RAN Translation of the Expanded CAG Repeats in the SCA3 Disease Context.

Spinocerebellar ataxia type 3 (SCA3) is a progressive neurodegenerative disorder caused by a CAG repeat expansion in the ATXN3 gene encoding the ataxin-3 protein. Despite extensive research the exact pathogenic mechanisms of SCA3 are still not understood in depth. In the present study, to gain insight into the toxicity induced by the expanded CAG repeats in SCA3, we comprehensively investigated repeat-associated non-ATG (RAN) translation in various cellular models expressing translated or non-canonically translated ATXN3 sequences with an increasing number of CAG repeats. We demonstrate that two SCA3 RAN proteins, polyglutamine (polyQ) and polyalanine (polyA), are found only in the case of CAG repeats of pathogenic length. Despite having distinct cellular localization, RAN polyQ and RAN polyA proteins are very often coexpressed in the same cell, impairing nuclear integrity and inducing apoptosis. We provide for the first time mechanistic insights into SCA3 RAN translation indicating that ATXN3 sequences surrounding the repeat region have an impact on SCA3 RAN translation initiation and efficiency. We revealed that RAN translation of polyQ proteins starts at non-cognate codons upstream of the CAG repeats, whereas RAN polyA proteins are likely translated within repeats. Furthermore, integrated stress response activation enhances SCA3 RAN translation. Our findings suggest that the ATXN3 sequence context plays an important role in triggering SCA3 RAN translation and that SCA3 RAN proteins may cause cellular toxicity.

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

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.

Oleic acid increases the transcriptional activity of FoxO1 by promoting its nuclear translocation and ß-catenin binding in pancreatic ß-cells.

In the setting of metabolic overload, chronic elevations of free fatty acids in blood and tissues are associated with pancreatic ß-cell lipotoxicity and failure. Ultimately, obesity combined with insulin resistance increases the dysfunctional demand of ß-cells and contributes to the development of type 2 diabetes. Forkhead box O1 (FoxO1) is a potent transcriptional regulator of pancreatic ß-cell function and tolerance to lipid stress. The present study examined the effects of stearoyl-CoA desaturase 1 (SCD1)-metabolized precursors and products, notably oleic acid, on the compensatory capacity of ß-cells and their relationship with regulation of the FoxO1 and Wnt pathways. The trioleate-induced compromise of insulin sensitivity blunted the compensatory response of pancreatic ß-cells in primary rat islets. These events were associated with increases in the nuclear accumulation and transcriptional activity of FoxO1. Such effects were also observed in INS-1E cells that were subjected to oleate treatment. The overexpression of human SCD1 that was accompanied by endogenously generated oleic acid also led to an increase in the nuclear abundance of FoxO1. The mechanism of the oleate-mediated subcellular localization of FoxO1 was independent of the fatty acid receptor GPR40. Instead, the mechanism involved diversion of the active ß-catenin pool from an interaction with transcription factor 7-like 2 toward FoxO1-mediated transcription in ß-cells. Our findings identify a unique role for oleic acid in the compensatory response of pancreatic ß-cells and emphasize the importance of FoxO1 in ß-cell failure in obesity-induced insulin resistance.