C9ORF72 DIPEPTIDE REPEAT PROTEINS DISRUPT NUCLEOCYTOPLASMIC TRANSPORT

Abstract

Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are two debilitating neurodegenerative disorders with considerable clinical, genetic and pathological overlap. A hexanucleotide repeat expansion in a non-coding region of C9orf72 has been identified as the most common cause of both FTD and ALS. However, the pathogenesis of FTD/ALS remains unclear, and there are currently no effective treatments available. Despite its location in a non-coding region of the gene, the C9orf72 expansion is translated to produce five different dipeptide repeat proteins (DPRs) in patient tissue. This is known as repeat-associated non-ATG translation (RAN-translation), a phenomenon which allows repetitive RNA transcripts to be translated in the absence of a start codon. Immunohistochemical studies have shown that these peptides form intraneuronal inclusions in patient tissue. Since the expansion is only translated in disease, dipeptides arising from it are completely novel and their impact on neuronal function is unclear. However, recent studies in cell culture and Drosophila models suggest that DPRs are toxic, and may contribute to neurodegeneration in C9orf72-linked FTD/ALS. We have used alternative codons in a randomised fashion to generate less repetitive DNA sequences coding for each DPR. This allows us to express each peptide in human cell culture separately, without production of the repetitive RNA sequences found in disease. Importantly, our constructs express the DPRs at physiologically-relevant repeat-lengths of over 1000 units, and several length-dependent differences have been noted in the distribution and toxicity of each DPR. In our cellular models, three of the five DPRs (poly-GA, poly-GR and poly-PR) caused severe disruption to the nuclear membrane integrity, with breakages and gaps observed in lamin staining around the nucleus. In addition, nuclei were frequently misshapen or exhibited a blebbing phenotype. Furthermore, key nuclear proteins such as FUS were mislocalised to the cytoplasm in cells expressing poly-GA and poly-GR. Taken together, our findings suggest that DPRs cause defects in nucleocytoplasmic transport of proteins and perhaps mRNA species, which may have a variety of downstream consequences contributing to neuronal dysfunction and death in FTD/ALS.