Mechanisms of Glucocorticoid-Excitatory Amino Acid Synergy on Promoting Nerve Regeneration

• Main Authors: Shih-Ying Tsai, 蔡世音
• Other Authors: Yi-Hsuan Lee
• Format: Others
• Language: en_US
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/20812715370915885674

博士 === 臺北醫學大學 === 醫學研究所 === 95 === Elevation of glucocorticoids and excessive glutamate release are the two major stress responses that occur sequentially during traumatic CNS injury which can result in secondary neuronal damage. Nevertheless, the synthetic glucocorticoids methylprednisone (MP) and dexamethasone are frequently used for treating the acute phase of CNS injury. Activation of glutamate receptor (GluR) has also been shown to influence several aspects of neuronal function during developing nervous system and neurite regeneration. These observations imply that glucocorticoids and glutamate might be beneficial for nerve repair. However, the mechanisms of this assumption are still limited. In this study, we applied corticosterone (CORT), the main adrenal glucocorticoids in rat, and a glutamate receptor agonist, kainic acid (KA), in cultured axotomized rat dorsal root ganglion neurons (AX-DRG) to mimic an environment of nerve injury to investigate the effects of glucocorticoid and excitatory amino acid on neurite outgrowth. Our results revealed that combined treatment of CORT and KA (CORT+KA) resulted in synergistic enhancement of neurite outgrowth and expression of growth-associated protein 43 (GAP-43) when applied as early as 1 and 2 days in vitro culture (1 and 2 DIV), but not 3 and 4 DIV in a sequential application order. In addition, application of CORT+KA caused neurotoxicity at 3 and 4 DIV but not at 1 and 2 DIV. The neurotrophic effects mediated by CORT+KA were abolished by down-regulation of glucocorticoids receptor (GR), AMPA/KA receptor, and receptor tyrosine kinase A (TrkA) activities. GAP-43 is known to promote neurite extension when phosphorylated by protein kinase C (PKC). Furthermore, PKC can phosphorylate the signal transducer and activator of transcription 3 (STAT3), a cytokine-related transcription factor, at Ser727, which is phosphorylated primarily by Janus kinase (JAK) at Tyr705. In further study, we examine the role of PKC in this stress-induced growth-promoting effect. In the cultured DRG neurons, the JAK inhibitor AG-490 and the PKC inhibitor Ro-318220 reduced the CORT+KA-enhanced neurite growth when applied prior to CORT and KA treatment, respectively. Both AG-490 and Ro-318220 diminished the CORT+KA-enhanced GAP-43 expression, phosphorylation, and axonal localization. Furthermore, treatment the cell with CORT+KA synergistically phosphorylated STAT3 at Ser727 but not at Tyr705. Similar phenomena were observed in an animal model of acute spinal cord injury (SCI), in which phosphorylation of GAP-43 and phospho-Ser727-STAT3 was elevated in the injured DRG at 4 hr after the impact injury. Further treatment with the therapeutic glucocorticoid methylprednisolone enhanced the phosphorylation of GAP-43 in both the DRG and the spinal cord of SCI rats. NogoA has been identified as a myelin-associated inhibitory protein that interacts with Nogo-66 receptor (NgR) to activate downstream signaling RhoA as well as Rho-associated kinase (ROK) and plays an important role in limiting axonal growth. In this study we further elucidated the role of NogoA-mediated inhibitory pathway in the synergistic effect of CORT+KA on neurite growth. RT-PCR analysis and immunofluorescent staining revealed that the suppressed NogoA expression by CORT+KA was abolished by transfecting cultured DRG with GR siRNA. Using Y-27632 to block ROK, the downstream signaling of NogoA receptor, showed a similar effect as CORT and KA synergy in promoting neurite growth. Glucocorticoids not only decreased RhoA expression in DRG neurons and astrocyte but also suppressed NogoA expression in reactivated astrocyte. Taken together, these results suggest that the neurotrophic effects of glucocorticoids on axonal regeneration might require facilitation of excitatory stimulation at early stage of nerve injury. CORT acting on GR to down-regulate NogoA and RhoA might play a key role in facilitating the subsequent PKC-dependent neurite growth upon activation of AMPA/KA receptors.