Browsing by Subject "Tauopathies"
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Item Distinct Tau Strains: Exploring Variability in Cell Uptake and Seeding(2019-03-15) Prueitt, William Lloyd; Diamond, Marc; Stopschinski, Barbara; Joachimiak, LukaszBACKGROUND: 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.Item Distinct Tau Strains: Exploring Variability in Cell Uptake and Seeding Using Heparinoids(2018-01-23) Prueitt, William; Stopschinski, Barbara; Diamond, MarcBACKGROUND: Tauopathies (including Alzheimer's Disease) are incurable, progressive neurodegenerative diseases caused by tau protein aggregation. 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). Evidence shows tau aggregates enter cells through heparan sulfate proteoglycan (HSPG) mediated macropinocytosis. In this project, I explored whether distinct tau strains bind cell surface HSPGs uniquely or generically to trigger uptake and used heparinoids to measure the relative importance of heparin size and sulfation patterns. METHODS: I used a "biosensor" cell line responsive to tau aggregates that scores induction of intracellular aggregation based on FRET flow cytometry. Using cell lysate from various strains of tau, I measured (1) the ability of different heparin-like molecules to block tau aggregate uptake and seeding, and (2) seeding in HSPG gene knockout cells. RESULTS: All tau strains tested were highly sensitive to heparin inhibition of seeding and most maintained a highly similar dose response. Some strains, however, showed subtle differences. At maximal heparin concentrations, noticeably higher seeding vs baseline was observed in DS 5 & 6 (17%, 9%) as compared to the other strains (<5%). Heparinoid titrations revealed highly similar inhibition patterns between DS 9 and 10. Seeding reduction: DS 9: dp4= 21%; dp8= 27%; dp12= 70%; dp16= 63%; De-2-O=65%; De-6-O= 52%; De-N= 35%. For DS 10: dp4= 19%; dp8= 33%; dp12= 64%; dp16=46%; De-2-O= 53%; De-6-O= 25%; De-N= 13%. Seeding in HSPG genetic knockout cells was reduced substantially in two knockouts, but increased in another. 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. But the unique behavior of some strains suggests a one-size-fits-all treatment approach may not always be sufficient. Certain size and sulfation patterns on heparin have specific importance for tau binding. Larger heparinoids better inhibit tau seeding (dp16 & dp12 > dp8 & dp4) and the importance of 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 here (using strains) and by others in the lab (using recombinant tau). This data shows many similarities and some differences between strains of tau. Parsing these differences could have important implications for understanding the diversity of tauopathies and finding unique approaches to diagnosis and treatment.Item Donald W. Seldin, M.D., Research Symposium finalist presentations(2022-04-29) Almonte, Matthew; Duvalyan, Angela; McAdams, Meredith; Onyirioha, Kristeen; Saez-Calveras, Nil; Triana, TaylorThis edition of the UT Southwestern Internal Medicine Grand Rounds features presentations by the six Foster Fellows selected as finalists from the Seventh Annual Donald W. Seldin, M.D. Research Symposium, which was held on April 21, 2022. These Foster Fellows presented work that spanned the breadth and depth of scholarly activity across the department, and at the close of Grand Rounds, one will be selected as the 2022 Seldin Scholar, in honor of Dr. Donald W. Seldin. The Grand Rounds presentation includes additional award presentations recognizing Clinical Vignettes, as well as the Award for Research in Quality and Education at Parkland Hospital and the Social Impact Award.Item Mechanistic Investigation into the Regulation of Amyloid Motifs in Tau Aggregation and Disease(2022-08-01T05:00:00.000Z) Chen, Dailu; Lin, Milo; Diamond, Marc; Rizo-Rey, José; Rosen, Michael K.; Joachimiak, LukaszAmyloid formation of tau protein is a unifying theme in a multitude of neurodegenerative diseases, collectively called tauopathies. Missense mutations in the tau gene (MAPT) correlate with aggregation propensity and cause dominantly inherited tauopathies, but the molecular mechanism of how they promote tau assembly into amyloids is poorly understood. Many disease-associated mutations localize within tau's repeat domain proximal to amyloidogenic sequences, such as 306VQIVYK311. We use computational modeling, recombinant protein and synthetic peptide systems, cross-linking mass spectrometry, and cell models to investigate the biophysical mechanisms behind aggregation of the P301L/S and S320F mutants. We conclude that the aggregation prone 306VQIVYK311 motif forms metastable compact structures with its upstream sequence that modulates aggregation propensity. We report that disease-associated mutations such as P301L/S at the inter-repeat interface, isomerization of a critical proline, or alternative splicing are all sufficient to destabilize this local structure and trigger aggregation. In the study of S320F, we again demonstrate the role of local protective structures in the regulation of tau aggregation driven by amyloid motif and uncover that the S320F mutation allosterically exposes 306VQIVYK311 by retaining one of the protecting motifs in a stabilized local hydrophobic cluster. We show that rational design of this nonpolar cluster centered on position 320 based on tauopathy fibril structures maintains a spontaneous aggregation phenotype revealing new principles that govern tau aggregation. We uncover a nuanced balance of local protective structures that sequester amyloid motifs and how introduction of a hydrophobic mutation redistributes these interactions to drive spontaneous aggregation. Tau presents as an aggregation-resistant monomer and only in the presence of an inducer such as heparin, RNA or other polyanion will tau aggregate in vitro. Our studies use disease-causing mutations as a bridge to explain the basis of early conformational changes that may underlie genetic and sporadic tau pathogenesis. Our findings provide molecular insights into regulation of tau assembly, and we anticipate deeper knowledge of this process will begin control of tau aggregation into discrete structural polymorphs.Item Targeting Distinct Tau Strains and Tau Aggregate Sizes with Heparin and Heparinoids to Explore Differential Inhibition of Cell Uptake and Seeding(2017-01-17) Prueitt, William; Stopschinski, Barbara; Diamond, MarcBACKGROUND: Tauopathies (including Alzheimer's Disease) are incurable, progressive neurodegenerative diseases caused by tau protein aggregation. Evidence suggests that tau aggregates spread pathology as do prions, infectious proteins that transmit a pathologic conformation to native proteins via strains--disease-specific conformers that propagate indefinitely in living systems. Like prion protein, tau also forms strains. It is unknown whether each binds the cell surface heparan sulfate proteoglycans in a unique or generic fashion to trigger uptake. METHODS: I used a "biosensor" cell line responsive to tau aggregates that scores induction of intracellular aggregation based on FRET flow cytometry. I tested the ability of different heparin-like molecules to block tau aggregate uptake and seeding. I measured tau uptake and induction of intracellular aggregation of a reporter. RESULTS: Tau seeding was comparably inhibited by heparin regardless of aggregate size. Data from testing two strains (Clone 9 & 10) for heparin inhibition of cell seeding suggested that they have differential sensitivity (DS9: IC50 = 335.9nM & DS10: IC50 = 2.1uM). Testing 9 heparinoids for tau seeding inhibition indicated that they had highly variable inhibition, some having no effect and some having an effect nearly as strong as heparin. CONCLUSIONS: This data suggests that (1) tau seeding is similarly inhibited by heparin regardless of tau aggregate size, and that (2) seeding of different strains of tau may be variably inhibited by heparin, hinting that specificity and avidity may differ by strain. If true, this knowledge will be applicable across many tauopathies and may influence diagnosis (because tau strains can differentiate pathology) and treatment (strain-specific therapies may be required). This data also indicated that certain size and sulfation patterns of heparin affect seeding inhibition. This matches other data produced in the lab using genetic knockouts, and supports the idea that crucial binding domains on heparin are necessary for pathologic tau spread between cells.Item Tau Seeding in Health and Disease(2022-08-01T05:00:00.000Z) LaCroix, Michael Shane; Nam, Yunsun; Herz, Joachim; Joachimiak, Lukasz; Diamond, Marc; Rice, Luke M.An abundance of evidence supports that the protein tau adopts a wide variety of conformations with the ability to self-assemble and propagate in living systems, and that this prion behavior may drive neurodegeneration in tauopathies. However, the inciting events that lead to tau seed formation and aggregation are unknown. It remains possible that tau can act as a prion outside the context of disease, as part of its normal function, and the accumulation of tau prions in neurodegenerative diseases reflects a loss of control of this normal function. During my dissertation research, I completed a series of investigations on tau's ability to form seeds outside the context of classical tauopathies. I discovered that tau seeds are present in the cerebral cortex of healthy individuals. Tau seeds form in a region and species specific manner, being absent in the cerebellum of healthy individuals and undetectable in murine models. Seeding in healthy individuals was independent of age, implying it is not a result of emerging tauopathy but rather, that prion formation is a normal aspect of tau biology. This may be related to its interactions with RNA. I also surveyed for tau seeding in several inflammatory diseases with neurodegenerative components that have been reported to exhibit tau accumulation based on immunohistochemistry. I found seeds at levels beyond that of healthy individuals in temporal lobe epilepsy as well as multiple sclerosis. Thus, tau may be a target of many convergent pathways that lead to neurodegeneration. The work here highlights the significant role that tau plays in human health and disease. Further understanding of how normal biological processes, as well as inflammation, affect tau's prion state will be essential for the development of therapeutic strategies for the prevention and treatment of tauopathies.