Abstract
Mechanisms associated with cyclin-dependent kinase 5 (Cdk5)-mediated heat hyperalgesia induced by inflammation remain undefined. This study was designed to examine whether Cdk5 mediates heat hyperalgesia resulting from peripheral injection of complete Freund's adjuvant (CFA) in the spinal dorsal horns of rats by interacting with synaptophysin, a well known membrane protein mediating the endocytosis-exocytosis cycle of synaptic vesicles as a molecular marker associated with presynaptic vesicle membranes. The role of Cdk5 in mediating synaptophysin was examined through the combined use of behavioral approaches, imaging studies, and immunoprecipitation following CFA-induced inflammatory pain. Results showed that Cdk5 colocalized with both synaptophysin and soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptors (SNAREs) consisting of VAMP-2, SNAP-25, and syntaxin 1A in spinal dorsal horn of rats. Increased synaptophysin expression of spinal cord horn neurons post intraplantar injection of CFA coincided with increased duration of heat hyperalgesia lasting from 6 h to 3 d. Intrathecal administration of roscovitine, a Cdk5 specific inhibitor, significantly depressed synaptophysin expression during peak heat hyperalgesia and heat hyperalgesia induced by peripheral injection of CFA. Data presented in this report indicated that calpain activity was transiently upregulated 6 h post CFA-treatment despite previous reports suggesting that calpain was capable of cleaving p35 into p25. Results from previous studies obtained by other laboratories demonstrated that significant changes in p35 expression levels within spinal cord horn neurons were not observed in the CFA-treated inflammatory pain model although significant upregulation of Cdk5 kinase was observed between 2 h to 7 d. Therefore, generation of p25 occurred in a calpain-independent fashion in a CFA-treated inflammatory pain model. Our results demonstrated that increased synaptophysin levels were involved in heat hyperalgesia mediated by Cdk5 in spinal cord dorsal horns of CFA-treated rats, suggesting that inhibiting abnormal activation of Cdk5-synaptophysin may present a novel target for diminishing inflammatory pain.
Highlights
Accumulating evidence has shown that multiple protein kinases are involved in mediating inflammatory pain in the central nervous system (CNS) [1,2,3]
cyclindependent kinase 5 (Cdk5) activity does not appear to depend on association with cyclins and its activity seems to be confined to postmitotic neurons where it can be activated by p35 or p39 that are cleaved to p25 or p29, respectively, by calpain [6]
Cdk5 colocalized with vesicle-associated membrane protein-2 (VAMP-2) in the cytoplasm of neurons, indicating that Cdk5 may directly interact with synaptophysin/ VAMP-2
Summary
Accumulating evidence has shown that multiple protein kinases are involved in mediating inflammatory pain in the central nervous system (CNS) [1,2,3]. Previous studies showed that Cdk was mainly associated with mediating neuronal migration and apoptosis [6,7]. Emerging evidence has shown that Cdk plays an important role in mediating heat but not mechanical hyperalgesia associated with inflammatory pain in the dorsal root ganglion (DRG) and in spinal cords of both mice and rats [8,9,10,11]. In the spinal cords of rats challenged with CFA, studies by others showed that Cdk activity and cofactor p25 expression were significantly upregulated and that roscovitine, a selective inhibitor of Cdk family members in neurons, markedly inhibited Cdk activity as well as heat hyperalgesia elicited following intraplantar injection of CFA [11]. The mechanism of Cdk5-mediated inflammatory pain has remained undefined to date
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