Distinct C9orf72-Associated Dipeptide Repeat Structures Correlate with Neuronal Toxicity
Fig 5
DPRs are internalized by neurons.
(A) Fluorescence micrographs of neurons treated with the indicated peptide and stained with antibodies recognizing the pan-neuronal marker (MAP2, red), the indicated DPR (GA, GP or GR; green), and a nuclear dye (4’,6-diamidino-2-phenylindole (DAPI), blue). Scale bar, 20 μm. (B) Frequency of DPR internalization detected by immunocytochemistry. (C) Quantification of the data in panels D-G, showing internalization of FITC-labeled DPRs. (D-G) Confocal microscopy of neurons treated with the indicated peptide shows intracellular accumulations of FITC-(GA)6 and -(GR)6, but not -(GP)6 or buffer. Representations of the y-z axis appear above, and x-z plots to the right of each micrograph. Scale bar in (D), 20 μm, applies to (D-G). (H-K) Intensity plots of fluorescence across a line that intercepts the inclusions shown in D-G, demonstrating overlapping MAP2 and FITC signals for (GA)6 and (GR)6 but not (GP)6. For (B) and (C), ***, p<0.05 one-way ANOVA with Dunnett’s test. n = 100–150 neurons per condition, pooled from 3 replicates and 2 independent experiments. ***, p<0.05 one-way ANOVA with Dunnett’s test.