IRON INDUCED APOPTOSIS IN NEURODEGENERATIVE DISEASE

描述(摘自申请者的摘要)：神经退行性疾病的病因学与氧化应激造成的损害有关。氧化应激调节的一个机制是过量的活性铁，它催化自由基的形成。铁储存在大脑中会导致神经元死亡的机制尚不清楚，因为细胞通常会受到保护，免受铁的有害影响。研究人员提纯了一种诱导胚胎第8天(E8)雏鸡睫状神经节神经元凋亡的活性。相比之下，更成熟的(&gt；E10)睫状神经节神经元没有被杀死。N-末端测序表明该活性为卵转铁蛋白。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EC50为5 nm，完全在胚胎细胞外液和血液中的水平(30-40微米)。FETR的这种作用并不局限于睫状神经节神经元：腰交感神经节含有两种神经元，一种在神经生长因子中存活，在FeTf作用下不死亡，另一种在临床神经营养因子(CNTF)中存活，并被FeTf杀死。CG神经元对FeTf敏感性的发育变化表明，神经元对转铁蛋白介导的细胞凋亡的敏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。这位研究人员建议研究转铁蛋白介导的铁运输引起的神经元对杀戮的差异敏感性的分子基础。这些研究可能会导致重要的线索，即神经退行性疾病的这一过程可能会出现问题。其具体目的是：(1)检验当FeTf不能下调转铁蛋白受体时，神经元对FeTf杀伤敏感的假说，人转铁蛋白受体在不敏感的神经元群体中过表达，以测试它是否增加了对人FeTf杀伤的敏感性：(2)检验FeTf易感性是由铁蛋白水平降低介导的，细胞内铁结合蛋白铁蛋白重链将过度表达，以检验是否对FeTf诱导的神经元死亡有保护作用；(3)为了验证细胞内铁通过降低线粒体中铁蛋白(一种刺激铁从线粒体外运输出去的蛋白质)的水平而诱导细胞凋亡的假设，Frataxin将在神经元中过度表达，以确定是否可以保护细胞免受FeTf的杀伤；以及(4)为了验证细胞内铁通过产生过量的自由基来杀死细胞的假设，它将确定：(1)FeTf产生自由基；(2)抗氧化剂从FeTf中拯救神经元；(3)氧化磷酸化抑制剂诱导具有与FeTf相同特征的细胞凋亡；(4)FeTf处理的神经元释放细胞色素c；(5)线粒体抗凋亡蛋白Bcl2的过表达保护细胞免受FeTf的伤害；(6)caspase抑制剂将神经元从FeTf中拯救出来。

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.