Distinct Tau Strains: Exploring Variability in Cell Uptake and Seeding

BACKGROUND: Tauopathies are neurodegenerative diseases characterized by the pathological aggregation of the microtubule-associated protein tau in neurons and glia. These conditions are incurable, progressive, and deadly. Alzheimer's Disease, the most common tauopathy, affects more than 30 million people worldwide and will afflict more than 120 million by 2050. Evidence suggests that tau aggregates spread pathology as do prions, infectious proteins that transmit a pathologic conformation to native proteins via disease-specific conformers (strains). Various tau strains have been identified which propagate stably in cultured cells over many generations. Additionally, evidence shows that tau aggregates enter cells through heparan sulfate proteoglycan (HSPG) mediated macropinocytosis. However, it is unknown if: 1) different tau strains bind HSPGs uniquely or generically to trigger uptake; 2) which HSPG size and sulfation patters are important for cellular uptake of tau. OBJECTIVE: Test for differential inhibition of cellular uptake using heparin, heparinoids, and HSPG modifications; and test effects of HSPG size and sulfation patterns on binding to tau. METHODS: A "biosensor" cell line responsive to tau aggregates was used to measure intracellular tau aggregation based on fluorescence resonance energy transfer (FRET). The biosensors were HEK-293T cells which overexpress the tau repeat domain (RD) with the disease-associated P301S mutation and were tagged with cyan or yellow fluorescent proteins (RD-CFP/YFP). Cell lysate from various strains of tau was used as source material for pathologic tau seeds to induce aggregation of native tau protein within the biosensors. Lysate was incubated with heparin or heparinoids (heparin-derived molecules of varying length and sulfation patterns) for 24-hours and then added to biosensor cells in culture. When incubated in this way, heparin and heparinoids block cellular uptake of tau by preventing its binding to HSPGs. In a separate assay, lysate was added to cultured biosensor cells with CRISPR/Cas9 knockouts of important genes in the HSPG synthesis pathway. In both assays, cells were harvested 48 hours after lysate/lysate-heparinoid addition and seeding was quantified using FRET flow cytometry. RESULTS: All tau strains tested (DS 5, 6, 8, 9, 10, 13, 14, 15, 16, 17) were highly sensitive to heparin inhibition of seeding and most maintained a highly similar dose response (IC50 of ~100 nM). Some strains, however, showed subtle differences. At maximal heparin concentrations (200 ug/mL), noticeably higher seeding vs baseline was observed in DS 5 and 6 (17%, 9%) as compared to the other strains (<5%). When using heparinoids of 4, 8, 12, and 16 disaccharide units to inhibit tau uptake, similar patterns were seen in DS 9 and 10 (seeding reduction: dp4 = 21% vs 19%; dp8 = 27% vs 33%; dp12 = 70% vs 64%; dp16 = 63% vs 46%). Heparinoids that were desulfated at the 2-O, 6-O, and N positions also showed similar patterns of tau uptake inhibition in DS 9 and 10 (De-2-O = 65% vs 53%; De-6-O = 52% vs 25%; De-N = 35% vs 13%). Finally, seeding in HSPG genetic knockout cells was reduced substantially across strains tested in two knockout cell lines (for genes EXT1 and NDST1). Interestingly, DS 5, DS 6, and DS 15 showed less reduction than the other strains in the knockout cell lines (-38%, -50%, and -51% respectively vs roughly -65% for other strains). Finally, seeding in the third knockout cell line (HS6ST2) increased across all strains tested ranging from +13% to +58%. CONCLUSIONS: Cellular uptake of many tau strains is similarly inhibited by heparin, hinting that the same heparinoid (or small molecule analog) could be used to treat diverse tauopathies. However, the unique behavior of some strains suggests that a one-size-fits-all treatment approach may not always be sufficient. Additionally, certain heparin size and sulfation patterns have specific importance for tau binding. Larger heparinoids better inhibited tau seeding (dp16 & dp12 > dp8 & dp4). Regarding sulfation patterns, the relative importance for tau binding of the sulfate moieties tested is: N-sulfation > 6-O-sulfation > 2-O-sulfation. This pattern remains consistent in recombinant tau, DS 9, DS 10, and in the genetic knockout data gathered in this project (using strains) and by others in the laboratory (using recombinant tau). Overall, this data shows many similarities and some differences in cellular uptake between strains of tau. Additional research to further characterize these differences could have important implications for understanding the diversity of tauopathies and finding unique approaches to diagnosis and treatment.