α-酮戊二酸去氫酶複合體在線蟲老化過程的角色與可能機制

α-酮戊二酸去氫酶複合體（α-ketoglutarate dehydrogenase complex，KGDHC）是三羧酸循環中重要步驟的催化酵素，過去研究證實多種神經退化性疾病皆可偵測到KGDHC活性降低與活性氧(reactive oxygen species；ROS)顯著的增加。而本研究室先前發現，利用parapyruvate抑制KGDHC的活性，具有顯著縮短人類Hs68細胞和線蟲壽命的作用，然而，KGDHC在老化過程所扮演的角色目前並不清楚？因此，我們使用KGDHC線蟲突變株Tm6589/nt1來探討KGDHC活性降低造成線蟲壽命縮短的能力及可能的機制，結果發現與野生型N2線蟲比較，Tm6589/nt1體內KGDHC顯著降低44%，平均壽命顯著的降低10-12%，並且ROS顯著的上升2-2.77倍。額外添加N-乙醯-L-半胱氨酸(N-acetyl-L-cysteine，NAC)及NADH皆顯示可以顯著延長Tm6589/nt1的壽命，並且顯著降低蟲體的ROS，同時體內NADPH和GSH亦顯著的升高，顯示NAC和NADH能有效的降低Tm6589/nt1體內氧化壓力，並且能延長Tm6589/nt1的壽命；此外，Tm6589/nt1粒線體中NADH濃度則顯著的較野生型線蟲降低，而外加NAC及NADH亦能有效的增加Tm6589/nt1粒線體中的NADH濃度。最後我們測定不同日齡野生型線蟲體內KGDHC活性變化的情形，結果顯示KGDHC活性隨著線蟲日齡顯著的降低，並且12-16日齡的野生型線蟲其體內KGDHC活性與Tm6589/nt1突變株相當。綜合上述結果證實KGDHC活性會隨年齡逐漸降低，且KGDHC活性降低，會使得粒線體NADH的生成減少，造成NADPH及GSH的再生不足，導致ROS大量上升，最後造成線蟲壽命的縮短，顯示KGDHC在線蟲老化的過程扮演重要角色。

Alpha-ketoglutarate dehydrogenase complex (KGDHC) is a catalytic enzyme for an important step of the TCA cycle. Studies have confirmed that a decrease in KGDHC activity and a significant increase in reactive oxygen species (ROS) can be detected in a variety of neurodegenerative diseases. Our previous studies found that the use of parapyruvate to inhibit the activity of KGDHC could significantly shorten the lifespan of human Hs68 cells and C. elegans. However, the role of KGDHC in the aging prcess remains unclear? Therefore, we used Tm6589/nt1, a KGDHC mutant of C. elegans to explore the ability and the possible mechanism of reduced KGDHC activity to shorten the C. elegans lifespan and the possible mechanism. The results showed that compared with wild-type N2 C. elegans, KGDHC in Tm6589/nt1 was significantly reduced by 44%, and the mean lifespan was significant reduced by 10-12%, and ROS was significantly increased by 2-2.77 times. The addition of N-acetyl-L-cysteine (NAC) and NADH showed that these two molecules could significantly extend the lifespan of Tm6589/nt1 and significantly reduced the ROS level in the Tm6589/nt1 mutants. At the same time, NADPH and GSH in the mutants also increased significantly, demonstrating that NAC and NADH can effectively reduce the oxidative stress of Tm6589/nt1, and can extend the lifespan of Tm6589/nt1. In addition, the concentration of NADH in Tm6589/nt1 mitochondria was significantly lower than that in wild-type C. elegans, and the addition of NAC and NADH could also effectively increase the concentration of NADH in Tm6589/nt1 mitochondria. Finally, we determined the changes in KGDHC activity in wild-type C. elegans of different ages. The results showed that KGDHC activity decreased significantly with the age of the C. elegans, and the KGDHC activity in wild-type C. elegans at 12-16 days of age was equivalent to that of the Tm6589/nt1 mutants. In summary, the results in this study have confirmed that KGDHC activity will gradually decrease with age, and the decrease of KGDHC activity will reduce the NADH levels in mitochondrial, resulting in insufficient regeneration of NADPH and GSH, leading to a large increase in ROS, and finally shortening the lifespan of the C. elegans. The results have demonstrated that KGDHC plays an important role in the aging of C. elegans.