Friedreich's ataxia (FRDA) is caused by the expansion of a trinucleotide repeat located in the first intron of the Frataxin (FXN) gene, which leads to reduced FXN gene transcription. The extent of Frataxin reduction depends on the GAA repeat length. The GAA repeats continue to expand in FRDA patients, aggravating symptoms and contributing to disease progression. The mechanism for repeat expansion is not clearly understood. It is proposed that an altered DNA replication program and replication fork stalling at secondary repeat structures could cause DNA polymerase slippage and repeat expansion. However, it remains unclear whether secondary repeat structures are formed in vivo at the endogenous FXN gene locus in patients. Furthermore it is unknown whether an altered replication program plays a role in repeat expansion in FRDA cells. Using a unique approach, which monitors the DNA replication in single DNA molecules by multi-color immunofluorescence microscopy (SMARD), our preliminary results show inaccuracies in the DNA replication process in FRDA cells. The goals of this proposal are to determine the cause of replication fork stalling at the expanded GAA repeats and to release the replication fork block to stabilize the repeats in FRDA cells.
These aims will address several fundamental aspects of the molecular mechanisms causing repeat expansion in FRDA patients. Can replication be detected in differentiate cells such as cardiomyocytes and if yes, is the replication program altered and can the replication program be corrected at the endogenous FXN locus in FRDA cells? Does the replication fork stall at the proposed secondary repeat structures at the endogenous disease locus in FRDA cells in vivo? Can small molecules release the stalled replication forks? In order to answer these questions we will use the newly established FRDA induced pluripotent stem cells (iPSCs) that contain expanding GAA repeats. FRDA iPSCs can be differentiated to neuronal cell and cardiomyocytes; which makes these cells a perfect tool to study the reason for the repeat instability and decrease FXN transcription in undifferentiated and differentiated FRDA cells. We will employ SMARD to determine the DNA replication at the endogenous disease locus in FRDA cells. In addition, we will test whether small molecules can release the replication fork stall and stabilize the GAA repeats in FRDA cells. Understanding how repeat expansion occurs in the native chromosomal context in FRDA cells is an important prerequisite for the development of effective therapeutic treatments for FRDA.

Public Health Relevance

Friedreich's ataxia, the most common form of inherited ataxia in Caucasians, for which there is yet no cure, is a severe neurological disease caused by a trinucleotide repeat expansion. The proposed studies will determine errors in the DNA replication, which drive GAA repeat expansion, and identify components, which could prevent repeat expansion and improve the disease pathology in Friedreich's ataxia cells. This work will uncover new avenues for therapy for Friedreich's ataxia patients. !

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Small Research Grants (R03)
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Molecular Neurogenetics Study Section (MNG)
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Gubitz, Amelie
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Weill Medical College of Cornell University
Obstetrics & Gynecology
Schools of Medicine
New York
United States
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