A Comprehensive Transcriptome Analysis Identifies FXN and BDNF as Novel Targets of miRNAs in Friedreich’s Ataxia Patients

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.

Targeting 3′ and 5′ untranslated regions with antisense oligonucleotides to stabilize frataxin mRNA and increase protein expression

“…A plausible alternative, at least for ONs targeting the 3′ UTR, would be interference with miRNAs regulating the FXN transcript. However, analysis of our comprehensive miRNA-seq data from FRDA and control fibroblasts ( 45 ) indicates that none of the miRNAs expressed in fibroblasts is likely to compete with ET3 or ET4 for binding of the FXN transcript 3′ UTR. Further studies designed to dissect mechanism(s) responsible for extension of the mRNA half-life may result in development of more efficient strategies.…”

“…Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Cat# 10074) and treated with DNase I (TURBO DNA-free; Thermo Fisher, Cat# AM1907) for 1 h according to the manufacturer’s protocol. The qRT-PCR reactions were assembled using the Power SYBR Green RNA-to-CT 1-Step Kit (Thermo Fisher, Cat# 4391178) and run on a Step-One Plus System (Applied Biosystems) as we described in ( 41 , 45 ). Reverse transcription was conducted at 48°C for 30 min, followed by 40 cycles of denaturation at 95°C for 15 s, annealing at 55°C for 20 s and elongation for 1 min at 60°C.…”

Recent Advances on the Role of Brain-Derived Neurotrophic Factor (BDNF) in Neurodegenerative Diseases

“…This suggests that the SCA6 pathogenic mechanism is associated with the abnormal localization of BDNF proteins and a reduction in BDNF mRNA expression. A comprehensive transcriptome analysis in Friedreich’s ataxia (FRDA) patients, a hereditary neurodegenerative disease characterized by guanine-adenine-adenine (GAA) nucleotide repeat expansion in the first intron of the frataxin (FXN) gene, identified BDNF and FXN as novel targets of miRNAs [ 137 ].…”

Future Prospects of Gene Therapy for Friedreich’s Ataxia

“…Furthermore, BDNF is secreted, which implies that not only the transduced cells will increase their BDNF levels, but also the neighboring cells. Moreover, it was recently demonstrated that an miRNA targeting BDNF is increased in fibroblasts from FA patients and that, coherently, BDNF levels are reduced [ 98 ]. Therefore, BDNF is a potential candidate for the treatment of neurodegenerative diseases, and especially for FA.…”