VCP regulates early tau seed amplification via specific cofactors.

Background: Neurodegenerative tauopathies may progress based on seeding by pathological tau assemblies, whereby an aggregate is released from one cell, gains entry to an adjacent or connected cell, and serves as a specific template for its own replication in the cytoplasm. In vitro seeding reactions typically take days, yet seeding into the complex cytoplasmic milieu happens within hours, implicating a machinery with unknown players that controls this process in the acute phase. Methods: We used proximity labeling to identify factors that control seed amplification within 5h of seed exposure. We fused split-APEX2 to the C-terminus of tau repeat domain (RD) to reconstitute peroxidase activity 5h after seeded intracellular tau aggregation. Valosin containing protein (VCP/p97) was the top hit. VCP harbors dominant mutations that underlie two neurodegenerative diseases, multisystem proteinopathy and vacuolar tauopathy, but its mechanistic role is unclear. We used immortalized cells and human neurons to study the effects of VCP on tau seeding. We exposed cells to fibrils or brain homogenates in cell culture media and measured effects on uptake and induction of intracellular tau aggregation following various genetic and chemical manipulations of VCP. Results: VCP knockdown reduced tau seeding. Chemical inhibitors had opposing effects on aggregation in HEK293T tau biosensor cells and human neurons alike: ML-240 increased seeding efficiency, whereas NMS-873 decreased it. The inhibitors were effective only when administered within 8h of seed exposure, indicating a role for VCP early in seed processing. We screened 30 VCP co-factors in HEK293T biosensor cells by genetic knockout or knockdown. Reduction of ATXN3, NSFL1C, UBE4B, NGLY1, and OTUB1 decreased tau seeding, as did NPLOC4, which also uniquely increased soluble tau levels. By contrast, reduction of FAF2 increased tau seeding. Conclusions: Divergent effects on tau seeding of chemical inhibitors and cofactor reduction indicate that VCP regulates this process. This is consistent with a dedicated cytoplasmic processing complex based on VCP that directs seeds acutely towards degradation vs. amplification.

Differentiation and characterization of healthy versus pathological tau using chemical exchange saturation transfer.

Neurofibrillary tangles of tau constitute one of the key biological hallmarks of Alzheimer's disease. Currently, the assessment of regional tau accumulation requires intravenous administration of radioactive tracers for PET imaging. A noninvasive MRI-based solution would have significant clinical implications. Herein, we utilized an MRI technique known as chemical exchange saturation transfer (CEST) to determine the imaging signature of tau in both its monomeric and pathologic fibrillated conformations. Three sets of purified recombinant full-length (4R) tau protein were prepared for collection of CEST spectra using a 9.4 T NMR spectrometer at varying temperatures (25, 37, and 42 °C) and RF intensities (0.7, 1.0, 1.5, and 2.2 µT). Monomeric and fibrillated tau were readily distinguished based on their Z-spectrum profiles. Fibrillated tau demonstrated a less prominent peak at 3.5 ppm with additional peaks near 0.5 and 1.5 ppm. No significant differences were identified between fibrillated tau prepared using heparin versus seed-competent tau. In conclusion, monomeric and fibrillated tau can be readily detected and distinguished based on their CEST-derived Z-spectra, pointing to the potential utility of CEST-MRI as a noninvasive biomarker of regional pathologic tau accumulation in the brain. Further testing and validation in vitro and in vivo will be necessary before this can be applied clinically.

VCP increases or decreases tau seeding using specific cofactors.

Background: Neurodegenerative tauopathies may progress based on seeding by pathological tau assemblies, whereby an aggregate is released from one cell, gains entry to an adjacent or connected cell, and serves as a specific template for its own replication in the cytoplasm. In vitro seeding reactions typically take days, yet seeding into the complex cytoplasmic milieu can happen within hours. A cellular machinery might regulate this process, but potential players are unknown. Methods: We used proximity labeling to identify factors that control seed amplification. We fused split-APEX2 to the C-terminus of tau repeat domain (RD) to reconstitute peroxidase activity upon seeded intracellular tau aggregation. We identified valosin containing protein (VCP/p97) 5h after seeding. Mutations in VCP underlie two neurodegenerative diseases, multisystem proteinopathy and vacuolar tauopathy, but its mechanistic role is unclear. We utilized tau biosensors, a cellular model for tau aggregation, to study the effects of VCP on tau seeding. Results: VCP knockdown reduced tau seeding. However, distinct chemical inhibitors of VCP and the proteasome had opposing effects on aggregation, but only when given <8h of seed exposure. ML-240 increased seeding efficiency ~40x, whereas NMS-873 decreased seeding efficiency by 50%, and MG132 increased seeding ~10x. We screened VCP co-factors in HEK293 biosensor cells by genetic knockout or knockdown. Reduction of ATXN3, NSFL1C, UBE4B, NGLY1, and OTUB1 decreased tau seeding, as did NPLOC4, which also uniquely increased soluble tau levels. Reduction of FAF2 and UBXN6 increased tau seeding. Conclusions: VCP uses distinct cofactors to determine seed replication efficiency, consistent with a dedicated cytoplasmic processing complex that directs seeds towards dissolution vs. amplification.

RNA induces unique tau strains and stabilizes Alzheimer's disease seeds.

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.

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

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.

Ultrasensitive tau biosensor cells detect no seeding in Alzheimer's disease CSF.

Tau protein forms self-replicating assemblies (seeds) that may underlie progression of pathology in Alzheimer's disease (AD) and related tauopathies. Seeding in recombinant protein preparations and brain homogenates has been quantified with "biosensor" cell lines that express tau with a disease-associated mutation (P301S) fused to complementary fluorescent proteins. Quantification of induced aggregation in cells that score positive by fluorescence resonance energy transfer (FRET) is accomplished by cell imaging or flow cytometry. Several groups have reported seeding activity in antemortem cerebrospinal fluid (CSF) using various methods, but these findings are not yet widely replicated. To address this question, we created two improved FRET-based biosensor cell lines based on tau expression, termed version 2 low (v2L) and version 2 high (v2H). We determined that v2H cells are ~ 100-fold more sensitive to AD-derived tau seeds than our original lines, and coupled with immunoprecipitation reliably detect seeding from samples containing as little as 100 attomoles of recombinant tau fibrils or ~ 32 pg of total protein from AD brain homogenate. We tested antemortem CSF from 11 subjects with a clinical diagnosis of AD, 9 confirmed by validated CSF biomarkers. We used immunoprecipitation coupled with seed detection in v2H cells and detected no tau seeding in any sample. Thus we cannot confirm prior reports of tau seeding activity in the CSF of AD patients. This next generation of ultra-sensitive tau biosensors may nonetheless be useful to the research community to quantify tau pathology as sensitively and specifically as possible.

A synthetic heparinoid blocks Tau aggregate cell uptake and amplification.

Tau aggregation underlies neurodegeneration in Alzheimer's disease and related tauopathies. We and others have proposed that transcellular propagation of pathology is mediated by Tau prions, which are ordered protein assemblies that faithfully replicate in vivo and cause specific biological effects. The prion model predicts the release of aggregates from a first-order cell and subsequent uptake into a second-order cell. The assemblies then serve as templates for their own replication, a process termed "seeding." We have previously observed that heparan sulfate proteoglycans on the cell surface mediate the cellular uptake of Tau aggregates. This interaction is blocked by heparin, a sulfated glycosaminoglycan. Indeed, heparin-like molecules, or heparinoids, have previously been proposed as a treatment for PrP prion disorders. However, heparin is not ideal for managing chronic neurodegeneration, because it is difficult to synthesize in defined sizes, may have poor brain penetration because of its negative charge, and is a powerful anticoagulant. Therefore, we sought to generate an oligosaccharide that would bind Tau and block its cellular uptake and seeding, without exhibiting anticoagulation activity. We created a compound, SN7-13, from pentasaccharide units and tested it in a range of assays that measured direct binding of Tau to glycosaminoglycans and inhibition of Tau uptake and seeding in cells. SN7-13 does not inhibit coagulation, binds Tau with low nanomolar affinity, and inhibits cellular Tau aggregate propagation similarly to standard porcine heparin. This synthetic heparinoid could facilitate the development of agents to treat tauopathy.

Tau local structure shields an amyloid-forming motif and controls aggregation propensity.

Tauopathies are neurodegenerative diseases characterized by intracellular amyloid deposits of tau protein. Missense mutations in the tau gene (MAPT) correlate with aggregation propensity and cause dominantly inherited tauopathies, but their biophysical mechanism driving amyloid formation is poorly understood. Many disease-associated mutations localize within tau's repeat domain at inter-repeat interfaces proximal to amyloidogenic sequences, such as 306VQIVYK311. We use cross-linking mass spectrometry, recombinant protein and synthetic peptide systems, in silico modeling, and cell models to 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, isomerization of a critical proline, or alternative splicing are all sufficient to destabilize this local structure and trigger spontaneous aggregation. These findings provide a biophysical framework to explain the basis of early conformational changes that may underlie genetic and sporadic tau pathogenesis.

Tau monomer encodes strains.

Tauopathies have diverse presentation, progression, and neuropathology. They are linked to tau prion strains, self-replicating assemblies of unique quaternary conformation, whose origin is unknown. Strains can be propagated indefinitely in cultured cells, and induce unique patterns of transmissible neuropathology upon inoculation into mice. DS9 and DS10 cell lines propagate different synthetic strains that derive from recombinant tau. We previously observed that tau monomer adopts two conformational states: one that is inert (Mi) and one that is seed-competent (Ms) (Mirbaha et al., 2018). We have now found that Ms itself is comprised of multiple stable ensembles that encode unique strains. DS9 monomer inoculated into naive cells encoded only DS9, whereas DS10 monomer encoded multiple sub-strains. Sub-strains each induced distinct pathology upon inoculation into a tauopathy mouse model (PS19). Ms purified from an Alzeimer's disease brain encoded a single strain. Conversely, Ms from a corticobasal degeneration brain encoded three sub-strains, in which monomer from any one re-established all three upon inoculation into cells. Seed competent tau monomer thus adopts multiple, stable seed-competent conformations, each of which encodes a limited number of strains. This provides insight into the emergence of distinct tauopathies, and may improve diagnosis and therapy.