C9orf72 BAC Transgenic Mice Display Typical Pathologic Features of ALS/FTD

Abstract

Summary Noncoding expansions of a hexanucleotide repeat (GGGGCC) in the C9orf72 gene are the most common cause of familial amyotrophic lateral sclerosis and frontotemporal dementia. Here we report transgenic mice carrying a bacterial artificial chromosome (BAC) containing the full human C9orf72 gene with either a normal allele (15 repeats) or disease-associated expansion (~100-1000 repeats; C9-BACexp). C9-BACexp mice displayed pathologic features seen in C9orf72 expansion patients, including widespread RNA foci and repeat associated non-ATG (RAN) translated dipeptides, which were suppressed by antisense oligonucleotides targeting human C9orf72. Nucleolin distribution was altered supporting that either C9orf72 transcripts or RAN dipeptides promote nucleolar dysfunction. Despite early and widespread production of RNA foci and RAN dipeptides in C9-BACexp mice, behavioral abnormalities and neurodegeneration were not observed even at advanced ages, supporting the hypothesis that RNA foci and RAN dipeptides occur presymptomatically, and are not sufficient to drive neurodegeneration in mice at levels seen in patients.

Haploinsufficiency leads to neurodegeneration in C9ORF72 ALS/FTD human induced motor neurons

“…Although it was not previously reported 58 , we observed significantly higher levels of GR+ punctae in hippocampal neurons in C9-BAC mice than controls (Fig. 6j) using a previously-validated poly(GR) antibody 11 .…”

“…After permeabilization, the samples were equilibrated in 1x SSC buffer for 10 min at room temperature and then transferred into 50% formamide (2x SSC) for 10 min at 60°C. The repeat expansion-targeting probe and the negative control probe were ordered from Exiqon 58 . During this step, the probe mixture (1µl salmon sperm (10 µg/µl), 0.5 µl E. coli tRNA (20 µg/µl), 0.4 µl probe (25 µM), 25 µl 80% formamide/per sample) was made and placed at 95°C for at least 10 min.…”

“…To determine if Pikfyve inhibition rescues gain-of-function processes in vivo , we measured DPR levels in C9-BAC transgenic mice 58 with or without Apilimod treatment. Although it was not previously reported 58 , we observed significantly higher levels of GR+ punctae in hippocampal neurons in C9-BAC mice than controls (Fig.…”

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

“…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).…”

Genetics insight into the amyotrophic lateral sclerosis/frontotemporal dementia spectrum

“…GGGGCC repeats and some DPRs colocalise in the nucleolus, the site where rRNAs are processed and assembled 54. Nucleolar dysfunction has been reported in patients and several models (figure 2),54 55 although the specific mechanisms mediating this toxicity are unknown at this time.…”