Huang Hsueh-Meei, Ou Hsiu-Chong, Xu Hui, Chen Huan-Lian, Fowler Corinne, Gibson Gary E
Weill Medical College of Cornell University, Burke Medical Research Institute, White Plains, New York 10605, USA.
J Neurosci Res. 2003 Oct 15;74(2):309-17. doi: 10.1002/jnr.10756.
Mitochondrial dysfunction has been implicated in cell death in many neurodegenerative diseases. Diminished activity of the alpha-ketoglutarate dehydrogenase complex (KGDHC), a key and arguably rate-limiting enzyme of the Krebs cycle, occurs in these disorders and may underlie decreased brain metabolism. The present studies used alpha-keto-beta-methyl-n-valeric acid (KMV), a structural analogue of alpha-ketoglutarate, to inhibit KGDHC activity to test effects of reduced KGDHC on mitochondrial function and cell death cascades in PC12 cells. KMV decreased in situ KGDHC activity by 52 +/- 7% (1 hr) or 65 +/- 4% (2 hr). Under the same conditions, KMV did not alter the mitochondrial membrane potential (MMP), as assessed with a method that detects changes as small as 5%. KMV also did not alter production of reactive oxygen species (ROS). However, KMV increased lactate dehydrogenase (LDH) release from cells by 100 +/- 4.7%, promoted translocation of mitochondrial cytochrome c to the cytosol, and activated caspase-3. Inhibition of the mitochondrial permeability transition pore (MPTP) by cyclosporin A (CsA) partially blocked this KMV-induced change in cytochrome c (-40%) and LDH (-15%) release, and prevented necrotic cell death. Thus, impairment of this key mitochondrial enzyme in PC12 cells may lead to cytochrome c release and caspase-3 activation by partial opening of the MPTP before the loss of mitochondrial membrane potentials.
线粒体功能障碍与许多神经退行性疾病中的细胞死亡有关。在这些疾病中，α-酮戊二酸脱氢酶复合体（KGDHC）——三羧酸循环中的一种关键且可能是限速酶——的活性降低，这可能是脑代谢下降的原因。本研究使用α-酮-β-甲基-n-戊酸（KMV）——α-酮戊二酸的结构类似物——来抑制KGDHC活性，以测试KGDHC活性降低对PC12细胞线粒体功能和细胞死亡级联反应的影响。KMV使原位KGDHC活性在1小时内降低了52±7%，在2小时内降低了65±4%。在相同条件下，用一种能检测低至5%变化的方法评估，KMV并未改变线粒体膜电位（MMP）。KMV也未改变活性氧（ROS）的产生。然而，KMV使细胞中乳酸脱氢酶（LDH）的释放增加了100±4.7%，促进了线粒体细胞色素c向细胞质的转位，并激活了半胱天冬酶-3。环孢素A（CsA）对线粒体通透性转换孔（MPTP）的抑制作用部分阻断了这种由KMV诱导的细胞色素c释放（降低40%）和LDH释放（降低15%）的变化，并防止了坏死性细胞死亡。因此，PC12细胞中这种关键线粒体酶的损伤可能在线粒体膜电位丧失之前通过MPTP的部分开放导致细胞色素c释放和半胱天冬酶-3激活。
