Iron in the pathogenesis of Friedreich's ataxia

描述（由申请人提供）：弗里德赖希共济失调（FRDA）是由于亲本共济失调蛋白（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所致。结果是缺乏共济失调蛋白。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异性成熟。其假定的正常功能是铁-硫（Fe-S）簇的生物发生，将铁稳态传递到整个细胞。Fe-S簇的供应不足严重损害线粒体电子传递链的复合物I、II和III以及柠檬酸循环酶乌头酸酶的活性。除了高能磷酸盐的次优生物合成外，缺乏共济失调蛋白还提高了对氧化应激的敏感性，推测这是由游离或松散结合的铁介导的。FRDA中的组织损伤非常多样。在心脏中，该疾病导致心肌细胞中铁的有限积累，但在中枢或外周神经系统中尚未证明类似的限制性铁过量。总铁的测定不能确定产生有毒氧物质所需的少量催化量的金属。人类尸检组织也不太可能适合直接测定这一小部分高活性铁。然而，失败的铁稳态仍然可以通过铁对铁响应蛋白的“下游”效应来评估，其中铁蛋白、线粒体铁蛋白和膜铁转运蛋白最有可能发生变化。在FRDA中，小脑齿状核和脊髓背根神经节（DRG）是高度脆弱的。 这项研究将测试这一假设，即这些解剖部位的frataxin缺乏的不良影响是弥漫性或局部铁过量的结果。区域增加可以通过一种新技术，高清晰度X射线荧光（HDXRF）直接确定;间接通过铁反应蛋白的系统检查。研究者将联合收割机HDXRF与载玻片技术和铁蛋白和膜铁蛋白的生化测定相结合。HDXRF“映射”组织块中的铁，并允许其基于标准的定量。显示铁反应蛋白的免疫细胞化学反应产物和铁“地图”的同一块的部分将被精确匹配，以达到铁和蛋白质的相关性。铁蛋白和膜铁转运蛋白的水平预期与共济失调蛋白的浓度负相关。患者死亡时的年龄或疾病持续时间也可能存在相关性。线粒体铁蛋白的表达响应于共济失调蛋白缺乏是更复杂的，并且结果预计是全或无，如先前报道的FRDA心脏。FRDA也可能由于不正确的线粒体内共济失调蛋白成熟而引起铁代谢障碍。这种潜在的影响因素将通过蛋白质印迹法检测共济失调蛋白前体和成熟的功能蛋白，并在一个更强大的方法，同位素编码的亲和标签技术和串联质谱。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，以量化神经细胞损伤和增加铁区域的突触末端损失;和电子显微镜，以揭示胞质和线粒体铁蛋白。 给定组织（如齿状核）中正常的高铁浓度并不一定会增加对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部对照。此外，一些脊髓小脑共济失调（SCA）的神经病理表型包括DRG病变。SCA样本将进行平行研究，以确定FRDA患者DRG的变化对遗传性共济失调蛋白缺乏症具有特异性。 这项工作是临床相关的，因为它将解决有关铁的正式发病机制和FRDA的自然史，以及铁螯合的潜在价值的问题。 公共卫生相关性：弗里德赖希共济失调（FRDA）是最常见的遗传性共济失调，在美国任何给定时间影响5，000 - 6，000名儿童和年轻人。载波频率在1：50到1：100之间。这种疾病是由于frataxin基因突变引起的，该基因控制所有细胞能量和铁代谢的重要步骤。心脏、外周和中枢神经系统最脆弱。突变由异常的鸟嘌呤-腺嘌呤-腺嘌呤三核苷酸重复扩增组成，并且在双亲等位基因上都具有这种扩增的人在他们的线粒体中缺乏足够的共济失调蛋白。本研究致力于研究小脑齿状核和脊髓背根神经节的铁代谢紊乱，因为这些解剖结构非常容易在FRDA中受损。这项研究预计将对我们理解FRDA的自然史和未来的治疗产生重大影响。目前正在进行几项实验性药物的临床试验。拟议的研究将为它们的有效性提供科学依据。药物艾地苯醌旨在抵消铁介导的氧化损伤，可能对FRDA患者的心脏病有益。螯合疗法，努力消除铁从受影响的组织仍然存在争议。拟议的调查将有助于澄清螯合疗法在FRDA中的潜在益处。

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. PUBLIC HEALTH RELEVANCE: Friedreich's ataxia (FRDA) is the most common hereditary ataxia, affecting 5,000-6,000 children and young adults in the United States at any given time. Carrier frequency is high at 1:50 to 1:100. The disease is due to a mutation in the frataxin gene, which controls important steps in energy and iron metabolism of all cells. Heart and peripheral and central nervous systems are most vulnerable. The mutation consists of an abnormal guanine-adenine-adenine trinucleotide repeat expansion, and persons with such an expansion on both parental alleles lack sufficient frataxin in their mitochondria. This research is devoted to the study of disturbed iron metabolism in the dentate nucleus of the cerebellum and dorsal root ganglia of the spinal cord because these anatomical structures are very susceptible to damage in FRDA. The research is expected to have high impact on our understanding of the natural history of FRDA and future therapy. Several clinical trials with experimental drugs are currently underway. The proposed research will provide the scientific basis for their effectiveness. The drug idebenone seeks to counteract iron-mediated oxidative damage and may be beneficial for the heart disease of patients with FRDA. Chelation therapy in an effort to remove iron from affected tissues remains controversial. The proposed investigation will help clarify the potential benefit of chelation therapy in FRDA.