弗里德赖希氏共济失调病人神经元丢失的病理机制研究

Friedreich ataxia (FRDA) is a progressive disease caused by expansion of a GAA trinucleotide repeat in the gene that encodes frataxin (FXN). Patients develop ataxia, sensory loss and cardiomyopathy, and symptoms are limited mainly to the central nervous system, e.g. cerebellum and spinal cord, and heart. FXN is important in iron-sulfur cluster assembly where it likely functions as a chaperone that provides iron in a bioavailable form in the early steps of iron-sulfur cluster biosynthesis. Our previous study showed that patients manifest mitochondrial iron-sulfur cluster assembly deficiency, with decreased activity of iron-sulfur proteins such as mitochondrial aconitase and succinate dehydrogenase. Because of the mitochondrial iron overload as disease progresses, it has been considered that iron chelation therapy should ameliorate disease by protecting mitochondria from the adverse effects of iron overload and have shown positive results. However, we found that cells of FRDA patients have evidence of cytosolic iron depletion, as indicated by increased levels of iron regulatory protein 2 (IRP2) and/or increased IRE-binding activity of IRP1. A link to oxidative stress was anticipated and confirmed by the treatment of FRDA patients with idebenone, a free-radical scavenger. In fact, idebenone remains the best drug for the treatment of FRDA. And FXN-deficient cells showed change of glutathione-dependent redox status in a yeast and cell culture model of FRDA. Therefore, the misregulation of intracellular iron imposed by FXN deficiency could not only elicit a Fenton chemistry-based mechanism of ROS toxicity leading to prominent abnormalities of the neuronal cytoskeleton, but also change of glutathione-dependent redox status causing abnormally increased protein glutathionylation, both of which result in neuronal cell death. However, the detail signaling pathway of the mechanism is unknown. Very recently reported an iron-dependent form of nonapoptotic cell death, ferroptosis, presents a similar characters of FXN-deficient cells. A specific inhibitor, Fer-1, of ferroptosis was identified. We demonstrate that overexpression of human frataxin or alpha-lipoic acid treatment of cells derived from FRDA patients increased cellular antioxidant defense via elevation of reduced thiols/glutathione. We hypothesize that ferroptosis is the pathway for the death of FXN-deficient neuronal cells and interventions of Fer-1 and alpha-lipoic acids will reverse or slow down the cell death, further clinically should reduce the severity of at least some aspects of FRDA disease. We will test the validity of this hypothesis by using the FXN-deficient cell and mouse models and combining IRP2 knockout mice to reveal the molecular pathological mechanism of neuronal cell death. Identification and characterization of the pathway may aid in the development of new therapeutic avenues for the treatment of Friedreich ataxia and other neurodegenerative disease.

弗里德赖希共济失调(FRDA）是由FXN表达量减少引起中枢神经系统中小脑和脊髓神经元的丢失造成的神经退行性疾病。这种特定部位的神经元是如何丢失的问题还未见报道。前期研究中我们发现，FXN表达量减少后，引起细胞内铁代谢异常，胞质内铁缺乏，依赖于铁硫簇的酶活性下降，从而导致线粒体功能减退；另有文献报道，FXN减少引起细胞氧化应激增加，总GSH含量下降，细胞内膜结构发生聚集。以上特点与最新报道的一种依赖于铁的细胞死亡方式ferroptosis的特征非常吻合。我们推测FXN减少导致神经元的丢失是ferroptosis的结果。本项目拟采用多学科的技术手段，研究FXN表达量减少后神经元死亡的分子通路及可能的干预途径。计划利用细胞模型、动物模型及临床病人的资料和药物干预， 阐明FXN的缺失导致FRDA疾病的分子病理机制，为临床上治疗FRDA和其他神经退行性疾病的药物筛选提供有的放矢的靶点。

弗里德赖希共济失调是FXN的表达量减少引起中枢神经系统中小脑和脊髓神经元的丢失造成的神经退行性疾病。但关于在特定病变部位处神经元是如何丢失的问题还没有人报道。前期研究中我们发现，FXN表达量减少后，引起细胞内铁代谢异常，胞质内铁缺乏，线粒体内铁的累积，依赖于铁硫簇的酶活性下降，从而导致线粒体功能减退；另有文献报道，FXN表达减少引起氧化应激增加，细胞膜脂质氧化，结构发生聚集。FXN表达减少引起的这些变化符合2012年报道的一种依赖于铁的细胞死亡方式ferroptosis（“铁死亡”）的特点。我们推测FXN减少导致神经元的丢失是铁死亡的结果。本项目在执行过程中发现当我们使用铁死亡的特定诱导剂和抑制剂处理来自FRDA病人的细胞时， 发现FRDA病人细胞并没有表现对erastin诱导的铁死亡更加敏感。这一结果动摇了我们的研究假说。由于本问题不是技术难题，而是一个科学现象，提示我们的研究课题不能完全按课题设计的技术路线进行，而是有所改变。改变1：研究FXN与低氧诱导因子的关系：.这部分的研究是基于我们在本课题中的一个新的发现：FXN缺失启动了细胞的低氧应答；.改变2：抗氧化剂硫辛酸LA对FXN缺失细胞的保护作用；这部分内容虽然原计划有所涉及，但当时的考虑是基于铁诱灭的立场考虑，现在就是基于LA的抗氧化功能角度进行；改变3：在本课题的支持下，开展了可用于弗里德赖氏共济失调（FRDA）疾病治疗相关的研究，通过使用靶向线粒体的药物SS-31处理FRDA病人细胞，结果发现不仅可提高细胞抵抗氧化应激的作用，特别值得关注的是SS-31提高了FXN蛋白的表达量，这从根本上具有治疗FRDA病人由于FXN减少造成的FRDA疾病的潜力；改变4：研究铁调节蛋白1和2的功能分化；改变5：转化医学相关的研究（比较杂）。