pubmed.ncbi.nlm.nih.gov

Internalization mechanisms of brain-derived tau oligomers from patients with Alzheimer's disease, progressive supranuclear palsy and dementia with Lewy bodies - PubMed

  • ️Wed Jan 01 2020

Internalization mechanisms of brain-derived tau oligomers from patients with Alzheimer's disease, progressive supranuclear palsy and dementia with Lewy bodies

Nicha Puangmalai et al. Cell Death Dis. 2020.

Abstract

Tau aggregates propagate in brain cells and transmit to neighboring cells as well as anatomically connected brain regions by prion-like mechanisms. Soluble tau aggregates (tau oligomers) are the most toxic species that initiate neurodegeneration in tauopathies, such as Alzheimer's disease (AD), progressive supranuclear palsy (PSP), and dementia with Lewy bodies (DLB). Exogenous tau aggregates have been shown to be internalized by brain cells; however, the precise cellular and molecular mechanisms that underlie the internalization of tau oligomers (TauO) remain elusive. Using brain-derived tau oligomers (BDTOs) from AD, PSP, and DLB patients, we investigated neuronal internalization mechanisms of BDTOs, including the heparan sulfate proteoglycan (HSPG)-mediated pathway, clathrin-mediated pathway, and caveolae-mediated pathway. Here, we demonstrated that the HSPG-mediated pathway regulates internalization of BDTOs from AD and DLB, while HSPG-mediated and other alternative pathways are involved in the internalization of PSP-derived tau oligomers. HSPG antagonism significantly reduced the internalization of TauO, prevented tau translocation to the endosomal-lysosomal system, and decreased levels of hyperphosphorylated tau in neurons, the well-known contributor for neurofibrillary tangles (NFT) accumulation, degeneration of neurons, and cognitive decline. Furthermore, siRNA-mediated silencing of heparan sulfate (HS)-synthesizing enzyme, exostosin-2, leads to decreased internalization of BDTOs, prevented tau-induced autophagy-lysosomal pathway impairment, and decreased hyperphosphorylated tau levels. Collectively, these findings suggest that HSPG-mediated endocytosis and exostsin-2 are involved in neuronal internalization of TauO and subsequent tau-dependent neuropathology in AD and DLB.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. HSPG antagonist reduces BDTOs internalization in neurons.

a–c Primary cortical neurons were pretreated with internalization inhibitors for 30 min before treating with 0.5 μM BDTOs for 1 h. AD TauO (a), PSP TauO (b), and DLB TauO (c) internalized into cells ~55%, 60%, and 45%, respectively. The percentage of AF568-tagged tau-positive cells and median fluorescence intensity (right panel) from AD TauO (a), PSP TauO (b), and DLB TauO (c) applications were significantly diminished in a concentration-dependent fashion by Heparin (a HSPG antagonist), whereas Dynasore (a clathrin inhibitor) and Nystatin (a caveolae inhibitor) did not show inhibitory effects. A total of 10,000 cells were analyzed for each condition in triplicate. Error bars show SEM. ***p < 0.001, ****p < 0.0001 vs vehicle control (Veh. Con; 0.02% (v/v) DMSO), ##p < 0.01, ###p < 0.001, ####p < 0.0001 vs TauO-treated. All histograms (right panel) represent integrated median fluorescence intensity of BDTOs in cells pretreated with 26–38 μg/ml Dynasore, 1–200 μg/ml Heparin, or 1–40 μg/ml Nystatin. AF568-tagged tau fibrils were used as the negative control in all experiments. See also Supplementary Fig. S3a. d–g Dextran and BDTOs uptake were associated with HSPG. A total of 20 μg/ml of 70,000 MW lysine-flexible Texas Red-conjugated dextran was applied to cells for 1 h. d Cells were fixed and immunostained for HSPG (green) and a mature neuronal marker (βIII-tubulin, blue). Representative orthogonal images indicated AF488-tagged AD TauO (e), PSP TauO (f), and DLB TauO (g) co-localized to dextran, indicating the HSPG-mediated uptake. Scale bar: 10 μm.

Fig. 2
Fig. 2. Exogenous tau oligomers bind to postsynaptic marker.

Neurons were exposed to AF568-tagged TauO (Red) from AD, PSP, or DLB for 1 h. Cells were immunolabeled with a presynaptic marker (Synapsin I, green) (a), a postsynaptic marker (PSD-95, green) (b), and a mature neuronal marker (βIII-tubulin, blue). Representative regions of interest are depicted in white rectangles with inserted high-magnification below. Scale bar is indicated. AD, PSP, or DLB TauO are located near the presynaptic marker. White arrows indicate AD, PSP, or DLB TauO co-localized with the postsynaptic marker. c Pearson’s correlation coefficient analysis of exogenous AD, PSP, and DLB TauO with pre- and postsynaptic markers over 1 h. Each treatment group was randomly imaged in five different regions of interest, and performed in triplicate. Image analyses were calculated by one-way ANOVA with Tukey’s multiple comparison test. Results showed as the value of mean ± SEM, ****p < 0.0001.

Fig. 3
Fig. 3. Localization of internalized tau oligomers with the endosomal– lysosomal system.

a, e Neurons were exposed to AF568-tagged TauO (Red) from AD, PSP, or DLB for 1 h with (+) or without (−) Heparin pretreatment. Cells were fixed and immunostained for a mature neuronal marker (βIII-tubulin, blue), an early endosomal marker (Rab5, green) (a) and a lysosomal marker (LAMP-2, green) (e). Representative orthogonal images indicate AF568-tagged TauO co-localized to early endosomes and lysosomes, indicated by arrows. Scale bar: 2 and 10 μm. b–d Pearson’s correlation coefficient analysis of internalized AD TauO (b), PSP TauO (c), DLB TauO (d) with early endosome over 1 h was demonstrated with similar experimental conditions and parameters as for (a). Each treatment group was randomly imaged in five different regions of interest, and performed in duplicate. f–h Pearson’s correlation coefficient analysis of lysosome with internalized AD TauO (f), PSP TauO (g), and DLB TauO (h) was demonstrated, using the same experimental conditions as for (e). Image analyses were calculated by unpaired and two-tailed Student’s t test. Results showed as the value of mean ± SEM, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs without (−) Heparin.

Fig. 4
Fig. 4. Exostosin-2 knockdown prevented internalization and accumulation of extracellular tau species in neurons.

a, b Primary neurons (DIV2) were applied AccellTM SMARTpool siRNA for non-targeting (NT) or Ext2 gene (NM_001355075) at concentrations of 50–500 nM for 96 h. 18s rRNA was used for the reference gene and normalization in gene expression analysis. Ext2 siRNA (500 nM) significantly reduced the exostosin-2 mRNA (a) and protein (b) levels compared with NT siRNA. Results were from three independent experiments and shown as fold difference expression relative to control (mean ± SEM). Statistical analyses were measured by one-way ANOVA with Tukey’s test (a) or unpaired, two-tailed Student’s t test (b) from three biological independent experiment. Results showed as the value of mean ± SEM, a ****p < 0.0001 vs UT group, ##p < 0.01, ####p < 0.0001 vs NT group, b **p < 0.01 vs NT group. c–e Neurons (DIV2) were preincubated with NT or Ext2 siRNA for 96 h followed by AF568-tagged TauO from AD (c), PSP (d), or DLB (e) treatment for 18 h. Cells were immunolabeled with a mature neuronal marker (βIII-tubulin, blue), and an early endosomal marker (Rab5, green). Representative orthogonal images depicted AF568-tagged TauO co-localized to early endosomes (arrows). Scale bar: 2 and 10 μm. f–h Neurons were untreated (UT) or treated with 0.1 μM biotin-tagged TauO (+) from AD (f), PSP (g), or DLB (h) with similar experimental conditions and parameters as for (c–e). Internalized tau was detected using anti-Streptavidin antibody. Representative Western blot images depicted the appearance of exogenously applied TauO. Internal controls from the same blot were probed with anti-βIII-tubulin. Analysis of internalized tau levels was on the lower panel of each immunoblot showing as Streptavidin band intensity (HMW: 75–250 kDa) normalized to internal control and presented as the percentage of UT group. Statistical analyses were measured by one-way ANOVA with Tukey’s test from three biological independent experiments. Results showed as the value of mean ± SEM, **p < 0.01, ***p < 0.001 vs UT group. ##p < 0.01 vs TauO-treated group. Western blot analyses of TauO from AD, PSP, or DLB were performed on separate membranes. The same membranes were re-probed for marker proteins of autophagy–lysosomal pathway as shown in Fig. 5a–c. The immunoblots for internal controls shown in Fig. 4f–h were reused in Fig. 5a–c.

Fig. 5
Fig. 5. Exogenous tau oligomers mediated alternations of autophagy–lysosomal pathway (ALP) in neurons.

Cells were pretreated with NT or Ext2 siRNA followed by the presence or absence of 0.1 μM or 0.5 μM biotin-tagged TauO from AD (a), PSP (b), or DLB (c). All experiments were performed with similar conditions and parameters as for Fig. 4f–h (cells pretreated with NT or Ext2 siRNA followed by the presence or absence of 0.1 μM biotin-tagged TauO), together with samples from cells preincubated with NT or Ext2 siRNA followed by 0.5 μM biotin-tagged TauO. Representative Western blot images revealed the expression of p62 (autophagy receptor), LC3B-II (autophagosome membrane formation), and LAMP-2 (lysosome). Quantification of band intensity shown below was normalized to βIII-tubulin. The same immunoblots probed with loading controls shown in Fig. 4f–h were reused in Fig. 5a–c. a AD TauO-exposed cells showed a significant increase in p62 and LAMP-2 levels, yet a drastic decrease was seen in LC3B-II/LC3B-I ratio. Reverse effects were observed in Ext2 siRNA-pretreated group. b PSP TauO-treated cells slightly changed the expression of p62, but did not alter LAMP-2 expression. LCB-II/LC3B-I ratio was significantly reduced in PSP TauO-treated, while Ext2 siRNA exposure alleviated the PSP TauO effect without reaching statistical significance. c Reductions of p62, LCB-II/LC3B-I ratio, and LAMP-2 were found in DLB TauO-exposed neurons, while the reverse effects from Ext2 siRNA-pretreated were not observed. Statistical analyses were measured by one-way ANOVA with Tukey’s test from three biological independent experiments. Results showed as the value of mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs UT group. #p < 0.05, ##p < 0.01, ###p < 0.001 vs the TauO-treated group.

Fig. 6
Fig. 6. Ext2 knockdown mitigates extracellular tau oligomer-induced phosphorylated tau in neurons.

Cells were pretreated with NT or Ext2 siRNA followed by the presence or absence of TauO from AD (a), PSP (b), or DLB (c). All experiments were performed with similar conditions and parameters to experiments indicated in Figs. 4 and 5. Representative Western blot analyses and quantifications showed levels of p-Tau detected by monoclonal antibodies AT180 and AT8, and total tau indicated by total Tau antibody. βIII-tubulin was used for loading control and normalization similar to Figs. 4 and 5. a AD TauO-treated cells (0.1 or 0.5 μM) revealed a significant increase of p-Tau (AT180 and AT8) in a concentration-dependent manner, which were reduced in Ext2 siRNA-pretreated group. b PSP TauO (0.5 μM) significantly elevated the level of p-Tau (AT8), as well as increased the p-Tau (AT180). c DLB TauO-exposed cells altered p-Tau (AT180) levels, while pretreatment of Ext2 siRNA drastically reduced the p-Tau (AT180) expression. Changes were not observed in p-Tau (AT8) after DLB TauO treatment. Statistical analyses were measured by one-way ANOVA with Tukey’s test from three biological independent experiments. Results showed as the value of mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs the UT group. ##p < 0.01, ####p < 0.0001 vs the TauO-treated group.

Similar articles

Cited by

References

    1. Braak H, Braak E. Demonstration of amyloid deposits and neurofibrillary changes in whole brain sections. Brain Pathol. 1991;1:213–216. doi: 10.1111/j.1750-3639.1991.tb00661.x. - DOI - PubMed
    1. Dujardin S, et al. Neuron-to-neuron wild-type tau protein transfer through a trans-synaptic mechanism: relevance to sporadic tauopathies. Acta Neuropathol. Commun. 2014;2:14. doi: 10.1186/2051-5960-2-14. - DOI - PMC - PubMed
    1. Liu L, et al. Trans-synaptic spread of tau pathology in vivo. PLoS ONE. 2012;7:e31302. doi: 10.1371/journal.pone.0031302. - DOI - PMC - PubMed
    1. Kaufman SK, et al. Tau prion strains dictate patterns of cell pathology, progression rate, and regional vulnerability in vivo. Neuron. 2016;92:796–812. doi: 10.1016/j.neuron.2016.09.055. - DOI - PMC - PubMed
    1. Xu D, Esko JD. Demystifying heparan sulfate-protein interactions. Annu. Rev. Biochem. 2014;83:129–157. doi: 10.1146/annurev-biochem-060713-035314. - DOI - PMC - PubMed

Publication types

MeSH terms

Substances