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Scaling analysis reveals the mechanism and rates of prion replication in vivo

Nature Structural & Molecular Biology volume 28, pages 365–372 (2021)Cite this article

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Abstract

Prions consist of pathological aggregates of cellular prion protein and have the ability to replicate, causing neurodegenerative diseases, a phenomenon mirrored in many other diseases connected to protein aggregation, including Alzheimer’s and Parkinson’s diseases. However, despite their key importance in disease, the individual processes governing this formation of pathogenic aggregates, as well as their rates, have remained challenging to elucidate in vivo. Here we bring together a mathematical framework with kinetics of the accumulation of prions in mice and microfluidic measurements of aggregate size to dissect the overall aggregation reaction into its constituent processes and quantify the reaction rates in mice. Taken together, the data show that multiplication of prions in vivo is slower than in in vitro experiments, but efficient when compared with other amyloid systems, and displays scaling behavior characteristic of aggregate fragmentation. These results provide a framework for the determination of the mechanisms of disease-associated aggregation processes within living organisms.

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Fig. 1: Principle of mechanistic analysis and increase of infectivity over time.
Fig. 2: PrPC and PrPSc concentrations over time for different mouse lines.
Fig. 3: Scaling exponents of the rate of infectivity and of PrPSc increase.
Fig. 4: Reaction network and consistent mechanisms.
Fig. 5: Sizing prions in brain homogenate.
Fig. 6: Individual rates in vivo and in vitro.

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Data availability

All data generated or analyzed during this study are included in this published article (and its Supplementary information files). Source data are provided with this paper.

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Acknowledgements

We thank to P. Schwarz and R. Moos for technical help. We acknowledge funding from Sidney Sussex College Cambridge (G.M.), the Mexican National Council of Science and Technology (I.C.-M.) and Cambridge Trust (I.C.-M.), the Synapsis Foundation (S.S.), the ERC (T.P.J.K.), the Amyloidosis Foundation (M.N.) and Peterhouse College Cambridge (T.C.T.M.).

Author information

Author notes

  1. These authors contributed equally: Georg Meisl, Timothy Kurt, Itzel Condado-Morales.

Authors and Affiliations

  1. Centre for Misfolding Diseases, Yusuf Hamied Department of Chemistry, University of Cambridge, Cambridge, UK

    Georg Meisl, Itzel Condado-Morales, Thomas C. T. Michaels, Samuel I. A. Cohen, Christopher M. Dobson & Tuomas P. J. Knowles

  2. Department of Pathology, UC San Diego, San Diego, CA, USA

    Timothy Kurt, Cyrus Bett & Christina J. Sigurdson

  3. Institute of Neuropathology, University of Zurich, Zurich, Switzerland

    Itzel Condado-Morales, Silvia Sorce, Mario Nuvolone, Daniel Heinzer, Merve Avar, Simone Hornemann & Adriano Aguzzi

  4. Amyloidosis Research and Treatment Center, Foundation IRCCS Policlinico San Matteo, Department of Molecular Medicine, University of Pavia, Pavia, Italy

    Mario Nuvolone

  5. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, USA

    Thomas C. T. Michaels

  6. Wren Therapeutics, Cambridge, UK

    Samuel I. A. Cohen

  7. Cavendish Laboratory, University of Cambridge, Cambridge, UK

    Tuomas P. J. Knowles

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Contributions

A.A., C.M.D., C.J.S. and T.P.J.K. conceived the study. T.K. and C.B. performed the PrPC and PrPSc measurement time courses in the four mouse lines. S.S., M.N., D.H. and M.A. performed the measurements of the infectivity by SSCA. I.C.-M. and S.H. designed and performed the prion size determination experiments. G.M. developed the theory and analyzed the data. G.M., S.I.A.C., T.C.T.M. and T.P.J.K. interpreted the data. G.M. wrote the manuscript. All authors contributed to editing the manuscript.

Corresponding authors

Correspondence to Christina J. Sigurdson or Tuomas P. J. Knowles.

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The authors declare no competing interests.

Additional information

Peer review information Nature Structural and Molecular Biology thanks Byron Caughey, Suzanne Sindi and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Inês Chen was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Comparison of infectivity and PrPSc amounts from Sandberg et al.

The data (open and filled circles) were obtained from Sandberg et al.27 Fig. 1 (PrP measurements, filled circles) and Fig. 2 (infectivity measurement, open circles). The infectivity data are given on a logarithmic scale but are here plotted on a linear scale, with the corresponding values given on the left axis. The PrPSc measurements are plotted on the right axis. Dotted lines connect the PrPSc measurements and are a guide to the eye. There is no clear systematic difference between PrPSc and infectivity when they are rescaled and both plotted in linear space. Data behind graphs are available as Source Data.

Source data

Extended Data Fig. 2 Sigmoidal and exponential fits of PrPSc measurements obtained here.

Data as shown in the main text (a-d), as well as the data obtained without PK digestion (e-g), fitted to both a sigmoidal function (solid line) and a simple exponential (dotted line). All data points (filled and open circles) are used in the sigmoidal fits, only pre-plateau data points (filled circles) are used in the exponential fits. The data include samples from different mice as well as technical repeats of the ELISA measurements (3-4 at each timepoint). Data behind graphs are available as Source Data.

Source data

Extended Data Fig. 3 Sigmoidal and exponential fits of data from Mays et al.

The data (open and filled circles) were obtained from Mays et al. 26 Fig. 2 (PrP measurements) and Fig. 4 (infectivity measurement). In the original paper the data are given for 10 different size fractions, the data here are a sum of all fractions. Fits to both a sigmoidal function (solid line) and a simple exponential (dotted line) are shown. All data points (filled and open circles) are used in the sigmoidal fits, only pre-plateau data points (filled circles) are used in the exponential fits. Data behind graphs are available as Source Data online.

Source data

Extended Data Fig. 4 Sigmoidal and exponential fits of data from Sandberg et al.

The data (open and filled circles) were obtained from Sandberg et al.27 Fig. 1. The infectivity data are given on a logarithmic scale and are analysed separately in Fig. 1 of the main text. Fits to both a sigmoidal function (solid line) and a simple exponential (dotted line) are shown. All data points (filled and open circles) are used in the sigmoidal fits, only pre-plateau data points (filled circles) are used in the exponential fits. Data behind graphs are available as Source Data.

Source data

Extended Data Fig. 5 Simulation and validation of microfluidic sizing.

a,b, Linking the ratio of concentrations in the diffused and the non-diffused halves of the channel to the hydrodynamic radius was accomplished by solving the diffusion equation for the relevant device geometry using finite element integration software. Shown are the results of simulations of the diffusion profiles (inset) and ratio of intensities from the non-diffused channel and the diffused channel, fnd/fd, for species with a range of hydrodynamic radii, for the device with 200 µm and 80 µm channel width, respectively. c. Validation was performed using lyophilised human insulin (Sigma-Aldrich UK). An insulin stock of 10 mg/ml was prepared in 50 mM HCl, filtered through a 22 µm filter. The concentration was measured in the NanoDrop 2000c (ThermoFisher Scientific) by UV absorbance at 276 nm, using an extinction coeffcient value of 1 for 1 mg/ml60. Insulin hexamer was prepared as described previously61. Monomeric or hexameric samples were injected into the microuidic device at a total flow rate of 400 µl/h, using a flow ratio of 19:21 protein to auxiliary buffer. For detection, the commercial HTRF immunoassay kit was used (Cisbio Bioassays, Codolet, France). Samples after diffusion (2 µl per well) were mixed with the antibody-pair (18 µl per well) and incubated for 30 min at room temperature. The TR-FRET readings were performed in Clariostar (BMG Labtech) in the time-resolved fluorescence mode, simultaneously with a standard curve made of 1:2 serial dilutions starting from 2 nM insulin. Quoted values are hydrodynamic radii, errors are standard deviations from 3 repeats, literature values from Oliva et al.62.

Extended Data Fig. 6 Separation and quantification of PrPC and PrPSc from prion-infected animals by centrifugation and size exclusion chromatography.

a, Brain homogenate from a WT mouse at the terminal stage of disease was subjected to centrifugation followed by FPLC. Aliquots (10 µl each) from fractions 2–12 and 13–23 were analysed by SDS-PAGE and western blotting without PK digestion was used to monitor PrP elution from the column, and revealed two distinct populations (indicated at top of panel a). b, PK digestion (20 µg/mL) of aliquots from fractions 3–6 (PrPSc) and 14–19 (PrPC) in (A) was used to reveal proteinase-resistant PrPSc. c, Aliquots (10 µl total) from fractions 15–19 of mice (genotype indicated) from shortly after inoculation and at the terminal stage of disease, labelled ‘early’ and ‘late’ respectively, were assessed by semi-quantitative dot blotting. PrPC was found primarily in fractions 16–19. d, Levels of PrPSc in aliquots (10 µl total) from (a) as assessed by peptide ELISA. Values were interpolated from a standard curve (R2=0.96) generated using recombinant mouse PrP. e, Aliquots (10 µl total) from (a) were mixed with an equal volume of 8 M Gdn-HCl or PBS and heated for 5 min at 80 °C prior to ELISA. Gdn-HCl denaturation increases the signal intensity of PrPSc by its disaggregation but does not alter the PrPC signals63,64. Samples were run in triplicate and the values were interpolated from a standard curve (R2=0.98) generated using recombinant mouse PrP.

Supplementary information

Supplementary Information

Supplementary Notes 1–5.

Reporting Summary

Supplementary Table 1

Summary of numbers of mice used. Figure 2a of the main text shows the averages of the PrPC measurements at early time and terminal disease. Figure 2b–e shows all technical repeats of the PrPSc ELISA measurements. Three technical repeats for each mouse are performed for the Prnp0/+ line, and four technical repeats for all the others. The table gives the time-point and the number of mice used in PrPSc ELISA measurements at that time-point in round brackets for each of the lines. The numbers of mice used to determine the early time and terminal disease levels of PrPC are also given in square brackets.

Supplementary Table 2

Summary of scaling exponents. The scaling exponents determined from all three datasets by the different methods, as well as their standard errors from a linear regression analysis, are given. Both the mean value for the scaling and the mean errors are calculated. Details are in Supplementary Note 2.

Source data

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Meisl, G., Kurt, T., Condado-Morales, I. et al. Scaling analysis reveals the mechanism and rates of prion replication in vivo. Nat Struct Mol Biol 28, 365–372 (2021). https://doi.org/10.1038/s41594-021-00565-x

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