D178N prion protein mutation endows RML prions with new strain properties that do not mimic human genetic prion diseases

Abstract

Genetic prion diseases are caused by mutant prion protein (PrP) misfolding, eventually leading to the formation of PrP^Sc, the infectious prion isoform that propagates by inducing misfolding of native PrP. Different mutations are thought to generate distinct prion strains with unique self-replicating and neurotoxic properties, contributing to the phenotypic diversity of genetic prion diseases. We previously showed that transgenic mice expressing the mouse PrP homologs of the D178N–M129 and D178N–V129 mutations linked to fatal familial insomnia (FFI) and genetic Creutzfeldt–Jakob disease (CJD¹⁷⁸) accumulate misfolded, mildly proteinase-K (PK)-resistant PrP in their brains. These mice develop spontaneous neurological illnesses resembling FFI and CJD¹⁷⁸, but their diseases have not been found to be transmissible to various mouse lines. In this study, we further assessed their prion propagation potential by inoculating bank voles—shown here to be susceptible to human FFI and CJD¹⁷⁸ prions—and by using RT-QuIC. Negative results from both approaches corroborate the idea that these mice do not generate infectious prions. However, when brain homogenates from Tg(FFI) and Tg(CJD) mice were subjected to protein misfolding cyclic amplification with RML PrP^Sc as a seed, they generated highly PK-resistant mutant prions (RML^FFI and RML^CJD) able to propagate in Tga20 mice overexpressing wild-type PrP. To determine whether these in vitro-converted prions modeled the human diseases better, we examined their transmissibility, biochemical traits, and neuropathological features. Despite successful serial propagation in Tga20 mice, RML^FFI and RML^CJD displayed long incubation times, poor transmissibility to C57BL/6 mice, identical PK-resistant PrP fragments, and distinctive neuropathological changes including large submeningeal and perivascular plaques enriched in endogenous proteolytically shed PrP lacking membrane anchorage. These findings indicate that, regardless of the M129V polymorphism, the D178N mutation imparts novel, stable strain properties to RML that do not recapitulate the features of FFI and CJD¹⁷⁸. Our results offer new insights into how genetic PrP mutations influence prion strain characteristics and suggest that spontaneous and templated prionogenesis may follow distinct mechanistic pathways.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00401-026-02976-w.

Introduction

Prions are proteinaceous infectious agents responsible for fatal neurodegenerative diseases in humans and other mammals [43]. They are composed of PrP^Sc, a misfolded, aggregation-prone, and often protease-resistant isoform of the cellular prion protein (PrP^C), which propagates by inducing misfolding and aggregation of native PrP^C. The best-known prion infections are scrapie in sheep, bovine spongiform encephalopathy, and chronic wasting disease (CWD) in cervids. In humans, prion diseases acquired by infection are extremely rare (< 1%), mostly transmitted iatrogenically through medical and surgical procedures. Approximately, 85% of human prion diseases occur spontaneously with no known genetic or environmental trigger, primarily as sporadic Creutzfeldt–Jakob disease (sCJD). Nearly 15% are dominantly inherited due to point mutations or insertions in the PRNP gene encoding PrP^C. Point mutations are mainly clustered in the protein’s C-terminus, leading to amino acid substitutions or protein truncations. The insertions consist of additional copies of an octapeptide repeat in the N-terminal region, which normally contains four octapeptides. Mutant PrP is thought to misfold and aggregate spontaneously, eventually acquiring the PrP^Sc structure.

Different PRNP mutations cause clinically and neuropathologically distinct diseases, including genetic CJD, fatal familial insomnia (FFI), and Gerstmann–Sträussler–Scheinker (GSS) syndrome. The disease phenotype is also influenced by the PRNP polymorphic codon 129, where either methionine (M) or valine (V) can be encoded. The most striking example is the prion disease linked to the substitution of aspartic acid (D) with asparagine (N) at codon 178, which, depending on the amino acid encoded at codon 129, segregates with either FFI (D178N–M129), primarily characterized by severe sleep disorders and autonomic dysfunction, or CJD¹⁷⁸ (D178N–V129), clinically identified by global cortical dementia and motor abnormalities. This phenotypic diversity is thought to arise from conformational differences between the two mutants, which eventually misfold into distinct PrP^Sc isoforms with unique self-replicating and neurotoxic properties (prion strains).

The existence of prion strains was initially suggested by the observations that infectious isolates of sheep scrapie could be serially transmitted to rodents, causing diseases with different incubation times to clinical disease, tissue tropism, and subcellular patterns of pathology [4]. Because multiple prion strains can be maintained in inbred mice with identical PrP genes, prion strains cannot be encoded by differences in PrP primary structure. Rather, cryo-electron microscopy data provide evidence that they represent structurally different PrP^Sc types that are maintained upon serial passage in susceptible hosts [23, 29].

Supporting the idea that D178N–M129 and D178N–V129 PrPs generate different prion strains, the brains of FFI and CJD¹⁷⁸ patients contain proteinase-K (PK)-resistant (PrP^res) isoforms with different glycosylation patterns which, after removal of N-glycans by enzymatic deglycosylation with peptide-N-glycosidase F (PNGase F), have apparent molecular masses of 19 and 21 kDa, respectively [32]. Additionally, brain homogenates from FFI patients transmitted disease to transgenic mice expressing a human–mouse PrP chimera, inducing the formation of the 19-kDa PrP^res fragment, whereas CJD brain extracts induced the formation of the 21-kDa PrP^res fragment [52]. The FFI- and CJD-inoculated mice showed different incubation times, patterns of PrP^res accumulation, and neuropathological lesions [52].

We previously generated transgenic (Tg) mice expressing the mouse (mo) PrP homologs of the FFI and CJD¹⁷⁸ mutations (moPrP D177N–M128 and D177N–V128) on a PrP knockout (Prnp^0/0) genetic background [5, 17]. We found that Tg(FFI) and Tg(CJD) mice developed slowly progressive, invariably fatal neurodegenerative diseases, with clinical and neuropathological characteristics reminiscent of the corresponding human disorders [5, 14, 17]. The mutant PrPs extracted from the brains of Tg(FFI) and Tg(CJD) mice were aggregated and resistant to mild PK digestion. However, they both yielded identical PrP^res fragments of 19 kDa and failed to transmit disease to several types of recipient mice, including C57BL/6 mice and Tga20 mice, which overexpress moPrP and are highly sensitive to mouse prions [5]. Additionally, no prion seeding activity was detected in the brains of Tg(FFI) and Tg(CJD) mice by serial protein misfolding cyclic amplification (PMCA), which allows highly efficient PrP^Sc amplification in vitro [5]. Thus, these mice recapitulate disease-specific features of FFI and CJD¹⁷⁸ that are independent of the conversion of mutant PrP to a replication-competent PrP^Sc state [11, 14].

In the present study, we first carried out additional experiments to check for the possible presence of low prion infectivity in the brains of spontaneously ill Tg(FFI) and Tg(CJD) mice. This was done by real-time quaking-induced conversion (RT-QuIC) and inoculation in bank voles, which are remarkably susceptible to a wide range of prions [8, 35]. These experiments confirmed the absence of replication-competent PrP^Sc in the brains of Tg(FFI) and Tg(CJD) mice. Next, we tested whether highly PK-resistant forms of D177N–M128 and D177N–V128 PrPs generated in vitro by subjecting Tg(FFI) and Tg(CJD) brain homogenates to serial PMCA reactions seeded with the mouse-adapted RML scrapie strain were transmissible to mice and induced more rapidly progressing diseases that recapitulated more closely the biochemical and neuropathological features of human FFI and CJD¹⁷⁸.

We found that in vitro-generated FFI and CJD mouse prions transmitted to mice much less efficiently than RML, with very long incubation times even after five passages in Tga20 mice, and very inefficient transmission to C57BL/6 mice. They induced similar biochemical and neuropathological profiles that did not resemble those of FFI and CJD¹⁷⁸, and were markedly different from RML. Thus, the D178N mutation endows RML with novel strain properties that are not influenced by the amino acid encoded at codon 129 and persist upon serial passage in wild-type PrP-expressing mice.

Methods

Animals

C57BL/6J (RRID: IMSR_JAX:000664) mice were obtained from Charles River Laboratories. Tga20 mice that overexpress moPrP at 8X and are highly sensitive to prions [20] were obtained from the European Mouse Mutant Archive, Monterotondo, Italy (EM:00181). Bank voles carrying methionine at the polymorphic Prnp codon 109, known to be susceptible to sporadic and genetic human prion diseases [37] were obtained from the breeding colony of Istituto Superiore di Sanità (ISS). Animals were housed at a controlled temperature (22 ± 2 °C) with a 12-/12-h light/dark cycle and free access to pelleted food and water. The health and home-cage behavior of the animals were monitored daily, according to guidelines for health evaluation of experimental laboratory animals [7].

Ethics statement

Procedures involving mice and their care were conducted in conformity with the institutional guidelines at the Mario Negri Institute for Pharmacological Research IRCCS. They were reviewed and approved by the Mario Negri Institute Animal Care and Use Committee, which includes ad hoc members for ethical issues, and by the Italian Ministry of Health (Decreto no. 822/2020-PR). For the experiments with bank voles, the experimental protocol was approved and supervised by the Service for Biotechnology and Animal Welfare of the Istituto Superiore di Sanità and authorized by the Italian Ministry of Health (Decreto no. 1119/2015-PR).

All procedures were carried out in compliance with national (D.lgs 26/2014; Authorization no. 19/2008-A issued March 6, 2008, by Ministry of Health) and international laws and policies (EEC Council Directive 2010/63/UE; the NIH Guide for the Care and Use of Laboratory Animals, 2011 edition). Animal facilities meet international standards and are regularly checked by a certified veterinarian responsible for health monitoring, animal welfare supervision, experimental protocols, and review of procedures.

Human samples

Brain specimens were dissected from the frontal cortex of an Alzheimer’s disease patient, the temporal cortex and thalamus of an FFI patient [45] and the temporal cortex of a CJD¹⁷⁸ patient [30]. Written informed consent for participation in research and all procedures for sample collection and experimental studies were in accordance with the 1964 Declaration of Helsinki and its later amendments and were approved by the Ethical Committees of Fondazione IRCCS Istituto Neurologico Carlo Besta and National Prion Disease Surveillance Center (Case Western Reserve University).

Bank vole prion protein expression and purification

Bank vole (residues 23 to 230; methionine at residue 109; accession no. AF367624) recombinant PrP substrate was purified as previously described [22]. Briefly, PrP DNA sequences were ligated into the pET41 vector (EMD Biosciences). Vectors were transformed into Rosetta (DE3) Escherichia coli and were grown in Luria broth medium in the presence of kanamycin and chloramphenicol. The autoinduction system was used to induce protein expression. Bank vole PrP was purified from inclusion bodies using denaturing conditions and Ni–nitrilotriacetic acid (NTA) Superflow resin (Qiagen) and an ÄKTA fast protein liquid chromatographer (GE Healthcare Life Sciences). Refolding of the substrate was achieved on the column by a guanidine HCl reduction gradient. Elution was done using an imidazole gradient as previously described [22]. The protein was dialyzed into 10 mM sodium phosphate buffer (pH 5.8), filtered using a 0.22-μm syringe filter (Fisher), and stored at − 80 °C. Protein concentration was measured by absorbance at 280 nm.

RT-QuIC protocol

RT-QuIC assays were performed as described previously [39]. The reaction mix was composed of 10 mM phosphate buffer (pH 7.4), 300 mM NaCl, 0.1 mg/ml bank vole PrP 23–230, 10 μM thioflavin T (ThT), 1 mM ethylenediaminetetraacetic acid tetrasodium salt (EDTA), and 0.001% SDS. Aliquots of the reaction mix (98 μL) were loaded into each well of a black 96-well plate with a clear bottom (Nunc). Reactions were seeded with 2 μL of indicated brain homogenate (BH) dilutions. A plate sealer film (Nalgene Nunc International) was used to seal the plate, which was then incubated at 42 °C in a BMG FLUOstar Omega plate reader with cycles of 1 min shaking (700 rpm double orbital) and 1 min rest. ThT fluorescence measurements (450 ± 10 nm excitation and 480 ± 10 nm emission; bottom read) were taken every 45 min.

The fluorescence threshold for a positive reaction was 10% of the maximum value of any reaction within the experiment’s cutoff time per experiment. Evaluations were done for each plate to account for differences across experiments and between plate readers. A sample was identified as positive if at least 50% of the replicate wells (e.g., ≥ 2 of 4) were positive. We calculated inverse time to thresholds for each reaction, and the wells that did not cross the fluorescence threshold were calculated as 1 h past the time cutoff (e.g., 1/51 h). End-point dilution analyses were analyzed using the Spearman–Kärber calculation [38] to provide estimates of the concentrations of seeding activity units giving positive reactions in 50% of replicate reactions, i.e., the 50% “seeding doses” or SD50s as previously described [55].

Transmission studies

Ten percent (w/v) BHs were prepared in ice-cold phosphate buffer saline (PBS–Gibco) and centrifuged for 5 min at 900×g, and the supernatant was diluted to 1% in PBS. 25 μL was injected intracerebrally into the right parietal lobe of recipient mice 40–100 days of age using a 25-gauge needle. Mice were monitored weekly for neurological dysfunction based on established objective criteria. Inoculated mice were observed weekly for symptoms of neurological dysfunction. The onset of disease was scored as the time at which at least three of the following neurological signs were observed: i) foot clasp reflex, evaluated by suspending mice by the tail for 30 seconds and observing if they clasped their hind limbs together; ii) kyphosis; iii) unbalanced body posture, assessed by visual inspection; iv) difficulty to walk on a horizontal metal grid (45 × 45 cm) consisting of steel rods (3 mm diameter) with a center-to-center spacing of 7 mm. Terminal disease was identified when mice were unable to right themselves from a supine position within 60 s, walk on the horizontal metal grid, or experience cumulative weight loss amounting to 20% of their initial body weight at disease onset. Mice in the terminal stage were euthanized via cervical dislocation, and their brains were collected, with one-half frozen and the other half fixed in formalin.

For experiments with bank voles, 10% (w/v) BHs in PBS were prepared from human or mouse brain tissues. Six- to eight-week-old bank voles were inoculated with 20 μL of BH into the left cerebral hemisphere, under ketamine anesthesia (ketamine, 0.1 μg/g). The animals were examined twice a week until the appearance of neurological signs, and daily thereafter. Neurological signs ranged from mild behavioral alterations and the disappearance of the typical behavior of hiding under the sawdust lining the cage at disease onset, to dorsal kyphosis, severe ataxia, and head bobbing in full-blown disease. Diseased animals were culled with carbon dioxide at the terminal stage of the disease, but before the severity of neurological impairment compromised their welfare, in particular their ability to drink and feed adequately. At postmortem, brains from inoculated bank voles were removed and divided sagittally. Half the brain was frozen, and half was fixed in formol saline for histological analyses. The attack rate was calculated as the number of animals scoring positive by Western blot (WB) and pathological diagnosis at postmortem/number inoculated. Animals found dead or culled for intercurrent disease before 200 days post-inoculation and scoring negative at postmortem were excluded from analyses. The survival time for animals scoring positive at postmortem was calculated as the time from inoculation to culling or death.

Protease resistance assay and Western blot analysis

For the experiments shown in Figs. 3, 4, and 5, 10% (w/v) BHs prepared in PBS were diluted in 20 mM Trs-HCl containing 0.5%, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, and incubated for 20 min at 4 °C on a rotating wheel. After brief centrifugation to remove debris, lysates corresponding to 100 μg of total protein were incubated with proteinase-K (Merck) at final concentrations of 25 or 50 μg/mL for 1 h at 37 °C in a total volume of 100 μL. PK digestion was stopped by addition of either 5 mM phenylmethylsulfonyl fluoride (PMSF; Merck) or 0.5 mg/mL Pefabloc (Roche), followed by incubation for 5 min at 4 °C.

Fig. 3.

PrP^res from RML^FFI−WT- and RML^CJD−WT-infected Tga20 mice show a predominance of di-glycosylated PrP. a, b Brain protein extracts from terminally ill Tga20 mice inoculated with RML^WT(I), RML^FFI−WT(I), or RML^CJD−WT(I) (a) or with RML^{FFI−WT(III)} or RML^{CJD−WT(III)} (b) were incubated with the concentrations of PK indicated. PrP was visualized by WB using antibody 12B2 (a) or 6D11 (b). The undigested samples (0 μg/ml PK) represent 25 μg of protein, and the other samples 100 μg. c Percentages of di-glycosylated, mono-glycosylated, and unglycosylated PrP determined by quantitative densitometry of WB like those shown in a and b. Data are from three to nine animals per inoculum and represent pooled first and third passages. p < 0.0001 RML^FFI−WT and RML^CJD−WT vs RML^WT by two-way ANOVA, Tukey’s post hoc test.

Fig. 4.

C57BL6/J mice inoculated with RML^FFI−WT and RML^CJD−WT produce PrP^res glycoforms distinct from those seen in RML^WT-inoculated mice. a Brain protein extracts from C57BL6/J mice inoculated with brain homogenates of RML^WT(I)-, RML^FFI−WT(I)-, and RML^CJD−WT(I)-inoculated Tga20 mice were digested with the concentrations of PK indicated, and PrP was visualized by WB using antibody 12B2. b Western blot analysis of PK-resistant PrP after an additional passage in C57BL/6J mice using antibody 6D11. The undigested samples (0 μg/ml PK) represent 25 μg of protein, and the other samples 100 μg. c Percentages of di-glycosylated, mono-glycosylated, and unglycosylated PrP determined by quantitative densitometry of WB like those shown in a and b. Data are from four to six animals per inoculum and represent pooled first and second passages. p < 0.0001 RML^FFI−WT and RML^CJD−WT vs RML^WT by two-way ANOVA, Tukey’s post hoc test.

Fig. 5.

RML^FFI and RML^CJD are biochemically similar, but different from RML^WT. Protein extracts from uninoculated Tga20 mice (normal brain homogenate, NBH) and from Tga20 mice inoculated with RML^{FFI−WT(III)}, RML^{CJD−WT(III)}, and RML^WT(I) were digested with PK (50 μg/mL), left untreated (upper panels), or incubated with PNGase F (lower panels), and analyzed by WB. PrP^res was detected using anti-PrP antibodies 12B2 (epitope 88–92; a), 6H4 (epitope 143–151; b), SAF84 (epitope 162–168; c), and sPrP²²⁷ (d), which selectively recognizes ADAM10-cleaved shed PrP.

For deglycosylation, 1.1 μL of 10 × denaturing buffer (New England Biolabs) was added to 10 μL of PK-digested lysate, and samples were incubated at 95 °C for 10 min. Subsequently, 1.5 μL of GlycoBuffer 2 (New England Biolabs), 1.5 μL of Nonidet P-40, 0.75 μL of PNGase F (500,000 U/mL; New England Biolabs), and 0.15 μL of H₂O were added, and samples were incubated overnight at 37 °C. Proteins were then mixed with 5 μL of 4 × Laemmli sample buffer (Bio-Rad) and heated at 95 °C for 10 min.

Proteins were resolved on 12% SDS-PAGE gels and transferred onto nitrocellulose membranes (Bio-Rad) using the Trans-Blot Turbo system (Bio-Rad). Transfer efficiency was verified by Ponceau S staining (Sigma-Aldrich). Membranes were washed in Tris-buffered saline containing 0.1% Tween-20 (TTBS) and blocked for 1 h at room temperature in TTBS supplemented with 5% non-fat dry milk (Sigma-Aldrich). Primary antibodies diluted in blocking buffer were incubated for either 1 h at room temperature or overnight at 4 °C. After washing with TTBS, membranes were incubated for 1 h at room temperature with HRP-conjugated goat anti-mouse or anti-rabbit IgG secondary antibodies (Bio-Rad), diluted 1:5000 in blocking buffer. Chemiluminescent signals were detected using Clarity Western ECL substrate (Bio-Rad), acquired with a ChemiDoc MP imaging system (Bio-Rad), and quantified using Image Lab software (Bio-Rad).

The experiments shown in Supplementary Figs. S1 and S2 were performed as follows. Brain homogenates (20% w/v) were prepared in 100 mM Tris–HCl, pH 7.4, containing Complete protease inhibitor cocktail (Roche), as previously described [42]. Mouse BHs were prepared at 10% (w/v) in PBS. After the addition of an equal volume of 100 mM Tris–HCl containing 4% sarkosyl, samples were incubated for 30 min at 37 °C with gentle shaking. PK (Sigma-Aldrich) was then added to a final concentration of 50 μg/mL, and samples were incubated for 1 h at 55 °C with gentle shaking. Proteolysis was terminated by the addition of 6 mM PMSF (Sigma-Aldrich).

Samples were mixed with an equal volume of isopropanol/butanol (1:1, v/v) and centrifuged at 20,000×g for 10 min. Supernatants were discarded, and pellets were resuspended in NuPAGE LDS sample buffer (Invitrogen) supplemented with 50 mM dithiothreitol (NuPAGE Sample Reducing Agent; Invitrogen) and 3 mM PMSF, then heated at 90 °C for 10 min. Proteins were separated on 12% Bis–Tris polyacrylamide gels (Invitrogen) and transferred to polyvinylidene difluoride (PVDF) membranes using the Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were incubated with anti-PrP primary antibodies and HRP-conjugated anti-mouse (Pierce Biotechnology; 1:20,000) or anti-rabbit (Thermo Fisher Scientific; 1:10,000) secondary antibodies using the SNAP i.d. 2.0 system (Millipore). PrP signals were visualized by enhanced chemiluminescence using the ChemiDoc imaging system (Bio-Rad) and quantified with Image Lab software (Bio-Rad).

The following anti-PrP primary antibodies were used: mouse monoclonal antibodies 12B2 (0.5 μg/mL, Wageningen University and Research; epitope moPrP 88–92), 6D11 (0.4 μg/mL, provided by Dr. R.J. Kascsak, New York State Institute for Basic Research, Staten Island, NY, U.S.A.; epitope moPrP 97–100) [25], 9A2 (2.7 μg/mL, Wageningen University and Research; epitope moPrP 98–100), SAF84 (1.2 μg/mL, Bertin Bioreagent; epitope moPrP 159–169), 6H4 (0.4 μg/mL, Prionics; epitope moPrP 144–151) [26]. Rabbit polyclonal sPrP^G227 raised against peptide 221QAYYDG227-COOH (G-COOH represents G227 as the PrP C-terminus exposed after ADAM10-mediated cleavage) was used as described [28].

Conformational stability assay

The conformational stability assay (CSA) was performed as described [40], with minor modifications. Brain homogenates (10% w/v) were mixed 1:1 with 100 mM Tris–HCl, pH 7.4, containing 4% sodium lauroyl sarcosinate (sarcosyl) and incubated 30 min at 37 °C with gentle shaking. Aliquots of 10 μL were mixed with 10 μL of GdnHCl to yield final GdnHCl concentrations of 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, and 4.0 M. Samples were incubated 1 h at 37 °C, then diluted to 0.4 M GdnHCl with buffer. Proteinase-K was added to 50 μg/mL, and samples were incubated 1 h at 37 °C with gentle shaking. The reaction was stopped with 6 mM PMSF. Samples were mixed 1:1 with isopropanol:butanol (1:1 v/v) and centrifuged at 20,000×g for 5 min. Pellets were resuspended in NuPAGE LDS sample buffer containing 50 mM dithiothreitol (NuPAGE Sample Reducing Agent) and 3 mM PMSF, heated 10 min at 90 °C with gentle shaking, and analyzed by WB. Densitometry values from at least three independent experiments were normalized and plotted as a fraction of PrP^res remaining versus [GdnHCl]. Individual denaturation curves were fitted to a four‑parameter logistic equation with variable slope using GraphPad Prism. The [GdnHCl]_1/2 and 95% confidence intervals were obtained from the fits. To compare [GdnHCl]_1/2 values between groups, data sets were fitted either independently or with constrained/shared parameters and compared using an extra sum‑of‑squares F test; the simpler model was retained unless p < 0.05.

Neuropathology

Histology, immunohistochemistry, and thioflavin-S staining were performed on formalin-fixed, paraffin-embedded tissues as previously described [35, 41]. Briefly, brains were fixed in 10% formol saline, embedded in paraffin wax, cut at 6 μm, and stained with hematoxylin and eosin (H&E) and 0.05% thioflavin S.

PrP immunolabeling in immunohistochemistry was performed using polyclonal sPrP^G227 antibody (1:1000) and EP1802Y antibody (Abcam; 1 μg/mL; epitope moPrP 218–227).

Results

RT-QuIC does not detect prion seeding activity in spontaneously ill Tg(FFI) and Tg(CJD) mice

To assess prion seeding activity in the brains of Tg(FFI) and Tg(CJD) mice, we employed an ultrasensitive RT-QuIC assay using recombinant bank vole PrP as substrate [39]. This assay detected all tested types of mammalian brain-derived prions (n > 28) [39]. Brain homogenates from spontaneously ill Tg(FFI) and Tg(CJD) mice, their non-transgenic littermates (Non-Tg), and Tg(WT) mice overexpressing wild-type PrP (two mice per genotype) [5, 12] were tested at a 10^–3 dilution. All samples were negative by the criteria described in the Methods section (i.e., < 50% positive replicate wells) (Fig. 1a). In contrast, human FFI and CJD¹⁷⁸ brain samples, two of which were used for the bank vole inoculation experiments described below, were positive out to 10^–8 (FFI) and 10^–7 (CJD¹⁷⁸) dilutions (Fig. 1a–c). These results indicate that the Tg(FFI) and Tg(CJD) brains had at least 10⁴- or 10⁵-fold less seeding activity than human FFI and CJD¹⁷⁸ brain samples.

Fig. 1.

Lack of bank vole RT-QuIC prion seeding activity in spontaneously ill Tg(FFI) and Tg(CJD) mice. a Comparison of indicated human and mouse brain tissue samples tested at 10^–4 (human; downward triangles) and 10^–3 (mouse; upward triangles) dilutions. The Y-axis represents the inverse of the time taken for a given reaction to reach the positivity threshold, i.e., 1/h to threshold or 1/TTT, used as a metric of amyloid formation rate. Human samples included Alzheimer’s disease frontal cortex (AD; negative control), FFI temporal cortex (TC), FFI thalamus (Th), and CJD¹⁷⁸ TC; the latter two were also used as the bank vole inocula. Mouse samples included (two mice per genotype, labeled a and b): Tg(WT-E1^+/−)/Prnp^0/0 mice, expressing moPrP WT at ~ 2 × physiological level; Tg(FFI-26^+/−)/Prnp^0/0 and Tg(CJD-66^+/−)/Prnp^0/0 mice expressing, respectively, moPrP D177N–M128 and moPrP D177N–V128 also at ~ 2 ×, and their non-transgenic Non-Tg/Prnp^0/0 littermates. The dotted blue line indicates the 1/TTT positivity threshold (0.033), which was assigned to reactions that did not become positive within a 30-h assay time. Bars show the mean ± SD of individual data points. Data are from two independent experiments, each with four replicate reactions, except for human FFI and CJD¹⁷⁸, which were tested twice, each with three replicates. Serial dilution RT-QuIC analyses of FFI Th (b) and CJD¹⁷⁸ (c) human brain homogenates, with four replicate reactions per indicated dilution (relative to solid tissue, not brain homogenate).

Bioassay in bank voles does not detect prion infectivity in spontaneously ill Tg(FFI) and Tg(CJD) mice

We previously found that brain homogenates from Tg(FFI) and Tg(CJD) mice had no detectable infectivity when inoculated into different mouse hosts, including C57BL/6J and Tga20 mice, as well as Tg mice which express low levels of D177N PrP and do not spontaneously become ill [5]. We tested whether prion infectivity could be detected by bioassay in bank voles, which can be infected with a wide range of prions, including those that are difficult to transmit to other laboratory animals, such as sCJD, GSS, variably protease-sensitive prionopathy, and CWD prions [16, 35–37, 41].

Bank voles were intracerebrally inoculated with BH from terminally ill Tg(FFI) and Tg(CJD) mice, and from Non-Tg mice as a negative control inoculum. None of the animals developed neurological dysfunction and either died from intercurrent illness or were euthanized near the end of their normal lifespan (Table 1, line 1–3). Neuropathological and biochemical analyses did not show evidence of neurodegeneration or PK-resistant PrP in the brains of the inoculated bank voles (Supplementary Fig. S1, lanes 1–6).

Table 1.

Transmission assay for infectivity in the brains of spontaneously ill Tg(FFI) and Tg(CJD) mice

In contrast, bank voles inoculated with brain homogenates from FFI and CJD¹⁷⁸ patients developed disease (Table 1, lines 4 and 5) and had PrP^res in their brains (Supplementary Fig. S1, lanes 8–11). The same BH from spontaneously ill Tg(FFI) and Tg(CJD) mice failed to transmit disease to Tga20 mice (Table 1, lines 7 and 8), confirming our previous findings [5].

In vitro-generated RML^FFI and RML^CJD prions can be serially transmitted in Tga20 mice with extended incubation times and cause subclinical infections in C57BL/6J mice

Our previous work demonstrated that RML prions, propagated through serial PMCA using brain homogenates from Tg(FFI) and Tg(CJD) mice as substrates, generated forms of D177N–M128 and D177N–V128 PrPs (hereafter referred to as RML^FFI and RML^CJD) that were highly PK resistant [5]. Tga20 mice intracerebrally inoculated with these PMCA reaction products did not develop neurological signs and were killed more than 540 days post-inoculation (d.p.i.) (Table 2, lines 3 and 8) [5]. Despite the absence of clinical signs, PrP^res was detected in the brains of four out of seven mice inoculated with RML^FFI and five out of nine mice inoculated with RML^CJD (Table 2, lines 3 and 8) [5], indicating the presence of subclinical prion infections. In contrast, RML amplified using C57BL/6J BH as a source of PrP^C (hereafter RML^WT) induced clinical prion disease in Tga20 mice 78 ± 3 d.p.i. with a 100% attack rate (Table 2, line 1) [5].

Table 2.

Serial transmission of RML^WT, RML^FFI and RML^CJD in Tga20 and C57BL/6J mice

The inefficient propagation of RML^FFI and RML^CJD in Tga20 mice may stem from differences in the primary structure of the mutant PrP^Sc in the infecting inoculum and the wild-type (WT) PrP^C in the recipient animals. Alternatively, it could reflect the generation of new prion strains, characterized by slower replication rates.

Serial transmissions may help distinguish between these possibilities: if the incubation time decreases and phenotypically aligns with RML upon further passage, this would suggest a primary role of the sequence mismatch. Conversely, if the incubation time does not decrease, or the phenotype does not converge on RML, this would provide evidence for the emergence of new prion strains.

Therefore, we carried out a secondary transmission experiment in Tga20 mice, which have been shown to faithfully propagate different prion strains, similar to C57BL/6 mice [24]. We inoculated Tga20 mice with 1% BH from RML^FFI- and RML^CJD-inoculated Tga20 mice culled at 611 d.p.i., with clearly detectable PrP^res in their brains (Fig. 2 and Table 2). These WT-adapted inocula were named, respectively, RML^FFI−WT(I) and RML^CJD−WT(I).

Fig. 2.

Scheme of the serial inoculations of PMCA-generated RML prions in Tga20 and C57BL/6J mice. The brains of Tga20 mice inoculated with the PMCA reaction products were used to prepare the inocula for serial passage in Tga20 and C57BL/6J mice. The progressive Roman numerals in parentheses refer to the names of the inocula used for serial passage (see nomenclature in Table 2).

Despite complete sequence identity between PrP^Sc in the infecting inoculum and PrP^C in the recipient mice—both are wild-type moPrP—transmission took very long, with incubation times of 478 ± 19 for RML^FFI−WT(I) and 357 ± 10 for RML^CJD−WT(I) (Table 2, lines 4 and 9). For comparison, RML^WT(I) induced disease 61 ± 1 d.p.i. (Table 2, line 2). Incubation times of both RML^FFI−WT and RML^CJD−WT significantly shortened during the two subsequent passages in Tga20 mice, then stabilized to approximately 320 d.p.i. (Table 2, lines 5–7 and 10–12). However, with subsequent passages in Tga20 mice, there was a prolongation of the symptomatic phase of the disease from ~ 30 to ~ 75 days.

Analysis of PK-resistant PrP indicated that RML^FFI−WT and RML^CJD−WT prions had a glycosylation pattern clearly distinct from that of RML^WT, with a predominance of the di-glycosylated band that was maintained in subsequent passages (Fig. 3).

RML^WT(I), RML^FFI−WT(I), and RML^CJD−WT(I) were also inoculated in C57BL/6J mice (Fig. 2). Inoculation of RML^WT(I) induced disease at 139 ± 1 d.p.i., with a 100% attack rate (Table 2, line 13). In contrast, none of the animals inoculated with RML^FFI−WT(I) and RML^CJD−WT(I) developed clinical signs and were killed at > 650 d.p.i. (Table 2, lines 14 and 17).

Biochemical analysis found PK-resistant PrP in the brains of five out of six mice inoculated with RML^FFI−WT(I) and one out of three inoculated with RML^CJD−WT(I) (Table 2, lines 14 and 17), showing an overrepresentation of the di-glycosylated band (Fig. 4A), similar to what was observed in Tga20 mice (Fig. 3). Brain homogenates from these mice were used for secondary inoculation in C57BL/6J mice. Again, the inoculated mice did not develop clinical signs and were killed at > 620 d.p.i. (Table 2, lines 15 and 18). Tertiary inoculation likewise failed to produce clinical disease, although PrP^res was detectable in the brains of most mice analyzed (Table 2, lines 16 and 19).

Collectively, these data indicate that D177N–M128 and D177N–V128 PrPs are not inherently resistant to PrP^Sc conversion, as they can adopt infectious conformations through RML-seeded PMCA and be serially transmitted to Tga20 and C57BL/6J mice. However, the prolonged incubation times and distinct PrP^res glycosylation patterns suggest that RML^FFI−WT and RML^CJD−WT may constitute new prion strains.

RML^FFI−WT and RML^CJD prions are biochemically similar, but markedly different from RML

Brain extracts from FFI and CJD¹⁷⁸ patients contain PrP^res isoforms with different glycosylation patterns and PK-resistant fragments of different sizes [32], consistent with the idea that PrP D178N–M129 and D178N–V129 generate distinct prion strains. To test whether PMCA-generated RML^FFI and RML^CJD represented different prion strains, we analyzed the PK-resistant PrP fragments generated after inoculation in Tga20 mice (hereafter, the specific passage is indicated only in the figure legends) by WB using anti-PrP antibodies with experimentally verified epitopes [53].

Brain protein extracts from RML^FFI-, RML^CJD-, and RML^WT-inoculated Tga20 mice were PK digested, and either left untreated or incubated with PNGase F to enzymatically remove all N-linked glycans, and analyzed by WB using anti-PrP antibodies 12B2 (epitope 88–92), 6H4 (epitope 143–151), SAF84 (epitope 162–168), and sPrP²²⁷, which selectively recognize ADAM 10-cleaved shed PrP [28]. After deglycosylation, a characteristic ∼ 18 kDa band corresponding to the typical N-terminally cleaved PrP^res (∼ 90–230) was readily detectable in all inoculated mice by 12B2, 6H4, and SAF84 (Fig. 5a–c, lower panels). Notably, SAF84 also detected an additional ∼ 14 kDa band exclusively in RML^FFI- and RML^CJD-inoculated mice, clearly distinguishing them from RML^WT (Fig. 5c). The sPrP²²⁷ antibody identified a ∼ 16 kDa band in RML^FFI- and RML^CJD-, but not in RML^WT-inoculated animals (Fig. 5d, lower panel). Quantitative analysis demonstrated that RML^CJD- and RML^FFI-accumulated 20- to 30-fold higher levels of shed PrP^res than RML^WT-inoculated mice (Supplementary Fig. S2).

A further comparison of the biochemical properties of RML^WT, RML^FFI, and RML^CJD was made using CSA, which measures the decrease in resistance to PK of PrP^Sc after exposure to increasing concentrations of GdnHCl [40]. To detect both the typical ~ 18 kDa N-terminally cleaved fragment and the ~ 14 kDa C-terminal fragment prominent in RML^FFI and RML^CJD, PrP^res was detected with mAbs 9A2 (epitope 98–100; detects PrP^res cleaved N-terminal to residue 100) and SAF84 (epitope 162–168; detects C-terminal PrP^res fragments).

Unexpectedly, the PrP^res profile of RML^WT differed from previous experiments because of a prominent presence of C-terminal fragments produced by PK treatment of GdnHCl-pretreated samples (Fig. 6). This indicates that low doses of GdnHCl can partially destabilize the N-terminal portion of the PK-resistant PrP^Sc core, producing region-dependent resistance to denaturation in RML^WT: residues ~ 80–90 to ~ 140–150 became sensitive to PK and destabilized at 1.5 M GdnHCl (Fig. 6, 9A2), while the C-terminal region remained highly resistant (Fig. 6, SAF84). Similar behavior has been reported for the parental scrapie strain Chandler [47].

Fig. 6.

Conformational stability assay of PrP^Sc from Tga20 mice infected with RML^WT, RML^FFI, or RML^CJD. a Representative WBs of brain homogenates of RML^{FFI−WT(III)}-, RML^{CJD−WT(III)}- and RML^WT(I)-inoculated Tga20 mice probed with 9A2 (top panels) or SAF84 (bottom panels). GdnHCl concentrations: 1, 1.5, 2, 2.5, 3, 3.5, and 4 M. b Dose–response curves best fitted with a four-parameter logistic equation. Data points are mean ± SD of at least three independent determinations. [GdnHCl]_1/2 values (95% CI): with 9A2, RML^WT 1.4 (0.8–2.0), RML^FFI 2.7 (2.4–3.0), RML^CJD 2.6 (2.1–3.0); with SAF84, RML^WT 3.1 (2.9–3.4), RML^FFI 2.8 (2.6–3.0), RML^CJD 2.6 (2.3–2.9). For RML^FFI and RML^CJD, [GdnHCl]_1/2 values did not differ significantly between antibodies (p = 0.75 for RML^FFI; p = 0.39 for RML^CJD) or between the two strains (p = 0.17 with 9A2; p = 0.18 with SAF84). For RML^WT, significant differences were detected between the two antibodies (p < 0.0001) and in comparison with RML^FFI or RML^CJD (p < 0.0001 for both strains with both antibodies).

By contrast, the PrP^res profiles of RML^FFI and RML^CJD were not substantially affected by GdnHCl pre-treatment. The prominent ~ 14 kDa PrP^res observed without GdnHCl pre-treatment was preserved after GdnHCl exposure (Figs. 5, 6). RML^FFI and RML^CJD showed overlapping denaturation profiles when probed with 9A2 or SAF84 (Fig. 6), and with sPrPG227, which selectively detects the shed PrP^res component (Supplementary Fig. S3). There was no evidence of region-specific susceptibility to denaturation for these strains.

Comparing conformational stability with 9A2 and SAF84 showed that [GdnHCl]_1/2 values were not significantly different between RML^FFI and RML^CJD, whereas both strains differed significantly from RML^WT (Fig. 6).

RML^FFI and RML^CJD induce similar neuropathological lesions markedly different from RML^WT

Neuropathological and immunohistochemical evaluation of RML^FFI and RML^CJD serially passaged in Tga20 mice showed the hallmarks of prion diseases, such as vacuolation (spongiosis), neuronal loss, gliosis, and PrP^Sc deposition. RML^FFI- and RML^CJD-inoculated Tga20 mice were characterized by similar spongiform change distribution and PrP^Sc deposition patterns, which were distinct from RML^WT-inoculated Tga20 mice.

Intense spongiosis was observed in the cerebral and cerebellar cortices of RML^FFI- and RML^CJD-inoculated Tga20 mice (Fig. 7a, b), while the cortices and cerebellum were unaffected in RML^WT-inoculated Tga20 mice (Fig. 7c). Subcortical regions, such as the thalamus, hippocampus, and midbrain, were characterized by moderate spongiform change in all groups.

Fig. 7.

Neuropathological features of RML^FFI-, RML^CJD-, RML^WT-inoculated Tga20 mouse brains. Brain sections from Tga20 mice inoculated with RML^{FFI−WT(III)} (a, d, g, l, n), RML^{CJD−WT(III)} (b, e, h), and RML^WT(I) (c, f, i, o, m) were analyzed by H&E staining (a–f), PrP immunohistochemistry (g–i, m–o), and thioflavin-S staining (l). Spongiform changes affecting the molecular layer of the cerebellum are visible in RML^{FFI−WT(III)}- and RML^{CJD−WT(III)}-inoculated Tga20 mice (a, b, respectively). Large submeningeal and perivascular plaques (black arrows) were observed in RML^{FFI−WT(III)}-inoculated mice (d, g, l) and RML^{CJD−WT(III)}-inoculated mice (e, h) by H&E staining (d, e), PrP immunohistochemistry (g, h), and thioflavin-S staining (l). Insets in panels d, e, g, h, and l show higher magnification views of the areas marked by asterisks. In the cerebellum of RML^WT(I)-inoculated Tga20 mice, no pathological changes (c, f) or PrP^Sc deposition (i, m) were detected. Small deposits (red arrows in n), intraneuronal (arrowheads in o), and glia-associated (black arrows in o) PrP^Sc accumulations were found in the cerebellum of RML^{FFI−WT(III)}-inoculated mice (n) and in the midbrain of RML^WT(I)-inoculated mice (o). Scale bars: 50 μm in a–c, m–o; 100 μm in d–l. ML, molecular layer; GL, granular layer

Strikingly, RML^FFI- and RML^CJD-inoculated Tga20 mice accumulated numerous and large submeningeal and perivascular plaques, readily observed by H&E staining (Fig. 7d, e). These plaques showed intense immunoreactivity to anti-PrP antibody (Fig. 7g, h) and were thioflavin S positive (Fig. 7l). No plaques were observed in RML-inoculated Tga20 mice (Fig. 7f, i).

A variety of PrP^Sc deposition patterns was observed in the brain parenchyma of all groups, including diffuse/punctate, small deposits, glial-associated, peri- and intraneuronal areas (Fig. 7n, o). However, the brain areas affected differed between the RML^FFI/RML^CJD and RML^WT groups. For example, intense PrP^Sc deposition was observed in the granular and molecular layers of the cerebellar cortex in RML^FFI- and RML^CJD-inoculated Tga20 mice (Fig. 7n), yet absent in RML^WT-inoculated Tga20 mice (Fig. 7m).

Given the unexpectedly high amount of shed PrP^Sc in the brain extracts of RML^FFI- and RML^CJD-inoculated Tga20 mice (Supplementary Fig. S2), we assessed the in situ deposition of shed PrP^Sc by immunohistochemistry using the anti-sPrP^G227 antibody. Abundant shed PrP^Sc deposits were readily detectable in the brains of RML^FFI- and RML^CJD-inoculated Tga20 mice, whereas immunoreactivity was less intense in the brains of RML^WT-inoculated Tga20 mice. The pattern of shed PrP^Sc deposition differed from that of total PrP^Sc, as assessed using the EP1802Y antibody. Specifically, in the brains of RML^WT-inoculated Tga20 mice, only PrP^Sc deposits along the alveus were labeled with anti-sPrP^G227 antibody (Fig. 8a). In contrast, in the brains of RML^FFI- and RML^CJD-inoculated mice, all the submeningeal and perivascular plaques observed with the EP1802Y antibody showed intense immunoreactivity to anti-sPrP^G227 antibody (Fig. 8b, c). Conversely, PrP^Sc deposits in the brain parenchyma were either unlabeled or only weakly stained with anti-sPrP^G227 antibody in certain brain regions of RML^FFI- and RML^CJD-inoculated mice (Fig. 8d, e).

Fig. 8.

Comparison between in situ deposition of total PrP^Sc detected with EP1802Y antibody and shed PrP^Sc detected with the anti-sPrP^G227 antibody. In each panel, the left image represents the EP1802Y-labeled brain section, while the right image shows the section labeled with sPrP^G227. In RML^WT(I)-inoculated Tga20 mouse brains, shed PrP^Sc deposits were detected along the alveus (a). Submeningeal and perivascular plaques were readily detected with both antibodies in the midbrain (b) and cerebellum (c) of RML^{FFI−WT(III)}-inoculated mice. In contrast, the parenchyma of the piriform cortex (d) and the prefrontal cortex (e) of RML^{FFI−WT(III)}-inoculated mice displayed weak or no immunoreactivity to the anti-sPrP^G227 antibody, respectively. In RML^{CJD−WT(III)}-inoculated mice, the immunolabeling patterns observed with both EP1802Y and sPrP^G227 antibodies were similar to those of RML^{FFI−WT(III)}-inoculated mice (not shown). In panels d–e, intraneuronal PrP^Sc deposits are indicated by arrows. Scale bars: 50 μm in a, d and e; 100 μm in b and c. Ctx, cortex; alv, alveus; CA1, Ammon’s horn 1; CA3, Ammon’s horn 3; GENd, geniculate group, dorsal thalamus; ML, molecular layer; GL, granular layer

Discussion

In this study, we investigated the transmission properties of prions generated in vitro through RML-templated conversion of mouse PrP homologs of the D178N–M129 and D178N–V129 mutations linked to FFI and genetic CJD¹⁷⁸. Serial transmission studies indicated that the D178N mutation drives the emergence of novel prion strains with transmission, biochemical, and neuropathological properties markedly different from RML that do not mimic FFI and CJD¹⁷⁸. Unlike spontaneously formed D178N prions in the brains of FFI and CJD¹⁷⁸ patients, whose biochemical characteristics and pathogenic properties are influenced by the M129V polymorphism, RML-seeded D178N prions are unaffected by this sequence variation. This suggests that spontaneous and templated conversion of PrP D178N may follow different mechanistic pathways.

Our previous studies showed that Tg(FFI) and Tg(CJD) mice accumulate aggregated, mildly PK-resistant forms of mutant PrP in their brains and spontaneously develop neurological illnesses that mimic key phenotypic aspects of human FFI and CJD¹⁷⁸ [5, 17]. However, these diseases were not horizontally transmissible to other mice, and no spontaneously generated PrP^Sc was detectable in Tg(FFI) and Tg(CJD) brains using PMCA [5, 14]. Here, we confirmed the absence of spontaneously generated prions by RT-QuIC and bioassay in bank voles. In contrast, PMCA and RT-QuIC readily detected PrP^Sc seeding activity in brain homogenates from FFI and CJD¹⁷⁸ patients ([39, 44] and this study), and bioassay in bank voles confirmed the infectious nature of these genetically determined diseases, consistent with previous inoculation studies in wild-type and transgenic mice [6, 51, 52].

To investigate whether acquiring self-replication competency could endow moPrP D177N–M128 and D177N–V128 with FFI- and CJD¹⁷⁸-specific biochemical features, potentially improving their capacity to more accurately model the human diseases, we sought to induce their conversion to a bona fide PrP^Sc state.

Previous studies showed that inoculating RML into Tg(PG14) mice that express a mutant PrP with a nine-octapeptide repeat insertion transformed their spontaneous, non-infectious neurological illness into a transmissible prion disease [13]. This transformation was marked by a shorter incubation time, accelerated disease progression, and additional neuropathological changes, including spongiform degeneration and larger, coarser PrP^res accumulations, absent in spontaneously ill Tg(PG14) mice. These changes were accompanied by the conversion of mildly PK-resistant PG14 PrP into a highly PK-resistant, more tightly aggregated form capable of seeding wild-type PrP conversion in vivo and in vitro [3, 13].

Attempts to transmit RML to Tg(FFI) and Tg(CJD) mice by intracerebral inoculation, however, were unsuccessful. RML-inoculated Tg(FFI) and Tg(CJD) mice showed no clear differences in disease course compared to uninoculated controls, and no spongiosis, PrP deposits, or highly PK-resistant PrP^Sc were detectable in their brains when killed at an advanced stage of their spontaneous disease (Tg(FFI): 619 ± 15 days of age, i.e., 570 ± 16 days post‑RML inoculation, n = 5; Tg(CJD): 297 ± 0 days of age, i.e., 262 ± 0 days post‑RML inoculation, n = 5) (Supplementary Fig. S4). In contrast, RML-seeded PMCA of Tg(FFI) and Tg(CJD) brain homogenates generated highly PK-resistant D177N–M128 and D177N–V128 PrP species that efficiently converted wild-type PrP in Tga20 mice [5]. Thus, although the D177N mutation strongly impairs RML infection in vivo, these PrP variants are not intrinsically conversion incompetent.

Several factors may explain the discrepancy between the efficient RML‑templated conversion of D177N PrP in PMCA and the inability of RML to infect Tg(FFI) and Tg(CJD) mice. PMCA creates a highly permissive environment that can amplify rare or metastable conformers that cannot propagate under physiological conditions [9, 21], so RML may template D177N PrP in vitro despite strong conformational barriers in vivo. Host‑specific cellular factors, for example, chaperones, cofactors, clearance pathways, and membrane contexts, likely constrain prion replication in living animals, but are absent in PMCA. Finally, the aggregated, mildly PK‑resistant mutant PrP species that accumulate in Tg(FFI) and Tg(CJD) brains may interfere with or competitively inhibit RML replication in vivo, whereas in vitro they may instead facilitate conformational templating events.

Serial transmission studies showed that the in vitro-generated RML^FFI and RML^CJD prions caused transmissible diseases that bore no resemblance to human FFI and CJD¹⁷⁸, and diverged substantially from the parental RML strain. RML^FFI and RML^CJD displayed prolonged incubation times in Tga20 mice, limited transmissibility to C57BL/6J mice, and produced similar PrP^Sc types regardless of the M129V polymorphism. Both strains generated large amounts of shed PrP that accumulated in large submeningeal and perivascular amyloid plaques—a feature not seen in RML^WT-inoculated mice, and reminiscent of mouse-adapted CWD prions [1, 46]. These findings suggest preferential recruitment of ADAM10-cleaved shed PrP during extracellular conversion.

The atypical characteristics of RML^FFI and RML^CJD were not attributable to de novo prion formation during PMCA, as no highly PK-resistant PrP^Sc capable of replicating in Tga20 mice was produced in unseeded PMCA reactions of Tg(FFI) and Tg(CJD) brain homogenates run in parallel [5]. They were also unlikely to be artifacts of the in vitro replication process, as PMCA-propagated RML^WT retained the canonical biochemical profile and neuropathological features of standard RML, consistent with evidence that PMCA with homologous PrP^C substrates preserves strain-specific characteristics [10, 21]. Instead, the distinctive features of RML^FFI and RML^CJD arose from RML replication on the moPrP D177N substrate, a process reminiscent of prion strain mutation during interspecies transmission or in vitro adaptation to non-homologous PrP^C substrates [2, 9, 21]. Mechanistically, this strain shift could result from a selection process where RML conformers that replicate more efficiently on moPrP D177N become dominant [15]. Alternatively, it may reflect structural remodeling of RML PrP^Sc during conversion, leading to novel conformers with altered pathogenic and transmission properties. Notably, the Tg(FFI) and Tg(CJD) brains used as PMCA substrates were from ~ 200-day-old animals, and contained substantial amounts of aggregated, mildly PK-resistant mutant PrP [5]; moreover, even monomeric D177N PrP adopts an abnormal structure [50]. Such abnormal substrates likely facilitated the emergence of novel PrP^Sc conformers.

A notable distinctive characteristic of RML^FFI and RML^CJD is the predominance of di-glycosylated PrP^res, already evident in the PMCA products [5] and maintained upon serial transmission in Tga20 and C57BL/6J mice. This suggests the selection of a strain that preferentially recruits di-glycosylated PrP^C. Previous studies indicate that host PrP^C glycoform stoichiometry influences PrP^Sc formation efficiency in vitro, with RML typically favoring unglycosylated PrP^C [33]. Since the mutant PrPs in Tg(FFI) and Tg(CJD) mice are predominantly di-glycosylated with minimal unglycosylated species [5, 17], this glycoform profile likely favored the emergence of a strain that preferentially converts di-glycosylated PrP^C. Notably, di-glycosylated PrP^C has also been shown to be the preferred substrate for ADAM10 in the physiological shedding process at the cell surface [28]. The persistence of this glycoform preference, and the inability of classical RML to re-emerge, indicates a stable strain shift.

While RML^FFI and RML^CJD prions successfully replicated in both Tga20 and C57BL/6J mice, they did not induce clinical disease in the latter. This may be due to their slow replication rate, as these strains require over 300 days to cause disease in Tga20 mice, compared to approximately 60 days for RML^WT. Additionally, the large amyloid plaques they produce may be less neurotoxic than smaller, more diffusible PrP^Sc oligomers [19, 48] or may even be neuroprotective by sequestering toxic oligomers in a biologically inert state—a process in which shed PrP may play a role [27, 31, 49].

The differences between spontaneous D178N conversion in FFI and CJD¹⁷⁸ patients and the RML-seeded conversion observed here suggest that these processes may follow distinct misfolding pathways. Spontaneous prion formation in genetic prion disease occurs without an exogenous seed and reflects the intrinsic misfolding trajectory of the mutant PrP in its physiological environment. In this setting, cellular determinants such as interacting cofactors, glycoform availability, and proteostatic handling likely determine which misfolded conformers emerge and persist, helping explain why D178N-M129 and D178N-V129 produce the distinct phenotypes of FFI and CJD¹⁷⁸.

By contrast, seeded conversion is initiated by the conformational information imposed by the exogenous PrP^Sc seed. The mutant D177N substrate does not simply adopt the RML fold; instead, it constrains and reshapes the range of permissible misfolded conformers, yielding novel PMCA-derived strains (RML^FFI and RML^CJD) that diverge from both RML and the native conformers associated with human D178N disease. Thus, infectious conversion reflects an interplay between seed-driven templating and substrate-imposed structural restrictions, whereas spontaneous conversion reflects the intrinsic folding preferences of the mutant protein modulated by the cellular milieu.

Interestingly, recombinant PMCA studies show that the parameters of the in vitro conversion process can profoundly influence misfolding outcomes of mutant PrP substrates, generating either convergent or sequence-specific PrP^Sc conformations [34, 54]. It would be interesting to test whether subjecting Tg(FFI) and Tg(CJD) brain homogenates to PMCA conditions different from those used in this study [18] might generate prion strains that more closely recapitulate human FFI and CJD¹⁷⁸. Such strains would enable the development of “infectious” mouse models of these diseases and allow direct comparison with spontaneously ill Tg(FFI) and Tg(CJD) mice, providing insights into how mutant PrP^Sc replication directs disease phenotypes.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

A.M., A.G., L.C., and G.L. performed clinical examinations and postmortem sampling in mice. R.B. and M.A.D.B. performed neuropathological analyses. A.M., A.G., L.C., I.V., and R.N. performed biochemical analyses of PrP. C.D.A. performed inoculations, clinical examinations, and postmortem sampling in bank voles. C.D.O. performed RT-QuIC analyses. B.C. supervised RT-QuIC studies. H.C.A. provided expertise in the analysis of shed PrP and the sPrP^G227 antibody. J.C. supervised PMCA studies. G.G. and F.T. characterized the FFI and CJD¹⁷⁸ cases and provided brain specimens for bank vole inoculations and RT-QuIC analyses. R.N. supervised transmission studies in bank voles. R.C. performed inoculations and supervised transmission studies in mice. R.N. and R.C. conceived and directed the study. A.M., M.A.D.B., R.N., and R.C. wrote the manuscript. R.B., C.D.O., B.C., H.C.A., and J.C. edited the manuscript.

Funding

This work was supported by the Italian Ministry of Health (RF-2016-02362950) and in part by the Division of Intramural Research of the NIAID.

Data availability

Data is provided within the manuscript or supplementary information files.

Declarations

Conflict of interest

The authors declare no competing interests.