Background
The most frequent genetic cause of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) is the expansion of a GGGGCC hexanucleotide repeat in a non-coding region of the chromosome 9 open reading frame 72 (C9orf72) locus. The pathological hallmarks observed in C9orf72 repeat expansion carriers are the formation of RNA foci and deposition of dipeptide repeat (DPR) proteins derived from repeat associated non-ATG (RAN) translation. Currently, it is unclear whether formation of RNA foci, DPR translation products, or partial loss of C9orf72 predominantly drive neurotoxicity in vivo. By using a transgenic approach in zebrafish we address if the most frequently found DPR in human ALS/FTLD brain, the poly-Gly-Ala (poly-GA) protein, is toxic in vivo. Method: We generated several transgenic UAS responder lines that express either 80 repeats of GGGGCC alone, or together with a translation initiation ATG codon forcing the translation of GA80-GFP protein upon crossing to a Gal4 driver. The GGGGCC repeat and GA80 were fused to green fluorescent protein (GFP) lacking a start codon to monitor protein translation by GFP fluorescence.
Conclusion
These novel transgenic C9orf72 associated repeat zebrafish models demonstrate poly-GA toxicity in zebrafish. Reduction of poly-GA protein rescues toxicity validating this therapeutic approach to treat C9orf72 repeat expansion carriers. These novel animal models provide a valuable tool for drug discovery to reduce DPR associated toxicity in ALS/FTLD patients with C9orf72 repeat expansions.
Results
Zebrafish transgenic for the GGGGCC repeat lacking an ATG codon showed very mild toxicity in the absence of poly-GA. However, strong toxicity was induced upon ATG initiated expression of poly-GA, which was rescued by injection of an antisense morpholino interfering with start codon dependent poly-GA translation. This morpholino only interferes with GA80-GFP translation without affecting repeat transcription, indicating that the toxicity is derived from GA80-GFP.