Advertisement

  • Loading metrics

Open Access

Peer-reviewed

Research Article

The axonal transport velocity of prions is independent of prion formation

  • Sam M. Koshy,

    Roles Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing

    Affiliation Department of Medical Microbiology and Immunology, School of Medicine, Creighton University, Omaha, Nebraska, United States of America

    ⨯
  • Jay Hrdlicka,

    Roles Data curation, Formal analysis, Investigation, Validation, Writing – review & editing

    Affiliation Department of Medical Microbiology and Immunology, School of Medicine, Creighton University, Omaha, Nebraska, United States of America

    ⨯
  • Michael G. Nichols,

    Roles Formal analysis, Funding acquisition, Methodology, Writing – review & editing

    Affiliation Department of Physics, College of Arts and Sciences, Creighton University, Omaha, Nebraska, United States of America

    ⨯
  • Anthony S. Stender,

    Roles Formal analysis, Methodology, Writing – review & editing

    Affiliation Integrated Biomedical Imaging Facility (IBIF), Creighton University, Omaha, Nebraska, United States of America

    ⨯
  • Jack H. Taylor,

    Roles Formal analysis, Writing – review & editing

    Affiliation Biostatistical core facility, Creighton University, Omaha, Nebraska, United States of America

    ⨯
  • Joaquín Castilla,

    Roles Funding acquisition, Investigation, Resources, Writing – review & editing

    Affiliations Center for Cooperative Research in Biosciences (CIC BioGUNE), Basque Research and Technology Alliance (BRTA), Derio, Spain, Centro de Investigación Biomédica en Red de Enfermedades infecciosas (CIBERINFEC), Carlos III National Health Institute, Madrid, Spain, IKERBASQUE, Basque Foundation for Science, Bilbao, Spain

    ⨯
  • Jason C. Bartz

    Roles Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Project administration, Supervision, Writing – original draft, Writing – review & editing

    jbartz@creighton.edu

    Affiliations Department of Medical Microbiology and Immunology, School of Medicine, Creighton University, Omaha, Nebraska, United States of America, Department of Microbiology, Immunology, and Pathology, Prion Research Center, Colorado State University, Fort Collins, Colorado, United States of America

    ⨯

Abstract

Prion diseases are caused by PrPSc, the self-templating infectious conformation of the normal host-encoded prion protein, PrPC. Spread of PrPSc in the peripheral and central nervous system follows a highly predictable pattern consistent with slow axonal transport along defined neuroanatomical pathways. The rate of PrPSc transport, the contribution of prion strain diversity, and the requirement for prion conversion during transport are poorly understood, in part, due to a lack of direct measurement of PrPSc transport under defined conditions. Here, using a combination of live animal and sciatic nerve explant culture experiments, and determined the velocity of PrPSc ranged from rates consistent with both slow and fast axonal transport. Interestingly, transport of PrPSc was not dependent upon prion conversion as it was observed using prion strain and host combinations that do not support prion formation. The rate of PrPSc transport of prion strains and synthetic prions suggest that prions can use both slow and fast modes of axonal transport. Overall, converging lines of evidence suggest that prions are transported at a range of velocities consistent with both slow and fast axonal transport that is independent prion formation.

Author summary

Prion diseases are fatal neurodegenerative diseases caused by PrPSc, the self-templating conformation of the normal host-encoded prion protein, PrPC. Transport of PrPSc in the nervous system follows a highly predictable spatial pattern consistent with spread along defined neuroanatomical pathways. The velocity of PrPSc transport and the requirement for prion conversion during transport are poorly understood. To address this problem, we developed a method to determine the velocity of fluorescently tagged PrPSc particles in the sciatic nerve of animals. We measured the velocity of PrPSc particles that ranged from rates consistent with both slow and fast axonal transport. Additionally, we found transport of PrPSc was not dependent upon prion conversion as it was observed under conditions that do not support prion formation. Overall, these results suggest that prions are transported at a range of velocities consistent with both slow and fast axonal transport that is independent prion formation.

Citation: Koshy SM, Hrdlicka J, Nichols MG, Stender AS, Taylor JH, Castilla J, et al. (2026) The axonal transport velocity of prions is independent of prion formation. PLoS Pathog 22(7): e1014456. https://doi.org/10.1371/journal.ppat.1014456

Editor: Neil A. Mabbott, University of Edinburgh, UNITED KINGDOM OF GREAT BRITAIN AND NORTHERN IRELAND

Received: March 1, 2026; Accepted: July 8, 2026; Published: July 24, 2026

Copyright: © 2026 Koshy et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: This work was supported by grants from the NIH (NS107246) to JCB. The integrated biomedical imaging facility at Creighton University is supported by the School of Medicine and grants from the NIH (GM103427, GM139762) to MGN. The present work was partially funded by two different grants awarded by the Agencia Estatal de Investigación, Ministerio de Ciencia e Innovación (Spanish Government), grant numbers PDC2025-165940-I00 and PID2024-160022OB-I00 to JC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Prion diseases are invariably fatal, transmissible, neurological disorders that affect a wide variety of mammalian species. Prion diseases are unique in biology in that they can have three etiologies, sporadic, familial and infectious. Prions are comprised of PrPSc, the self-templating conformation of the host encoded prion protein, PrPC [1–5]. Prion formation occurs when PrPC binds to PrPSc and, through an unknown process, PrPSc directs a global rearrangement of the helical structure of PrPC to a parallel in register intermolecular beta sheet structure of PrPSc [6–8]. Fragmentation of the growing PrPSc fibril results in generation of new free ends that support prion formation [9,10]. Repeated cycles of PrPSc formation and fibril fragmentation can result in exponential amplification of PrPSc and prion infectivity that is thought to be recapitulated in vitro using protein misfolding cyclic amplification (PMCA) [11–13].

Prion pathogenesis is characterized by prion spread in the nervous system [14–17]. Following intraperitoneal and oral routes of infection, prions can establish infection in Peyer’s patches, gut-associated lymphoid tissues, spleen and lymph nodes [18–21]. From these tissues, through an unknown process, prions gain access to sympathetic and parasympathetic nerves where they are transported to the central nervous system [22–26]. Direct inoculation of peripheral nerves (e.g., optic, sciatic nerve) results in spread of PrPSc along synaptically connected neuroanatomical pathways that include peripheral ganglia, peripheral nerves (somatic and autonomic), spinal cord tracts, and brainstem nuclei [27–33]. Overall, prions are transported along synaptically connected structures where they can cause neurotoxicity, resulting in neuronal death.

The rate of prion transport within neural pathways is poorly understood. Initial studies tracked prion spread using the presence of vacuolation pathology or infectivity of target tissues, leveraging the predictable pattern of neuroanatomical spread of PrPSc. The rate of prion infectivity is consistent with slow axonal transport at a rate of approximately 0.8-4.0 mm/day (0.01-0.05 µm/s) that was calculated after inoculations via the ocular, intraperitoneal, sciatic, or oral routes of infection [29,34–36]. Subsequent studies utilizing immunohistochemistry or immunoblot detection of PrPSc similarly observed that the rate of PrPSc spread was consistent with slow axonal transport [37]. Additionally, the rate of PrPSc spread was measured between three well-characterized hamster-adapted prion strains, the hyper (HY) strain of hamster-adapted transmissible mink encephalopathy (TME), the drowsy (DY) strain of hamster-adapted TME and the 139H strain of hamster-adapted scrapie. These three hamster-adapted prion strains differ in incubation period, clinical signs and distribution of PrPSc and/or pathology in the host [38–41]. Interestingly, strain-specific rates of PrPSc spread were observed between HY TME, DY TME, and 139H suggesting strain-specific differences in axonal transport [42–44]. The methods used in the aforementioned studies, however, cannot distinguish between the inoculated PrPSc and newly formed PrPSc. It is possible, therefore, that the previously reported PrPSc velocities are instead a combination of both PrPSc transport and conversion where PrPSc is only identified once it has accumulated above the detection threshold [17].

Overall, bona fide axonal transport of PrPSc particles has not been observed or measured directly. In this study, we investigated the rate of PrPSc axonal transport, the effect of prion strain on PrPSc velocity, and if transport requires prion formation..

Materials and methods

Ethics statement

All procedures involving animals comply with the Guide for the Care and Use of Laboratory Animals and were approved by the Creighton University Institutional Animal Care and Use Committee.

Prion inocula

Brains from terminally-ill mice or hamsters infected with either the RML, 263K, HY TME, DY TME or 139H strains of prions were collected and homogenized to 10% w/v in Dulbecco’s phosphate buffered saline (DPBS).

Sciatic nerve inoculation, tissue collection and processing

Inoculation of the sciatic nerve (ScN) was performed as previously described [44]. Briefly, mice were anesthetized and maintained in the plane of anesthesia with inhaled isoflurane. The right ScN was exposed using sterilized surgical instruments and separated from the surrounding fascia and musculature. Closed spring scissors were used to bring the nerve into the superficial field out of the surrounding structures taking care not to damage the nerve, and a 30-gauge Hamilton syringe (Hamilton Company, Reno, NV) was utilized to inject 1 µl of the prion sample into the sciatic nerve after the needle had penetrated the epineurium. The nerve was returned to its location within the musculature, and the surgical incision was closed with surgical staples. Mice were then given 30 µl of butorphanol (2 mg/kg; Patterson Veterinary, Loveland, CO) and allowed to recover in their home cage. At 24 h post infection (p.i.), mice were anesthetized with isoflurane and transcardially perfused with DPBS. Both the inoculated (ipsilateral) and the uninoculated (contralateral) ScN were removed. The lumbar (L3-L4 vertebral segments) spinal cord (SC) was also removed from the vertebral column. To limit cross contamination, tissues were harvested from the least infectious to the most infectious (in order: contralateral ScN, lumbar SC, ipsilateral ScN). Tissues were frozen at -80oC.

Protein misfolding cyclic amplification

Protein misfolding cyclic amplification (PMCA) was performed as previously described [45]. Briefly, dissected tissues were homogenized in a bead mill homogenizer (OMNI International, Kennesaw, GA) in sterile DPBS to produce 10% w/v tissue homogenates. In a thin-walled PCR tube, 5 µl of tissue homogenates were seeded into 45 µl of 10% w/v uninfected (Un) hamster or Un mouse brain homogenate (BH) in PMCA conversion buffer (1% Triton X-100, 1 tablet Complete protease inhibitor, 6 mM EDTA, 150 mM NaCl, 100 µg/ml Heparin, 0.05% Digitonin, sterile DPBS) before being subjected to 2 rounds of PMCA using a Q700 sonicator with a microplate horn (Qsonica, Newtown, CT). In the second serial round of PMCA, 5 µl of the round 1 PMCA reaction was added to 45 µl of Un hamster BH in PMCA conversion buffer. Each round of PMCA had a duration of 72 h at 37oC with 1 second of active sonication every 10 min (rest for 9:59 min). Sonicator amplitude was set at 37–40 with a wattage of 220–280 W. Unseeded samples containing only Un hamster BH in PMCA conversion buffer served as negative controls, and HY TME BH dilutions seeded into Un hamster BH in PMCA conversion buffer served as positive controls for each PMCA experiment. All samples were seeded in triplicate.

Western Blot analysis

Western blot analysis was performed as previously described [45]. Briefly, 10 µl of PMCA reaction was combined with 10 µl of proteinase K (PK; 0.05 mg/ml; MilliporeSigma, Burlington, MA), and the reaction was incubated at 37oC with constant shaking. Afterwards, 20 µl of 2x sample buffer (4% w/v SDS, 2% v/v β-mercaptoethanol, 40% v/v glycerol, 0.004% w/v Bromophenol blue, and 0.5 M Tris buffer pH 6.8) was added to the reaction mixture and incubated at 100oC for 10 min. The reaction mixture was placed on ice, and 10 µl of the mixture was size fractionated on 4–12% Bis-Tris NuPage polyacrylamide gels (Invitrogen, Carlsbad, CA). Gels were transferred onto polyvinylidene difluoride (PVDF) membranes (MilliporeSigma, Burlington, MA). Membranes were blocked with 5% w/v blotto (Bio-Rad Laboratories, Hercules, CA) in 0.05% v/v tween tris-buffered saline (TTBS; BioRad Laboratories, Hercules, CA) and then incubated overnight with anti-PrP antibody 3F4 or anti-PrP 8H4 monoclonal anti-PrP primary antibody (1:10000). Membranes were then washed five times in TTBS and incubated in goat anti-mouse HRP conjugated secondary antibody for 1 hour (1:4000). Membranes were then washed five times in TTBS, exposed with ECL reagent, and imaged via chemiluminescence in the Li-Cor Odyssey XF imager (Li-Cor Biosciences, Lincoln, NE).

Production of infectious synthetic mouse PrPSc

Spontaneous generation of recombinant bona fide prions was performed as described previously [46,47]. Briefly, 100 mg of acid-washed 1 mm glass beads (Sigma-Aldrich) were placed in clean, labelled 2-ml tubes with screw caps, containing 500–800 µl of fresh substrate. Protein misfolding shaking amplification (PMSA) was performed for 24 h, 39°C and with continuous shaking at 700 rpm using programmable thermoblocks (Digital Shaking Drybath, Thermo Scientific). Up to four 24 h serial PMSA rounds were performed, diluting 1:10 the PMSA product from the previous round into a new tube with freshly thawed rec mouse PrPC substrate and their corresponding glass beads. To produce the Mo recPrPSc batch employed in this study, a two-step propagation was performed by PMSA. First, four 2-ml screw cap tubes with 1 ml of fresh PMSA substrate each, were complemented with three clean zirconia-silicate beads of 2.3 mm (BioSpec Products Inc., Bartlesville, OK) and 3 prion-coated beads, that were subjected to PMSA for 5 h. Then, the product of this first round was used at 1:5 dilution to seed 5-ml screw cap tubes each containing 4 ml of fresh PMSA substrate and 15 clean zirconia-silicate beads of 2.3 mm. These were then submitted to a 19-h PMSA reaction. The product of this reaction was analysed by electrophoresis and total protein staining to confirm efficient propagation and conservation of the original electrophoretic mobility pattern.

PrPSc enrichment

Enrichment of PrPSc from BH was performed as previously described [48]. Briefly, 2 μl of PK (2.5 mg/mL) was added to 200 μl aliquots of 10% w/v BH from animals terminally infected with various prion strains and incubated with shaking at 37oC for 30 min. Afterwards, 4.1 μl of 0.5M EDTA (pH 8.0), 206 μl of 4% w/v sarkosyl in DPBS, and 0.83 μl of benzonase (MilliporeSigma, Burlington, MA) were added and were mixed by inverting the tube 10 times and incubated with shaking at 37oC for 10 min. Subsequently, 33.5 μl of 4% w/v sodium phosphotungstate in ultrapure H2O (NaPTA; pH 7.4) was added, mixed by inverting the tube 10 times, and incubated with shaking at 37oC for 30 min. Then, 705.3 μl iodixanol and 57.2 μl of NaPTA was added. Samples were mixed by inverting the tube 10 times, and centrifuged at 16,100g for 90 min. After centrifugation, 500 μl of the clarified supernatant was added to a durapore-PVDF 0.45 μm filtration column (Merck Millipore. Burlington, Massachusetts) and centrifuged at 12,000g for 30 s (each starting tube generated two 500 μl aliquots). The filtered sample (~480 μl) was combined with 480 μl of 2% w/v sarkosyl/0.3% w/v NaPTA in ultrapure H2O (pH 7.4), samples were mixed by inverting the tube 10 times and incubated with shaking at 37oC for 10 min. Samples were then centrifuged at 16,100g for 90 min. After centrifugation, the supernatant was removed, and the pellet was resuspended in 10 μl of wash buffer (17.5% iodixanol and 0.1% sarkosyl in DPBS). Samples were sonicated in a cup horn sonicator at 50% power for 10 s twice with resting on ice for 15 s in between. Split samples were pooled into a single 20 µl aliquots, and 180 μl of wash buffer and 16.2 μl of 4% w/v NaPTA was added. Samples were mixed by inverting the tube 10 times, and centrifuged at 16,100g for 30 min. The supernatant was removed, and samples were resuspended in 200 μl of wash buffer and 16.2 μl of 4% w/v NaPTA. Samples were again mixed by inverting the tube 10 times, and centrifuged at 16,100g for 30 min. The supernatant was removed, and samples were resuspended in 20 μl of 0.1% sarkosyl in DPBS. To concentrate the PrPSc, 12 purified 20 µL PrPSc aliquots in 0.1% sarkosyl from the previous step were combined and centrifuged for 30 min at 16,100g. The supernatant was removed, and samples were resuspended in 20 μl of 0.1% sarkosyl. Successful isolation of PrPSc was confirmed by WB, and the purity of PrPSc was determined by Sypro Ruby (Thermo Fisher Scientific, Waltham, MA) staining of size fractionated PrPSc following the manufacturer’s protocol. Gels were imaged with the Cytivia typhoon scanner (Marlborough, MA) using the Cy3 laser and filter set (excitation: 532 nm, emission: 560–580 nm, PMT: 500–600 V).

Fluorophore conjugation

Enriched brain derived or synthetic PrPSc was labeled with Alexa Fluor 647 dye (AF647; Thermo Fisher Scientific, Waltham, MA) or pHrodo Deep Red tetrafluorophenyl (TFP) ester (pHrodo; Thermo Fischer Scientific, Waltham, MA) according to the outlined protocol. Briefly, 20 μl of enriched, concentrated PrPSc was combined with 12 μl of AF647 dissolved in DMSO, incubated at room temperature for 1 hour with shaking, and then incubated at 4oC for 48–72 h. Samples were shielded from light at all steps. After incubation, samples were centrifuged for 30 min at 16,100g and the supernatant containing unconjugated AF647 was removed. The pellet was resuspended in 100 μl of 10 mM glycine to quench any remaining unconjugated AF647 and centrifuged for 30 min at 16,100g. This was repeated two more times, and the final pellet was resuspended in 20 μl of 10% sarkosyl in DPBS. Successful conjugation of PrPSc was confirmed by fractionating the samples on 4–12% Bis-Tris NuPage polyacrylamide gels and imaging with the Cytivia Typhoon scanner using the Cy5 laser and filter set (excitation: 635 nm, emission: 655–685 nm, PMT: 500 V).

Live sciatic nerve explant imaging

Sciatic nerve explant imaging was adapted from protocols described previously [49,50]. Anesthetized male or female FvB or PrP-/- on FvB background mice [51] were bilaterally inoculated in the ScN with either 1–2 μl of, DPBS, unconjugated AF647 dye, purified uninfected brain homogenate conjugated to AF647 (Un-AF647), or purified PrPSc conjugated to AF647 (PrPSc-AF647) or to pHrodo (PrPSc-pHrodo). The ScN was excised and placed into an FCS3 live cell chamber (Bioptechs, Butler, PA) heated and maintained at 37oC, and perfused continuously with oxygenated glucose solution (98 mM NaCl, 1 mM KCl, 2 mM KH2PO4, 1 mM MgSO4, 1.5 mM CaCl2, 5.6 mM D-glucose, 24 mM NaHCO3, 95% O2/5% CO2). The ScN samples were imaged starting approximately 20 minutes after inoculation on a Leica TCS SP8 multiphoton confocal microscope (Leica Microsystems, Wetzlar, Germany) using the Mai-Tai Deep-See ultrafast laser (Spectra-Physics, Milpitas, CA) as an excitation source (excitation: 840 nm). The emission bandpass was set to detect from 660-720 nm to encompass the peak emission of AF647. Using an HC PL APO 63X/1.4 oil immersion objective, Second Harmonic Generation (SHG) from collagen at 420 nm was used to localize the nerve and bring the axons into focus, and then confocal/multiphoton excitation was used to focus on groups of axons containing fluorescent particles. Axons were identified as the structures containing fluorescent particles that ran parallel to the body of the nerve and spanned the imaging field. Fluorescence specific to the AF647 labeled PrPSc was confirmed through lambda spectral scans (excitation: 840 nm; emission: 600–750 nm, consecutive 10 nm intervals). Time series images were acquired for 1–3 min at 63X with 3–5 s intervals between consecutive frames.

Time series images were analyzed using the Fiji/ImageJ plugin, Trackmate [52]. The smooth function was used to reduce noise, and regions of interest (ROI) were drawn around individual axons in the first frame of the time series. Particles were detected utilizing the DOG algorithm which is suitable for particles < 5 μm in diameter. Most particles observed within axons were fine and punctate with diameters < 5 µm. Particle diameter was set to 1 μm to ensure all possible particles in the axon were detected, and particles with a quality score at or above 5 were selected for track analysis. Most particles with a quality score > 5 were within the main body of the axon and displayed strong fluorescent signal that could be easily tracked. The nearest neighbor tracking algorithm was used and the distance between frames was set to the maximal possible distance a particle could travel in the given frame interval. Track velocity data was saved in Microsoft Excel. The mean velocity of each particle > 0.1 μm/sec was selected and analyzed statistically.

Live animal sciatic nerve imaging

Male FvB/N or PrP-/- mice on a FvB background were anesthetized with inhaled isoflurane and unilaterally inoculated in the sciatic nerve as described above [51]. A triangular piece of parafilm was slid between the lifted nerve and the muscular fascia to physically separate the sciatic nerve from the body plane of the animal. Mice were intraperitoneally anesthetized with a ketamine/xylazine cocktail (87.5/12.5 mg/kg) using an insulin syringe. To stabilize the anesthetized animal, the animal was placed onto the adjustable platform of a purpose-built live animal imaging system with the exposed nerve in contact with the coverslip. The platform was maintained at 37oC by a temperature control unit (TC 324B, Warner Instruments, Holliston, MA). The ScN of the live mouse was imaged beginning approximately 30 minutes after inoculation to identify fluorescent particles using the Leica SP8 multiphoton microscope, and the images were analyzed as described above.

Statistical analysis

A multifactorial ANOVA was performed comparing PrPSc particle velocities in sciatic nerve explants between strains, mouse PrP genotype, and mouse sex (4 strains x 2 genotype x 2 sex). A multifactorial ANOVA was performed comparing PrPSc particle velocities for live animal imaging between strains and mouse PrP genotype (5 strains x 2 genotype). To reduce the possibility of statistical significance solely due to large n in each group, all recorded particle velocities > 0.1 µm/s were averaged within each axon to get a single average particle velocity for each individual axon. A White-Huber correction was done to account for heteroscedasticity, and significant main effects were probed with Fisher’s LSD post hoc tests. All omnibus and post hoc tests were considered significant if p < 0.05.

Results

Spatiotemporal detection of inoculum PrPSc in anatomically connected locations consistent with spread by fast axonal transport

The distance from the site of inoculation in the sciatic nerve to the lumbar spinal cord was determined as previously described [44]. Briefly, the spinal cord was exposed by removal of all vertebral laminae and the site of sciatic nerve inoculated was dissected proximally to the location at which the spinal nerves entered the intervertebral foramen and followed proximally in the vertebral canal to the location at which they entered the spinal cord. The distance between the site of inoculation in the sciatic nerve and where the sciatic nerve entered the spinal cord is 25mm. Following sciatic inoculation of prions, if PrPSc utilized slow axonal transport, PrPSc would be detected in the ipsilateral sciatic nerve and not in the lumbar spinal cord at 24 h postinfection (Fig 1, Panel A). Alternatively, if PrPSc utilizes fast axonal transport, inoculum PrPSc would be detected in the lumbar spinal cord in addition to the ipsilateral sciatic nerve (Fig 1, Panel B).

thumbnail
Download:
Fig 1. Temporal spread of inoculum PrPSc from the site of inoculation in the sciatic nerve to the lumbar spinal cord.

Predicted spread of PrPSc following sciatic nerve inoculation via either A) slow or B) fast axonal transport rates at 24 h post sciatic nerve inoculation. C) Western blot analysis of proteinase K digested PMCA reactions of sciatic nerve PrP+/+ (1st and 3rd panel from the top) or PrP-/- mice (2nd and 4th panel from the top) inoculated with either uninfected (UN, top two panels) or 263K-infected hamster brain homogenate (bottom two panels) at 24 h post inoculation. Arrows indicate the migration of the 29 kDa molecular weight marker. Created with BioRender. www.biorender.com.

https://doi.org/10.1371/journal.ppat.1014456.g001

The PMCA protocol used was able to detect as little as 5 x 10-12 μg eq of starting HY infected brain homogenate seeded into uninfected hamster brain homogenate substrate after two rounds of PMCA (S1 Fig, Panel A). In contrast, similar amounts of HY TME did not seed uninfected mouse brain homogenate after two serial rounds of PMCA (S1 Fig, Panel B). Our unseeded negative controls, containing only uninfected hamster or mouse PMCA substrate, did not amplify PrPSc after two serial rounds of PMCA (S1 Fig).

As negative controls, PrP+/+ or PrP-/- mice were inoculated in the sciatic nerve with uninfected hamster brain homogenate (Fig 1, Panels A, B). Following two serial rounds of PMCA, PrPSc was not detected in the ipsilateral sciatic nerve, the lumbar spinal cord or the contralateral sciatic nerve, from either the PrP+/+ or PrP-/- mice (Fig 1, Panel C). Sciatic nerve inoculation of PrP+/+ mice with 263K prions resulted in PMCA detection of PrPSc in the ipsilateral sciatic nerve and lumbar spinal cord, but not in the contralateral sciatic nerve (Fig 1, panel C). Although there is a large species barrier between 263K hamster prions and mice as determined by PMCA (S1 Fig, Panel B) and bioassay (S5 Fig), and the 3F4 anti-PrP antibody recognizes hamster, but not mouse PrP, we could not formally exclude the possibility that detected PrPSc is newly formed PrPSc rather than inoculum. To exclude this possibility, 263K prions were inoculated into the sciatic nerve of PrP-/- mice, which do not support prion formation, that resulted in PMCA detection of PrPSc in the ipsilateral sciatic nerve and lumbar spinal cord, but not in the contralateral sciatic nerve at 24 h postinfection (Fig 1, panel C). For each group, a minimum of three mice were examined with similar results. Overall, the rate of PrPSc transport was at least 25 mm/day.

Purification of PrPSc and conjugation to AF647 retains prion infectivity

PrPSc from multiple prion strains were purified from hamster and mouse brain homogenates and the purified PrPSc was size fractionated on SDS-PAGE and stained with Sypro Ruby (S2 Fig). The bands <25 kDa correspond to PrPSc glyco-isoforms including the strong di-glycosylated form as well as the mono-glycosylated and un-glycosylated forms (S2 Fig). Next, the purified PrPSc conjugated to AF647 dye were size fractionated on SDS-PAGE and imaged on a Typhoon imager, revealing detection of fluorescent bands corresponding to the di-, mono-, and unglycosylated forms of PrPSc conjugated to AF647 (PrPSc-AF647) (S3 Fig). To confirm biological activity of PrPSc-AF647, 10-fold dilutions of HY PrPSc-AF647 were seeded into PMCA reactions (S4 Fig). HY PrPSc-AF647 were able to template efficient conversion after 2 rounds of PMCA (S4 Fig). Overall, the purified PrPSc retains biological activity following conjugation to AF647. To investigate if this material was infectious, uninfected mouse BH, purified uninfected mouse BH, RML-infected BH, and purified RML were unilaterally inoculated into mouse sciatic nerve. All (n = 5) RML BH and purified RML preparation inoculated mice succumbed to prion disease in 146 ± 4 and 145 ± 4 days postinfection respectively (S5 Fig, Panel A). The incubation periods were not statistically different (p > 0.05). All (n = 5) mice inoculated with either the uninfected mouse BH or purified uninfected mouse BH did not develop clinical signs of prion disease by 600 days postinfection (S5 Fig, panel A). Western blot analysis of PK digested brain homogenate from each of these groups were consistent with the clinical diagnosis of disease (S5 Fig, panel B).

Determination of PrPSc-AF647 axonal transport velocity in PrP+/+ mouse sciatic nerve explant cultures

After sciatic nerve inoculation of DPBS, unconjugated AF647, and Un-AF647, individual axons could not be demarcated with fluorescent particles, and the distribution of fluorescence was random (S6 Fig). Lambda spectral scans of these random particles did not have a distinct peak in fluorescence intensity and were easily distinguishable from the lambda spectral scan pattern of AF647 (S6 Fig) and indicated that the low molecular weight fluorescent material that was detected in the Sypro Ruby gel of the uninfected preparations (S3 Fig) did not contribute to measurable signal in the sciatic nerve explant cultures.

To investigate PrPSc axonal transport, purified RML, HY, DY, or 139H PrPSc strains conjugated to AF647 were injected into the sciatic nerve and the explant was imaged using two photon confocal microscopy. Fluorescence, with all prion strains examined, in both PrP+/+ and PrP-/-, and in both male and female animals, localized to multiple sciatic nerve axons within the imaging field and throughout the nerve body (Fig 2). Lambda scans confirmed the presence of PrPSc-AF647 with an intensity peak at ~675 nm (Fig 2). Particles from each strain had a heterogenous size distribution that contained fine particles and larger, brighter aggregates (Fig 2). Within each axon, multiple particles were localized by Trackmate and tracked over multiple frames to calculate mean velocity. To investigate PrPSc transport, we assembled a distribution profile of PrPSc particle velocities between the prion strains, host genotype and animal sex (Fig 3) and represented the average velocities of these particles, plus the percentage of the total particles that had a measured mean velocity of > 2 μm/sec (Table 1) and compared statistically (S1 Table). For each group, a minimum of three mice were examined and velocities of a minimum of 5000 particles were measured.

thumbnail
Download:
Table 1. Average velocities of PrPSc particles in mouse sciatic nerve explant cultures.

https://doi.org/10.1371/journal.ppat.1014456.t001

thumbnail
Download:
Fig 2. Time series images of PrPSc-AF647 in sciatic nerve explant cultures.

Murine sciatic nerve explants were inoculated with either Rocky Mountain Laboratory (RML) prions, hyper (HY), drowsy (DY), or 139H PrPSc conjugated to AF647. A particle is followed over 12 s with 3 selected frames showing its movement along the axon (3 s/frame). Lambda scans (top row) were consistent with AF647 bound particles. Still images of the nerve containing multiple axon fibers (second row from the top) with AF647 bound particles, and example motile particles within an individual axon (bottom three rows) were used to measure the velocity of the AF647 conjugated PrPSc. Scale bars represent either 20 (top row) or 5 μm (bottom three rows).

https://doi.org/10.1371/journal.ppat.1014456.g002

thumbnail
Download:
Fig 3. Velocities of PrPSc-AF647 particles in sciatic nerve explant cultures.

Violin plot representation of PrPSc particle velocities from either male or female PrP+/+ or PrP-/- mice inoculated in the sciatic nerve with either, Rocky Mountain Laboratory (RML) prions, hyper (HY), drowsy (DY), or 139H PrPSc-AF647.

https://doi.org/10.1371/journal.ppat.1014456.g003

Determination of PrPSc-AF647 axonal transport velocity in the intact sciatic nerve of live mice

To measure PrPSc axonal velocity in live animals, the sciatic nerve was inoculated with PrPSc-AF647 from RML, HY, DY, 139H or Mo recPrPSc. Similar to what was observed in the explant culture, fluorescent particles localized to axons with a heterogenous particle size distribution and lambda scans confirmed the presence of AF647 (Fig 4). To investigate PrPSc transport velocities, we assembled a distribution profile of PrPSc between the prion strains and host genotype (Fig 5) and represented the average velocities of these particles plus the percentage of the total particles that had a measured mean velocity of > 2 μm/sec (Table 2) and compared statistically (S2 Table). For each group, a minimum of three mice were examined and velocities of a minimum of 5000 particles were measured.

thumbnail
Download:
Table 2. Average velocities of PrPSc particles in mouse sciatic nerve from live animals.

https://doi.org/10.1371/journal.ppat.1014456.t002

thumbnail
Download:
Fig 4. Time series images of PrPSc-AF647 in the sciatic nerve of live animals.

Murine sciatic nerve explants were inoculated with either Rocky Mountain Laboratory (RML) prions, hyper (HY), drowsy (DY), 139H or synthetic mouse recombinant (Mo rec) PrPSc conjugated to AF647. A particle is followed over 12 s with 3 selected frames showing its movement along the axon (3 s/frame). Lambda scans (top row) were consistent with AF647 bound particles. Still images of the nerve containing multiple axon fibers (second row from the top) with AF647 bound particles, and example motile particles within an individual axon (bottom three rows) were used to measure the velocity of the AF647 conjugated PrPSc. Scale bars represent either 20 (top row) or 5 μm (bottom three rows).

https://doi.org/10.1371/journal.ppat.1014456.g004

thumbnail
Download:
Fig 5. Velocities of PrPSc-AF647 particles in the sciatic nerve of live animals.

Violin plot representation of PrPSc particle velocities from male PrP+/+ or PrP-/- mice inoculated in the sciatic nerve with either, Rocky Mountain Laboratory (RML) prions, hyper (HY), drowsy (DY), 139H or synthetic mouse recombinant (Mo rec) PrPSc-AF647.

https://doi.org/10.1371/journal.ppat.1014456.g005

Synthetic mouse recombinant PrPSc (Mo recPrPSc) is infectious in animals [46,47]. Following sciatic nerve inoculation of Mo recPrPSc-AF647, similar to what is observed in brain derived fluorescently labeled PrPSc in live animal sciatic nerves, the fluorescent Mo recPrPSc-AF647 particles localized to axons (Fig 4) with similar distribution profiles and velocities (Fig 5, Table 2). As the AF647 labeled (Mo recPrPSc-AF647) is only generated from PrP, this excludes the possibility that contaminants in the purified PrPSc from brain, and not PrPSc, is being measured in the live animal imaging studies. A minimum of three mice were examined and velocities of a minimum of 5000 particles were measured.

The velocities measured for some of the particles suggested they may associate with vesicles [53]. To begin to investigate if PrPSc is utilizing vesicle movement along axons, the sciatic nerve of wild type mice was inoculated with HY TME PrPSc-pHrodo, a pH-sensitive dye, only emitting fluorescent signal in acidic environments. The particles had an emission spectrum that was consistent with the emission profile of pHrodo (Fig 6, panel A). Additionally, we found evidence for PrPSc particles where the net movement of a particle was observed in one direction (Fig 6, panel B, triangle 1 to triangle 6), the particle could be observed moving in the opposite direction (Fig 6, panel B, triangle 1 to triangle 2) and then resume movement in the original direction (Fig 6, panel B, triangle 2 to triangle 3). Finally, we found the velocity profile of the particles ranged from 0.1 to 5µm/s (Fig 6, panel C) like what was measured for HY PrPSc-AF647 (Fig 5). A minimum of three mice were examined and velocities of a minimum of 5000 particles were measured. Overall, in the sciatic nerve of live animals, both brain-derived PrPSc and synthetic murine PrPSc were transported at velocities up to 5µm/s.

thumbnail
Download:
Fig 6. Time course image of HY TME PrPSc-pHrodo labeled PrPSc particles in male PrP+/+ mice.

A) Lambda scan of particles measured is consistent with pHrodo emission spectra. B) Image of a sciatic nerve inoculated with PrPSc-pHrodo from a live animal showing the final position (number 6, yellow triangle) of an individual PrPSc-pHrodo particle. The five previous positions (numbered 1 through 5, blue triangles) of the PrPSc-pHrodo particle at 3 s intervals. C) Violin plot representation of PrPSc particle velocities of HY TME PrPSc-pHrodo labeled PrPSc particles. Scale bar represents 5 μm.

https://doi.org/10.1371/journal.ppat.1014456.g006

Discussion

In whole animal experiments, inoculum PrPSc was detected by PMCA in mouse lumbar spinal cord after unilateral sciatic nerve inoculation at 24 h post-inoculation (Fig 1). Because the PMCA and Western blot conditions were optimized to detect hamster and not mouse PrPSc (S1 Fig), this indicates that the hamster inoculum PrPSc was detected in the lumbar spinal cord. Additionally, PrPSc was not detected in the contralateral sciatic nerve (Fig 1). This is consistent with previous studies where, following sciatic nerve inoculation with HY TME, PrPSc was not detected in the contralateral sciatic nerve until nine weeks post infection [44]. This also suggests that the detection of inoculum PrPSc in the lumbar cord is not due to spillover. Based on these observations, we therefore hypothesize that the detection of PrPSc in the lumbar spinal cord is due to transport along sciatic nerve axons to ventral motor neurons (VMNs), consistent with our previous studies [42–44,54,55]. However, as this is an indirect measurement of prion spread between the site of inoculation and the lumbar spinal cord, we cannot exclude the possibility that inoculum PrPSc uses an alternative pathway to gain access to the lumbar spinal cord. Based on the average distance from the inoculation location in the sciatic nerve to the lumbar spinal cord, we calculated the rate of PrPSc transport to be a minimum of 25 mm/day. This calculated rate of transport is consistent with a recent report of 263K prion transport along the sciatic nerve following footpad inoculation that suggested rates of axonal prion transport reaching 50 mm/day [56]. These rates of PrPSc transport are more than 10-fold faster than what has been reported suggesting that PrPSc may be using previously unrecognized mechanisms of axonal transport [57–60]. To further explore this observation, we directly measured the velocities of fluorescently tagged PrPSc particles in the sciatic nerve of explant cultures and in live animals.

In sciatic nerve explant cultures and in vivo sciatic nerves, we observed velocities of PrPSc-AF647 that ranged from 0.1 to 5 μm/sec. We did not detect fluorescent particles in the nerves of uninfected PrPSc enriched preparations conjugated to AF647 (negative control, S6 Fig). Importantly, the autofluorescence excitation-emission spectra (lambda scans) of negative controls were not consistent with AF647 (S6 Fig). This is in stark contrast to what was observed for AF647 labeled PrPSc where a precise emission spectrum was observed and allowed positive identification of AF647 labeled PrPSc (Figs 2 and 4). In sciatic nerve explant cultures and in vivo intact sciatic nerves inoculated with AF647 labeled PrPSc we reasoned that if PrPSc exclusively used the previously reported slow axonal transport rate of spread, the measured velocities would not exceed 2 µm/sec. However, we observed that approximately 20% of the PrPSc particles measured had a mean velocity exceeding 2 µm/sec that reached up to 5 µm/sec, which is inconsistent with previous studies and suggests that a subset of PrPSc utilizes fast axonal transport (Figs 3 and 5; Tables 1 and 2). However, the relative contribution of PrPSc particles transported greater than 2 µm/sec to prion pathogenesis is unknown. To use an additional method of particle detection and investigate if PrPSc is hijacking vesicle movement along axons, we used PrPSc labeled with pHrodo Deep Red TFP Ester, a pH-sensitive dye, only emitting fluorescent signal in acidic environments. This pH-sensitivity has been utilized in cultured neurons to provide mechanistic details of intracellular trafficking of pathological proteins [61,62]. We identified PrPSc particles traveling at a velocity ranging from 0.1 to 5 µm/s, with the observed particles undergoing bidirectional transport with variable run lengths between each directional change (Fig 6, panel B). These transport dynamics are consistent with vesicle-associated kinesin and dynein motors driving transport in a coordinated effort [63], however, more work is required to determine if PrPSc associates with these vesicles. Additionally, synthetic PrPSc was found to have a similar transport velocity profile as brain derived PrPSc (Fig 5). As synthetic PrPSc lacks glycosylation, this indicates that glycosylation is not required for axonal transport consistent with previous studies showing genetic removal of N-linked PrPC glycosylation sites are not required for prion disease development [64,65]. However, a lower percentage of synthetic PrPSc was observed to be transported at mean velocities of >2 μm/sec compared to brain derived PrPSc (Table 2). This biological significance of this difference is unknown. Overall, in both our in vivo animal studies and the in vitro explant culture systems, multiple converging lines of evidence suggest that a subpopulation of PrPSc may utilize fast axonal transport.

Strain-specific rates of PrPSc transport were not observed. Previous studies determined a strain-specific rate of prion transport following sciatic nerve inoculation with HY having the fastest rate at 4.14 mm/day, DY having the slowest rate at 1.10 mm/day and 139H having an intermediate rate of spread of 1.80 mm/day [42,43]. Based on this previous work, we hypothesized that HY PrPSc particles should have a velocity of nearly 4 times that of DY PrPSc. What was observed, however, is that all three prion strains had nearly indistinguishable transport velocity profiles (Figs 3 and 5, Tables 1 and 2). In the systems used in this study, PrPSc transport and replication are decoupled, while in our previous studies both transport and prion replication can occur [42,43]. Based on these observations, we hypothesize that the previously observed strain-specific differences in prion spread reported in hamsters is due to differences in the rate of prion formation and not due to differences in the axonal transport velocity of PrPSc [17]. Supporting this hypothesis are PMCA studies examining the rate of prion formation where HY PrPSc formation is more efficient compared to DY and 139H [45,66,67]. Alternatively, of the range of PrPSc particles measured, or of particles that were not measured in this study (e.g., PK sensitive PrPSc) it is unknown which ones contribute to the strain specific differences in pathogenesis of disease. Therefore, we cannot exclude the possibility that strain-specific differences in PrPSc transport may contribute to the pathogenesis of disease in a yet undiscovered way.

In the explant and live animal axonal transport experiments, small, but significant differences in particle velocities were observed between various prion strains, host genotype and sex (S1 and S2 Tables). However, the observed differences do not have a consistent pattern across prion strain, host genotype, or sex of the animal. It is unclear if these differences are biologically significant or are due to the large number of particles compiled and analyzed, potentially allowing small variations in mean axonal transport velocity to display significance. Overall, all strains tested are transported at similar velocities, and we hypothesize that prion strains are transported using similar mechanisms.

Transport of PrPSc was independent of PrPC expression. In whole animal studies, PrPSc was detected in the lumbar spinal cord at 24 h p.i. in PrP-/- mice, similar to what was observed in WT mice (Fig 1). Using both the in vitro sciatic nerve explant culture system and the in vivo sciatic nerves from PrP-/- mice, we detected PrPSc particle velocities similar to what was observed in WT animals from all mouse and hamster prion strains tested (Figs 3 and 5; Tables 1 and 2). These findings are in contrast with previous work indicating that PrPC expression is required for axonal prion transport [68]. In these studies, intraocular (i.o) inoculation of prions into PrP-/- mice containing a PrPC overexpressing brain graft failed to result in the establishment of prion infection in the grafted material [68]. The authors showed that intraocular inoculation of PrPC expressing mice with the PrPC expressing graft resulted in prion pathology, suggesting that the graft was synaptically connected with the retino-tectal pathway. Therefore, the failure to establish infection in the PrP-/- mice was not due to a lack of anatomically connected structures but due to the lack of PrPC. The authors suggest that PrPC is required for a “domino” mechanism of prion conversion and spread along axons and/or PrPC is required for crossing of synapses. The studies presented here are inconsistent with the “domino” hypothesis and instead point to the potential requirement of PrPC at the synapse for transsynaptic prion transport.

The experimental paradigm used in this study has several limitations. First, the contribution of PrPSc that is detected by PMCA in the lumbar cord 24 h after sciatic nerve inoculation to subsequent transsynaptic spread and disease development is unknown. As PMCA can detect minute levels of PrPSc, it is possible the amount of PrPSc that is rapidly transported to VMNs cannot establish infection and therefore does not contribute to the pathogenesis of disease [29,69]. Second, sciatic nerve inoculation is an artificial means of introducing prions to the nervous system. While sciatic nerve inoculation can cause disease and the previously measured rate of PrPSc spread from the inoculation site in the sciatic nerve to VMNs is the same as what is measured within the CNS, it is possible that the mechanisms used differ [42,43]. Third, while the purified material is infectious following sciatic nerve inoculation and the labeling does not affect PMCA seeding activity (S4 and S5 Figs), it is possible that in natural prion disease PrPSc may associate with other proteins that affect axonal transport properties that are not accounted for in this experimental paradigm [53]. Additionally, these studies do not capture the contributions of PK sensitive forms of PrPSc or newly formed PrPSc in axonal transport.

Overall, the findings reported here provide evidence for axonal transport of PrPSc that is independent of prion strain or host PrPC expression. These findings provide a clearer view of prion axonal transport and clarify an important variable in understanding strain-specific differences in the tempo of prion pathogenesis. These results suggest the potential commonalities in the axonal transport mechanisms of PrPSc, consistent with previous research showing the common neuroanatomical pathways of prion spread, and strain independent uptake of prions [43,70,71]. Finally, as the similarities between prion and prion-like diseases is becoming increasingly clear, it is possible that prion-like proteins may share a similar axonal transport properties [72–76].

Supporting information

S1 Fig. Sensitivity and specificity of PMCA for detection of HY TME prions.

A) Western blot analysis of proteinase K digested PMCA reactions seeded with ten-fold serial dilutions of HY TME brain homogenate into uninfected (Un) hamster brain homogenate substrate after two serial rounds of PMCA. Each panel represents a technical replicate. B) Western blot analysis of proteinase K digested PMCA reactions ten-fold serial dilutions of HY TME brain homogenate in uninfected mouse brain homogenate substrate after two serial rounds of PMCA. Each panel represents a technical replicate. The Western blots in panels A and B were probed with the monoclonal anti-PrP antibody 3F4, which recognizes hamster, but not murine PrP. Arrows indicate the migration of the 29 kDa molecular weight marker.

https://doi.org/10.1371/journal.ppat.1014456.s001

(TIF)

S2 Fig. Enrichment of PrPSc from prion-infected brain homogenates.

Sypro Ruby gel stain of proteinase K (PK) digested (+) or undigested (-) brain homogenates, or enriched preparations (prep, highlighted by green box) from either A) Rocky Mountain Laboratory (RML) prions, B) hyper (HY), C) drowsy (DY), or D) 139H infected animals. Migration of the 10, 15, 20 and 25 kDa molecular weight markers is indicated on the left of the panel. The PK band is indicated by a red arrow.

https://doi.org/10.1371/journal.ppat.1014456.s002

(TIF)

S3 Fig. Conjugation of purified PrPSc to AF647.

Uninfected brain and brains terminally infected with either hyper (HY), drowsy (DY), 139H or Rocky Mountain Laboratory (RML) prions purified according to the protocol from Wenborn et al., 2015 [48] and conjugated to AF647. Purified and fluorescently conjugated isolates were gel fractionated, and fluorescence was detected by using the Typhoon laser scanning imager.

https://doi.org/10.1371/journal.ppat.1014456.s003

(TIF)

S4 Fig. Conjugation of AF647 to HY PrPSc does not reduce its biological activity.

Western blot analysis of proteinase K digested brain homogenates from first and second serial rounds of PMCA reactions seeded with either uninfected brain homogenate (UN) or serial dilutions of either HY TME-infected brain homogenates or HY PrPSc conjugated to Alexa Fluor 647 (HY AF647).

https://doi.org/10.1371/journal.ppat.1014456.s004

(TIF)

S5 Fig. Hamster prions fail to cause disease or establish infection following sciatic nerve inoculation of mice.

A) Kaplan-Meyer survival curve of mice inoculated in the sciatic nerve with either uninfected (UN) mouse brain homogenate (green circles), enriched PrP preparations (prep) from uninfected mouse brain (blue squares), rocky mountain laboratory (RML) infected mouse brain homogenate (BH)(purple triangles), enriched PrP preparations from RML-infected mouse brain (orange upside down triangles), or brain homogenate from 263K-infected hamster brain (red diamonds). Mice inoculated with either UN mouse BH, UN mouse prep or 263K-infected hamster BH failed to develop clinical signs of prion infection by 600 days post inoculation. B) Western blot analysis of proteinase K digested brain homogenates from mice infected with the inoculums listed in panel A.

https://doi.org/10.1371/journal.ppat.1014456.s005

(TIF)

S6 Fig. Two-photon confocal microscopy analysis of negative controls.

Still images from mouse sciatic nerves inoculated with either A) DPBS, B) Unconjugated AF647 quenched by glycine, or C) Uninfected purified preparations conjugated to AF647. Lambda spectral scans (D) of fluorescence from A-C are inconsistent with the AF647 spectral fingerprint. Scale bar represents 20 μm.

https://doi.org/10.1371/journal.ppat.1014456.s006

(TIF)

S1 File. Uncropped gel and blot images.

https://doi.org/10.1371/journal.ppat.1014456.s009

(PDF)

S1 Table. Comparison of average axonal velocities of PrPSc strains between PrP+/+ and PrP-/- mouse sciatic nerve explant genotypes within mouse sex.

https://doi.org/10.1371/journal.ppat.1014456.s007

(DOCX)

S2 Table. Comparison of average axonal velocities of PrPSc strains between male PrP+/+ and male PrP-/- in live mouse sciatic nerve axons.

https://doi.org/10.1371/journal.ppat.1014456.s008

(DOCX)

Acknowledgments

We thank Dr. Miguel Ángel Pérez-Castro for his valuable contribution in generating the synthetic prion strain utilized throughout this work. We thank the Integrated Biomedical Imaging Facility at Creighton University, Omaha, NE (RRID:SCR_023806). We would like to thank the Creighton University Animal Resource Facility for excellent animal care and Johsette Witt for outstanding technical assistance.

References

  1. 1. Oesch B, Westaway D, Wälchli M, McKinley MP, Kent SB, Aebersold R, et al. A cellular gene encodes scrapie PrP 27-30 protein. Cell. 1985;40(4):735–46. pmid:2859120
  2. 2. Bolton DC, McKinley MP, Prusiner SB. Identification of a protein that purifies with the scrapie prion. Science. 1982;218(4579):1309–11. pmid:6815801
  3. 3. Prusiner SB. Novel proteinaceous infectious particles cause scrapie. Science. 1982;216(4542):136–44. pmid:6801762
  4. 4. Deleault NR, Harris BT, Rees JR, Supattapone S. Formation of native prions from minimal components in vitro. Proc Natl Acad Sci U S A. 2007;104(23):9741–6. pmid:17535913
  5. 5. Wang F, Wang X, Yuan C-G, Ma J. Generating a prion with bacterially expressed recombinant prion protein. Science. 2010;327(5969):1132–5. pmid:20110469
  6. 6. Kraus A, Hoyt F, Schwartz CL, Hansen B, Artikis E, Hughson AG, et al. High-resolution structure and strain comparison of infectious mammalian prions. Mol Cell. 2021;81(21):4540–4551.e6. pmid:34433091
  7. 7. Caughey B, Raymond GJ. The scrapie-associated form of PrP is made from a cell surface precursor that is both protease- and phospholipase-sensitive. J Biol Chem. 1991;266(27):18217–23. pmid:1680859
  8. 8. Manka SW, Zhang W, Wenborn A, Betts J, Joiner S, Saibil HR, et al. 2.7 Å cryo-EM structure of ex vivo RML prion fibrils. Nat Commun. 2022;13(1):4004. pmid:35831275
  9. 9. Sun Y, Jack K, Ercolani T, Sangar D, Hosszu L, Collinge J, et al. Direct Observation of Competing Prion Protein Fibril Populations with Distinct Structures and Kinetics. ACS Nano. 2023;17(7):6575–88. pmid:36802500
  10. 10. Cohen FE, Pan KM, Huang Z, Baldwin M, Fletterick RJ, Prusiner SB. Structural clues to prion replication. Science. 1994;264(5158):530–1. pmid:7909169
  11. 11. Green KM, Castilla J, Seward TS, Napier DL, Jewell JE, Soto C, et al. Accelerated high fidelity prion amplification within and across prion species barriers. PLoS Pathog. 2008;4(8):e1000139. pmid:18769716
  12. 12. Castilla J, Saá P, Hetz C, Soto C. In vitro generation of infectious scrapie prions. Cell. 2005;121(2):195–206. pmid:15851027
  13. 13. Saborio GP, Permanne B, Soto C. Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding. Nature. 2001;411(6839):810–3. pmid:11459061
  14. 14. Glatzel M, Aguzzi A. Peripheral pathogenesis of prion diseases. Microbes Infect. 2000;2(6):613–9. pmid:10884612
  15. 15. Mabbott N. How do PrPSc Prions Spread between Host Species, and within Hosts? Pathogens. 2017;6(4):60.
  16. 16. Heikenwalder M, Julius C, Aguzzi A. Prions and peripheral nerves: a deadly rendezvous. J Neurosci Res. 2007;85(12):2714–25. pmid:17393538
  17. 17. Koshy SM, Kincaid AE, Bartz JC. Transport of Prions in the Peripheral Nervous System: Pathways, Cell Types, and Mechanisms. Viruses. 2022;14(3):630. pmid:35337037
  18. 18. McCulloch L, Brown KL, Bradford BM, Hopkins J, Bailey M, Rajewsky K, et al. Follicular dendritic cell-specific prion protein (PrP) expression alone is sufficient to sustain prion infection in the spleen. PLoS Pathog. 2011;7(12):e1002402. pmid:22144895
  19. 19. Glaysher BR, Mabbott NA. Role of the draining lymph node in scrapie agent transmission from the skin. Immunol Lett. 2007;109(1):64–71. pmid:17292972
  20. 20. Mohan J, Bruce ME, Mabbott NA. Follicular dendritic cell dedifferentiation reduces scrapie susceptibility following inoculation via the skin. Immunology. 2005;114(2):225–34. pmid:15667567
  21. 21. Mabbott NA, Mackay F, Minns F, Bruce ME. Temporary inactivation of follicular dendritic cells delays neuroinvasion of scrapie. Nat Med. 2000;6(7):719–20. pmid:10888894
  22. 22. McBride PA, Schulz-Schaeffer WJ, Donaldson M, Bruce M, Diringer H, Kretzschmar HA, et al. Early spread of scrapie from the gastrointestinal tract to the central nervous system involves autonomic fibers of the splanchnic and vagus nerves. J Virol. 2001;75(19):9320–7. pmid:11533195
  23. 23. Beekes M, McBride PA. Early accumulation of pathological PrP in the enteric nervous system and gut-associated lymphoid tissue of hamsters orally infected with scrapie. Neurosci Lett. 2000;278(3):181–4. pmid:10653023
  24. 24. Beekes M, McBride PA, Baldauf E. Cerebral targeting indicates vagal spread of infection in hamsters fed with scrapie. J Gen Virol. 1998;79 (Pt 3):601–7. pmid:9519840
  25. 25. Prinz M, Heikenwalder M, Junt T, Schwarz P, Glatzel M, Heppner FL, et al. Positioning of follicular dendritic cells within the spleen controls prion neuroinvasion. Nature. 2003;425(6961):957–62. pmid:14562059
  26. 26. Montrasio F, Frigg R, Glatzel M, Klein MA, Mackay F, Aguzzi A, et al. Impaired prion replication in spleens of mice lacking functional follicular dendritic cells. Science. 2000;288(5469):1257–9. pmid:10818004
  27. 27. Buyukmihci N, Goehring-Harmon F, Marsh RF. Neural pathogenesis of experimental scrapie after intraocular inoculation of hamsters. Exp Neurol. 1983;81(2):396–406. pmid:6683661
  28. 28. Fraser H, Dickinson AG. Targeting of scrapie lesions and spread of agent via the retino-tectal projection. Brain Res. 1985;346(1):32–41. pmid:4052769
  29. 29. Kimberlin RH, Walker CA. Pathogenesis of scrapie (strain 263K) in hamsters infected intracerebrally, intraperitoneally or intraocularly. J Gen Virol. 1986;67(Pt 2):255–63. pmid:3080549
  30. 30. Scott JR, Davies D, Fraser H. Scrapie in the central nervous system: neuroanatomical spread of infection and Sinc control of pathogenesis. J Gen Virol. 1992;73(Pt 7):1637–44. pmid:1629695
  31. 31. Scott JR, Fraser H. Transport and targeting of scrapie infectivity and pathology in the optic nerve projections following intraocular infection. Prog Clin Biol Res. 1989;317:645–52. pmid:2513583
  32. 32. Scott JV, Stowring L, Haase AT, Narayan O, Vigne R. Antigenic variation in visna virus. Cell. 1979;18(2):321–7. pmid:227603
  33. 33. Kimberlin RH, Hall SM, Walker CA. Pathogenesis of mouse scrapie. Evidence for direct neural spread of infection to the CNS after injection of sciatic nerve. J Neurol Sci. 1983;61(3):315–25. pmid:6418861
  34. 34. Kimberlin RH, Walker CA. Pathogenesis of mouse scrapie: patterns of agent replication in different parts of the CNS following intraperitoneal infection. J R Soc Med. 1982;75(8):618–24. pmid:6809940
  35. 35. Bassant MH, Baron H, Gumpel M, Cathala F, Court L. Spread of scrapie agent to the central nervous system: study of a rat model. Brain Res. 1986;383(1–2):397–401. pmid:3094830
  36. 36. Glatzel M, Aguzzi A. PrP(C) expression in the peripheral nervous system is a determinant of prion neuroinvasion. J Gen Virol. 2000;81(Pt 11):2813–21. pmid:11038396
  37. 37. Beekes M, Baldauf E, Diringer H. Sequential appearance and accumulation of pathognomonic markers in the central nervous system of hamsters orally infected with scrapie. J Gen Virol. 1996;77 (Pt 8):1925–34. pmid:8760444
  38. 38. Ye X, Carp RI. Histopathological changes in the pituitary glands of female hamsters infected with the 139H strain of scrapie. J Comp Pathol. 1996;114(3):291–304. pmid:8762587
  39. 39. Carp RI, Kim YS, Callahan SM. Pancreatic lesions and hypoglycemia-hyperinsulinemia in scrapie-injected hamsters. J Infect Dis. 1990;161(3):462–6. pmid:2313125
  40. 40. Bessen RA, Marsh RF. Distinct PrP properties suggest the molecular basis of strain variation in transmissible mink encephalopathy. J Virol. 1994;68(12):7859–68. pmid:7966576
  41. 41. Shikiya RA, Langenfeld KA, Eckland TE, Trinh J, Holec SAM, Mathiason CK, et al. PrPSc formation and clearance as determinants of prion tropism. PLoS Pathog. 2017;13(3):e1006298. pmid:28355274
  42. 42. Langenfeld KA, Shikiya RA, Kincaid AE, Bartz JC. Incongruity between Prion Conversion and Incubation Period following Coinfection. J Virol. 2016;90(12):5715–23. pmid:27053546
  43. 43. Ayers JI, Kincaid AE, Bartz JC. Prion strain targeting independent of strain-specific neuronal tropism. J Virol. 2009;83(1):81–7. pmid:18971281
  44. 44. Bartz JC, Kincaid AE, Bessen RA. Retrograde transport of transmissible mink encephalopathy within descending motor tracts. J Virol. 2002;76(11):5759–68. pmid:11992004
  45. 45. Steadman BS, Bian J, Shikiya RA, Bartz JC. Minor prion substrains overcome transmission barriers. mBio. 2024;15(11):e0272124. pmid:39440977
  46. 46. Pérez-Castro MÁ, Eraña H, Vidal E, Charco JM, Lorenzo NL, Gonçalves-Anjo N, et al. Cofactors facilitate bona fide prion misfolding in vitro but are not necessary for the infectivity of recombinant murine prions. PLoS Pathog. 2025;21(1):e1012890. pmid:39841704
  47. 47. Eraña H, Sampedro-Torres-Quevedo C, Charco JM, Díaz-Domínguez CM, Peccati F, San-Juan-Ansoleaga M, et al. A Protein Misfolding Shaking Amplification-based method for the spontaneous generation of hundreds of bona fide prions. Nat Commun. 2024;15(1):2112. pmid:38459071
  48. 48. Wenborn A, Terry C, Gros N, Joiner S, D’Castro L, Panico S, et al. A novel and rapid method for obtaining high titre intact prion strains from mammalian brain. Sci Rep. 2015;5:10062. pmid:25950908
  49. 49. Walker CL, Uchida A, Li Y, Trivedi N, Fenn JD, Monsma PC, et al. Local Acceleration of Neurofilament Transport at Nodes of Ranvier. J Neurosci. 2019;39(4):663–77. pmid:30541916
  50. 50. Breuer AC, Lynn MP, Atkinson MB, Chou SM, Wilbourn AJ, Marks KE, et al. Fast axonal transport in amyotrophic lateral sclerosis: an intra-axonal organelle traffic analysis. Neurology. 1987;37(5):738–48. pmid:2437494
  51. 51. Angers RC, Seward TS, Napier D, Green M, Hoover E, Spraker T, et al. Chronic wasting disease prions in elk antler velvet. Emerg Infect Dis. 2009;15(5):696–703. pmid:19402954
  52. 52. Ershov D, Phan M-S, Pylvänäinen JW, Rigaud SU, Le Blanc L, Charles-Orszag A, et al. TrackMate 7: integrating state-of-the-art segmentation algorithms into tracking pipelines. Nat Methods. 2022;19(7):829–32. pmid:35654950
  53. 53. Shearin H, Bessen RA. Axonal and transynaptic spread of prions. J Virol. 2014;88(15):8640–55. pmid:24850738
  54. 54. Shikiya RA, Ayers JI, Schutt CR, Kincaid AE, Bartz JC. Coinfecting prion strains compete for a limiting cellular resource. J Virol. 2010;84(11):5706–14. pmid:20237082
  55. 55. Bartz JC, Kramer ML, Sheehan MH, Hutter JAL, Ayers JI, Bessen RA, et al. Prion interference is due to a reduction in strain-specific PrPSc levels. J Virol. 2007;81(2):689–97. pmid:17079313
  56. 56. Cardone F, Porreca F, Sbriccoli M, Poleggi A, Ladogana A, Lu M, et al. Sciatic Integrity Is Necessary for Fast and Efficient Scrapie Infection After Footpad Injection. Int J Mol Sci. 2025;26(15):7273. pmid:40806406
  57. 57. Goldstein LS, Yang Z. Microtubule-based transport systems in neurons: the roles of kinesins and dyneins. Annu Rev Neurosci. 2000;23:39–71. pmid:10845058
  58. 58. Black MM, Lasek RJ. Axonal transport of actin: slow component b is the principal source of actin for the axon. Brain Res. 1979;171(3):401–13. pmid:89886
  59. 59. Hoffman PN, Lasek RJ. The slow component of axonal transport. Identification of major structural polypeptides of the axon and their generality among mammalian neurons. J Cell Biol. 1975;66(2):351–66. pmid:49355
  60. 60. Ochs S, Johnson J. Fast and slow phases of axoplasmic flow in ventral root nerve fibres. J Neurochem. 1969;16(3):845–53. pmid:4186225
  61. 61. Karpowicz RJ Jr, Haney CM, Mihaila TS, Sandler RM, Petersson EJ, Lee VM-Y. Selective imaging of internalized proteopathic α-synuclein seeds in primary neurons reveals mechanistic insight into transmission of synucleinopathies. J Biol Chem. 2017;292(32):13482–97. pmid:28611062
  62. 62. Evans LD, Wassmer T, Fraser G, Smith J, Perkinton M, Billinton A, et al. Extracellular Monomeric and Aggregated Tau Efficiently Enter Human Neurons through Overlapping but Distinct Pathways. Cell Rep. 2018;22(13):3612–24. pmid:29590627
  63. 63. Encalada SE, Szpankowski L, Xia C, Goldstein LSB. Stable kinesin and dynein assemblies drive the axonal transport of mammalian prion protein vesicles. Cell. 2011;144(4):551–65. pmid:21335237
  64. 64. Tuzi NL, Cancellotti E, Baybutt H, Blackford L, Bradford B, Plinston C, et al. Host PrP glycosylation: a major factor determining the outcome of prion infection. PLoS Biol. 2008;6(4):e100. pmid:18416605
  65. 65. Sevillano AM, Aguilar-Calvo P, Kurt TD, Lawrence JA, Soldau K, Nam TH, et al. Prion protein glycans reduce intracerebral fibril formation and spongiosis in prion disease. J Clin Invest. 2020;130(3):1350–62. pmid:31985492
  66. 66. Eckland TE, Shikiya RA, Bartz JC. Independent amplification of co-infected long incubation period low conversion efficiency prion strains. PLoS Pathog. 2018;14(10):e1007323. pmid:30335854
  67. 67. Ayers JI, Schutt CR, Shikiya RA, Aguzzi A, Kincaid AE, Bartz JC. The strain-encoded relationship between PrP replication, stability and processing in neurons is predictive of the incubation period of disease. PLoS Pathog. 2011;7(3):e1001317. pmid:21437239
  68. 68. Brandner S, Raeber A, Sailer A, Blättler T, Fischer M, Weissmann C, et al. Normal host prion protein (PrPC) is required for scrapie spread within the central nervous system. Proc Natl Acad Sci U S A. 1996;93(23):13148–51. pmid:8917559
  69. 69. Chen B, Soto C, Morales R. Peripherally administrated prions reach the brain at sub-infectious quantities in experimental hamsters. FEBS Lett. 2014;588(5):795–800. pmid:24492001
  70. 70. Kincaid AE, Ayers JI, Bartz JC. Specificity, Size, and Frequency of Spaces That Characterize the Mechanism of Bulk Transepithelial Transport of Prions in the Nasal Cavities of Hamsters and Mice. J Virol. 2016;90(18):8293–301. pmid:27384659
  71. 71. Greil CS, Vorberg IM, Ward AE, Meade-White KD, Harris DA, Priola SA. Acute cellular uptake of abnormal prion protein is cell type and scrapie-strain independent. Virology. 2008;379(2):284–93. pmid:18692214
  72. 72. Andersen KB, Krishnamurthy A, Just MK, Van Den Berge N, Skjærbæk C, Horsager J, et al. Sympathetic and parasympathetic subtypes of body-first Lewy body disease observed in postmortem tissue from prediagnostic individuals. Nat Neurosci. 2025;28(5):925–36. pmid:40082617
  73. 73. Borghammer P, Van Den Berge N. Brain-First versus Gut-First Parkinson’s Disease: A Hypothesis. J Parkinsons Dis. 2019;9(s2):S281–95. pmid:31498132
  74. 74. Ayers JI, Brooks MM, Rutherford NJ, Howard JK, Sorrentino ZA, Riffe CJ, et al. Robust Central Nervous System Pathology in Transgenic Mice following Peripheral Injection of α-Synuclein Fibrils. J Virol. 2017;91(2):e02095–16. pmid:27852849
  75. 75. Ayers JI, Fromholt SE, O N VM, Diamond JH. Prion-like propagation of mutant SOD1 misfolding and motor neuron disease spread along neuroanatomical pathways. Acta. 2016. pmid:5983784982764462838
  76. 76. Braak H, Del Tredici K, Rüb U, de Vos RAI, Jansen Steur ENH, Braak E. Staging of brain pathology related to sporadic Parkinson’s disease. Neurobiol Aging. 2003;24(2):197–211. pmid:12498954