Extracellular Monomeric Tau Protein Is Sufficient to Initiate the Spread of Tau Protein Pathology

Michel, Claire H.; Kumar, Satish; Pinotsi, Dorothea; Tunnacliffe, Alan; George-Hyslop, Peter St; Mandelkow, Eckhard; Mandelkow, Eva‐Maria; Kaminski, Clemens F.; Schierle, Gabriele S. Kaminski

doi:10.1074/jbc.m113.515445

Cited by 144 publications

(167 citation statements)

References 42 publications

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“…Preventing disulfide linkage reduces spontaneous aggregation of tau monomer in vitro, enabling isolation of stable tau monomer (26). Tau C→A monomer (50 kD) as well as high molecular-weight tau oligomer fractions (∼500 kDa) were purified by size-exclusion chromatography and transduced into the biosensor cells.…”

Section: Resultsmentioning

confidence: 99%

Proteopathic tau seeding predicts tauopathy in vivo

Holmes

Furman

Mahan

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

501

792

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Transcellular propagation of protein aggregates, or proteopathic seeds, may drive the progression of neurodegenerative diseases in a prion-like manner. In tauopathies such as Alzheimer's disease, this model predicts that tau seeds propagate pathology through the brain via cell-cell transfer in neural networks. The critical role of tau seeding activity is untested, however. It is unknown whether seeding anticipates and correlates with subsequent development of pathology as predicted for a causal agent. One major limitation has been the lack of a robust assay to measure proteopathic seeding activity in biological specimens. We engineered an ultrasensitive, specific, and facile FRET-based flow cytometry biosensor assay based on expression of tau or synuclein fusions to CFP and YFP, and confirmed its sensitivity and specificity to tau (∼300 fM) and synuclein (∼300 pM) fibrils. This assay readily discriminates Alzheimer's disease vs. Huntington's disease and aged control brains. We then carried out a detailed time-course study in P301S tauopathy mice, comparing seeding activity versus histological markers of tau pathology, including MC1, AT8, PG5, and Thioflavin S. We detected robust seeding activity at 1.5 mo, >1 mo before the earliest histopathological stain. Proteopathic tau seeding is thus an early and robust marker of tauopathy, suggesting a proximal role for tau seeds in neurodegeneration.amyloid | neuropathology | dementia | aging P rotein aggregation characterizes many neurodegenerative disorders, including Alzheimer's disease (AD) and the related tauopathies. These disorders feature the accumulation of fibrillar deposits of the microtubule-associated protein tau with progressive deterioration of the central nervous system. Tau pathology and its associated brain atrophy do not appear randomly throughout the brain, but rather progress along distinct neural networks (1-5). This aspect suggests a role for transcellular spread of a pathogenic agent via neural connections. Our laboratory and others have previously hypothesized that tau aggregates-or seeds-serve as this agent of spread, transmitting the aggregated state from cell to cell via prion-like mechanisms (6-15).Mounting fundamental insights support this hypothesis. Tau seeds applied to the outside of cells bind the cell surface by attaching to heparan sulfate proteoglycans, triggering uptake by macropinocytosis (13). Upon internalization, tau seeds nucleate the fibrillization of endogenous tau monomer via templated conformational change, or seeding (8, 10). Tau seeding requires a critical unit of size for activity, as only particular species propagate the aggregated state (16). In vivo studies have described tau protein spreading from local sites to distant regions, presumably via transsynaptic movement (11,12,(17)(18)(19). Finally, our laboratory and another recently demonstrated that tau propagates discrete amyloid conformations through the brains of animals that give rise to unique neuropathologies (18,20).Despite this evidence, it remains unclear wheth...

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Section: Resultsmentioning

confidence: 99%

Proteopathic tau seeding predicts tauopathy in vivo

Holmes

Furman

Mahan

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

501

792

View full text Add to dashboard Cite

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“…Cells surrounded by fibrils are likely to be excluded from cell-cell contact, a vital mechanism for maintaining cell viability. Cell treatment with trypsin (40,48,59), chondroitinase ABC, and heparinase II (60, 61) did not detach the external amyloid from the cell membrane (data not shown). We propose that membrane surfaces facilitate fibril attachment and act as an anchor point for cell-mediated seeding mechanism.…”

Section: Journal Of Biological Chemistry 19819mentioning

confidence: 97%

Cell Damage in Light Chain Amyloidosis

Argany¹,

Lin²,

Misra³

et al. 2016

Journal of Biological Chemistry

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Light chain (AL) amyloidosis is an incurable human disease characterized by the misfolding, aggregation, and systemic deposition of amyloid composed of immunoglobulin light chains (LC). This work describes our studies on potential mechanisms of AL cytotoxicity. We have studied the internalization of AL soluble proteins and amyloid fibrils into human AC16 cardiomyocytes by using real time live cell image analysis. Our results show how external amyloid aggregates rapidly surround the cells and act as a recruitment point for soluble protein, triggering the amyloid fibril elongation. Soluble protein and external aggregates are internalized into AC16 cells via macropinocytosis. AL amyloid fibrils are shown to be highly cytotoxic at low concentrations. Additionally, caspase assays revealed soluble protein induces apoptosis, demonstrating different cytotoxic mechanisms between soluble protein and amyloid aggregates. This study emphasizes the complex immunoglobulin light chain-cell interactions that result in fibril internalization, protein recruitment, and cytotoxicity that may occur in AL amyloidosis. Light chain (AL)2 amyloidosis is a protein misfolding disease characterized by extracellular deposition of immunoglobulin light chains (LC) as amyloid fibrils. LC proteins are comprised of two distinct domains: the variable (V L ) and constant (C L ) domains (also called LC full-length (FL) protein to differentiate with the V L domain). In patients with AL amyloidosis, the LC aggregate and deposit in vital organs, causing organ failure and death (1). The factors governing deposition in individual tissues are unknown. Patients with cardiac AL amyloidosis have the worst prognosis, with a median survival of less than a year (2, 3).The finding that the V L was the primary component of amyloid fibrils influenced previous biophysical studies (4,5). Recent proteomic studies have demonstrated that amyloid deposits are likely heterogeneous in nature and can be formed by FL, V L , C L , or mixtures of all types of LC fragments (6 -8). Thermodynamic studies proposed a stabilizing role for the 3C L domain in the stability and a modulating effect on fibril formation (9). Recently, our laboratory has demonstrated that the C L domain modulates the amyloid formation reaction but has no effect on the stability of the protein (10).Soluble monoclonal LC, isolated from patients with amyloidosis, can impair rat cardiomyocyte function (11) and induce apoptotic events in mouse cardiomyocytes (12, 13). Also, urinederived LC can be internalized into primary rat cardiac fibroblasts (14) and primary human renal mesangial cells (15) through a pinocytic pathway (16) or via receptor, clathrin-mediated mechanisms, respectively (15).Within the amyloid field, it is widely accepted that oligomeric species are potentially more toxic than mature fibrils (17-20). However, toxicity associated with amyloid fibrils may also be pathologically relevant. Engel et al. (21) described a mechanism in which growth of islet amyloid associated polypeptides fibrils is re...

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“…For applications in cell biology and molecular self-assembly reactions [7][8][9][10][11][12] that require imaging with high temporal resolution over many time points, structured illumination microscopy (SIM) can be well-suited as it is not dependent on the photophysical properties of a particular fluorescent probe. Despite this inherent advantage of SIM, up to now its use has been mainly confined to imaging fixed cells or slow moving processes.…”

Section: Introductionmentioning

confidence: 99%

A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors

Young

Ströhl

Kaminski

2016

JoVE

Self Cite

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Optical super-resolution imaging with structured illumination microscopy (SIM) is a key technology for the visualization of processes at the molecular level in the chemical and biomedical sciences. Although commercial SIM systems are available, systems that are custom designed in the laboratory can outperform commercial systems, the latter typically designed for ease of use and general purpose applications, both in terms of imaging fidelity and speed. This article presents an in-depth guide to building a SIM system that uses total internal reflection (TIR) illumination and is capable of imaging at up to 10 Hz in three colors at a resolution reaching 100 nm. Due to the combination of SIM and TIRF, the system provides better image contrast than rival technologies. To achieve these specifications, several optical elements are used to enable automated control over the polarization state and spatial structure of the illumination light for all available excitation wavelengths. Full details on hardware implementation and control are given to achieve synchronization between excitation light pattern generation, wavelength, polarization state, and camera control with an emphasis on achieving maximum acquisition frame rate. A step-by-step protocol for system alignment and calibration is presented and the achievable resolution improvement is validated on ideal test samples. The capability for video-rate super-resolution imaging is demonstrated with living cells.

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Extracellular Monomeric Tau Protein Is Sufficient to Initiate the Spread of Tau Protein Pathology

Cited by 144 publications

References 42 publications

Proteopathic tau seeding predicts tauopathy in vivo

Proteopathic tau seeding predicts tauopathy in vivo

Cell Damage in Light Chain Amyloidosis

A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors

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