Translation of dipeptide repeat proteins from the C9ORF72 expanded repeat is associated with cellular stress

Abstract

Expansion of a hexanucleotide repeat (HRE), GGGGCC, in the C9ORF72 gene is recognized as the most common cause of familial amyotrophic lateral sclerosis (FALS), frontotemporal dementia (FTD) and ALS-FTD, as well as 5-10% of sporadic ALS. Despite the location of the HRE in the non-coding region (with respect to the main C9ORF72 gene product), dipeptide repeat proteins (DPRs) that are thought to be toxic are translated from the HRE in all three reading frames from both the sense and antisense transcript. Here, we identified a CUG that has a good Kozak consensus sequence as the translation initiation codon. Mutation of this CTG significantly suppressed polyglycine-alanine (GA) translation. GA was translated when the G₄C₂ construct was placed as the second cistron in a bicistronic construct. CRISPR/Cas9-induced knockout of a non-canonical translation initiation factor, eIF2A, impaired GA translation. Transfection of G₄C₂ constructs induced an integrated stress response (ISR), while triggering the ISR led to a continuation of translation of GA with a decline in conventional cap-dependent translation. These in vitro observations were confirmed in chick embryo neural cells. The findings suggest that DPRs translated from an HRE in C9ORF72 aggregate and lead to an ISR that then leads to continuing DPR production and aggregation, thereby creating a continuing pathogenic cycle.

Keywords: C9ORF72; Dipeptide protein repeats (DPRs); Hexanucleotide repeat expansions (HREs); Integrated stress response (ISR); Internal ribosome entry site (IRES); Repeat associated non-AUG (RAN) translation; Unconventional translation; eIF2A.

Fig. 1.

GA is the most abundantly translated DPR from G₄C₂ expanded repeat. (A) Schematic diagram showing ΔC9-nLuc, GA-nLuc, GP-nLuc, and GR-nLuc plasmids. NSC34 (B, D) and HEK293 (C, E) cells were cotransfected with fLuc plasmid along with either ΔC9-nLuc, GA-nLuc, GP-nLuc, or GR-nLuc plasmids. (B, C) Cells were harvested after 48 h and processed for dual luciferase assays. Unless noted in other figure legends, the nLuc/fLuc ratio was normalized to that of ΔC9-nLuc, which was set to 1. (D, E) Cell lysates were processed for western blots and immunostained with poly-GA and β-actin antibody. Molecular weight in kDa is shown on the side of the western blot.

Fig. 2.

Translation of GA is dependent on a CUG codon located upstream of the G₄C₂ expanded repeat. (A) Part of DNA sequence of CTG-GA-nLuc, TAG-GA-nLuc, CTA-GA-nLuc or CCC-GA-nLuc showing either the underlined putative CUG translation start codon upstream of the expanded repeat or with mutated CTG shown in red. NSC34 (B, D) and HEK293 (C, E) cells were cotransfected with fLuc plasmid along with either ΔC9-nLuc, CTG-GA-nLuc, TAG-GA-nLuc, CTA-GA-nLuc or CCC-GA-nLuc plasmids. Cells were harvested after 48 h and assessed by dual luciferase assays (B, C) or western blot (D, E) of cell lysates that were immunostained with poly-GA and β-actin antibody. Molecular weight in kDa is shown on the side of the western blot. The ratio of the GA band to the β-actin band is shown at the bottom of the western blots with the ratios normalized to the ratio in the CTG lane, which was set to 1.

Fig. 3.

GA is translated from an IRES. (A) Schematic diagram of ΔC9, AUGnLuc, and C9 bicistronic constructs. NSC34 (B, D, F, H) and HEK293 (C, E, G, I) cells were transfected with ΔC9, AUGnLuc, or C9 bicistronic plasmids (B–E) or in vitro derived transcripts from ΔC9 and C9 bicistronic plasmids (F–I). Cells were harvested 48 h later for dual luciferase assays (B, C, F, G) or western blots (D, E, H, I). Western blots were immunostained with poly-GA and β-actin antibody. Molecular weight in kDa is shown on the side of the western blot.

Fig. 4.

Translation of GA utilizes eIF2A. (A) Part of DNA sequence of non-targeted (NT) HEK293 cells and EIF2A-knockout (EIF2A-KO) cell lines showing protospacer adjacent motif (PAM) and sgRNA sequence. (B) Western blot of lysates of NT and EIF2A-KO cell lines immunostained with eIF2A, eIF2α, and β-actin antibody. NT and EIF2A-KO cells were transfected with either ΔC9 (black) or C9 (gray) bicistronic plasmids (C), or with in vitro derived transcripts from ΔC9 (black), or C9 (gray) bicistronic plasmids (D).

Fig. 5.

Translation of GA and the ISR. (A) Western blot of HEK293 cells that were either untreated or pretreated with 500 nM thapsigargin (TG), 1 mM 1,4-dithiothreitol (DTT), or 100 μM sodium arsenite (SA) for 30 min. Western blots of lysates were immunostained with antibody against phosphorylated eIF2α (p-eIF2α) or eIF2α. The ratio of p-eIF2α/eIF2α is shown at the bottom of the eIF2α western blot, and the ratio of p-eIF2α/β-actin is shown at the bottom of the β-actin western blot, with the ratios normalized to the ratio of untreated HEK293 cells, which was set to 1. (B, C) Thirty minutes following treatment with TG, DTT or SA or no treatment, HEK293 cells were transfected with transcripts in vitro derived from the C9 bicistronic construct. After 2 h the cells were harvested for dual luciferase assays. In (B), the nLuc/fLuc ratio is shown normalized to those in untreated samples. In (C), the level of fLuc (black) and nLuc (gray) in the untreated cells is normalized to 1, and the level for each treated sample is normalized to the level in the untreated sample of nLuc and fLuc respectively. (D) Double immunofluorescent staining for p-eIF2α (green) and poly-GA (red) in HEK293 cells transfected with ΔC9-nluc (upper panel) or GA-nluc plasmid (lower panel). Cells transfected with GAnluc plasmid that had poly-GA staining also had p-eIF2α staining. The p-eIF2α staining was significantly above the background staining seen in cells that were not transfected or cells that were transfected with ΔC9-nluc plasmid. All images shown have the same magnification, laser intensity, gain and offset values, and pinhole setting. Scale bars, 10 μm.

Fig. 6.

Translation of poly-GA in chick embryo spinal cord neural cells is dependent on a CUG codon and mediated by eIF2A. (A–C) The fLuc plasmid was coelectropolated into the central canal of chick embryos along with either: ΔC9-nLuc, CTG-GA-nLuc (C9), TAG-GA-nLuc (C9 with a TAG mutation replacing the upstream CTG), GP-nLuc, or GR-nLuc plasmids. The samples were harvested after 24 h for dual luciferase assays (A, C) or western blotted with immunostaining with anti-polyGA antibody (B). (D) The ΔC9 or C9 bicistronic plasmid was electroporated into the central canal of chick embryos. The samples were harvested after 48 h and assessed by dual luciferase assays. The nLuc/fLuc ratio was normalized to that of ΔC9, which was set to 1. (E) The control or anti-eIF2A shRNA was transfected into DF-1 chick fibroblast cell line. The levels of β-actin and eIF2A mRNAs were assessed by RT-PCR. (F) C9 bicistronic plasmid along with either control or anti-eIF2A shRNA plasmids were coelectroporated into the central canal of chick embryos. The samples were harvested after 48 h and assessed by dual luciferase assays. The nLuc/fLuc ratio was normalized to that of ΔC9, which was set to 1.