Abstract
Friedreich ataxia (FA) is an autosomal recessive disease characterized by progressive damage to the nervous system and severe cardiac abnormalities. The disease is caused by a GAA•TTC triplet repeat expansion in the first intron of the FXN gene, which results in epigenetic repression of FXN transcription and reduction in FXN (frataxin) protein which results in mitochondrial dysfunction. Factors and pathways that promote FXN repression represent potential therapeutic targets whose inhibition would restore FXN transcription and frataxin protein levels. Here, we performed a candidate-based RNAi screen to identify kinases, a highly druggable class of proteins, that when knocked down upregulate FXN expression. Using this approach, we identified Rho kinase ROCK1 as a critical factor required for FXN repression. ShRNA-mediated knockdown of ROCK1, or the related kinase ROCK2, increases FXN mRNA and frataxin protein levels in FA patient-derived induced pluripotent stem cells (iPSCs) and differentiated neurons and cardiomyocytes to levels observed in normal cells. We demonstrate that small molecule ROCK inhibitors, including the FDA-approved drug belumosudil and fasudil, reactivate FXN expression in cultured FA iPSCs, neurons, cardiomyocytes, and FA patient primary fibroblasts, and ameliorate the characteristic mitochondrial defects in these cell types. Remarkably, treatment of transgenic FA mice of both sexes with belumosudil or fasudil upregulates FXN expression, ameliorates the mitochondrial defects in the brain and heart tissues, and improves motor coordination and muscle strength. Collectively, our study identifies ROCK kinases as critical repressors of FXN expression and provides preclinical evidence that FDA approved ROCK inhibitors may be repurposed for treatment of FA.
Significance Statement Friedreich ataxia is a debilitating disorder caused by epigenetic repression of the frataxin (FXN) gene, leading to neurodegeneration and cardiomyopathy. Through an RNA interference screen, we identified ROCK1 and ROCK2 kinases as critical repressors of FXN expression, making them promising therapeutic targets for upregulating FXN in patient-derived cells. Treatment with small-molecule ROCK inhibitors, including the FDA-approved drug belumosudil and clinically advanced fasudil, restores frataxin levels, alleviates mitochondrial defects, and improves disease phenotypes in cells and animal models. These findings establish ROCK kinases as targets for Friedreich ataxia therapy and open new avenues for repurposing existing ROCK inhibitors, warranting clinical exploration.
Footnotes
M.F. and M.R.G. are listed as inventors on a PCT application (WO2020047229A1) filed by the University of Massachusetts Chan Medical School describing methods that include inhibition of ROCK1/2 and other protein kinases to treat Friedreich ataxia. The remaining authors declare no competing interests.
We thank Marek Napierala for providing the isogenic pair of FA iPSC lines and FA patient fibroblasts, the UMass Chan Medical School RNAi Core Facility for providing shRNA libraries and clones, and Cole Haynes for assistance with experiments measuring mitochondrial oxygen consumption rate, Jonathan K Watts for assistance and sharing transgenic mice.
↵†These authors contribute equally to this work and share first authorship
