&lt;em&gt;In Vitro&lt;/em&gt; Aggregation Assays Using Hyperphosphorylated Tau Protein

Sui, Dexin; Liu, Mengyu; Kuo, Min Hao

doi:10.3791/51537

Cited by 16 publications

(13 citation statements)

References 54 publications

(52 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…ThT assay, which is a common marker of amyloid fibrils, was performed as described before [42] with some modifications. Briefly, samples of 30 μM Tau alone or Tau with 8-fold excess of ZnCl 2 were mixed with ThT.…”

Section: Fluorescence Tht Assaymentioning

confidence: 99%

Zinc Induces Temperature-Dependent Reversible Self-Assembly of Tau

Roman

Devred

Byrne

et al. 2019

Journal of Molecular Biology

View full text Add to dashboard Cite

Tau is an intrinsically disordered microtubule-associated protein that is implicated in several neurodegenerative disorders called tauopathies. In these diseases, Tau is found in the form of intracellular inclusions that consist of aggregated paired helical filaments (PHFs) in neurons. Given the importance of this irreversible PHF formation in neurodegenerative disease, Tau aggregation has been extensively studied. Several different factors, such as mutations or post translational modifications, have been shown to influence the formation of late-stage non-reversible Tau aggregates. It was recently shown that zinc ions accelerated heparin-induced oligomerization of Tau constructs. Indeed, in vitro studies of PHFs have usually been performed in the presence of additional co-factors, such as heparin, in order to accelerate their formation. Using turbidimetry, we investigated the impact of zinc ions on Tau in the absence of heparin and found that zinc is able to induce a temperature-dependent reversible oligomerization of Tau. The obtained oligomers were not amyloid-like and dissociated instantly following zinc chelation or a temperature decrease. Finally, a combination of isothermal titration calorimetry and dynamic light scattering experiments showed zinc binding to a high-affinity binding site and three low-affinity sites on Tau, accompanied by a change in Tau folding. Altogether, our findings stress the importance of zinc in Tau oligomerization. This newly identified Zn-induced oligomerization mechanism may be a part of a pathway different of and concurrent to Tau aggregation cascade leading to PHF formation.

show abstract

Section: Fluorescence Tht Assaymentioning

confidence: 99%

Zinc Induces Temperature-Dependent Reversible Self-Assembly of Tau

Roman

Devred

Byrne

et al. 2019

Journal of Molecular Biology

View full text Add to dashboard Cite

show abstract

“…Despite numerous efforts, the tau aggregation kinetics reported in the literature to date lack the desired level of reproducibility and/or some of the features of a nucleation dependent polymerization 19,20,21,22,23,25 . This is often emphasized by the lack of a lag phase, inefficient seeding and non-fibrillar nature of tau aggregates.…”

Section: Discussionmentioning

confidence: 99%

“…In most cases, the tau aggregation kinetics was lacking the initial lag phase associated with tau nucleation. This might have been the consequence of using very high tau protein concentrations, presence of aggregates in the starting tau protein preparations and/or use of tau fragments with much higher aggregation propensity than the more physiological full length tau protein 19,20,21,22,23 . Furthermore, previous studies did not address the reproducibility and robustness aspect of tau aggregation kinetics.…”

Section: Introductionmentioning

confidence: 99%

<em>In Vitro</em> Assay for Studying the Aggregation of Tau Protein and Drug Screening

Crespo

Koudstaal

Apetri

2018

JoVE

View full text Add to dashboard Cite

Aggregation of tau protein and formation of paired helical filaments is a hallmark of Alzheimer's disease and other tauopathies. Compared to other proteins associated with neurodegenerative diseases, the reported in vitro aggregation kinetics for tau protein are less consistent presenting a relatively high variability. Here we describe the development of an in vitro aggregation assay that mimics the expected steps associated with tau misfolding and aggregation in vivo. The assay uses the longest tau isoform (huTau441) which contains both N-terminal acidic inserts as well as four microtubule binding domains (MBD). The in vitro aggregation is triggered by addition of heparin and followed continuously by thioflavin T fluorescence in a 96 well microplate format. The tau aggregation assay is highly reproducible between different wells, experimental runs and batches of the protein. The aggregation leads to tau PHF-like morphology which is very efficient in seeding the formation of de novo fibrillar structures. In addition to its application in studying the mechanism of tau misfolding and aggregation, the current assay is a robust tool for screening drugs that could interfere with the pathogenesis of tau.

show abstract

“…Other seeding assays, such as Thioflavin Twhich exhibits enhanced fluorescence when bound to beta sheet structure -are laborious and require a pure, recombinant protein substrate. Additionally, in vitro seeding assays for tau are only semi-quantitative and generally insensitive to subnanomolar levels of seed material 23,24 . FRET flow cytometry, however, provides a quantitative and exquisitely sensitive measure of seeding activity from either recombinant or biological samples.…”

Section: Discussionmentioning

confidence: 99%

Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry

Furman

Holmes

Diamond

2015

JoVE

View full text Add to dashboard Cite

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and progression of pathology in several neurodegenerative diseases, including Alzheimer's disease and the related tauopathies. The potentially critical role of tau seeds in disease progression strongly supports the need for a sensitive assay that readily detects seeding activity in biological samples. By combining the specificity of fluorescence resonance energy transfer (FRET), the sensitivity of flow cytometry, and the stability of a monoclonal cell line, an ultra-sensitive seeding assay has been engineered and is compatible with seed detection from recombinant or biological samples, including human and mouse brain homogenates. The assay employs monoclonal HEK 293T cells that stably express the aggregation-prone repeat domain (RD) of tau harboring the disease-associated P301S mutation fused to either CFP or YFP, which produce a FRET signal upon protein aggregation. The uptake of proteopathic tau seeds (but not other proteins) into the biosensor cells stimulates aggregation of RD-CFP and RD-YFP, and flow cytometry sensitively and quantitatively monitors this aggregation-induced FRET. The assay detects femtomolar concentrations (monomer equivalent) of recombinant tau seeds, has a dynamic range spanning three orders of magnitude, and is compatible with brain homogenates from tauopathy transgenic mice and human tauopathy subjects. With slight modifications, the assay can also detect seeding activity of other proteopathic seeds, such as α-synuclein, and is also compatible with primary neuronal cultures. The ease, sensitivity, and broad applicability of FRET flow cytometry makes it useful to study a wide range of protein aggregation disorders.

show abstract

<em>In Vitro</em> Aggregation Assays Using Hyperphosphorylated Tau Protein

Cited by 16 publications

References 54 publications

Zinc Induces Temperature-Dependent Reversible Self-Assembly of Tau

Zinc Induces Temperature-Dependent Reversible Self-Assembly of Tau

<em>In Vitro</em> Assay for Studying the Aggregation of Tau Protein and Drug Screening

Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry

Contact Info

Product

Resources

About