32^nd European Neurology Congress

A hexanucleotide repeat expansion in the C9orf72 gene is the most common genetic cause of frontotemporal dementia and motor neuron disease. Carriers present with both conditions concurrently, so they are considered a continuum. Th e RNA from the hexanucleotide expansion is translated by repeat associated non-ATG (RAN) translation; producing five dipeptide repeat (DPR) proteins. These are the alanine rich polyAP and polyGA, the arginine-rich polyGR and polyPR, and polyGP. Previous in vitro studies show that the arginine-rich DPRs are toxic because they localise to the nucleolus, causing nucleolar stress. However, preliminary research showed sequestration of polyGR by polyGA when they were co-expressed in HeLa cells, Drosophila and human neurons. Our aim was to determine whether alanine-rich DPR proteins sequester arginine-rich DPR proteins when the two are co-expressed in vitro. This was investigated using alternative coding sequences for the DPR proteins, cloned into mCherrytagged plasmids. Next, two DPR proteins with different fl uorescent tags were co-transfected into HeLa cells and the subcellular locations of the DPR proteins were visualised using immunofluorescence. Our results showed cytoplasmic co-localisation of the arginine-rich DPRs with polyGA. This was replicated 3 times in HeLa cells and once in SH-SY5Y cells. Co-transfection, but no colocalisation, was seen when two alanine-rich or two argininerich DPR proteins were co-expressed. Overall, our findings suggest that the arginine-rich DPRs are sequestered by polyGA in the cytoplasm, meaning they are unlikely to cause nucleolar stress. This demonstrates that single transfections of DPR proteins may not be a good model to study DPR protein function or toxicity.