Elucidation of contributions of telomere damage and non-cell autonomy to the pathophysiology of Friedreich ataxia using a zebrafish model

Friedreich共济失调(FA)是一种常染色体隐性遗传的神经和心脏退行性疾病，患病率为 欧洲人口中每40,000人中就有1人死亡。FA由FXN基因的隐性突变引起，FXN基因编码 Frataxin，一种参与铁-硫簇(ISC)生物发生的蛋白质。Frataxin缺乏症影响线粒体 含有ISC的酶，以及线粒体外的ISC酶，包括参与DNA的酶 复制和修复，以及端粒维护。端粒损伤和/或缩短可能导致 FA病理生理学。FA患者的白细胞和小脑尸检组织平均较短 端粒长度高于正常对照组。DNA损伤，特别是关键的端粒缩短，与 一种衰老相关分泌表型(SASP)，我们在FA中描述过。DNA损伤被激活 P38MAPK应激反应通路，我们发现该通路在原发疾病中呈结构性过度激活 在人FA成纤维细胞和我们的FA斑马鱼模型中，但在FA小鼠模型的细胞中不存在。小白鼠 端粒是人类端粒的5-10倍，这可能解释了为什么目前的小鼠模型没有端粒。 明显的心脏表型和神经学表型，症状轻微，需要数月时间才能形成。这 使用小鼠模型研究端粒缩短对FA病理生理学的影响是有问题的。 相比之下，斑马鱼有人类长度的端粒，这使得低Frataxin对端粒的影响 在体内发育过程中表现出来。条件培养和共培养实验表明 FA病理生理学中非细胞自主性的组成部分。我们的初步数据也表明非细胞 FA的自主性，例如我们在人FA细胞中观察到的明显的SASP。一种更间接的形式 低密度脂蛋白内皮细胞明显异常提示FA非细胞自主性 Frataxin，特别是在FA患者的肺血管中。以前的研究发现内皮细胞- FA患者心脏血管中的细胞异常，我们的初步数据显示 在我们的FA斑马鱼模型中，大脑中央动脉的内皮细胞异常。这些结果表明 血管系统疾病，由内皮细胞中低Frataxin引起，可能在FA的发病机制中起重要作用。 生理学。我们的具体目标是：目标1.量化斑马鱼FA中的Frataxin(ZFXN)水平 模特们。目的2.探讨端粒损伤在FA病理生理学中的作用。我们将建造 斑马鱼端粒酶基因(TERT)表达受斑马鱼驱动的转基因斑马鱼品系 泛素启动子(Ubi)，我们将把这条线与我们的zFXN-突变体系交叉，并评估pho-1基因的逆转。 类型。目的3.探讨非细胞自主性在FA病理生理学中的作用。我们将建造 转基因斑马鱼品系中，斑马鱼Frataxin的表达由内皮细胞启动子驱动- 细胞特异性基因flk1，其中斑马鱼Frataxin的表达是由胶质细胞启动子驱动的。 特异性基因，GFAP。我们将把这些品系与我们的zFXN突变品系杂交，并评估表型的逆转。

Friedreich ataxia (FA) is an autosomal recessive, neuro- and cardio-degenerative disorder, with a prevalence of ~1 in 40,000 in European populations. FA is caused by recessive mutations in the FXN gene, which encodes frataxin, a protein involved in iron-sulfur-cluster (ISC) biogenesis. Frataxin deficiency affects mitochondrial ISC-containing enzymes, as well as extra-mitochondrial ISC enzymes, including enzymes involved in DNA replication and repair, and in telomere maintenance. Telomere damage and/or shortening likely contributes to FA pathophysiology. White blood cells and cerebellar autopsy tissue from FA patients have shorter average telomere lengths than normal controls. DNA damage, especially critical telomere shortening, is associated with a senescence associated secretion phenotype (SASP), which we have described in FA. DNA damage activates the p38 MAPK stress-response pathway, which we have found to be constitutively hyperactivated in primary human FA fibroblasts and in our FA zebrafish models, but not in cells from FA mouse models. Mouse telomeres are 5-10x longer than human telomeres, which may explain why current mouse models have no significant cardiac phenotype and neurologic phenotypes that are mild and take many months to develop. This makes problematic the use of mouse models to study the effects of telomere shortening on FA pathophysiology. In contrast, zebrafish have human-length telomeres, which allows the effects of low frataxin on telomeres to manifest over the course of in vivo development. Conditioned-media and co-culture experiments suggest a component of non-cell autonomy in FA pathophysiology. Our preliminary data also implicate non-cell autonomy in FA, for example the pronounced SASP we have observed in human FA cells. A more indirect form of non-cell autonomy in FA is suggested by the finding of significant abnormalities in endothelial cells with low frataxin, especially in the pulmonary vasculature of patients with FA. Previous studies identified endothelial- cell abnormalities in the cardiac vasculature of patients with FA, and our preliminary data show significant endothelial-cell abnormalities in the central artery of the brain in our FA zebrafish model. These results suggest that vasculature disease, caused by low frataxin in endothelial cells, may contribute significantly to FA patho- physiology. Our Specific Aims are: Aim 1. To quantify zebrafish frataxin (zFXN) levels in our FA zebrafish models. Aim 2. To test the contribution of telomere damage to the pathophysiology of FA. We will construct transgenic zebrafish lines in which zebrafish telomerase gene (Tert) expression is driven by the zebrafish ubiquitin promoter (ubi), and we will cross this line with our zFXN-mutant line and assess reversal of pheno- types. Aim 3. To test the contribution of non-cell autonomy to the pathophysiology of FA. We will construct transgenic zebrafish lines in which zebrafish frataxin expression is driven by the promoter of the endothelial- cell-specific gene, flk1, and in which zebrafish frataxin expression is driven by the promoter of the glial-cell- specific gene, gfap. We will cross these lines with our zFXN-mutant line and assess reversal of phenotypes.