2 in the spinal dorsal horn, which subsequently triggers SHP2/PSD-95/GluN2B cascade and induces spinal LTP at C-fiber synapses via activation of GluN2B-NMDA receptors, thereby playing a vital role in the development of central sensitization and pain hypersensitivity in neuropathic rats.…”

“…In an in vitro study, Lin and colleagues have reported that SHP2 participates in BDNF-mediated GluN2B-NMDA receptors signaling at the postsynaptic site, and BDNF enhances association of SHP2 with the NMDA receptor subunit GluN2B in the cortical postsynaptic density (PSD) (Lin et al, 1999). Recently, Peng et al have revealed that phosphorylation of spinal SHP2 via signal regulatory protein alpha 1 (SIRPα1) induces SIRPα1-SHP2 interaction, which subsequently triggers SHP2/PSD-95/GluN2B signaling, and thereby playing a role in neuropathic pain development in a rat model (Peng et al, 2012).…”