Antisense, but not sense, repeat expanded RNAs activate PKR/eIF2α-dependent ISR in C9ORF72 FTD/ALS

Abstract

GGGGCC (G₄C₂) hexanucleotide repeat expansion in the C9ORF72 gene is the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). The repeat is bidirectionally transcribed and confers gain of toxicity. However, the underlying toxic species is debated, and it is not clear whether antisense CCCCGG (C₄G₂) repeat expanded RNAs contribute to disease pathogenesis. Our study shows that C9ORF72 antisense C₄G₂ repeat expanded RNAs trigger the activation of the PKR/eIF2α-dependent integrated stress response independent of dipeptide repeat proteins that are produced through repeat-associated non-AUG-initiated translation, leading to global translation inhibition and stress granule formation. Reducing PKR levels with either siRNA or morpholinos mitigates integrated stress response and toxicity caused by the antisense C₄G₂ RNAs in cell lines, primary neurons, and zebrafish. Increased phosphorylation of PKR/eIF2α is also observed in the frontal cortex of C9ORF72 FTD/ALS patients. Finally, only antisense C₄G₂, but not sense G₄C₂, repeat expanded RNAs robustly activate the PKR/eIF2α pathway and induce aberrant stress granule formation. These results provide a mechanism by which antisense C₄G₂ repeat expanded RNAs elicit neuronal toxicity in FTD/ALS caused by C9ORF72 repeat expansions.

Keywords: C9ORF72; FTD/ALS; PKR; antisense RNA; integrated stress response; neuroscience; zebrafish.

Figure 1.. C9ORF72 antisense C₄G₂ expanded repeats activate PKR/eIF2α-dependent integrated stress response and cause neuronal toxicity.

(A) Schematic illustration of the in_(C₄G₂)75 repeat construct including 6× stop codons, 450 bp of human intronic sequences at the N-terminus and 3× protein tags at the C- terminus of the repeats to monitor the DPR proteins in each frame. (B) Representative images of antisense RNA foci in HEK293T cells and in primary cortical neurons expressing in_(C₄G₂)75 detected by RNA FISH. Red, foci; blue, DAPI; magenta, MAP2. (C) Kaplan–Meier curves showing increased risk of cell death in in_(C₄G₂)75-expressing primary cortical neurons compared with neurons expressing 2 repeats. Statistical analyses were performed using Mantel–Cox test (replicated three times with similar results). (D, E) Immunoblotting analysis of phosphorylated PKR (p-PKR) and total PKR in HEK293T cells expressing in_(C₄G₂)75 or 2 repeats. p-PKR levels were detected using anti-p-PKR (T446) and normalized to total PKR. GAPDH was used as a loading control. Error bars represent SD (n = 3 independent experiments). Statistical analyses were performed using Student’s t-test. (F, G) Immunoblotting analysis of phosphorylated eIF2α (p-eIF2α) and total eIF2α in HEK293T cells expressing in_(C₄G₂)75 or 2 repeats. p-eIF2α levels were detected using anti-phosphor eIF2α (Ser51) and normalized to total eIF2α. GAPDH was used as a loading control. Error bars represent SD (n = 3 independent experiments). Statistical analyses were performed using Student’s t-test. (H, I) Immunoblotting analysis of p-PKR and p-eIF2α in HEK293T cells expressing in_(C₄G₂)75, with or without co-expression of wild type PKR, or treatment of a PKR inhibitor, C16. Error bars represent SD (n = 3 independent experiments). Statistical analyses were performed using one-way ANOVA with Tukey’s post hoc test. Scale bars: 10 µm (neurons), 20 µm (HEK293T).

Figure 1—figure supplement 1.. C9ORF72 C₄G₂ repeat expanded repeats produce antisense DPR proteins in HEK293T cells and primary neurons.

(A) Representative images of DPR protein staining in HEK293T expressing in_(C₄G₂)75 repeats using DPR antibodies. Red, GP, PA, and PR; blue, DAPI. (B) Immunoblotting of DPR proteins in HEK293T expressing in_(C₄G₂)75 repeats using TAG antibodies. GAPDH was used as a loading control. (C) Representative images of DPR protein staining in primary neurons expressing in_(C₄G₂)75 repeats using DPR antibodies. Red, GP, PA, and PR; blue, DAPI; MAP2, magenta. Scale bars, 10 µm (neurons), 20 µm (HEK293T).

Figure 1—figure supplement 2.. C9ORF72 C₄G₂ repeat expanded repeats activate PKR/eIF2α-dependent integrated stress response in SH-SY5Y cells.

(A) Immunoblotting of p-PERK in HEK293T cells expressing in_(C₄G₂)75 or (C₄G₂)2 repeats. Phosphorylated PERK levels were quantified and normalized to total PERK. GAPDH was used as a loading control. Error bars represent SD (n = 2 independent experiments). Statistical analyses were performed using Student’s t-test. (B, C) Immunoblotting of p-PKR and p-eIF2α in SH-SY5Y cells expressing (C₄G₂)75 or (C₄G₂)2 repeats. p-PKR (T446) and p-eIF2α (Ser51) were normalized to total PKR and eIF2α, respectively. GAPDH was used as a loading control. Error bars represent SD (n = 2 independent experiments). Statistical analyses were performed using Student’s t-test.

Figure 2.. C9ORF72 antisense C₄G₂ expanded repeats inhibit global protein synthesis and induce stress granule assembly.

(A) Immunoblotting of puromycin in HEK293T cells expressing in_(C₄G₂)75 or 2 repeats. Cells were incubated with puromycin for 30 min before harvesting. The level of puromycin was normalized to GAPDH. Error bars represent SD (n = 3 independent experiments). Statistical analyses were performed using Student’s t-test (B) Representative images and (C) quantification of primary neurons expressing either (C₄G₂)75 or 2 repeats stained with anti-puromycin (red), anti-FLAG (green), DAPI (blue), and MAP2 (magenta). The puromycin intensity was quantified using ImageJ. Error bars represent SD (n = 40–50 neurons/group; similar results were obtained from two independent experiments). Statistical analyses were performed using Student’s t-test. (D) Representative images of G3BP1 and FMRP staining in HEK293T cells expressing in_(C₄G₂)75 and in_(C₄G₂)2 repeats. (E) Quantification of antisense foci-positive cells with G3BP1 and FMRP granules. Error bars represent SD (n = 150 cells/condition and three independent experiments). Statistical analyses were performed using Student’s t-test. Scale bars, 10 µm (neurons), 20 µm (HEK293T).

Figure 3.. Antisense C₄G₂ repeat expanded RNAs activate the PKR/eIF2α pathway independent of DPR proteins.

(A) (Top) Schematic illustration of (C₄G₂)75 repeats without the human intronic sequences. 3× protein tags were included at the C- terminus of the repeats to monitor the DPR proteins in each frame. (Bottom) Schematic illustration of antisense (C₄G₂)108RO repeats with stop codons inserted in every 12 repeats to prevent the translation of DPR proteins from all reading frames. (B) Immunoblotting of DPR proteins in HEK293T cells expressing in_(C₄G₂)75, (C₄G₂)75, or 2 repeats. DPR protein levels were detected using anti-FLAG (frame with GP), anti-MYC (frame with PR), and anti-HA (frame with PA). GAPDH was used as a loading control. (C) mRNA levels were measured by quantitative qPCR in cell expressing in_(C₄G₂)75, (C₄G₂)75, or 2 repeats. Error bars represent SD (n = 3). (D, E) Immunoblotting of p-PKR and p-eIF2α in HEK293T cells expressing in_(C₄G₂)75, (C₄G₂)75, (C₄G₂)108RO, or 2 repeats. p-PKR (T446) and p-eIF2α (Ser51) were normalized to total PKR and eIF2α, respectively. GAPDH was used as a loading control. Error bars represent SD (n = 3 independent experiments). Statistical analyses were performed using one-way ANOVA with Tukey’s post hoc test. (F, G) Representative images (F) and quantitation (G) of p-eIF2α in primary neurons expressing antisense (C₄G₂)108RO or 2 repeats in the presence and absence of PKR siRNA. Scale bars, 10 µm.

Figure 3—figure supplement 1.. Antisense DPR proteins do not activate PKR/eIF2α-dependent integrated stress response.

(A) Representative images and (B) quantification of p-eIF2α staining in HEK293T cells expressing in_(C₄G₂)75, PR50, PA50, or GP80. Green, GP, PA, or PR; red, p-eIF2α; blue, DAPI. Error bars represent SD (n = 2 independent experiments). (C) Representative images of antisense RNA foci in HEK293T cells expressing in_(C₄G₂)75, (C₄G₂)75 or (C₄G₂)108RO repeats. Foci were detected by RNA FISH. Red, foci; blue, DAPI. (D) Immunoblotting of DPR proteins in HEK293T cells expressing in_(C₄G₂)75, (C₄G₂)75 and (C₄G₂)108RO repeats. DPR protein levels were detected using anti-PR and anti-GP antibodies. GAPDH was used as a loading control. (E) Representative images of antisense RNA foci in primary cortical neurons expressing (C₄G₂)108 detected by RNA FISH. Red, foci; blue, DAPI; Green, MAP2. (F) Immunoblotting and (G) quantification of PKR in primary neurons expressing (C₄G₂)2 and (C₄G₂)108RO together with control or PKR siRNA. PKR were normalized to GADPH. Error bars represent SD. (H) The levels of PKR and p-eIF2α (Ser51) in HEK293T cells expressing (C₄G₂)108RO together with control or PKR siRNA. PKR and p-eIF2α (Ser51) were normalized to GADPH and total eIF2α, respectively. Error bars represent SD (n = 2 independent experiments). Statistical analyses were performed using Student’s t-test. (I) Representative images and quantification of puromycin staining in HEK293T cells expressing (C₄G₂)108RO together with control or PKR siRNA. Error bars represent SD (n = 3 independent experiments). mApple was co-transfected to identify cells with (C₄G₂)108RO expression. Statistical analyses were performed using Student’s t-test. Scale bars, 20 µm (HEK293T), 10 µm (neurons).

Figure 4.. Antisense C₄G₂ repeat expanded RNAs themselves induce stress granules and lead to neuronal toxicity.

(A) Representative images of FMRP and G3BP1 staining in HEK293T cells expressing (C₄G₂)108RO or 2 repeats. (B, C) Quantification of antisense foci-positive cells with FMRP and G3BP1 granules. Error bars represent SD (n = 180 cells/condition and three independent experiments). Statistical analyses were performed using Student’s t-test. (D) Immunoblotting of PKR and p-eIF2α (Ser51) in HEK293T cells expressing (C₄G₂)108RO together with control or PKR siRNA. GAPDH was used as a loading control. (E) Representative images of FMRP staining in HEK293T cells expressing (C₄G₂)108 repeats together with either control or PKR siRNA. (F) Quantification of antisense foci-positive cells with FMRP granules. Error bars represent SD (n = 150 cells/condition and three independent experiments). (F) Kaplan–Meier curves showing the risk of cell death in (C₄G₂)108RO-expressing neurons compared with 2 repeats in the presence and absence of PKR siRNA (replicated three times with similar results). Statistical analyses were performed using Mantel–Cox test (*p<0.05, **p<0.01). Scale bars, 20 µm.

Figure 5.. Increased levels of phosphorylated PKR and eIF2α in C9FTD/ALS patients.

(A) Representative immunohistochemistry images of phosphorylated PKR staining in control and C9FTD/ALS patient’s frontal cortex (FCX) using anti-p-PKR (T446) (n = 4 per genotype). (B, C) Immunoblotting of p-eIF2α in proteins extracted from control (C1–C6) and C9FTD/ALS patient’s frontal cortex (P1–P6). p-eIF2α (Ser51) was normalized to total eIF2α. GAPDH was used as a loading control. Error bars represent SD (control n = 6 and C9FTD/ALS n = 6). Statistical analyses were performed using unpaired Student’s t-test. Scale bars, 10 µm.

Figure 5—figure supplement 1.. Phosphorylated PKR and eIF2α levels are unchanged in C9FTD/ALS iPSC-derived motor neurons.

(A) Immunoblotting and (B) quantification of p-PKR and p-eIF2α in two different lines of iPSC-derived motor neurons of C9FTD/ALS patient and their isogenic controls (38 d post differentiation). P-PKR levels were detected using anti-p-PKR (T446) and normalized to total PKR. P-eIF2α levels were detected using anti-phosphor eIF2α (Ser51) and normalized to total eIF2α. Error bars represent SD (n = 3).

Figure 6.. Sense G₄C₂ repeat expanded RNAs cannot activate the PKR/eIF2α pathway.

(A) Immunoblotting of p-PKR and p-eIF2α in HEK293T cells expressing (G₄C₂)75 or (G₄C₂)2 repeats. (B) Representative images of sense and antisense RNA foci in HEK293T expressing (G₄C₂)75 repeats together with either control antisense oligonucleotides (ASOs), ASOs targeting sense G₄C₂RNAs or ASOs targeting antisense C₄G₂ repeat expanded RNAs. Foci were detected by RNA FISH. Red, foci; blue, DAPI. (C) Immunoblotting and (D) quantification of p-PKR and p-eIF2α in HEK293T cells expressing (G₄C₂)75 or (G₄C₂)2 repeats together with either control ASO or ASOs targeting sense G₄C₂ repeat expanded RNAs. Phosphorylated PKR levels were detected using anti-p-PKR (phosphor T446) and normalized to total PKR. Phosphorylated eIF2α levels were detected using anti-peIF2α (Ser51) and normalized to total eIF2α. GAPDH was used as a loading control. Error bars represent SD (n = 3 independent experiments). Statistical analyses were performed using unpaired Student’s t-test. (E, F) Immunoblotting of p-PKR and p-eIF2α in HEK293T cells expressing (G₄C₂)75 or (G₄C₂)2 repeats together with either control ASO or ASOs targeting antisense G₄C₂ repeat expanded RNAs. P-PKR (T446) and p-eIF2α (Ser51) were normalized to total PKR and eIF2α, respectively. GAPDH was used as a loading control. Error bars represent SD (n = 3 independent experiments). Statistical analyses were performed using one-way ANOVA with Tukey’s post hoc test. (G) Representative images and (H) quantification of FMRP and G3BP1 staining in HEK293T cells expressing (G₄C₂)75 or (G₄C₂)2 repeats in the presence and absence of antisense ASO2. Error bars represent SD (n = 150 cells/condition and three independent experiments). Statistical analyses were performed using one-way ANOVA with Tukey’s post hoc test. Scale bars, 20 µm.

Figure 6—figure supplement 1.. Sense G₄C₂ repeat expanded RNAs cannot activate the PKR/eIF2α pathway.

(A) Schematic illustration of the sense (G₄C₂)75 repeat construct. (B) Quantification of p-PKR and p-eIF2α in HEK293T cells expressing (G₄C₂)75 or (G₄C₂)2 repeats. p-PKR (T446) and p-eIF2α (Ser51) were normalized to total PKR and eIF2α, respectively. GAPDH was used as a loading control. Error bars represent SD (n = 2 independent experiments). Statistical analyses were performed using Student’s t-test. (C) Representative images of sense and antisense RNA foci in HEK293T cells expressing sense (G₄C₂)75 and interrupted antisense (C₄G₂)108RO repeats. Foci were detected by RNA FISH. Red, foci; blue, DAPI. (D, E) Quantification of RNA foci in HEK293T expressing (G₄C₂)75 together with either control antisense oligonucleotide (ASO) or ASOs targeting sense G₄C₂ and antisense C₄G₂ repeat expanded RNAs. Error bars represent SD (n = 80–100 cells/condition from three independent experiments). (F) Immunoblotting of p-PKR and p-eIF2α in HEK293T cells expressing interrupted sense (G₄C₂)108RO and control repeats. p-PKR levels were detected using anti-p-PKR (T446) and normalized to total PKR. P-eIF2α levels were detected using anti-phosphor eIF2α (Ser51) and normalized to total eIF2α. Error bars represent. GAPDH was used as a loading control. Error bars represent SD Scale bars, 20 µm (HEK293T).

Figure 7.. Reduction of PKR mitigates antisense C₄G₂, but not sense G₄C₂, RNA- mediated toxicity in zebrafish.

(A) Schematic illustration of splice-blocking morpholino (SB-MO) targeting the exon 3/intron 3 junction in zebrafish eif2ak2 pre-mRNA. The correctly spliced (wild type transcripts) and intron 3 retained transcripts are shown. The translation-blocking morpholino targets the AUG start codon in post-spliced eif2ak2 mRNA. (B) SV2 immunostaining of motor axons in 30 hpf zebrafish embryos injected with GFP control mRNA and antisense (C₄G₂)70 RNAs. Scale bar = 50 µm. (C, D) Effects of SB-MO on axonal length (C) and branching (D) of zebrafish expressing antisense (C₄G₂)70 RNAs. Embryos were injected with 0.844 µM GFP mRNA, 0.844 µM antisense (C₄G₂)70 RNAs, 0.844 µM antisense (C₄G₂)70 RNAs plus 0.25 mM control morpholino, or 0.844 µM antisense (C₄G₂)70 RNAs plus 0.25 mM eif2ak2 morpholino. Error bars represent SD (n = 4 independent experiments). Statistical analyses were performed using one-way ANOVA with Tukey’s post hoc test. (E, F) Effects of SB-MO on axonal length and branching of zebrafish expressing sense (G₄C₂)70 RNAs. Embryos were injected with 0.844 µM GFP mRNA, 0.844 µc sense (G₄C₂)70 RNAs, 0.844 µM sense (G₄C₂)70 RNAs plus 0.25 mM control morpholino or 0.844 µM sense (G₄C₂)70 RNAs plus 0.25 mM eif2ak2 morpholino. Error bars represent SD (n = 4 independent experiments). Statistical analyses were performed using one-way ANOVA with Tukey’s post hoc test.

Figure 7—figure supplement 1.. Translation-blocking morpholino of PKR mitigates antisense C₄G₂ repeat RNA-induced axonopathy in zebrafish.

(A) Dose-dependent knockdown of WT transcripts and increase of transcripts with intron 3 retention as determined by RT-PCR analysis. Eif2ak2 splice variants in non-injected, standard control and eif2ak2 targeting MO-injected embryos 30 hpf are shown. (B, C) Effect of translation-blocking morpholino (TB-MO)-mediated reduction of Eif2ak2 on axonal length (B) and branching (C), using a dose of 0.25 mM for TB-MO and standard control MO in GFP and antisense (C₄G₂)70 RNAs expressing zebrafish. Error bars represent SD (n = 4 independent experiments). Statistical analyses were performed using one-way ANOVA with Tukey’s post hoc test.

Figure 8.. Proposed pathogenic mechanisms caused by C9ORF72 antisense C₄G₂ repeat expanded RNAs.

C9ORF72 antisense C₄G₂ repeat expanded RNAs activate PKR/eIF2α-dependent integrated stress response and lead to neurotoxicity independent of DPR proteins.