C9ORF72 hexanucleotide repeat exerts toxicity in a stable, inducible motor neuronal cell model, which is rescued by partial depletion of Pten

Abstract

Amyotrophic lateral sclerosis (ALS) is a devastating and incurable neurodegenerative disease, characterised by progressive failure of the neuromuscular system. A (G4C2)n repeat expansion in C9ORF72 is the most common genetic cause of ALS and frontotemporal dementia (FTD). To date, the balance of evidence indicates that the (G4C2)n repeat causes toxicity and neurodegeneration via a gain-of-toxic function mechanism; either through direct RNA toxicity or through the production of toxic aggregating dipeptide repeat proteins. Here, we have generated a stable and isogenic motor neuronal NSC34 cell model with inducible expression of a (G4C2)102 repeat, to investigate the gain-of-toxic function mechanisms. The expression of the (G4C2)102 repeat produces RNA foci and also undergoes RAN translation. In addition, the expression of the (G4C2)102 repeat shows cellular toxicity. Through comparison of transcriptomic data from the cellular model with laser-captured spinal motor neurons from C9ORF72-ALS cases, we also demonstrate that the PI3K/Akt cell survival signalling pathway is dysregulated in both systems. Furthermore, partial knockdown of Pten rescues the toxicity observed in the NSC34 (G4C2)102 cellular gain-of-toxic function model of C9ORF72-ALS. Our data indicate that PTEN may provide a potential therapeutic target to ameliorate toxic effects of the (G4C2)n repeat.

Figure 1

NSC34 (G4C2)102 cells have tetracycline inducible (G4C2)102 RNA expression, which forms RNA foci. (A) Sequence of the (G4C2)102 construct. (B) Gel electrophoresis sizing the (G4C2)102 repeat constructs in the pcDNA5/FRT/TO-(G4C2)102 plasmids. (C) NSC34 sham and NSC34 (G4C2)102 cells were cultured for 3 days with (or without) 0.5 µg/ml tetracycline. NSC34 (G4C2)102 cells were additionally treated with RNAse A as a control. Cells were stained with a locked nucleic acid (C4G2)3 sense probe (Red) and Dapi (Blue). Foci magnified 5× inset. Scale bar = 10 µm. (D) Average number of RNA foci per cell (***p < 0.001; two-way ANOVA with Tukey’s multiple comparisons post hoc test; data shown are mean and SD; n = 3).

Figure 2

NSC34 (G4C2)n cells do not transcribe (G4C2)n in the antisense direction. NSC34 sham and NSC34 (G4C2)102 cells were cultured for 3 days with 0.5 µg/ml tetracycline. Cells were stained with a locked nucleic acid (G4C2)3 antisense probe (Red) and Dapi (Blue). HEK293 cells transfected with an antisense (C4G2)102 plasmid contain (C4G2)102 foci and serve as a positive control. Scale bar = 10 µm.

Figure 3

The (G4C2)102 construct undergoes RAN translation in the NSC34 (G4C2)102 (G4C2)102 cells. (A) Schematic of translation products (including Stop codons) from interrupted (G4C2)102 RNA construct in all three reading frames. GA, GP and GR repeats are highlighted in red, yellow and green, respectively. (B–F) NSC34 sham and NSC34 (G4C2)102 cells were cultured for 7 days with (or without) 0.5 µg/ml tetracycline. HEK293 cells were transfected with (GA)68, (GR)100, (AP)100 and (PR)100 constructs to serve as a positive control for the respective DPR antibody. Cells were lysed and immunoblotted with anti-poly-GA (B), anti-poly-GR (C), anti-poly-GP (D), anti-poly-AP (E) and anti-poly-PR (F). Arrows indicate the RAN translation products in the NSC34 (G4C2)102 cells. α-Tubulin was used as a loading control for blots. Molecular weight markers are indicated (kDa).

Figure 4

NSC34 (G4C2)102 cells recapitulate pathological features of C9ORF72-ALS. (A–C, E) NSC34 (G4C2)102 cells were induced with 0.5 µg/ml tetracycline for 5 days. Cells were then stained with a locked nucleic acid (C4G2)3 sense probe (Red), anit-SRSF1 (A), anti-SRSF2 (B) or anti-NCL (C) (all green), and Dapi (Blue). Scale bar = 10 µm. (D) Cerebellar slices from C9ORF72-ALS cases were stained with a locked nucleic acid (C4G2)3 sense probe (Red), anti-NCL (green) and Dapi (Blue). Scale bar = 3 µm. (E) NSC34 sham and NSC34 (G4C2)102 cells were stained for NCL (green) and DAPI (blue). Scale bar = 10 µm. (F) Quantification of NCL area as a percentage of nuclear area in NSC34 sham and (G4C2)102 cells (*p < 0.05; **p < 0.01; two-way ANOVA with Tukey’s multiple comparisons post hoc test; n = 3). (G) Ventral horn from C9ORF72-ALS was stained for NCL and DAPI. Motor neurons (indicated with arrows and nuclei outlined) show disrupted NCL staining, while glial cells do not show disrupted NCL staining. Scale bar = 3 µm.

Figure 5

(G4C2)102 repeat is toxic in NSC34 cells. (A) NSC34 sham and NSC34 (G4C2)102 cells were cultured for 7 days and were induced for various lengths of time with 0.5 µg/ml tetracycline. Cell viability was measured using an MTT assay (*p < 0.05; two-way ANOVA with Tukey’s multiple comparisons post hoc test; data shown are mean and SD; n = 3). (B) NSC34 sham and NSC34 (G4C2)102 cells were cultured for 16 days with or without 0.5 µg/ml tetracycline. The cells were counted every 4 days, and then 1.5 × 10⁶ cells were reseeded (***p < 0.001; ****p < 0.0001; two-way ANOVA with Tukey’s multiple comparisons post hoc test; data shown are mean and SD; n = 4).

Figure 6

Pten knockdown rescues NSC34 cells from (G4C2)102-induced toxicity. (A) Cells were lysed and immunoblotted with anti-Pten. β-Actin was used as a loading control for blots. Molecular weight markers are indicated (kDa). (B) Quantification of Pten normalised to β-actin (*p < 0.05; **p < 0.01; one-way ANOVA with Tukey’s multiple comparisons post hoc test; data shown are mean and SD; n = 3). (C) NSC34 sham GFP, NSC34 (G4C2)102 GFP, NSC34 sham Pten shRNA and NSC34 (G4C2)102 Pten shRNA cells were cultured for 7 days, with (or without) 0.5 µg/ml tetracycline. Cell viability was measured using an MTT assay, and the viability of the tetracycline induced cells was normalised to the non-induced control for each individual cell line (**p < 0.01; two-way ANOVA with Sidak’s multiple comparisons post hoc test; data shown are mean and SD; n = 3).