Figure 7.

Cholesterol extraction promotes seeded aggregation in organotypic hippocampal slice cultures

(A–C) Levels of seeded aggregation in brain slice cultures from P301S tau transgenic mice treated with the relevant solvent control or (A) endocytosis inhibitors (Pitstop 2, 10 μM; Dyngo 4a, 2 μM), (B) cyclodextrins (γCD and MβCD, 200 μM), and (C) 25-HC (10 μM) for 24 h prior to addition of fresh drug and 100 nM tau assemblies for 3 days. Medium was exchanged, and slice cultures were incubated for another 3 weeks prior to fixation and immunostaining. Slices are from N = 6 mice.

(D) Quantification of seeded aggregation in slice cultures from P301S transgenic mice treated with 100 nM tau assemblies in the presence of 200 μM or 1 mM MβCD as in (A). Slices are from N = 6 mice.

(E) Fluorescence microscopy images of entire slice cultures with details of hippocampal (blue) and cortical (orange) areas. OHSCs were seeded with 100 nM tau assemblies in the presence or absence of 1 mM MβCD. The cortex; hippocampal CA1, CA2, and CA3; and the dentate gyrus (DG) are labeled. Scale bar, 500 μm and 50 μm (hippocampus and cortex, respectively).

(F) Percent maximum seeding in the presence of MβCD (200 μM) or solvent (water). Seeded aggregation was quantified in OHSCs from P301S tau transgenic mice. A black line indicates a single-hit curve. Slices are from N = 6 mice.

All error bars indicate mean ± SEM. ∗p < 0.05 by Student's t test (C), ∗∗p < 0.01, ∗∗∗∗p < 0.0001 by Kruskal-Wallis test with Dunn's comparisons (A, B, and D)