IRON INDUCED APOPTOSIS IN NEURODEGENERATIVE DISEASE

描述（改编自申请人摘要）：神经退行性疾病的病因与氧化应激引起的损伤有关。氧化应激的一种介导机制是过量的活性铁，其催化自由基的形成。 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，免受铁的有害影响。 研究者已经纯化了诱导胚胎第8天（E8）鸡睫状神经节神经元凋亡的活性。 相反，更成熟（>E10）的睫状神经节神经元没有被杀死。 N-末端测序表明，这种活性是卵转铁蛋白。 死亡诱导活性需要铁与转铁蛋白结合。 二铁重组转铁蛋白诱导细胞凋亡的EC_（50）为5 nM，完全在胚胎细胞外液和血液中发现的水平（30-40 μ M）内。 FeTr的这种作用不限于睫状神经节神经元：腰交感神经节含有两个神经元群体，一个在神经生长因子中存活，暴露于FeTf时不会死亡，另一个在睫状神经营养因子（CNTF）中存活，并被FeTf杀死。 CG神经元对FeTf敏感性的发育开关表明神经元对转铁蛋白介导的凋亡的易感性可能是由细胞-细胞相互作用调节的正常发育事件。 研究者建议研究神经元对转铁蛋白介导的铁转运所引起的杀伤的不同敏感性的分子基础。 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。 具体目标是：（1）为了检验当神经元不能下调转铁蛋白受体时神经元变得对FeTf的杀伤敏感的假设，将人转铁蛋白受体在不敏感的神经元群体中过表达以检验其是否赋予对人FeTf的杀伤的敏感性;（2）检验对FeTf的易感性是由铁蛋白（细胞内铁结合蛋白）水平降低介导的假设，将过表达铁蛋白重链以测试神经元是否受到保护免于FeTf诱导的死亡;（3）为了检验细胞内铁通过降低共济失调蛋白（一种刺激铁转运出线粒体的蛋白质）的水平在线粒体中积累而诱导凋亡的假设，frataxin将在神经元中过表达，以确定它们是否可以被保护免受FeTf的杀伤;和（4）为了检验细胞内铁通过产生过量的自由基而杀死细胞的假设，所述自由基破坏线粒体，将确定是否：（1）FeTf产生自由基;（2）抗氧化剂从FeTf中拯救神经元;（3）氧化磷酸化抑制剂以与FeTf相同的特征诱导凋亡;（4）细胞色素c由FeTf处理的神经元释放;（5）线粒体抗凋亡蛋白Bcl-2的过表达保护细胞免受FeTf的侵害;半胱天冬酶抑制剂从FeTf中拯救神经元。

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.