Human C9ORF72 Hexanucleotide Expansion Reproduces RNA Foci and Dipeptide Repeat Proteins but Not Neurodegeneration in BAC Transgenic Mice

Abstract

Summary A non-coding hexanucleotide repeat expansion in the C9ORF72 gene is the most common mutation associated with familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Patients harboring this expansion develop several unique histopathological hallmarks, including intranuclear foci composed of either sense or antisense RNA transcripts from the expanded repeats and dipeptide repeat proteins generated by non-canonical translation of the expanded RNA transcripts. To further investigate the pathological role of C9ORF72 in these diseases, we generated a line of mice carrying a bacterial artificial chromosome containing exons 1 to 6 of the human C9ORF72 gene with approximately 500 repeats of the GGGGCC motif. The mice showed no overt behavioral phenotype but recapitulated distinctive histopathological features that are the hallmark of C9ORF72 ALS/FTD, including sense and antisense intranuclear RNA foci and poly(glycine-proline) dipeptides repeat proteins. Finally, using a synthetic microRNA that targets human C9ORF72 in cultures of primary cortical neurons from the C9BAC mice, we have attenuated expression of the C9BAC transgene and the poly(GP) dipeptides. The C9ORF72 BAC transgenic mice will be a valuable tool in the study of ALS/FTD pathobiology and therapy.

There has been an awakening: Emerging mechanisms of C9orf72 mutations in FTD/ALS

“…This mouse model recapitulates the cardinal features seen in human disease, including the accumulation of RNA foci transcribed from the sense strand of the GGGGCC repeat, production of RAN translation products (GP, GA, GR) from the sense strand, neuronal loss and astrogliosis, and behavioral and locomotor impairments (Chew et al, 2015). Strikingly, these mice also exhibit robust TDP-43 pathology, a key feature of c9FTD/ALS, not recapitulated in the BAC models (O’Rourke et al, 2015; Peters et al, 2015). Given the ease and reproducibility of this viral vector model, it can now be used, in a way similar to the fly experiments, to test relative roles of RNA and DPRs towards neurodegenerative phenotypes.…”

“…Another formal test of loss- vs. gain-of-function involves the use of C9orf72 knockout mice and some of the recently described viral-mediated and BAC transgenic c9FTD/ALS models (Chew et al, 2015; O’Rourke et al, 2015; Peters et al, 2015). These models employ expression of human C9orf72 transgenes harboring various GGGGCC repeat lengths either via adeno-associated virus mediated somatic transgenesis (Chew et al, 2015) or in transgenic mice generated from a bacterial artificial chromosomes (BAC) that expresses a fragment of human C9orf72 containing an expanded hexanucleotide repeat (O’Rourke et al, 2015; Peters et al, 2015) or the full length C9orf72 gene harboring an expanded repeat (O’Rourke et al, 2015).…”

“…These models employ expression of human C9orf72 transgenes harboring various GGGGCC repeat lengths either via adeno-associated virus mediated somatic transgenesis (Chew et al, 2015) or in transgenic mice generated from a bacterial artificial chromosomes (BAC) that expresses a fragment of human C9orf72 containing an expanded hexanucleotide repeat (O’Rourke et al, 2015; Peters et al, 2015) or the full length C9orf72 gene harboring an expanded repeat (O’Rourke et al, 2015). These mice exhibit various phenotypes and pathological features reminiscent of c9FTD/ALS (Chew et al, 2015; O’Rourke et al, 2015; Peters et al, 2015). Breeding these mice to C9orf72 knockout mice (heterozygous and homozygous) or injecting the C9orf72 transgene into the central nervous system of the knockout animals will test if disease features are or are not accelerated by the reduction of wild type C9orf72 (Figure 2B, C).…”

“…Bacterial artificial chromosomes (BAC) harboring various fragments (Peters et al, 2015) or the full-length (O’Rourke et al, 2015) human C9orf72 locus containing GGGGCC repeat expansions have been generated. All of these mice recapitulate pathological features, especially sense and antisense RNA foci and DPR production (O’Rourke et al, 2015; Peters et al, 2015). However, these mice do not seem to recapitulate the neurodegenerative disease features seen in ALS and FTLD, although future studies to analyze contributions of strain background and other factors are needed.…”

C9orf72 Protein Plasmatic Concentrations Are Similar between <b><i>C9ORF72</i></b> Expansion Carriers and Noncarriers in Frontotemporal Dementia

“…RNA aggregates or foci were found in different brain regions of C9ORF72 FTLD patients [19]. These foci might not cause a direct cellular dysfunction [20], but lead to a sequestration of RNA binding proteins and disrupt their normal activity [21, 22]. Finally, an unconventional translation of C9ORF72 expansions into specific polypeptides was discovered in 2013 [23].…”