IRON INDUCED APOPTOSIS IN NEURODEGENERATIVE DISEASE

描述（改编自申请人摘要）：神经退行性命令的病因与氧化应激引起的损伤有关。氧化应激介导的一种机制是过量的活性铁，它催化自由基的形成。铁储存在大脑中导致神经元死亡的机制尚不清楚，因为细胞通常受到保护，不受铁的有害影响。研究者纯化了一种诱导胚胎第8天（E8）鸡睫状神经节神经元凋亡的活性。相比之下，更成熟的（>E10）睫状神经节神经元未被杀死。n端测序显示该活性为卵转铁蛋白。诱导死亡的活性需要铁与转铁蛋白结合。异源重组转铁蛋白诱导细胞凋亡的EC50值为5nM，与胚胎细胞外液和血液中的EC50值（30 ~ 40 μ m）相当。fer的影响并不局限于睫状神经节神经元：腰交感神经节包含两类神经元，一类神经元在神经生长因子中存活，暴露于fef时不会死亡，另一类神经元在睫状神经营养因子（CNTF）中存活，并被fef杀死。CG神经元对FeTf敏感性的发育转换表明，神经元对转铁蛋白介导的凋亡的易感性可能是一种正常的发育事件，受细胞间相互作用的调节。研究者拟研究转铁蛋白介导的铁转运引起的神经元杀伤差异敏感性的分子基础。这些研究可能会为神经退行性疾病的过程如何出错提供重要线索。具体目的是：(1)验证当神经元不能下调转铁蛋白受体时，转铁蛋白受体会在不敏感的神经元群体中过表达，从而验证其是否对人转铁蛋白杀伤具有敏感性；(2)为了验证fef易感性是由铁蛋白水平降低介导的假设，细胞内铁结合蛋白、铁蛋白重链会过表达，以检验神经元是否能保护fef诱导的死亡；(3)为了验证细胞内铁通过降低frataxin（一种刺激铁从线粒体运输出来的蛋白质）水平在线粒体中积累而诱导细胞凋亡的假设，frataxin将在神经元中过度表达，以确定它们是否可以免受FeTf的杀死；(4)为了验证细胞内铁通过产生过量的自由基来杀死细胞并破坏线粒体的假设，将确定：(1)FeTf是否产生自由基；(2)抗氧化剂可使神经元从FeTf中恢复；(3)氧化磷酸化抑制剂诱导细胞凋亡的特点与FeTf相同；(4) fet处理的神经元释放细胞色素c；(5)线粒体抗凋亡蛋白Bcl-2过表达可保护细胞免受FeTf；(6) caspase抑制剂可拯救FeTf神经元。

DESCRIPTION (Adapted from the applicant's abstract): The etiology of neurodegenerative orders has been linked to damage caused by oxidative stress. One mechanism by which oxidative stress can be mediated is an excess of reactive iron, which catalyzes the formation of free radicals. The mechanism by which iron is stored in the brain can induce neuronal death has not been clear because cells are normally protected against the deleterious effects of iron. The investigator have purified an activity that induces apoptosis in embryonic day 8 (E8) chick ciliary ganglion neurons. In contrast more mature (>E10) ciliary ganglion neurons were not killed. N-terminal sequencing revealed that this activity was ovotransferrin. Death inducing activity required that iron be bound to the transferrin. The EC50 of diferric recombinant transferrin in inducing apoptosis was 5nM, well within the levels found in embryonic extracellular fluid and blood (30-40 uM). This effect of FeTr was not limited to ciliary ganglion neurons: lumbar sympathetic ganglia contain two populations of neurons, one which survives in nerve growth factor and does not die when exposed to FeTf, and another which survives in clilary neurotrophic factor (CNTF) and is killed by FeTf. The developmental switch in CG neuron sensitivity to FeTf suggests that susceptibility of neurons to transferrin-mediated apoptosis is likely to be a normal developmental event that is regulated by cell-cell interactions. The investigator proposes to study the molecular basis for the differential sensitivity of neurons to killing caused by transferrin-mediated iron transport. These studies are likely to lead to important clues as to how the process may go awry in neurodegenerative disease. The specific aims are: (1) to test the hypothesis that neurons become sensitive to killing by FeTf when they fail to downregulate transferrin receptor, human transferrin receptor will be overexpressed in insensitive neuronal populations in order to test whether it confers sensitivity to killing by human FeTf; (2) to test the hypothesis that susceptibility to FeTf is mediated by reduced levels of ferritin, the intracellular iron binding protein, ferritin heavy chain will be overexpressed in order test whether neurons are protected from death induced by FeTf; (3) to test the hypothesis that intracellular iron induces apoptosis by accumulating in mitochondria through reduced levels of frataxin, a protein that stimulates iron transport out of mitochondria, frataxin will be overexpressed in neurons in order to determine if they can be protected from killing by FeTf; and (4) to test the hypothesis that intracellular iron kills cells by generating excess free radicals which damage mitochondria, it will be determined if: (1) FeTf generates free radicals; (2) anti-oxidants rescue neurons from FeTf; (3) inhibitors of oxidative phosphorylation induce apoptosis with the same characteristics as FeTf; (4) cytochrome c is released by FeTf treated neurons; (5) overexpression of the mitochondrial anti-apoptotic protein Bcl-2 protects cells from FeTf; and (6) caspase inhibitors rescue neurons from FeTf.