RAN proteins and RNA foci from antisense transcripts in C9ORF72 ALS and frontotemporal dementia

Abstract

The finding that a GGGGCC (G4C2) hexanucleotide repeat expansion in the chromosome 9 ORF 72 (C9ORF72) gene is a common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) links ALS/FTD to a large group of unstable microsatellite diseases. Previously, we showed that microsatellite expansion mutations can be bidirectionally transcribed and that these mutations express unexpected proteins by a unique mechanism, repeat-associated non-ATG (RAN) translation. In this study, we show that C9ORF72 antisense transcripts are elevated in the brains of C9ORF72 expansion-positive [C9(+)] patients, and antisense GGCCCC (G2C4) repeat-expansion RNAs accumulate in nuclear foci in brain. Additionally, sense and antisense foci accumulate in blood and are potential biomarkers of the disease. Furthermore, we show that RAN translation occurs from both sense and antisense expansion transcripts, resulting in the expression of six RAN proteins (antisense: Pro-Arg, Pro-Ala, Gly-Pro; and sense: Gly-Ala, Gly-Arg, Gly-Pro). These proteins accumulate in cytoplasmic aggregates in affected brain regions, including the frontal and motor cortex, hippocampus, and spinal cord neurons, with some brain regions showing dramatic RAN protein accumulation and clustering. The finding that unique antisense G2C4 RNA foci and three unique antisense RAN proteins accumulate in patient tissues indicates that bidirectional transcription of expanded alleles is a fundamental pathologic feature of C9ORF72 ALS/FTD. Additionally, these findings suggest the need to test therapeutic strategies that target both sense and antisense RNAs and RAN proteins in C9ORF72 ALS/FTD, and to more broadly consider the role of antisense expression and RAN translation across microsatellite expansion diseases.

Keywords: clustered aggregates; cytoplasmic inclusions; noncoding RNA.

Fig. 1.

G₂C₄ antisense transcripts accumulate as RNA foci in C9ORF72 patient tissues. (A) Schematic diagram of C9ORF72 gene and antisense transcripts and relative location of primers for strand-specific RT-PCR and RACE primers. (B) Strand-specific RT-PCR of sense (S) and antisense (AS) transcripts (across intron 1b and exon 1a) from FCX of C9(+) and C9(−) ALS patients. (C) Strand-specific qRT-PCR showing elevated antisense mRNA in C9(+) compared with C9(−) ALS patients and a healthy control (HC). (D) FISH with G₄C₂-Cy3 probe showing G₂C₄ antisense RNA foci (red) in C9(+)FCX and PBLs which are absent in C9(−) cases. Nuclear foci in FCX are indicated by arrowheads.

Fig. 2.

In vitro evidence for RAN translation of antisense G₂C₄ expansion and dual immunological detection strategy. (A) Diagram of putative proteins translated from sense and antisense transcripts. Sequences highlighted in red were used to generate antibodies. CT, C-terminal; f1–3, reading frames 1–3; *, stop codon. (B) Abbreviated example of validation of α-PA_AS rabbit polyclonal antibody. Immunoblots (Lower Left) and IF staining (Lower Right) of HEK293T cells transfected with Flag-PA_AS construct (Upper) and probed with α-Flag and α-PA_AS antibodies. See Figs. S4 and S5 for additional controls and validation of eight additional antibodies generated against RAN repeat motifs and CT regions. (C) Immunoblots (Lower Left) and IF staining (Lower Right) of HEK293T cells 48 h posttransfection with the AS-(G₂C₄)_EXP-3T construct (Upper). PR_AS and GP_AS expansion proteins were detected by Western blot and PA_AS, PR_AS and GP_AS proteins were detected by IF in transfected cells.

Fig. 3.

In vivo evidence for RAN-translation of the G₂C₄ AS repeat and toxicity studies. (A) Dot blots containing frontal cortex lysates from four different C9(−) and four different C9(+) ALS/FTD patients were probed with α-PA_AS, α-PA-CT_AS, α-PR_AS, and α-PR-CT_AS antibodies. Results show evidence of RAN protein accumulation with all four antibodies in two of four C9(+) samples and weaker positive staining in one sample with α-PA_AS. (B) Immunoblots of C9(+) and C9(−) ALS frontal cortex 2% SDS insoluble fractions with α-PR_AS, α-PA_AS, and α-GP_S/AS. (C) IHC detection of PA_AS, PR_AS, and GP_S/AS protein aggregates in hippocampal neurons from C9(+) ALS patients detected with α-PA_AS, α-PA-CT_AS, α-PR_AS, α-PR-CT_AS, and α-GP_S/AS antibodies. (D) IF staining with mouse α-GP_S/AS (Cy3, red) and rabbit α-GP-CT_S (Alexa Fluor 488, green) of C9(+)hippocampal tissue with sense inclusions positive for both antibodies (Upper) and antisense inclusions positive for only GP repeat antibody (Lower). Colocalization of α-GP_S/AS and α-GP-CT_S staining was confirmed by confocal z-stacks. (E) IF staining of larger region. Arrowheads indicate background lipofuscin autofluroescense, which can be distinguished from the real signal because it fluoresces in all three channels and when merged appears as brownish-orange signal that is distinct from the Cy3 and Alexa Fluor 488 signals shown (31). (F) Percentages of double- (sense) and single- (antisense) labeled aggregates were quantitated among 1,389 cells from three different tiled areas and a total of 88 aggregates. (G–J) RAN and PR_AS toxicity studies. (G) G₂C₄ expansion constructs (+/−ATG-PR-3T) with or without an ATG initiation codon in PR_AS frame and 3′ epitope tags. (H) Western blots showing levels of PR_AS and GP_AS in cells transfected with constructs in G. (I) LDH and (J) MTT assays of transfected HEK293T cells. *P ≤ 0.05, **P ≤ 0.01), ***P ≤ 0.001, n = 6 independent experiments).

Fig. 4.

In vivo evidence for RAN translation in both antisense and sense directions of C9ORF72. Cytoplasmic inclusions detected by IHC using antibodies against sense (α-GR_S, α-GR-CT_S, α-GA-CT_S, α-GP-CT_S) and antisense (α-PA_AS, α-PA-CT_AS, α-PR_AS, α-PR-CT_AS), and α-GP_S/AS, which recognizes GP proteins made in both the sense and antisense directions. Aggregates are found in neurons of the CA and DG regions of the hippocampus and the motor cortex (MC) of C9(+) ALS autopsy tissue.

Fig. 5.

Clustered RAN-positive cells in hippocampus and motor cortex and RAN aggregates in motor neurons. IHC showing cytoplasmic α-GP_S/AS aggregates in: (A) layer III of motor cortex; (B) upper motor neuron in layer V of the motor cortex; (C) lower motor neurons in the lumbar spinal cord (LSC); (D) in CA and (E) DG regions of the hippocampus. (F and G) Abundant PA-CT_AS and PR-CT_AS cytoplasmic inclusions in the presubiculum (PrSub) from one patient.

Fig. 6.

Clustered staining of RAN proteins. (A) Low-power image of IHC staining with α-PA-CT_AS shows variations in staining intensity (red is positive) in regions I–IV, with Insets showing higher-power images. (B) Examples of aggregates from region I show immunoreactivity against all nine antibodies with similar staining for antibodies against repeat and unique C-terminal epitopes.