The dual fates of exogenous tau seeds: Lysosomal clearance versus cytoplasmic amplification

Abstract

Tau assembly movement from the extracellular to intracellular space may underlie transcellular propagation of neurodegenerative tauopathies. This begins with tau binding to cell surface heparan sulfate proteoglycans, which triggers macropinocytosis. Pathological tau assemblies are proposed then to exit the vesicular compartment as “seeds” for replication in the cytoplasm. Tau uptake is highly efficient, but only ∼1 to 10% of cells that endocytose aggregates exhibit seeding. Consequently, we studied fluorescently tagged full-length (FL) tau fibrils added to native U2OS cells or “biosensor” cells expressing FL tau or repeat domain. FL tau fibrils bound tubulin. Seeds triggered its aggregation in multiple locations simultaneously in the cytoplasm, generally independent of visible exogenous aggregates. Most exogenous tau trafficked to the lysosome, but fluorescence imaging revealed a small percentage that steadily accumulated in the cytosol. Intracellular expression of Gal3-mRuby, which binds intravesicular galactosides and forms puncta upon vesicle rupture, revealed no evidence of vesicle damage following tau exposure, and most seeded cells had no evidence of endolysosome rupture. However, live-cell imaging indicated that cells with pre-existing Gal3-positive puncta were seeded at a slightly higher rate than the general population, suggesting a potential predisposing role for vesicle instability. Clearance of tau seeds occurred rapidly in both vesicular and cytosolic fractions. The lysosome/autophagy inhibitor bafilomycin inhibited vesicular clearance, whereas the proteasome inhibitor MG132 inhibited cytosolic clearance. Tau seeds that enter the cell thus have at least two fates: lysosomal clearance that degrades most tau, and entry into the cytosol, where seeds amplify, and are cleared by the proteasome. Tau assembly movement from the extracellular to intracellular space may underlie transcellular propagation of neurodegenerative tauopathies. This begins with tau binding to cell surface heparan sulfate proteoglycans, which triggers macropinocytosis. Pathological tau assemblies are proposed then to exit the vesicular compartment as “seeds” for replication in the cytoplasm. Tau uptake is highly efficient, but only ∼1 to 10% of cells that endocytose aggregates exhibit seeding. Consequently, we studied fluorescently tagged full-length (FL) tau fibrils added to native U2OS cells or “biosensor” cells expressing FL tau or repeat domain. FL tau fibrils bound tubulin. Seeds triggered its aggregation in multiple locations simultaneously in the cytoplasm, generally independent of visible exogenous aggregates. Most exogenous tau trafficked to the lysosome, but fluorescence imaging revealed a small percentage that steadily accumulated in the cytosol. Intracellular expression of Gal3-mRuby, which binds intravesicular galactosides and forms puncta upon vesicle rupture, revealed no evidence of vesicle damage following tau exposure, and most seeded cells had no evidence of endolysosome rupture. However, live-cell imaging indicated that cells with pre-existing Gal3-positive puncta were seeded at a slightly higher rate than the general population, suggesting a potential predisposing role for vesicle instability. Clearance of tau seeds occurred rapidly in both vesicular and cytosolic fractions. The lysosome/autophagy inhibitor bafilomycin inhibited vesicular clearance, whereas the proteasome inhibitor MG132 inhibited cytosolic clearance. Tau seeds that enter the cell thus have at least two fates: lysosomal clearance that degrades most tau, and entry into the cytosol, where seeds amplify, and are cleared by the proteasome. Multiple lines of experimental evidence suggest that tau protein triggers neurodegeneration after intracellular accumulation in ordered assemblies. Myriad tauopathies are linked to distinct assembly structures and include Alzheimer’s disease (AD), frontotemporal dementia, and chronic traumatic encephalopathy, among many others (1Lee V.M.-Y. Goedert M. Trojanowski J.Q. Neurodegenerative tauopathies.Annu. Rev. Neurosci. 2001; 24: 1121-1159Crossref PubMed Scopus (2087) Google Scholar). The transcellular propagation and faithful replication of unique assembly structures, or “strains,” appears to underlie the characteristic progression patterns of specific tauopathies (2Vaquer-Alicea J. Diamond M.I. Propagation of protein aggregation in neurodegenerative diseases.Annu. Rev. Biochem. 2019; 88: 785-810Crossref PubMed Scopus (102) Google Scholar). Tau propagation presumably involves three steps: uptake of an assembly into the cell; amplification and maintenance of the aggregated state; and exit from the cell. We originally observed that tau binding to heparan sulfate proteoglycans on the cell surface underlies uptake via macropinocytosis, and is mediated by specific heparan sulfate proteoglycans sulfation patterns (3Holmes B.B. DeVos S.L. Kfoury N. Li M. Jacks R. Yanamandra K. et al.Heparan sulfate proteoglycans mediate internalization and propagation of specific proteopathic seeds.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: E3138-E3147Crossref PubMed Scopus (512) Google Scholar, 4Stopschinski B.E. Holmes B.B. Miller G.M. Manon V.A. Vaquer-Alicea J. Prueitt W.L. et al.Specific glycosaminoglycan chain length and sulfation patterns are required for cell uptake of tau versus α-synuclein and β-amyloid aggregates.J. Biol. Chem. 2018; 293: 10826-10840Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). After uptake into macropinosomes, an assembly must make contact with endogenous tau in the cytoplasm to serve as a template and amplify a specific structure. Several reports suggest that this might happen after vesicle rupture (5Chen J.J. Nathaniel D.L. Raghavan P. Nelson M. Tian R. Tse E. et al.Compromised function of the ESCRT pathway promotes endolysosomal escape of tau seeds and propagation of tau aggregation.J. Biol. Chem. 2019; 294: 18952-18966Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 6Calafate S. Flavin W. Verstreken P. Moechars D. Loss of Bin1 promotes the propagation of tau pathology.Cell Rep. 2016; 17: 931-940Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), but the details are unclear. Finally, when tau seeds enter the cytoplasm, it is unknown how they are degraded. In this study, we used cultured cells to dynamically visualize tau uptake and seeding, to track the relationship of seeding to vesicle processing and rupture, and to determine mechanisms of seed degradation in the vesicular versus cytoplasmic compartments. Wildtype full-length (FL) tau undergoes seeded aggregation inefficiently in cultured cells, and thus, we studied FL (2N4R) tau containing a disease-associated P301S mutation, fused to mClover3 (FL tau-Clo) (Fig. 1A). We stably expressed FL tau-Clo in U2OS cells, where it colocalized with tubulin (Fig. 1B). We then directly imaged U2OS biosensor cells that were exposed to exogenous FL wildtype (2N4R) tau fibrils covalently labeled with Alexa Fluor 647 (AF647), tracking FL tau-Clo puncta formation over time. Similar to our original observations (7Frost B. Jacks R.L. Diamond M.I. Propagation of tau misfolding from the outside to the inside of a cell.J. Biol. Chem. 2009; 284: 12845-12852Abstract Full Text Full Text PDF PubMed Scopus (827) Google Scholar), a minority of cells exhibited induced aggregation of tau, which predominated in the cytoplasm (Fig. 1C). Time-lapse imaging (IN Cell Analyzer 6000, GE) also revealed intracellular aggregation in the cytoplasm (Fig. 1D), and in imaging dynamic inclusion formation in ∼50 cells, we often observed aggregates forming simultaneously throughout the cytoplasm (Movie S1). For nascent intracellular aggregates, we observed no significant colocalization with AF647-labeled exogenous tau. This raised the question of how tau seeds traffic into the cytoplasm. To study the fate of internalized tau, we exposed U2OS to FL tau fibrils labeled with AF647, imaging them repeatedly over 2 days with high-content microscopy. We first tested for colocalization with vesicles by stably expressing mRuby3 fusions to Rab5 (to mark early endosomes, Figs. 1A and 2A), Rab7 (to mark late endosomes, Figs. 1A and 2B), and lysosomal-associated membrane protein 1 (LAMP1) (to mark lysosomes, Figs. 1A and 2C). Tau progressively colocalized with these markers, especially LAMP1. We concluded that most internalized tau entered the endolysosomal pathway. When we tracked seeding in U2OS biosensor cells expressing FL tau-Clo using LAMP1, we observed no significant colocalization of emergent tau puncta with the lysosome, which was largely in a separate compartment versus the induced FL-tau-Clo aggregates (Fig. 3A). FL tau seeding is inefficient enough to make capture of large numbers of seeding events relatively difficult. We therefore performed the same experiment using repeat domain tau-Clo containing a P301S mutation (RD tau-Clo) to image a high number of seeded cells (Fig. 3B). Similar to our observation with FL tau, we observed no significant colocalization between RD tau-Clo aggregates and LAMP1 (Fig. 3C). Thus, although most internalized tau wound up in the lysosome, this seemed unlikely to be the primary location of seeding.Figure 3Tau inclusion formation is independent of the lysosome. A, U2OS cells expressing FL tau-Clo and LAMP1-Rub were treated with exogenous fibrils. Cells were observed over time for coincidence of LAMP1-Rub signal and FL tau-Clo inclusion formation. A representative image shows the seeded tau (white arrowhead) and LAMP1 signal (magenta). No colocalization was observed between tau aggregates (white arrow) and lysosome (yellow arrow). Time after tau fibril addition is indicated above each column. The images are representative of five seeded cells studied. Scale bars = 20 μm. B, U2OS cells expressing RD tau-Clo and LAMP1-Rub were treated with tau fibril and followed over time for coincidence of LAMP1 signal and RD tau-Clo aggregation. No colocalization was observed between tau aggregates (white arrowhead) and the LAMP1 marking lysosome (yellow arrowhead). The images are representative of 50 seeded cells. Scale bars = 20 μm. C, quantification of colocalization of LAMP1-Rub with RD tau-Clo aggregates. The graph shows colocalization coefficient (Mander’s overlap coefficient) from 50 seeded cells, and the bar indicates the mean. LAMP1, lysosomal-associated membrane protein 1; RD, repeat domain.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Since we did not observe significant colocalization of newly formed tau puncta with vesicle markers, we hypothesized that tau seeds might be released into the cytoplasm from a vesicular pool. After exposing U2OS cells to FL tau fibrils labeled with AF647, we detected diffuse fluorescence in the cytoplasm at 20 h (Fig. 4A). We next used live-cell imaging to monitor hundreds of cells exposed to FL tau fibrils tagged with AF647. We observed a small but steady increase of cytosolic AF647 signal, whereas transferrin-AF647 did not increase in signal following a similar exposure protocol (Fig. 4B). To test our observations by a different approach, we used cell fractionation based on differential centrifugation to measure tau seed levels in cytosol versus organelle (vesicle) fractions (Fig. 4C). We confirmed the accuracy of the fractionations using Western blot against GAPDH (cytosol), voltage-dependent anion channel (organelle), LAMP1 (organelle), and lamin B1 (nucleus) (Fig. 4D). We attempted to quantify tau protein levels via Western blot, ELISA, and mass but they were for We next seeding in the cytoplasm by into a biosensor based on a cell B.B. et tau seeding in Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, Vaquer-Alicea J. Manon V.A. et tau biosensor cells no seeding in Alzheimer’s disease PubMed Scopus Google Scholar). We observed a steady increase in cytosol seeding over time (Fig. Tau seeds thus steadily from vesicles to the cytosol. have proposed that damage to vesicles might into the cytoplasm to seeding S. Flavin W. Verstreken P. Moechars D. Loss of Bin1 promotes the propagation of tau pathology.Cell Rep. 2016; 17: 931-940Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, B. J. Goedert M. against seeded tau aggregation.J. Biol. Chem. 2018; 293: Full Text Full Text PDF PubMed Scopus Google Scholar). To test this we expressed the protein fused to mRuby3 to observe the relationship of vesicle rupture and tau seeding. to the of the cell membrane and are in the of expressed is However, an is binds puncta M. N. J. J. et a for by PubMed Scopus Google Scholar, J.J. in and Neurosci. 2018; PubMed Scopus Google Scholar). We stably expressed in U2OS cells and tracked puncta formation using high-content microscopy. in most cells (Fig. exposure of cells to FL tau fibrils tagged with AF647, we observed tau in cell. However, we observed no in puncta formation (Fig. This was confirmed by of hundreds of cells (Fig. We this with to which induced puncta formation (Fig. In we observed no in vesicle upon tau exposure, suggesting that tau fibrils not damage We used live-cell imaging to track seeding into cells using expression of FL tau-Clo as a We observed seeding into cells with and puncta (Fig. A and FL tau is relatively to quantify puncta in to seeding we used the RD This of hundreds of seeding We by in an that of cells exhibited puncta (Fig. of cells with seeding exhibited whereas did not (Fig. The higher of puncta with seeded cells to test the relationship We used dynamic imaging of cells in to that tau after exogenous seeding. We then tracked the cells h to the of tau to determine the percentage that no puncta at time puncta or pre-existing puncta (Fig. The large of seeded cells did not evidence of vesicle rupture. The number with pre-existing evidence of vesicle was slightly higher than the we observed in cells To directly the seeding after rupture of we tested the of vesicle rupture by versus seed via We observed a increase in seeding after to which presumably aggregates directly to the cytoplasm, seeding to (Fig. We concluded that vesicle may the of seeding this is not the primary observations were with escape of tau seeds from the vesicular compartment to the cytosol, where seeding The of tau in each compartment thus determine the of seeding. Consequently, we the of tau seed clearance using of seeds from each with using biosensor cells Vaquer-Alicea J. Manon V.A. et tau biosensor cells no seeding in Alzheimer’s disease PubMed Scopus Google Scholar). We exposed U2OS cells to FL tau followed by fractionation of cells into organelle (vesicle) versus cytosol fractions. We used to test for seeding the organelle (to on biosensor and directly the cytosol In the organelle tau had by h (Fig. In the cytosol clearance was slightly with tau seeding at h (Fig. We attempted to and mass to monitor tau clearance but levels were for using large of tau in cultured cells. We next tested the of of the lysosome/autophagy and proteasome on seed clearance over a time to degradation in the organelle (Fig. but had no on the cytosol (Fig. MG132 had no on organelle clearance (Fig. but clearance of tau seeds from the cytosol (Fig. We observed a seed clearance in the organelle In the cytosol, to be a clearance with of h and a of to the two of seed clearance for in the seeds are via the lysosome, whereas in the cytoplasm, seeds are cleared rapidly via the proteasome. is unknown how a tau assembly a unique from the outside to the inside of a cell. 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Full Text Full Text PDF PubMed Scopus Google have used to cytoplasmic of tau assemblies. In biosensor cells expressing FL we observed seeding in of exposed cells, of the cells up labeled tau assemblies into the endolysosomal We expressed FL tau-Clo that the cytoplasmic tubulin after tau seed exposure, we observed of tau puncta throughout the cytoplasm. The of puncta the cell that may be a pathway this In not we tested for a role for the cell but did not observe We have concluded that seeds not visible by the endolysosomal compartment to endogenous FL tau-Clo from the tubulin our following macropinocytosis, only a small of tau assemblies up in the cytosol, where they serve as for have that tau the cytosol by vesicles S. Flavin W. Verstreken P. Moechars D. Loss of Bin1 promotes the propagation of tau pathology.Cell Rep. 2016; 17: 931-940Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, B. J. Goedert M. against seeded tau aggregation.J. Biol. 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When we treated cells with to rupture following with tau, we only a increase in seeding, from to In with seeding This that the by which tau the cytosol may its seeding and against vesicle rupture as a primary The of our to of others S. Flavin W. Verstreken P. Moechars D. Loss of Bin1 promotes the propagation of tau pathology.Cell Rep. 2016; 17: 931-940Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, B. J. Goedert M. against seeded tau aggregation.J. Biol. Chem. 2018; 293: Full Text Full Text PDF PubMed Scopus Google may our of different and tau fibrils. a study using cells and primary also concluded that tau not damage vesicles Miller S. M. et assemblies enter the cytosol in a to seeded Scopus Google Scholar). We are to the by which assemblies might the vesicle membrane in the of rupture, via suggest of tau seeds into two the endolysosome and the cytoplasm. 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