Quantitative Methods for the Detection of Tau Seeding Activity in Human Biofluids

Lathuilière, Aurélien; Hyman, Bradley T.

doi:10.3389/fnins.2021.654176

Cited by 7 publications

(7 citation statements)

References 66 publications

(50 reference statements)

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“…It has been reported that the phosphorylated high-molecular-weight tau is responsible for tau propagation 62 , and we observed its increase in the cells co-exposed to blue light and nocodazole (see figure 4B and C). To test the promotable effect of the blue light-induced insoluble tau oligomers on tau seeding activity, a seed amplification assay 63,64 was carried out. Recombinant tau aggregation having a β-sheet structure was significantly increased by treatment of lysates from blue light-irradiated cells, compared with no irradiation (figure 5F).…”

Section: Resultsmentioning

confidence: 99%

Intracellular Tau Fragment Droplets Serve as Seeds for Tau Fibrils

Soeda,

Yoshimura,

Bannai

et al. 2023

Preprint

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Intracellular tau aggregation requires a local protein concentration increase, referred to as droplets. However, the cellular mechanism remains insufficiently understood. We expressed OptoTau, a fusion of P301L mutant tau with CRY2olig, a light-sensitive protein that forms homo-oligomers. Under blue light exposure, OptoTau increased tau phosphorylation and was sequestered in aggresomes. Suppressing aggresome formation by nocodazole formed tau granular clusters in the cytoplasm. The granular clusters disappeared by discontinuing blue light exposure or 1,6-hexanediol treatment, which can dissolve droplets, suggesting that intracellular tau droplet formation requires the microtubules collapse. Expressing OptoTau-ΔN, a species of N-terminal cleaved tau that was observed in the Alzheimer’s disease brain, formed 1,6-hexanediol and detergent-resistant tau clusters in the cytoplasm by blue light stimulation. This intracellular stable tau clusters acted as seeds for tau fibrils in vitro. These results suggest that both tau droplet formation and N-terminal cleavage are necessary for neurofibrillary tangles formation in neurodegenerative diseases.

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Section: Resultsmentioning

confidence: 99%

Intracellular Tau Fragment Droplets Serve as Seeds for Tau Fibrils

Soeda,

Yoshimura,

Bannai

et al. 2023

Preprint

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“…In general, proteopathic seeds recruit, misfold, and template the aggregation of tau monomers, leading to their use in a wide range of seeding activity assays. So far, tau seeding activity can be quantified by in vitro seed amplification assays, such as Real-Time Quaking-Induced Conversion-based assay, RT-QuIC-based assay [ 12 , 46 ], cell-based assays in cultured cells, such as FRET-biosensor assay [ 31 ], and in vivo seed amplification assays [ 9 , 40 ]. Tau in AD and most other tauopathies is not mutated [ 22 ].…”

Section: Discussionmentioning

confidence: 99%

“…The seeding activity of proteopathic tau has been evaluated by seed amplification assay in vitro, cell-based assay, and in vivo seed amplification assay [ 40 ]. Tau in AD and most tauopathies is not mutated [ 22 , 55 ].…”

Section: Introductionmentioning

confidence: 99%

“…Tau in AD and most tauopathies is not mutated [ 22 , 55 ]. Most tau seeding activity assays use tau with FTDP-17–associated mutations [ 40 ]. Here, by using truncated tau 151-391 , we report two new assays, an in vitro tau capture assay and a seeded-tau aggregation assay in cultured cells, for the assessment of tau seeding activity.…”

Section: Introductionmentioning

confidence: 99%

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Tau seeding activity in various regions of down syndrome brain assessed by two novel assays

Jin

et al. 2022

acta neuropathol commun

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Propagation of tau pathology via the seeding of naive tau aggregation underlies the progression of Alzheimer’s disease (AD) and related tauopathies. Individuals with Down syndrome (DS) develop tau pathology at the fourth decade of life, but tau seeding activity in DS brain has not yet been determined. To measure tau seeding activity, we developed capture assay and seeded-tau aggregation assay with truncated tau151-391. By using brain extracts from AD and related tauopathies, we validated these two methods and found that the brain extracts from AD and related tauopathies, but not from controls and the diseases in which tau was not hyperphosphorylated, captured in vitro and seeded 3R-tau151-391 and 4R-tau151-391 to aggregate in cultured cells similarly. Captured tau151-391 levels were strongly correlated with the seeded-tau151-391 aggregation. Employing these two newly developed assays, we analyzed tau seeding activity in the temporal (TC), frontal (FC), and occipital cortex (OC); corpus callosum (CC); and cerebellar cortex (CBC) of DS and control brains. We found that the extracts of TC, FC, or OC, but not the CC or CBC of DS or the corresponding brain regions of control cases, captured tau151-391. Levels of the captured tau151-391 by brain extracts were positively correlated with their levels of phosphorylated tau. Extracts of cerebral cortex and CC, but not CBC of DS with a similar tau level, induced more tau151-391 aggregation than did the corresponding samples from the control cases. Thus, higher tau seeding activity associated with tau hyperphosphorylation was found in the TC, FC, and OC of DS compared with the corresponding control regions as well as with the CBC and CC of DS. Of note, these two assays are sensitive, specific, and repeatable at a low cost and provide a platform for measuring tau seeding activity and for drug screening that targets tau propagation.

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“…Tau seeding activity has been assessed by in vitro seed amplification assays, such as the Real-Time Quaking-Induced Conversion-based (RT-QuIC-based) assay ( Dujardin et al, 2020 ; Metrick et al, 2020 ), by cell-based assays in cultured cells, e.g., the fluorescence resonance energy transfer (FRET)-biosensor assay ( Holmes et al, 2014 ), and by in vivo seed amplification assays ( Clavaguera et al, 2009 ; Lathuiliere and Hyman, 2021 ). We previously found that truncated tau 151-391 is highly prone to being captured and templated to aggregation by oligomeric tau derived from AD brain (AD O-tau) ( Gu et al, 2020 ).…”

Section: Introductionmentioning

confidence: 99%

Two simple assays for assessing the seeding activity of proteopathic tau

Liu

Jin

et al. 2023

Front. Aging Neurosci.

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The regional distribution of neurofibrillary tangles of hyperphosphorylated tau aggregates is associated with the progression of Alzheimer’s disease (AD). Misfolded proteopathic tau recruits naïve tau and templates its misfolding and aggregation in a prion-like fashion, which is believed to be the molecular basis of propagation of tau pathology. A practical way to assess tau seeding activity is to measure its ability to recruit/bind other tau molecules and to induce tau aggregation. Based on the properties of proteopathic tau, here we report the development of two simple assays to assess tau seeding activity ----- capture assay in vitro and seeded-tau aggregation assay in cultured cells. In the capture assay, proteopathic tau was applied onto a nitrocellulose membrane and the membrane was incubated with cell lysate containing HA-tagged tau151-391 (HA-tau151-391). The captured tau on the membrane was determined by immuno-blots developed with anti-HA. For the seeded-tau aggregation assay, HEK-293FT cells transiently expressing HA-tau151-391 were treated with proteopathic tau in the presence of Lipofectamine 2000 and then lysed with RIPA buffer. RIPA-insoluble fraction containing aggregated tau was obtained by ultracentrifugation and analyzed by immuno-blot developed with anti-HA. To validate these two assays, we assessed the seeding activity of tau in the middle frontal gyrus, middle temporal gyrus and basal forebrain of AD and control brains and found that AD, but not control, brain extracts effectively captured and seeded tau151-391 aggregation. Basal forebrain contained less phospho-tau and tau seeding activity. The levels of captured tau or seeded-tau aggregates were positively correlated to the levels of phospho-tau, Braak stages and tangle sores. These two assays are specific and sensitive and can be carried out in a regular biomedical laboratory setting by using routine biochemical techniques.

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Quantitative Methods for the Detection of Tau Seeding Activity in Human Biofluids

Cited by 7 publications

References 66 publications

Intracellular Tau Fragment Droplets Serve as Seeds for Tau Fibrils

Intracellular Tau Fragment Droplets Serve as Seeds for Tau Fibrils

Tau seeding activity in various regions of down syndrome brain assessed by two novel assays

Two simple assays for assessing the seeding activity of proteopathic tau

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