Figure 2.

Stabilization of tau fibrils and seeds in vitro.A, tau fibrils preformed with polyA RNA or heparin were treated with RNase (T1 and A, 316 U/μl and 3.2 mg/ml), DNase (I, 633 U/μl), or heparinase I (3797 U/ml) for 24 h before transduction of biosensors. (+) signifies RNase treatment added to fibrils at same time as seeding, with no pre-incubation. Seeds formed with polyA were sensitive to RNase treatment (p < 0.0001). RNase added directly to seeds at the time of seeding had no effect (p = 0.49). DNase and heparinase treatment did not affect seeds formed by polyA (p = 0.99 and 0.97). Seeds formed with heparin were sensitive to heparinase treatment (p < 0.0001), but not RNase or DNase (p = 0.09 RNase, p > 0.99 immediate RNase, p = 0.24 DNase). p values were calculated with one-way repeated measures ANOVA, post-hoc Dunnett’s multiple comparisons test, comparing control to enzyme treated conditions for both polyA and heparin fibrils. Seeding data represents two experimental replicates, each performed in technical triplicate. These experiments are representative of similar studies performed 15 times overall. Error bars = S.D. B, TEM 400CF grids containing tau (4 μM) stained with 2% UA. All grids were scanned; representative images are shown. Scale bar = 200 nm. Fibrils formed by polyA lose integrity after exposure to RNase, but not heparinase. Heparin fibrils were undetectable after heparinase treatment, but not RNase. FRET, fluorescence resonance energy transfer. ∗∗∗∗p < 0.0001.