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LetterMarch 28, 2018

Measurement of Tau Filament Fragmentation Provides Insights into Prion-like Spreading
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Franziska Kundel
Franziska Kundel
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom
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http://orcid.org/0000-0001-5013-0004
Liu Hong
Liu Hong
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom
More by Liu Hong
Benjamin Falcon
Benjamin Falcon
MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, United Kingdom
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William A. McEwan
William A. McEwan
MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, United Kingdom
More by William A. McEwan
Thomas C. T. Michaels
Thomas C. T. Michaels
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom
Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts 02138, United States
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Georg Meisl
Georg Meisl
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom
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http://orcid.org/0000-0002-6562-7715
Noemi Esteras
Noemi Esteras
Department of Molecular Neuroscience, UCL Institute of Neurology, London WC1N 3BG, United Kingdom
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Andrey Y. Abramov
Andrey Y. Abramov
Department of Molecular Neuroscience, UCL Institute of Neurology, London WC1N 3BG, United Kingdom
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Tuomas J. P. Knowles*
Tuomas J. P. Knowles
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom
*E-mail: [email protected]. Tel.: +44 1223 336344.
More by Tuomas J. P. Knowles
http://orcid.org/0000-0002-7879-0140
Michel Goedert*
Michel Goedert
MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, United Kingdom
*E-mail: [email protected]
More by Michel Goedert
David Klenerman*
David Klenerman
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom
UK Dementia Research Institute, University of Cambridge, Cambridge CB2 0XY, United Kingdom
*E-mail: [email protected]. Tel.: +44 1223 336481.
More by David Klenerman

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ACS Chemical Neuroscience

Cite this: ACS Chem. Neurosci. 2018, 9, 6, 1276–1282

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https://doi.org/10.1021/acschemneuro.8b00094

Published March 28, 2018

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Abstract

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The ordered assembly of amyloidogenic proteins causes a wide spectrum of common neurodegenerative diseases, including Alzheimer’s and Parkinson’s diseases. These diseases share common features with prion diseases, in which misfolded proteins can self-replicate and transmit disease across different hosts. Deciphering the molecular mechanisms that underlie the amplification of aggregates is fundamental for understanding how pathological deposits can spread through the brain and drive disease. Here, we used single-molecule microscopy to study the assembly and replication of tau at the single aggregate level. We found that tau aggregates have an intrinsic ability to amplify by filament fragmentation, and determined the doubling times for this replication process by kinetic modeling. We then simulated the spreading time for aggregates through the brain and found this to be in good agreement with both the observed time frame for spreading of pathological tau deposits in Alzheimer’s disease and in experimental models of tauopathies. With this work we begin to understand the physical parameters that govern the spreading rates of tau and other amyloids through the human brain.

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Keywords

For many years, cell autonomous mechanisms were believed to account for human neurodegenerative diseases. However, mounting evidence suggests that the protein assemblies, which are involved in various neurodegenerative diseases, including aggregated tau, are capable of self-sustaining amplification. (1) The latter is believed to enable the propagation of pathological assemblies along connected brain cells, resulting in progression from restricted areas to large numbers of brain regions. This phenomenon is commonly referred to as prion-like spreading. According to the prion hypothesis, upon entry into the cytoplasm of cells, tau assemblies can seed the aggregation of native monomers, thereby initiating the formation of additional aggregates, which can be released and spread to neighboring cells. (2,3) Synthetic tau filaments made from recombinant protein as well as filamentous material extracted from tau mouse models or Alzheimer’s disease (AD) brains have been shown to act as seeds in various model systems and initiate the spreading of tau pathology. (4)

On a molecular level, to achieve propagation of protein aggregates through the brain, an amplification of seeds is required. This requires secondary aggregation processes, by which new seeds are produced through the fragmentation of filaments and/or new assemblies form on the surface of existing filaments. (5) For prion diseases, amplification appears to occur predominantly through fragmentation. (6,7) Thus, an inverse correlation between the stability of prions and their infectivity has been described. (7−9) The mechanisms of amplification of tau seeds are less well studied. In experimental studies of tau transmission or propagation in cells or animal models, seed material is often amplified artificially by sonication before administration to the model system to increase the efficiency of templated seeding. Here, we set out to quantify the intrinsic ability of tau to amplify in order to gain insights into the key mechanisms governing prion-like spreading of tau in vivo. For this purpose, we characterized the aggregation kinetics of unlabeled full-length wild-type and mutant P301S tau using the recently developed single-molecule technique SAVE (Single Aggregate Visualization by Enhancement) imaging. (10)

Results and Discussion

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As the kinetic rates for the elongation and fragmentation of amyloid proteins can be derived from the length distributions of filaments during aggregation, (11,12) we employed SAVE imaging to follow the evolution of fibrillar tau aggregates. In more detail, wild-type and P301S tau (both 0N4R isoform) were aggregated in vitro using heparin as an inducer; at regular time points, aliquots of these aggregation reactions were mixed with the luminescent conjugated oligothiophene pFTAA (13) and imaged on a total internal reflection fluorescence (TIRF) microscope (see scheme in Figure 1a). As pFTAA has a high affinity for tau aggregates (14) and becomes highly fluorescent upon binding, we could readily detect the evolution of single tau fibrils after the induction of aggregation using this approach (see Figure 1b-i for representative images). It should be noted that our method was not able to detect small fibrillar species (<100 nm), either due to the reduced number of dye binding sites or reduced binding of small aggregates to the glass surface, and therefore fewer species were visible on our images at the early and late time-points.

Figure 1. Tau aggregation is a two stage process of elongation and fragmentation. (a) Observing the aggregation of full length tau by SAVE imaging. Wild-type and P301S tau (0N4R) were incubated under aggregating conditions for up to 2 months. At regular time points, aliquots of the reaction mixtures were stained with pFTAA, adsorbed onto a glass cover slide and imaged on a TIRF microscope. (b) (i) Representative images of fibrils during midelongation phase, at maximum length and at the end point. Scale bar: 15 μm. Inset: 3-fold magnification of boxed areas. Scale bar: 2.5 μm. (ii) The apparent average length of fibrils was analyzed and plotted as a function of time. To account for the diffraction limited resolution, the “apparent” average length was plotted whereby diffraction limited spots are counted as fibrils with a length of 1 pixel (204 nm). N = 3 (3 different batches of protein, each in triplicates). Error bars: SEM. Solid lines: fit to fragmentation model. (c) Representative electron micrographs of the 2 μM P301S aggregation reaction after 24 h and after 672 h. Scale bar: 500 nm. Dotted areas were magnified and shown on the right. Scale bar: 100 nm.

In order to obtain quantitative mechanistic insights from our SAVE images, we extracted the average lengths of fibrillar aggregates present at each time point and plotted these as a function of aggregation time. Aggregates smaller than the diffraction limit were counted as fibrils with the length of 1 pixel (204 nm). This revealed two stages of aggregation for both wild-type and mutant tau: first a relatively short stage (<200 h for the wild-type protein and <24 h for the P301S mutant) dominated by an increase of the average fibril length by elongation, and a long second phase during which the average fibril length decreased (see Figure 1b-ii). Wild-type tau aggregated slowly into straight fibrils with a maximum apparent length of 900 to 1,000 nm. By contrast, mutant P301S tau elongated markedly faster into long fibrils with curly appearance, reaching an apparent average length of 2,000 to 3,000 nm. In the late stage of aggregation, fibrils from both protein variants decreased in length over time and this effect was more apparent for P301S tau than for wild-type tau. At 10 μM aggregation concentration, wild-type fibrils decreased in size from around 900 nm at maximum to around 570 nm at the last measurement and mutant fibrils decreased from around 3000 nm to around 1300 nm. A comparison of the size histograms of P301S tau fibrils at the end of the elongation phase (24 h/1 day) with late fibrils (672 h/28 days) showed a redistribution of long fibrils toward small fibrils with a length less than 400 nm (Supporting Figure 1a). To corroborate this finding and to get an estimate of the fragment lengths by a nondiffraction limited method, transmission electron microscopy (TEM) was performed on P301S tau aggregates after 1 day and 28 days of aggregation (see representative electron micrographs in Figure 1c). The length histograms obtained from these images confirmed an increase of small fragments with a length of less than 100 nm after 28 days (Supporting Figure 1b). Taken together, this data shows that tau undergoes slow spontaneous fibril fragmentation, leading to a buildup of small fibrillar fragments over long periods of time. No proteolysis of tau was observed over the course of aggregation studied here, indicating that the protein stayed intact (see Supporting Figure 2).

Next, the experimentally determined average fibril lengths were fitted to a kinetic model of filamentous aggregation. Only a kinetic model describing aggregation as a nucleation-elongation mechanism with fibril fragmentation (15,16) (solid lines in Figure 1b-ii; kinetic model Figure 2a) was able to fully reproduce the data (see Supporting Information for details). By contrast, a model in which the fibrils self-replicate via secondary nucleation and do not fragment was unable to reproduce the data (see supporting Figure 3). The rate constants obtained from the fits are shown in Figure 2b. Both the elongation and fragmentation kinetics of tau determine the efficiency by which new fibrillar seeds are formed from existing aggregates. Here, we found that P301S tau has a 64 times faster elongation rate compared to wild type tau, possibly due to P301S tau existing in a more favorable conformation for addition to fibril ends than wild-type tau. The fragmentation rates of both protein variants are the same within experimental error. The primary nucleation rate constant and the critical nucleus size do not influence the amplification rate of existing aggregates and are therefore not relevant for aggregate spreading in vivo.

Figure 2. Kinetic model. (a) Schematic of microscopic steps during tau aggregation and corresponding rate constants. (b) Rate constants obtained from fits. K_m is the critical concentration for saturated elongation; n_c is the critical nucleus size. It is assumed that only the nucleation step is heparin dependent. Errors are allowed relative deviation.

Importantly, it was shown that the phenotype of different yeast prion strains can be predicted by a simple analytical model which takes into account the availability of monomeric protein, the rate of cell division, and the elongation and fragmentation rates of the different prion strains. (8) Here, we employ a similar model based on the experimentally determined rates for tau fibril growth and fragmentation to obtain estimates for the rate of prion-like replication of tau in vivo (see Figure 3a), since recombinant tau fibrils similar to the ones studied here have previously been shown to seed endogenous tau in recipient cells (3,17−20) and in PS19 mice (transgenic for 1N4R P301S tau). (21) If one assumes that the elongation and fragmentation rates are unaltered in vivo, we can derive the doubling time of tau aggregates from stochastic simulations of fibril growth and fragmentation. Notably, these processes are dependent on the availability of free monomer which can be added onto fibril ends during elongation. In the htau P301S mouse model overexpressing human P301S tau, we estimate the cytosolic human tau concentration to be ≈6 μM, assuming negligible binding of human tau to microtubules in the presence of mouse tau (see Supporting Figure 4). As shown in our simulations, fibril amplification is efficient at this concentration of free monomer (Figure 3b). On average, the doubling time t₂ for a tau seed is given by

(1)

where m is the concentration of monomer, k₊ is the elongation rate, k_f is the fragmentation rate, and K_m is the critical concentration for saturated elongation. (15) Under these conditions, we obtain a doubling time of 8 days for wild-type tau and 1.7 days for P301S tau. In order to put these doubling times into the context of tau propagation in a mouse brain, containing around 70 million neurons, (22) we calculated the time it would take to obtain one aggregate per cell (i.e., 70 million aggregates). According to our simple model, this would take around 7 months for wild-type tau and 1.5 months for P301S tau, values which are within an order of magnitude agreement of the experimentally observed life span of transgenic mice. (23)

Figure 3. Simulations of tau fibril replication. (a) Hypothetical mechanism for tau fibril amplification and spreading in tissues. (b,c) Simulations of stochastic fibril amplification for wild-type (green) and P301S tau (blue) aggregates (b) in the htau P301S mouse model or (c) in humans. On the left, representative simulations are shown for each condition; average amplification times of tau aggregates are shown on the right. For each condition, 100 independent simulations were performed to obtain a reliable amplification time.

In human neuronal cells, the cytosolic concentration of monomeric tau is markedly lower as the protein is expressed at endogenous levels and binds with nanomolar affinity to microtubules. (24,25) Therefore, when we lowered the cytosolic tau concentration in our simulations to 100 nM, the doubling times for wild-type and P301S tau aggregates increased distinctly to 38 days and 5 days, respectively (see Figure 3c). Based on these doubling times and the larger number of cells in a human brain (∼90 billion cells (26)), we obtain estimates of approximately 4 years for the spreading of wild-type tau through the brain or approximately 6 months for the frontotemporal dementia (FTD) associated mutant P301S tau. By comparison, the age of onset for sporadic AD is around 65, (27) while early onset FTD in individuals with the P301S mutation starts in their 20s or 30s. (28,29) The time between diagnosis and death is similar in both cases with typically 8–10 years.

It is important to note that the spreading of aggregates in vivo is dependent on many factors such as the efficiency of uptake, the rate of aggregate clearance, the release of new seeds from the cytosol, and so forth. These and other factors likely reduce the spreading efficiency, i.e., the fraction of aggregates which successfully enter a cell and amplify. Thus, seeding efficiencies in vitro, where recombinant aggregates are added to cultured cells, are usually much lower than 100%. (17,30) However, due to the exponential nature of fibril replication, our model is relatively insensitive to changes in spreading efficiencies, with estimated spreading times remaining within an order of magnitude of a human life span even at very low spreading efficiencies such as 0.0001% (see Supporting Table 1).

We have recently also measured the elongation and fragmentation rate for murine PrP and human α-synuclein (31) under similar experimental conditions (see Supporting Table 2), and these are plotted in Figure 4 for comparison. Since the doubling time depends on the product of the elongation and fragmentation rate, it is possible to have a slow fragmentation rate but a fast elongation rate for efficient aggregate amplification as is the case for P301S tau. Murine PrP has high values of both rate constants and is therefore predicted to amplify and hence spread much faster than the other proteins. Both wild-type tau and α-synuclein are predicted to amplify more slowly and appear to have doubling times close to the limit for the disease to occur in the human lifetime (Figure 4, gray area). Intriguingly, these values seem to correlate with the time of disease progression in the respective diseases, with the most common prion disease Creutzfeldt–Jakob disease having the shortest survival time of a few months to 2 years, (32) followed by tauopathies such as AD and FTD with a survival time of around 10 years (27−29) and Parkinson’s disease (the most common α-synucleinopathy) where disease worsens over long periods of time (10–20 years). (33)

Figure 4. Elongation and fragmentation rates for murine PrP, tau, and α-synuclein. Elongation and fragmentation rates of several disease associated proteins. The conditions used to determine these rate constants are shown in Supporting Table 2. The shaded area represents the minimum product of rate constants that would yield 90 billion aggregates in 100 years at a protein concentration of 100 nM.

Based on our findings, we suggest that the exponential amplification of aggregates by fragmentation is the basis for prion-like spreading in tauopathies such as AD and could play an important role in other neurodegenerative conditions. The exponential phase may be preceded by an initial lag phase during which the first tau aggregates are formed. Subsequently, a long silent period would follow, during which tau pathology slowly spreads but remains undetectable by common staining protocols. This hypothesis is supported by the recent finding that abnormal tau is indeed present in subcortical regions in a large fraction of young asymptomatic individuals. (34,35) Furthermore, brain tissue derived from mice transgenic for P301S tau shows seeding activity long before pathological tau deposits can be detected by immunohistological staining, (36,37) suggesting that seeds are present well before they can be detected by common staining protocols. Similarly, in tissue from human AD subjects at early Braak stages, seeding activity was observed including cortical regions which lack overt neurofibrillary pathology. (38) If correct, this makes it very hard to study this process directly due to the long doubling times and modeling enables extrapolation from experiments performed under conditions where seeding is more favorable and can hence be more easily measured. While not fully quantitative, our simple model provides insights into the key factors that will likely affect spreading rates. Higher expression of tau in specific cells would lead to faster doubling times in these cells while conversely efficient removal of aggregates by the cell would extend the doubling time. These factors in combination could provide a simple explanation for why certain cells are more susceptible to aggregate formation. Furthermore, different tau aggregate strains may have different fragmentation or elongation rates and thus different doubling times as is the case for prion strains, (8) which could account for the varying rates of disease progression in different tauopathies and patients. (27−29) It is worth noting that tau aggregates present in cells will continue to grow and fragment with time resulting in a loss of function and several aggregates being present in a cell, which could be toxic to cells and ultimately contribute to neuronal death. Cell division will reduce the number of aggregates, offering a possible explanation for why spreading occurs via connected neurons since they do not divide.

In summary, we have quantified the growth and amplification processes of full length tau and found that it can sustain a slow prion-like spreading mechanism. Further, we have developed a simple model that is able to make semiquantitative predictions about the replication of tau aggregates in the brain, which are in good agreement with experimental observations. Finally, extending the doubling time by reducing the rate of elongation or fragmentation may be a valid strategy for the treatment of tauopathies.

Methods

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Tau Purification

Human 0N4R tau and human 0N4R P301S tau were expressed as previously described. (19,29) Induced cells were cultured for 3–4 h at 37 °C, then harvested by centrifugation, and finally resuspended in buffer A (50 mM MES, 0.1 mM PMSF, 2 mM TCEP, pH 6.5), containing one EDTA-free complete protease inhibitor cocktail tablet per liter of culture. The cells were lysed by sonication (1 × 1.5 min, 5 s on, 10 s off, 40% amplitude), and the soluble fraction was cleared from cell debris by centrifugation at 4 °C, 15 000g (JA-20 rotor, Beckman Coulter). The cleared lysate was then filtered through a 0.45 μM syringe driven filter and loaded onto a HiTrap Capto S column (5 mL, GE Healthcare) previously equilibrated with buffer A. After any unbound proteins were removed from the column by washing with 10 column volumes of buffer A, tau was eluted using a linear gradient from 0 to 50% buffer B (buffer A + 1 M NaCl) over 10 column volumes. Fractions containing tau protein were then pooled and precipitated with 20% ammonium sulfate at 4 °C for 1h and the precipitate collected by centrifugation at 4 °C, 15,000 g (JA-20 rotor, Beckman Coulter). This pellet was either stored at −20 °C overnight or used directly for the next purification step. Tau was further purified by size-exclusion chromatography on a Superdex 200 Increase 10/300 GL size exclusion column (GE Healthcare Life Sciences), previously equilibrated with 1x SSPE and 1 mM TCEP. The fractions collected were analyzed by SDS-PAGE, and those showing the purest bands corresponding to tau were pooled. The protein concentration was determined by measuring its absorbance at 280 nm on a Nanodrop 2000 using an extinction coefficient of 7450 M^–1 cm^–1 and by BCA assay. Aliquots were frozen in liquid nitrogen and stored at −80 °C.

TIRF Microscope

The samples were imaged using a home-built total internal reflection fluorescence microscope. The total internal reflection mode restricts the fluorescence illumination to within 200 nm from the sample slide. A 488 nm laser (Cobolt MLD) was aligned and directed parallel to the optical axis at the edge of a 1.49 NA TIRF objective (APON60XO TIRF, Olympus), mounted on an inverted Nikon Eclipse Ti microscope. Fluorescence was collected by the same objective and separated from the returning TIRF beam by a dichroic mirror (Di01-R405/488/532/635, Semrock), and passed through an emission filters (FF03-525/50-25, Semrock). The control of the hardware was performed using custom-written scripts (bean-shell) for MicroManager (NIH). The images were recorded on an EMCCD camera (Evolve 512 Delta, Photometrics) operating in frame transfer mode (EMGain of 4.4 e^–/ADU and 250 ADU/photon). Each pixel was 204 nm in length. Images were recorded for 30 frames with an exposure time of 50 ms and averaged using ImageJ (NIH) software.

Sample Preparation for TIRF Imaging

Borosilicate glass coverslips (VWR international, Ø 50 mm) were cleaned using an argon plasma cleaner (PDC-002, Harrick Plasma) for 30 min to remove any fluorescent residues. Multiwell slide chambers (CultureWell chambered cover glass 50 well, Grace Bio-Laboratories) were separated from the original cover glass and affixed to the cleaned cover slides. To stain aggregates for imaging, samples were diluted into 30 nM pFTAA to a final protein concentration of 50 nM tau. Then 10 μL of each sample was adsorbed to the cover slides for 15 min before imaging.

Analyzing Number and Length of Aggregates

To analyze the number and length of tau aggregates, a Hessian based filament detection algorithm was employed as described by Salji et al. (39)

Electron Microscopy

Samples were placed on glow-discharged 400 meshed Formvar/carbon film-coated copper grids (Sigma-Aldrich) for 3 min and stained with uranyl acetate. Images were taken on a Philips Spirit transmission electron microscope at a magnification of 16 000.

Model

In order to explain the concentration dependent aggregation of tau proteins, we propose a three-step mechanism: nucleation, saturated elongation and fragmentation, as shown in Figure 2a. Here, we need to take the saturation effect into consideration, since for wild-type tau and P301S tau the maximal average fibril length under 2 μM and 10 μM conditions varies by a factor of less than 1.4 times, in contrast to the prediction of 2.2 times for simple first order elongation. (40) This is a clear sign that monomer saturation needs to be included.

Accordingly, the following kinetic equation can be used to quantify the time dependent fiber length distribution of tau fibrils,

(2)

where [F_j](t) (j ≥ n_c) is the concentration of tau fibrils constituted by j monomers, m(t) is the concentration of free tau monomers, and m_tot = m(t) + Σ_{j=n_c}^∞j[F_j](t) is the total concentration of tau proteins in the system. k_n, k_e, and k_f are the rate constants governing the primary nucleation, elongation, and fragmentation of tau fibrils, respectively. n_c is the critical nucleus size for primary nucleation, and K_m is the critical concentration for saturated elongation. With respect to [F_j](t), we can further introduce P_obs(t) = Σ_{j=n_b}^∞[F_j](t) and M_obs(t) = Σ_{j=n_b}^∞j·[F_j](t) as the number P and mass concentration M of tau fibrils which are observable in our single molecule measurement. The smallest observable aggregate was defined as an aggregate with a length of 100 nm, corresponding to n_b = 340 monomers, calculated from the mass-per length for full length tau fibrils of around 160 kDa/nm. (41)

Determining the Spreading Time Based on Stochastic-Deterministic Combined Simulations

In order to assess the potential of tau to propagate in a prion-like manner, i.e., by cycles of seeded growth and amplification, and spread over the whole mouse/human brain under normal physiological conditions, numerical investigations were performed on the basis of the elongation-fragmentation mechanism with kinetic rates given in eq 3,

(3)

with m₀ as the initial monomer concentration. Instead of direct stochastic simulations, which cannot reach a sufficiently large number of fibrils within an adequate computational time (more than 500 years on a personal computer), we adopted an alternative efficient algorithm by combining stochastic and deterministic simulations. In detail, starting from a single tau fibril of an initial length L₀ = 1000 monomers (≅300 nm) in a cell-like volume (10 μm)³, stochastic simulations using a Gillespie algorithm implementation were first performed. Then we switched to the deterministic ODE solver and continued the calculation from the end point of stochastic simulations, once there were enough fibrils generated in the system (fibril mass exceeds 10 million monomers, corresponding to ∼1000 fibrils, in our simulation). For each case, 100 independent simulations were performed to obtain a reliable spreading time.

Determining Mouse Tau Concentration by ELISA

Whole brains from P301S tau transgenic mice or wild-type C57BL/6 mice were snap frozen, suspended in 10x brain mass of A68 buffer (10 mM Tris-HCl, pH 7.4, 0.8 M NaCl, 1 mM EGTA, and 10% sucrose) and homogenized using a hand-held homogenizer (Millipore). Debris was pelleted by centrifugation at 21 000g for 20 min at 4 °C. The concentration of tau in homogenates was determined using a sandwich ELISA approach with HT7 (Life Technologies) as capture antibody and BR133 (42) as detection antibody. A standard curve was obtained using recombinant human P301S tau. To calculate the average brain concentration of tau, it was assumed that brains had a density of 1.1 g/mL.

Supporting Information

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschemneuro.8b00094.

Detailed description of the experimental procedures, data analysis methods, and kinetic modeling; figures illustrating fibril length distributions of P301S tau aggregates by TIRFM and TEM, SDS-PAGE analysis of tau to show absence of degradation during aggregation, misfits to a model which includes secondary nucleation, concentration measurements of P301S tau in P301S Tg mouse brains by ELISA; tables showing hypothetical tau spreading times at different spreading efficiencies and experimental conditions used for mPrP, α-synuclein, and tau aggregations from which data was derived for Figure 5 (PDF)

cn8b00094_si_001.pdf (611.31 kb)

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Author Information

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Corresponding Authors
- Tuomas J. P. Knowles - Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom; http://orcid.org/0000-0002-7879-0140; Email: [email protected]
- Michel Goedert - MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, United Kingdom; Email: [email protected]
- David Klenerman - Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom; UK Dementia Research Institute, University of Cambridge, Cambridge CB2 0XY, United Kingdom; Email: [email protected]
Authors
- Franziska Kundel - Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom; http://orcid.org/0000-0001-5013-0004
- Liu Hong - Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom; Present Address: L.H.: Zhou-Pei Yuan Center for Applied Mathematics, Tsinghua University, Beijing, P. R. China
- Benjamin Falcon - MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, United Kingdom
- William A. McEwan - MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, United Kingdom; Present Address: W.A.M.: Department of Clinical Neurosciences, UK Dementia Research Institute, University of Cambridge, Cambridge CB2 0XY, UK
- Thomas C. T. Michaels - Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom; Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts 02138, United States
- Georg Meisl - Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom; http://orcid.org/0000-0002-6562-7715
- Noemi Esteras - Department of Molecular Neuroscience, UCL Institute of Neurology, London WC1N 3BG, United Kingdom
- Andrey Y. Abramov - Department of Molecular Neuroscience, UCL Institute of Neurology, London WC1N 3BG, United Kingdom
Author Contributions
D.K. jointly conceived the study with M.G. and T.J.P.K. F.K., W.A.M., B.F., and N.E. performed the experiments and analyzed the data. L.H., T.C.T.M., and G.M. conducted the kinetics fits and simulations. F.K., D.K., and M.G. wrote the manuscript. All authors discussed the results and implications and commented on the manuscript at all stages. All authors have given approval to the final version of the manuscript.
Funding
D.K. acknowledges funding from the ERC (Grant Number 669237) and the Royal Society. F.K. acknowledges funding from the Augustus Newman foundation and the ERC. L.H. was supported by the Tsinghua University Initiative Scientific Research Programme (Grants 20151080424) and the program of China Scholarships Council (CSC). W.A.M. is supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (grant number 206248/Z/17/Z). T.C.T.M. acknowledges funding from Peterhouse College Cambridge and the Swiss National Science Foundation. G.M. was supported by Sidney Sussex College Cambridge. M.G. is an Honorary Professor in the Department of Clinical Neurosciences of the University of Cambridge. His work is supported by the UK Medical Research Council (MC_U105184291) and the European Union (Joint Programme-Neurodegeneration Research, JPND-REfrAME, and Horizon 2020 IMPRiND).
Notes
The authors declare no competing financial interest.

Acknowledgments

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The authors would like to thank Dr. Mathew Horrocks for help with the image acquisition, Dr. Therése Klingstedt for providing pFTAA, and Jason Sang, Dr. Rohan Ranasinghe, and David Wirthensohn for helpful discussions.

Abbreviations

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AD	Alzheimer’s disease
ELISA	enzyme-linked immunosorbent assay
FTD	frontotemporal dementia
pFTAA	pentameric formyl thiophene acetic acid
SAVE	Single Aggregate Visualization by Enhancement, TEM transmission electron microscopy
TIRF(M)	total internal reflection fluorescence (microscopy)

References

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This article references 42 other publications.

1
Goedert, M. (2015) Alzheimer’s and Parkinson’s diseases: The prion concept in relation to assembled Aβ, tau, and α-synuclein. Science 349 (6248), 1255555, DOI: 10.1126/science.1255555
Google Scholar
1
NEURODEGENERATION. Alzheimer's and Parkinson's diseases: The prion concept in relation to assembled Aβ, tau, and α-synuclein
Goedert Michel
Science (New York, N.Y.) (2015), 349 (6248), 1255555 ISSN:.
The pathological assembly of Aβ, tau, and α-synuclein is at the heart of Alzheimer's and Parkinson's diseases. Extracellular deposits of Aβ and intraneuronal tau inclusions define Alzheimer's disease, whereas intracellular inclusions of α-synuclein make up the Lewy pathology of Parkinson's disease. Most cases of disease are sporadic, but some are inherited in a dominant manner. Mutations frequently occur in the genes encoding Aβ, tau, and α-synuclein. Overexpression of these mutant proteins can give rise to disease-associated phenotypes. Protein assembly begins in specific regions of the brain during the process of Alzheimer's and Parkinson's diseases, from where it spreads to other areas.
2
Clavaguera, F. (2009) Transmission and spreading of tauopathy in transgenic mouse brain. Nat. Cell Biol. 11 (7), 909– 913, DOI: 10.1038/ncb1901
Google Scholar
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Transmission and spreading of tauopathy in transgenic mouse brain
Clavaguera, Florence; Bolmont, Tristan; Crowther, R. Anthony; Abramowski, Dorothee; Frank, Stephan; Probst, Alphonse; Fraser, Graham; Stalder, Anna K.; Beibel, Martin; Staufenbiel, Matthias; Jucker, Mathias; Goedert, Michel; Tolnay, Markus
Nature Cell Biology (2009), 11 (7), 909-913, S909/1-S909/8CODEN: NCBIFN; ISSN:1465-7392. (Nature Publishing Group)
Hyperphosphorylated tau makes up the filamentous intracellular inclusions of several neurodegenerative diseases, including Alzheimer's disease. In the disease process, neuronal tau inclusions first appear in the transentorhinal cortex from where they seem to spread to the hippocampal formation and neocortex. Cognitive impairment becomes manifest when inclusions reach the hippocampus, with abundant neocortical tau inclusions and extracellular β-amyloid deposits being the defining pathol. hallmarks of Alzheimer's disease. An abundance of tau inclusions, in the absence of β-amyloid deposits, defines Pick's disease, progressive supranuclear palsy, corticobasal degeneration and other diseases. Tau mutations cause familial forms of frontotemporal dementia, establishing that tau protein dysfunction is sufficient to cause neurodegeneration and dementia. Thus, transgenic mice expressing mutant (for example, P301S) human tau in nerve cells show the essential features of tauopathies, including neurodegeneration and abundant filaments made of hyperphosphorylated tau protein. By contrast, mouse lines expressing single isoforms of wild-type human tau do not produce tau filaments or show neurodegeneration. Here we have used tau-expressing lines to investigate whether exptl. tauopathy can be transmitted. We show that injection of brain ext. from mutant P301S tau-expressing mice into the brain of transgenic wild-type tau-expressing animals induces assembly of wild-type human tau into filaments and spreading of pathol. from the site of injection to neighboring brain regions.
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Frost, B., Jacks, R. L., and Diamond, M. I. (2009) Propagation of tau misfolding from the outside to the inside of a cell. J. Biol. Chem. 284 (19), 12845– 12852, DOI: 10.1074/jbc.M808759200
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Goedert, M., Eisenberg, D. S., and Crowther, R. A. (2017) Propagation of tau aggregates and neurodegeneration. Annu. Rev. Neurosci. 40, 189– 210, DOI: 10.1146/annurev-neuro-072116-031153
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Propagation of Tau Aggregates and Neurodegeneration
Goedert, Michel; Eisenberg, David S.; Crowther, R. Anthony
Annual Review of Neuroscience (2017), 40 (), 189-210CODEN: ARNSD5; ISSN:0147-006X. (Annual Reviews)
A pathway from the natively unfolded microtubule-assocd. protein Tau to a highly structured amyloid fibril underlies human Tauopathies. This ordered assembly causes disease and represents the gain of toxic function. In recent years, evidence has accumulated to suggest that Tau inclusions form first in a small no. of brain cells, from where they propagate to other regions, resulting in neurodegeneration and disease. Propagation of pathol. is often called prion-like, which refers to the capacity of an assembled protein to induce the same abnormal conformation in a protein of the same kind, initiating a self-amplifying cascade. In addn., prion-like encompasses the release of protein aggregates from brain cells and their uptake by neighboring cells. In mice, the intracerebral injection of Tau inclusions induces the ordered assembly of monomeric Tau, followed by its spreading to distant brain regions. Conformational differences between Tau aggregates from transgenic mouse brain and in vitro assembled recombinant protein account for the greater seeding potency of brain aggregates. Short fibrils constitute the major species of seed-competent Tau in the brains of transgenic mice. The existence of multiple human Tauopathies with distinct fibril morphologies has led to the suggestion that different mol. conformers (or strains) of aggregated Tau exist.
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Knowles, T. P. J., Vendruscolo, M., and Dobson, C. M. (2014) The amyloid state and its association with protein misfolding diseases. Nat. Rev. Mol. Cell Biol. 15 (6), 384– 396, DOI: 10.1038/nrm3810
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The amyloid state and its association with protein misfolding diseases
Knowles, Tuomas P. J.; Vendruscolo, Michele; Dobson, Christopher M.
Nature Reviews Molecular Cell Biology (2014), 15 (6), 384-396CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)
A review. The phenomenon of protein aggregation and amyloid formation has become the subject of rapidly increasing research activities across a wide range of scientific disciplines. Such activities have been stimulated by the assocn. of amyloid deposition with a range of debilitating medical disorders, from Alzheimer's disease to type II diabetes, many of which are major threats to human health and welfare in the modern world. It has become clear, however, that the ability to form the amyloid state is more general than previously imagined, and that its study can provide unique insights into the nature of the functional forms of peptides and proteins, as well as understanding the means by which protein homeostasis can be maintained and protein metastasis avoided.
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Bett, C. (2012) Biochemical properties of highly neuroinvasive prion strains. PLoS Pathog. 8 (2), e1002522, DOI: 10.1371/journal.ppat.1002522
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Prusiner, S. B. (2013) Biology and genetics of prions causing neurodegeneration. Annu. Rev. Genet. 47, 601– 623, DOI: 10.1146/annurev-genet-110711-155524
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Biology and genetics of prions causing neurodegeneration
Prusiner, Stanley B.
Annual Review of Genetics (2013), 47 (), 601-623CODEN: ARVGB7; ISSN:0066-4197. (Annual Reviews)
A review. Prions are proteins that acquire alternative conformations that become self-propagating. Transformation of proteins into prions is generally accompanied by an increase in β-sheet structure and a propensity to aggregate into oligomers. Some prions are beneficial and perform cellular functions, whereas others cause neurodegeneration. In mammals, more than a dozen proteins that become prions have been identified, and a similar no. has been found in fungi. In both mammals and fungi, variations in the prion conformation encipher the biol. properties of distinct prion strains. Increasing evidence argues that prions cause many neurodegenerative diseases (NDs), including Alzheimer's, Parkinson's, Creutzfeldt-Jakob, and Lou Gehrig's diseases, as well as the tauopathies. The majority of NDs are sporadic, and 10% to 20% are inherited. The late onset of heritable NDs, like their sporadic counterparts, may reflect the stochastic nature of prion formation; the pathogenesis of such illnesses seems to require prion accumulation to exceed some crit. threshold before neurol. dysfunction manifests.
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Tanaka, M., Collins, S. R., Toyama, B. H., and Weissman, J. S. (2006) The physical basis of how prion conformations determine strain phenotypes. Nature 442 (7102), 585– 589, DOI: 10.1038/nature04922
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The physical basis of how prion conformations determine strain phenotypes
Tanaka, Motomasa; Collins, Sean R.; Toyama, Brandon H.; Weissman, Jonathan S.
Nature (London, United Kingdom) (2006), 442 (7102), 585-589CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
A principle that has emerged from studies of protein aggregation is that proteins typically can misfold into a range of different aggregated forms. Moreover, the phenotypic and pathol. consequences of protein aggregation depend critically on the specific misfolded form. A striking example of this is the prion strain phenomenon, in which prion particles composed of the same protein cause distinct heritable states. Accumulating evidence from yeast prions such as [PSI+] and mammalian prions argues that differences in the prion conformation underlie prion strain variants. Nonetheless, it remains poorly understood why changes in the conformation of misfolded proteins alter their physiol. effects. Here, we present and exptl. validate an anal. model describing how [PSI+] strain phenotypes arise from the dynamic interaction among the effects of prion diln., competition for a limited pool of sol. protein, and conformation-dependent differences in prion growth and division rates. Anal. of three distinct prion conformations of yeast Sup35 (the [PSI+] protein determinant) and their in vivo phenotypes reveals that the Sup35 amyloid causing the strongest phenotype surprisingly shows the slowest growth. This slow growth, however, is more than compensated for by an increased brittleness that promotes prion division. The propensity of aggregates to undergo breakage, thereby generating new seeds, probably represents a key determinant of their physiol. impact for both infectious (prion) and non-infectious amyloids.
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Derdowski, A., Sindi, S. S., Klaips, C. L., DiSalvo, S., and Serio, T. R. (2010) A size threshold limits prion transmission and establishes phenotypic diversity. Science 330 (6004), 680– 683, DOI: 10.1126/science.1197785
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Horrocks, M. H. (2016) Single-molecule imaging of individual amyloid protein aggregates in human biofluids. ACS Chem. Neurosci. 7 (3), 399– 406, DOI: 10.1021/acschemneuro.5b00324
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Single-Molecule Imaging of Individual Amyloid Protein Aggregates in Human Biofluids
Horrocks, Mathew H.; Lee, Steven F.; Gandhi, Sonia; Magdalinou, Nadia K.; Chen, Serene W.; Devine, Michael J.; Tosatto, Laura; Kjaergaard, Magnus; Beckwith, Joseph S.; Zetterberg, Henrik; Iljina, Marija; Cremades, Nunilo; Dobson, Christopher M.; Wood, Nicholas W.; Klenerman, David
ACS Chemical Neuroscience (2016), 7 (3), 399-406CODEN: ACNCDM; ISSN:1948-7193. (American Chemical Society)
The misfolding and aggregation of proteins into amyloid fibrils characterizes many neurodegenerative disorders such as Parkinson's and Alzheimer's diseases. We report here a method, termed SAVE (single aggregate visualization by enhancement) imaging, for the ultrasensitive detection of individual amyloid fibrils and oligomers using single-mol. fluorescence microscopy. We demonstrate that this method is able to detect the presence of amyloid aggregates of α-synuclein, tau, and amyloid-β. In addn., we show that aggregates can also be identified in human cerebrospinal fluid (CSF). Significantly, we see a twofold increase in the av. aggregate concn. in CSF from Parkinson's disease patients compared to age-matched controls. Taken together, we conclude that this method provides an opportunity to characterize the structural nature of amyloid aggregates in a key biofluid, and therefore has the potential to study disease progression in both animal models and humans to enhance our understanding of neurodegenerative disorders.
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Michaels, T. C. T. (2015) The length distribution of frangible biofilaments. J. Chem. Phys. 143 (16), 164901, DOI: 10.1063/1.4933230
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The length distribution of frangible biofilaments
Michaels, Thomas C. T.; Yde, Pernille; Willis, Julian C. W.; Jensen, Mogens H.; Otzen, Daniel; Dobson, Christopher M.; Buell, Alexander K.; Knowles, Tuomas P. J.
Journal of Chemical Physics (2015), 143 (16), 164901/1-164901/14CODEN: JCPSA6; ISSN:0021-9606. (American Institute of Physics)
A no. of different proteins possess the ability to polymerize into filamentous structures. Certain classes of such assemblies can have key functional roles in the cell, such as providing the structural basis for the cytoskeleton in the case of actin and tubulin, while others are implicated in the development of many pathol. conditions, including Alzheimer's and Parkinson's diseases. In general, the fragmentation of such structures changes the total no. of filament ends, which act as growth sites, and hence is a key feature of the dynamics of filamentous growth phenomena. Here, the authors present an anal. study of the master equation of breakable filament assembly and derive closed-form expressions for the time evolution of the filament length distribution for both open and closed systems with infinite and finite monomer supply, resp. The authors used this theor. framework to analyze exptl. data for length distributions of insulin amyloid fibrils and showed that their theory allows insights into the microscopic mechanisms of biofilament assembly to be obtained beyond those available from the conventional anal. of filament mass only. (c) 2015 American Institute of Physics.
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Xue, W.-F. and Radford, S. E. (2013) An imaging and systems modeling approach to fibril breakage enables prediction of amyloid behavior. Biophys. J. 105 (12), 2811– 2819, DOI: 10.1016/j.bpj.2013.10.034
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An Imaging and Systems Modeling Approach to Fibril Breakage Enables Prediction of Amyloid Behavior
Xue, Wei-Feng; Radford, Sheena E.
Biophysical Journal (2013), 105 (12), 2811-2819CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
Delineating the nanoscale properties and the dynamic assembly and disassembly behaviors of amyloid fibrils is key for technol. applications that use the material properties of amyloid fibrils, as well as for developing treatments of amyloid-assocd. disease. However, quant. mechanistic understanding of the complex processes involving these heterogeneous supramol. systems presents challenges that have yet to be resolved. Here, we develop an approach that is capable of resolving the time dependence of fibril particle concn., length distribution, and length and position dependence of fibril fragmentation rates using a generic math. framework combined with exptl. data derived from at. force microscopy anal. of fibril length distributions. By application to amyloid assembly of β2-microglobulin in vitro under const. mech. stirring, we present a full description of the fibril fragmentation and growth behavior, and demonstrate the predictive power of the approach in terms of the samples' fibril dimensions, fibril load, and their efficiency to seed the growth of new amyloid fibrils. The approach developed offers opportunities to det., quantify, and predict the course and the consequences of amyloid assembly.
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Klingstedt, T. (2011) Synthesis of a library of oligothiophenes and their utilization as fluorescent ligands for spectral assignment of protein aggregates. Org. Biomol. Chem. 9 (24), 8356– 8370, DOI: 10.1039/c1ob05637a
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Synthesis of a library of oligothiophenes and their utilization as fluorescent ligands for spectral assignment of protein aggregates
Klingstedt, Therese; Aaslund, Andreas; Simon, Rozalyn A.; Johansson, Leif B. G.; Mason, Jeffrey J.; Nystroem, Sofie; Hammarstroem, Per; Nilsson, K. Peter R.
Organic & Biomolecular Chemistry (2011), 9 (24), 8356-8370CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
Mol. probes for selective identification of protein aggregates are important to advance the understanding of the mol. pathogenesis underlying protein aggregation diseases. Here the authors report the chem. design of a library of anionic luminescent conjugated oligothiophenes (LCOs), which can be utilized as ligands for detection of protein aggregates. Certain mol. requirements were shown to be necessary for detecting (i) early nonthioflavinophilic protein assemblies of Aβ1-42 and insulin preceding the formation of amyloid fibrils and (ii) for obtaining distinct spectral signatures of the two main pathol. hallmarks obsd. in human Alzheimer's disease brain tissue (Aβ plaques and neurofibrillary tangles). The authors' findings suggest that a superior anionic LCO-based ligand should have a backbone consisting of five to seven thiophene units and carboxyl groups extending the conjugated thiophene backbone. Such LCOs will be highly useful for studying the underlying mol. events of protein aggregation diseases and could also be utilized for the development of novel diagnostic tools for these diseases.
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Brelstaff, J. (2015) The fluorescent pentameric oligothiophene pFTAA identifies filamentous tau in live neurons cultured from adult P301S tau mice. Front. Neurosci. 9, 184, DOI: 10.3389/fnins.2015.00184
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The fluorescent pentameric oligothiophene pFTAA identifies filamentous tau in live neurons cultured from adult P301S tau mice
Brelstaff Jack; Ossola Bernardino; Spillantini Maria Grazia; Tolkovsky Aviva M; Neher Jonas J; Klingstedt Therese; Goedert Michel; Nilsson K Peter R
Frontiers in neuroscience (2015), 9 (), 184 ISSN:1662-4548.
Identification of fluorescent dyes that label the filamentous protein aggregates characteristic of neurodegenerative disease, such as β-amyloid and tau in Alzheimer's disease, in a live cell culture system has previously been a major hurdle. Here we show that pentameric formyl thiophene acetic acid (pFTAA) fulfills this function in living neurons cultured from adult P301S tau transgenic mice. Injection of pFTAA into 5-month-old P301S tau mice detected cortical and DRG neurons immunoreactive for AT100, an antibody that identifies solely filamentous tau, or MC1, an antibody that identifies a conformational change in tau that is commensurate with neurofibrillary tangle formation in Alzheimer's disease brains. In fixed cultures of dorsal root ganglion (DRG) neurons, pFTAA binding, which also identified AT100 or MC1+ve neurons, followed a single, saturable binding curve with a half saturation constant of 0.14 μM, the first reported measurement of a binding affinity of a beta-sheet reactive dye to primary neurons harboring filamentous tau. Treatment with formic acid, which solubilizes filamentous tau, extracted pFTAA, and prevented the re-binding of pFTAA and MC1 without perturbing expression of soluble tau, detected using an anti-human tau (HT7) antibody. In live cultures, pFTAA only identified DRG neurons that, after fixation, were AT100/MC1+ve, confirming that these forms of tau pre-exist in live neurons. The utility of pFTAA to discriminate between living neurons containing filamentous tau from other neurons is demonstrated by showing that more pFTAA+ve neurons die than pFTAA-ve neurons over 25 days. Since pFTAA identifies fibrillar tau and other misfolded proteins in living neurons in culture and in animal models of several neurodegenerative diseases, as well as in human brains, it will have considerable application in sorting out disease mechanisms and in identifying disease-modifying drugs that will ultimately help establish the mechanisms of neurodegeneration in human neurodegenerative diseases.
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Knowles, T. P. J. (2009) An analytical solution to the kinetics of breakable filament assembly. Science 326 (5959), 1533– 1537, DOI: 10.1126/science.1178250
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An Analytical Solution to the Kinetics of Breakable Filament Assembly
Knowles, Tuomas P. J.; Waudby, Christopher A.; Devlin, Glyn L.; Cohen, Samuel I. A.; Aguzzi, Adriano; Vendruscolo, Michele; Terentjev, Eugene M.; Welland, Mark E.; Dobson, Christopher M.
Science (Washington, DC, United States) (2009), 326 (5959), 1533-1537CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
We present an anal. treatment of a set of coupled kinetic equations that governs the self-assembly of filamentous mol. structures. Application to the case of protein aggregation demonstrates that the kinetics of amyloid growth can often be dominated by secondary rather than by primary nucleation events. Our results further reveal a range of general features of the growth kinetics of fragmenting filamentous structures, including the existence of generic scaling laws that provide mechanistic information in contexts ranging from in vitro amyloid growth to the in vivo development of mammalian prion diseases.
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Hong, L. and Yong, W.-A. (2013) Simple moment-closure model for the self-assembly of breakable amyloid filaments. Biophys. J. 104 (3), 533– 540, DOI: 10.1016/j.bpj.2012.12.039
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There is no corresponding record for this reference.
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Guo, J. L. and Lee, VM-Y (2011) Seeding of normal tau by pathological tau conformers drives pathogenesis of Alzheimer-like tangles. J. Biol. Chem. 286 (17), 15317– 15331, DOI: 10.1074/jbc.M110.209296
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Seeding of Normal Tau by Pathological Tau Conformers Drives Pathogenesis of Alzheimer-like Tangles
Guo, Jing L.; Lee, Virginia M.-Y.
Journal of Biological Chemistry (2011), 286 (17), 15317-15331CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
Neurofibrillary tangles (NFTs) in Alzheimer disease and related tauopathies are composed of insol. hyperphosphorylated Tau protein, but the mechanisms underlying the conversion of highly sol. Tau into insol. NFTs remain elusive. Here, we demonstrate that introduction of minute quantities of misfolded preformed Tau fibrils (Tau pffs) into Tau-expressing cells rapidly recruit large amts. of sol. Tau into filamentous inclusions resembling NFTs with unprecedented efficiency, suggesting a "seeding"-recruitment process as a highly plausible mechanism underlying NFT formation in vivo. Consistent with the emerging concept of prion-like transmissibility of disease-causing amyloidogenic proteins, we found that spontaneous uptake of Tau pffs into cells is likely mediated by endocytosis, suggesting a potential mechanism for the propagation of Tau lesions in tauopathy brains. Furthermore, sequestration of sol. Tau by pff-induced Tau aggregates attenuates microtubule overstabilization in Tau-expressing cells, supporting the hypothesis of a Tau loss-of-function toxicity in cells harboring NFTs. In summary, our study establishes a cellular system that robustly develops authentic NFT-like Tau aggregates, which provides mechanistic insights into NFT pathogenesis and a potential tool for identifying Tau-based therapeutics.
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Nonaka, T., Watanabe, S. T., Iwatsubo, T., and Hasegawa, M. (2010) Seeded aggregation and toxicity of α-synuclein and tau - Cellular models of neurodegenerative diseases. J. Biol. Chem. 285 (45), 34885– 34898, DOI: 10.1074/jbc.M110.148460
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There is no corresponding record for this reference.
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Falcon, B. (2015) Conformation determines the seeding potencies of native and recombinant tau aggregates. J. Biol. Chem. 290 (2), 1049– 1065, DOI: 10.1074/jbc.M114.589309
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Conformation Determines the Seeding Potencies of Native and Recombinant Tau Aggregates
Falcon, Benjamin; Cavallini, Annalisa; Angers, Rachel; Glover, Sarah; Murray, Tracey K.; Barnham, Luanda; Jackson, Samuel; O'Neill, Michael J.; Isaacs, Adrian M.; Hutton, Michael L.; Szekeres, Philip G.; Goedert, Michel; Bose, Suchira
Journal of Biological Chemistry (2015), 290 (2), 1049-1065CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
Intracellular Tau inclusions are a pathol. hallmark of several neurodegenerative diseases, collectively known as the tauopathies. They include Alzheimer disease, tangle-only dementia, Pick disease, argyrophilic grain disease, chronic traumatic encephalopathy, progressive supranuclear palsy, and corticobasal degeneration. Tau pathol. appears to spread through intercellular propagation, requiring the formation of assembled "prion-like" species. Several cell and animal models have been described that recapitulate aspects of this phenomenon. However, the mol. characteristics of seed-competent Tau remain unclear. Here, we have used a cell model to understand the relationships between Tau structure/phosphorylation and seeding by aggregated Tau species from the brains of mice transgenic for human mutant P301S Tau and full-length aggregated recombinant P301S Tau. Deletion of motifs 275VQIINK280 and 306VQIVYK311 abolished the seeding activity of recombinant full-length Tau, suggesting that its aggregation was necessary for seeding. We describe conformational differences between native and synthetic Tau aggregates that may account for the higher seeding activity of native assembled Tau. When added to aggregated Tau seeds from the brains of mice transgenic for P301S Tau, sol. recombinant Tau aggregated and acquired the mol. properties of aggregated Tau from transgenic mouse brain. We show that seeding is conferred by aggregated Tau that enters cells through macropinocytosis and seeds the assembly of endogenous Tau into filaments.
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Clavaguera, F. (2013) “Prion-like” templated misfolding in tauopathies. Brain Pathol. 23 (3), 342– 349, DOI: 10.1111/bpa.12044
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Prion-like templated misfolding in tauopathies
Clavaguera, Florence; Lavenir, Isabelle; Falcon, Ben; Frank, Stephan; Goedert, Michel; Tolnay, Markus
Brain Pathology (2013), 23 (3), 342-349CODEN: BRPAE7; ISSN:1015-6305. (Wiley-Blackwell)
A review. The sol. microtubule-assocd. protein tau forms hyperphosphorylated, insol. and filamentous inclusions in a no. of neurodegenerative diseases referred to as "tauopathies.". In Alzheimer's disease, tau pathol. develops in a stereotypical manner, with the first lesions appearing in the locus coeruleus and entorhinal cortex, from where they appear to spread to the hippocampus and neocortex. Propagation of tau pathol. is also a characteristic of argyrophilic grain disease, where the tau lesions spread throughout the limbic system. Significantly, isoform compn. and morphol. of tau filaments can differ between tauopathies, suggesting the existence of distinct tau strains. Extensive exptl. findings indicate that prion-like mechanisms underly the pathogenesis of tauopathies.
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Iba, M. (2013) Synthetic tau fibrils mediate transmission of neurofibrillary tangles in a transgenic mouse model of Alzheimer’s-like tauopathy. J. Neurosci. 33 (3), 1024– 1037, DOI: 10.1523/JNEUROSCI.2642-12.2013
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Synthetic tau fibrils mediate transmission of neurofibrillary tangles in a transgenic mouse model of Alzheimer's-like tauopathy
Iba, Michiyo; Guo, Jing L.; McBride, Jennifer D.; Zhang, Bin; Trojanowski, John Q.; Lee, Virginia M.-Y.
Journal of Neuroscience (2013), 33 (3), 1024-1037CODEN: JNRSDS; ISSN:0270-6474. (Society for Neuroscience)
Tauopathies, including Alzheimer's disease (AD) and frontotemporal lobar degeneration with tau pathologies, are neurodegenerative diseases characterized by neurofibrillary tangles (NFTs) comprising filamentous tau protein. Although emerging evidence suggests that tau pathol. may be transmitted, we demonstrate here that synthetic tau fibrils are sufficient to transmit tau inclusions in a mouse model. Specifically, intracerebral inoculation of young PS19 mice overexpressing mutant human tau (P301S) with synthetic preformed fibrils (pffs) assembled from recombinant full-length tau or truncated tau contg. four microtubule binding repeats resulted in rapid induction of NFT-like inclusions that propagated from injected sites to connected brain regions in a time-dependent manner. Interestingly, injection of tau pffs into either hippocampus or striatum together with overlaying cortex gave rise to distinct pattern of spreading. Moreover, unlike tau pathol. that spontaneously develops in old PS19 mice, the pff-induced tau inclusions more closely resembled AD NFTs because they were Thioflavin S pos., acetylated, and more resistant to proteinase K digestion. Together, our study demonstrates that synthetic tau pffs alone are capable of inducing authentic NFT-like tau aggregates and initiating spreading of tau pathol. in a tauopathy mouse model.
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Herculano-Houzel, S., Mota, B., and Lent, R. (2006) Cellular scaling rules for rodent brains. Proc. Natl. Acad. Sci. U. S. A. 103 (32), 12138– 12143, DOI: 10.1073/pnas.0604911103
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Cellular scaling rules for rodent brains
Herculano-Houzel, Suzana; Mota, Bruno; Lent, Roberto
Proceedings of the National Academy of Sciences of the United States of America (2006), 103 (32), 12138-12143CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
How do cell no. and size det. brain size Here, we show that, in the order Rodentia, increased size of the cerebral cortex, cerebellum, and remaining areas across six species is achieved through greater nos. of neurons of larger size, and much greater nos. of nonneuronal cells of roughly invariant size, such that the ratio between total neuronal and nonneuronal mass remains const. across species. Although relative cerebellar size remains stable among rodents, the no. of cerebellar neurons increases with brain size more rapidly than in the cortex, such that the cerebellar fraction of total brain neurons increases with brain size. In contrast, although the relative cortical size increases with total brain size, the cortical fraction of total brain neurons remains const. We propose that the faster increase in av. neuronal size in the cerebral cortex than in the cerebellum as these structures gain neurons and the rapidly increasing glial nos. that generate glial mass to match total neuronal mass at a fixed glia/neuron total mass ratio are fundamental cellular constraints that lead to the relative expansion of cerebral cortical vol. across species.
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Allen, B. (2002) Abundant tau filaments and nonapoptotic neurodegeneration in transgenic mice expressing human P301S tau protein. J. Neurosci. 22 (21), 9340– 9351
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Abundant tau filaments and nonapoptotic neurodegeneration in transgenic mice expressing human P301S Tau protein
Allen, Bridget; Ingram, Esther; Takao, Masaki; Smith, Michael J.; Jakes, Ross; Virdee, Kanwar; Yoshida, Hirotaka; Holzer, Max; Craxton, Molly; Emson, Piers C.; Atzori, Cristiana; Migheli, Antonio; Crowther, R. Anthony; Ghetti, Bernardino; Spillantini, Maria Grazia; Goedert, Michel
Journal of Neuroscience (2002), 22 (21), 9340-9351CODEN: JNRSDS; ISSN:0270-6474. (Society for Neuroscience)
The identification of mutations in the Tau gene in frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) has made it possible to express human tau protein with pathogenic mutations in transgenic animals. Here the authors report on the prodn. and characterization of a line of mice transgenic for the 383 aa isoform of human tau with the P301S mutation. At 5-6 mo of age, homozygous animals from this line developed a neurol. phenotype dominated by a severe paraparesis. According to light microscopy, many nerve cells in brain and spinal cord were strongly immunoreactive for hyperphosphorylated tau. According to electron microscopy, abundant filaments made of hyperphosphorylated tau protein were present. The majority of filaments resembled the half-twisted ribbons described previously in cases of FTDP-17, with a minority of filaments resembling the paired helical filaments of Alzheimer's disease. Sarkosyl-insol. tau from brains and spinal cords of transgenic mice ran as a hyperphosphorylated 64 kDa band, the same apparent mol. mass as that of the 383 aa tau isoform in the human tauopathies. Perchloric acid-sol. tau was also phosphorylated at many sites, with the notable exception of serine 214. In the spinal cord, neurodegeneration was present, as indicated by a 49% redn. in the no. of motor neurons. No evidence for apoptosis was obtained, despite the extensive colocalization of hyperphosphorylated tau protein with activated MAP kinase family members. The latter may be involved in the hyperphosphorylation of tau.
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Ackmann, M., Wiech, H., and Mandelkow, E. (2000) Nonsaturable binding indicates clustering of tau on the microtubule surface in a paired helical filament-like conformation. J. Biol. Chem. 275 (39), 30335– 30343, DOI: 10.1074/jbc.M002590200
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Makrides, V., Massie, M. R., Feinstein, S. C., and Lew, J. (2004) Evidence for two distinct binding sites for tau on microtubules. Proc. Natl. Acad. Sci. U. S. A. 101 (17), 6746– 6751, DOI: 10.1073/pnas.0400992101
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Azevedo, F. A. C. (2009) Equal numbers of neuronal and nonneuronal cells make the human brain an isometrically scaled-up primate brain. J. Comp. Neurol. 513 (5), 532– 541, DOI: 10.1002/cne.21974
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Equal numbers of neuronal and nonneuronal cells make the human brain an isometrically scaled-up primate brain
Azevedo Frederico A C; Carvalho Ludmila R B; Grinberg Lea T; Farfel Jose Marcelo; Ferretti Renata E L; Leite Renata E P; Jacob Filho Wilson; Lent Roberto; Herculano-Houzel Suzana
The Journal of comparative neurology (2009), 513 (5), 532-41 ISSN:.
The human brain is often considered to be the most cognitively capable among mammalian brains and to be much larger than expected for a mammal of our body size. Although the number of neurons is generally assumed to be a determinant of computational power, and despite the widespread quotes that the human brain contains 100 billion neurons and ten times more glial cells, the absolute number of neurons and glial cells in the human brain remains unknown. Here we determine these numbers by using the isotropic fractionator and compare them with the expected values for a human-sized primate. We find that the adult male human brain contains on average 86.1 +/- 8.1 billion NeuN-positive cells ("neurons") and 84.6 +/- 9.8 billion NeuN-negative ("nonneuronal") cells. With only 19% of all neurons located in the cerebral cortex, greater cortical size (representing 82% of total brain mass) in humans compared with other primates does not reflect an increased relative number of cortical neurons. The ratios between glial cells and neurons in the human brain structures are similar to those found in other primates, and their numbers of cells match those expected for a primate of human proportions. These findings challenge the common view that humans stand out from other primates in their brain composition and indicate that, with regard to numbers of neuronal and nonneuronal cells, the human brain is an isometrically scaled-up primate brain.
27
Bird, T. D. (1993) Alzheimer Disease Overview. In GeneReviews (Adam, M. P., Ardinger, H. H., and Pagon, R. A., et al., Eds.), University of Washington, Seattle, WA, available at: http://www.ncbi.nlm.nih.gov/books/NBK1161/ [accessed November 17, 2017].
Google Scholar
There is no corresponding record for this reference.
28
Sperfeld, A. D. (1999) FTDP-17: an early-onset phenotype with parkinsonism and epileptic seizures caused by a novel mutation. Ann. Neurol. 46 (5), 708– 715, DOI: 10.1002/1531-8249(199911)46:5<708::AID-ANA5>3.0.CO;2-K
Google Scholar
28
FTDP-17: an early-onset phenotype with parkinsonism and epileptic seizures caused by a novel mutation
Sperfeld A D; Collatz M B; Baier H; Palmbach M; Storch A; Schwarz J; Tatsch K; Reske S; Joosse M; Heutink P; Ludolph A C
Annals of neurology (1999), 46 (5), 708-15 ISSN:0364-5134.
Recently, mutations in the tau gene on chromosome 17 were found causative for autosomal dominantly inherited frontotemporal dementia and parkinsonism (FTDP-17). We describe a family carrying a missense mutation at nucleotide 1137 C --> T, resulting in the amino acid substitution P301S. Methods of investigations include clinical, electrophysiological, and imaging techniques. This kindred presents with a novel phenotype characterized by an early onset of rapidly progressive frontotemporal dementia and parkinsonism in combination with epileptic seizures. We define the dopaminergic deficits as being predominantly presynaptic by the use of single-photon emission computed tomography with a dopamine transporter ligand. The association of this early-onset phenotype with P301S mutation is not entirely consistent with current criteria for the diagnosis of frontotemporal dementias and may encourage the search for tau mutations in diseases similar but not identical to FTDP-17. Also, the change from proline to serine suggests that this mutation might contribute to tau hyperphosphorylation.
29
Bugiani, O. (1999) Frontotemporal dementia and corticobasal degeneration in a family with a P301S mutation in tau. J. Neuropathol. Exp. Neurol. 58 (6), 667– 677, DOI: 10.1097/00005072-199906000-00011
Google Scholar
29
Frontotemporal dementia and corticobasal degeneration in a family with a P301S mutation in tau
Bugiani O; Murrell J R; Giaccone G; Hasegawa M; Ghigo G; Tabaton M; Morbin M; Primavera A; Carella F; Solaro C; Grisoli M; Savoiardo M; Spillantini M G; Tagliavini F; Goedert M; Ghetti B
Journal of neuropathology and experimental neurology (1999), 58 (6), 667-77 ISSN:0022-3069.
The tau gene has been found to be the locus of dementia with rigidity linked to chromosome 17. Exonic and intronic mutations have been described in a number of families. Here we describe a P301S mutation in exon 10 of the tau gene in a new family. Two members of this family were affected. One individual presented with frontotemporal dementia, whereas his son has corticobasal degeneration, demonstrating that the same primary gene defect in tau can lead to 2 distinct clinical phenotypes. Both individuals developed rapidly progressive disease in the third decade. Neuropathologically, the father presented with an extensive filamentous pathology made of hyperphosphorylated tau protein. Biochemically, recombinant tau protein with the P301S mutation showed a greatly reduced ability to promote microtubule assembly.
30
McEwan, W. A. (2017) Cytosolic Fc receptor TRIM21 inhibits seeded tau aggregation. Proc. Natl. Acad. Sci. U. S. A. 114 (3), 574– 579, DOI: 10.1073/pnas.1607215114
Google Scholar
There is no corresponding record for this reference.
31
Sang, J. C., Direct observation of protein aggregate replication in vitro to model prion and prion-like diseases; submitted.
Google Scholar
There is no corresponding record for this reference.
32
Pocchiari, M. (2004) Predictors of survival in sporadic Creutzfeldt–Jakob disease and other human transmissible spongiform encephalopathies. Brain 127 (10), 2348– 2359, DOI: 10.1093/brain/awh249
Google Scholar
There is no corresponding record for this reference.
33
Kalia, L. V. and Lang, A. E. (2015) Parkinson’s disease. Lancet 386 (9996), 896– 912, DOI: 10.1016/S0140-6736(14)61393-3
Google Scholar
33
Parkinson's disease
Kalia, Lorraine V.; Lang, Anthony E.
Lancet (2015), 386 (9996), 896-912CODEN: LANCAO; ISSN:0140-6736. (Elsevier Ltd.)
Parkinson's disease is a neurol. disorder with evolving layers of complexity. It has long been characterised by the classical motor features of parkinsonism assocd. with Lewy bodies and loss of dopaminergic neurons in the substantia nigra. However, the symptomatol. of Parkinson's disease is now recognized as heterogeneous, with clin. significant non-motor features. Similarly, its pathol. involves extensive regions of the nervous system, various neurotransmitters, and protein aggregates other than just Lewy bodies. The cause of Parkinson's disease remains unknown, but risk of developing Parkinson's disease is no longer viewed as primarily due to environmental factors. Instead, Parkinson's disease seems to result from a complicated interplay of genetic and environmental factors affecting numerous fundamental cellular processes. The complexity of Parkinson's disease is accompanied by clin. challenges, including an inability to make a definitive diagnosis at the earliest stages of the disease and difficulties in the management of symptoms at later stages. Furthermore, there are no treatments that slow the neurodegenerative process. In this Seminar, we review these complexities and challenges of Parkinson's disease.
34
Braak, H. and Del Tredici, K. (2011) The pathological process underlying Alzheimer’s disease in individuals under thirty. Acta Neuropathol. 121 (2), 171– 181, DOI: 10.1007/s00401-010-0789-4
Google Scholar
34
The pathological process underlying Alzheimer's disease in individuals under thirty
Braak Heiko; Del Tredici Kelly
Acta neuropathologica (2011), 121 (2), 171-81 ISSN:.
Brains of 42 individuals between the ages of 4 and 29 were examined with antibodies (AT8, 4G8) and silver stains for the presence of intraneuronal and extracellular protein aggregates associated with Alzheimer's disease. Thirty-eight of 42 (38/42) cases displayed abnormally phosphorylated tau protein (pretangle material) in nerve cells or in portions of their cellular processes, and 41/42 individuals showed no extracellular amyloid-β protein deposition or neuritic plaques-an individual with Down syndrome was the only exception. In 16/42 cases abnormal tau was found in the transentorhinal region, and in 3/42 cases this site was Gallyas-positive for isolated NFTs (NFT stage I). Of 26 cases that lacked abnormal tau in the transentorhinal region, 4 did not show pretangle material at subcortical sites. The remaining 22 of these same 26 cases, however, had subcortical lesions confined to non-thalamic nuclei with diffuse projections to the cerebral cortex, and, remarkably, in 19/22 individuals the pretangle material was confined to the noradrenergic coeruleus/subcoeruleus complex. Assuming the pretangle alterations are not transient and do not regress, these findings may indicate that the Alzheimer's disease-related pathological process leading to neurofibrillary tangle formation does not begin in the cerebral cortex but, rather, in select subcortical nuclei, and it may start quite early, i.e., before puberty or in early young adulthood.
35
Elobeid, A., Soininen, H., and Alafuzoff, I. (2012) Hyperphosphorylated tau in young and middle-aged subjects. Acta Neuropathol. 123 (1), 97– 104, DOI: 10.1007/s00401-011-0906-z
Google Scholar
There is no corresponding record for this reference.
36
Holmes, B. B. (2014) Proteopathic tau seeding predicts tauopathy in vivo. Proc. Natl. Acad. Sci. U. S. A. 111 (41), E4376– E4385, DOI: 10.1073/pnas.1411649111
Google Scholar
36
Proteopathic tau seeding predicts tauopathy in vivo
Holmes, Brandon B.; Furman, Jennifer L.; Mahan, Thomas E.; Yamasaki, Tritia R.; Mirbaha, Hilda; Eades, William C.; Belaygorod, Larisa; Cairns, Nigel J.; Holtzman, David M.; Diamond, Marc I.
Proceedings of the National Academy of Sciences of the United States of America (2014), 111 (41), E4376-E4385CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
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 pathol. through the brain via cell-cell transfer in neural networks. The crit. role of tau seeding activity is untested, however. It is unknown whether seeding anticipates and correlates with subsequent development of pathol. as predicted for a causal agent. One major limitation has been the lack of a robust assay to measure proteopathic seeding activity in biol. 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 vs. histol. markers of tau pathol., including MC1, AT8, PG5, and Thioflavin S. We detected robust seeding activity at 1.5 mo, >1 mo before the earliest histopathol. stain. Proteopathic tau seeding is thus an early and robust marker of tauopathy, suggesting a proximal role for tau seeds in neurodegeneration.
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Kaufman, S. K., Thomas, T. L., Del Tredici, K., Braak, H., and Diamond, M. I. (2017) Characterization of tau prion seeding activity and strains from formaldehyde-fixed tissue. Acta Neuropathol. Commun. 5 (1), 41, DOI: 10.1186/s40478-017-0442-8
Google Scholar
37
Characterization of tau prion seeding activity and strains from formaldehyde-fixed tissue
Kaufman Sarah K; Thomas Talitha L; Diamond Marc I; Kaufman Sarah K; Del Tredici Kelly; Braak Heiko
Acta neuropathologica communications (2017), 5 (1), 41 ISSN:.
Tauopathies such as Alzheimer's disease (AD) feature progressive intraneuronal deposition of aggregated tau protein. The cause is unknown, but in experimental systems trans-cellular propagation of tau pathology resembles prion pathogenesis. Tau aggregate inoculation into mice produces transmissible pathology, and tau forms distinct strains, i.e. conformers that faithfully replicate and create predictable patterns of pathology in vivo. The prion model predicts that tau seed formation will anticipate neurofibrillary tau pathology. To test this idea requires simultaneous assessment of seed titer and immunohistochemistry (IHC) of brain tissue, but it is unknown whether tau seed titer can be determined in formaldehyde-fixed tissue. We have previously created a cellular biosensor system that uses flow cytometry to quantify induced tau aggregation and thus determine seed titer. In unfixed tissue from PS19 tauopathy mice that express 1 N,4R tau (P301S), we have measured tau seeding activity that precedes the first observable histopathology by many months. Additionally, in fresh frozen tissue from human AD subjects at early to mid-neurofibrillary tangle stages (NFT I-IV), we have observed tau seeding activity in cortical regions predicted to lack neurofibrillary pathology. However, we could not directly compare the same regions by IHC and seeding activity in either case. We now describe a protocol to extract and measure tau seeding activity from small volumes (.04 mm(3)) of formaldehyde-fixed tissue immediately adjacent to that used for IHC. We validated this method with the PS19 transgenic mouse model, and easily observed seeding well before the development of phospho-tau pathology. We also accurately isolated two tau strains, DS9 and DS10, from fixed brain tissues in mice. Finally, we have observed robust seeding activity in fixed AD brain, but not controls. The successful coupling of classical IHC with seeding and strain detection should enable detailed study of banked brain tissue in AD and other tauopathies.
38
Furman, J. L. (2017) Widespread tau seeding activity at early Braak stages. Acta Neuropathol. 133 (1), 91– 100, DOI: 10.1007/s00401-016-1644-z
Google Scholar
There is no corresponding record for this reference.
39
Salji, C. J. (2015) The JCMT Gould Belt Survey: properties of star-forming filaments in Orion A North. Mon. Not. R. Astron. Soc. 449 (2), 1782– 1796, DOI: 10.1093/mnras/stv369
Google Scholar
39
The JCMT Gould Belt Survey: properties of star-forming filaments in Orion A North
Salji, C. J.; Richer, J. S.; Buckle, J. V.; Di Francesco, J.; Hatchell, J.; Hogerheijde, M.; Johnstone, D.; Kirk, H.; Ward-Thompson, D.
Monthly Notices of the Royal Astronomical Society (2015), 449 (2), 1782-1796CODEN: MNRAA4; ISSN:0035-8711. (Oxford University Press)
We develop and apply a Hessian-based filament detection algorithm to submillimetre continuum observations of Orion A North. The resultant filament radial d. profiles are fitted with beam-convolved line-of-sight Plummer-profiles using Markov chain Monte Carlo techniques. The posterior distribution of the radial decay parameter demonstrates that the majority of filaments exhibit p = 1.5-3, with a mode at p = 2.2, suggesting deviation from the Ostriker p = 4 isothermal, equil., self-gravitating cylinder. The spatial distribution of young stellar objects relative to the high column d. filaments is investigated, yielding a lower limit on the star-forming age of the integral-shaped filament ∼1.4 Myr. Addnl., inferred lifetimes of filaments are examd. which suggest long-term filament accretion, varying rates of star formation, or both. Theor. filament stability measures are detd. with the aid of HARP C18O J=3-2 observations and indicate that the majority of filaments are gravitationally subcrit., despite the presence of young protostars. The results from this investigation are consistent with the one-dimensional accretion flow filament model recently obsd. in numerical simulations.
40
Hong, L., Qi, X., and Zhang, Y. (2012) Dissecting the kinetic process of amyloid fiber formation through asymptotic analysis. J. Phys. Chem. B 116 (23), 6611– 6617, DOI: 10.1021/jp205702u
Google Scholar
There is no corresponding record for this reference.
41
von Bergen, M. (2006) The Core of Tau - Paired Helical Filaments Studied by Scanning Transmission Electron Microscopy and Limited Proteolysis. Biochemistry 45 (20), 6446– 6457, DOI: 10.1021/bi052530j
Google Scholar
There is no corresponding record for this reference.
42
Goedert, M., Spillantini, M. G., Jakes, R., Rutherford, D., and Crowther, R. A. (1989) Multiple isoforms of human microtubule-associated protein tau: sequences and localization in neurofibrillary tangles of Alzheimer’s disease. Neuron 3 (4), 519– 526, DOI: 10.1016/0896-6273(89)90210-9
Google Scholar
42
Multiple isoforms of human microtubule-associated protein tau: sequences and localization in neurofibrillary tangles of Alzheimer's disease
Goedert, M.; Spillantini, M. G.; Jakes, R.; Rutherford, D.; Crowther, R. A.
Neuron (1989), 3 (4), 519-26CODEN: NERNET; ISSN:0896-6273.
This study detd. the sequences of isoforms of human tau protein, which differ from previously reported forms by insertions of 29 or 58 amino acids in the amino-terminal region. CDNA cloning shows that the insertions occur in combination with both three and 4 tandem repeats. RNAase protection assays indicate that transcripts encoding isoforms with the insertions are expressed in an adult-specific manner. Transcripts encoding 4 tandem repeats are also expressed in an adult-specific manner, whereas mRNAs encoding 3 tandem repeats are expressed throughout life, including in fetal brain. The levels of transcripts encoding the 29 or 58 amino acid inserts were ot significantly changed in cerebral cortex from patients with Alzheimer's disease. Antisera raised against synthetic peptides corresponding to these different human tau isoforms demonstrate that multiple tau protein isoforms are incorporated into the neurofibrillary tangles of Alzheimer's disease.

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Georg Meisl, Timothy Kurt, Itzel Condado-Morales, Cyrus Bett, Silvia Sorce, Mario Nuvolone, Thomas C. T. Michaels, Daniel Heinzer, Merve Avar, Samuel I. A. Cohen, Simone Hornemann, Adriano Aguzzi, Christopher M. Dobson, Christina J. Sigurdson, Tuomas P. J. Knowles. Scaling analysis reveals the mechanism and rates of prion replication in vivo. Nature Structural & Molecular Biology 2021, 28 (4) , 365-372. https://doi.org/10.1038/s41594-021-00565-x
T. B. Thompson, G. Meisl, T. P. J. Knowles, A. Goriely. The role of clearance mechanisms in the kinetics of pathological protein aggregation involved in neurodegenerative diseases. The Journal of Chemical Physics 2021, 154 (12) https://doi.org/10.1063/5.0031650
Farhang Aliakbari, Hossein Mohammad‐Beigi, Shahsanam Abbasi, Nasrollah Rezaei‐Ghaleh, Frederik Lermyte, Soha Parsafar, Stefan Becker, Azita Parvaneh Tafreshi, Peter B. O'Connor, Joanna F. Collingwood, Gunna Christiansen, Duncan S. Sutherland, Poul Henning Jensen, Dina Morshedi, Daniel E. Otzen. Multiple Protective Roles of Nanoliposome‐Incorporated Baicalein against Alpha‐Synuclein Aggregates. Advanced Functional Materials 2021, 31 (7) https://doi.org/10.1002/adfm.202007765
Jie Yang, Sarah Perrett, Si Wu. Single Molecule Characterization of Amyloid Oligomers. Molecules 2021, 26 (4) , 948. https://doi.org/10.3390/molecules26040948
Lien Veys, Jessie Van houcke, Jeroen Aerts, Sophie Van Pottelberge, Michel Mahieu, Audrey Coens, Ronald Melki, Dieder Moechars, Louis De Muynck, Lies De Groef. Absence of Uptake and Prion-Like Spreading of Alpha-Synuclein and Tau After Intravitreal Injection of Preformed Fibrils. Frontiers in Aging Neuroscience 2021, 12 https://doi.org/10.3389/fnagi.2020.614587
Emma E. Cawood, Theodoros K. Karamanos, Andrew J. Wilson, Sheena E. Radford. Visualizing and trapping transient oligomers in amyloid assembly pathways. Biophysical Chemistry 2021, 268 , 106505. https://doi.org/10.1016/j.bpc.2020.106505
Michiel Bertsch, Bruno Franchi, Ashish Raj, Maria Carla Tesi. Macroscopic modelling of Alzheimer’s disease: difficulties and challenges. Brain Multiphysics 2021, 2 , 100040. https://doi.org/10.1016/j.brain.2021.100040
Lauren J. Rice, Heath Ecroyd, Antoine M. van Oijen. Illuminating amyloid fibrils: Fluorescence-based single-molecule approaches. Computational and Structural Biotechnology Journal 2021, 19 , 4711-4724. https://doi.org/10.1016/j.csbj.2021.08.017
Noemi Esteras, Franziska Kundel, Giuseppe F. Amodeo, Evgeny V. Pavlov, David Klenerman, Andrey Y. Abramov. Insoluble tau aggregates induce neuronal death through modification of membrane ion conductance, activation of voltage‐gated calcium channels and NADPH oxidase. The FEBS Journal 2021, 288 (1) , 127-141. https://doi.org/10.1111/febs.15340
Liam D. Aubrey, Ben J. F. Blakeman, Liisa Lutter, Christopher J. Serpell, Mick F. Tuite, Louise C. Serpell, Wei-Feng Xue. Quantification of amyloid fibril polymorphism by nano-morphometry reveals the individuality of filament assembly. Communications Chemistry 2020, 3 (1) https://doi.org/10.1038/s42004-020-00372-3
André L. B. Bitencourt, Raquel M. Campos, Erika N. Cline, William L. Klein, Adriano Sebollela. Antibody Fragments as Tools for Elucidating Structure-Toxicity Relationships and for Diagnostic/Therapeutic Targeting of Neurotoxic Amyloid Oligomers. International Journal of Molecular Sciences 2020, 21 (23) , 8920. https://doi.org/10.3390/ijms21238920
Travis B. Thompson, Pavanjit Chaggar, Ellen Kuhl, Alain Goriely, , . Protein-protein interactions in neurodegenerative diseases: A conspiracy theory. PLOS Computational Biology 2020, 16 (10) , e1008267. https://doi.org/10.1371/journal.pcbi.1008267
Alain Goriely, Ellen Kuhl, Christian Bick. Neuronal Oscillations on Evolving Networks: Dynamics, Damage, Degradation, Decline, Dementia, and Death. Physical Review Letters 2020, 125 (12) https://doi.org/10.1103/PhysRevLett.125.128102
Andrey Y. Abramov, Elena V. Potapova, Viktor V. Dremin, Andrey V. Dunaev. Interaction of Oxidative Stress and Misfolded Proteins in the Mechanism of Neurodegeneration. Life 2020, 10 (7) , 101. https://doi.org/10.3390/life10070101
Alexander J. Dear, Thomas C. T. Michaels, Georg Meisl, David Klenerman, Si Wu, Sarah Perrett, Sara Linse, Christopher M. Dobson, Tuomas P. J. Knowles. Kinetic diversity of amyloid oligomers. Proceedings of the National Academy of Sciences 2020, 117 (22) , 12087-12094. https://doi.org/10.1073/pnas.1922267117
Donald E. Moss. Improving Anti-Neurodegenerative Benefits of Acetylcholinesterase Inhibitors in Alzheimer’s Disease: Are Irreversible Inhibitors the Future?. International Journal of Molecular Sciences 2020, 21 (10) , 3438. https://doi.org/10.3390/ijms21103438
Qiong-Qiong Yao, Liu Hong, Si Wu, Sarah Perrett. Distinct microscopic mechanisms for the accelerated aggregation of pathogenic Tau mutants revealed by kinetic analysis. Physical Chemistry Chemical Physics 2020, 22 (14) , 7241-7249. https://doi.org/10.1039/C9CP06083A
Georg Meisl, Tuomas PJ Knowles, David Klenerman. The molecular processes underpinning prion-like spreading and seed amplification in protein aggregation. Current Opinion in Neurobiology 2020, 61 , 58-64. https://doi.org/10.1016/j.conb.2020.01.010
Sveva Fornari, Amelie Schäfer, Ellen Kuhl, Alain Goriely. Spatially-extended nucleation-aggregation-fragmentation models for the dynamics of prion-like neurodegenerative protein-spreading in the brain and its connectome. Journal of Theoretical Biology 2020, 486 , 110102. https://doi.org/10.1016/j.jtbi.2019.110102
Christopher M. Dobson, Tuomas P.J. Knowles, Michele Vendruscolo. The Amyloid Phenomenon and Its Significance in Biology and Medicine. Cold Spring Harbor Perspectives in Biology 2020, 12 (2) , a033878. https://doi.org/10.1101/cshperspect.a033878
Marcella Catania, Giuseppe Di Fede. One or more β-amyloid(s)? New insights into the prion-like nature of Alzheimer's disease. 2020, 213-237. https://doi.org/10.1016/bs.pmbts.2020.07.003
Michel Goedert. Tau proteinopathies and the prion concept. 2020, 239-259. https://doi.org/10.1016/bs.pmbts.2020.08.003
Sveva Fornari, Amelie Schäfer, Mathias Jucker, Alain Goriely, Ellen Kuhl. Prion-like spreading of Alzheimer’s disease within the brain’s connectome. Journal of The Royal Society Interface 2019, 16 (159) , 20190356. https://doi.org/10.1098/rsif.2019.0356
Suman De, David Klenerman. Imaging individual protein aggregates to follow aggregation and determine the role of aggregates in neurodegenerative disease. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics 2019, 1867 (10) , 870-878. https://doi.org/10.1016/j.bbapap.2018.12.010
Thomas W. Rösler, Amir Tayaranian Marvian, Matthias Brendel, Niko-Petteri Nykänen, Matthias Höllerhage, Sigrid C. Schwarz, Franziska Hopfner, Thomas Koeglsperger, Gesine Respondek, Kerstin Schweyer, Johannes Levin, Victor L. Villemagne, Henryk Barthel, Osama Sabri, Ulrich Müller, Wassilios G. Meissner, Gabor G. Kovacs, Günter U. Höglinger. Four-repeat tauopathies. Progress in Neurobiology 2019, 180 , 101644. https://doi.org/10.1016/j.pneurobio.2019.101644
Nima N. Naseri, Hong Wang, Jennifer Guo, Manu Sharma, Wenjie Luo. The complexity of tau in Alzheimer’s disease. Neuroscience Letters 2019, 705 , 183-194. https://doi.org/10.1016/j.neulet.2019.04.022
Christoph Weber, Thomas Michaels, L Mahadevan. Spatial control of irreversible protein aggregation. eLife 2019, 8 https://doi.org/10.7554/eLife.42315
Eftychia Vasili, Antonio Dominguez-Meijide, Tiago Fleming Outeiro. Spreading of α-Synuclein and Tau: A Systematic Comparison of the Mechanisms Involved. Frontiers in Molecular Neuroscience 2019, 12 https://doi.org/10.3389/fnmol.2019.00107
Carol J. Huseby, Ralf Bundschuh, Jeff Kuret. The role of annealing and fragmentation in human tau aggregation dynamics. Journal of Biological Chemistry 2019, 294 (13) , 4728-4737. https://doi.org/10.1074/jbc.RA118.006943
Airi Tarutani, Masato Hasegawa. Prion-like propagation of α-synuclein in neurodegenerative diseases. 2019, 323-348. https://doi.org/10.1016/bs.pmbts.2019.07.005

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ACS Chemical Neuroscience

Cite this: ACS Chem. Neurosci. 2018, 9, 6, 1276–1282

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Abstract
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Figure 1
Figure 1. Tau aggregation is a two stage process of elongation and fragmentation. (a) Observing the aggregation of full length tau by SAVE imaging. Wild-type and P301S tau (0N4R) were incubated under aggregating conditions for up to 2 months. At regular time points, aliquots of the reaction mixtures were stained with pFTAA, adsorbed onto a glass cover slide and imaged on a TIRF microscope. (b) (i) Representative images of fibrils during midelongation phase, at maximum length and at the end point. Scale bar: 15 μm. Inset: 3-fold magnification of boxed areas. Scale bar: 2.5 μm. (ii) The apparent average length of fibrils was analyzed and plotted as a function of time. To account for the diffraction limited resolution, the “apparent” average length was plotted whereby diffraction limited spots are counted as fibrils with a length of 1 pixel (204 nm). N = 3 (3 different batches of protein, each in triplicates). Error bars: SEM. Solid lines: fit to fragmentation model. (c) Representative electron micrographs of the 2 μM P301S aggregation reaction after 24 h and after 672 h. Scale bar: 500 nm. Dotted areas were magnified and shown on the right. Scale bar: 100 nm.
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Figure 2
Figure 2. Kinetic model. (a) Schematic of microscopic steps during tau aggregation and corresponding rate constants. (b) Rate constants obtained from fits. K_m is the critical concentration for saturated elongation; n_c is the critical nucleus size. It is assumed that only the nucleation step is heparin dependent. Errors are allowed relative deviation.
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Figure 3
Figure 3. Simulations of tau fibril replication. (a) Hypothetical mechanism for tau fibril amplification and spreading in tissues. (b,c) Simulations of stochastic fibril amplification for wild-type (green) and P301S tau (blue) aggregates (b) in the htau P301S mouse model or (c) in humans. On the left, representative simulations are shown for each condition; average amplification times of tau aggregates are shown on the right. For each condition, 100 independent simulations were performed to obtain a reliable amplification time.
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Figure 4
Figure 4. Elongation and fragmentation rates for murine PrP, tau, and α-synuclein. Elongation and fragmentation rates of several disease associated proteins. The conditions used to determine these rate constants are shown in Supporting Table 2. The shaded area represents the minimum product of rate constants that would yield 90 billion aggregates in 100 years at a protein concentration of 100 nM.
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References
This article references 42 other publications.
1. 1
  Goedert, M. (2015) Alzheimer’s and Parkinson’s diseases: The prion concept in relation to assembled Aβ, tau, and α-synuclein. Science 349 (6248), 1255555, DOI: 10.1126/science.1255555
  1
  NEURODEGENERATION. Alzheimer's and Parkinson's diseases: The prion concept in relation to assembled Aβ, tau, and α-synuclein
  Goedert Michel
  Science (New York, N.Y.) (2015), 349 (6248), 1255555 ISSN:.
  The pathological assembly of Aβ, tau, and α-synuclein is at the heart of Alzheimer's and Parkinson's diseases. Extracellular deposits of Aβ and intraneuronal tau inclusions define Alzheimer's disease, whereas intracellular inclusions of α-synuclein make up the Lewy pathology of Parkinson's disease. Most cases of disease are sporadic, but some are inherited in a dominant manner. Mutations frequently occur in the genes encoding Aβ, tau, and α-synuclein. Overexpression of these mutant proteins can give rise to disease-associated phenotypes. Protein assembly begins in specific regions of the brain during the process of Alzheimer's and Parkinson's diseases, from where it spreads to other areas.
2. 2
  Clavaguera, F. (2009) Transmission and spreading of tauopathy in transgenic mouse brain. Nat. Cell Biol. 11 (7), 909– 913, DOI: 10.1038/ncb1901
  2
  Transmission and spreading of tauopathy in transgenic mouse brain
  Clavaguera, Florence; Bolmont, Tristan; Crowther, R. Anthony; Abramowski, Dorothee; Frank, Stephan; Probst, Alphonse; Fraser, Graham; Stalder, Anna K.; Beibel, Martin; Staufenbiel, Matthias; Jucker, Mathias; Goedert, Michel; Tolnay, Markus
  Nature Cell Biology (2009), 11 (7), 909-913, S909/1-S909/8CODEN: NCBIFN; ISSN:1465-7392. (Nature Publishing Group)
  Hyperphosphorylated tau makes up the filamentous intracellular inclusions of several neurodegenerative diseases, including Alzheimer's disease. In the disease process, neuronal tau inclusions first appear in the transentorhinal cortex from where they seem to spread to the hippocampal formation and neocortex. Cognitive impairment becomes manifest when inclusions reach the hippocampus, with abundant neocortical tau inclusions and extracellular β-amyloid deposits being the defining pathol. hallmarks of Alzheimer's disease. An abundance of tau inclusions, in the absence of β-amyloid deposits, defines Pick's disease, progressive supranuclear palsy, corticobasal degeneration and other diseases. Tau mutations cause familial forms of frontotemporal dementia, establishing that tau protein dysfunction is sufficient to cause neurodegeneration and dementia. Thus, transgenic mice expressing mutant (for example, P301S) human tau in nerve cells show the essential features of tauopathies, including neurodegeneration and abundant filaments made of hyperphosphorylated tau protein. By contrast, mouse lines expressing single isoforms of wild-type human tau do not produce tau filaments or show neurodegeneration. Here we have used tau-expressing lines to investigate whether exptl. tauopathy can be transmitted. We show that injection of brain ext. from mutant P301S tau-expressing mice into the brain of transgenic wild-type tau-expressing animals induces assembly of wild-type human tau into filaments and spreading of pathol. from the site of injection to neighboring brain regions.
3. 3
  Frost, B., Jacks, R. L., and Diamond, M. I. (2009) Propagation of tau misfolding from the outside to the inside of a cell. J. Biol. Chem. 284 (19), 12845– 12852, DOI: 10.1074/jbc.M808759200
  There is no corresponding record for this reference.
4. 4
  Goedert, M., Eisenberg, D. S., and Crowther, R. A. (2017) Propagation of tau aggregates and neurodegeneration. Annu. Rev. Neurosci. 40, 189– 210, DOI: 10.1146/annurev-neuro-072116-031153
  4
  Propagation of Tau Aggregates and Neurodegeneration
  Goedert, Michel; Eisenberg, David S.; Crowther, R. Anthony
  Annual Review of Neuroscience (2017), 40 (), 189-210CODEN: ARNSD5; ISSN:0147-006X. (Annual Reviews)
  A pathway from the natively unfolded microtubule-assocd. protein Tau to a highly structured amyloid fibril underlies human Tauopathies. This ordered assembly causes disease and represents the gain of toxic function. In recent years, evidence has accumulated to suggest that Tau inclusions form first in a small no. of brain cells, from where they propagate to other regions, resulting in neurodegeneration and disease. Propagation of pathol. is often called prion-like, which refers to the capacity of an assembled protein to induce the same abnormal conformation in a protein of the same kind, initiating a self-amplifying cascade. In addn., prion-like encompasses the release of protein aggregates from brain cells and their uptake by neighboring cells. In mice, the intracerebral injection of Tau inclusions induces the ordered assembly of monomeric Tau, followed by its spreading to distant brain regions. Conformational differences between Tau aggregates from transgenic mouse brain and in vitro assembled recombinant protein account for the greater seeding potency of brain aggregates. Short fibrils constitute the major species of seed-competent Tau in the brains of transgenic mice. The existence of multiple human Tauopathies with distinct fibril morphologies has led to the suggestion that different mol. conformers (or strains) of aggregated Tau exist.
5. 5
  Knowles, T. P. J., Vendruscolo, M., and Dobson, C. M. (2014) The amyloid state and its association with protein misfolding diseases. Nat. Rev. Mol. Cell Biol. 15 (6), 384– 396, DOI: 10.1038/nrm3810
  5
  The amyloid state and its association with protein misfolding diseases
  Knowles, Tuomas P. J.; Vendruscolo, Michele; Dobson, Christopher M.
  Nature Reviews Molecular Cell Biology (2014), 15 (6), 384-396CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)
  A review. The phenomenon of protein aggregation and amyloid formation has become the subject of rapidly increasing research activities across a wide range of scientific disciplines. Such activities have been stimulated by the assocn. of amyloid deposition with a range of debilitating medical disorders, from Alzheimer's disease to type II diabetes, many of which are major threats to human health and welfare in the modern world. It has become clear, however, that the ability to form the amyloid state is more general than previously imagined, and that its study can provide unique insights into the nature of the functional forms of peptides and proteins, as well as understanding the means by which protein homeostasis can be maintained and protein metastasis avoided.
6. 6
  Bett, C. (2012) Biochemical properties of highly neuroinvasive prion strains. PLoS Pathog. 8 (2), e1002522, DOI: 10.1371/journal.ppat.1002522
  There is no corresponding record for this reference.
7. 7
  Prusiner, S. B. (2013) Biology and genetics of prions causing neurodegeneration. Annu. Rev. Genet. 47, 601– 623, DOI: 10.1146/annurev-genet-110711-155524
  7
  Biology and genetics of prions causing neurodegeneration
  Prusiner, Stanley B.
  Annual Review of Genetics (2013), 47 (), 601-623CODEN: ARVGB7; ISSN:0066-4197. (Annual Reviews)
  A review. Prions are proteins that acquire alternative conformations that become self-propagating. Transformation of proteins into prions is generally accompanied by an increase in β-sheet structure and a propensity to aggregate into oligomers. Some prions are beneficial and perform cellular functions, whereas others cause neurodegeneration. In mammals, more than a dozen proteins that become prions have been identified, and a similar no. has been found in fungi. In both mammals and fungi, variations in the prion conformation encipher the biol. properties of distinct prion strains. Increasing evidence argues that prions cause many neurodegenerative diseases (NDs), including Alzheimer's, Parkinson's, Creutzfeldt-Jakob, and Lou Gehrig's diseases, as well as the tauopathies. The majority of NDs are sporadic, and 10% to 20% are inherited. The late onset of heritable NDs, like their sporadic counterparts, may reflect the stochastic nature of prion formation; the pathogenesis of such illnesses seems to require prion accumulation to exceed some crit. threshold before neurol. dysfunction manifests.
8. 8
  Tanaka, M., Collins, S. R., Toyama, B. H., and Weissman, J. S. (2006) The physical basis of how prion conformations determine strain phenotypes. Nature 442 (7102), 585– 589, DOI: 10.1038/nature04922
  8
  The physical basis of how prion conformations determine strain phenotypes
  Tanaka, Motomasa; Collins, Sean R.; Toyama, Brandon H.; Weissman, Jonathan S.
  Nature (London, United Kingdom) (2006), 442 (7102), 585-589CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
  A principle that has emerged from studies of protein aggregation is that proteins typically can misfold into a range of different aggregated forms. Moreover, the phenotypic and pathol. consequences of protein aggregation depend critically on the specific misfolded form. A striking example of this is the prion strain phenomenon, in which prion particles composed of the same protein cause distinct heritable states. Accumulating evidence from yeast prions such as [PSI+] and mammalian prions argues that differences in the prion conformation underlie prion strain variants. Nonetheless, it remains poorly understood why changes in the conformation of misfolded proteins alter their physiol. effects. Here, we present and exptl. validate an anal. model describing how [PSI+] strain phenotypes arise from the dynamic interaction among the effects of prion diln., competition for a limited pool of sol. protein, and conformation-dependent differences in prion growth and division rates. Anal. of three distinct prion conformations of yeast Sup35 (the [PSI+] protein determinant) and their in vivo phenotypes reveals that the Sup35 amyloid causing the strongest phenotype surprisingly shows the slowest growth. This slow growth, however, is more than compensated for by an increased brittleness that promotes prion division. The propensity of aggregates to undergo breakage, thereby generating new seeds, probably represents a key determinant of their physiol. impact for both infectious (prion) and non-infectious amyloids.
9. 9
  Derdowski, A., Sindi, S. S., Klaips, C. L., DiSalvo, S., and Serio, T. R. (2010) A size threshold limits prion transmission and establishes phenotypic diversity. Science 330 (6004), 680– 683, DOI: 10.1126/science.1197785
  There is no corresponding record for this reference.
10. 10
  Horrocks, M. H. (2016) Single-molecule imaging of individual amyloid protein aggregates in human biofluids. ACS Chem. Neurosci. 7 (3), 399– 406, DOI: 10.1021/acschemneuro.5b00324
  10
  Single-Molecule Imaging of Individual Amyloid Protein Aggregates in Human Biofluids
  Horrocks, Mathew H.; Lee, Steven F.; Gandhi, Sonia; Magdalinou, Nadia K.; Chen, Serene W.; Devine, Michael J.; Tosatto, Laura; Kjaergaard, Magnus; Beckwith, Joseph S.; Zetterberg, Henrik; Iljina, Marija; Cremades, Nunilo; Dobson, Christopher M.; Wood, Nicholas W.; Klenerman, David
  ACS Chemical Neuroscience (2016), 7 (3), 399-406CODEN: ACNCDM; ISSN:1948-7193. (American Chemical Society)
  The misfolding and aggregation of proteins into amyloid fibrils characterizes many neurodegenerative disorders such as Parkinson's and Alzheimer's diseases. We report here a method, termed SAVE (single aggregate visualization by enhancement) imaging, for the ultrasensitive detection of individual amyloid fibrils and oligomers using single-mol. fluorescence microscopy. We demonstrate that this method is able to detect the presence of amyloid aggregates of α-synuclein, tau, and amyloid-β. In addn., we show that aggregates can also be identified in human cerebrospinal fluid (CSF). Significantly, we see a twofold increase in the av. aggregate concn. in CSF from Parkinson's disease patients compared to age-matched controls. Taken together, we conclude that this method provides an opportunity to characterize the structural nature of amyloid aggregates in a key biofluid, and therefore has the potential to study disease progression in both animal models and humans to enhance our understanding of neurodegenerative disorders.
11. 11
  Michaels, T. C. T. (2015) The length distribution of frangible biofilaments. J. Chem. Phys. 143 (16), 164901, DOI: 10.1063/1.4933230
  11
  The length distribution of frangible biofilaments
  Michaels, Thomas C. T.; Yde, Pernille; Willis, Julian C. W.; Jensen, Mogens H.; Otzen, Daniel; Dobson, Christopher M.; Buell, Alexander K.; Knowles, Tuomas P. J.
  Journal of Chemical Physics (2015), 143 (16), 164901/1-164901/14CODEN: JCPSA6; ISSN:0021-9606. (American Institute of Physics)
  A no. of different proteins possess the ability to polymerize into filamentous structures. Certain classes of such assemblies can have key functional roles in the cell, such as providing the structural basis for the cytoskeleton in the case of actin and tubulin, while others are implicated in the development of many pathol. conditions, including Alzheimer's and Parkinson's diseases. In general, the fragmentation of such structures changes the total no. of filament ends, which act as growth sites, and hence is a key feature of the dynamics of filamentous growth phenomena. Here, the authors present an anal. study of the master equation of breakable filament assembly and derive closed-form expressions for the time evolution of the filament length distribution for both open and closed systems with infinite and finite monomer supply, resp. The authors used this theor. framework to analyze exptl. data for length distributions of insulin amyloid fibrils and showed that their theory allows insights into the microscopic mechanisms of biofilament assembly to be obtained beyond those available from the conventional anal. of filament mass only. (c) 2015 American Institute of Physics.
12. 12
  Xue, W.-F. and Radford, S. E. (2013) An imaging and systems modeling approach to fibril breakage enables prediction of amyloid behavior. Biophys. J. 105 (12), 2811– 2819, DOI: 10.1016/j.bpj.2013.10.034
  12
  An Imaging and Systems Modeling Approach to Fibril Breakage Enables Prediction of Amyloid Behavior
  Xue, Wei-Feng; Radford, Sheena E.
  Biophysical Journal (2013), 105 (12), 2811-2819CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
  Delineating the nanoscale properties and the dynamic assembly and disassembly behaviors of amyloid fibrils is key for technol. applications that use the material properties of amyloid fibrils, as well as for developing treatments of amyloid-assocd. disease. However, quant. mechanistic understanding of the complex processes involving these heterogeneous supramol. systems presents challenges that have yet to be resolved. Here, we develop an approach that is capable of resolving the time dependence of fibril particle concn., length distribution, and length and position dependence of fibril fragmentation rates using a generic math. framework combined with exptl. data derived from at. force microscopy anal. of fibril length distributions. By application to amyloid assembly of β2-microglobulin in vitro under const. mech. stirring, we present a full description of the fibril fragmentation and growth behavior, and demonstrate the predictive power of the approach in terms of the samples' fibril dimensions, fibril load, and their efficiency to seed the growth of new amyloid fibrils. The approach developed offers opportunities to det., quantify, and predict the course and the consequences of amyloid assembly.
13. 13
  Klingstedt, T. (2011) Synthesis of a library of oligothiophenes and their utilization as fluorescent ligands for spectral assignment of protein aggregates. Org. Biomol. Chem. 9 (24), 8356– 8370, DOI: 10.1039/c1ob05637a
  13
  Synthesis of a library of oligothiophenes and their utilization as fluorescent ligands for spectral assignment of protein aggregates
  Klingstedt, Therese; Aaslund, Andreas; Simon, Rozalyn A.; Johansson, Leif B. G.; Mason, Jeffrey J.; Nystroem, Sofie; Hammarstroem, Per; Nilsson, K. Peter R.
  Organic & Biomolecular Chemistry (2011), 9 (24), 8356-8370CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
  Mol. probes for selective identification of protein aggregates are important to advance the understanding of the mol. pathogenesis underlying protein aggregation diseases. Here the authors report the chem. design of a library of anionic luminescent conjugated oligothiophenes (LCOs), which can be utilized as ligands for detection of protein aggregates. Certain mol. requirements were shown to be necessary for detecting (i) early nonthioflavinophilic protein assemblies of Aβ1-42 and insulin preceding the formation of amyloid fibrils and (ii) for obtaining distinct spectral signatures of the two main pathol. hallmarks obsd. in human Alzheimer's disease brain tissue (Aβ plaques and neurofibrillary tangles). The authors' findings suggest that a superior anionic LCO-based ligand should have a backbone consisting of five to seven thiophene units and carboxyl groups extending the conjugated thiophene backbone. Such LCOs will be highly useful for studying the underlying mol. events of protein aggregation diseases and could also be utilized for the development of novel diagnostic tools for these diseases.
14. 14
  Brelstaff, J. (2015) The fluorescent pentameric oligothiophene pFTAA identifies filamentous tau in live neurons cultured from adult P301S tau mice. Front. Neurosci. 9, 184, DOI: 10.3389/fnins.2015.00184
  14
  The fluorescent pentameric oligothiophene pFTAA identifies filamentous tau in live neurons cultured from adult P301S tau mice
  Brelstaff Jack; Ossola Bernardino; Spillantini Maria Grazia; Tolkovsky Aviva M; Neher Jonas J; Klingstedt Therese; Goedert Michel; Nilsson K Peter R
  Frontiers in neuroscience (2015), 9 (), 184 ISSN:1662-4548.
  Identification of fluorescent dyes that label the filamentous protein aggregates characteristic of neurodegenerative disease, such as β-amyloid and tau in Alzheimer's disease, in a live cell culture system has previously been a major hurdle. Here we show that pentameric formyl thiophene acetic acid (pFTAA) fulfills this function in living neurons cultured from adult P301S tau transgenic mice. Injection of pFTAA into 5-month-old P301S tau mice detected cortical and DRG neurons immunoreactive for AT100, an antibody that identifies solely filamentous tau, or MC1, an antibody that identifies a conformational change in tau that is commensurate with neurofibrillary tangle formation in Alzheimer's disease brains. In fixed cultures of dorsal root ganglion (DRG) neurons, pFTAA binding, which also identified AT100 or MC1+ve neurons, followed a single, saturable binding curve with a half saturation constant of 0.14 μM, the first reported measurement of a binding affinity of a beta-sheet reactive dye to primary neurons harboring filamentous tau. Treatment with formic acid, which solubilizes filamentous tau, extracted pFTAA, and prevented the re-binding of pFTAA and MC1 without perturbing expression of soluble tau, detected using an anti-human tau (HT7) antibody. In live cultures, pFTAA only identified DRG neurons that, after fixation, were AT100/MC1+ve, confirming that these forms of tau pre-exist in live neurons. The utility of pFTAA to discriminate between living neurons containing filamentous tau from other neurons is demonstrated by showing that more pFTAA+ve neurons die than pFTAA-ve neurons over 25 days. Since pFTAA identifies fibrillar tau and other misfolded proteins in living neurons in culture and in animal models of several neurodegenerative diseases, as well as in human brains, it will have considerable application in sorting out disease mechanisms and in identifying disease-modifying drugs that will ultimately help establish the mechanisms of neurodegeneration in human neurodegenerative diseases.
15. 15
  Knowles, T. P. J. (2009) An analytical solution to the kinetics of breakable filament assembly. Science 326 (5959), 1533– 1537, DOI: 10.1126/science.1178250
  15
  An Analytical Solution to the Kinetics of Breakable Filament Assembly
  Knowles, Tuomas P. J.; Waudby, Christopher A.; Devlin, Glyn L.; Cohen, Samuel I. A.; Aguzzi, Adriano; Vendruscolo, Michele; Terentjev, Eugene M.; Welland, Mark E.; Dobson, Christopher M.
  Science (Washington, DC, United States) (2009), 326 (5959), 1533-1537CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
  We present an anal. treatment of a set of coupled kinetic equations that governs the self-assembly of filamentous mol. structures. Application to the case of protein aggregation demonstrates that the kinetics of amyloid growth can often be dominated by secondary rather than by primary nucleation events. Our results further reveal a range of general features of the growth kinetics of fragmenting filamentous structures, including the existence of generic scaling laws that provide mechanistic information in contexts ranging from in vitro amyloid growth to the in vivo development of mammalian prion diseases.
16. 16
  Hong, L. and Yong, W.-A. (2013) Simple moment-closure model for the self-assembly of breakable amyloid filaments. Biophys. J. 104 (3), 533– 540, DOI: 10.1016/j.bpj.2012.12.039
  There is no corresponding record for this reference.
17. 17
  Guo, J. L. and Lee, VM-Y (2011) Seeding of normal tau by pathological tau conformers drives pathogenesis of Alzheimer-like tangles. J. Biol. Chem. 286 (17), 15317– 15331, DOI: 10.1074/jbc.M110.209296
  17
  Seeding of Normal Tau by Pathological Tau Conformers Drives Pathogenesis of Alzheimer-like Tangles
  Guo, Jing L.; Lee, Virginia M.-Y.
  Journal of Biological Chemistry (2011), 286 (17), 15317-15331CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
  Neurofibrillary tangles (NFTs) in Alzheimer disease and related tauopathies are composed of insol. hyperphosphorylated Tau protein, but the mechanisms underlying the conversion of highly sol. Tau into insol. NFTs remain elusive. Here, we demonstrate that introduction of minute quantities of misfolded preformed Tau fibrils (Tau pffs) into Tau-expressing cells rapidly recruit large amts. of sol. Tau into filamentous inclusions resembling NFTs with unprecedented efficiency, suggesting a "seeding"-recruitment process as a highly plausible mechanism underlying NFT formation in vivo. Consistent with the emerging concept of prion-like transmissibility of disease-causing amyloidogenic proteins, we found that spontaneous uptake of Tau pffs into cells is likely mediated by endocytosis, suggesting a potential mechanism for the propagation of Tau lesions in tauopathy brains. Furthermore, sequestration of sol. Tau by pff-induced Tau aggregates attenuates microtubule overstabilization in Tau-expressing cells, supporting the hypothesis of a Tau loss-of-function toxicity in cells harboring NFTs. In summary, our study establishes a cellular system that robustly develops authentic NFT-like Tau aggregates, which provides mechanistic insights into NFT pathogenesis and a potential tool for identifying Tau-based therapeutics.
18. 18
  Nonaka, T., Watanabe, S. T., Iwatsubo, T., and Hasegawa, M. (2010) Seeded aggregation and toxicity of α-synuclein and tau - Cellular models of neurodegenerative diseases. J. Biol. Chem. 285 (45), 34885– 34898, DOI: 10.1074/jbc.M110.148460
  There is no corresponding record for this reference.
19. 19
  Falcon, B. (2015) Conformation determines the seeding potencies of native and recombinant tau aggregates. J. Biol. Chem. 290 (2), 1049– 1065, DOI: 10.1074/jbc.M114.589309
  19
  Conformation Determines the Seeding Potencies of Native and Recombinant Tau Aggregates
  Falcon, Benjamin; Cavallini, Annalisa; Angers, Rachel; Glover, Sarah; Murray, Tracey K.; Barnham, Luanda; Jackson, Samuel; O'Neill, Michael J.; Isaacs, Adrian M.; Hutton, Michael L.; Szekeres, Philip G.; Goedert, Michel; Bose, Suchira
  Journal of Biological Chemistry (2015), 290 (2), 1049-1065CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
  Intracellular Tau inclusions are a pathol. hallmark of several neurodegenerative diseases, collectively known as the tauopathies. They include Alzheimer disease, tangle-only dementia, Pick disease, argyrophilic grain disease, chronic traumatic encephalopathy, progressive supranuclear palsy, and corticobasal degeneration. Tau pathol. appears to spread through intercellular propagation, requiring the formation of assembled "prion-like" species. Several cell and animal models have been described that recapitulate aspects of this phenomenon. However, the mol. characteristics of seed-competent Tau remain unclear. Here, we have used a cell model to understand the relationships between Tau structure/phosphorylation and seeding by aggregated Tau species from the brains of mice transgenic for human mutant P301S Tau and full-length aggregated recombinant P301S Tau. Deletion of motifs 275VQIINK280 and 306VQIVYK311 abolished the seeding activity of recombinant full-length Tau, suggesting that its aggregation was necessary for seeding. We describe conformational differences between native and synthetic Tau aggregates that may account for the higher seeding activity of native assembled Tau. When added to aggregated Tau seeds from the brains of mice transgenic for P301S Tau, sol. recombinant Tau aggregated and acquired the mol. properties of aggregated Tau from transgenic mouse brain. We show that seeding is conferred by aggregated Tau that enters cells through macropinocytosis and seeds the assembly of endogenous Tau into filaments.
20. 20
  Clavaguera, F. (2013) “Prion-like” templated misfolding in tauopathies. Brain Pathol. 23 (3), 342– 349, DOI: 10.1111/bpa.12044
  20
  Prion-like templated misfolding in tauopathies
  Clavaguera, Florence; Lavenir, Isabelle; Falcon, Ben; Frank, Stephan; Goedert, Michel; Tolnay, Markus
  Brain Pathology (2013), 23 (3), 342-349CODEN: BRPAE7; ISSN:1015-6305. (Wiley-Blackwell)
  A review. The sol. microtubule-assocd. protein tau forms hyperphosphorylated, insol. and filamentous inclusions in a no. of neurodegenerative diseases referred to as "tauopathies.". In Alzheimer's disease, tau pathol. develops in a stereotypical manner, with the first lesions appearing in the locus coeruleus and entorhinal cortex, from where they appear to spread to the hippocampus and neocortex. Propagation of tau pathol. is also a characteristic of argyrophilic grain disease, where the tau lesions spread throughout the limbic system. Significantly, isoform compn. and morphol. of tau filaments can differ between tauopathies, suggesting the existence of distinct tau strains. Extensive exptl. findings indicate that prion-like mechanisms underly the pathogenesis of tauopathies.
21. 21
  Iba, M. (2013) Synthetic tau fibrils mediate transmission of neurofibrillary tangles in a transgenic mouse model of Alzheimer’s-like tauopathy. J. Neurosci. 33 (3), 1024– 1037, DOI: 10.1523/JNEUROSCI.2642-12.2013
  21
  Synthetic tau fibrils mediate transmission of neurofibrillary tangles in a transgenic mouse model of Alzheimer's-like tauopathy
  Iba, Michiyo; Guo, Jing L.; McBride, Jennifer D.; Zhang, Bin; Trojanowski, John Q.; Lee, Virginia M.-Y.
  Journal of Neuroscience (2013), 33 (3), 1024-1037CODEN: JNRSDS; ISSN:0270-6474. (Society for Neuroscience)
  Tauopathies, including Alzheimer's disease (AD) and frontotemporal lobar degeneration with tau pathologies, are neurodegenerative diseases characterized by neurofibrillary tangles (NFTs) comprising filamentous tau protein. Although emerging evidence suggests that tau pathol. may be transmitted, we demonstrate here that synthetic tau fibrils are sufficient to transmit tau inclusions in a mouse model. Specifically, intracerebral inoculation of young PS19 mice overexpressing mutant human tau (P301S) with synthetic preformed fibrils (pffs) assembled from recombinant full-length tau or truncated tau contg. four microtubule binding repeats resulted in rapid induction of NFT-like inclusions that propagated from injected sites to connected brain regions in a time-dependent manner. Interestingly, injection of tau pffs into either hippocampus or striatum together with overlaying cortex gave rise to distinct pattern of spreading. Moreover, unlike tau pathol. that spontaneously develops in old PS19 mice, the pff-induced tau inclusions more closely resembled AD NFTs because they were Thioflavin S pos., acetylated, and more resistant to proteinase K digestion. Together, our study demonstrates that synthetic tau pffs alone are capable of inducing authentic NFT-like tau aggregates and initiating spreading of tau pathol. in a tauopathy mouse model.
22. 22
  Herculano-Houzel, S., Mota, B., and Lent, R. (2006) Cellular scaling rules for rodent brains. Proc. Natl. Acad. Sci. U. S. A. 103 (32), 12138– 12143, DOI: 10.1073/pnas.0604911103
  22
  Cellular scaling rules for rodent brains
  Herculano-Houzel, Suzana; Mota, Bruno; Lent, Roberto
  Proceedings of the National Academy of Sciences of the United States of America (2006), 103 (32), 12138-12143CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
  How do cell no. and size det. brain size Here, we show that, in the order Rodentia, increased size of the cerebral cortex, cerebellum, and remaining areas across six species is achieved through greater nos. of neurons of larger size, and much greater nos. of nonneuronal cells of roughly invariant size, such that the ratio between total neuronal and nonneuronal mass remains const. across species. Although relative cerebellar size remains stable among rodents, the no. of cerebellar neurons increases with brain size more rapidly than in the cortex, such that the cerebellar fraction of total brain neurons increases with brain size. In contrast, although the relative cortical size increases with total brain size, the cortical fraction of total brain neurons remains const. We propose that the faster increase in av. neuronal size in the cerebral cortex than in the cerebellum as these structures gain neurons and the rapidly increasing glial nos. that generate glial mass to match total neuronal mass at a fixed glia/neuron total mass ratio are fundamental cellular constraints that lead to the relative expansion of cerebral cortical vol. across species.
23. 23
  Allen, B. (2002) Abundant tau filaments and nonapoptotic neurodegeneration in transgenic mice expressing human P301S tau protein. J. Neurosci. 22 (21), 9340– 9351
  23
  Abundant tau filaments and nonapoptotic neurodegeneration in transgenic mice expressing human P301S Tau protein
  Allen, Bridget; Ingram, Esther; Takao, Masaki; Smith, Michael J.; Jakes, Ross; Virdee, Kanwar; Yoshida, Hirotaka; Holzer, Max; Craxton, Molly; Emson, Piers C.; Atzori, Cristiana; Migheli, Antonio; Crowther, R. Anthony; Ghetti, Bernardino; Spillantini, Maria Grazia; Goedert, Michel
  Journal of Neuroscience (2002), 22 (21), 9340-9351CODEN: JNRSDS; ISSN:0270-6474. (Society for Neuroscience)
  The identification of mutations in the Tau gene in frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) has made it possible to express human tau protein with pathogenic mutations in transgenic animals. Here the authors report on the prodn. and characterization of a line of mice transgenic for the 383 aa isoform of human tau with the P301S mutation. At 5-6 mo of age, homozygous animals from this line developed a neurol. phenotype dominated by a severe paraparesis. According to light microscopy, many nerve cells in brain and spinal cord were strongly immunoreactive for hyperphosphorylated tau. According to electron microscopy, abundant filaments made of hyperphosphorylated tau protein were present. The majority of filaments resembled the half-twisted ribbons described previously in cases of FTDP-17, with a minority of filaments resembling the paired helical filaments of Alzheimer's disease. Sarkosyl-insol. tau from brains and spinal cords of transgenic mice ran as a hyperphosphorylated 64 kDa band, the same apparent mol. mass as that of the 383 aa tau isoform in the human tauopathies. Perchloric acid-sol. tau was also phosphorylated at many sites, with the notable exception of serine 214. In the spinal cord, neurodegeneration was present, as indicated by a 49% redn. in the no. of motor neurons. No evidence for apoptosis was obtained, despite the extensive colocalization of hyperphosphorylated tau protein with activated MAP kinase family members. The latter may be involved in the hyperphosphorylation of tau.
24. 24
  Ackmann, M., Wiech, H., and Mandelkow, E. (2000) Nonsaturable binding indicates clustering of tau on the microtubule surface in a paired helical filament-like conformation. J. Biol. Chem. 275 (39), 30335– 30343, DOI: 10.1074/jbc.M002590200
  There is no corresponding record for this reference.
25. 25
  Makrides, V., Massie, M. R., Feinstein, S. C., and Lew, J. (2004) Evidence for two distinct binding sites for tau on microtubules. Proc. Natl. Acad. Sci. U. S. A. 101 (17), 6746– 6751, DOI: 10.1073/pnas.0400992101
  There is no corresponding record for this reference.
26. 26
  Azevedo, F. A. C. (2009) Equal numbers of neuronal and nonneuronal cells make the human brain an isometrically scaled-up primate brain. J. Comp. Neurol. 513 (5), 532– 541, DOI: 10.1002/cne.21974
  26
  Equal numbers of neuronal and nonneuronal cells make the human brain an isometrically scaled-up primate brain
  Azevedo Frederico A C; Carvalho Ludmila R B; Grinberg Lea T; Farfel Jose Marcelo; Ferretti Renata E L; Leite Renata E P; Jacob Filho Wilson; Lent Roberto; Herculano-Houzel Suzana
  The Journal of comparative neurology (2009), 513 (5), 532-41 ISSN:.
  The human brain is often considered to be the most cognitively capable among mammalian brains and to be much larger than expected for a mammal of our body size. Although the number of neurons is generally assumed to be a determinant of computational power, and despite the widespread quotes that the human brain contains 100 billion neurons and ten times more glial cells, the absolute number of neurons and glial cells in the human brain remains unknown. Here we determine these numbers by using the isotropic fractionator and compare them with the expected values for a human-sized primate. We find that the adult male human brain contains on average 86.1 +/- 8.1 billion NeuN-positive cells ("neurons") and 84.6 +/- 9.8 billion NeuN-negative ("nonneuronal") cells. With only 19% of all neurons located in the cerebral cortex, greater cortical size (representing 82% of total brain mass) in humans compared with other primates does not reflect an increased relative number of cortical neurons. The ratios between glial cells and neurons in the human brain structures are similar to those found in other primates, and their numbers of cells match those expected for a primate of human proportions. These findings challenge the common view that humans stand out from other primates in their brain composition and indicate that, with regard to numbers of neuronal and nonneuronal cells, the human brain is an isometrically scaled-up primate brain.
27. 27
  Bird, T. D. (1993) Alzheimer Disease Overview. In GeneReviews (Adam, M. P., Ardinger, H. H., and Pagon, R. A., et al., Eds.), University of Washington, Seattle, WA, available at: http://www.ncbi.nlm.nih.gov/books/NBK1161/ [accessed November 17, 2017].
  There is no corresponding record for this reference.
28. 28
  Sperfeld, A. D. (1999) FTDP-17: an early-onset phenotype with parkinsonism and epileptic seizures caused by a novel mutation. Ann. Neurol. 46 (5), 708– 715, DOI: 10.1002/1531-8249(199911)46:5<708::AID-ANA5>3.0.CO;2-K
  28
  FTDP-17: an early-onset phenotype with parkinsonism and epileptic seizures caused by a novel mutation
  Sperfeld A D; Collatz M B; Baier H; Palmbach M; Storch A; Schwarz J; Tatsch K; Reske S; Joosse M; Heutink P; Ludolph A C
  Annals of neurology (1999), 46 (5), 708-15 ISSN:0364-5134.
  Recently, mutations in the tau gene on chromosome 17 were found causative for autosomal dominantly inherited frontotemporal dementia and parkinsonism (FTDP-17). We describe a family carrying a missense mutation at nucleotide 1137 C --> T, resulting in the amino acid substitution P301S. Methods of investigations include clinical, electrophysiological, and imaging techniques. This kindred presents with a novel phenotype characterized by an early onset of rapidly progressive frontotemporal dementia and parkinsonism in combination with epileptic seizures. We define the dopaminergic deficits as being predominantly presynaptic by the use of single-photon emission computed tomography with a dopamine transporter ligand. The association of this early-onset phenotype with P301S mutation is not entirely consistent with current criteria for the diagnosis of frontotemporal dementias and may encourage the search for tau mutations in diseases similar but not identical to FTDP-17. Also, the change from proline to serine suggests that this mutation might contribute to tau hyperphosphorylation.
29. 29
  Bugiani, O. (1999) Frontotemporal dementia and corticobasal degeneration in a family with a P301S mutation in tau. J. Neuropathol. Exp. Neurol. 58 (6), 667– 677, DOI: 10.1097/00005072-199906000-00011
  29
  Frontotemporal dementia and corticobasal degeneration in a family with a P301S mutation in tau
  Bugiani O; Murrell J R; Giaccone G; Hasegawa M; Ghigo G; Tabaton M; Morbin M; Primavera A; Carella F; Solaro C; Grisoli M; Savoiardo M; Spillantini M G; Tagliavini F; Goedert M; Ghetti B
  Journal of neuropathology and experimental neurology (1999), 58 (6), 667-77 ISSN:0022-3069.
  The tau gene has been found to be the locus of dementia with rigidity linked to chromosome 17. Exonic and intronic mutations have been described in a number of families. Here we describe a P301S mutation in exon 10 of the tau gene in a new family. Two members of this family were affected. One individual presented with frontotemporal dementia, whereas his son has corticobasal degeneration, demonstrating that the same primary gene defect in tau can lead to 2 distinct clinical phenotypes. Both individuals developed rapidly progressive disease in the third decade. Neuropathologically, the father presented with an extensive filamentous pathology made of hyperphosphorylated tau protein. Biochemically, recombinant tau protein with the P301S mutation showed a greatly reduced ability to promote microtubule assembly.
30. 30
  McEwan, W. A. (2017) Cytosolic Fc receptor TRIM21 inhibits seeded tau aggregation. Proc. Natl. Acad. Sci. U. S. A. 114 (3), 574– 579, DOI: 10.1073/pnas.1607215114
  There is no corresponding record for this reference.
31. 31
  Sang, J. C., Direct observation of protein aggregate replication in vitro to model prion and prion-like diseases; submitted.
  There is no corresponding record for this reference.
32. 32
  Pocchiari, M. (2004) Predictors of survival in sporadic Creutzfeldt–Jakob disease and other human transmissible spongiform encephalopathies. Brain 127 (10), 2348– 2359, DOI: 10.1093/brain/awh249
  There is no corresponding record for this reference.
33. 33
  Kalia, L. V. and Lang, A. E. (2015) Parkinson’s disease. Lancet 386 (9996), 896– 912, DOI: 10.1016/S0140-6736(14)61393-3
  33
  Parkinson's disease
  Kalia, Lorraine V.; Lang, Anthony E.
  Lancet (2015), 386 (9996), 896-912CODEN: LANCAO; ISSN:0140-6736. (Elsevier Ltd.)
  Parkinson's disease is a neurol. disorder with evolving layers of complexity. It has long been characterised by the classical motor features of parkinsonism assocd. with Lewy bodies and loss of dopaminergic neurons in the substantia nigra. However, the symptomatol. of Parkinson's disease is now recognized as heterogeneous, with clin. significant non-motor features. Similarly, its pathol. involves extensive regions of the nervous system, various neurotransmitters, and protein aggregates other than just Lewy bodies. The cause of Parkinson's disease remains unknown, but risk of developing Parkinson's disease is no longer viewed as primarily due to environmental factors. Instead, Parkinson's disease seems to result from a complicated interplay of genetic and environmental factors affecting numerous fundamental cellular processes. The complexity of Parkinson's disease is accompanied by clin. challenges, including an inability to make a definitive diagnosis at the earliest stages of the disease and difficulties in the management of symptoms at later stages. Furthermore, there are no treatments that slow the neurodegenerative process. In this Seminar, we review these complexities and challenges of Parkinson's disease.
34. 34
  Braak, H. and Del Tredici, K. (2011) The pathological process underlying Alzheimer’s disease in individuals under thirty. Acta Neuropathol. 121 (2), 171– 181, DOI: 10.1007/s00401-010-0789-4
  34
  The pathological process underlying Alzheimer's disease in individuals under thirty
  Braak Heiko; Del Tredici Kelly
  Acta neuropathologica (2011), 121 (2), 171-81 ISSN:.
  Brains of 42 individuals between the ages of 4 and 29 were examined with antibodies (AT8, 4G8) and silver stains for the presence of intraneuronal and extracellular protein aggregates associated with Alzheimer's disease. Thirty-eight of 42 (38/42) cases displayed abnormally phosphorylated tau protein (pretangle material) in nerve cells or in portions of their cellular processes, and 41/42 individuals showed no extracellular amyloid-β protein deposition or neuritic plaques-an individual with Down syndrome was the only exception. In 16/42 cases abnormal tau was found in the transentorhinal region, and in 3/42 cases this site was Gallyas-positive for isolated NFTs (NFT stage I). Of 26 cases that lacked abnormal tau in the transentorhinal region, 4 did not show pretangle material at subcortical sites. The remaining 22 of these same 26 cases, however, had subcortical lesions confined to non-thalamic nuclei with diffuse projections to the cerebral cortex, and, remarkably, in 19/22 individuals the pretangle material was confined to the noradrenergic coeruleus/subcoeruleus complex. Assuming the pretangle alterations are not transient and do not regress, these findings may indicate that the Alzheimer's disease-related pathological process leading to neurofibrillary tangle formation does not begin in the cerebral cortex but, rather, in select subcortical nuclei, and it may start quite early, i.e., before puberty or in early young adulthood.
35. 35
  Elobeid, A., Soininen, H., and Alafuzoff, I. (2012) Hyperphosphorylated tau in young and middle-aged subjects. Acta Neuropathol. 123 (1), 97– 104, DOI: 10.1007/s00401-011-0906-z
  There is no corresponding record for this reference.
36. 36
  Holmes, B. B. (2014) Proteopathic tau seeding predicts tauopathy in vivo. Proc. Natl. Acad. Sci. U. S. A. 111 (41), E4376– E4385, DOI: 10.1073/pnas.1411649111
  36
  Proteopathic tau seeding predicts tauopathy in vivo
  Holmes, Brandon B.; Furman, Jennifer L.; Mahan, Thomas E.; Yamasaki, Tritia R.; Mirbaha, Hilda; Eades, William C.; Belaygorod, Larisa; Cairns, Nigel J.; Holtzman, David M.; Diamond, Marc I.
  Proceedings of the National Academy of Sciences of the United States of America (2014), 111 (41), E4376-E4385CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
  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 pathol. through the brain via cell-cell transfer in neural networks. The crit. role of tau seeding activity is untested, however. It is unknown whether seeding anticipates and correlates with subsequent development of pathol. as predicted for a causal agent. One major limitation has been the lack of a robust assay to measure proteopathic seeding activity in biol. 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 vs. histol. markers of tau pathol., including MC1, AT8, PG5, and Thioflavin S. We detected robust seeding activity at 1.5 mo, >1 mo before the earliest histopathol. stain. Proteopathic tau seeding is thus an early and robust marker of tauopathy, suggesting a proximal role for tau seeds in neurodegeneration.
37. 37
  Kaufman, S. K., Thomas, T. L., Del Tredici, K., Braak, H., and Diamond, M. I. (2017) Characterization of tau prion seeding activity and strains from formaldehyde-fixed tissue. Acta Neuropathol. Commun. 5 (1), 41, DOI: 10.1186/s40478-017-0442-8
  37
  Characterization of tau prion seeding activity and strains from formaldehyde-fixed tissue
  Kaufman Sarah K; Thomas Talitha L; Diamond Marc I; Kaufman Sarah K; Del Tredici Kelly; Braak Heiko
  Acta neuropathologica communications (2017), 5 (1), 41 ISSN:.
  Tauopathies such as Alzheimer's disease (AD) feature progressive intraneuronal deposition of aggregated tau protein. The cause is unknown, but in experimental systems trans-cellular propagation of tau pathology resembles prion pathogenesis. Tau aggregate inoculation into mice produces transmissible pathology, and tau forms distinct strains, i.e. conformers that faithfully replicate and create predictable patterns of pathology in vivo. The prion model predicts that tau seed formation will anticipate neurofibrillary tau pathology. To test this idea requires simultaneous assessment of seed titer and immunohistochemistry (IHC) of brain tissue, but it is unknown whether tau seed titer can be determined in formaldehyde-fixed tissue. We have previously created a cellular biosensor system that uses flow cytometry to quantify induced tau aggregation and thus determine seed titer. In unfixed tissue from PS19 tauopathy mice that express 1 N,4R tau (P301S), we have measured tau seeding activity that precedes the first observable histopathology by many months. Additionally, in fresh frozen tissue from human AD subjects at early to mid-neurofibrillary tangle stages (NFT I-IV), we have observed tau seeding activity in cortical regions predicted to lack neurofibrillary pathology. However, we could not directly compare the same regions by IHC and seeding activity in either case. We now describe a protocol to extract and measure tau seeding activity from small volumes (.04 mm(3)) of formaldehyde-fixed tissue immediately adjacent to that used for IHC. We validated this method with the PS19 transgenic mouse model, and easily observed seeding well before the development of phospho-tau pathology. We also accurately isolated two tau strains, DS9 and DS10, from fixed brain tissues in mice. Finally, we have observed robust seeding activity in fixed AD brain, but not controls. The successful coupling of classical IHC with seeding and strain detection should enable detailed study of banked brain tissue in AD and other tauopathies.
38. 38
  Furman, J. L. (2017) Widespread tau seeding activity at early Braak stages. Acta Neuropathol. 133 (1), 91– 100, DOI: 10.1007/s00401-016-1644-z
  There is no corresponding record for this reference.
39. 39
  Salji, C. J. (2015) The JCMT Gould Belt Survey: properties of star-forming filaments in Orion A North. Mon. Not. R. Astron. Soc. 449 (2), 1782– 1796, DOI: 10.1093/mnras/stv369
  39
  The JCMT Gould Belt Survey: properties of star-forming filaments in Orion A North
  Salji, C. J.; Richer, J. S.; Buckle, J. V.; Di Francesco, J.; Hatchell, J.; Hogerheijde, M.; Johnstone, D.; Kirk, H.; Ward-Thompson, D.
  Monthly Notices of the Royal Astronomical Society (2015), 449 (2), 1782-1796CODEN: MNRAA4; ISSN:0035-8711. (Oxford University Press)
  We develop and apply a Hessian-based filament detection algorithm to submillimetre continuum observations of Orion A North. The resultant filament radial d. profiles are fitted with beam-convolved line-of-sight Plummer-profiles using Markov chain Monte Carlo techniques. The posterior distribution of the radial decay parameter demonstrates that the majority of filaments exhibit p = 1.5-3, with a mode at p = 2.2, suggesting deviation from the Ostriker p = 4 isothermal, equil., self-gravitating cylinder. The spatial distribution of young stellar objects relative to the high column d. filaments is investigated, yielding a lower limit on the star-forming age of the integral-shaped filament ∼1.4 Myr. Addnl., inferred lifetimes of filaments are examd. which suggest long-term filament accretion, varying rates of star formation, or both. Theor. filament stability measures are detd. with the aid of HARP C18O J=3-2 observations and indicate that the majority of filaments are gravitationally subcrit., despite the presence of young protostars. The results from this investigation are consistent with the one-dimensional accretion flow filament model recently obsd. in numerical simulations.
40. 40
  Hong, L., Qi, X., and Zhang, Y. (2012) Dissecting the kinetic process of amyloid fiber formation through asymptotic analysis. J. Phys. Chem. B 116 (23), 6611– 6617, DOI: 10.1021/jp205702u
  There is no corresponding record for this reference.
41. 41
  von Bergen, M. (2006) The Core of Tau - Paired Helical Filaments Studied by Scanning Transmission Electron Microscopy and Limited Proteolysis. Biochemistry 45 (20), 6446– 6457, DOI: 10.1021/bi052530j
  There is no corresponding record for this reference.
42. 42
  Goedert, M., Spillantini, M. G., Jakes, R., Rutherford, D., and Crowther, R. A. (1989) Multiple isoforms of human microtubule-associated protein tau: sequences and localization in neurofibrillary tangles of Alzheimer’s disease. Neuron 3 (4), 519– 526, DOI: 10.1016/0896-6273(89)90210-9
  42
  Multiple isoforms of human microtubule-associated protein tau: sequences and localization in neurofibrillary tangles of Alzheimer's disease
  Goedert, M.; Spillantini, M. G.; Jakes, R.; Rutherford, D.; Crowther, R. A.
  Neuron (1989), 3 (4), 519-26CODEN: NERNET; ISSN:0896-6273.
  This study detd. the sequences of isoforms of human tau protein, which differ from previously reported forms by insertions of 29 or 58 amino acids in the amino-terminal region. CDNA cloning shows that the insertions occur in combination with both three and 4 tandem repeats. RNAase protection assays indicate that transcripts encoding isoforms with the insertions are expressed in an adult-specific manner. Transcripts encoding 4 tandem repeats are also expressed in an adult-specific manner, whereas mRNAs encoding 3 tandem repeats are expressed throughout life, including in fetal brain. The levels of transcripts encoding the 29 or 58 amino acid inserts were ot significantly changed in cerebral cortex from patients with Alzheimer's disease. Antisera raised against synthetic peptides corresponding to these different human tau isoforms demonstrate that multiple tau protein isoforms are incorporated into the neurofibrillary tangles of Alzheimer's disease.
Supporting Information
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschemneuro.8b00094.
- Detailed description of the experimental procedures, data analysis methods, and kinetic modeling; figures illustrating fibril length distributions of P301S tau aggregates by TIRFM and TEM, SDS-PAGE analysis of tau to show absence of degradation during aggregation, misfits to a model which includes secondary nucleation, concentration measurements of P301S tau in P301S Tg mouse brains by ELISA; tables showing hypothetical tau spreading times at different spreading efficiencies and experimental conditions used for mPrP, α-synuclein, and tau aggregations from which data was derived for Figure 5 (PDF)
- cn8b00094_si_001.pdf (611.31 kb)
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Measurement of Tau Filament Fragmentation Provides Insights into Prion-like SpreadingClick to copy article linkArticle link copied!

ACS Chemical Neuroscience

Abstract

Results and Discussion

Figure 1

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Methods

Tau Purification

TIRF Microscope

Sample Preparation for TIRF Imaging

Analyzing Number and Length of Aggregates

Electron Microscopy

Model

Determining the Spreading Time Based on Stochastic-Deterministic Combined Simulations

Determining Mouse Tau Concentration by ELISA

Supporting Information

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Author Information

Acknowledgments

Abbreviations

References

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Supporting Information

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Measurement of Tau Filament Fragmentation Provides Insights into Prion-like Spreading
Click to copy article linkArticle link copied!