Knock in of a hexanucleotide repeat expansion in the C9orf72 gene induces ALS in rats

Background: The GGGGCC(G4 C2) repeat expansion in the human open reading frame 72 on chromosome 9, C9 orf72, is the most common cause of amyotrophic lateral sclerosis(ALS). Studies in transgenic mouse models have linked the pathogenic mechanism of G4 C2 repeat expansion to RNA foci or the accumulation of unnatural dipeptide repeats in neurons. However, only one of the existing transgenic mouse lines developed typical ALS.Methods: C9 orf72 knockin rats were generated by knockin of 80 G4 C2 repeats with human flanking fragments within exon1 a and exon1 b at the rat C9 orf72 locus. Protein expression was detected by western blot. Motor coordination and grip force were measured using a Rotarod test and a grip strength test. Neurodegeneration was assessed by Nissl staining with cresyl violet.Results: C9 orf72 haploinsufficiency reduced C9 orf72 protein expression 40% in the cerebrum, cerebellum and spinal cords from knockin rats(P <.05). The knockin(KI) rats developed motor deficits from 4 months of age. Their falling latencies and grip force were decreased by 67%(P <.01) and 44%(P <.01), respectively, at 12 months of age compared to wild-type(WT) mice. The knockin of the hexanucleotide repeat expansion(HRE) caused a 47% loss of motor neurons in the spinal cord(P <.001) and 25%(5/20) of female KI rats developed hind limb paralysis at 13 to 24 months.Conclusion: Motor defects in KI rats may result from neurotoxicity caused by HRE and the resulting reduction in C9 orf72 protein due to haploinsufficiency. These KI rats could be a useful model for investigating the contributions of loss-of-function to neurotoxicity in C9 orf72-related ALS.