The Mechanism Underlying RAN Translation-mediated Cytotoxicity In C9orf72-linked ALS/FTD

Aim: An expanded hexanucleotide GGGGCC repeat within the C9orf72 gene causes amyotrophic lateral sclerosis and frontotemporal dementia(C9FTD/ALS), but the molecular mechanism is unknown. It has been previously reported that through unconventional repeat-associated non-ATG(RAN) translation, sense and antisense transcripts of those repeats produce dipeptide-repeat proteins(referred to as RAN proteins). It is unclear whether RAN proteins induce neurotoxicity. In our study, we investigated whether toxicity induced by these repeat protein in neural cells and non-neural cells and explored the roles of them in the pathogenic mechanism of ALS and FTD.Methods: We construct plasmids containing 30 GGGGCC repeats that can express poly Gly-Ala(poly-GA) and poly glycine-proline(poly-GP) and poly glycine arginine(poly-GR) according to different reading frames respectively. Through the same method we generated the GFP-tagged constructs that encoded poly Gly-Ala( poly-GA) and poly glycine proline(poly-GP) and poly glycine arginine(poly-GR), poly proline arginine(poly-PR) and poly proline- alanine(poly-PA). Of note, all of the tag is located in the N terminal of these plasmids. We also generated GFP-tagged or FALG-tagged ‘synthetic’ constructs encoding RAN proteins that contained non-GGGGCC repeats, such as GGAGCA repeats encoding poly-GA and GGAAGA repeats encoding poly-GR. Similarly, the tag is also located in the N terminal of these plasmids. The synthetic constructs were predicted to lack the ability to produce RNA foci and other RAN proteins because they could not form putative hairpin structures[1]. All of the above-mentioned GFP-tagged constructs and a GFP-tagged construct containing a repeated GGGGCC sequence lacking an ATG(No ATG) were transfected into neural cells and non-neural cells to identify the toxicity of these RAN proteins. In order to further explore the mechanism of toxicity, we observed the location of the RAN proteins within the cell and effects on cell morphology. After transfection, we treated cells by the ubiquitin proteasome inhibitor MG132 and autophagy inhibitor Bafilomycin A1 to observe the effects of these proteins on ubiquitin-proteasome system(UPS) and autophagy pathway. We also evaluated the maturation rRNA in RAN protein expressing cells by Quantitative Real-time PCR. To further insight into the abnormal nucleolar changes induced by arginine-enriched RAN proteins, we identify the binding partners of the arginine-enriched RAN proteins through mass spectrometry-based proteomic analysis. The effects of these RAN proteins to stress granule formation in HEK293 cells were also observed by means of immunofluorescence.Results: In our cell model, we found that all five types of RAN proteins are not prone to aggregate and the turnover of these RAN proteins was not regulated by the ubiquitin–proteasome system or autophagy. In addition, only poly-GR and poly-PR located in the nucleolus causing critical nucleoprotein B23(nucleophosmin) transported from the nucleolus to nucleoplasm, leading to nucleolar stress and cell death.Conclusion: The results of the study suggest a simple model of the molecular mechanisms underlying RAN translation-mediated cytotoxicity in C9orf72-linked ALS/FTD in which nucleolar stress, but not protein aggregation, is the primary contributor to C9orf72-linked neurodegeneration.