PubMed: 29590627

Extracellular Monomeric and Aggregated Tau Efficiently Enter Human Neurons through Overlapping but Distinct Pathways.
Cell reports
Billinton A | Evans LD | Fraser G | Livesey FJ | Perkinton M | Smith J | Wassmer T

Evidence 7c20488891

Inhibition of actin polymerization with Cytochalasin D disrupts several clathrin-independent endocytic processes, including bulk endocytosis/ macropinocytosis (Mooren et al., 2012)

Evidence 7d49e2c5fd

Entry of monomeric tau was markedly reduced in the presence of 1 mM Cytochalasin D, as reflected in the 95% reduction in the number of monomeric tau-pHrodo-positive objects after 3-hr incubation in the presence of 1 mMCytochalasinD (Figure 5C)

Evidence 286b52c124

In contrast, disruption of actin polymerization with Cytochalasin D had little effect on the entry of aggregated tau (total fluorescent intensity and number of objects; Figures 5D–5F; Figure S5)

Evidence 78886be917

Conversely, dynamin inhibition significantly reduced the entry of aggregated tau, with no significant effects of Cytochalasin D (Figure S6) at this relatively high molar concent of aggregated tau

Evidence 6eb732dfe7

These kinetics of aggregated tau-pHrodo entry are similar to that of both lower concentrations of monomeric tau (2.5 nM) and of low-molecular weight (10-kDa) dextran-pHrodo (same molarity as monomeric tau samples; Figures S5A–S5C

Evidence bb60ae5108

The amount of monomeric tau entering neurons, as measured by total fluorescent intensity of intracellular monomeric tau-pHrodo vesicles, was significantly reduced by dynamin inhibition, as shown at 1 and 3 hr after the addition of extracellular tau (Figure 4B)

Evidence 76e41952c7

The effect of dynamin inhibition on the entry of aggregated tau was more pronounced than on monomeric tau (Figures 4D–4F). The total fluorescent intensity of intracellular aggregated taupHrodo was consistently reduced by more than 70% at 1 and 3 hr after tau addition (Figure 4E), and the number of taupHrodo- positive objects was reduced by 95% (Figure 4F)

Evidence 16a5384c45

We confirmed that heparin within the aggregated tau preparations did not contribute to tau entry into neurons, finding that uptake of both monomeric and aggregated tau was unaffected in the presence of heparin (Figures S4C and S4D

Evidence 658f6161cf

As early as 1 hr after the addition of extracellular tau, monomeric and aggregated tau-Dylight were co-localized in EEA1+ early endosomes

Evidence 5998bdf778

Monomeric and aggregated tau-Dylight were also detected in LAMP1+late endosomes and lysosomes, consistent with endocytosed proteins first reaching early endosomes, before late endosomes and lysosomes

Evidence d06b6c0f01

Thus, monomeric and aggregated tau both efficiently enter neurons via the endosome/lysosome system, and they are actively trafficked within vesicles over long distances within neurons over several hours

Evidence 326635e03b

After extensive washing, monomeric and aggregated tau-Dylight were both detected within cells expressing the neuron-specific microtubule-associated protein MAP2, confirming that both forms of tau enter neurons

Evidence 4126d89d11

Tau-Dylight was found predominantly within the somatic compartment of neurons (Figure 1A)

Evidence 40d0201959

After a 4-hr incubation with extracellular tau, flow cytometry analysis (Figures 1B and 1C) revealed that 83% and 73% of dissociated cells contained monomeric or aggregated tau-Dylight, respectively, demonstrating that extracellular tau efficiently enters human neurons in culture

Evidence ffd8929c4b

Internalization of aggregated tau-pHrodo (Figure 2D) was also found to be concentration dependent (Figure 2E)

Evidence da6b228f6e

Disruption of actin polymerization has previously been shown to inhibit entry of fibrils formed of the tau repeat domain (Holmes et al., 2013)

Evidence 6aabff68ec

These data indicate that, at the concentrations studied, aggregated tau enters neurons almost entirely via endocytosis

Evidence 3e45fa17a6

These independent assays confirmed the same differential effects of the two inhibitors observed by live imaging of pHrodo-tau: at the 3-hr assay point, dynamin inhibition had no effect on the number of monomeric tau-Dylightpositive punctae within neurons, whereas inhibition of actin polymerization reduced the amount of intracellular tau by over half (Figure S6).

Evidence ddb41b5004

We used the tau P301S variant, an autosomal dominant mutation that causes early onset FTD with high penetrance (Bugiani et al., 1999; Guo et al., 2017)

Evidence b52d591674

Internalization of monomeric tau (P301S) and wild-type tau was comparable and concentration dependent (Figure S3A), confirming that the P301S mutation does not confer the ability to efficiently enter neurons, nor is this form of tau likely to aggregate in extracellular media during the 3- to 4-hr incubation period

Evidence 695c9a99f7

Incubation of tau-pHrodo with human neurons at a range of concentrations from 2.5 to 25 nM (0.12–1.2 µg.mL-1, diluted in culture medium) showed that tau entry to neurons is rapid, as visualized by live imaging

Evidence 205ea7fa06

We found that FLAG-tagged tau enters neurons efficiently and that internalized tau persists at detectable levels within neurons for at least 4 days (Figure S4A)

Evidence 4b9d9dbb0d

Intracellular fluorescent punctae were observed within the first 10 min of exposure of neurons to monomeric tau-pHrodo (Figure 2A; Video S1). Tau-pHrodo-positive structures increased in size and intensity over the 4-hr course of the assay. These structures were present within neurites and accumulated in the cell bodies of neurons

Evidence 9be37637d0

In the presence of 15 and 25 nM monomeric tau-pHrodo, the number of tau-pHrodo-positive objects approached a plateau (>90% of final measurement) after approximately 1 hr (Figure 2C)

Evidence 4d2e726166

In agreement with the monomeric tau-pHrodo experiments, monomeric tau-Dylight was rapidly taken up into neurons (Figure 3C


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