Title: RNA induces unique tau strains and stabilizes Alzheimer’s disease seeds
Abstract: Tau aggregation underlies neurodegenerative tauopathies, and transcellular propagation of tau assemblies of unique structure, i.e., strains, may underlie the diversity of these disorders. Polyanions have been reported to induce tau aggregation in vitro, but the precise trigger to convert tau from an inert to a seed-competent form in disease states is unknown. RNA triggers tau fibril formation in vitro and has been observed to associate with neurofibrillary tangles in human brain. Here, we have tested whether RNA exerts sequence-specific effects on tau assembly and strain formation. We found that three RNA homopolymers, polyA, polyU, and polyC, all bound tau, but only polyA RNA triggered seed and fibril formation. In addition, polyA:tau seeds and fibrils were sensitive to RNase. We also observed that the origin of the RNA influenced the ability of tau to adopt a structure that would form stable strains. Human RNA potently induced tau seed formation and created tau conformations that preferentially formed stable strains in a HEK293T cell model, whereas RNA from other sources, or heparin, produced strains that were not stably maintained in cultured cells. Finally, we found that soluble, but not insoluble seeds from Alzheimer’s disease brain were also sensitive to RNase. We conclude that human RNA specifically induces formation of stable tau strains and may trigger the formation of dominant pathological assemblies that propagate in Alzheimer’s disease and possibly other tauopathies. Tau aggregation underlies neurodegenerative tauopathies, and transcellular propagation of tau assemblies of unique structure, i.e., strains, may underlie the diversity of these disorders. Polyanions have been reported to induce tau aggregation in vitro, but the precise trigger to convert tau from an inert to a seed-competent form in disease states is unknown. RNA triggers tau fibril formation in vitro and has been observed to associate with neurofibrillary tangles in human brain. Here, we have tested whether RNA exerts sequence-specific effects on tau assembly and strain formation. We found that three RNA homopolymers, polyA, polyU, and polyC, all bound tau, but only polyA RNA triggered seed and fibril formation. In addition, polyA:tau seeds and fibrils were sensitive to RNase. We also observed that the origin of the RNA influenced the ability of tau to adopt a structure that would form stable strains. Human RNA potently induced tau seed formation and created tau conformations that preferentially formed stable strains in a HEK293T cell model, whereas RNA from other sources, or heparin, produced strains that were not stably maintained in cultured cells. Finally, we found that soluble, but not insoluble seeds from Alzheimer’s disease brain were also sensitive to RNase. We conclude that human RNA specifically induces formation of stable tau strains and may trigger the formation of dominant pathological assemblies that propagate in Alzheimer’s disease and possibly other tauopathies. Tau forms highly ordered assemblies, termed amyloids, that underlie Alzheimer’s disease (AD) and related tauopathies (1Lee V.M. Goedert M. Trojanowski J.Q. Neurodegenerative tauopathies.Annu. Rev. Neurosci. 2001; 24: 1121-1159Crossref PubMed Scopus (2237) Google Scholar), which may progress based on trans-cellular propagation (2Frost 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 (921) Google Scholar, 3Clavaguera F. Bolmont T. Crowther R.A. Abramowski D. Frank S. Probst A. et al.Transmission and spreading of tauopathy in transgenic mouse brain.Nat. Cell Biol. 2009; 11: 909-913Crossref PubMed Scopus (1340) Google Scholar). The fundamental origin of tauopathy is unknown, but systemic triggers include amyloid beta, trauma, and inflammation. Tau assemblies adopt faithfully self-replicating conformations in cells, termed strains. Strains may be propagated indefinitely in cell culture (4Sanders D.W. Kaufman S.K. DeVos S.L. Sharma A.M. Mirbaha H. Li A. et al.Distinct tau prion strains propagate in cells and mice and define different tauopathies.Neuron. 2014; 82: 1271-1288Abstract Full Text Full Text PDF PubMed Scopus (693) Google Scholar, 5Calafate S. Buist A. Miskiewicz K. Vijayan V. Daneels G. de Strooper B. et al.Synaptic contacts enhance cell-to-cell tau pathology propagation.Cell Rep. 2015; 11: 1176-1183Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar) and induce specific patterns of neuropathology when inoculated into mouse models (6Kaufman S.K. Sanders D.W. Thomas T.L. Ruchinskas A.J. Vaquer-Alicea J. Sharma A.M. et al.Tau prion strains dictate patterns of cell pathology, progression rate, and regional vulnerability in vivo.Neuron. 2016; 92: 796-812Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 7Clavaguera F. Akatsu H. Fraser G. Crowther R.A. Frank S. Hench J. et al.Brain homogenates from human tauopathies induce tau inclusions in mouse brain.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: 9535-9540Crossref PubMed Scopus (555) Google Scholar). We have observed that certain strains faithfully transmit neuropathology as prions (4Sanders D.W. Kaufman S.K. DeVos S.L. Sharma A.M. Mirbaha H. Li A. et al.Distinct tau prion strains propagate in cells and mice and define different tauopathies.Neuron. 2014; 82: 1271-1288Abstract Full Text Full Text PDF PubMed Scopus (693) Google Scholar). However, so far, stable amplification and propagation of defined strains in vitro has proven difficult. It is unknown why tau forms unique strains, but the observation that a seed-competent tau monomer encodes limited strain ensembles (8Sharma A.M. Thomas T.L. Woodard D.R. Kashmer O.M. Diamond M.I. Tau monomer encodes strains.Elife. 2018; 7e37813Crossref Scopus (48) Google Scholar) suggests that there may be very specific molecular inducers of different strains. Specific posttranslational modifications of insoluble tau are reported to correlate with neuropathological diagnosis (9Wesseling H. Mair W. Kumar M. Schlaffner C.N. Tang S. Beerepoot P. et al.Tau PTM profiles identify patient heterogeneity and stages of Alzheimer's disease.Cell. 2020; 183: 1699-1713.e13Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar), but it is unknown whether phosphorylation triggers the conversion of inert to seed-competent tau monomer in tauopathy. Recent work from our lab has failed to find any detected posttranslational modification of tau that correlates with this event (10Mirbaha H. 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Tau was initially described to bind RNA, which sequestered it and prevented spontaneous tubulin assembly (17Bryan J.B. Nagle B.W. Doenges K.H. Inhibition of tubulin assembly by RNA and other polyanions: Evidence for a required protein.Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 3570-3574Crossref PubMed Scopus (115) Google Scholar). RNA has also been observed in association with tau tangles in brain samples (18Ginsberg S.D. Crino P.B. Lee V.M. Eberwine J.H. Trojanowski J.Q. Sequestration of RNA in Alzheimer's disease neurofibrillary tangles and senile plaques.Ann. Neurol. 1997; 41: 200-209Crossref PubMed Scopus (132) Google Scholar, 19Ginsberg S.D. Galvin J.E. Chiu T.S. Lee V.M. Masliah E. Trojanowski J.Q. RNA sequestration to pathological lesions of neurodegenerative diseases.Acta Neuropathol. 1998; 96: 487-494Crossref PubMed Scopus (108) Google Scholar) and in association with induced tau aggregates in HEK293 cells (20Lester E. Ooi F.K. Bakkar N. Ayers J. Woerman A.L. Wheeler J. et al.Tau aggregates are RNA-protein assemblies that mislocalize multiple nuclear speckle components.Neuron. 2021; 109: 1675-1691.e9Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Because tau has multiple positively charged residues, especially lysines, it was logical to assume that polyanions trigger tau assembly formation in vitro by neutralizing charge interactions and somehow unfolding the protein (21Gustke N. Trinczek B. Biernat J. Mandelkow E.M. Mandelkow E. Domains of tau protein and interactions with microtubules.Biochemistry. 1994; 33: 9511-9522Crossref PubMed Scopus (549) Google Scholar, 22Mukrasch M.D. von Bergen M. Biernat J. Fischer D. Griesinger C. Mandelkow E. et al.The "jaws" of the tau-microtubule interaction.J. Biol. Chem. 2007; 282: 12230-12239Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar) to facilitate its self-assembly. The development of cell lines that amplify tau seeds, termed biosensors, has transformed our ability to characterize prion-like activity of relatively small amounts of soluble tau species (23Holmes B.B. Furman J.L. Mahan T.E. Yamasaki T.R. Mirbaha H. Eades W.C. et al.Proteopathic tau seeding predicts tauopathy in vivo.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: E4376-E4385Crossref PubMed Scopus (369) Google Scholar, 24Hitt B.D. Vaquer-Alicea J. Manon V.A. Beaver J.D. Kashmer O.M. Garcia J.N. et al.Ultrasensitive tau biosensor cells detect no seeding in Alzheimer's disease CSF.Acta Neuropathol. Commun. 2021; 9: 99Crossref PubMed Scopus (22) Google Scholar). Biosensors are based on expression of full-length (FL) or repeat domain (RD)-segments of tau (with or without disease-associated mutations) fused to suitable fluorescent protein tags. They respond to exogenous tau seeds by forming thioflavin positive inclusions (4Sanders D.W. Kaufman S.K. DeVos S.L. Sharma A.M. Mirbaha H. Li A. et al.Distinct tau prion strains propagate in cells and mice and define different tauopathies.Neuron. 2014; 82: 1271-1288Abstract Full Text Full Text PDF PubMed Scopus (693) Google Scholar). Biosensor cell lines can indefinitely and faithfully propagate myriad tau strains (4Sanders D.W. Kaufman S.K. DeVos S.L. Sharma A.M. Mirbaha H. Li A. et al.Distinct tau prion strains propagate in cells and mice and define different tauopathies.Neuron. 2014; 82: 1271-1288Abstract Full Text Full Text PDF PubMed Scopus (693) Google Scholar, 5Calafate S. Buist A. Miskiewicz K. Vijayan V. Daneels G. de Strooper B. et al.Synaptic contacts enhance cell-to-cell tau pathology propagation.Cell Rep. 2015; 11: 1176-1183Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar), suggesting innate mechanisms of specific replication. Strains derived from cells create specific, transmissible forms of neuropathology after inoculation in a transgenic mouse model (PS19) that expresses tau (1N4R) containing a disease-associated mutation (P301S) (4Sanders D.W. Kaufman S.K. DeVos S.L. Sharma A.M. Mirbaha H. Li A. et al.Distinct tau prion strains propagate in cells and mice and define different tauopathies.Neuron. 2014; 82: 1271-1288Abstract Full Text Full Text PDF PubMed Scopus (693) Google Scholar, 6Kaufman S.K. Sanders D.W. Thomas T.L. Ruchinskas A.J. Vaquer-Alicea J. Sharma A.M. et al.Tau prion strains dictate patterns of cell pathology, progression rate, and regional vulnerability in vivo.Neuron. 2016; 92: 796-812Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). It is thus feasible to create and study tau strains induced in vitro and propagated in cultured cells. Given our interest in physiologic inducers of seed and strain formation, for which RNA seemed a plausible candidate, we investigated its role in this process. We first tested whether homopolymers of RNA would differentially bind recombinant tau. We used 40-mers of adenine (A), cytidine (C), and uracil (U), omitting guanidine because of its difficulty to synthesize. We purified recombinant, FL tau monomer (2N4R) according to our prior methods (25Frost B. Ollesch J. Wille H. Diamond M.I. Conformational diversity of wild-type Tau fibrils specified by templated conformation change.J. Biol. Chem. 2009; 284: 3546-3551Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar), termed “tau” henceforth. We immobilized tau on amine reactive second-generation biolayer interferometry biosensors (ForteBio) and with exposure to increasing half-log concentrations of single-stranded RNA (0.13 μM, 0.4 μM, 1.3 μM, 4 μM, 13 μM, and 41 μM). We measured avidity based on changes in surface interferometry (Octet, ForteBio). Binding data were interpolated according to a 2:1 interaction model. PolyA, polyU, and polyC strands each bound tau with similar avidities in the nanomolar range, as did a randomly generated single strand of DNA (Figs. 1A and S1, Table 1).Table 1Affinity of tau for nucleic acid sequencesInducerKDAPPkon (M−1 s−1)koff (s−1)polyA20 nM1.0 X 1041.4 X 10−4polyU254 nM1.7 X 1034.2 X 10−4polyC13 nM3.7 X 1044.5 X 10−4ssDNA seq112 nM2.8 X 1043.4 X 10−4Abbreviations: KDAPP, apparent binding affinity; kon, association rate; koff, dissociation rate.Results were calculated as the mean of three independent experiments. Open table in a new tab Abbreviations: KDAPP, apparent binding affinity; kon, association rate; koff, dissociation rate. Results were calculated as the mean of three independent experiments. We next tested the ability of RNA homopolymers to induce tau fibril formation in vitro. We incubated tau (8 μM) in the presence of polyA, polyU, and polyC RNA (50 μg/ml, or 4 μM) for 24 h before measuring seeding with v2L biosensor cells that stably expressed the tau repeat domain with the P301S mutation fused to cerulean and mClover fluorescent proteins. After 48 h, we quantified intracellular aggregation by fluorescence resonance energy transfer (FRET) as per prior studies (23Holmes B.B. Furman J.L. Mahan T.E. Yamasaki T.R. Mirbaha H. Eades W.C. et al.Proteopathic tau seeding predicts tauopathy in vivo.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: E4376-E4385Crossref PubMed Scopus (369) Google Scholar). PolyA generated seeding activity from tau monomer, while polyU and polyC did not (Fig. 1B). A polyU/polyA hybrid also failed to elicit seeding. Two single-stranded DNA homopolymers (polydA, polydT) did not induce seeding (Fig. S2A). Two randomly generated single-stranded, complementary DNA sequences of the same length and similar molecular weight as the RNA both effectively seeded, while the annealed double stranded complex did not, nor did an RNA/DNA complex of polyA hybridized with polydT (Fig. 1B). To rule out kinetic differences in seed induction, we incubated tau and RNA for up to 2 weeks. While polyA induced seed-competent forms, polyC and polyU never did (Fig. S2, A–C). We observed fibril formation via transmission electron microscopy (TEM) that correlated with seeding activity after 48 h incubation (Fig. 1C). Thus, despite universal binding of RNA to tau, only certain single-stranded nucleotide sequences induced seed-competent conformations. The optimal ratio of RNA:tau to induce seeding was 1:2 (Fig. 1D). We defined the minimal length of RNA necessary to induce seeding by incubating tau with increasing sizes of polyA ranging from 10 to 40 nucleotides (nt) for various time periods. At 48 h, the smallest length of RNA capable of inducing seed-competent tau to a significant degree was 40 nt (p < 0.0001, one-way ANOVA, post-hoc Dunnett’s multiple comparisons test) (Fig. 1E). After 7 days, we observed minimal increases in seed formation with 30 nt (4.1% FRET positive), which were still not significant compared to buffer control (p = 0.1451, one-way ANOVA, post-hoc Dunnett’s multiple comparisons test), while 40 nt seeding increased to 55% FRET positive. Amyloid fibrils are thought to occupy a particularly stable, low-energy state (26Hartl F.U. Protein misfolding diseases.Annu. Rev. Biochem. 2017; 86: 21-26Crossref PubMed Scopus (327) Google Scholar), which might predict their persistence after inducers are removed. We tested this idea for RNA induction. We first formed tau fibrils by incubation with polyA RNA. We then exposed fibrils to a mixture of RNase A and RNase T1 for 24 h to degrade the RNA and tested the effect on seeding and fibril integrity. Tau seeds induced by RNA lost all seeding activity after RNase exposure, while incubation with DNase or heparinase had no effect (Fig. 2A). To exclude a direct effect of RNase on biosensor activity, we added RNase directly to the seeds without preincubation and detected no loss in seeding activity (Fig. 2A). We next used TEM to test fibril stability in the presence of nucleases. RNase eliminated all detectable fibrils, but not DNase or heparinase (Fig. 2B). Conversely, fibrils induced by heparin remained unchanged after DNase and RNase treatment but disassembled and lost 74% of seeding after incubation with heparinase (Fig. 2, A and B). Incomplete loss of seeding after removal of heparin is consistent with our prior observations that it converts tau monomer to a highly stable, seed-competent conformation (27Mirbaha H. Chen D. Morazova O.A. Ruff K.M. Sharma A.M. Liu X. et al.Inert and seed-competent tau monomers suggest structural origins of aggregation.Elife. 2018; 7e36584Crossref PubMed Scopus (138) Google Scholar, 28Hou Z. Chen D. Ryder B.D. Joachimiak L.A. Biophysical properties of a tau seed.Sci. Rep. 2021; 1113602Crossref Scopus (15) Google Scholar). Tau adopts multiple, faithfully propagated assembly structures that produce unique, transmissible patterns of neuropathology in vivo, termed strains (4Sanders D.W. Kaufman S.K. DeVos S.L. Sharma A.M. Mirbaha H. Li A. et al.Distinct tau prion strains propagate in cells and mice and define different tauopathies.Neuron. 2014; 82: 1271-1288Abstract Full Text Full Text PDF PubMed Scopus (693) Google Scholar, 6Kaufman S.K. Sanders D.W. Thomas T.L. Ruchinskas A.J. Vaquer-Alicea J. Sharma A.M. et al.Tau prion strains dictate patterns of cell pathology, progression rate, and regional vulnerability in vivo.Neuron. 2016; 92: 796-812Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). Given that RNA sequence dictated seeding activity, we tested if it might also control strain composition. We previously developed the DS1 biosensor cell line, which expresses tau RD containing two disease-associated mutations (P301L and V337M) fused to YFP (4Sanders D.W. Kaufman S.K. DeVos S.L. Sharma A.M. Mirbaha H. Li A. et al.Distinct tau prion strains propagate in cells and mice and define different tauopathies.Neuron. 2014; 82: 1271-1288Abstract Full Text Full Text PDF PubMed Scopus (693) Google Scholar). Although it represents a rarified experimental system, this line has proved very useful because it readily propagates myriad tau strains. Depending on their replication efficiency, some strains propagate faithfully, whereas others will sector, i.e., inclusions will steadily disappear from the cell population. Thus, one simple and robust classifier of strain identity is the ability to maintain itself in dividing cells, or to sector. Tau was incubated in the presence of total human RNA, total yeast RNA, polyA RNA, or ssDNA before treating DS1 cells with induced tau seeds to initiate inclusion formation. After 72 h, we used fluorescence activated cell sorting to isolate 384 single aggregate-containing cells for each condition in the individual wells of 96-well plates (which was possible based simply on gating for the high fluorescence intensity that occurred when RD-YFP aggregates (8Sharma A.M. Thomas T.L. Woodard D.R. Kashmer O.M. Diamond M.I. Tau monomer encodes strains.Elife. 2018; 7e37813Crossref Scopus (48) Google Scholar)). After 2 weeks of cell growth, we counted amplified colonies (n = 130 derived from human RNA, n = 228 from polyA, n = 220 from DNA, n = 98 from yeast RNA) that still propagated aggregates or had sectored. Sixty percent of colonies induced by human RNA:tau and 43% of colonies induced by polyA:tau still contained inclusions, while only 1% of colonies induced by yeast RNA:tau and 3% of colonies induced by ssDNA:tau contained inclusions (Fig. 3A). Hence, despite starting with 100% inclusion-bearing cells derived from tau seeds, across multiple inducers, human RNA most efficiently induced conformations of tau that created stable strains. To further explore the effect of RNA origin on strain identity, we maintained remaining stable colonies (n = 78 human RNA, 99 polyA RNA, 7 ssDNA, and 1 yeast RNA) for characterization of inclusion morphology, a surrogate for strain identity (4Sanders D.W. Kaufman S.K. DeVos S.L. Sharma A.M. Mirbaha H. Li A. et al.Distinct tau prion strains propagate in cells and mice and define different tauopathies.Neuron. 2014; 82: 1271-1288Abstract Full Text Full Text PDF PubMed Scopus (693) Google Scholar). Colonies were assessed via fluorescence microscopy and blindly scored for morphology. After unblinding and quantification, human RNA–derived colonies largely had speckle morphology (79%), while remaining colonies were ordered (21%) (Fig. 3, B and C). Colonies derived from polyA RNA were primarily disordered (65%), in addition to thread (17%), speckle (12%), and ordered (6%) morphologies (Fig. 3, B and C). While relatively fewer ssDNA colonies stably maintained aggregates, we observed a distinct spiked morphology in 71% of colonies and additional colonies with speckle (14.5%) and ordered (14.5%) patterns (Fig. 3, B and C). The sole remaining yeast RNA–derived colony produced speckle morphology (Fig. 3, B and C). Although distinct inducers of intracellular aggregates generally displayed a heterogenous profile of intracellular morphologies, the predominant morphology for each inducer was unique. Given the ability of RNA to facilitate tau seeding in vitro and strains in cells, we tested its stabilization of seeds derived from the most common tauopathy, Alzheimer’s Disease (AD) (29Holtzman D.M. Morris J.C. Goate A.M. Alzheimer's disease: The challenge of the second century.Sci. Transl Med. 2011; 3: 77sr1Crossref PubMed Scopus (1091) Google Scholar). We homogenized frontal lobe tissue from an AD patient and fractionated the tau based on sarkosyl-insoluble material, which produced fibrils (Fig. S3) and soluble fractions. We used size exclusion chromatography to resolve soluble assemblies of different sizes. Because each sample had different amounts of seeding activity, to ensure that we evaluated seeding accurately across samples, we first titrated each to empirically determine a dose-response curve and be sure we used extract amounts within the linear range (Fig. S4). We then exposed each fraction to RNase, DNase, or buffer. After 24 h, we quantified seeding activity using biosensor cells. In total and soluble fractions, we observed a ∼70 to 85% reduction in seeding after RNase treatment (Fig. 4). By contrast, fibrillar tau exhibited no change in seeding in presence of RNase. DNase had no effect on seeding for any fraction. These data indicated that soluble seeds in AD require RNA for stability. This study has examined the role of RNA in the development and maintenance of tau seeds and strains in vitro and in AD. This preliminary work provides a conceptual framework for further tests of the origins of strains in humans. We initially found that tau bound multiple forms of single-stranded RNA with relatively high avidity, yet formation of seeds and fibrils depended on RNA sequence and size, with a maximally effective size >40 nt. RNA was critical to maintaining induced seed integrity in vitro. While RNA from a variety of sources induced seed-competent tau, when this was used to transduce a human biosensor cell system, strain stability was far more robust when seeds had been produced by induction with human RNA. Finally, soluble tau seeds from AD brain were highly sensitive to RNase but not DNase, indicating a critical role for RNA in the maintenance of AD-derived seeds. While more studies are required, these observations suggest that certain RNAs might specifically trigger the formation and maintenance of unique tau assemblies or strains, contributing to the diversity of tauopathies. Our finding that RNA modulates the conformation of tau assemblies builds on many prior reports that have linked tau and RNA-binding proteins (RBPs) in disease (reviewed in (30Koren S.A. Galvis-Escobar S. Abisambra J.F. Tau-mediated dysregulation of RNA: evidence for a common molecular mechanism of toxicity in frontotemporal dementia and other tauopathies.Neurobiol. 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