Role Of GABA_AR/STEP61Signaling Pathway In The Modification Of Spinal NMDA Receptor Functions

Objective:The removal of GABA (y-aminobutyric acid) type A receptor (GABAAR)-mediated inhibition and the hyperfunction of.NMDA (N-Methyl-D-Aspartate)-subtype glutamate receptors (NMDARs) in spinal dorsal horn have been considered as two key mechanisms undelying chronic pathological pain. The present study investigated the relationship between these two pathological processes, and probed the possible role of Striatal-enriched protein tyrosine phosphatase (STEP) in NMDARs hyperfunction after impairing GABAergic inhibition (GABAergic disinhibition).Method:Nociceptive behavioral tests, immunoblotting, co-immunoprecipitatian and immunohistochemistry were conducted to explore whether and how STEP relayed signalings from GABAergic disinhibition to NMDARs hyperfunction.Results:(1) Immunoblotting analysis demonstrated that61kD isoform of STEP (STEP61) was the only STEP varient that was present in spinal cord. And immunohistochemistry experiments illustrated that STEP61predominantly distributed at superficial dorsal horn neurons of adult mice, which might provide the possibility for STEP61to regulate the nociceptive conveyance and intergration.(2) Intrathecal injection of recombinant adenovirus encoding wild-type STEP61[STEP61(WT)] generated little effects on the basal nociceptive responses in intact mice, while viral expression of a dominant negative STEP61mutant STEP61(C472S) to interfere with endogenous STEP61function elicited a pronounced mechanical allodynia in intact mice, suggesting that STEP61exerted a tonic inhibition of nociceptive transmission within spinal dorsal horn under physiological conditions.(3) Intrathecal injection of GABAAR antagonist bicuculline (0.1μg) to mimick GABAergic disinhibition evoked an evident mechanical allodynia in intact mice, which, however, could be blocked by STEP61(WT). Meanwhile, spinal expression of STEP61(C472S) to decrease the PWT values completely occluded the pronociceptive action of subsequent application of bicuculline, suggesting that disturbance of endogenous STEP61function played a critical role for GABAergic disinhibition to induce pain hypersensitivity.(4) Co-immunoprecipitation experiments illustrated that intrathecal injection of GABAAR antagonist bicuculline (0.1μg), via activation of cAMP-dependent protein kinase (PKA), disrupted STEP61interaction with Src-family protein tyrosine kinases (SFKs) member Fyn and with extracellular-signal regulated protein kinase1/2(ERK1/2), leading to the activation of Fyn and ERK as well as Fyn/ERK-dependent mechanical allodynia.(5) Overexpression of exogenous STEP61(WT) totally blocked the activation of Fyn and ERK by GABAergic disinhibition.(6) Importantly, direct expression of enzymatically inactive STEP61(C472S) generated a similar effect of GABAergic disinhibition by enhancing Fyn/ERK phosphorylation and inducing Fyn/ERK-dependent pain sensitization. Intrathecal application of MEK inhibitor U-0126(0.5μg) or SFKs inhibitor PP2(1.5μg) greatly alleviated the pain hypersensitivity in STEP61(C472S)-expressing mice, providing further evidence that STEP61acted downstream of GABAergic disinihibtion.(7) Intrathecal injection of GABAAR antagonist bicuculline (0.1u.g) dramatically increased the tyrosine phosphorylation of NMDA receptor NR2B subunit at Tyr1472and promoted the accumuylation of NMDA receptor NR1and NR2B subunit at synaptosomal membrane fraction, which could be abolished by spinal expression of STEP61(WT). More importantly, direct antagonism of spinal NR2B receptors by intrathecal application of NR2B-selective blocker ifenprodil (0.15～2.0μg) dose-dependently reversed the reduction of PWT values in bicuculline-treated mice, implicating that GABAergic disinhibition might disturb STEP61activity to specifically induce NR2B receptor hyperfunction and pain sensitization.Conclusion:GABAergic disinhibition destroyed STEP61function in spinal dorsal horn to trigger a series of intracellular signalings, which led to NMDARs hyperfunction and mechanical allodynia.