C9orf72 poly(glycine-alanine) knock-in mice exhibit mild rotarod and proteomic changes consistent with amyotrophic lateral sclerosis/frontotemporal dementia.

A GGGGCC repeat expansion in C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The repeat expansion is translated into five different dipeptide repeat proteins: poly(glycine-alanine) (polyGA), poly(glycine-proline) (polyGP), poly(glycine-arginine) (polyGR), poly(alanine-proline) (polyAP) and poly(proline-arginine) (polyPR). To investigate the effect of polyGA, which is the most abundant dipeptide repeat protein in patient brains, we used clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated nuclease 9 (Cas9) to insert 400 codon-optimized polyGA repeats immediately downstream of the mouse C9orf72 start codon. This generated (GA)400 knock-in mice driven by the endogenous mouse C9orf72 promoter, coupled with heterozygous C9orf72 reduction. PolyGA remains soluble up to 18 months of age and (GA)400 mice develop subtle dysfunction characterized by impaired rotarod performance, without overt neuropathological alterations. Quantitative proteomics revealed polyGA expression caused protein alterations in the spinal cord, including changes in previously identified polyGA interactors. Our findings show that (GA)400 mice are a complementary in vivo model to better understand C9orf72 ALS/FTD pathology and determine the specific role of individual DPRs in disease.