Anti-prion drugs do not improve survival in novel knock-in models of inherited prion disease

Daniel J. Walsh; Judy R. Rees; Surabhi Mehra; Matthew E. C. Bourkas; Lech Kaczmarczyk; Erica Stuart; Walker S. Jackson; Joel C. Watts; Surachai Supattapone

doi:10.1371/journal.ppat.1012087

Abstract

Prion diseases uniquely manifest in three distinct forms: inherited, sporadic, and infectious. Wild-type prions are responsible for the sporadic and infectious versions, while mutant prions cause inherited variants like fatal familial insomnia (FFI) and familial Creutzfeldt-Jakob disease (fCJD). Although some drugs can prolong prion incubation times up to four-fold in rodent models of infectious prion diseases, no effective treatments for FFI and fCJD have been found. In this study, we evaluated the efficacy of various anti-prion drugs on newly-developed knock-in mouse models for FFI and fCJD. These models express bank vole prion protein (PrP) with the pathogenic D178N and E200K mutations. We applied various drug regimens known to be highly effective against wild-type prions in vivo as well as a brain-penetrant compound that inhibits mutant PrP^Sc propagation in vitro. None of the regimens tested (Anle138b, IND24, Anle138b + IND24, cellulose ether, and PSCMA) significantly extended disease-free survival or prevented mutant PrP^Sc accumulation in either knock-in mouse model, despite their ability to induce strain adaptation of mutant prions. Our results show that anti-prion drugs originally developed to treat infectious prion diseases do not necessarily work for inherited prion diseases, and that the recombinant sPMCA is not a reliable platform for identifying compounds that target mutant prions. This work underscores the need to develop therapies and validate screening assays specifically for mutant prions, as well as anti-prion strategies that are not strain-dependent.

Author summary

We treated two mouse models of inherited prion disease with a variety of drug treatments, including several which have been previously shown to be highly effective against infectious prion diseases and another that biochemically inhibits the formation of mutant prion proteins in a test tube assay. Surprisingly none of the treatments improved lifespans in the either mouse model even though several treatments changed the distribution pattern of prion pathology in the brains of treated mice. Our results show that alternative strategies are needed to develop treatments for inherited prion diseases.

Citation: Walsh DJ, Rees JR, Mehra S, Bourkas MEC, Kaczmarczyk L, Stuart E, et al. (2024) Anti-prion drugs do not improve survival in novel knock-in models of inherited prion disease. PLoS Pathog 20(4): e1012087. https://doi.org/10.1371/journal.ppat.1012087

Editor: Suzette A. Priola, National Institute of Allergy and Infectious Diseases Division of Intramural Research, UNITED STATES

Received: October 25, 2023; Accepted: March 1, 2024; Published: April 1, 2024

Copyright: © 2024 Walsh 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 contained within the paper, Supporting Information files, and the Mendeley Data repository at DOI: 10.17632/225jyctt6f.1.

Funding: This study was funded by the National Institute for Neurological Diseases and Stroke https://www.ninds.nih.gov/ (R37NS125431, R01NS117276, and R01NS118796 to SS) and the National Institutes of Health https://www.nih.gov/ (P20-GM113132 to Dean Madden, the PI of an NIH COBRE award that funds a core facility with equipment used that was used for this project and NIH requires acknowledgment of the COBRE award.). The funders played no role in the 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 fatal neurodegenerative diseases that uniquely occur in inherited, sporadic, and infectious forms [1]. All forms of prion diseases are caused by autocatalytic misfolding of the prion protein (PrP), a host-encoded glycoprotein. Inherited prion diseases such as familial Creutzfeldt-Jakob disease (fCJD), fatal familial insomnia (FFI), and Gerstmann-Sträussler-Scheinker disease (GSS) are all caused by pathogenic PrP mutations [2–4], whereas sporadic and infectious forms of prion disease are caused by the misfolding of normal wild-type PrP (PrP^C) into an infectious conformer (PrP^Sc) [5,6]. Interestingly, mutant prions from patients with inherited prion diseases can also be infectious; both FFI and fCJD prions have been experimentally transmitted to normal hosts [7,8]. (Note: the term “mutant” in this manuscript refers to PrP sequences containing point mutations associated with inherited forms of human prion disease).

Several easily administered, non-toxic drug treatments have been shown to prolong lifespan in mice inoculated with scrapie, an infectious form of prion disease. For instance, IND24, a 2-aminothiazole compound, and Anle138b, a 3,5-diphenyl-pyrazole (DPP) derivative, both extend scrapie incubation times >2-fold when administered orally immediately after prion inoculation [9,10,11]; and prophylactic subcutaneous administration of polymeric cellulose ether extends scrapie incubation times 4-fold [12]. Despite these advances, there are inherent difficulties in treating infectious and sporadic forms of prion diseases. First, wild type PrP^Sc molecules can adapt into drug-resistant conformations during treatment, limiting drug efficacy [10]. Remarkably, even combination and alternating chemotherapy regimens cannot prevent the emergence of drug-resistant PrP^Sc molecules [11,13]. Second, patients with sporadic and infectious forms of prion disease typically present with neurological symptoms at late clinical stages when the majority of PrP^Sc accumulation and irreversible neurodegeneration has already occurred.

On the other hand, inherited prion diseases represent a more attractive target for therapeutic intervention. First, patients with inherited prion disease are usually diagnosed by genetic testing many years before the onset of clinical symptoms, and therefore treatment for inherited prion disease can be initiated much earlier than for sporadic or infectious forms of prion diseases. Although experiments using genetically engineered CRE-Lox mice have shown that, in principle, the process of neuronal dysfunction during the symptomatic phase of prion disease is reversible if PrP^Sc production can be completely halted [14], other studies indicate that anti-prion drug therapy is typically more effective if administered before the onset of clinical symptoms [15]. For instance, Giles et al. reported that prophylactic administration of IND24 to prion-infected animals extended lifespan ~4-fold, whereas administration of IND24 1 day after inoculation extended lifespan ~1.7-fold [16]. Second, it is possible that mutant prions are less malleable than wild-type prions, based on the following observations: (1) each pathogenic PrP mutation appears to cause a characteristic strain phenotype and mutant PrP^Sc conformation in vivo [17–21], as discussed above (and notably even E200G produces a different phenotype than E200K [22]); (2) mutations and polymorphisms have been shown to distort the folding landscape of PrP molecules, thermodynamically favoring conversion into a sequence-specific misfolded conformation [23]; and (3) unlike wild-type prions, mutant prions can be propagated in vitro without cofactor molecules [24], and therefore may not be able to harness cofactor diversity to create alternative conformers. If mutant prions are truly not malleable, it is possible that inherited prion diseases could be indefinitely suppressed by static drug therapy without strain adaptation or the emergence of drug-resistant strains.

Although treatment of inherited prion diseases appears to be a tractable goal, there have been no drugs specifically developed to target mutant prions. Recently, Vallabh et al. accurately noted that a longstanding barrier to developing and testing such drugs pre-clinically has been the lack of animal models of inherited prion disease that exhibit shortened lifespans without transgene overexpression [25]. Here, we evaluate several drug regimens that can either inhibit wild-type PrP^Sc accumulation and prolong incubation time in scrapie-infected animals or inhibit mutant PrP^Sc propagation in sPMCA reactions for their ability to treat new knock-in mouse models of FFI and fCJD with shortened lifespans.

Materials and methods

Ethics statement

The Guide for the Care and Use of Laboratory Animals of the National Research Council was strictly followed for animal experiments. The mouse bioassay experiment in this study was conducted in accordance with protocol supa.su.1 as reviewed and approved by Dartmouth College’s Institutional Animal Care and Use Committee, operating under the regulations/guidelines of the NIH Office of Laboratory Animal Welfare (assurance number A3259-01).

Mouse models

The methods used to design and produce kiBVI^D178N and kiBVI^E200K mice are described in a complementary manuscript [26]. Briefly, gene targeting in V6.5 embryonic stem cells was performed at the DZNE/Bonn University using CRISPR technology as described previously [27]. Plasmids containing the open reading frames of either wild-type, D178N-mutant, or E200K-mutant BVPrP (I109 polymorphic variant) were used as a starting point [28]. Targeting constructs were generated by ligating the respective BVPrP open reading frame variants between EagI and ClaI sites of the intermediate vector pWJPrP101 [27] containing homology regions and a neomycin selection cassette removable by Flp recombinase. The Cas9 vector used for double-strand break generation in the Prnp gene is available from Addgene (plasmid #78621) [27]. Expansion of gene-edited embryonic stem cells and aggregation with diploid CD-1(ICR) mouse embryos was performed at The Centre for Phenogenomics (Toronto, Canada). Chimeric mice were identified by their mixed coat colors and then bred with B6(Cg)-Tyr^c-2J/J mice (“B6-albino mice”; Jackson Lab #000058) to identify those with germline transmission of the gene-edited Prnp allele. Successful chimeras were crossed with a Flp deleter strain (B6.129S4-Gt(ROSA)26Sor^tm1(FLP1)Dym/RainJ; Jackson Lab #009086) to remove their selectable marker and then backcrossed with wild-type C57BL/6 mice to remove the Flp transgene. Mice that were positive for the BVPrP knock-in allele and negative for the Flp transgene were then intercrossed to create homozygous knock-in mice. All knock-in lines were maintained by crossing homozygous female with homozygous male mice.

Drug treatment, diagnosis, and neuropathology

Female mice were used for this study and housed in groups of 4. Each group contained 8 mice at the beginning of the experiment, and mice with incidental disorders unrelated to prion disease (i.e. large tumors, dermatitis, or non-healing fight wounds) were euthanized and excluded from subsequent survival curve and neuropathological analyses. Starting at 1 month of age, mice were fed Teklad chow (Envigo, Madison, WI) formulated by Envigo to contain either 280 mg compound/kg body weight/day IND24 (1.4 gm IND24/kg chow), 400 mg/kg/day Anle138b (2 gm Anle138b/kg chow), or 280 mg/kg/day IND24 plus 400 mg/kg/day Anle138b. IND24 and Anle138b were synthesized by Sundia (Sacramento, CA). Control animals were fed Teklad chow alone. On average, each mouse consumed ~4 gm food per day and weighed ~20 gm. Mice were administered Metolose 60SH-50 (cellulose ether viscosity 50) (Shin Etsu, Akron, OH) by subcutaneous injections (4 g/kg body weight/injection) every 4 months starting at 1 month of age. Mice were administered (poly [4-styrenesulfonic acid-co-maleic acid] (PSCMA) (Sigma Aldrich, St. Louis, MO) (~480 mg/kg body weight/day) starting at 1 month of age through drinking water (2mg/mL). We did not perform any pharmacokinetic assays for this study, but we previously reported that all of the same chows (containing IND24, Anle138b, or IND24 plus Anle138b) doubled incubation times in mice infected with wild type prions [11]. In addition, the oral dose of PSCMA used in this study was 12 times higher than the oral dose previously shown to yield a brain concentration of 40 nM PSCMA (K_D of PSCMA/PrP^C binding = 540 pM) [29].

Mice were monitored daily for the appearance and progression of neurological symptoms associated with prion disease, including ataxia, kyphosis, limb paralysis, and slow gait. Clinical diagnosis of prion disease was made when a prion-associated symptom persisted or progressed over a period of 3 consecutive days. Neuropathology was performed as previously described [30]. Tissue blocks and microscopic sections stained with hematoxylin and eosin were commercially prepared by iHisto (Salem, MA), and vacuolation in various brain regions was scored on a 0–5 scale. Additional sections were stained for glial fibrillary acidic protein (GFAP) immunohistochemistry by the Dartmouth Hitchcock Research Pathology Service Core. Slides were cut at 4μm thickness and air-dried at room temperature before baking at 60°C for 30 min. An automated protocol was performed on the Leica Bond Rx, which included paraffin dewax, antigen retrieval and staining steps. Heat-induced epitope retrieval using Bond Epitope Retrieval 2, pH 9.0 (Leica Biosystems, Cat# AR9640) was performed at 100°C for 20 min. Primary anti-GFAP antibody (Agilent, Cat# Z0334) was applied and incubated for 15 min at room temperature at 1:800 dilution. Primary antibody binding was detected and visualized using the Leica Bond Polymer Refine Detection Kit (DS9800) with DAB chromogen and Hematoxylin counterstain.

Mean vacuolation and GFAP density scores were plotted using the radarchart function of the fmsb package (https://cran.r-project.org/web/packages/fmsb/) in R (version 4.1.3).

Detection of mutant PrP^Sc in brain homogenates

Brains for biochemical analysis were homogenized to 10% (w/v) in sterile PBS. Sodium deoxycholate and NP-40 were each added to final concentration of 0.5% and samples were incubated for 20 min with intermittent vortexing. After a 5 min centrifugation at 1,000 x g, supernatants were recovered, and total protein was quantified using a Micro BCA Protein Assay Kit (ThermoFisher). The remaining supernatant was either treated with thermolysin (TL) protease (Sigma) at a ratio of 50 μg protein to 1 μg protease or water (-TL control), shaking for 1 hr at 600 rpm and 37°C. Digests were quenched by addition of 5 mM EDTA. Sarkosyl was then added to a final concentration of 2% (w/v) and samples were incubated for 5 min before centrifugation at 100,000 x g for 45 min. Supernatants were aspirated and pellets were resuspended in Laemmli SDS sample buffer (Bioland Scientific) and boiled for 15 min at 95°C. Unless otherwise specified, all steps above were performed on ice or at 4°C.

Denatured protein pellets were run on 12% SDS-PAGE gels and transferred to PVDF membrane using a semi-dry blotting apparatus. Western blots were probed using either EP1802Y (Abcam, 1:10,000 dilution) or 27/33 (in-house mAb, epitope = 142–149 mouse numbering, 1:25000 dilution) primary antibodies and HRP-linked goat anti-rabbit or sheep anti-mouse secondary antibodies, respectively.

Mutant recombinant PrP^Sc in vitro propagation assays

Unseeded sPMCA reactions using recombinant D177N mouse (Mo) PrP substrate (without cofactor) were performed and analyzed as previously described [24] with the modification that the final PrP concentration in each reaction was 40 μg/mL. Alternatively, two serial rounds of continuous shaking reactions containing recombinant I109 D178N BV PrP^Sc substrate (without cofactor) and initially seeded with protein-only I109 D178N Mo PrP^Sc were performed as previously described [31].

QuIC assay

Quaking-induced conversion reactions were performed as described previously [32], with the following modifications. Recombinant PrP substrates containing the D178N or E200K mutations were created by site-directed mutagenesis with the GeneTailor system (Invitrogen) using the bank vole (BV) I109 recPrP expression plasmid [32] as template. The D178N mutation was inserted using 5’-AGAACAACTTCGTGCACAATTGCGTCAACATCACC-3’ and 5’-GGTGATGTTGACGCAATTGTGCACGAAGTTGTTCT-3’ as forward and reverse primers, respectively. The E200K mutation was inserted using 5’-AGGGGGAGAACTTCACGAAGACCGACGTCAAGATG-3’ and 5’-CATCTTGACGTCGGTCTTCGTGAAGTTCTCCCCCT-3’ as forward and reverse primers, respectively. The mutant genes were cloned into the pET-22b plasmid for bacterial expression and purified, as described previously [31–33].

Reactions were allowed to misfold spontaneously by the omission of PrP^Sc seeds, and were supplemented with either 1 μM Anle13b, 1 μM IND24, 1 μM Anle13b + 1 μM IND24, or DMSO as a vehicle control (“Untreated”). Shaking was performed in an Allsheng MSC-100 Thermo-Shaker set at 42°C and 900 rpm for 30 hours. At 2 hr increments, Thioflavin T fluorescence was measured in a SpectraMax iD5 plate reader (Molecular Devices) pre-warmed to 42°C.

t_1/2 was measured as the time from the start of the experiment to the point at which half-maximal fluorescence was reached. Average t_1/2 and standard error of the mean (SEM) was calculated for each treatment condition across 3 replicate reactions.

Statistical analyses

We used a nonparametric approach to compare the characteristics of treated and untreated animals in six brain regions of kiBVI^D178N and kiBV^E200K mice. Specifically, we used Mann–Whitney tests with statistical significance set at alpha = 0.05 for all statistical comparisons. Three types of comparisons were evaluated: (i) age (days) at onset of prion disease in each treatment group when compared with untreated controls, (ii) vacuolation scores in each region of the brain in each treatment group when compared with untreated controls, and (iii) t_1/2 values from QuIC assays in each treatment group when compared with untreated controls. Analyses were conducted using Stata 15.1 (StataCorp, Texas, USA). All raw data and Stata log statistical analysis files for survival curves, vacuolation scores, and QuIC assays are publicly available in the Mendeley Data repository at DOI: 10.17632/225jyctt6f.1.

Results

For this study, we used recently developed knock-in mouse models of inherited prion diseases with shortened lifespans [34]. These models express I109 bank vole (BV) PrP with either the D178N or E200K pathogenic mutations (hereafter, these models are respectively termed “kiBVI^D178N mice” and “kiBVI^E200K mice” for brevity, but is important to note the caveat that these models do not produce the same mutant PrP^Sc conformation and neuropathological patterns seen in human patients.) In choosing drug regimens to test in kiBVI^D178N and kiBVI^E200K mice, we included a variety of oral and sub-cutaneous treatments previously shown to be highly effective in treating prion-infected mice (Table 1). These regimens (Anle138b, IND24, a combination regimen of Anle138b plus IND24, and Metolose cellulose ether) all produce survival indices [35] between 2-4-fold in scrapie-infected rodents [10–12,36] (summarized in Table 2, column 2). We also included PSCMA, an oral, brain-penetrant compound previously shown to inhibit PrP^C-dependent Aβ oligomer toxicity in vivo [29] because we found that it could inhibit spontaneous D177N Mo PrP^Sc propagation in sPMCA reactions (Fig 1A) as well as seeded I109 D178N BV PrP^Sc propagation in continuous shaking reactions (Fig 1B).

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Fig 1. Effect of various drug treatments on mutant recombinant PrP^Sc propagation in vitro.

(A) Western blots of unseeded 3-round sPMCA reactions using recombinant D177N Mo PrP substrate. Where indicated, reactions contained 20 μM quinacrine, PSCMA, or doxycycline. Blot probed with anti-PrP mAb 27–33. (B) Western blots of seeded continuous shaking reactions using D178N BV PrP^Sc as substrate. Reactions contained varying concentrations of PSCMA, as indicated. Only samples from the second round of serial propagation are shown. Blot probed with anti-PrP mAb EP1802Y. -PK = control sample not subjected to protease digestion. All other samples treated with 25 μg/mL proteinase K.

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Table 1. Drug regimens used in this study.

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Table 2. Effect of drug treatments on infectious and inherited prion diseases in mice.

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Untreated kiBVI^D178N and kiBVI^E200K mice spontaneously died of prion disease at ~520 days and ~650 days of age, respectively (Fig 2A and 2B, black triangles). Paradoxically, kiBVI^D178N mice treated with a combination of Anle138b + IND24 developed disease more quickly (~380 days) compared to untreated controls (~520 days) (P = 0.0017). There was no statistically significant difference in the survival of any other treatment groups compared to untreated controls (Fig 2 and Table 1, columns 3–4). Thus, none of the regimens tested provided significant therapeutic benefit in either kiBVI^D178N or kiBVI^E200K mice, despite displaying strong efficacy in mice infected with wild-type prions (Table 1, compare column 2 vs. columns 3–4) or inhibitory activity in mutant recombinant sPMCA reactions (Fig 1).

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Fig 2. Effect of various drug treatments on survival in kiBVI^D178N and kiBVI^E200K mice.

Survival curves of knock-in mice treated with various anti-prion drug regimens, as specified in the legend. (A) Survival curves of kiBVI^D178N (n = 5–7), and (B) kiBVI^E200K mice (n = 5–8). Survival index values are shown in Table 2.

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To investigate how the combination of Anle138b + IND24 might have decreased survival in kiBVI^D178N mice, we tested the effect of these two compounds on the kinetics of amyloid formation in unseeded QuIC assays with mutant recombinant BV PrP substrates. The results show that the combination of Anle138b + IND24 significantly accelerated D178N BV PrP amyloid formation compared to untreated reactions (Table 3) (P = 0.0495).

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Table 3. Effect of Anle138b and IND24 on QuIC assays with mutant PrP substrates.

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QuIC assays were performed in biological triplicates as described in Methods. Anle138b and IND24 were each used at a final concentration of 1 μM alone or in combination, as indicated. t_1/2 = time to reach half-maximal fluorescence. SEM = standard error of the mean. *Statistically significant vs. untreated control (P = 0.0495) Mann-Whitney test.

Exposure to anti-prion drug regimens, including several of the regimens tested here, can lead to the selection and emergence of wild-type prions with altered biochemical and neuropathological strain properties [10,11]. To examine whether anti-prion drug therapy can also change the strain properties of mutant prions, we compared the migration patterns of protease-resistant PrP^Sc molecules as well as regional distributions of vacuolation in the brains of kiBVI^D178N and kiBVI^E200K mice in our control vs. treatment groups.

To detect protease-resistant PrP^Sc molecules in the brains of mutant mice, we treated brain homogenates of kiBVI^D178N and kiBVI^E200K mice from all the treatment groups with thermolysin (TL) and performed western blots with two different anti-PrP monoclonal antibodies, 27–33 (epitope = residues 142–149 mouse numbering) and EP1802Y (epitope = residues 217–226). We only detected TL-resistant PrP^Sc bands in a subset of knock-in mouse that developed spontaneous disease. In general, the majority of kiBVI^D178N mice had TL-resistant PrP^Sc in their brains, whereas few kiBVI^E200K mice did (Fig 3A versus 3B and S1 Text). When we compared the electrophoretic mobility of TL-resistant PrP^Sc bands from kiBVI^D178N mice in different treatment groups, we observed several differences in Western blots probed with mAb 27–33 (Fig 3A, top blot). Most strikingly, whereas a ~14 kDa TL-resistant band is present in untreated kiBVI^D178N brains, IND24- and PSCMA-treated kiBVI^D178N brains contain a ~16 kDa TL-resistant band instead (Fig 3A, top blot). Interestingly, the ~14 kDa and ~16 kDa band are only detected by mAb 27/33 (epitope 134–144) (Fig 3A) but not by EP1802Y (epitope 217–226) (Fig 3B), suggesting that both of these PK-resistant bands may be C-terminally truncated. In addition, a smaller band (~10 kDa) can also by detected by mAb 27/33 in IND24- and PSCMA-treated kiBVI^D178N brains, both before and after PK digestion (Fig 3A). The identity of this smaller band is unclear, but its presence only in IND24- and PSCMA-treated kiBVI^D178N brains correlates with the presence of the ~16 kDa band and therefore provides additional evidence that drug-induced PrP^Sc conformational change likely occurred. In contrast, TL-resistant PrP^Sc bands showed similar electrophoretic mobility between individual kiBVI^D178N mice within each treatment group (S1 Text). These results suggest that treatment with either IND24 or PSCMA induced the emergence of an alternative PrP^Sc conformation in kiBVI^D178N mice.

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Fig 3. Mutant PrP^Sc molecules in brains of kiBVI^D178N and kiBVI^E200K mice treated with various anti-prion drug regimens.

Western blot showing insoluble and thermolysin-resistant PrP^Sc molecules in brain homogenates of (A) kiBVI^D178N and (B) kiBVI^E200K mice treated with various drug regimens, as indicated. Samples were either treated with thermolysin (TL) or water, as indicated. All samples were centrifuged to collect insoluble PrP. Within each panel, the top blot is probed with anti-PrP mAb 27–33 (epitope = residues 142–149 mouse numbering) and the lower blot is probed with mAb EP1802Y (epitope = residues 217–226).

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We also observed differences in vacuolation profiles between various treatment groups and untreated controls in both kiBVI^D178N and kiBVI^E200K mice (Fig 4). Specifically, kiBVI^D178N mice treated with either Anle138b + IND24 or PSCMA displayed different vacuolation profiles than untreated kiBVI^D178N mice, and kiBVI^E200K mice treated with IND24, Anle138b + IND24, PSCMA, or Metolose displayed different vacuolation profiles than untreated kiBVI^E200K mice (Table 4). A particularly striking example of drug-induced change in neuropathology was observed in the corpus callosum of kiBVI^E200K mice. Whereas untreated kiBVI^E200K mice displayed abundant vacuolation in the corpus callosum, kiBVI^E200K mice treated with either IND24 alone or Anle138b + IND24 showed little to no vacuolation in the corpus callosum (Fig 5). A similar difference in reactive astrocytosis in the corpus callosum was observed between these groups, as determined by GFAP immunohistochemistry (S1 and S2 Figs). Taken together, the biochemical and neuropathological strain typing assays provide evidence that all the drug regimens used in this study except Anle138b monotherapy altered the strain properties of mutant prions in both kiBVI^D178N and kiBVI^E200K mice.

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Fig 4. Regional vacuolation profiles in brains of kiBVI^D178N and kiBVI^E200K mice treated with various anti-prion drug regimens.

Mean vacuolation scores (n = 2–7) in various brain regions of mice treated with various drug regimens, as specified in the legends. T = Thalamus, Ctx = cerebral cortex, CC = corpus callosum, Cb = cerebellum, H = hippocampus, P = pons. (A) Regional vacuolation profiles of kiBVI^D178N mice, and (B) regional vacuolation profiles of kiBVI^E200K mice. Statistical analyses are shown in Table 4.

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Fig 5. Neuropathology of corpus callosum in kiBVI^E200K mice treated with various anti-prion drug regimens.

Representative microscopic images of brain sections of kiBVI^E200K mice treated with various drug regimens, as specified, stained with hematoxylin and eosin. Square brackets indicate the location of the corpus callosum (in between the cerebral cortex and hippocampus). Horizontal scale bar = 200 μm.

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Table 4. P-values of significant differences in vacuolation scores of treatment groups versus untreated controls in different brain regions.

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Discussion

Here we report the first drug trial in knock-in animal models of FFI and fCJD with shortened lifespans. We tested four drug regimens that have previously been shown to inhibit the accumulation of wild-type PrP^Sc and significantly prolong incubation time in scrapie-infected animals [10–12,36] and one compound that potently inhibits mutant PrP^Sc propagation in recombinant sPMCA reactions. Surprisingly, we observed that none of these drugs inhibited the accumulation of mutant PrP^Sc in or significantly extended the lifespan of either kiBVI^D178N or kiBVI^E200K mice (Anle138b and PSCMA increased lifespan modestly in fCJD). Notably, all the compounds we tested belong to different chemical classes, cross the blood-brain barrier, and are well-tolerated by mice. Three of the compounds tested (IND24, Anle138b, and Metolose cellulose ether) represent the most efficacious prophylactic drugs currently known for wild-type scrapie prions, extending scrapie incubation time between 2- to 4-fold [9–12,36]. A fourth compound, PSCMA, has not yet been tested in prion-infected animals, but potently inhibits D178N propagation in recombinant sPMCA reactions in vitro and blocks PrP^C-dependent Aβ oligomer toxicity in vivo [29].

It has been previously shown that non-specific clearance therapies (rapamycin and IVIG) but not Anle138b can delay the onset of disease in an A117V transgenic mouse model of Gerstmann-Sträussler-Scheinker (GSS) syndrome [39–41]. Our current study differs from these prior studies because (1) GSS is a phenotypically distinct inherited prion disease characterized by the accumulation of amyloid plaques not seen in FFI or fCJD, and (2) we used knock-in [34] rather than overexpression animal models [42]. Theoretically, using animal models with lower expression levels and fewer plaques should improve our ability to detect a therapeutic effect and therefore increase our confidence about the negative results observed in this study. A more recent study testing Anle138b in alternative knock-in mouse models of FFI and fCJD expressing mutant mouse PrP molecules rather than bank vole PrP molecules was limited by the relatively normal lifespan of those mutant mice [25].

It has been previously shown that drug-resistant wild-type prions with altered PrP^Sc conformation and strain characteristics can emerge in response to anti-prion drug therapy [10, 11]. In this study, we observed that mutant prions can also undergo strain adaptation in response to drug therapy in vivo, based on changes in biochemical and neuropathological strain-typing assays. At the same time, we did not observe a survival benefit for any of the anti-prion therapies tested. Together, these results suggest that drug-resistant conformers may emerge more rapidly during the replication of mutant prions compared to wild-type prions. It is worth considering, as an alternative to the strain selection hypothesis, the possibility that each drug may preferentially inhibit prion replication in specific brain regions, and that lifespan is determined by disease progression in the regions not impacted by each drug. However, this alternative hypothesis would not explain the differences in mobility of TL-resistant PrP^Sc bands in IND24-treated and combination-treated mice vs. untreated mice. Also, in the case of cellulose ethers, it is possible that these compounds are more effective at combating peripherally administered prions than prions that form exclusively within the brain [12]. In addition, it is worth noting that cellulose ethers are less effective in mice with C57Bl/6 background [12], and the mice used in this study have been backcrossed into the C57Bl/6 genetic background [34].

An unexpected result in our study was that combination therapy with Anle138b and IND24 paradoxically accelerated disease in kiBVI^D178N mice. This effect is surprising because this same combination regimen does not accelerate disease in wild-type mice [11]. We observed that the combination of Anle138b and IND24 accelerated mutant PrP aggregation in QuIC reactions, suggesting that the combination regimen may also increase the kinetics of mutant PrP^Sc formation in vivo. Alternatively, it is possible that strain adaptation of mutant prions induced by combination therapy selects for a conformer that is either more toxic or difficult to degrade in neurons. Finally, the shortened lifespans may be due to drug toxicity caused by the high dosages used in the combination regimen; however, the observation that combination regimen-treated kiBVI^E200K mice survive ~100 days longer than combination-treated kiBVI^E200K mice argues somewhat against this explanation. This result suggests that where mechanism of action is unknown, caution is warranted in extrapolating anti-prion effects across strains.

A potential limitation of our disease models is that they use bank vole rather than human PrP as the sequence backbone. However, it has generally been difficult to model inherited human prion diseases in mice using the human PrP backbone [43], and to our knowledge A117V is the only mutation that has successfully modeled on the human PrP backbone in transgenic mice [44]. The models we used for this study are the first knock-in models of inherited prion disease that show reduced lifespans and transmissibility (without PrP overexpression)[34].

The major conclusion of our study is that drugs that effectively treat wild type prion disease are inactive against two different mutant prions in knock-in mouse models of inherited prion disease. One hypothesis for the ineffectiveness of the repurposed drugs is that the spontaneous formation of mutant prions and templated formation of wild-type prions may use different mechanisms. Mutant prions can form without seed or cofactor molecules, whereas cofactor molecules are required to form and propagate wild-type prions [24]. Cofactor molecules are even required by wild-type PrP^C substrate molecules to propagate the conformation of mutant PrP^Sc seeds [24]. Consistent with this hypothesis, the host translational response of mice expressing mutant prions is strikingly different from the translation response of mice infected with wild-type prions [45,46]. An alternative hypothesis is that none of the three repurposed drugs tested work on the mutant prion strains in kiBVI^D178N and kiBVI^E200K mice. Each of the three of these drugs has been previously shown to inhibit some prion strains but not others: IND24 inhibits RML, Me7, and chronic wasting disease (CWD), but not sCJD prions [10]; Metolose inhibits 263K and CWD, but not RML prions [12,47,48]; and Anle138b inhibits RML, but not 263K prions [49]. A third possible explanation is that mutant PrP^Sc and wild type PrP^Sc molecules may be formed in different cellular compartments and that the compounds tested may not be able to access the cellular compartments where mutant PrP^Sc forms.

A critical lesson from this study is that treatments for inherited prion diseases should be identified and/or developed by specifically targeting mutant prions rather than repurposing drugs that work on wild-type prions. Developing valid cell-based assays to screen candidate compounds for their ability to inhibit mutant prions would greatly facilitate such efforts since biochemical assays may not accurately predict in vivo efficacy. We observed that PSCMA potently inhibited D178N PrP^Sc propagation in recombinant sPMCA reactions, but PSCMA had no therapeutic effect in vivo. Our results also suggest that the novel knock-in models of FFI and fCJD that we used for these studies may be generally advantageous for pre-clinical in vivo testing because they have physiological PrP expression levels and shortened lifespans, thereby providing an objective and quantifiable endpoint for evaluating therapeutic efficacy, as recently suggested by Vallabh and Minikel [25]. Finally, our results suggest that less easily administered therapies which lower PrP levels (such as anti-sense oligonucleotides delivered by lumbar puncture) may ultimately be required to escape the ability of both wild-type and mutant prions to undergo strain-adaptation leading to anti-prion drug failure [31,50,51].

In summary, our findings show that several anti-prion drugs with activity against wild-type prions have no beneficial effect on disease-free survival of two different knock-in mouse models of inherited prion disease. Our work highlights the importance of developing valid drug screening assays and alternative therapies specifically for inherited prion disease, as well as anti-prion strategies that not strain-dependent, such as PrP-lowering therapies [50,51].

Supporting information

S1 Fig. Regional GFAP immunohistochemistry profiles in brains of kiBVI^E200K mice treated with various anti-prion drug regimens.

Mean GFAP immunohistochemistry scores (n = 2–7) in various brain regions of mice treated with various drug regimens, as specified in the legends. T = Thalamus, Ctx = cerebral cortex, CC = corpus callosum, Cb = cerebellum, H = hippocampus, P = pons.

https://doi.org/10.1371/journal.ppat.1012087.s001

(TIF)

S2 Fig. GFAP immunohistochemistry of corpus callosum in kiBVI^E200K mice treated with various anti-prion drug regimens.

Representative microscopic images of brain sections of kiBVI^E200K mice treated with various drug regimens, as specified, stained with hematoxylin and eosin. Square brackets indicate the location of the corpus callosum (in between the cerebral cortex and hippocampus). Horizontal scale bar = 500 μm.

https://doi.org/10.1371/journal.ppat.1012087.s002

(TIF)

S1 Text. Anti-prion drugs do not improve survival in knock-in models of inherited prion disease.

https://doi.org/10.1371/journal.ppat.1012087.s003

(DOC)

Acknowledgments

The authors thank Rachel Pepin and Emma Fiske for assistance with protease-digestion experiments, and Ciara Groesbeck and Eric DuFour for their assistance with veterinary care and tissue harvests.

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Figures

Abstract

Author summary

Introduction

Materials and methods

Ethics statement

Mouse models

Drug treatment, diagnosis, and neuropathology

Detection of mutant PrPSc in brain homogenates

Mutant recombinant PrPSc in vitro propagation assays

QuIC assay

Statistical analyses

Results

Discussion

Supporting information

S1 Fig. Regional GFAP immunohistochemistry profiles in brains of kiBVIE200K mice treated with various anti-prion drug regimens.

S2 Fig. GFAP immunohistochemistry of corpus callosum in kiBVIE200K mice treated with various anti-prion drug regimens.

S1 Text. Anti-prion drugs do not improve survival in knock-in models of inherited prion disease.

Acknowledgments

References

Detection of mutant PrP^Sc in brain homogenates

Mutant recombinant PrP^Sc in vitro propagation assays

S1 Fig. Regional GFAP immunohistochemistry profiles in brains of kiBVI^E200K mice treated with various anti-prion drug regimens.

S2 Fig. GFAP immunohistochemistry of corpus callosum in kiBVI^E200K mice treated with various anti-prion drug regimens.