Fig. 7: HTTex1 fibrils with disentangled PRDs are more efficient in seeding soluble HTTex1.

a Time-dependent CW EPR intensities of unseeded HTTex1(Q46) 35R1 (blue) and HTTex1(Q46) 35R1 seeded with N- (red), T- (black), and P-fibrils (purple). b EPR intensity of HTTex1(Q46) 35R1 4 h after initiating fibril formation of three separate experiments (n = 3). The decrease in intensity, which is a reporter of fibril formation, is fastest when seeding with P-fibrils followed by seeding with T-, N-fibrils, and unseeded protein. c Puncta formation in Neuro2a cells transfected with HTTex1 with varying polyQ length, and a C-terminal EGFP tag and EGFP as control. Cells were co-transfected with N-, T-, and P-fibril seeds and BSA fibril seeds as control. Fluorescence microscopy images were taken 48 h after transfection. Representative images for HTTex1Q39 (red) and Q72 (burgundy) are shown. Scale bars denotes 40 μm. Puncta formation was measured in 3–11 biological replicates and was reproducible as illustrated in (d). d Fractions of transfected cells containing puncta depending on HTTex1 polyQ length and nature of co-transfected fibrils seeds. Fractions of separate experiments are reported together with boxplots that show 25th to 75th percentiles, the median in orange, and whiskers from minimum to maximum. Significant P values from a two-sided t-test are indicated. P-fibril seeds led to the strongest induction of puncta in cells transfected with HTTex1Q72 (burgundy) and Q39 (red) followed by T-fibrils. The effect of N-fibril and BSA fibril seeds on puncta formation was roughly the same. Few puncta were observed for HTTex1Q16 (orange) and EGFP (yellow) independent of co-transfected fibril seeds. e Fibril toxicity using STHDH Q7/111 cells. Representative images of DAPI-labeled cells to which fibril types were exogenously added. White arrows point DAPI-labeled brighter small puncta, which are recognized as dead cells. Scale bars denote 20 μm. Data were reproducible as illustrated in (f). f Percent of dead cells detected in ten images coming from three biological replicates. Each individual percentage together with a boxplot is shown. Boxplots indicate 25th to 75th percentiles, the median in orange, and whiskers from minimum to maximum. Significant P values from two tailed t-tests are indicated.