Abstract
Friedreich’s ataxia (FRDA) is a genetic neurodegenerative disease that is caused by guanine-adenine-adenine (GAA) nucleotide repeat expansions in the first intron of the frataxin (FXN) gene. Although present in the intron, this mutation leads to a substantial decrease in protein expression. Currently, no effective treatment is available for FRDA, and, in addition to FXN, other targets with therapeutic potential are continuously sought. As miRNAs can regulate the expression of a broad spectrum of genes, are used as biomarkers, and can serve as therapeutic tools, we decided to identify and characterize differentially expressed miRNAs and their targets in FRDA cells compared to unaffected control (CTRL) cells. In this study, we performed an integrated miRNAseq and RNAseq analysis using the same cohort of primary FRDA and CTRL cells. The results of the transcriptome studies were supported by bioinformatic analyses and validated by qRT-PCR. miRNA interactions with target genes were assessed by luciferase assays, qRT-PCR, and immunoblotting. In silico analysis identified the FXN transcript as a target of five miRNAs upregulated in FRDA cells. Further studies confirmed that miRNA-224-5p indeed targets FXN, resulting in decreases in mRNA and protein levels. We also validated the ability of miRNA-10a-5p to bind and regulate the levels of brain-derived neurotrophic factor (BDNF), an important modulator of neuronal growth. We observed a significant decrease in the levels of miRNA-10a-5p and increase in the levels of BDNF upon correction of FRDA cells via zinc-finger nuclease (ZFN)-mediated excision of expanded GAA repeats. Our comprehensive transcriptome analyses identified miRNA-224-5p and miRNA-10a-5p as negative regulators of the FXN and BDNF expression, respectively. These results emphasize not only the importance of miRNAs in the pathogenesis of FRDA but also their potential as therapeutic targets for this disease.
Highlights
Friedreich’s ataxia (FRDA, FA) is an autosomal recessive neurodegenerative disease that affects about 15,000 people worldwide, making it the most common inherited ataxia [1,2,3,4]
The analysis was performed on primary fibroblasts from both FRDA and non-disease carriers, CTRL, each group consisting of 15 cell lines, which were deposited in our laboratory [34]
We conducted a comparison of the FRDA transcriptome by integrating the results of mRNA and miRNA sequencing experiments performed using a set of well characterized primary FRDA and CTRL fibroblast lines
Summary
Friedreich’s ataxia (FRDA, FA) is an autosomal recessive neurodegenerative disease that affects about 15,000 people worldwide, making it the most common inherited ataxia [1,2,3,4]. The vast majority of FRDA patients are homozygous for expanded GAA trinucleotide repeats in the first intron of the frataxin (FXN) gene. FXN deficiency disturbs intracellular iron metabolism [7] but, even more importantly, has broad cellular consequences via affecting the functions of numerous proteins requiring Fe-S clusters as prosthetic groups [8]. Processes such as mitochondrial respiration, energy metabolism, and DNA replication or repair are affected by decreased levels of FXN [9, 10]. The age of disease onset varies among patients and inversely correlates with GAA expansion length [13]
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