C9orf72 dipeptides disrupt the nucleocytoplasmic transport machinery and cause TDP-43 mislocalisation to the cytoplasm.

A repeat expansion in C9orf72 is the major cause of both frontotemporal dementia and amyotrophic lateral sclerosis, accounting for approximately 1 in 12 cases of either disease. The expansion is translated to produce five dipeptide repeat proteins (DPRs) which aggregate in patient brain and are toxic in numerous models, though the mechanisms underlying this toxicity are poorly understood. Recent studies highlight nucleocytoplasmic transport impairments as a potential mechanism underlying neurodegeneration in C9orf72-linked disease, although the contribution of DPRs to this remains unclear. We expressed DPRs in HeLa cells, in the absence of repeat RNA. Crucially, we expressed DPRs at repeat-lengths found in patients (> 1000 units), ensuring our findings were relevant to disease. Immunofluorescence imaging was used to investigate the impact of each DPR on the nucleus, nucleocytoplasmic transport machinery and TDP-43 localisation. DPRs impaired the structural integrity of the nucleus, causing nuclear membrane disruption and misshapen nuclei. Ran and RanGAP, two proteins required for nucleocytoplasmic transport, were also mislocalised in DPR-expressing cells. Furthermore, DPRs triggered mislocalisation of TDP-43 to the cytoplasm, and this occurred in the same cells as Ran and RanGAP mislocalisation, suggesting a potential link between DPRs, nucleocytoplasmic transport impairments and TDP-43 pathology.

CRISPR/Cas9 does not facilitate stable expression of long C9orf72 dipeptides in mice.

A C9orf72 repeat expansion is the most common cause of both frontotemporal dementia and motor neuron disease. The expansion is translated to produce dipeptide repeat proteins (DPRs), which are toxic in vivo and in vitro. However, the mechanisms underlying DPR toxicity remain unclear. Mouse models which express DPRs at repeat lengths found in human disease are urgently required to investigate this. We aimed to generate transgenic mice expressing DPRs at repeat lengths of >1000 using alternative codon sequences, to reduce the repetitive nature of the insert. We found that although these inserts did integrate into the mouse genome, the alternative codon sequences did not protect from instability between generations. Our findings suggest that stable integration of long DPR sequences may not be possible. Administration of viral vectors after birth may be a more effective delivery method for long repeats.