Gain of Toxicity from ALS/FTD-Linked Repeat Expansions in C9ORF72 Is Alleviated by Antisense Oligonucleotides Targeting GGGGCC-Containing RNAs

Abstract

Hexanucleotide expansions in C9ORF72 are the most frequent genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Disease mechanisms were evaluated in mice expressing C9ORF72 RNAs with up to 450 GGGGCC repeats or with one or both C9orf72 alleles inactivated. Chronic 50% reduction of C9ORF72 did not provoke disease, while its absence produced splenomegaly, enlarged lymph nodes, and mild social interaction deficits, but not motor dysfunction. Hexanucleotide expansions caused age-, repeat-length-, and expression-level-dependent accumulation of RNA foci and dipeptide-repeat proteins synthesized by AUG-independent translation, accompanied by loss of hippocampal neurons, increased anxiety, and impaired cognitive function. Single-dose injection of antisense oligonucleotides (ASOs) that target repeat-containing RNAs but preserve levels of mRNAs encoding C9ORF72 produced sustained reductions in RNA foci and dipeptide-repeat proteins, and ameliorated behavioral deficits. These efforts identify gain of toxicity as a central disease mechanism caused by repeat-expanded C9ORF72 and establish the feasibility of ASO-mediated therapy.

Figure 1. Reduction of C9ORF72 is well tolerated, but complete loss of C9ORF72 induces premature death and splenomegaly

(A) Targeting strategy to generate C9orf72 null mice with exons 2–6 replaced with β-galactosidase and neomycin (Neo) genes. Schematic shows (upper panel) the genomic region, (middle panel) targeting construct, and (lower panel) final targeted C9orf72 gene. Filled boxes correspond to exons. Open box represents the 3′ untranslated region (3′-UTR). (B) Quantitative RT-PCR measurement of mouse C9orf72 RNAs in brains of C9orf72^+/+, C9orf72^+/− and C9orf72^−/− mice (n=6 per group). (C) Immunoblot demonstrating the levels of the 54 kD mouse C9ORF72 protein. GAPDH was used as a loading control. (D) Survival curve up to 600 days. (E) Body weights at 3, 6 and 12 months of age (n=25 per genotype). (F) Spleen sizes at 12 months of age. (G–I) Behavioral performance measured at 3, 6 and 12 months of age (n=25 animals per group). (G) Social interactions measured by the mean time spent with an object, in the center, or with a mouse. (H) Social recognition measured by the mean time spent with a familiar mouse, in the center, or with a novel mouse. (I) Hindlimb grip strength. (J) Average number of ChAT-positive neurons per section in the anterior horn of lumbar spinal cord in 12 month old mice (n=4–5 per group). (K) Resting electromyographic (EMG) recordings, and (L) myogenic motor evoked potentials (MMEPs) in 12 month old C9orf72 mice (n=5 per genotype) or in end-stage transgenic mice expressing mutant SOD1^G37R (crosshatched bars, n=2). Error bars represent s.e.m in biological replicates, n.s. not significant, * p<0.05, ** p<0.01 using one-way ANOVA. (See also Figure S1 and S2).

Figure 2. Generation of multiple BAC transgenic mouse lines expressing different levels of a human C9ORF72 transgene with 100 –700 GGGGCC repeats

(A) Schematic of the human BAC containing 450 GGGGCC repeats in the first intron of a truncated human C9ORF72 gene. The coordinates of the BAC sequence on the University of California Santa Cruz Genome Browser (Hg19) are indicated. No other gene is on the BAC. (B–D) Genomic DNA blot analysis of tail DNA from (B) founder (F0) and F1 transgenic mice from lines 9 or 15 and DNA from human fibroblasts (Hu) with normal C9ORF72 alleles, (C) twelve different mice of lines 1, 8, 10 and 11 (re-designated C9^450A, C9¹¹⁰, C9^450B and C9^450C, respectively), and (D) mice from F0, F2 and F5 generations in Line C9^450B. (E) Repeat lengths determined by genomic DNA blotting using DNA from the CNS and peripheral tissues of a C9^450B mouse. (F) Human C9ORF72 RNA in cortex of transgenic mice measured by qRT-PCR, normalized to C9^450A mice. Numbers above bars are repeat lengths measured by genomic DNA blots. (G) Expression levels of total (mouse plus human) C9ORF72 RNAs in the cortex of C9¹¹⁰, C9^450A, C9^450B and C9^450C mice normalized to the level of endogenous C9orf72 RNA. (H) Level of repeat-containing C9ORF72 RNA variants in the cortex measured by qRT-PCR and normalized to levels in C9^450A mice. (I–J) Levels of (I) total C9ORF72 RNAs (human plus mouse) or (J) repeat-containing C9ORF72 RNA measured by qRT-PCR in the cortex of heterozygous and homozygous C9^450C mice, normalized to C9orf72 levels in wild type littermates. Error bars represent s.e.m. from 3–5 biological replicates per group. (See also Figure S3).

Figure 3. Repeat size- and dose-dependent accumulation of sense and antisense RNA foci in C9ORF72 transgenic mice

(A) FISH detection of sense and antisense RNA foci (arrows) in 2 month old C9^450B mice. DNA was stained with DAPI. (B–E) Numbers of sense and antisense foci (per 100 nuclei) in hippocampal dentate gyrus of (B,C) 2 month old C9¹¹⁰, C9^450A and C9^450B mice, and (D,E) 6 month old heterozygous and homozygous C9^450C mice. Error bars represent s.e.m in 3–5 biological replicates. * p<0.05, ** p<0.01 using student t test. (F) Quantification of sense RNA foci per nucleus in hippocampal dentate gyrus of 6 month old heterozygous and homozygous C9^450C mice. (G) qRT-PCR measurement of human and mouse C9ORF72 RNAs in cortex of 2, 6 and 16 month old C9^450B mice, normalized to wild type littermates (n=2–4 per group). (H) Numbers of sense foci (per 100 nuclei) in hippocampal dentate gyrus of 2, 6 and 16 month old C9^450B mice. Error bars represent s.e.m in 2–4 biological replicates. n.s., not significant using one way ANOVA. (See also Figure S3).

Figure 4. Repeat size- and expression-dependent production of sense strand encoded DPR proteins is associated with age-dependent formation of cytoplasmic inclusions

(A) Poly(GA), poly(GP) and poly(GR) perinuclear aggregates (arrows) detected by immunohistochemistry in the retrosplenial cortex of 6 month old homozygous C9^450C mice. Nuclei were stained with haemalum. (B) Poly(GA) aggregates (arrows) in different CNS regions of 6 month old homozygous C9^450C mice. (C) Percent of cells containing poly(GA) or poly(GP) inclusions in frontal cortex (Ftx), hippocampus (Hip) and retrosplenial cortex (RSC) of 22 month old heterozygous C9^450C mice (n=2–4 biological replicates). (D) Aggregates positive for (green) poly(GP), (red) poly(GA) or (yellow) both, and (E) (Green) Poly(GP) and (red) P62-positive inclusions identified by immunofluorescence in retrosplenial cortex of 6 month old homozygous C9^450C mice. DNA is stained with DAPI. (F–H) Levels of poly(GP) soluble in 2% SDS measured by immunoassay in (F) cerebellum of 3 month old C9¹¹⁰, C9^450A and C9^450B mice, (G) cerebellum, cortex and spinal cord of 6 month old heterozygous and homozygous C9^450C mice, and (H) during aging in the cerebellum of C9^450A and C9^450B mice (n=2–5 biological replicates). (I) Poly(GA) aggregates (arrows) identified by immunohistochemistry in retrosplenial cortex of heterozygous or homozygous C9^450C mice or wild type mice at the noted ages. (J) (left panel) Percent of cells containing poly(GA) aggregates and (right panel) average size of poly(GA) inclusions in retrosplenial cortex of C9^450C mice (n=2–3 biological replicates). (K) Percent of cells with poly(GA) inclusions in retrosplenial cortex of heterozygous and homozygous C9^450C mice (n=3 per group). Error bars represent s.e.m. * p<0.05, ** p<0.01 using student t-test. (See also Figure S4).

Figure 5. No motor neuron loss and motor deficits in C9ORF72 transgenic mice with 450 repeats

(A) Motor performance on a Rotarod measured by latency to fall, (B) hindlimb grip strength, and (C) body weight of 4, 12 and 18 month old wild type (WT), C9^450B and C9^450C mice. (D) Choline Acetyltransferase (ChAT) positive motor neurons detected by immunofluorescence in the anterior horn of lumbar spinal cord of 12 month old WT, C9¹¹⁰ and C9^450C mice. (E) Average number of ChAT-positive neurons per section. Error bars represent s.e.m in n=4–5 animals per group. n.s., not significant using one way ANOVA. (See also Figure S5).

Figure 6. Age-dependent increased anxiety and impaired cognitive function in C9ORF72 mice with 450 repeats

(A–B) Behavioral performances in WT, C9^450B and C9^450C mice at 4, 12 and 18 months of age [n=25 mice per group at 4 months and n=16 (WT), n=14 (C9^450B) and n=10 (C9^450C) at 12 and 18 months of age]. (A) Spatial learning and memory performance on a Barnes maze showing the number of errors in finding the escape chamber at days 1–3, 4–6 and 7–9. (B) Working memory performance on a radial maze showing errors per trial over 10 days of testing. (C–D) Anxiety-related behaviors in WT, C9^450B and C9^450C male mice at 4, 12 and 18 months of age [n=11 (WT) and n=13 (C9^450B) at 4 months, and n=9 (WT), n=4 (C9^450B) and n=7 (C9^450C) at 12 and 18 months of age]. (C) Anxiety-related behavior determined by marble burying test showing the percent of marbles buried during a 20-minute trial, and (D) elevated plus maze showing the percent of time spent on the open arm. (E) Representative images and (F) quantification of DAPI-positive nuclei in the hippocampal dentate gyrus and CA1 region in 4 and 12 month old WT, C9^450B and C9^450C mice (n=4–5 per group). Error bars represent s.e.m, n.s., not significant, * p<0.05 and ** p<0.01 using one-way ANOVA. (See also Figure S6 and S7).

Figure 7. Sustained reduction in RNA foci and DPR proteins from a single dose of ASOs targeting C9ORF72 repeat-containing RNAs

(A) Schematic of injected ASOs targeting the sense strand C9ORF72 transcripts for degradation in 3 month old C9^450B mice. (B) Schematic of the C9ORF72 gene showing the GGGGCC repeats within the first intron, the two transcription initiation sites (arrows), the positions of five ASOs, and primers for detection (by qRT-PCR) of various C9ORF72 RNAs. (C–E) Expression of (C) repeating-containing, (D) total and (E) exon 1b-containing C9ORF72 RNAs determined by qRT-PCR in mice treated either with ASO1 targeting only the repeat-containing RNAs, ASO2 targeting all C9orf72 variants, or a control ASO. (F) Number of sense and antisense foci (per 100 nuclei) determined by FISH and (G) quantification of sense foci per nucleus in hippocampal dentate gyrus. (H–I) Levels of (H) poly(GP) or (I) poly(GA) in the cortex and spinal cord of C9^450B mice treated with ASO1, ASO2 or a control ASO, as measured by immunoassay. Error bars represent s.e.m in n=5–6 biological replicates. * p<0.05, ** p<0.01, n.s. not significant using one-way ANOVA. (See also Figure S8).

Figure 8. Single dose ASO treatment alleviates age-dependent behavioral deficits in C9ORF72 mice expressing 450 repeats

(A) Schematic of the experimental procedure for a single dose ASO targeting degradation of the sense strand C9ORF72 RNA in 9 month old C9^450B mice. (B) Expression of repeat-containing C9ORF72 RNAs was determined by qRT-PCR 2 weeks after injection of ASO5 or control ASO (n=6 per group). (C–D) Anxiety-related behaviors determined by (C) marble burying test and (D) elevated plus maze in 12 and 15 month old WT and C9^450B mice treated with either control ASO or ASO5 (n=5–7 per group). (E–F) Cognition-related behaviors determined on (E) a Barnes maze and (F) a radial maze in 12 and 15 month old WT and C9^450B mice treated with either control ASO or ASO5 (n=10–13 per group). (G) Expression of repeat-containing C9ORF72 RNAs and (H) levels of poly(GP) or poly(GA) proteins in the cortex of 16 month old C9^450B mice treated at 9 months with ASO5 or control ASO. Error bars represent s.e.m. Error bars represent s.e.m in biological replicates * p<0.05, ** p<0.01, n.s. not significant, using student t test.