Subtle change of fibrillation condition leads to substantial alteration of recombinant Tau fibril structure

Li, Xiang; Zhang, Shenqing; Liu, Zhengtao; Tao, Youqi; Xia, Wencheng; Sun, Yunpeng; Liu, Cong; Sun, Binyong; Li, Dan

doi:10.1016/j.isci.2022.105645

Cited by 16 publications

(29 citation statements)

References 40 publications

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“…In another work, doubling the MgCl 2 concentration from 200 mM to 400 mM increased the fraction of PHF-like fibrils by negative stain EM from 8 to 30%. 16 When we increased MgCl 2 to 400 mM, we observed faster and more consistent aggregation based on the time to half the maximum fluorescence of ThT than when no MgCl 2 was included (Figure 3C). Despite this change in aggregation kinetics, the relative abundance of PHF-like fibrils did not change significantly from 14% (range 9−23%) at 200 mM MgCl 2 to 15% (range 12−21%) at 400 mM MgCl 2 after 30 h of shaking.…”

Section: ■ Resultsmentioning

confidence: 64%

“…Dextran Sulfate and Other Conditions Reported to Form PHFs. Since other conditions were also reported to form PHF structures from this same fragment in vitro, 14,16 we assessed these conditions for the formation of PHF-like structures as well. Similar to 200 mM MgCl 2 , addition of 0.1 μg/mL dextran sulfate also favored the formation of PHF-like structures, although with a similar degree of structural impurity and prep-to-prep variation (Figure 4).…”

Section: ■ Resultsmentioning

confidence: 99%

“…However, fibrils with a single C-shaped protofilament per layer can be found upon seeding human neuroblastoma SH-SY5Y cells expressing 1N3R tau with AD-derived tau fibrils. 21 Tau filaments composed of varying numbers of these same C-shaped building blocks can be readily formed from truncated tau under a variety of conditions 14,16 including here, but generating the AD tau ultrastructure where exactly two C-shaped building blocks per layer come together in a specific arrangement robustly is still a challenge. Taken together, our work demonstrates that variation in polymorph relative abundance can exist both between laboratories and between experiments performed in the same lab.…”

Section: ■ Discussionmentioning

confidence: 99%

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Reconstitution of the Alzheimer’s Disease Tau Core Structure from Recombinant Tau^297–391 Yields Variable Quaternary Structures as Seen by Negative Stain and Cryo-EM

Glynn,

Chun,

Donahue

et al. 2023

Biochemistry

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The protein tau misfolds into disease-specific fibrillar structures in more than 20 neurodegenerative diseases collectively referred to as tauopathies. To understand and prevent disease-specific mechanisms of filament formation, in vitro models for aggregation that robustly yield these different end point structures will be necessary. Here, we used cryo-electron microscopy (cryo-EM) to reconstruct fibril polymorphs taken on by residues 297−391 of tau under conditions previously shown to give rise to the core structure found in Alzheimer's disease (AD). While we were able to reconstitute the AD tau core fold, the proportion of these paired helical filaments (PHFs) was highly variable, and a majority of filaments were composed of PHFs with an additional identical C-shaped protofilament attached near the PHF interface, termed triple helical filaments (THFs). Since the impact of filament layer quaternary structure on the biological properties of tau and other amyloid filaments is not known, the applications for samples of this morphology are presently uncertain. We further demonstrate the variation in the proportion of PHFs and PHF-like fibrils compared to other morphologies as a function of shaking time and AD polymorph-favoring cofactor concentration. This variation in polymorph abundance, even under identical experimental conditions, highlights the variation that can arise both within a lab and in different laboratory settings when reconstituting specific fibril polymorphs in vitro.

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Section: ■ Resultsmentioning

confidence: 64%

Section: ■ Resultsmentioning

confidence: 99%

Section: ■ Discussionmentioning

confidence: 99%

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Reconstitution of the Alzheimer’s Disease Tau Core Structure from Recombinant Tau^297–391 Yields Variable Quaternary Structures as Seen by Negative Stain and Cryo-EM

Glynn,

Chun,

Donahue

et al. 2023

Biochemistry

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“…This alleviates concerns in using recombinant proteins for drug candidate screening in order to evaluate potential antiaggregation activity using endogenous aggregates from diseased brain. Prior studies demonstrated differences in the ultrastructure of fibrils isolated from AD brains or generated from recombinant proteins. − The protein quantification of the plaques isolated from AD brains was measured with NanoDrop. Three replicates resulted in a concentration of 0.27 mg/mL of proteins.…”

Section: Results and Discussionmentioning

confidence: 99%

Evaluation of N- and O-Linked Indole Triazines for a Dual Effect on α-Synuclein and Tau Aggregation

Ramirez,

Ganegamage,

Min

et al. 2023

ACS Chem. Neurosci.

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Alzheimer’s disease (AD) is the most prevalent neurodegenerative disorder underlying dementia in the geriatric population. AD manifests by two pathological hallmarks: extracellular amyloid-β (Aβ) peptide-containing senile plaques and intraneuronal neurofibrillary tangles comprised of aggregated hyperphosphorylated tau protein (p-tau). However, more than half of AD cases also display the presence of aggregated α-synuclein (α-syn)-containing Lewy bodies. Conversely, Lewy bodies disorders have been reported to have concomitant Aβ plaques and neurofibrillary tangles. Our drug discovery program focuses on the synthesis of multitarget-directed ligands to abrogate aberrant α-syn, tau (2N4R), and p-tau (1N4R) aggregation and to slow the progression of AD and related dementias. To this end, we synthesized 11 compounds with a triazine-linker and evaluated their effectiveness in reducing α-syn, tau isoform 2N4R, and p-tau isoform 1N4R aggregation. We utilized biophysical methods such as thioflavin T (ThT) fluorescence assays, transmission electron microscopy (TEM), photoinduced cross-linking of unmodified proteins (PICUP), and M17D intracellular inclusion cell-based assays to evaluate the antiaggregation properties and cellular protection of our best compounds. We also performed disaggregation assays with isolated Aβ-plaques from human AD brains. Our results demonstrated that compound 10 was effective in reducing both oligomerization and fibril formation of α-syn and tau isoform 2N4R in a dose-dependent manner via ThT and PICUP assays. Compound 10 was also effective at reducing the formation of recombinant α-syn, tau 2N4R, and p-tau 1N4R fibrils by TEM. Compound 10 reduced the development of α-syn inclusions in M17D neuroblastoma cells and stopped the seeding of tau P301S using biosensor cells. Disaggregation experiments showed smaller Aβ-plaques and less paired helical filaments with compound 10. Compound 10 may provide molecular scaffolds for further optimization and preclinical studies for neurodegenerative proteinopathies.

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“…Additionally, cryo-EM aids in categorizing various conformations, thus enabling the examination of diverse sample populations . It has played a crucial role in unravelling the polymorphic characteristics of amyloid fibrils formed by proteins like α-syn, tau, and TDP-43. − Cryo-EM has also assisted in the exploration of dynamic regulatory mechanisms underlying amyloid fibril formation and aggregation. − Furthermore, cryo-EM has enhanced our knowledge of our understanding of the principles of protein misfolding and aggregation, and provided mechanistic insights into pathogenic protein strains implicated in NDs.…”

Section: Cryo-electron Microscopy (Cryo-em)-based Technologymentioning

confidence: 99%

Advanced Techniques for Detecting Protein Misfolding and Aggregation in Cellular Environments

Bai,

Zhang,

Dong

et al. 2023

Chem. Rev.

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Protein misfolding and aggregation, a key contributor to the progression of numerous neurodegenerative diseases, results in functional deficiencies and the creation of harmful intermediates. Detailed visualization of this misfolding process is of paramount importance for improving our understanding of disease mechanisms and for the development of potential therapeutic strategies. While in vitro studies using purified proteins have been instrumental in delivering significant insights into protein misfolding, the behavior of these proteins in the complex milieu of living cells often diverges significantly from such simplified environments. Biomedical imaging performed in cell provides cellular-level information with high physiological and pathological relevance, often surpassing the depth of information attainable through in vitro methods. This review highlights a variety of methodologies used to scrutinize protein misfolding within biological systems. This includes optical-based methods, strategies leaning on mass spectrometry, in-cell nuclear magnetic resonance, and cryo-electron microscopy. Recent advancements in these techniques have notably deepened our understanding of protein misfolding processes and the features of the resulting misfolded species within living cells. The progression in these fields promises to catalyze further breakthroughs in our comprehension of neurodegenerative disease mechanisms and potential therapeutic interventions.

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Subtle change of fibrillation condition leads to substantial alteration of recombinant Tau fibril structure

Cited by 16 publications

References 40 publications

Reconstitution of the Alzheimer’s Disease Tau Core Structure from Recombinant Tau^297–391 Yields Variable Quaternary Structures as Seen by Negative Stain and Cryo-EM

Reconstitution of the Alzheimer’s Disease Tau Core Structure from Recombinant Tau^297–391 Yields Variable Quaternary Structures as Seen by Negative Stain and Cryo-EM

Evaluation of N- and O-Linked Indole Triazines for a Dual Effect on α-Synuclein and Tau Aggregation

Advanced Techniques for Detecting Protein Misfolding and Aggregation in Cellular Environments

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Subtle change of fibrillation condition leads to substantial alteration of recombinant Tau fibril structure

Cited by 16 publications

References 40 publications

Reconstitution of the Alzheimer’s Disease Tau Core Structure from Recombinant Tau297–391 Yields Variable Quaternary Structures as Seen by Negative Stain and Cryo-EM

Reconstitution of the Alzheimer’s Disease Tau Core Structure from Recombinant Tau297–391 Yields Variable Quaternary Structures as Seen by Negative Stain and Cryo-EM

Evaluation of N- and O-Linked Indole Triazines for a Dual Effect on α-Synuclein and Tau Aggregation

Advanced Techniques for Detecting Protein Misfolding and Aggregation in Cellular Environments

Contact Info

Product

Resources

About

Reconstitution of the Alzheimer’s Disease Tau Core Structure from Recombinant Tau^297–391 Yields Variable Quaternary Structures as Seen by Negative Stain and Cryo-EM

Reconstitution of the Alzheimer’s Disease Tau Core Structure from Recombinant Tau^297–391 Yields Variable Quaternary Structures as Seen by Negative Stain and Cryo-EM