Superoxide anion- and nitric oxide-dependent mitochondrial dysfunction in neuronal apoptosis after spinal cord injury
| Autor: | Kay Li-Hui Wu, 吳俐慧 |
|---|---|
| Rok vydání: | 2007 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 95 The mitochondrion participates in caspase-independent or caspase-dependent apoptotic pathways through the release of apoptosis-inducing factor (AIF) or cytochrome c. Whether both mitochondrial apoptotic cascades are triggered in the injured spinal cord remains unknown. In this PhD thesis, I demonstrated for the first time that neurons, astrocytes and microglia in spinal segments proximal to a complete spinal cord transection (SCT) underwent two phases of apoptotic cell death. The early phase of high-molecular weight (HMW) DNA fragmentation was associated with nuclear translocation of AIF, reduction in mitochondrial respiratory chain enzyme activity and decrease in cellular ATP content. The delayed phase of low-molecular weight (LMW) DNA fragmentation was accompanied by cytosolic release of cytochrome c, activation of caspases 9 and 3, and resumption of mitochondrial respiratory functions and ATP contents. Microinfusion via osmotic minipumps of coenzyme Q10 (CoQ10), an electron carrier in mitochondrial respiratory chain, into the epicenter of the transected spinal cord attenuated both phases of apoptosis, and reversed the mitochondrial dysfunction, bioenergetic failure, and activation of AIF, cytochrome c, or caspases 9 and 3 after transection. These results suggest that mitochondrial dysfunction after SCT initiates the sequential activation of AIF-dependent and caspase-dependent signaling cascades, leading to apoptotic cell death in the injured spinal cord. Dysfunction of mitochondrial electron transport triggers the production of ROS, which play an important role in inducing apoptosis. Therefore, I hypothesized that ROS accumulation due to mitochondrial dysfunction leads to apoptosis in the injury spinal cord after transection. I found that nitric oxide (NO) content in the injured spinal cord was significantly elevated, alongside an increase in superoxide anion (O2-) level and an up-regulation of the inducible NO synthase (iNOS) expression. Interestingly, the SCT-evoked O2- increase in the injured spinal cord was attenuated by continuous microinfusion of an iNOS inhibitor, S-methylisothiourea (SMT) into the epicenter of the transected spinal cord via the osmotic minipumps. In addition, I also found poly(ADP-ribose) polymerase-1 (PARP-1) activation and translocation of the pro-apoptotic Bax from cytoplasm to mitochondria in the injury spinal cord during the same post-operation intervals. On the other hand, microinfusion of NO trapping agent, 2-(4-carboxyphenyl)-4,4,5,5- tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO), or the SOD mimetic, 4-Hydroxy-2,2,6,6- tetramethylpiperidine-1-oxyl (tempol), into the epicenter of the transected spinal cord significantly attenuated the reduction in mitochondrial respiratory enzyme activity or ATP content induced by SCT. Carboxy-PTIO also effectively inhibited the PARP-1 activation, nuclear translocation of AIF, and the early phase of apoptosis after spinal cord injury. Tempol also inhibited Bax translocation and mitochondrial release of cytochrome c, cellular events that are related to the late phase of apoptosis after SCT. The early phase of SCT-induced apoptosis was also inhibited by 5,10,15,20-Tetrakis(N-methyl-4′-pyridyl) porphinato Iron (III) Chloride (FeTMPyP), a scavenger of peroxynitrite formed by reaction between NO and O2-. Taken together, these results support my hypothesis that NO overproduction and O2- accumulation resultant from the mitochondrial dysfunction initiate the AIF-dependent and caspase-dependent apoptotic cascades and lead to SCT-induced apoptosis. Furthermore, NO may participate in the AIF-dependent apoptosis PARP-1 activation, whereas O2- may initiate caspase-dependent pathway via translocation of Bax to mitochondria. These novel findings of differential roles of NO, O2-, as well as mitochondrial dysfunction in the manifestation of early and late stages of apoptosis in the spinal cord after SCT will shed new lights on the therapeutic strategies in spinal cord injury. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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