Cofactors are essential constituents of stable and seeding-active tau fibrils.
Ontology highlight
ABSTRACT:
SUBMITTER: Fichou Y
PROVIDER: S-EPMC6310788 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Several studies have proposed that fibrillary aggregates of tau and other amyloidogenic proteins are neurotoxic and result in numerous neurodegenerative diseases. However, these studies usually involve sonication or extrusion through needles before experimentation. As a consequence, these methods may fragment large aggregates producing a mixture of aggregated species rather than intact fibrils. Therefore, the results of these experiments may be reflective of other amyloidogenic species, such as oligomers and/or protofibrils/short fibrils. To investigate the effects of sonication on the aggregation of tau and other amyloidogenic proteins, fibrils were prepared and well characterized, then sonicated and evaluated by various biochemical and biophysical methods to identify the aggregated species present. We found that indeed a mixture of aggregated species was present along with short fibrils indicating that sonication leads to impure fibril samples and should be analyzed with caution. Our results corroborate the previous studies showing that sonication of prion and Aβ fibrils leads to the formation of toxic, soluble aggregates. We also show that the oligomeric forms are the most toxic species although it is unclear how sonication causes oligomer formation. Recent results suggest that these small toxic oligomers produced by sonication, rather than the stable fibrillar structures, are prion-like in nature displaying seeding and cross-seeding behavior.
| S-EPMC6201727 | biostudies-literature
Project description:Tau protein fibrillization is implicated in the pathogenesis of several neurodegenerative diseases collectively known as Tauopathies. For decades, investigating Tau fibrillization in vitro has required the addition of polyanions or other co-factors to induce its misfolding and aggregation, with heparin being the most commonly used. However, heparin-induced Tau fibrils exhibit high morphological heterogeneity and a striking structural divergence from Tau fibrils isolated from Tauopathies patients' brains at ultra- and macro-structural levels. To address these limitations, we developed a quick, cheap, and effective method for producing completely co-factor-free fibrils from all full-length Tau isoforms and mixtures thereof. We show that Tau fibrils generated using this ClearTau method - ClearTau fibrils - exhibit amyloid-like features, possess seeding activity in biosensor cells and hiPSC-derived neurons, retain RNA-binding capacity, and have morphological properties and structures more reminiscent of the properties of the brain-derived Tau fibrils. We present the proof-of-concept implementation of the ClearTau platform for screening Tau aggregation-modifying compounds. We demonstrate that these advances open opportunities to investigate the pathophysiology of disease-relevant Tau aggregates and will facilitate the development of Tau pathology-targeting and modifying therapies and PET tracers that can distinguish between different Tauopathies.
| S-EPMC10319797 | biostudies-literature
Project description:Amyloid aggregates of Tau protein have been implicated in etiology of many neurodegenerative disorders including Alzheimer's disease (AD). When amyloid growth is induced by seeding with preformed fibrils assembled from the same protein, structural characteristics of the seed are usually imprinted in daughter generations of fibrils. This so-called conformational memory effect may be compromised when the seeding involves proteins with non-identical sequences leading to the emergence of distinct structural variants of fibrils (amyloid 'strains'). Here, we investigate cross-seeding of full-length human Tau (FL Tau) with fibrils assembled from K18 and K18?K280 fragments of Tau in the presence of poly-L-glutamate (poly-Glu) as an enhancer of Tau aggregation. To study cross-seeding between Tau polypeptides and the role of the conformational memory effect in induction of Tau amyloid polymorphism, kinetic assays, transmission electron microscopy, infrared spectroscopy and limited proteolysis have been employed. The fastest fibrillization was observed for FL Tau monomers seeded with preformed K18 amyloid yielding daughter fibrils with unique trypsin digestion patterns. Morphological features of daughter FL Tau fibrils induced by K18 and K18?K280 seeds were reminiscent of the mother fibrils (i.e. straight paired fibrils and paired helical filaments (PHFs), respectively) but disappeared in the following generations which became similar to unpaired FL Tau amyloid fibrils formed de novo. The structural evolution observed in our study was accompanied by disappearance of the unique proteolysis profile originated from K18. Our findings may have implications for understanding molecular mechanisms of the emergence and stability of Tau amyloid strains.
| S-EPMC6053212 | biostudies-literature
Project description:Pathological aggregation of the protein tau into insoluble aggregates is a hallmark of neurodegenerative diseases. The emergence of disease-specific tau aggregate structures termed tau strains, however, remains elusive. Here we show that full-length tau protein can be aggregated in the absence of co-factors into seeding-competent amyloid fibrils that sequester RNA. Using a combination of solid-state NMR spectroscopy and biochemical experiments we demonstrate that the co-factor-free amyloid fibrils of tau have a rigid core that is similar in size and location to the rigid core of tau fibrils purified from the brain of patients with corticobasal degeneration. In addition, we demonstrate that the N-terminal 30 residues of tau are immobilized during fibril formation, in agreement with the presence of an N-terminal epitope that is specifically detected by antibodies in pathological tau. Experiments in vitro and in biosensor cells further established that co-factor-free tau fibrils efficiently seed tau aggregation, while binding studies with different RNAs show that the co-factor-free tau fibrils strongly sequester RNA. Taken together the study provides a critical advance to reveal the molecular factors that guide aggregation towards disease-specific tau strains.
| S-EPMC8270918 | biostudies-literature
Project description:Despite much effort, antibody therapies for Alzheimer's disease (AD) have shown limited efficacy. Challenges to the rational design of effective antibodies include the difficulty of achieving specific affinity to critical targets, poor expression, and antibody aggregation caused by buried charges and unstructured loops. To overcome these challenges, we grafted previously determined sequences of fibril-capping amyloid inhibitors onto a camel heavy chain antibody scaffold. These sequences were designed to cap fibrils of tau, known to form the neurofibrillary tangles of AD, thereby preventing fibril elongation. The nanobodies grafted with capping inhibitors blocked tau aggregation in biosensor cells seeded with postmortem brain extracts from AD and progressive supranuclear palsy (PSP) patients. The tau capping nanobody inhibitors also blocked seeding by recombinant tau oligomers. Another challenge to the design of effective antibodies is their poor blood-brain barrier (BBB) penetration. In this study, we also designed a bispecific nanobody composed of a nanobody that targets a receptor on the BBB and a tau capping nanobody inhibitor, conjoined by a flexible linker. We provide evidence that the bispecific nanobody improved BBB penetration over the tau capping inhibitor alone after intravenous administration in mice. Our results suggest that the design of synthetic antibodies that target sequences that drive protein aggregation may be a promising approach to inhibit the prion-like seeding of tau and other proteins involved in AD and related proteinopathies.
| S-EPMC10576031 | biostudies-literature
Project description:The intracellular deposition of fibrils composed of the microtubule-associated protein Tau is a characteristic feature of Alzheimer's disease (AD) and other fatal neurodegenerative disorders collectively known as tauopathies. Short Tau fibrils spread intracerebrally through transfer between interconnected neurons. Once taken up by a recipient cell, Tau fibrils recruit Tau monomers onto their ends. Based on the number of microtubule-binding repeats, there are two distinct groups of Tau isoforms: three-repeat (3R) Tau and four-repeat (4R) Tau. In AD, all Tau isoforms are deposited, whereas in other tauopathies, only 3R or 4R Tau isoforms are deposited. The molecular basis for these isoform-specific depositions is poorly understood, although conformation-based cross-seeding barriers are key. Here, we used sedimentation assays, EPR spectroscopy, and other structural readouts to better understand the cross-seeding barriers of 4R Tau fibrils. We observed that fibrils formed from truncated Tau (K18), but not full-length Tau (htau40), exhibit a barrier that inhibits 3R Tau recruitment. Investigating an array of differently sized fragments, we found that the Tau C terminus modulates the cross-seeding barrier and that the N terminus plays a synergistic role. Two disease-associated Tau variants, P301S and P301L, also established strong cross-seeding barriers. EPR analysis indicated that fibrils seeded with truncated and mutated Tau, but not htau40, are structurally disordered in the second half of repeat four and onward. These findings suggest that the disorder in this region diminishes the ability of 4R Tau fibrils to recruit 3R Tau monomers, revealing a new mechanism for Tau cross-seeding barriers.
| S-EPMC6231118 | biostudies-literature
Project description:Tau aggregates are pleiotropic and exhibit differences in conformation, structure, and size. These aggregates develop endogenously but are also propagated among neurons in disease. We explored the actions of two distinct types of tau aggregates, tau oligomers (oTau) and tau fibrils (fTau), using a seeding assay in primary neuron cultures expressing human 4R0N tau. We find that oTau and fTau elicit distinct patterns of tau inclusions in the neurons and distinct molecular interactions. The exogenously applied oTau and fTau both clear rapidly from the neurons, but both also seed intracellular inclusions composed of endogenously produced tau. The two types of seeds elicit differential dose-response relationships for seed uptake and the number of resulting intracellular inclusions. Immunocytochemical studies show that co-localization with RNA binding proteins associated with stress granules is much greater for seeds composed of oTau than fTau. Conversely, co-localization with p62/SQSTM1 and thioflavine S is much greater for fTau than oTau. These results suggest that oTau seeds inclusions that modulate the translational stress response and are physiologically active, whereas fTau seeds inclusions that are fibrillar and shunted to the autolysosomal cascade.
| S-EPMC7554625 | biostudies-literature
Project description:The accumulation of amyloid Tau aggregates is implicated in Alzheimer's disease (AD) and other tauopathies. Molecular chaperones are known to maintain protein homeostasis. Here, we show that an ATP-dependent human chaperone system disassembles Tau fibrils in vitro We found that this function is mediated by the core chaperone HSC70, assisted by specific cochaperones, in particular class B J-domain proteins and a heat shock protein 110 (Hsp110)-type nucleotide exchange factor (NEF). The Hsp70 disaggregation machinery processed recombinant fibrils assembled from all six Tau isoforms as well as Sarkosyl-resistant Tau aggregates extracted from cell cultures and human AD brain tissues, demonstrating the ability of the Hsp70 machinery to recognize a broad range of Tau aggregates. However, the chaperone activity released monomeric and small oligomeric Tau species, which induced the aggregation of self-propagating Tau conformers in a Tau cell culture model. We conclude that the activity of the Hsp70 disaggregation machinery is a double-edged sword, as it eliminates Tau amyloids at the cost of generating new seeds.
| S-EPMC7363153 | biostudies-literature
Project description:Neurodegenerative tauopathies are caused by accumulation of toxic tau protein assemblies. This appears to involve template-based seeding events, whereby tau monomer changes conformation and is recruited to a growing aggregate. Several large families of chaperone proteins, including Hsp70s and J domain proteins (JDPs), cooperate to regulate the folding of intracellular proteins such as tau, but the factors that coordinate this activity are not well known. The JDP DnaJC7 binds tau and reduces its intracellular aggregation. However, it is unknown whether this is specific to DnaJC7 or if other JDPs might be similarly involved. We used proteomics within a cell model to determine that DnaJC7 co-purified with insoluble tau and colocalized with intracellular aggregates. We individually knocked out every possible JDP and tested the effect on intracellular aggregation and seeding. DnaJC7 knockout decreased aggregate clearance and increased intracellular tau seeding. This depended on the ability of the J domain (JD) of DnaJC7 to stimulate Hsp70 ATPase activity, as JD mutations that block this interaction abrogated the protective activity. Disease-associated mutations in the JD and substrate binding site of DnaJC7 also abolished its protective activity. DnaJC7 thus specifically regulates tau aggregation in cooperation with Hsp70.
| S-EPMC10473839 | biostudies-literature
Project description:Neurodegenerative tauopathies are caused by accumulation of toxic tau protein assemblies. This appears to involve template-based seeding events, whereby tau monomer changes conformation and is recruited to a growing aggregate. Several large families of chaperone proteins, including Hsp70s and J domain proteins (JDPs) cooperate to regulate the folding of intracellular proteins such as tau, but the factors that coordinate this activity are not well known. The JDP DnaJC7 binds tau and reduces its intracellular aggregation. However, it is unknown whether this is specific to DnaJC7 or if other JDPs might be similarly involved. We used proteomics within a cell model to determine that DnaJC7 co-purified with insoluble tau and colocalized with intracellular aggregates. We individually knocked out every possible JDP and tested the effect on intracellular aggregation and seeding. DnaJC7 knockout decreased aggregate clearance and increased intracellular tau seeding. This depended on the ability of the J domain (JD) of DnaJC7 to bind to Hsp70, as JD mutations that block binding to Hsp70 abrogated the protective activity. Disease-associated mutations in the JD and substrate binding site of DnaJC7 also abrogated its protective activity. DnaJC7 thus specifically regulates tau aggregation in cooperation with Hsp70.
| S-EPMC10055123 | biostudies-literature