Iron in the pathogenesis of Friedreich's ataxia

描述(申请人提供)：Friedreich‘s共济失调(FRDA)是由于亲代Frataxin(FXN)基因中鸟嘌呤-腺嘌呤-腺嘌呤(GAA)三核苷酸重复扩增的纯合传递。其结果是缺乏Frataxin。Frataxin是一种线粒体蛋白，在转移到线粒体内部期间和之后经历特定的成熟。它的正常功能是铁-硫簇(Fe-S)的生物发生，将铁的动态平衡输送到整个细胞。铁-S簇的供应不足严重损害了线粒体电子传递链上的复合体I、II和III以及柠檬酸循环酶乌头酸酶的活性。除了高能磷酸盐的生物合成不佳外，缺乏Frataxin还会增加对氧化应激的敏感性，据推测，氧化应激是由自由或松散结合的铁介导的。FRDA的组织损伤是多种多样的。在心脏，这种疾病会导致心肌细胞铁的有限积累，但在中枢或外周神经系统中还没有表现出类似的限制性铁过量。对总铁的分析不能确定产生有毒氧物种所需的少量催化金属。人类尸检组织也不太可能适合直接测定这一小块高活性铁的含量。然而，铁稳态的失败仍然可以通过铁对铁反应蛋白的“下游”影响来评估，其中铁蛋白、线粒体铁蛋白和铁蛋白最有可能发生变化。在FRDA中，小脑齿状核和脊髓的背根节(DRG)是非常脆弱的。这项研究将检验这样一种假设，即Frataxin缺乏对这些解剖部位的不利影响是弥漫性或局限性铁过量的结果。区域增长可以通过一种新技术--高清晰度X射线荧光(HDXRF)直接确定；也可以间接通过对铁反应蛋白的系统检查来确定。研究人员将把HDXRF与载玻片技术以及铁蛋白和铁蛋白的生化分析结合起来。HDXRF“映射”组织块中的铁，并允许根据标准对其进行量化。同一块中显示铁反应蛋白的免疫细胞化学反应产物的部分将被精确匹配，以达到铁和蛋白质的关联。铁蛋白和铁蛋白的水平预计与Frataxin的浓度呈负相关。患者死亡时的年龄或他(她)的病程也可能存在相关性。线粒体铁蛋白在Frataxin缺乏的反应中的表达更为复杂，结果可能是全有或全无，就像之前报道的FRDA心脏。FRDA还可能由于错误的线粒体内Frataxin成熟而导致铁代谢障碍。对Frataxin前体和成熟功能蛋白进行Western blotting，并采用更可靠的方法--同位素编码亲和标记技术和串联质谱仪--来检验这一潜在的促成因素。这项研究还利用先进的载玻片技术，如双标记免疫荧光显微镜，对铁增加区域的神经细胞损伤和突触终末的丢失进行量化；并利用电子显微镜显示胞浆和线粒体铁蛋白。在特定的组织中，如齿状核，正常铁浓度高并不一定会增加FRDA的易感性。因此，将添加铁和铁蛋白水平同样高的苍白球作为内控。此外，一些脊髓小脑性共济失调(SCA)的神经病理表型包括背根节病变。SCA的样本将进行平行研究，以确定FRDA患者DRG的变化是遗传性Frataxin缺乏症的特异性。这项工作具有临床意义，因为它将解决有关铁在FRDA的正式发病机制和自然病程中的问题，以及铁螯合的潜在价值。

DESCRIPTION (provided by applicant): Friedreich's ataxia (FRDA) is due to homozygous transmission of guanine-adenine-adenine (GAA) trinucleotide repeat expansions in parental frataxin (FXN) genes. The result is deficiency of frataxin. Frataxin is a mitochondrial protein that undergoes specific maturation during and after transfer into the mitochondrial interior. Its putative normal function is the biogenesis of iron- sulfur (Fe-S) clusters conveying iron homeostasis to the entire cell. An inadequate supply of Fe- S clusters seriously impairs the activities of complexes I, II, and III of the mitochondrial electron transport chain, and the citric acid cycle enzyme, aconitase. In addition to suboptimal biosynthesis of high-energy phosphates, lack of frataxin also heightens sensitivity to oxidative stress, presumed to be mediated by free or loosely bound iron. Tissue damage in FRDA is very diverse. In the heart, the disease causes limited accumulation of iron in cardiomyocytes but similar restricted iron excess has not been demonstrated in central or peripheral nervous systems. Assays of total iron do not identify the small catalytic amounts of the metal that are required for the generation of toxic oxygen species. It is also unlikely that human autopsy tissues are suitable for the direct determination of this small pool of highly reactive iron. Nevertheless, failing iron homeostasis can still be assessed by the "downstream" effects of iron on iron-responsive proteins among which ferritin, mitochondrial ferritin, and ferroportin are most likely to undergo changes. In FRDA, the cerebellar dentate nuclei and the dorsal root ganglia (DRG) of the spinal cord are highly vulnerable. This research will test the hypothesis that the adverse effect of frataxin deficiency on these anatomical sites is the result of diffuse or localized iron excess. Regional increase can be determined directly by a new technology, high-definition X-ray fluorescence (HDXRF); and indirectly by a systematic examination of iron-responsive proteins. The investigator will combine HDXRF with slide techniques and biochemical assays of ferritin and ferroportin. HDXRF "maps" iron in tissue blocks and allows its quantification based on standards. Sections of the same block displaying immunocytochemical reaction products of iron-responsive proteins and iron "maps" will be matched precisely to reach a correlation of iron and proteins. Levels of ferritin and ferroportin are expected to be inversely correlated with concentrations of frataxin. A correlation may also exist with the age of the patient at the time of death or the duration of his (her) disease. The expression of mitochondrial ferritin in response to frataxin deficiency is more complex, and results are expected to be all-or-none, as previously reported for FRDA heart. FRDA may also cause iron dysmetabolism due to incorrect intramitochondrial frataxin maturation. This potential contributing factor will be examined by Western blotting of frataxin precursors and the mature functional protein, and in a more robust approach, isotope-coded affinity tag technology and tandem mass spectrometry. This research also utilizes advanced slide technology such as double-label immunofluorescence microscopy to quantify nerve cell damage and loss of synaptic terminals in regions of increased iron; and electron microscopy to reveal cytosolic and mitochondrial ferritin. High normal iron concentration in a given tissue, such as the dentate nucleus, does not necessarily convey increased vulnerability to FRDA. Therefore, the globus pallidus with its equally high iron and ferritin levels will be added as an internal control. Furthermore, the neuropathological phenotype of some spinocerebellar ataxias (SCA) includes lesions of DRG. Samples of SCA will be studied in a parallel effort to determine that the changes in DRG of FRDA patients are specific for inherited frataxin deficiency. The work is clinically relevant because it will resolve questions about iron in the formal pathogenesis and natural history of FRDA, and the potential value of iron chelation.