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

弗里德赖希共济失调（FA）是一种常染色体隐性遗传的神经和心脏退行性疾病， 在欧洲人口中约为1/40，000。FA是由FXN基因的隐性突变引起的，FXN基因编码 frataxin是一种参与铁硫簇（ISC）生物发生的蛋白质。Frataxin缺乏影响线粒体 含ISC的酶，以及线粒体外ISC酶，包括参与DNA合成的酶。 复制和修复，以及端粒的维护。端粒损伤和/或缩短可能有助于 FA病理生理学。FA患者的白色血细胞和小脑尸检组织的平均值较短， 端粒长度比正常对照组长。DNA损伤，特别是关键的端粒缩短，与 衰老相关分泌表型（SASP），我们已经在FA中描述过。DNA损伤激活 p38 MAPK应激反应途径，我们发现在原发性肝癌中是组成性过度激活的。 人FA成纤维细胞和我们的FA斑马鱼模型中，但不是在来自FA小鼠模型的细胞中。鼠标 端粒比人类端粒长5 - 10倍，这可以解释为什么目前的小鼠模型没有 显著的心脏表型和神经表型，这些表型是轻微的，需要数月才能形成。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。条件培养基和共培养实验表明， FA病理生理学中非细胞自主性的组成部分。我们的初步数据还表明， FA中的自主性，例如我们在人FA细胞中观察到的明显的SASP。更间接的形式 FA中的非细胞自主性是通过发现内皮细胞的显著异常而提出的， 在FA患者的肺血管中，尤其是在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，脑中央动脉的内皮细胞异常。这些结果表明 血管系统疾病，由内皮细胞中的低共济失调蛋白引起，可能显著促进FA病理性， physiology.我们的具体目标是：目标1。为了量化我们的FA斑马鱼中的斑马鱼共济失调蛋白（zFXN）水平， 模型目标2.检测端粒损伤在FA病理生理学中的作用。了建设一 转基因斑马鱼系，其中斑马鱼端粒酶基因（Tert）表达由斑马鱼驱动 泛素启动子（ubi），我们将这个线与我们的zFXN突变体线交叉，并评估表型逆转。 类型目标3。检测非细胞自主性对FA病理生理学的贡献。了建设一 转基因斑马鱼系，其中斑马鱼共济失调蛋白的表达由内皮细胞的启动子驱动， 细胞特异性基因flk1，并且其中斑马鱼共济失调蛋白的表达由神经胶质细胞的启动子驱动， 特异性基因gfap。我们将这些品系与我们的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.