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. 2023 Apr 19;12:e85902. doi: 10.7554/eLife.85902

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—source data 1. Original western blot results for Figure 1D and F.
Figure 1—source data 2. Original western blot results for Figure 1H.

Figure 1.

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

Figure 1—figure supplement 1.

(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 1—source data 1. Original western blot results for Figure 1—figure supplement 1B.
Figure 1—figure supplement 2. C9ORF72 C4G2 repeat expanded repeats activate PKR/eIF2α-dependent integrated stress response in SH-SY5Y cells.

Figure 1—figure supplement 2.

(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 1—figure supplement 2—source data 1. Original western blot results for Figure 1—figure supplement 2B and C.