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 EC Bourkas, Lech Kaczmarczyk, Erica Stuart, Walker S. Jackson, Joel C. Watts, Surachai Supattapone
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 PrPSc propagation in vitro. None of the regimens tested (Anle138b, IND24, Anle138b + IND24, cellulose ether, and PSCMA) significantly extended disease-free survival or prevented mutant PrPSc 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.
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 (PrPC) into an infectious conformer (PrPSc) [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).
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 kiBVID178N and kiBVIE200K 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].
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 “kiBVID178N mice” and “kiBVIE200K mice” for brevity, but is important to note the caveat that these models do not produce the same mutant PrPSc conformation and neuropathological patterns seen in human patients.) In choosing drug regimens to test in kiBVID178N and kiBVIE200K 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 PrPC-dependent Aβ oligomer toxicity in vivo [29] because we found that it could inhibit spontaneous D177N Mo PrPSc propagation in sPMCA reactions (Fig 1A) as well as seeded I109 D178N BV PrPSc propagation in continuous shaking reactions (Fig 1B).
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 PrPSc and significantly prolong incubation time in scrapie-infected animals [10–12,36] and one compound that potently inhibits mutant PrPSc propagation in recombinant sPMCA reactions. Surprisingly, we observed that none of these drugs inhibited the accumulation of mutant PrPSc in or significantly extended the lifespan of either kiBVID178N or kiBVIE200K 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 PrPC-dependent Aβ oligomer toxicity in vivo [29].
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.
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.
https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1012087#abstract0
