Figure 1. Neurotoxicity of C9RAN poly-dipeptides.

(A) Representative live-cell images of cortical neurons co-transfected at DIV10 with control-GFP + Td-tomato plasmids (4:1) or PR₅₀-GFP + Td-Tomato (4:1). The same neurons were imaged for 9 days (8 days post-transfection) at 24-hour intervals. Calibration bar is 100 μm. (B) Kaplan-Meier survival analysis of cortical neurons co-transfected with constructs encoding different DRPs + Td-Tomato. At least 80 neurons were followed/group; n=5 experiments; ***P<0.001. The insert shows the schematic of the construct. The sequence of the insert is the following: ATG-DYKDDK-KLGR-DPR₅₀-GYRARIHYSSVVEFM-EGFP-TAA. The insert was subcloned into pcDNA3.1 vector and its expression driven by the CMV promoter. The DRP sequence has been constructed with a randomized codon strategy. (C) PR₅₀ aggregates formation preceded neuronal death. Representative images show a group of cortical neurons expressing both PR₅₀ (GFP tagged) and Td-tomato. Neurons in which PR₅₀ formed aggregates underwent cell death within 24–36 hours (yellow arrows, day 1–2; white arrows, day 3–4). Conversely, neurons in which PR₅₀ stayed in a diffused, not aggregated pattern (orange arrow and insets) outlived the neurons with PR aggregates. Calibration bar is 50 μm. (D) Cortical neurons transfected with PR₅₀ that formed aggregates exhibited decreased survival and increased risk of death compared to those that didn’t form detectable PR₅₀ aggregates (***p<0.001). PR₅₀ transfected neurons with no detectable aggregates showed no difference in survival and risk of death compared to control transfected neurons (P = 0.08). (E) Length-dependent toxicity of DRPs in cortical neurons. Poly-GR displayed clear length-dependent toxicity (**P<0.01; one-way ANOVA), whereas the effect of poly-PR already peaked at 25 repeats, displaying no significant length-dependency (one-way ANOVA). (F) Cortical neuron toxicity of PR₅₀ diminished as amount of transfected cDNA was reduced suggesting a dose-dependency in the effect. Maximum amount of cDNA-PR₅₀ transfected was 0.8 μg/well (100%). Total amount of DNA transfected was kept constant across groups (1 μg/well). *P<0.05, **P<0.01, ***P<0.001. (G) NSC34 cells were transfected with indicated constrcuts. Expression levels of different lengths of DRP constructs were assessed using dot blots. Actin was used as a loading control. Comparable expression levels were observed for DRPs of different lengths.