Figure 2. Intronic G4C2 repeats are directly toxic to primary neurons.

(A) Representative time-lapse images of cortical neurons co-transfected with Td-Tomato and R0, R21 or R42 constructs (1:4 ratio; 1 μg cDNA/well total). Neurons that went on to die are pointed by arrows. More death events were observed in neurons transfected with R42. Calibration bar is 100 μm. (B, C) Kaplan-Meier survival and Cox proportional hazards analysis of cortical neurons transfected with R0, R21 or R42 showed that expanded intronic G4C2 repeats caused cell death and significantly increased the risk of death. Neurotoxicity of G4C2 decreased with reduced amount of R42 cDNA transfected into neurons, suggesting dose-dependency. Note that 50% of R42 cDNA (or 0.4 μg) is not sufficient to cause toxicity. Total amount DNA used in transfection remained the same across groups. At least 80 neurons were followed/group; n=3 experiments. *P<0.05, **P<0.01, ***P<0.001. (D) Representative live-cell images based on GFP fluorescence at day 1, 2 and 4 post-transfection of R42 construct in motor neurons show clear motor neuron loss. Calibration bar is 50 μm. (E) Risk-of-death analysis of motor neurons transfected at DIV5 with R0–42 constructs. At least 40 motor neurons are imaged/group; n = 3 experiments. **P<0.01. (F) Kaplan-Meier survival analysis showed that hippocampal neurons were not sensitive to expanded intronic G4C2 repeats. At least 80 hippocampal neurons were imaged/group; n = 3 experiments. P = 0.337