Poly-glycine\u2013alanine exacerbates C9orf72 repeat expansion-mediated DNA damage via sequestration of phosphorylated ATM and loss of nuclear hnRNPA3

Yoshihiro Nihei,Julia Japtok,Andreas Hermann,Kohji Mori,Georg Werner,Barham Khosravi,Christian Haass,Qihui Zhou,Brigitte Nuscher,Frits Kamp,Dieter Edbauer,Andreas Sommacal,Markus Weber,Thomas Arzberger

doi:10.1007/s00401-019-02082-0

Abstract

Repeat expansion in C9orf72 causes amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Expanded sense and antisense repeat RNA transcripts in C9orf72 are translated into five dipeptide-repeat proteins (DPRs) in an AUG-independent manner. We previously identified the heterogeneous ribonucleoprotein (hnRNP) A3 as an interactor of the sense repeat RNA that reduces its translation into DPRs. Furthermore, we found that hnRNPA3 is depleted from the nucleus and partially mislocalized to cytoplasmic poly-GA inclusions in C9orf72 patients, suggesting that poly-GA sequesters hnRNPA3 within the cytoplasm. We now demonstrate that hnRNPA3 also binds to the antisense repeat RNA. Both DPR production and deposition from sense and antisense RNA repeats are increased upon hnRNPA3 reduction. All DPRs induced DNA double strand breaks (DSB), which was further enhanced upon reduction of hnRNPA3. Poly-glycine–arginine and poly-proline-arginine increased foci formed by phosphorylated Ataxia Telangiectasia Mutated (pATM), a major sensor of DSBs, whereas poly-glycine–alanine (poly-GA) evoked a reduction of pATM foci. In dentate gyri of C9orf72 patients, lower nuclear hnRNPA3 levels were associated with increased DNA damage. Moreover, enhanced poly-GA deposition correlated with reduced pATM foci. Since cytoplasmic pATM deposits partially colocalized with poly-GA deposits, these results suggest that poly-GA, the most frequent DPR observed in C9orf72 patients, differentially causes DNA damage and that poly-GA selectively sequesters pATM in the cytoplasm inhibiting its recruitment to sites of DNA damage. Thus, mislocalization of nuclear hnRNPA3 caused by poly-GA leads to increased poly-GA production, which partially depletes pATM, and consequently enhances DSB.

Highlights

C9orf72 repeat expansion is the most common cause of autosomal dominant FTLD, FTLD/ALS, and ALS [14, 20, 50]
We investigated the association of hnRNPA3 expression, RNA foci formation, dipeptide-repeat proteins (DPRs) production, and DNA damage in cultured cells, including patient-derived human neurons and brains of C9orf72 carriers
We investigated if hnRNPA3 binds to the antisense repeat RNA and if reduction of hnRNPA3 increases the number of antisense repeat RNA foci

Summary

Introduction

C9orf repeat expansion is the most common cause of autosomal dominant FTLD, FTLD/ALS, and ALS [14, 20, 50]. Extended author information available on the last page of the article even thousands of repeats [50]. Sense and antisense repeat RNAs accumulate within intranuclear RNA foci [14]. Sense and antisense transcripts are translated in all reading frames into dipeptide-repeat proteins (DPRs) in an AUG-independent manner [2, 43]. Accumulating evidence suggests that neurotoxicity occurs via various cellular pathways, such as RNA mis-splicing and reduced transcription of the C9orf gene [27, 29], nucleocytoplasmic transport dysfunction [19, 28, 65, 66], nucleolar stress [23, 38, 60], and DNA damage [15, 32, 58]

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Conclusion