Purification and Characterization of Low-n Tau Oligomers

Kaniyappan, Senthilvelrajan; Chandupatla, Ram Reddy; Mandelkow, Eckhard

doi:10.1007/978-1-4939-7816-8_8

Cited by 5 publications

(5 citation statements)

References 20 publications

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“…AFM measurements were performed as described earlier [39]. Briefly, Tau fibril samples were diluted in PBS buffer for a final concentration of 0.5 -1 μM.…”

Section: Atomic Force Microscopy (Afm)mentioning

confidence: 99%

FRET-based Tau seeding assay does not represent prion-like templated assembly of Tau filaments

Kaniyappan

Tepper

Biernat

et al. 2020

Mol Neurodegeneration

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Tau aggregation into amyloid fibers based on the cross-beta structure is a hallmark of several Tauopathies, including Alzheimer Disease (AD). Trans-cellular propagation of Tau with pathological conformation has been suggested as a key disease mechanism. This is thought to cause the spreading of Tau pathology in AD by templated conversion of naive Tau in recipient cells into a pathological state, followed by assembly of pathological Tau fibers, similar to the mechanism of nucleated polymerization proposed for prion pathogenesis. In cell cultures, the process is often monitored by a FRET assay where the recipient cell expresses the Tau repeat domain (Tau RD) with a pro-aggregant mutation, fused to GFP-based FRET pairs. Since the size of the reporter GFP (barrel of~3 nm × 4 nm) is~7 times larger than the β-strand distance (0.47 nm), this points to a potential steric clash. Hence, we investigated the influence of the GFP tag on Tau FL or Tau RD aggregation. Using biophysical methods (light scattering, atomic force microscopy (AFM), and scanning-transmission electron microscopy (STEM)), we found that the assembly of Tau RD-GFP was severely inhibited and incompatible with that of Alzheimer filaments. These observations argue against the hypothesis that the propagation of Tau pathology in AD is caused by the prion-like templated aggregation of Tau protein, transmitted via cell-to-cell spreading of Tau. Thus, even though the observed local increase of FRET in recipient cells may be a valid hallmark of a pathological reaction, our data argue that it is caused by a process distinct from assembly of Tau RD filaments.

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“…AFM measurements were performed as described earlier [39]. Briefly, Tau fibril samples were diluted in PBS buffer for a final concentration of 0.5 -1 μM.…”

Section: Atomic Force Microscopy (Afm)mentioning

confidence: 99%

FRET-based Tau seeding assay does not represent prion-like templated assembly of Tau filaments

Kaniyappan

Tepper

Biernat

et al. 2020

Mol Neurodegeneration

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“…Non-adherent proteins were removed by washing twice with imaging buffer (10 mM Tris-HCl; 50 mM KCl, pH 7.4). Samples were imaged in oscillation mode in liquid (imaging buffer) as described(17) and acquired using the Nanoscope III microscope.…”

Section: Methodsmentioning

confidence: 99%

The plasma Factor XIII heterotetrameric complex structure: unexpected unequal pairing within a symmetric complex

Singh

Nazabal²,

Kaniyappan

et al. 2019

Preprint

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Factor XIII (FXIII) is a predominant determinant of clot stability, strength, and composition. Plasma FXIII circulates as a pro-transglutaminase with 2 catalytic A subunits and 2 carrierprotective B subunits in a heterotetramer (FXIII-A 2 B 2 ). FXIII-A 2 and -B 2 subunits are synthesized separately and then assembled in plasma. Following proteolytic activation by thrombin and calcium-mediated dissociation of the B-subunits, activated FXIII (FXIIIa) covalently cross-links fibrin, promoting clot stability. The zymogen and active states of the FXIII-A subunits have been structurally characterized; however, the structure of FXIII-B subunits and the FXIII-A 2 B 2 complex have remained elusive. Using integrative hybrid approaches including atomic force microscopy, cross-linking mass spectrometry, and computational approaches, we have constructed the first all-atom model of the FXIII-A 2 B 2 complex. We also used molecular dynamic simulations in combination with isothermal titration calorimetry to characterize FXIII-A 2 B 2 assembly, activation, and dissociation. Our data reveal unequal pairing of individual subunit monomers in an otherwise symmetric complex, and suggest this unusual structure is critical for both assembly and activation of this complex. Our findings enhance understanding of mechanisms associating FXIII-A 2 B 2 mutations with disease and have important implications for the rational design of molecules to alter FXIII assembly and/or activity to reduce bleeding and thrombotic complications.

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“…Tau RDΔK oligomers were prepared and purified as described previously, making use of hydrophobic interaction chromatography. 35 Briefly, 50 µM recombinant Tau RDΔK (Mr∼13.5 kDa) was diluted in tris buffered saline (TBS) pH 9.0 and incubated at 37 • C for 30 minutes for aggregation. The aggregated sample was lightly cross-linked using 0.01% glutaraldehyde (GA) for 10 minutes at 37…”

Section: Antibodiesmentioning

confidence: 99%

Novel antibody against low‐n oligomers of tau protein promotes clearance of tau in cells via lysosomes

Chandupatla

Flatley

Feederle

et al. 2020

A&D Transl Res & Clin Interv

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Introduction Tau, a natively unfolded soluble protein, forms abnormal oligomers and insoluble filaments in several neurodegenerative diseases, including Alzheimer disease (AD). Tau‐induced toxicity is mainly due to oligomers rather than monomers or fibrils. Methods We have developed monoclonal antibodies against purified low‐n tau oligomers of the tau repeat domain as a tool to neutralize tau aggregation and toxicity. In vitro aggregation inhibition was tested by thioflavin S, dynamic light scattering (DLS), and atomic force microscopy (AFM). Using a split‐luciferase complementation assay and fluorescence‐activated cell sorting (FACS), the inhibition of aggregation was analyzed in an N2a cell model of tauopathy. Results Antibodies inhibited tau aggregation in vitro up to ~90% by blocking tau at an oligomeric state. Some antibodies were able to block tau dimerization/oligomerization in cells, as measured by a split‐luciferase complementation assay. Antibodies applied extracellularly were internalized and led to sequestration of tau into lysosomes for degradation. Discussion Novel low‐n tau oligomer specific monoclonal antibody inhibits Tau oligomerization in cells and promotes toxic tau clearance.

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Purification and Characterization of Low-n Tau Oligomers

Cited by 5 publications

References 20 publications

FRET-based Tau seeding assay does not represent prion-like templated assembly of Tau filaments

FRET-based Tau seeding assay does not represent prion-like templated assembly of Tau filaments

The plasma Factor XIII heterotetrameric complex structure: unexpected unequal pairing within a symmetric complex

Novel antibody against low‐n oligomers of tau protein promotes clearance of tau in cells via lysosomes

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