| Summary: | Frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) are neurodegenerative diseases with considerable clinical, genetic and pathological overlap. A GGGGCC hexanucleotide repeat expansion in a non-coding region of C9orf72 on chromosome 9 is the major cause of both FTLD and ALS. An understanding of the mechanisms through which the expansion leads to neurodegeneration will therefore be vital for development of novel therapeutics. There are 3 possible mechanisms through which the GGGGCC expansion may cause toxicity: (i) through haploinsufficiency of C9orf72, (ii) repetitive RNA transcripts arising from the expansion may be toxic or (iii) translation of the expansion may produce toxic peptides. Whilst the expansion is located in a non-coding region of the gene, long GC-rich RNA transcripts may be translated in the absence of an ATG start codon, through a process known as repeat-associated non-ATG translation (RAN-translation). 5 distinct dipeptide repeat proteins (DPRs) have been found to arise from the expansion through RAN-translation of the sense and antisense strands in all frames: poly-GA, -GR, -PR, -AP and –GP. All 5 DPRs have been shown to aggregate in patient tissue, indicating that they may play a role in C9FTLD/ALS pathogenesis. This project aimed to generate a series of models to investigate the mechanisms of neurodegeneration in C9FTLD/ALS, using C. elegans and cell culture. A transgenic worm strain which does not express the C. elegans orthologue of C9orf72 was first characterised. No impairments were observed in motility or life-span, demonstrating that loss of C9orf72 function does not cause an ALS-like phenotype in C. elegans. When considered alongside recent literature, this finding suggests that toxic gain of function mechanisms may be more important in C9FTLD/ALS pathogenesis. The impact of DPRs on cellular function was next investigated. Constructs were generated containing alternative codon sequences for each of the 5 DPRs, to express each peptide in the absence of the repetitive GGGGCC RNA sequences found in disease. A step-wise cloning strategy was employed to progressively increase repeat-length in these constructs until physiologically-relevant lengths of > 1000 repeats were obtained. DPRs were then expressed in HeLa cells, in order to assess the individual effects of each peptide on cellular function. Poly-GA formed large, star-shaped cytoplasmic inclusions which co-localised with ubiquilin-2 and p62, closely resembling the inclusions observed in patient tissue. This implies a potential role of proteasome dysfunction in C9FTLD/ALS. Conversely, the alanine-rich DPRs, poly-GR and –PR, translocated to the nucleolus, where poly-GR in particular caused nucleolar stress. Furthermore, nucleolar poly-GR caused loss of Cajal bodies from the nucleus, and a loss or mislocalisation of survival motor neuron protein (SMN). This is of particular interest to C9ALS, since loss of SMN is selectively toxic to motor neurons. Furthermore, loss of Cajal bodies and nucleolar stress are likely to cause defects in RNA processing, which may contribute to neurotoxicity.
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