Transcription elongation factor AFF2/FMR2 regulates expression of expanded GGGGCC repeat-containing C9ORF72 allele in ALS/FTD

Yeliz Yuva-Aydemir,Tania F Gendron,Sandra Almeida,Fen-Biao Gao,Gopinath Krishnan

doi:10.1038/s41467-019-13477-8

Abstract

Expanded GGGGCC (G4C2) repeats in C9ORF72 cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). How RNAs containing expanded G4C2 repeats are transcribed in human neurons is largely unknown. Here we describe a Drosophila model in which poly(GR) expression in adult neurons causes axonal and locomotor defects and premature death without apparent TDP-43 pathology. In an unbiased genetic screen, partial loss of Lilliputian (Lilli) activity strongly suppresses poly(GR) toxicity by specifically downregulating the transcription of GC-rich sequences in Drosophila. Knockout of AFF2/FMR2 (one of four mammalian homologues of Lilli) with CRISPR-Cas9 decreases the expression of the mutant C9ORF72 allele containing expanded G4C2 repeats and the levels of repeat RNA foci and dipeptide repeat proteins in cortical neurons derived from induced pluripotent stem cells of C9ORF72 patients, resulting in rescue of axonal degeneration and TDP-43 pathology. Thus, AFF2/FMR2 regulates the transcription and toxicity of expanded G4C2 repeats in human C9ORF72-ALS/FTD neurons.

Highlights

Expanded GGGGCC (G4C2) repeats in C9ORF72 cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD)
Relative expression of the variant 3 (V3)-specific G-allele was much lower in AFF2 knockout lines than in the respective parental lines (Fig. 7i). These results suggest that AFF2 preferentially regulates the transcription of the C9ORF72 allele containing expanded G4C2 repeats in patient neurons
We found that the percentage of induced pluripotent stem cells (iPSCs) containing RNA foci, and the average number of foci per cell, were significantly lower in AFF2 knockout lines than in the respective parental lines (Supplementary Fig. 15)

Summary

Introduction

Expanded GGGGCC (G4C2) repeats in C9ORF72 cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Knockout of AFF2/FMR2 (one of four mammalian homologues of Lilli) with CRISPR-Cas[9] decreases the expression of the mutant C9ORF72 allele containing expanded G4C2 repeats and the levels of repeat RNA foci and dipeptide repeat proteins in cortical neurons derived from induced pluripotent stem cells of C9ORF72 patients, resulting in rescue of axonal degeneration and TDP-43 pathology. In pull-down experiments, proteins that interact with arginine-containing DPR proteins include cytoplasmic and mitochondrial ribosomal proteins, as well as other RNA-binding proteins such as components of stress granules with low complexity domains, spliceosomes, and nucleoli[19,22,23,24,25,26,27] Consistent with these findings, poly(GR) has been linked to defects in nuclear cytoplasmic transport, translation, stress granule dynamics, nucleolar function, splicing, mitochondrial function, DNA damage response, and other cellular processes[1,2]

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Conclusion