Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Summary

Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

Abstract

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitro prion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrP^C) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrP^C that converts to a proteinase K (PK)-resistant state is modulated by the original host’s species barrier to the TSE agent. In contrast, PrP^C in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host’s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

Introduction

Transmissible spongiform encephalopathies (TSEs, prion diseases) are a group of fatal neurodegenerative diseases with extended incubation periods that affect a variety of animals and humans. The putative etiological agent of TSEs is comprised of a misfolded isomer of the host prion protein (PrP) that is capable of self-propagation by template-driven conversion of the normal cellular form of PrP (PrP^C) into an infectious, disease-associated form (PrP^TSE) that accumulates in central nervous system tissues of the infected host¹. Infectious mammalian prions generally transmit from host-to-host in a species-specific manner, which has given rise to the concept of the “TSE species barrier” limiting interspecies transmission events². The biological determinants of the prion species barrier are not well understood. Amino acid sequence similarity between the infectious PrP^TSE and the host PrP^C can strongly influence whether conversion takes place^3-5, but remains insufficient to explain all prion transmission events observed in vivo^6,7.

Thus, characterization of TSE species barriers has largely relied upon animal challenge studies: exposing naïve animals from a given species to prions from another and measuring the resulting incubation time to disease onset and attack rate as indicators of transmission efficiency. Mice expressing PrP^C from heterologous species are also used for this type of study⁸. The costs associated with transgenic mouse production and protracted prion bioassays, as well as ethical considerations of animal use, are obstacles to experimental investigation of TSE species barriers. Assessment of the human species barrier to TSEs relies on mice engineered to express human PrP^C. These mice require long incubation periods to succumb to human TSEs or modifications to the human PrP^C molecule for rapid disease onset⁹. Incubation periods for non-human TSEs in these mice may extend beyond the normal mouse lifespan making interpretation of negative results challenging. Non-human primates have also been used as proxies for studying human species barriers, but these studies are fraught with the same challenges as other types of animal experimentation and non-human primates may not precisely recapitulate disease as it proceeds in the human host.

Animal bioassays remain the “gold standard” method for measuring the susceptibility of a species to a TSE, but the obstacles, costs and ethics of these live animal studies have compelled investigation into alternatives. A number of in vitro assays, based on assessing the conversion of host PrP^C to a proteinase K (PK)-resistant state (PrP^res) when seeded by PrP^TSE, have been developed and used to investigate TSE species barriers^10-12. Examples of in vitro assays include cell-free conversion assays, protein misfolding cyclic amplification (PMCA), and the conversion efficiency ratio (CER) assay^10-14. While none of these assays take into account peripheral factors involved in species barriers after natural infection, all can be useful to identify potentially susceptible hosts for TSEs.

Here we present the protocol for the CER assay, in which two denatured PrP^C substrates derived from normal brain homogenates are used in bench top prion conversion reactions (Figure 1)¹³. PrP^C in the substrate denatured at pH 7.4 can only be converted to PrP^res by PrP^TSE seeded into the reaction in the absence of a species barrier¹⁴. In contrast, denaturation of PrP^C in the other substrate at pH 3.5 allows it to be converted to PrP^res following incubation with PrP^TSE from any species and serves as a control for conversion. The ratio of conversion of PrP^C to PrP^res in pH 7.4 substrate relative to that of the pH 3.5 substrate provides a measure of the species barrier. We have found that the CER assay predicts known species barriers of laboratory mice to various TSEs and have used the assay in efforts to predict the species barrier of numerous mammalian species, including bighorn sheep, to chronic wasting disease (CWD) and other TSEs^14,15. Investigators interested in a tool allowing rapid screening of TSE species barriers or assessment of PrP^C-to-PrP^res conversion will find this methodology useful.

Protocol

Animal work conducted at the USGS National Wildlife Health Center was performed in accordance with the NIH Office of Laboratory Animal Welfare guidelines and under institutional animal care and use committee protocol #EP080716. Tissues from hunter-harvested animals were gifts from the Wisconsin or Michigan Departments of Natural Resources.

1. Solution Preparation

NOTE: The solutions listed below are required for preparation of the CER assay substrate in Section 2 below. Prepare each of these solutions from higher concentration stock solutions; recommended concentrations for stock solutions will be given in parentheses after each respective chemical name listed. Prepare all solutions fresh upon substrate preparation and store at 4 °C until used.

1. For lysis buffer, prepare a solution of the following in 10 mM Tris, pH 7.5 (1 M Tris, pH 7.5 stock solution recommended): 100 mM sodium chloride (NaCl, 1 M stock solution), 10 mM ethylenediaminetetraacetic acid (EDTA, 500 mM EDTA, pH 8.0 stock solution), 0.5% NP-40 (10% stock solution), 0.5% deoxycholate (DOC, 10% stock solution).
2. For conversion buffer, prepare a solution of 0.05% sodium dodecyl sulfate (SDS, 10% stock solution) and 0.5% Triton X-100 (10% stock solution) in 1 phosphate buffered saline (PBS, 10, pH 7.4 stock solution).
3. For chaotropic solutions, prepare two 3 M solutions of guanidine hydrochloride (GdnHCl, 8 M stock) in 1x PBS (10x stock solution). Adjust the pH of one of the solutions to 3.5 ± 0.05 using concentrated hydrochloric acid (HCl). Check that the pH of the second solution remains at pH 7.4 ± 0.05 and adjust using concentrated HCl or sodium hydroxide (NaOH) if necessary.

2. Prepare CER Assay Substrate Pairs

1. Obtain healthy, non-TSE-infected brain tissue from species of interest and prepare a 10% weight-per-volume (w/v) homogenate in lysis buffer using any of the following methods: dounce, bead-mill, or mortar and pestle homogenization. Further refine resulting homogenates by subjecting them to syringe-and-needle homogenization, using needles of increasing gauge until substrate can be aspirated and expelled with ease through a 27 G syringe needle.
   1. For substrates prepared from rodents and other small mammals, homogenize whole brain(s); for substrates prepared from larger mammals, use obex (brainstem) tissue to prepare homogenates. When selecting the quantity of brain homogenate, prepare no more than 50% of the capacity of the centrifuge used for protein precipitation in step 2.6.
   2. If the quality of acquired brain tissue is questionable, assess PrP^C levels by SDS-PAGE¹⁶ and immunoblot¹⁷ prior to substrate preparation.
2. Divide brain homogenate into two equal volumes in separate labelled plastic conical tubes. Add GdnHCl (3 M solutions in 1 PBS) at either pH 3.5 or 7.4 to each tube in a 1:1 volume ratio to brain homogenate.
3. Check the pH values of the two solutions. Adjust the pH of the pH 3.5 substrate to pH ± 0.05 using concentrated HCl. Ensure the pH of the other substrate remains at pH 7.4 ± 0.05 and adjust pH if necessary with HCl or NaOH as needed. Avoid overshooting pH values by judicious addition of acid or base.
4. Rotate solutions on an end-over-end mixer at room temperature (RT) for 5 hr.
5. Precipitate protein by adding four volumes of methanol to the substrate solutions in each conical tube, vortex to mix well, and incubate at -20 °C for 16-18 hr.
6. Sediment samples by centrifugation at 13,000 × g for 30 min at 4 °C. Carefully decant and discard the supernatants and allow methanol to evaporate from the pellets. Use cotton-tipped applicators to assist in absorbing methanol clung to the inside of the tube. Do not allow pellets to over-dry and once methanol is no longer visible, proceed to the next step.
7. Resuspend protein pellets in conversion buffer. Use a quantity of conversion buffer equal to the amount of brain homogenate starting material dispensed into each tube in step 2.2.
   NOTE: It is critical that the conversion buffer used to resuspend protein pellets from both pH 3.5 and 7.4-treated substrate preparations is the solution defined in step 1.2 above (which contains low levels of detergents and a physiological pH).
8. Briefly sonicate substrate solutions in a cuphorn sonicator (10 sec at ~30% maximal power), aliquot into 0.5 - 1.0 ml volumes in labeled 1.5 ml microcentrifuge tubes. Perform a quality control immunoblot (step 2.9) on an aliquot of each substrate. Store other aliquots at -80 °C until ready to perform CER assay (Section 4).
9. Perform quality control on CER substrate pairs prior to use (Figure 2). These steps ensure that CER substrates will provide optimal results in conversion studies.
   1. Ensure comparable amounts of PrP^C in the two substrates. Compare PrP levels in 10-25 µl of each substrate by SDS-PAGE¹⁶ and immunoblotting¹⁷. If protein levels in the pH 7.4 and 3.5 substrates are within ~10% of each other, the substrate pairs are appropriate for use in CER studies.
      NOTE: The PrP immunoblots should not show evidence of extensive PrP^C degradation. The PrP immunoreactivity should be mainly >20 kDa. Bands less than this molecular mass can be degradation products. Banding patterns should be roughly equivalent between substrate pairs.
   2. As the PrP^C in both substrates should be PK sensitive, treat 10-25 µl of each substrate with PK at a final concentration of 100 μg/ml and digest samples at 1,000 rpm at 37 °C for 1 hr in the thermo-shaker. Assess any remaining PrP signal by SDS-PAGE¹⁶ and immunoblotting¹⁷. Substrates with PrP^res are not appropriate for use in CER studies.

3. Prepare TSE Agent Seeds

1. Obtain TSE-infected brain tissue from species of interest and prepare a 10% (w/v) homogenate in 1x PBS pH 7.4 using the methods listed in step 2.1 above.
   NOTE: Seeds could also be produced from other TSE-infected tissues outside the central nervous system, however, efficiency of conversion of brain derived substrates by seeds derived from TSE-infected peripheral tissues has not yet been experimentally assessed in the published literature.
2. Aliquot resulting homogenate(s) into 100 - 300 µl volumes in 0.5 ml microcentrifuge tubes and store at -80 °C until ready to perform the CER assay.

4. CER Assay

NOTE: The CER assay involves addition of a relatively small amount of PrP^TSE (provided in the “seed”) from one species into a relative excess of PrP^C (provided in the uninfected brain “substrate”) from another species that has been partially denatured at either pH 3.5 or 7.4. Following an extended shaking incubation period, template-driven conversion of PrP^C by PrP^TSE in each reaction is assessed by the densitometric signal of PrP^res remaining after PK digestion and immunoblot detection. Comparing PrP^res levels in the substrates previously denatured at pH 3.5 vs. 7.4 provides a measure of the species barrier. An experimental overview of this assay procedure is provided in Figure 1.

1. Slowly thaw uninfected CER substrate pairs (assay requires equal volumes of substrates previously denatured at both pH 3.5 and 7.4) and TSE-infected seed solutions by placing them on top of a bed of ice (approximately 1 hr thaw time).
2. Once fully thawed, aspirate and then expel each substrate solution through a 27 G syringe needle several times to re-homogenize it. Briefly sonicate each TSE-infected seed solution for 10 sec at ~30% maximal power. Keep all solutions on ice while preparing conversion reactions.
3. Label 5 individual low binding, thin-walled PCR tubes per TSE agent being tested.
4. Prepare conversion reactions in these tubes by adding 5 μl of 10% TSE-infected seed solution to 95 μl of (a) CER substrate prepared at pH 7.4 and (b) CER substrate prepared at pH 3.5.
   1. For each experimental sample, prepare parallel reactions in CER substrate pairs previously denatured at both pH 3.5 and 7.4.
   2. Optimize ratios of TSE agent seed to uninfected brain substrate through empirical determination. Common seed:substrate ratios employed maintain a total reaction volume of 100 μl and include 1:99 μl, 2:98 μl and 5:95 μl. For most applications, the 5:95 μl seed:substrate volume ratio is appropriate and is what is presented in this protocol.
5. Prepare three control samples per TSE agent being tested as follows: (a) add 5 µl of 10% TSE-infected seed solution to 95 µl conversion buffer as a control for input PrP^res; add 5 µl conversion buffer to 95 µl substrate prepared at (b) pH 3.5 and (c) pH 7.4 as controls for non-specific, non-templated conversion.
6. Briefly vortex each sample in its closed PCR tube to mix seed and substrate well. Follow with a momentary pulse in a low-speed bench top mini-centrifuge to remove any volume of the reaction mixture trapped in the PCR tube lid.
7. Load samples in individually-labeled PCR tubes into a bench top thermo-shaker equipped to accept PCR tubes. Shake samples at 1,000 rpm at 37 °C for 24 hr.
8. Following shaking, add sarkosyl to a final concentration of 2% (w/v) and PK to a final concentration of 100 μg/ml. Digest samples at 1,000 rpm at 37 °C for 1 hr in the thermo-shaker.
   NOTE: Addition of sarkosyl to samples post-conversion aids in PK digestion of PrP^C. False positive signals in unseeded samples (Step 4.5) are virtually eliminated by addition of sarkosyl.
9. Add SDS-PAGE sample buffer, heat samples to 95°C for 5 minutes and resolve remaining PrP^res in each sample by SDS-PAGE¹⁶ followed by immunoblotting¹⁷.
   NOTE: After addition of sample buffer and heating, samples may be immediately processed or samples can be stored at -20 °C or -80 °C prior to immunoblotting. All data presented here were generated using the Novex NuPAGE gel and transfer systems. Individual samples from the CER assay were treated with sample buffer and reducing agent according to manufacturer’s instructions. Samples were subsequently heated at 95 °C for 5 min and 30 µl of each sample was loaded into a 12% bis-tris precast gels.
10. Calculate the conversion efficiency ratio (CER) as a measure of species barrier. Measure levels of PrP^res by densitometry¹⁷ and divide the total density of the pH 7.4 sample by that of the pH 3.5 sample. Multiply by 100 to produce a percentage.
11. Compile data from independent experimental replicates to generate a mean CER ± standard deviation for a species with a given TSE agent.

Results

Successful use of the CER assay is largely dependent upon the quality of the substrate pairs used in conversion reactions. For this reason, following procedures to prepare CER substrate pairs, a quality control immunoblot should be performed (Figure 2). For both substrates, assay 10-25 μl by immunoblotting. PrP^C should be easily detectable in each substrate and PrP^C levels must be approximately the same between the two. Immunoreactivity will be mainly visible above 20 kDa, but small...

Discussion

For successful completion of this protocol, attention should be paid to PrP^C levels in uninfected brain tissue used for substrate preparation (step 2.1.2) and the seed to substrate ratio for conversion reactions (step 4.4.2). In our experience, brains can be extracted for use as CER substrate after a substantial period post-mortem, as long as PrP^C is present by immunoblotting (Figure 4). In fact, some autolysis was observed in the brains of the bobcats used for CER studies. Neverthe...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
12% Bis-Tris SDS-PAGE gels	Life Technologies	NP0342
0.5 mm Zirconium oxide beads	Next Advance	ZROB05	Other varieties of beads are also effective
Antibodies	various suppliers		Select appropriate primary and secondary antibodies for immunoblot detection of PrPres from species of interest
Bead homogenizer	Next Advance	BBY24M
Centrifuge	Beckman Coulter	369434	High speed with temperature control
Conical tubes	any brand
Cotton-tipped applicator	Uline	S-18991
Cuphorn sonicator	Heat Systems-Ultrasonics	W-380	Heat Systems-Ultrasonics, Inc. is now Qsonica, LLC
Densitometry software program	UVP	Vision Works LS Image Acquisition and Analysis software	Other programs, such as NIH ImageJ, will also work
Dounce homogenizer	Kimble Chase	885300
End-over-end mixer	Labnet International	H5600
Ethylenediaminetetra acetic acid	Boston Bioproducts	P-770	Hazardous chemical: eye irritation
Guanidine hydrochloride, 8 M	Thermo Fisher Scientific	24115	Hazardous chemical: acute toxicity, skin irritation, eye irritation
Heating block	Fisher Scientific	11-718
Hydrochloric acid	Sigma-Aldrich	435570	Hazardous chemical: strong acid
Lithium dodecyl sulfate sample buffer, 4x	Life Technologies	NP0008	Hazardous chemical: skin & respiratory irritation, serious eye damage, flammable solid
Methanol	Fisher Scientific	A454-4	Hazardous chemical: acute toxicity, flammable liquid
Microcentrifuge tubes	any brand
Mini-centrifuge	Labnet International	C1301
N-lauroyl-sarcosine (sarkosyl)	Sigma-Aldrich	L-5125	Hazardous chemical: acute toxicity, skin irritation, eye damage
Nonidet P-40	Amresco	M158	Hazardous chemical: skin irritation, eye damage
SDS-PAGE gel system	Life Technologies	NuPAGE electrophoresis system	Other SDS-PAGE systems will also work
PCR tubes (low-binding)	Axygen	PCR-02-L-C
Pestle homogenizer	Fisher Scientific	03-392-106
pH meter	Sentron	SI600
Polyvinyldifluoride membrane	Millipore	IPVH00010
Proteinase K	Promega	V3021	Hazardous chemical: skin & eye irritation, respiratory sensitisation, organ toxicity
Reducing agent for SDS-PAGE samples, 10x	Life Technologies	NP0009
Sodium chloride	Fisher Scientific	7647-14-5
Sodium deoxycholate	Sigma-Aldrich	D6750	Hazardous chemical: acute toxicity
Sodium dodecyl sulfate	Thermo Fisher Scientific	28364	Hazardous chemical: acute toxicity, skin irritation, eye damage, flammable solid
Sodium hydroxide	Sigma-Aldrich	S5881	Hazardous chemical; strong base
Syringe	BD Biosciences	various	Use syringe size appropriate to volumes of substrate to be homogenized
Syringe needles	BD Biosciences	various
Thermoshaker (PCR tube shaker)	Hangzhou All Sheng Instruments	MS-100
Tris base	Bio Basic	77-86-1	Hazardous chemical: skin, eye, respiratory irritation
Triton X-100	Integra Chemical Company	T756.30.30	Hazardous chemical: acute toxicity, eye irritation
Vortexer	Fisher Scientific	12-812