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

使用斑马鱼模型阐明端粒损伤和非细胞自主性对弗里德赖希共济失调病理生理学的贡献

• 批准号: 10723485
• 负责人: ROBERT B WILSON
• 金额: $ 49.35万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10723485
• 关键词: Abnormal Endothelial Cell; Acceleration; Adult; Affect; Age Months; Antibodies; Arrhythmia; Autopsy; Behavioral; Biogenesis; Blood Cells; Brain; Cardiac; Cardiac Myocytes; Catalytic Domain; Cells; Central Artery; Cessation of life; Coculture Techniques; DNA Damage; DNA Polymerase III; DNA Repair; DNA biosynthesis; Data; Degenerative Disorder; Development; Disease; Drug Targeting; Endothelial Cells; Enzymes; European; Exhibits; Failure; Fibroblasts; Friedreich Ataxia; Functional disorder; Gene Expression Profiling; Genes; Genotype; Histologic; Human; Immunoprecipitation; Inherited; Inherited Spinocerebellar Degenerations; Iron; Length; Leukocytes; Lung; MAP Kinase Gene; Mass Spectrum Analysis; Mission; Mitochondria; Mitochondrial Matrix; Modeling; Mus; Mutation; Myocardial; N-terminal; National Heart, Lung, and Blood Institute; National Institute of Neurological Disorders and Stroke; Neuroglia; Neurologic; Neurons; Nuclear; Organ; Pathway interactions; Patients; Peptides; Phenotype; Population; Prevalence; Proteins; Public Health; Research; Single Strand Break Repair; Sulfur; Telomerase; Telomere Maintenance; Telomere Shortening; Testing; Tissues; Transgenic Organisms; Ubiquitin; United States National Institutes of Health; Wheelchairs; Zebrafish; autosome; biological adaptation to stress; cardiac vasculature; cell type; drug testing; experimental study; frataxin; helicase; in vivo; mouse model; mutant; p38 Mitogen Activated Protein Kinase; premature; promoter; repair enzyme; response; senescence; telomere; transgene expression

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.

Friedreich 共济失调 (FA) 是一种常染色体隐性遗传、神经和心脏退行性疾病，患病率 欧洲人口中约四万分之一。 FA 是由 FXN 基因隐性突变引起的，该基因编码 frataxin，一种参与铁硫簇（ISC）生物合成的蛋白质。 Frataxin 缺乏会影响线粒体 含有 ISC 的酶，以及线粒体外 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的平均值较短 端粒长度比正常对照组长。 DNA 损伤，尤其是关键的端粒缩短，与 衰老相关分泌表型（SASP），我们在 FA 中已经描述过。 DNA损伤激活 p38 MAPK 应激反应通​​路，我们发现该通路在原发性中过度激活 人类 FA 成纤维细胞和我们的 FA 斑马鱼模型中存在，但 FA 小鼠模型的细胞中没有。老鼠 端粒比人类端粒长 5-10 倍，这可以解释为什么当前的小鼠模型没有端粒 显着的心脏表型和神经表型是轻微的，需要几个月的时间才能形成。这 使用小鼠模型来研究端粒缩短对 FA 病理生理学的影响存在问题。 相比之下，斑马鱼具有人类长度的端粒，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非细胞 FA 的自主性，例如我们在人类 FA 细胞中观察到的明显的 SASP。更间接的形式 通过发现内皮细胞的显着异常，提示 FA 中的非细胞自主性 frataxin，特别是在 FA 患者的肺血管系统中。先前的研究发现内皮细胞 FA 患者心脏血管系统细胞异常，我们的初步数据显示显着 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 由内皮细胞中低 frataxin 引起的脉管系统疾病可能对 FA 病理有显着影响 生理。我们的具体目标是： 目标 1. 量化 FA 斑马鱼中的斑马鱼 frataxin (zFXN) 水平 模型。目标 2. 测试端粒损伤对 FA 病理生理学的影响。我们将建设 转基因斑马鱼品系，其中斑马鱼端粒酶基因 (Tert) 的表达由斑马鱼驱动 泛素启动子（ubi），我们将将此品系与我们的 zFXN 突变品系杂交并评估表型的逆转 类型。目标 3. 测试非细胞自主性对 FA 病理生理学的贡献。我们将建设 转基因斑马鱼品系，其中斑马鱼 frataxin 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 的表达由神经胶质细胞的启动子驱动 特定基因，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。