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播。其结果是缺乏卵黄蛋白。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定的成熟。其正常功能是铁硫（Fe-S）簇的生物生成，将铁稳态传递到整个细胞。铁- S簇供应不足严重损害线粒体电子传递链的复合物I、II和III的活性，以及柠檬酸循环酶和乌头酸酶的活性。除了高能磷酸盐的生物合成不理想外，缺乏卵黄蛋白还会增加对氧化应激的敏感性，这可能是由游离或松散结合的铁介导的。FRDA的组织损伤非常多样。在心脏，该疾病引起心肌细胞铁的有限积累，但在中枢或周围神经系统中尚未证实类似的限制性铁过量。总铁的测定不能确定产生有毒氧所需的少量金属催化量。人体解剖组织也不太可能适合直接测定这一小池高活性铁。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）是非常脆弱的。本研究将验证一种假设，即对这些解剖部位的脆性蛋白缺乏的不良影响是由弥漫性或局域性铁过量引起的。高清晰度x射线荧光（HDXRF）可以直接确定区域的增加；间接地通过对铁反应蛋白的系统检查。研究者将结合HDXRF与载玻片技术和铁蛋白和运铁蛋白的生化分析。HDXRF“映射”组织块中的铁，并允许根据标准进行量化。显示铁反应蛋白和铁“图”的免疫细胞化学反应产物的同一块的切片将精确匹配，以达到铁和蛋白质的相关性。铁蛋白和运铁蛋白的水平预计与卵黄蛋白的浓度呈负相关。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对frataxin缺乏症的反应更为复杂，正如之前报道的FRDA心脏一样，结果可能是全有或无。FRDA也可能由于线粒体内铁蛋白不正确成熟而导致铁代谢障碍。这一潜在的影响因素将通过卵黄蛋白前体和成熟功能蛋白的Western blotting，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测。本研究还利用先进的载玻片技术，如双标记免疫荧光显微镜来量化神经细胞损伤和突触终端在铁增加区域的损失；电镜显示细胞质和线粒体铁蛋白。特定组织（如齿状核）中高的正常铁浓度并不一定表示对FRDA的易感性增加。因此，苍白球具有同样高的铁和铁蛋白水平将被添加作为内部控制。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样本将被同时研究，以确定FRDA患者DRG的变化是遗传性纤维蛋白缺乏症的特异性。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史中的问题，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.