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

Children'S Hosp Of Philadelphia, Philadelphia PA

Investigators

Abstract

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.