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

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 polyglycine-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.

High content live profiling reveals concomitant gain and loss of function pathomechanisms in C9ORF72 amyotrophic lateral sclerosis

“…In this study, we wished to compare HRE C9ORF72 phenotypically and mechanistically against mutant FUS and TDP43, all of which are common genetic aberrations causing ALS (6,7). We sought to combine LOF of C9ORF72 with HRE-mediated GOF in a meaningful manner with no overexpression artifacts to clarify the role of both debated mechanisms (15,16,26,27). In contrast to FUS-and TDP43 ALS, proximal in parallel to distal axonal trafficking deficits were the hallmarks of C9ORF72 pathology in hiPSC- such as aggregate depositions along with altered nucleo-cytosolic shuttling of disease mediators (5,41), suppression of neuroprotective HSP induction (42,43), impaired DNA damage response and repair (5,44,45).…”

“…Control cells did only show traces of GP, GA and either raised the question whether either type of perturbation concurred in the same neuron or in two distinct parts of the population. This presents an important aspect of C9ORF72 pathology as the causative GOF of elevated DPR expression in DNA damage is hotly debated (26,27). As for the neuritic GP foci, we were unable to assign affected neurites…”

“…There is extensive discussion about the pathomechanisms of C9ORF72 HRE including mainly gain of toxic functions (GOF) (26,27) versus loss of C9ORF72 functions (LOF) (18,20). Thus, we wished to investigate the role of GOF and LOF mechanisms in HREmediated trafficking defects.…”

“…Subsequently, the discovery of non-canonical RAN-translation of neurotoxic DPRs from intronic HREs (24,25) led to the hypothesis of a DPR-mediated GOF (22). Specifically, DPRs are translated bidirectionally from both the sense and antisense HRE-RNA transcripts resulting in a whole spectrum of DPR variants, amongst them more abundant poly glycine-alanine (GA) and poly glycine-proline (GP) (26,27).…”

“…DPR expression confers its toxic GOF presumably through the formation of inclusion bodies which sequester phosphorylated Ataxia Telangiectasia Mutated (pATM), a key player of DNA damage response, and heterogeneous ribonucleoprotein (hnRNP) A3, normally limiting DPR expression (26,27). The resultant DNA damage accumulation eventually leads to neurodegeneration.…”