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. 2022 May 6;39(5):110776. doi: 10.1016/j.celrep.2022.110776

Figure 5.

Figure 5

Entry of tau assemblies into mouse primary neurons depends on receptor-mediated uptake and is controlled by cholesterol

(A) Entry of 50 nM tau-HiBiT assemblies into GPLN neurons in 1 h, 72 h after knockdown with Lrp1 or NTC siRNA; n = 3, N = 3 independent experiments.

(B) Western blot for LRP1 and GAPDH in cells from (A).

(C) Confocal microscopy images (z stack) of WT neurons (DIV 7) immunostained for LRP1 and MAP2 after a 1-h tau uptake assay with 200 nM tau-GFP assemblies. White arrows indicate examples of colocalization of tau assemblies and LRP1. Scale bar, 5 μm.

(D) Entry of 50 nM tau-HiBiT assemblies into GPLN-neurons in the presence of the indicated concentration of heparin, with entry measured 1 h after challenge; n = 3.

(E and F) Effect of cholesterol depletion in GPLN neurons (E) and GPLN-expressing human iNeurons (F). Neurons were pre-treated with γCD (2 mM), MβCD (2 mM), or solvent (water) for 2 h before addition of 50 nM tau-HiBiT assemblies for 1 h; n ≥ 4, N = 3 independent experiments/differentiations (E and F).

(G) Effect of exogenous cholesterol on tau entry in cholesterol-depleted GPLN neurons. Cells were pre-treated with MβCD (500 μM) or solvent (water) with or without 10 μM TopFluor-cholesterol (TF-cholesterol; TFC) for 2 h before challenge with 50 nM tau-HiBiT assemblies. Entry was measured 1 h after challenge with tau-HiBiT assemblies; n = 3.

(H) Confocal microscopy image of WT DIV 7 primary neurons with 10 μM TF-cholesterol added 16 h before. Scale bar, 5 μm.

(I) Titration of TF-cholesterol onto GPLN neurons for 1 h prior to a 1-h entry assay with 50 nM tau-HiBiT assemblies; n = 3. Pearson correlation coefficient (r)= −0.98, ∗∗p = 0.0041.

(J and K) Entry of 50 nM tau-HiBiT assemblies in GPLN neurons 1 h after challenge, which followed 1-h pre-treatment with 10 μM 24(s)-HC (J), 25-HC (K), or solvent (DMSO or ethanol [EtOH]); n = 3, N = 3–4 independent experiments.

(L) Entry of 50 nM tau-HiBiT assemblies into GPLN-expressing iNeurons in 1 h after 1-h pre-treatment with 10 μM 25-HC or solvent (EtOH); n = 3, N = 3 independent differentiations.

(M and N) Entry of 50 nM tau-HiBiT assemblies into GPLN neurons 1 h after 72-h treatments with siRNA against Npc1 or NTC siRNA (M) with western blot to confirm protein depletion (N); n = 3, N = 3 independent experiments.

All error bars indicate mean ± SEM. ∗p < 0.05 by Student's t test (L and J), ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 by one-way ANOVA with Tukey's comparisons (A, E, F, G, K, and M).