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所致。结果是frataxin缺乏。 Frataxin 是一种线粒体蛋白，在转移到线粒体内部期间和之后会经历特定的成熟。其假定的正常功能是铁硫 (Fe-S) 簇的生物发生，将铁稳态传递至整个细胞。 Fe-S簇供应不足会严重损害线粒体电子传递链复合物I、II和III以及柠檬酸循环酶、乌头酸酶的活性。除了高能磷酸盐的生物合成不理想之外，frataxin 的缺乏也会增加对氧化应激的敏感性，推测氧化应激是由游离或松散结合的铁介导的。 FRDA 中的组织损伤多种多样。在心脏中，该疾病导致心肌细胞中铁的有限积累，但在中枢或周围神经系统中尚未证明类似的限制性铁过量。总铁的测定无法确定产生有毒氧物质所需的少量催化金属。人体尸检组织也不可能适合直接测定这一小部分高活性铁。然而，铁稳态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) 非常脆弱。 这项研究将检验以下假设：frataxin 缺乏对这些解剖部位的不利影响是由于弥漫性或局部铁过量造成的。可以通过新技术——高清X射线荧光（HDXRF）直接测定区域的增加；并间接通过铁反应蛋白的系统检查。研究人员将 HDXRF 与载玻片技术以及铁蛋白和铁转运蛋白的生化测定结合起来。 HDXRF“映射”组织块中的铁并允许根据标准对其进行量化。显示铁响应蛋白和铁“图”的免疫细胞化学反应产物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。预计铁蛋白和铁转运蛋白的水平与frataxin的浓度呈负相关。患者死亡时的年龄或其疾病持续时间也可能存在相关性。线粒体铁蛋白的表达对 frataxin 缺乏的反应更为复杂，并且预期结果是全有或全无，正如之前针对 FRDA 心脏的报道。 FRDA 还可能由于线粒体内 frataxin 成熟不正确而导致铁代谢障碍。这种潜在的影响因素将通过 frataxin 前体和成熟功能蛋白的蛋白质印迹法以及更可靠的方法、同位素编码亲和标签技术和串联质谱法进行检查。这项研究还利用先进的载玻片技术，例如双标记免疫荧光显微镜，来量化铁含量增加区域的神经细胞损伤和突触末梢损失；和电子显微镜揭示细胞质和线粒体铁蛋白。 特定组织（例如齿状核）中正常铁浓度较高，并不一定会增加对 FRDA 的脆弱性。因此，将添加具有同样高铁和铁蛋白水平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。我们将同时研究 SCA 样本，以确定 FRDA 患者 DRG 的变化是否是遗传性 frataxin 缺乏症所特有的。 这项工作具有临床相关性，因为它将解决有关铁在 FRDA 的正式发病机制和自然史中的问题，以及铁螯合的潜在价值。 公共健康相关性：弗里德赖希共济失调 (FRDA) 是最常见的遗传性共济失调，在任何特定时间影响美国 5,000-6,000 名儿童和年轻人。载波频率较高，为1:50至1:100。这种疾病是由于 frataxin 基因突变造成的，该基因控制着所有细胞能量和铁代谢的重要步骤。心脏以及周围和中枢神经系统是最脆弱的。该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，双亲等位基因均出现这种扩增的人的线粒体中缺乏足够的frataxin。本研究致力于研究小脑齿状核和脊髓背根神经节铁代谢紊乱，因为这些解剖结构非常容易受到 FRDA 损伤。这项研究预计将对我们对 FRDA 自然史和未来治疗的理解产生重大影响。目前正在进行多项实验药物的临床试验。拟议的研究将为它们的有效性提供科学依据。艾地苯醌药物旨在抵消铁介导的氧化损伤，可能对 FRDA 患者的心脏病有益。从受影响组织中去除铁的螯合疗法仍然存在争议。拟议的研究将有助于阐明螯合疗法在 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.