Effects And The Potential Mechanisms Of APC-Cdhl On The Maintenance Of Mechanical Allodynia After Nerve Injury In Rats

BackgroundNeuropathic pain is an important public health problem that negatively impacts quality of life of affected individuals.Growing evidence supports that neuropathic pain is associated with the alteration in synaptic plasticities of spinal dorsal horn.A series of inflammatory cytokines,neurotransmitter,peptide and transduction signals are involved in plasticity regulation of synapse in the spinal dorsal horn,which comprise the pain matrices in spinal level.Anaphase-promoting complex(APC),a multi-subunit E3 ubiquitin-protein ligase,is initially identified as regulating cell cycle progression.Cdhl is an important co-activator protein that activates APC during late mitosis and G1 in proliferating cells.Meanwhile,the observation that both APC and Cdhl are expressed in terminally differentiated neurons has revealed unexpected functions of APC-Cdhl outside cell cycle.In recent years,there is firm evidence showing that APC-Cdhl signalling cascade in the central nervous system(CNS)is critical for regulation of synaptic differentiation and transmission and involvement of late-phase long-term potentiation(LTP),hippocampus-dependent memory and learning and behavioral flexibility,suggesting that APC-Cdhl signalling pathway in the CNS may play an important role in the modulation of neuronal activity and synaptic plasticity by,presumably,inducing the ubiquination and degradation of certain functional proteins or receptors of excitatory neurons.In addition,APC-Cdhl signalling pathway plays an important role in the involvement of some neurological disorders,such as ischemic brain injury and Alzheimer’s disease.However,little is known about the involvement of spinal APC-Cdhl in the neuropathic pain.In this study,a spared nerve injury(SNI)model of neuropathic pain and integrative approaches,including morphological,biochemical,behavioural and lentiviral transduction techniques,were used to address the effects and potential mechanisms of APC-Cdhl on the maintenance of mechanical allodynia after nerve injury.Methods and Results1.Alteration in APC-Cdhl signalling pathway expression in the ipsilateral spinal cord after SNIMethods:A SNI model was applied to induce peripheral neuropathic pain in rats.Paw withdrawal mechanical thresholds(PWMTs)of both the surgical ipsilateral and contralateral hind paws were assessed by von Frey filaments before and at 1,3,7,14 d after SNI surgey.The expression of c-Fos in the bilateral spinal dorsal horn was assessed by immunohistochemistry at 7 days after SNI surgey.The total expression of APC-Cdh1,its upstream signalling protein Cdk5/p35,its downstream substrate SnoN and Skp2 in the bilateral spinal dorsal horn was examined by Western-Blot at 14 days after SNI surgey.Results:PWMTs of the surgical ipsilateral hind paws in rats began to significantly decreased from 3 days to 14 days after SNI and developed to the lowest at 7 days after SNI,which showed that behavioural mechanical allodynia developed after SNI in rats.The number of c-Fos immunoreactive cells was increased in the ipsilateral spinal dorsal horn,which suggested the activation of spinal dorsal horn after SNI surgey.The expression levels of total Cdhl in the ipsilateral spinal cord in SNI animals were significantly lower while that of total p 35 were higher,suggested the downregulation of spinal APC-Cdhl signalling pathway induced by peripheral nerve injury.2.The effects of spinal Cdhl overexpression on the mechanical allodynia in SNI-treated ratsMethods:Immunofluorescent staining was performed to examine the distribution of Cdhl in the spinal cord.The expressions of cytoplasmic Cdhl were examined by Western blotting and immunofluorescent staining on 14 days post nerve injury,the time of maximal expression of mechanical allodynia for SNI-treated rats.GFP expression and quantitatively Cdhl expression through immunofluorescent staining and Western-Blot were assessed in the spinal cord tissue at 3 days after intrathecal injection of lentivirvus encoding Cdhl and GFP(Lenti-Cdh1-GFP),control lentivirvus encoding GFP(Lenti-GFP)or vehicle.PWMTs of the surgical ipsilateral hind paws were examined at 1,3,7,14 days after intrathecal injection of Lenti-Cdh1-GFP.Results:Cdhl immunoreactivity was distributed in the spinal dorsal horn in adult rats,with enriched distribution in neurons but not astrocyte of the superficial dorsal horn.At day 14 after peripheral nerve injury,Cdhl levels in the cytoplasmic fraction were significantly increased in the ipsilateral spinal cord.Double immunofluorescent staining revealed that after peripheral nerve injury,more nonoverlapping regions of Cdhl immunoreactivity and DAPI staining were observed in the ipsilateral spinal dorsal horn but not in the contralateral fraction.At 3 days after intrathecal injection of Lenti-Cdhl-GFP and Lenti-GFP,a wide GFP expression was observed in many cells of spinal cord.Intrathecal injection of Lenti-Cdhl-GFP induced a remarkable upregulation of total Cdhl protein in spinal cord and a fusion protein of Cdhl-GFP was detected.Furthermore,intrathecal treatment with LentiCdhl-GFP significantly attenuated SNI-induced mechanical hypersensitivity,starting on 7 days and persisting to 14 days after treatment.3.The effects of APC-Cdhl on synaptic ultrastructural changes and the surface expression changes of the GluR1 in the ipsilateral dorsal horn induced by peripheral nerve injuryMethods:Synaptic ultrastructural changes from the ipsilateral dorsal horn were detected with transmission electron microscopy at 14 days after intrathecal injection of Lenti-Cdhl-GFP.The expressions of cytoplasmic and membranal GluR1,EphA4-EphrinA in the dorsal horn were examined by Western-Blot on 14 days post nerve injury and 14 days after intrathecal injection of Lenti-Cdhl-GFP in SNI rats.The interaction of GluR1,APC2,Cdhl and EphA4-EphrinAl was assessed by co-immunoprecipitation.Results:The structural changes induced by SNI were reversed to normal levels after injection with Lenti-Cdhl-GFP but not after injection with Lenti-GFP.At 14 days after SNI,the surface expression of GluR1 in the ipsilateral spinal cord was significantly upregulated while the expression of EphA4-EphrinAl was downregulated.Co-immunoprecipitation revealed that GluR1 interacted with Cdhl,EphA4-EphrinAl in the ipsilateral spinal cord.At 14 days after intrathecal administration,Lenti-Cdhl-GFP,but not Lenti-GFP,inhibited the enhanced surface expression of GluR1 and prevented the decrease in the EphA4-EphrinAl signalling complex induced by SNI surgery in the ipsilateral spinal cord.Conclusion1.Downregulation of Cdhl activity in the ipsilateral spinal cord after SNI.2.Downregulation of spinal Cdhl activity is required for the development of mechanical allodynia in SNI-treated rats.3.Downregulation of spinal Cdhl activity is required for SNI-induced increase of the surface expression of the GluRl in the spinal cord which is related to interaction with EphA4-EphrinAl signalling pathway.