Vidal et al. Acta Neuropathologica Communications (2022) 10:179 https://doi.org/10.1186/s40478-022-01477-7 RESEARCH Open Access Bona fide atypical scrapie faithfully reproduced for the first time in a rodent model Enric Vidal1,2†, Manuel A. Sánchez‑Martín3†, Hasier Eraña4,5,6, Sonia Pérez Lázaro7, Miguel A. Pérez‑Castro4, Alicia Otero7, Jorge M. Charco4,5,6, Belén Marín7, Rafael López‑Moreno4, Carlos M. Díaz‑Domínguez4, Mariví Geijo8, Montserrat Ordóñez1,2, Guillermo Cantero1,2, Michele di Bari9, Nuria L. Lorenzo10, Laura Pirisinu9, Claudia d’Agostino9, Juan María Torres11, Vincent Béringue12, Glenn Telling13, Juan J. Badiola7, Martí Pumarola14, Rosa Bolea7, Romolo Nonno9, Jesús R. Requena10 and Joaquín Castilla4,6,15*    Abstract Atypical Scrapie, which is not linked to epidemics, is assumed to be an idiopathic spontaneous prion disease in small ruminants. Therefore, its occurrence is unlikely to be controlled through selective breeding or other strategies as it is done for classical scrapie outbreaks. Its spontaneous nature and its sporadic incidence worldwide is reminiscent of the incidence of idiopathic spontaneous prion diseases in humans, which account for more than 85% of the cases in humans. Hence, developing animal models that consistently reproduce this phenomenon of spontaneous PrP misfolding, is of importance to study the pathobiology of idiopathic spontaneous prion disorders. Transgenic mice overexpressing sheep ­PrPC with I112 polymorphism (TgShI112, 1–2 × PrP levels compared to sheep brain) manifest clinical signs of a spongiform encephalopathy spontaneously as early as 380 days of age. The brains of these animals show the neuropathological hallmarks of prion disease and biochemical analyses of the misfolded prion protein show ­ rPres pattern with a predominant 7–10 kDa band. Brain homogenates from spontaneously diseased a ladder-like P transgenic mice were inoculated in several models to assess their transmissibility and characterize the prion strain generated: TgShI112 (ovine I112 ARQ ­PrPC), Tg338 (ovine VRQ ­PrPC), Tg501 (ovine ARQ ­PrPC), Tg340 (human M129 P ­ rPC), C C C Tg361 (human V129 ­PrP ), TgVole (bank vole I109 ­PrP ), bank vole (I109I ­PrP ), and sheep (AHQ/ARR and AHQ/AHQ churra-tensina breeds). Our analysis of the results of these bioassays concludes that the strain generated in this model is indistinguishable to that causing atypical scrapie (Nor98). Thus, we present the first faithful model for a bona fide, transmissible, ovine, atypical scrapie prion disease. Keywords: Atypical, Scrapie, Spontaneous, Prion disease, Isoleucine Introduction Transmissible spongiform encephalopathies (TSE) or prion diseases are a group of transmissible neurodegen- erative diseases associated with the misfolding of the † endogenous cellular prion protein ­(PrPC) into a patho- Enric Vidal and Manuel A. Sánchez-Martín contributed equally to the manuscript genic isoform, termed P ­ rPres, which is partially resistant to proteases and induces neurotoxicity [64]. This misfold- *Correspondence: jcastilla@cicbiogune.es ing event can be spontaneous, either idiopathic (with- 4 Centro de Investigación Cooperativa en Biociencias (CIC BioGUNE), out a known cause), linked with a mutation in the gene Laboratorio de Investigación de Priones, Basque Research and Technology Alliance (BRTA), Derio, Bizkaia, Spain encoding ­PrPC (PRNP), or acquired through exposure to Full list of author information is available at the end of the article external sources of misfolded prions [9, 15, 16, 31, 38, 59]. © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. 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Acta Neuropathologica Communications (2022) 10:179 Page 2 of 22 Acquired animal prion diseases, such as scrapie in small phenomenon [9, 59], as reflected by the low incidence of ruminants or chronic wasting disease (CWD) in cervids, spontaneous idiopathic prion diseases and the difficul- can be transmitted horizontally with high efficiency, ties of reproducing genetic diseases in animals through while others, such as BSE, rely on human interventions introduction of human PRNP mutations. Nonetheless, (recirculation of meat and bone meal contaminated with a few models have succeeded in reproducing genetic prions, for instance) [77, 79]. Over the years, several sup- mutation-based prionopathies. These include the ki- posedly idiopathic spontaneous prion diseases have also 3F4-FFI mice that harbour the D177N mutation, equiva- been described in animals. These are known as atypical lent to the human mutation responsible for FFI [43] and BSE cases in cattle, namely BSE-L [18] and BSE-H [12], the D178N Tg(FFI) also reproducing FFI phenotype [14], and the atypical scrapie or Nor98-like cases in small the Tg(A116V) mice mimicking A117V GSS [84], the ruminants [11]. In the case of BSE-L, a clear potential for TgMHuME199K mimicking E200K genetic CJD [30], the zoonotic risk has been established [48, 81]. However, in 113LBoPrP-Tg mice that incorporated the P113L substi- contrast to human prion diseases, no genetic cases have tution, homologous to the P102L mutation causing GSS been described in animals to date. in humans in a bovine PRNP transgenic mouse model Nor98 cases are of particular interest since they have [70], the P102L mutation of GSS in the murine prnp [68] been linked to the emergence of the BSE agent when or the 117VVTg30 mice also mimicking A117V GSS inoculated in a host with bovine [41] or porcine ­PrPC [6]. Despite the fact that all these models succumb to a [27, 49]. The cause of Nor98 cases has not been yet estab- spontaneous neurological illness related to misfolded PrP lished, and although some cases are associated with accumulation, these misfolded products are poorly infec- specific polymorphisms in the PNRP gene, such as the tious, if infectious at all in wild type animals, where they presence of phenylalanine in codon 141 [53] or the pres- show incomplete attack rates or require multiple serial ence of histidine in codon 154 [21], these polymorphisms inoculations, raising doubts on their infectious or trans- do not explain all cases. Thus, an idiopathic spontaneous missible nature or indicating that homotypic mutant PrP origin is the current hypothesis. Atypical/Nor98 scra- expression is required for propagation. pie is a worldwide distributed disease occurring in small Moreover, in spite of the relative success of the previous ruminants even in classical scrapie-free regions such as models in mimicking genetic prion diseases, the study of Australia [22]. Thus, despite the apparently low horizon- spontaneous idiopathic prion disease in animal models, tal transmission capacity of these atypical prions, which which occurs in absence of mutations in the PRNP gene, do not seem to be related to outbreaks unlike those that is more challenging. The most successful approach to cause classical scrapie, their spontaneous occurrence date explores the particular susceptibility of bank voles remains a concern given their potential transmissibility (Myodes glareolus) to most prion strains [1, 7, 54, 75] by and potential alterations upon transmission. producing transgenic mouse models with the bank vole Apart from their origin and horizontal transmission ­PrPC. These models, particularly when ­PrPC is overex- ability, there are several other differences between clas- pressed, spontaneously develope a bona fide transmissi- sical and atypical scrapie regarding clinical, neuropatho- ble prion disease that is determined by the presence of logical, and biochemical features. For instance, instead of an isoleucine in position 109 [56, 57, 76]. The other natu- showing the classical three-banded pattern on Western rally occurring polymorphism at that position, M109, has blotting upon proteinase K (PK) digestion, corresponding not been linked to spontaneity [17, 76]. Other authors to the three 27–30 kDa ­PrPres glycoforms, Nor98-associ- however, demonstrated that the M109 polymorph, when ated ­PrPres is characterized by an enhanced PK sensitivity overexpressed, was also associated to ­PrPres deposition, and a multi-banded or ladder-like pattern with a predom- although this ­PrPres was not infectious, and proposed inant band of 7–14 kDa. A similar atypical biochemical other polymorphisms of the bank vole PRNP as responsi- signature, which reflects a different structural arrange- ble for its enhanced susceptibility and tendency towards ment [5], has also been described in other human disor- spontaneous misfolding [44]. ders of idiopathic or genetic origin [32, 55, 63, 67]. Interestingly, the presence of isoleucine in this position Although animal models and, most importantly, is a naturally occurring polymorphism not only in bank transgenic mouse models have been essential to study vole, but in at least 10 mammal species including cattle acquired prion disorders through experimental inocu- [86], horse [87], skunk [88] and also in sheep, described lations, their ability to model genetic or spontaneous in the Tibetan breed [85]. Albeit to date no cases of spon- idiopathic prion disorders is more limited. Mimicking taneous prion disease are reported in animals with this the main event underlying these diseases, which con- polymorphism. sists of the putatively spontaneous misfolding of P ­ rPC Due to the lack of animal models that mimic a sponta- res into ­PrP , is hindered by the low frequency of such neous prionopathy in domestic animals, our goal was to Vidal et al. Acta Neuropathologica Communications (2022) 10:179 Page 3 of 22 explore whether the equivalent polymorphism in sheep ACA​ GGT​ GGA​ TAA​ CC 3’). Those which tested posi- (i.e. I112) would yield an ovine ­PrPC more prone to spon- tive were bred to mice null for the mouse Prnp gene in taneous misfolding in a transgenic mouse model. order to avoid endogenous expression of mouse prion As predicted, our mouse model TgShI112 sponta- protein. Absence of the mouse endogenous Prnp was neously developed a prion disease that, interestingly, assessed using the following primers: 5’ ATG​GCG​AAC​ presented strain features indistinguishable to that of CTT​GGC​TAC​TGGC 3’ and 5’ GAT​TAT​GGG​TAC​CCC​ atypical (Nor98) scrapie, providing the first animal model CTC​CTTGG 3’. The sheep PrP expression levels of brain of clearly transmissible and atypical prion disease in homogenates from transgenic mouse founders were absence of pathology-associated mutations. determined by Western blot using anti-PrP mAb D18 [46] and compared with the PrP expression levels from Materials and methods sheep brain homogenates. Generation of TgShI112 mouse lines 460 and 445 The international code to identify these transgenic extracted using GeneJET™ Genomic DNA Purification After isolation by PCR amplification from genomic DNA, mouse lines are 129OLA-Prnptm2Edin-Tg(sheepPrnp)1/ Sal and 129OLA-Prnptm2Edin-Tg(sheepPrnp)2/Sal Kit (Fermentas) from a sheep brain tissue sample using although throughout the article they are referred to as 5’ CCG​GAA​TTC​CGG​CGT​ACG​ATG​GTG​AAA​AGC​ TgShI112 (L460) and TgShI112 (L456) mice, respectively. CAC​ATA​GGC 3’ and 5’ CTA​GTC​TAG​ACT​AGG​CCG​ ­ rPC expression lev- The two lines have slightly different P GCC​CTA​TCC​TAC​TAT​GAG​AAA​AAT​G 3’ as primers, els (Additional file 1: Fig. S1). the open reading frame (ORF) of the sheep PRNP gene with A136, R154 and Q171 polymorphisms was cloned Preparation of inocula into the pDrive vector (Qiagen), being the sequence 10% brain homogenates (w/V) from sheep diagnosed identical to that of the Genbank entry NP_001009481.1. of atypical scrapie in the PRIOCAT laboratory, IRTA- The genetic construct containing the sheep M112I sub- CReSA (Barcelona, Catalonia), sheep infected with stitution was carried out by two-step PCR site-directed SSBP/1 (TSE Resource Centre, University of Edinburgh) mutagenesis using the pDrive sheep construct as tem- and BSE infected sheep (SheepBSE, kindly provided by plate, using primers 5’ CCG​GAA​TTC​CGG​CGT​ACG​ Dr.Olivier Andreoletti, UMR INRAE-ENVT, Toulouse) ATG​GTG​AAA​AGC​CAC​ATA​GGC 3’ with 5’ GCT​CCT​ were prepared manually using a glazed mortar and pestle GCC​ACA​TGC​TTA​ATG​TTG​GTT​TTT​GG 3’ and 5’ in Phosphate buffered saline (PBS, Fisher Reagents) with CTA​G TC​TAG​ACT​AGG​C CG​G CC​C TA​TCC​TAC​TAT​ Complete Protease inhibitor cocktail (Roche) and further GAG​AAA​AAT​G 3’ with 5’ CCA​AAA​ACC​AAC​ATT​ diluted to 1% in Dulbecco’s PBS (DPBS, Gibco) for direct AAG​CAT​GTG​GCA​GGA​GC 3’. Then using the previous intracerebral inoculation. fragments as templates and primers 5’ CCG​GAA​TTC​ An inoculum was prepared by pooling the 10% CGG​CGT​ACG​ATG​GTG​AAA​AGC​CAC​ATA​GGC 3’ homogenates of five TgShI112 mouse brains with a con- and 5’ CTA​GTC​TAG​ACT​AGG​CCG​GCC​CTA​TCC​TAC​ firmed disease phenotype, which was also further diluted TAT​GAG​AAA​AAT​3’, the sheep M112I-PrP ORF was to 1% in DPBS for direct intracerebral inoculation, this generated and cloned into the pDrive vector.The ORF inoculum was named ShTgSPON. from sheep PRNP M112I was excised from the cloning vector by using the restriction enzymes BsiWI (Thermo Bioassays with ShTgSPON, atypical and classical scrapie Fisher Scientific Inc.) and FseI (New England Biolabs inocula in transgenic mice and bank voles Ltd.) and then inserted into a modified version of MoPrP. Mice of 42–56 days of age were intracerebrally inoculated Xho vector [13] as described previously [19], which was under gaseous anesthesia (Isoflurane) through the right also digested with BstWI and FseI. This vector contains parietal bone. A 50 µl SGC precision syringe was used the murine PrP promoter and exon-1, intron-1, exon-2 with a 25 G gauge needle to inoculate a final volume of and 3’ untranslated sequences. Finally, the construct was 20 µl per animal. A dose of buprenorphine was subcu- excised using NotI and purified with an Invisorb Spin taneously injected before recovery to consciousness to DNA Extraction Kit (Inviteck) according to the manufac- reduce post-inoculation pain. Mice were kept in a con- turer recommendations. trolled environment at a room temperature of 22 ºC, 12 h Transgenic mouse founders were generated by micro- light-darkness cycle and 60% relative humidity in HEPA injection of NotI excised DNA into pronuclei follow- filtered cages (both air inflow and extraction) in venti- ing standard procedures [20]. DNA extracted from tail lated racks. The mice were fed ad libitum, observed daily biopsies was analyzed by PCR using specific primers and their clinical status assessed twice a week. The pres- for the mouse exon 2 and 3’ untranslated sequences (5’ ence of TSE-associated clinical signs was scored (0–3) GAA​CTG​AAC​CAT​TTC​AAC​CGAG 3’ and 5’ AGA​GCT​ including: cyphosis, gait abnormalities, altered coat state, Vidal et al. Acta Neuropathologica Communications (2022) 10:179 Page 4 of 22 depressed mental state, flattened back, eye discharge, brains from inoculated voles were removed and divided hyperactivity, loss of body condition and incontinence. into two parts by a sagittal paramedian cut. The smaller Positive TSE diagnosis relied principally on the detec- portion (left part) was frozen for WB analysis of ­ rPres (by either immunohistochemistry and/or tion of P ­PrPSc and the larger one (right part) was fixed in formol- Western blotting, or ELISA) and associated spongiform saline for histological and PET-blot analyses. changes on stained histological sections of the brain parenchyma. Sheep bioassay The transgenic mouse models used in this paper ShTgSPON prions were intracerebrally inoculated into include Tg338 [23], Tg501 [2], TgVole (1x) [25, 29], TgV- four sheep of the churra-tensina breed, 2 of them express- ole (4x) [56, 57], TgHu340(Met129) and TgHu361(Val ing the AHQ/AHQ and another 2 expressing the AHQ/ 129) [58]. ARR PRNP genotype. To check whether the ShTgSPON For the bank vole inoculations, brain tissues from inoculum reproduced the features of atypical scrapie, one TSE affected animals were homogenized at 10% (w/v) AHQ/AHQ sheep was intracerebrally inoculated with an in phosphate buffered saline (PBS) and stored at -80 °C. atypical scrapie/Nor98 isolate. The intracerebral inocula- Groups of six-to-eight week old bank voles carrying iso- tion was performed as described previously[37]. Briefly, a leucine at PRNP codon 109 (Bv109I) were inoculated small perforation was made in the cranial bone with the intracerebrally with 20 μl of homogenate into the left aid of a special bone drill. The place of election was at the cerebral hemisphere, under ketamine anaesthesia (keta- level of the frontal sinus one centimetre lateral from the mine 0.1 μg/g). All voles were individually identified by midline. Subsequently, 0.5 mL of inoculum consisting of a passive integrated transponder. Animals were exam- a 10% homogenate of the ShTgSPON or atypical scarpie ined twice a week until neurological signs appeared, after agent in sterile saline solution was inoculated in the fron- which they were examined daily. The attack rate was tal cortex using a 25G diameter hypodermic needle. This calculated as the number of animals scoring positive at procedure was performed under a deep anaesthetic plane post-mortem/number inoculated. Animals found dead and subsequently to the inoculation, antibiotic therapy or culled for intercurrent disease before 200 days post was administered. inoculation and scoring negative at postmortem were excluded from analyses. The survival time for animals ELISA scoring positive at post-mortem was calculated as the Frozen mouse brains were thawed and homogenised 1:10 time from inoculation to culling or death. (weight volume) in sterile phosphate buffered saline. They were routinely tested by ELISA (IDEXX, Herdcheck) Sample processing and general procedures ultrashort protocol. It is a commercial ELISA based on (termed Seprion®). A new threshold was defined to When the clinical end-point criteria were reached (quan- the affinity of misfolded prions to an anionic substrate titatively a clinical score higher than 10, qualitatively an unresponsive mental state or the presence of invalidating adapt to the higher densitometry readings obtained when motor disturbances), mice were euthanized by decapita- working with samples with ­ PrPC overexpression: only tion under gaseous anesthesia (Isofluorane). The brain samples with a ratio spectrophotometry reading/cut-off was extracted immediately, divided longitudinally, and over 5 were considered positive. placed into 10% phosphate buffered formalin. Trans- versal sections of the brain were performed at the lev- Immunohistochemistry els of the medulla oblongata, piriform cortex and optic Immunohistochemistry (IHC) for detection of ­PrPres was chiasm. Samples were embedded in paraffin-wax after performed as described previously [66]. Briefly, depar- dehydration through increasing alcohol concentrations affinized sections were subjected to epitope unmask- and xylene. Four-micrometre sections were mounted on ing treatments: immersed in formic acid and boiled at glass microscope slides and stained with hematoxylin and low pH (6.15) in a pressure cooker and pre-treated with eosin for morphological evaluation. Additional sections proteinase K (4 µg/mL). Endogenous peroxidases were were mounted in 3-trietoxysilil-propilamine-coated glass blocked by immersion in a 3% H ­ 2O2 in methanol solu- microscope slides for immunohistochemistry. One half of tion. Sections were then incubated overnight with anti- the brain was separated prior to fixation and kept frozen PrP MAb 2G11 primary antibody (1:100, Bio-Rad) for for biochemical analysis. sheep ­PrPC models and 6C2 (1:1000, CVI-Wageningen For the experiments in bank voles, animals were culled UR) for Bank vole P ­ rPC models and 6H4 (1:100, Ther- with carbon dioxide before neurological impairment was mofisher) for human P ­rPC models, and subsequently such as to compromise their welfare, in particular their visualized using the Goat anti-mouse EnVision system ability to drink and feed adequately. At post-mortem, (DAKO) and 3,3’-diaminobenzidine (DAKO) as the Vidal et al. Acta Neuropathologica Communications (2022) 10:179 Page 5 of 22 chromogen substrate. As a background control, incuba- Graphs were plotted using Microsoft Office Excel tion with the primary antibody was omitted. software. PrPres immunodetection in sheep was performed as described previously [52]. Sheep brain sections were Proteinase K digestion and western blotting treated in 98% formic acid for 15 min followed by incu- Two different PK digestion procedures were applied bation with proteinase K (4 µg/ml, Roche, Switzerland) to the mouse and sheep brain homogenate samples in for 15 min at 37 ˚C and hydrated autoclaving in citrate order to evaluate the presence of P ­ rPres with a classi- buffer. Immunostaining was done in an automated auto- cal three-banded pattern or with an atypical ladder-like stainer (DAKO). After blocking endogenous peroxidase multi-banded pattern by Western blotting. To detect (ready-to-use solution, DAKO) immunodetection was ­PrPres with a classical biochemical signature, applied for performed using the 8G8 antibody (1:200, 1 h at RT; the digestion of SSBP/1 classical scrapie isolate used as Cayman Chemical). Goat anti-mouse EnVision system controls in the Western blot gels, the brain previously (DAKO) was used as the visualization system and diam- homogenized at 10% (w/V) in Phosphate buffered saline inobenzidine (DAKO) as the chromogen. Sections were (PBS) (Fisher Bioreagents) with Protease inhibitor cock- counterstained with hematoxylin. tail (Roche), was mixed with digestion buffer [2% (w/V For bank vole studies histology and PET-blot analy- Tween-20 (Sigma-Aldrich), 2% (V/V) NP-40 (Sigma- ses were performed on formalin-fixed tissues as previ- Aldrich) and 5% (w/V) Sarkosyl (Sigma-Aldrich) in PBS] ously described [54]. Briefly, coronal brain sections were at 1:1 (V/V). Proteinase K (Roche) was added to reach a obtained from four antero-posterior levels including final concentration of 85 µg/ml to each sample and these the following: (1) telencephalon at midlevel of caudate were incubated at 42 °C for 1 h with moderate shaking. nucleus, (2) diencephalon at midlevel of thalamus, (3) Digestion was stopped by adding loading buffer (NuPage midbrain, and (4) hindbrain at midlevel of medulla and 4X Loading Buffer, Invitrogen) 1:3 (V/V). For the detec- cerebellum. Sections of 5 µm from the above levels were ­ rPres with atypical biochemical signature, which tion of P prepared and stained either with hematoxylin and eosin allows also detection of classical ­PrPres banding pattern, to assess spongiosis or were subjected to PET-blot using we based on a procedure described previously [78]. This the 6C2 mAb (epitope on bank vole PrP 111–116). Neu- procedure was applied in all samples in which presence ropathological assessment was performed on sections of classical or atypical P­ rPres was uncertain: all TgShI112 stained with hematoxylin and eosin, and lesion profiles animals developing disease spontaneously or inoculated were constructed scoring the vacuolar degeneration in both with atypical scrapie, ShTgSPON inoculum and nine gray-matter areas of the brain. Vacuolation scores sheepBSE; and also in TgVole, Tg338 and Tg501 animals were derived from at least five individual animals per inoculated either with atypical scrapie isolates or ShTg- group and are reported as means ± SEM. SPON inoculum. Briefly, 10% brain homogenates were digested with Pronase E (Sigma-Aldrich) at 100 µg/ Semi‑quantification and data analysis ml for 30 min at 37 °C and vigorous shaking (800 rpm). Mouse histological lesions (i.e. spongiform change) and After addition of EDTA (Calbiochem) for a final con- ­PrPres immunolabeling were evaluated under a light centration of 10 mM and Sarkosyl for a final 2% (w/V) microscope by a single pathologist. A semi-quantitative concentration, Pronase E-digested samples were further approach was used as previously described [72]. Spongi- processed with Benzonase (Merck) at 50 U/ml for 10 min form lesion and P ­ rPres immunolabeling were separately at 37 °C and 800 rpm. Then, sodium phosphotungstic scored. Fourteen different brain regions were chosen: salt (NaPTA) (Sigma-Aldrich) was added at 0.3% (w/V) piriform cortex (Pfc), hippocampus (H), occipital cortex and samples incubated for 30 min at 37 °C and 800 rpm. (Oc), temporal cortex (Tc), parietal cortex (Pc), frontal Upon addition of iodixanol 60% (OptiPrep density gradi- cortex (Fc), striatum (S), thalamus (T), hypothalamus ent medium, Sigma-Aldrich) and NaPTA, for final con- (HT), mesencephalon (M), medulla oblongata (Mobl), centrations of 35% (w/V) and 0.3% (w/V), respectively, cerebellar nuclei (Cm), cerebellar vermis (Cv) and cer- samples were centrifuged at 16 100 g for 90 min and ebellar cortex (Cc). Scores ranging from (0) absence of supernatant (avoiding flocculants) transferred to a new spongiosis or immunolabeling: (1) mild, (2) moderate, (3) tube after filtration through 0.45 µm porus-size micro- intense and (4) maximum intensity of lesion or immuno- centrifuge filtration units (Millipore). These supernatants labeling were assigned to each brain area studied. Each were afterwards mixed 1:1 with a buffer composed by 2% area was investigated globally as region for the scoring. Sarkosyl (w/V) and 0.3% NaPTA diluted in PBS. After an Brain profiles were plotted as a function of the anatomi- additional 90 min centrifugation at 16 100 g the super- cal areas which were ordered along the horizontal axis natant was discarded and pellet resuspended in washing to represent the caudo-rostral axis of the encephalon. buffer [iodixanol 17.5% (w/V) and Sarkosyl 0.1% (w/V) in Vidal et al. Acta Neuropathologica Communications (2022) 10:179 Page 6 of 22 PBS]. The resuspended pellets were then digested with sheep numbering) or 9A2 (aa 102–104). The PrP was vis- Proteinase K at a final concentration of 10 µg/ml for 1 h ualized by enhanced chemiluminescent substrate (Super- at 37 °C and 800 rpm. After adding washing buffer and Signal Femto, Pierce) and the ChemiDoc imaging system NaPTA for a final concentration of 0.3% (w/V), samples (Bio-Rad). were once more centrifuged at for 30 min at 16 100 g and supernatants discarded. This step was repeated and the Ethics statement final pellet resuspended in loading buffer (NuPage 4X All experiments involving mice (TgShI112, Tg340, Loading Buffer, Invitrogen) 1:3 (V/V). Tg361, Tg338, and Tg501) were approved by the animal Prion protein immunodetection was performed by experimentation ethics committee of the Autonomous Western blotting as described previously [36]. Briefly, University of Barcelona (Reference numbers: 5767 and PK-digested samples were boiled for 10 min and loaded 1124M2R) in agreement with Article 28, sections a), b), c) on 4–12% acrylamide gels (NuPAGE Midi gel Invit- and d) of the “Real Decreto 214/1997 de 30 de Julio” and rogen Life Technologies) with the exception of the gel the European Directive 86/609/CEE and the European shown in Fig. 2, that was a 4–15% acrylamide gel (Cri- Council Guidelines included in the European Convention terion TGX, Bio-Rad), subjected to electrophoresis for for the Protection of Vertebrate Animals used for Experi- approximately 1 h and 20 min and transferred to a PVDF mental and Other Scientific Purposes. using the Trans-Blot® TurboTM transfer system (Bio- membrane (Trans-Blot Turbo Transfer Pack, Bio-Rad) TgVole (1x) and TgVole (4x) mice were obtained from the breeding colonies at CIC bioGUNE (Spain) and were Rad). After blocking non-specific antibody binding of inoculated at the University of Santiago de Compostela the membranes by incubation in 5% non-fat milk pow- and Neiker—Basque Institute for Agricultural Research der for 1 h at room temperature, monoclonal antibodies and Development. All experiments involving animals in D18 (1:5000) [80] or 12B2 (1:5000) (Central Veterinary Spain adhered to the guidelines included in the Spanish Institute, Wageningen UR) were added and incubated law “Real Decreto 1201/2005 de 10 de Octubre” on pro- for 1 h at room temperature, prior to washing. After tection of animals used for experimentation and other incubation with peroxidase-conjugated secondary goat scientific purposes, which is based on the European anti-human IgG (H+L, Thermo Scientific or anti-mouse Directive 86/609/EEC on Laboratory Animal Protection. antibody (m-IgGκ BP-HRP, Santa Cruz Biotechnology)), The project was approved by the Ethical Committees on membranes were washed again and developed with an Animal Welfare (project codes assigned by the Ethical enhanced chemiluminescent horseradish peroxidase Committee P-CBG-CBBA-0314 and 15,005/16/006) and substrate (West Pico Plus, Thermo Scientific), using a performed under their supervision. FluorChem Q (Alpha Innotech) for image acquisition Bank voles were obtained from the breeding colony and the software AlphaView (Alpha Innotech) for image at the Istituto Superiore di Sanità (ISS), Italy. Experi- processing. ments involving animals adhered to the guidelines con- For bank vole samples WB analysis, brain homogen- tained in the Italian Legislative Decree 116/92, which is ates (20% w/v) were prepared as previously described based on the European Directive 86/609/EEC on Labo- [62]. After adding an equal volume of 100 mM Tris–HCl ratory Animal Protection, and then in the Legislative containing 4% sarkosyl, the homogenates were incu- Decree 26/2014, which transposed the European Direc- bated for 30 min at 37 °C with gentle shaking. Protein- tive 2010/63/UE on Laboratory Animal Protection. The ase K (Sigma-Aldrich) was added at a final concentration research protocols were approved and supervised by the of 50 μg/ml and then the samples were incubated for Service for Biotechnology and Animal Welfare of the ISS 1 h at 55 °C with gentle shaking. Protease treatment was and were authorized by the Italian Ministry of Health stopped with 3 mM PMSF (Sigma-Aldrich). Aliquots (decree numbers 84/12.B and 1119/2015-PR). of samples were added with an equal volume of isopro- Experimental inoculation in sheep was approved by the panol/butanol (1:1 v/v) and centrifuged at 20 000 g for Advisory Ethics Commission for Animal Experimenta- 5 min. Supernatants were discarded and the pellets were tion of the University of Zaragoza with permit number resuspended in denaturing sample buffer (NuPAGE PI 04/17. The care and use of animals was carried out in LDS Sample Buffer, Invitrogen) and heated for 10 min accordance with the Spanish Protection Policy Animal at 90 °C. After electrophoresis on 12% bis–Tris poly- collected in RD 53/2013, which complies with the direc- acrylamide gels (Invitrogen) and WB on polyvinylidene tive of the European Union 2010/63/EU regarding the fluoride membranes using the Trans-Blot Turbo Transfer protection of animals used for experimentation and other System (Bio-Rad), the blots were processed with anti- scientific purposes. PrP mAbs by using the SNAP i.d. 2.0 system (Millipore). Membranes were probed with mAbs SAF84 (aa 167–173, Vidal et al. Acta Neuropathologica Communications (2022) 10:179 Page 7 of 22 Results Upon necropsy, the brains of the animals were har- Generation of a new transgenic mouse model with the ORF vested and a positive result to enzyme immunosorbent from sheep PRNP and the I112 polymorphism assay for the detection of ­PrPres (IDEXX herdcheck) was Based on previous models set up in our laboratory, obtained in all animals with clinical signs. Neuropatho- new mouse lines were generated by pronuclear injec- logical examination revealed lesions compatible with a tion of a construct consisting of the mouse PrP pro- prion disease, namely spongiosis, astro- and microgliosis, moter and the Open Reading Frame (ORF) from sheep and deposition of ­PrPres aggregates (Fig. 2). PRNP sequence with the I112 polymorphism, equiva- Spongiform change was prominent in the cerebellar lent to polymorphism I109 in bank voles and identical cortex, mesencephalon colliculi, thalamus, hippocam- to the PRNP from the Tibetan sheep breed. Five found- pal formation, neocortex (frontal, parietal, and occipital ers were obtained that transmitted the transgene to their lobes) and striatum. The medulla oblongata, mesenceph- progeny. After backcrossing to a line that did not express alon tegmentum, hypothalamus, and piriform cortex endogenous PrP (Prnp−/− mice)[47], expression levels of were mostly spared. Fine punctate P ­ rPres immunolabel- the transgene were analyzed by Western blotting (WB) ling was observed in the cerebellar cortex with seldom and two lines were selected: L456 and L460 with P ­ rPC coarse granular aggregates, in the thalamus, particularly expression levels of 1.5–2 × and 1–1.5 × in homozygosity, intense in the dorsolateral nuclei, and in the hippocam- respectively, with an unaltered glycoform ratio upon WB pal formation, selectively involving the alveus and lacu- (Additional file 1: Fig. S1). nosum moleculare layers. A certain degree of variation was observed in the intensity of the lesions and ­PrPres TgShI112 mice spontaneously develop a prion disease immunostaining. Some animals (mostly those of the After an incubation period ranging from 380 to 800 days L456 line) showed widespread involvement of the neo- of age, 27% (15/52) of the animals of the L456 line and cortical lobes whether in other animals a milder phe- 15% (7/48) of the L460 line developed a prion disease, notype was observed confined to the cerebellar cortex as confirmed by postmortem tests. All of them, except and mildly involving the thalamus and hippocampal 3 that were euthanized due to intercurrent pathologies formation. No correlation was found between the age at and were later confirmed as TSE positive, developed which signs appeared and this variability, since some of neurological signs characterized by a marked ataxia with the animals with widespread lesions were younger than titubation (when the animal attempted to walk a lateral those with the milder phenotype (Spearman r = 0.1690, swaying was observed that progressed to falling sideways P = 0,6216). The lesion type and distribution were very in more advances cases), this lesion was consistent with similar in both lines but lesions in animals from line L460 a cerebellar location of the neurological lesion. Indeed were milder, meaning that among the positive animals of moderate spongiosis was observed in the cerebellum this line, a lesser proportion of animals with widespread with loss of neurons in the granular cell layer (Fig. 1). lesions was found (40% vs. 28%) (Fig. 2). Interestingly, a Fig. 1 Graphical representation of the age of euthanasia of animals from TgShI112 L456 and L460 mouse lines. Every dot in the graph represents a mouse at the time it was euthanized. Dots with a red margin indicate TSE confirmed animals (by IDEXX ELISA, histopathology, and immunohistochemistry), all of which showed neurological clinical signs (except one animal that was found dead without previous signs, and two other animals that were euthanized due to a lung neoplasia and a massive bilateral hydrometra, respectively, and resulted positive in postmortem tests). A window (red rectangle) of time has been identified between 380 and 800 days of age (doa) in which clinical signs of a spontaneous TSE appear in both lines of the TgShI112 model. The remaining negative animals were euthanized for humanitarian reasons (i.e., final point criteria were reached due to signs unrelated to TSE) or in a programmed manner to obtain information at different time points Vidal et al. Acta Neuropathologica Communications (2022) 10:179 Page 8 of 22 Fig. 2 Brain lesions and P ­ rPres deposit distribution for TgShI112 mice. a Line 456 (n = 14) and b Line L460 (n = 7). Brain lesion profiles and ­PrPres deposition profiles represent the mean semi-quantitative scoring (0–4, vertical axis) of the spongiform lesions (continuous line, black) and the immunohistochemical labelling of ­PrPres deposits (dashed line, grey) against 14 brain regions (Pfc: piriform cortex, H: hippocampus, Oc: occipital cortex, Tc: temporal cortex, Pc: parietal cortex, Fc: frontal cortex, cc: corpus callosum; S: striatum, T: thalamus, HT: hypothalamus, M: mesencephalon, Mob: medulla oblongata, Cm: cerebellar nuclei, Cv: cerebellar vermis, Cc: cerebellar cortex). Lesions in line L460 are milder than those of L456 but follow a similar distribution. Bars: standard error of the mean. c Histopathological assessment of spongiform lesions and P ­ rPres deposits in TgShI112 res mice (line 456).Spongiform change can be observed in the top row (H&E staining). P ­ rP deposits are of punctiform and granular nature. In the bottom row a healthy L456 mouse brain is shown. Notice the intense ­PrPC backroad in the cerebellar molecular layer and the synaptic glomerules (but lacking the punctiform ­PrPres staining pattern). Mouse monoclonal antibody 2G11 (1:100). d Neuroinflammatory response. The animals that develop a disease also show a conspicuous neuroinflammatory response with activation of both astroglial (Glial fibrillary acidic protein – GFAP- immunohistochemistry, 1:1500) and microglial (Lycopersicum esculentum agglutinin –LEA- affinity histochemistry, 1:50) cell populations in comparison to age matched unaffected siblings. e Western blotting of a classical scrapie isolate (SSBP/1) and a healthy sheep brain homogenate (Ov NBH) digested for the detection of classical ­PrPres, compared to an isolate of atypical (Nor98) ovine scrapie and the prions extracted from spontaneously diseased TgShI112 mice digested according to the protocol described in [78], which allows detection of atypical P ­ rPSc pattern, and requires 20 times more brain homogenate. The same undigested samples from spontaneously diseased TgShI112 mice and atypical scrapie were also included. Anti PrP antibody 12B2 (1:5000). Spontaneous TgShI112 displays an atypical ladder-like pattern resembling the Nor98 atypical scrapie isolate. MW: Molecular weight mild punctate ­PrPres staining was observed in the white observed in Nor98 atypical scrapie isolates, character- matter. ized by a low molecular weight fragment of 7–10 kDa The biochemical analysis by WB of the prion protein (Fig. 2e). was performed after digestion with proteinase K of the brain homogenates from mice with clinical signs show- The prions generated in the TgShI112 model are ing an atypical ladder-like pattern resembling the one transmissible To assess whether the phenotype observed was a bona fide prion disease, we needed to demonstrate that the Vidal et al. Acta Neuropathologica Communications (2022) 10:179 Page 9 of 22 new spontaneous prions were transmissible. With this inoculum (Fig. 3. Again, the biochemical profile on WB goal in mind, an inoculum (termed ShTgSPON) was pre- matched the multi-band pattern observed in atypical pared from a pool of brain homogenates of five positive scrapie (Fig. 4d). animals form the L456 line, which was intracerebrally inoculated in both L460 and L456 lines of the same Propagation of classical prion diseases is not precluded TgShI112 model. All inoculated animals succumbed to by I112 polymorphism in ovine P ­ rPC a prion disease with an incubation period much shorter To assess whether the I112 polymorphism could restrict than the minimum 380 days of age required for the spon- prion propagation only to those with atypical features taneous phenotype to appear: 162 ± 12 days post inocu- or, on the contrary, could be also permissive to classical lation (dpi) (standard error of the mean -s.e.m.-); for the prion disease propagation and modelling, TgShI112 mice L460 line and a little shorter, 152 ± 11 dpi ± s.e.m., for the from lines L456 (n = 5) and L460 (n = 10) were intracer- L456 line (Table 1). ebrally inoculated with SSBP/1, a pool of brain homoge- The lesions and ­PrPres distribution were similar to the nates from sheep affected by classical scrapie and also milder phenotype but differed from the widespread pat- with a sheepBSE inoculum. SSBP/1 inoculated animals tern observed in the spontaneous phenotype, being were culled at 570 dpi without any clinical signs. No mostly restricted to the cerebellar cortex and vermis pathological or biochemical indication of a prion disease with an evident loss of granules from the granular layer was observed except for one animal (line 456), in which (Fig. 3). The immunostaining pattern observed also con- a focal area of punctate P ­rPres immunolabelling was sisted of fine granular to small clusters of coarse granular observed in the medulla oblongata. This animal had an ­PrPres deposits. ELISA positive result in absence of the spontaneous phe- notype described previously. This result is in accordance ShTgSPON is indistinguishable from atypical scrapie with other ovine models with the ARQ genotype that when bioassayed in TgShI112 mice show reduced susceptibility to the SSBP/1 classical scra- Since the biochemical signature of the P ­ rPres from spon- pie isolate [34, 40]. In contrast, the six TgShI112 (Line taneously sick animals was remarkably similar to that 460) animals inoculated with a sheep-BSE inoculum suc- of natural isolates from sheep with atypical scrapie, an cumbed to disease with a 100% attack rate and 590 ± 20 isolate of ovine atypical scrapie (a field case diagnosed dpi ± s.e.m.. Even though this prolonged incubation in PRIOCAT laboratory, IRTA-CReSA, Catalonia) was period could overlap with the spontaneous phenotype in inoculated in the same model TgShI112 (line 456) to some animals, the immunostaining pattern, consisting of compare it with the spontaneous disease. This bioassay multifocal round plaque-like P ­ rPres deposits, the distri- resulted in a 100% attack rate at 177 ± 14 dpi ± s.e.m. bution of lesions in the brain confirmed that a classical (Table 1, Fig. 4a), an incubation period similar to that prion strain was being replicated in the model (Table 1, observed for ShTgSPON (100% attack rate at 152 ± 11 Additional file 1: Fig. S2). The length of this incubation dpi ± s.e.m.). The brain lesion and P ­rPres distribution period is comparable to that reported in other ARQ ovine profiles were indistinguishable from those obtained in models such as Tg501 (485 ± 62 dpi ± s.e.m.)[3]. Since the the same model when inoculated with the ShTgSPON Tg501 model slightly overexpresses ovine ARQ ­PrPC it is reasonable that the incubation period is slightly shorter than in our model. Table 1 Summary of the bioassays performed in TgShI112 mice. Mean dpi of positive animals ± SEM (positive/inoculated). NP: Transmission of ShTgSPON into mouse models expressing Not performed wild type ovine PrP demonstrates it is indistinguishable from atypical scrapie TgShI112 L460 TgShI112 L456 ShTgSPON inoculation in the ovine VRQ PrPC Tg338 model 1st passage 1st passage The transgenic mouse model Tg338 [8, 69] bearing the ShTgSPON 162 ± 12 (8/8) 152 ± 11 (9/9) wild type ovine PRNP gene with VRQ polymorphisms, SSBP/1 > 570 (0/10)a 572 (1/5)b which easily propagates Nor98 isolates [24], was inocu- Atypical Scrapie ND 177 ± 14 (14/14) lated with both ShTgSPON and the atypical scrapie Sheep BSE 590 ± 20 (6/6c) ND isolate (Table 2). On first passage, a 100% attack rate a demonstrated that ShTgSPON prions were also transmis- One animal was euthanized at 380 dpi with spontaneous atypical profile. Endpoint established at 579 dpi sible to a wild type ovinized mouse model. b In one animal a fine punctate ­PrPres signalling was observed in the medulla The incubation period for the ShTgSPON isolate was oblongata, absence of atypical profile. IDEXX Positive slightly shorter than for the atypical scrapie isolate, both c A seventh animal was prematurely euthanized at 352 dpi and was negative, it with a 100% attack rate, observing a striking similarity was excluded from the calculations. ND: not determined Vidal et al. Acta Neuropathologica Communications (2022) 10:179 Page 10 of 22 Fig. 3 Brain lesion profiles and ­PrPres deposit distribution for the inoculum ShTgSPON in TgShI112 mice. a Line 456 (n = 9) and b line L460 (n = 8). Brain lesion and ­PrPres deposition profiles represent the mean semi-quantitative scoring (0–4, vertical axis) of the spongiform lesions (continuous line, red) and the immunohistochemical labelling of ­PrPres deposits (dashed line, red) against 14 brain regions (Pfc Piriform cortex, H Hippocampus, Oc Occipital cortex, Tc Temporal cortex, Pc Parietal cortex, Fc Frontal cortex, S Striatum, T Thalamus, HT Hypothalamus, M Mesencephalon, Mob Medulla oblongata, Cm Cerebellar nuclei, Cv Cerebellar vermis, Cc Cerebellar cortex). Lesions and ­PrPres deposition are mostly restricted to cerebellar cortex and vermis, a profile clearly distinguishable from the spontaneous phenotype. Bars: standard error of the mean, c Neuropathological characterisation of the lesions (H&E staining) and ­PrPres immunohistochemistry (2G11, 1:100) in the brains of ShTgSPON inoculated TgShI112 (L456) mice. Spongiosis and evident neuronal loss was observed in the granular cell layer of the cerebellar cortex (the cerebellar vermis -Cv- is depicted)) along with punctate-granular P ­ rPres deposit in the cerebellar cortex and vermis. A healthy L456 brain mouse is shown as a negative control, notice a strong ­PrPC background in the molecular layer and the synaptic glomerules and lack of granular P ­ rPres staining pattern regarding the spongiosis and ­PrPres deposition brain pro- strain, SSBP/1, the differences were substantial (Addi- files (Fig. 5) suggesting that both inocula contained the tional file 1: Fig. S3). Furthermore, on second passage of same prion strain. When compared to a classical scrapie the ShTgSPON inoculum, no change was observed on the Vidal et al. Acta Neuropathologica Communications (2022) 10:179 Page 11 of 22 incubation period indicating a lack of transmission bar- samples for the detection of the low molecular weight rier or adaptation phenomena (Additional file 1: Fig. S3). band, which requires large amounts of brain homogen- Biochemical analysis of the P ­rPres from ShTgSPON ate, low PK concentration and exhaustive purification inoculated animals further confirmed the transmission and concentration steps (Fig. 6). of an atypical prion disease in this model, characterized by the expected low molecular weight band of around Inoculation of ShTgSPON and atypical scrapie in bank 7–10 kDa (Fig. 6). vole‑based models reveals the facilitating role of isoleucine ShTgSPON inoculation in the ovine ARQ PrPC Tg501 model Despite the differences in P ­ rPC expression levels of the A second ovinized transgenic mouse model was used three ovine models inoculated with atypical scrapie bearing the wild type ovine PRNP gene with ARQ poly- and ShTgSPON, the disease was unexpectedly fast in morphisms, the Tg501 [2–4]. As with the previous TgShI112 (with the lowest P ­ rPC expression levels among model, a 100% attack rate was obtained after inoculation the three models used) pointing towards a relevant role with ShTgSPON and a slightly shorter incubation period of the I112 polymorphism. Since the particularity of the than that of atypical scrapie isolate (Table 2). The brain bank vole model relies on the presence of isoleucine at spongiosis and ­PrPres profiles were once again indistin- the equivalent position (position 109), which promotes guishable; spongiosis was minimal in both groups with a the spontaneous formation of an atypical prion strain severe loss of granular neurons in the cerebellar granular when overexpressed in transgenic mice and allows effi- cell layer, and the presence of ­PrPres plaque-like deposits cient transmission of atypical scrapie in bank voles [62, only in the cerebellar cortex and vermis (Fig. 7). These 75], we wondered whether the presence of this polymor- features contrast with the distribution of P­ rPres and lesion phism could facilitate propagation of atypical prions. For profile of a classical scrapie strain, such as SSBP/1, in that, we inoculated ShTgSPON and an atypical scrapie the same model, in which the cerebellar cortex is mostly isolate in different animal models bearing bank vole PrP spared, and lesions are centred in the brain stem (Addi- with I109 polymorphism (TgVole 1x, TgVole 4x, and bank tional file 1: Fig. S3). voles) (Table 2). As reported in previous studies, bank Biochemically, the ­PrPres pattern was also comparable voles with M109 polymorphism and wild type mice do to that obtained after inoculation with an atypical scrapie not succumb to disease upon intracerebral inoculation isolate in the same model, showing the characteristic low of atypical scrapie or do so in a highly inefficient manner molecular weight band that is absent in classical scrapie [35, 61]. Whereas the bank vole with I109 and our TgVole isolates. Differences in the intensity of such fragment and models, with 100% attack rate, manifested neurological the presence of higher molecular weight bands, reminis- signs in less than 260 dpi in non-overexpressing ones and cent of the electrophoretic migration profile of classical around 100 dpi in those overexpressing P ­ rPC fourfold. scrapie isolates, likely derive from the processing of the Moreover, the incubation periods of ShTgSPON were (See figure on next page.) Fig. 4 Analysis of ShTgSPON-inoculated compared to atypical scrapie-inoculated TgShI112 mice. a Kaplan-Meyer survival curves comparing ShTgSPON (red) innoculum and an atypical scrapie isolate (blue) incubation periods in the TgShI112 mouse model. Notice the overlapping curve. dpi Days post inoculation. b Brain lesion and ­PrPres deposit distribution for the atypical scrapie inoculum in TgShI112 mice (line 456, n = 14). Brain lesion profiles and ­PrPres deposition profiles represent the mean semi-quantitative scoring (0–4, vertical axis) of the spongiform lesions (continuous line, blue) and the immunohistochemical labelling of P ­ rPres deposits (dashed line, blue) against 14 brain regions (Pfc Piriform cortex, H Hippocampus, Oc Occipital cortex, Tc Temporal cortex, Pc Parietal cortex, Fc Frontal cortex, S Striatum, T Thalamus, HT Hypothalamus, M Mesencephalon, Mob Medulla oblongata, Cm Cerebellar nuclei, Cv Cerebellar vermis, Cc Cerebellar cortex). Lesions and ­PrPres deposits were mostly restricted to the cerebellar cortex and vermis. This pattern was identical to that observed in the same mice when inoculated with ShTgSPON inoculum (see Fig. 3a and b). Bars: standard error of the mean. c Neuropathological characterisation of the lesions (H&E staining) and P ­ rPres immunohistochemistry (2G11, 1:100) in the brains of atypical scrapie inoculated TgShI112 mice (L456). Spongiosis and evident neuronal loss was observed in the granular cell layer of the cerebellar cortex (the cerebellar vermis is depicted -Cv-) along with punctate-granular ­PrPres deposit in the cerebellar cortex and vermis. This pattern was identical to that observed in the same mice when inoculated with ShTgSPON inoculum (see Fig. 3c). A healthy L456 brain mouse is shown as a negative control, notice a strong ­PrPC background in the molecular layer and the synaptic glomerules and lack of granular ­PrPres staining pattern. (d) Western blotting comparison of a healthy sheep brain homogenate (Ov NBH), a classical scrapie isolate (SSBP/1), digested for detection of classical ­PrPres, and the prions extracted from spontaneously diseased TgShI112 mice or atypical scrapie inoculated TgShI112 mice after digestion with proteinase K (PK) following the protocol described in [78]. Anti PrP antibody 12B2 (1:5000). Electrophoretic migration profiles of both, the spontaneously developed prions in TgShI112 mice (ShTgSPON prions) and an ovine atypical scrapie isolate inoculated in TgShI112 animals, are indistinguishable and are characterized by a low molecular weight band of around 7–10 kDa, further indicating the similarity of the spontaneously generated prions with atypical scrapie isolates. The presence of higher molecular weight bands in these samples, apart from the characteristic 7–10 kDa fragment, could be due to the presence of mildly protease resistant forms that arise due to the low PK concentration and the requirement of using large amounts of brain homogenate for the detection of the small fragment. MW: Molecular weight Vidal et al. Acta Neuropathologica Communications (2022) 10:179 Page 12 of 22 Fig. 4 (See legend on previous page.) similar to those of atypical scrapie inocula in the three gliosis of the hippocampus, particularly involving the models (Table 2). CA1 pyramidal cell layer, as well as variable (patchy) loss As observed in the ovinized models, the neuropatho- of granules of the cerebellar granular layer. The thala- ­ rPres biochemical features from bank voles logical and P mus, but not the hypothalamus, the striatum and the (Fig. 8) and both TgVole mouse lines (Fig. 9 and Addi- neocortex are more severely affected in the overexpress- tional file 1: Fig. S4) were indistinguishable between ing model TgVole (4x) while in the TgVole (1x) model the ShTgSPON and atypical scrapie inocula. In the TgVole ­ rPres are almost restricted to the hippocam- lesions and P models, both inocula induce a severe neuronal loss and pus and cerebellar cortex (Additional file 1: Fig. S4). Vidal et al. Acta Neuropathologica Communications (2022) 10:179 Page 13 of 22 Table 2 Summary of the bioassays performed in mice to compare the ShTgSPON inoculum with atypical scrapie and SSPB/1. Mean dpi of positive animals ± SEM (positive/inoculated). NP: Not performed Tg338 (ovine VRQ ­PrPC) ­ rPC) Tg501 (ovine ARQ P TgVole 1x TgVole 4x Bank vole 1st passage 2nd passage 1st passage 1st passage 1st passage 1st passage ShTgSPON 172 ± 5 (11/11) 177 ± 8 (10/10) 455 ± 14 120 ± 1 (13/13) 59 ± 2 (5/5) 256 ± 14 (12/12) (12/12) SSBP/1 72 ± 1 (11/11) ND 502 ± 27 (5/5) ND ND 523 ± 62 (9/10) Atypical 239 ± 2 (12/12) 144 ± 3 [41]b 577 ± 7 (5/5)a 135 ± 6 (17/17) 80 ± 2 (4/4) 247 ± 24 (9/9)c scrapie a A sixth animal was found death at 104 dpi and was excluded from the study b Using 10 times more inoculum (10% vs. 1%) than in our inoculations. ND Not determined c Data published by Pirisinu et al. [61] Fig. 5 Anatomopathological analysis of ShTgSPON-inoculated compared to atypical scrapie-inoculated Tg338 mice (Ovine VRQ ­PrPC). a Kaplan-Meyer survival curves comparing ShTgSPON (red) innoculum and an atypical scrapie isolate (blue) incubation periods in the Tg338 mouse model. b Brain lesion and P ­ rPres deposit distribution for the inoculum ShTgSPON (n = 11) and c atypical scrapie in Tg338 mice (n = 12). Brain lesion profiles and ­PrPres deposition profiles represent the mean semi-quantitative scoring (0–4, vertical axis) of the spongiform lesions (continuous line, blue) and the immunohistochemical labelling of ­PrPres deposits (dashed line, blue) against 14 brain regions (Pfc piriform cortex, H Hippocampus, Oc Occipital cortex, Tc Temporal cortex, Pc Parietal cortex, Fc Frontal cortex, S Striatum, T Thalamus, HT Hypothalamus, M Mesencephalon, Mob Medulla oblongata, Cm Cerebellar nuclei, Cv Cerebellar vermis, Cc Cerebellar cortex). Notice the striking similarity of the brain profiles obtained with both isolates. Bars: standard error of the mean. d Neuropathological characterisation of the lesions (H&E staining) and P ­ rPres immunohistochemistry (2G11, 1:100) in the brains of ShTgSPON (left panel) and atypical Scrapie (right panel) inoculated Tg338 mice. Both inocula show an identical lesional and ­PrPres deposition pattern. Notice the selective involvement of both isolates of the different layers of the parietal cortex (III-IV and VI) and hippocampal formation (alveus and lacunosum moleculare) as well as that of the dorso-lateral thalamic nuclei Vidal et al. Acta Neuropathologica Communications (2022) 10:179 Page 14 of 22 ­ rPC the inoculum does not seem to transmit to human P expressing hosts, at least on first passage. ShTgSPON is transmissible to sheep and produces the atypical scrapie phenotype The last bioassay proving that a bona fide atypical scra- pie strain is spontaneously generated in the TgShI112 model was performed in its original host, the sheep. Four sheep of the churra-tensina breed (2 AHQ/AHQ and 2 AHQ/ARR) where intracerebrally inoculated with the ShTgSPON innoculum. One AHQ/AHQ sheep started showing clinical signs compatible with atypical scrapie 18 months post-inoculation (mpi). This animal reached end-point criteria and was euthanized at 30 mpi. The remaining sheep are still alive at the moment of writ- ing this report. Immunohistochemical and biochemical studies showed that the sheep had a disease phenotype identical to that of sheep intracerebrally inoculated with atypical scrapie (Fig. 10). Discussion The following discussion is based on the consideration of Fig. 6 Western blotting comparison of a classical scrapie isolate prion diseases into two broad categories: acquired (when (SSBP/1) and the prions extracted from diseased TgShI112 or the source of prions is an external one that seeds the mis- atypical scrapie inoculated Tg338 (Ovine VRQ ­PrPC) and Tg501 (Ovine ARQ ­PrPC) mouse brains after digestion with proteinase K (PK). folding of the host protein) versus spontaneous (where Electrophoretic migration profiles of prions from Tg338 and Tg501 the misfolding event occurs spontaneously in an indi- mouse lines inoculated intracerebrally with ShTgSPON are similar vidual either idiopathic in nature or favoured by the host and characterized by the presence of a 7–10 kDa band, also similar genotype, in terms of the presence of a pathogenic muta- to the ovine atypical scrapie isolate inoculated in the same models. tion on the PRNP gene and its expression levels). Model- The detection of mildly PK-resistant high molecular weight bands in some of the samples may respond to some degree of variability due ling prion diseases of genetic or idiopathic spontaneous to the differences between the models in terms of PrP sequence, origin in animal models is highly challenging. In the first expression levels and slight variations in disease stage at culling. case, because mutations associated to genetic human Along this line, variability due to the processing of the samples for the prion disorders in the context of other PrP sequences do detection of the low molecular weight band could also explain the not always faithfully mimic the human pathology [30, 43, occasional presence of high molecular weight PrP fragments, given the low PK concentration and high sample amounts required. Since 74, 84]. Regarding human idiopathic spontaneous prion anatomopathological analysis did not show significant differences on disorders, in which spontaneous misfolding of ­PrPC is plaque type and distribution between animals with or without them. believed to occur in the absence of mutations, their low Overall, all models challenged with ShTgSPON show the characteristic incidence (1–5 cases per million inhabitants/year) pre- 7–10 kDa fragment resembling that from atypical scrapie, indicating cludes their systematic investigation in animals with wild the spontaneous formation of an atypical ovine prion in TgSh112I model. Anti PrP antibody 12B2, 1:5000. MW: Molecular weight type PRNP gene and absence of ­PrPC overexpression. However, given that idiopathic spontaneous prion dis- eases are the most common in humans, accounting for 80–85% of the cases of transmissible spongiform enceph- Lack of transmission to humanized models alopathies diagnosed, it is important to develop animal To assess the zoonotic potential of our new spontaneous models for such diseases. This lack of models for spon- isolate, two transgenic mice expressing human ­PrPC were taneous prion diseases was nonetheless partially solved inoculated with ShTgSPON: Tg340 (Met129) and Tg361 by the finding of bank voles (Myodes glareolus) and their (Val129). Experimental transmission of atypical scrapie unusual behaviour towards prion infection. Evidences isolates to humanized transgenic mouse models has not have been published that the particular composition of been reported so far, at least on first passage [73, 82]. No the bank vole P ­ rPC, not only renders bank voles highly evidence of transmission to humanised models has been susceptible to most of all known prion strains [7, 44, 54, observed: 0/12 Tg340 (Met129) animals (> 959dpi) and 62, 75], but it is also responsible for generating a sponta- 0/10 Tg361 (Val129) (> 840 dpi). Based on these results, neous and transmissible prion disease when expressed in Vidal et al. Acta Neuropathologica Communications (2022) 10:179 Page 15 of 22 Fig. 7 Anatomopathological analysis of ShTgSPON-inoculated compared to atypical scrapie-inoculated Tg501 mice. a Kaplan-Meyer survival curves comparing ShTgSPON (red) innoculum and an atypical scrapie isolate (TOA3, blue) incubation periods in the Tg501 mouse model. b Brain lesion and ­PrPres deposit distribution for the inoculum ShTgSPON (n = 12) and c atypical scrapie (n = 5) in Tg501 mice. Brain lesion profiles and ­PrPres deposition profiles represent the mean semi-quantitative scoring (0–4, vertical axis) of the spongiform lesions (continuous line, blue) and the immunohistochemical labelling of ­PrPres deposits (dashed line, blue) against 14 brain regions (Pfc Piriform cortex, H Hippocampus, Oc Occipital cortex, Tc Temporal cortex, Pc Parietal cortex, Fc Frontal cortex, S Striatum, T Thalamus, HT Hypothalamus, M Mesencephalon, Mob Medulla oblongata, Cm Cerebellar nuclei, Cv Cerebellar vermis, Cc Cerebellar cortex). Notice the striking similarity of the brain profiles obtained with both isolates. Bars: standard error of the mean. d Neuropathological characterisation of the lesions (H&E staining) and P ­ rPres immunohistochemistry (2G11, 1:100) in the brains of ShTgSPON (left panel) and atypical scrapie (right panel) inoculated Tg501 mice. Both inocula show an identical lesional and ­PrPres deposition pattern. Notice the selective involvement of both isolates of the cerebellar cortex with focal massive granule cell loss and plaque-like ­PrPres deposits in the superficial molecular layer of the cerebellar cortex and vermis a transgenic mouse model [17, 29, 56, 57, 76]. Different spontaneously engenders a transmissible prion disease amino acid variants particular to bank voles have been and the length of its incubation period inversely corre- suggested to be the drivers of this enhanced susceptibil- lates with the expression levels of the 109I-PrPC [56, 57, ity and proneness to spontaneous misfolding, including 76]. That can explain why, to date, no spontaneous pri- D227E and R230S of the C-terminal domain [44] and, onopathies have been described in wild type bank voles particularly, isoleucine at position 109, a residue that with this polymorphism. This is the key of the success is also present in other species [56, 57, 76]. The latter of the overexpression approach of both the TgVole and (I109) when overexpressed in a transgenic mouse model our model, in which the presence of this polymorphism Vidal et al. Acta Neuropathologica Communications (2022) 10:179 Page 16 of 22 Fig. 8 Bank vole bioassay. a PET blot detection of protease resistant ­PrPSc in coronal sections of the brain, representing telencephalon (1), diencephalon (2), midbrain (3), and hindbrain (4) from representative Bv109I. Isolates indicated on the Left. ­PrPSc was detected with mAb 6C2. b Representative replica Western blots showing PK-treated (PK) ­PrPSc from the brains of voles inoculated with atypical scrapie (AS, n = 9), ShTgSPON (n = 12) and classical scrapie (SSBP/1, n = 9), as indicated on the Top of the blots, and analyzed with mAbs recognizing different epitopes on PrP, as indicated on the Left of the blots (SAF84 epitope at 167 to 173; 12B2 epitope at 89 to 93). Notice absence of labelling for the c-terminal epitope for both AS and ShTgSPON. c Lesion profiles in groups of Bv109I infected with atypical scrapie (AS), ShTgSPON and classical scrapie (SSBP/1). Data points represent the mean ± SEM of at least five voles per group. Brain-scoring areas: medulla (1), cerebellum (2), superior colliculus (3), hypothalamus (4), thalamus (5), hippocampus (6), septum (7), retrosplenial and adjacent motor cortex (8), and cingulate and adjacent motor cortex (9) is necessary but not sufficient to generate prions spon- polymorphism. The aim of our research was to assess taneously. It is only upon overexpression that prion dis- whether overexpression of I112 PRNP polymorphism ease develops, probably related to endoplasmic reticulum in a transgenic mouse model would also elicit the spon- stress and proteasome impairment [56]. A further con- taneous generation of a transmissible prion, in order to firmation of the relevance of the P­ rPC expression levels obtain a laboratory model to study the pathogenesis of is the report of a genetic CJD occurrence in a family in prionopathies in ruminants, avoiding exogenous prion which only the homozygous members developed the dis- administration. ease [83]. Thus, the TgShI112 transgenic mouse model was gen- The 109I polymorphism was of particular interest in erated and, indeed, it spontaneously developed a prion our line of research since the homologous polymorphism disease with all the neuropathological hallmarks of this in sheep (I112) actually exists in nature, particularly in group of diseases: neurological clinical signs, spongiform the Tibetan sheep breed [85]. Other polymorphisms have degeneration, astro and microgliosis and P ­ rPres deposi- been described in this locus, such as the T112 which is tion. A certain variability was observed in the incubation associated to certain resistance to classical scrapie [33, period and the penetrance was not a 100%, this is similar 42]. Nevertheless, no scrapie cases either classical or to human TSEs associated to PNRP mutations in which atypical have been reported in sheep with the I112 poly- a wide variability on the age of onset among individu- morphism, which can probably be explained due to insuf- als carrying the same mutation has been observed [51]. ficient expression levels and the low frequency of the Additionally, it was transmissible upon intracerebral Vidal et al. Acta Neuropathologica Communications (2022) 10:179 Page 17 of 22 which could indicate strain variability. However, the same difference was also observed in the atypical scrapie iso- late used as control, suggesting that it might be due to technical variability rather than real strain differences. In fact, freezing and thawing of individual samples seemed to have some effect on the presence or absence of such band and also regarding intensity of high molecular weight bands (data not shown). Moreover, the gel used in Fig. 2 was from a different brand than the rest, what could exacerbate the apparent differences, but since the lowest molecular weight band, the most significant sig- nature of these atypical prions, was unaltered we con- sider that the spontaneously generated strain was likely conserved upon passage. Taking into account that atypi- cal scrapie is the strain supposedly associated with idi- opathic spontaneous prion disease in small ruminants [10], the model presented here exceeds our expectations regarding faithful recreation of a sporadic transmissible spongiform encephalopathy. It provides the opportunity to study systematically the spontaneous development of Fig. 9 Western blotting comparison of a classical scrapie isolate an atypical and transmissible prion disorder neuropatho- (SSBP/1) and the prions extracted from spontaneous-diseased logically indistinguishable from that found in nature, in TgShI112 or atypical scrapie inoculated TgVole (4x) and TgVole (1x) an animal model bearing a naturally occurring sheep mouse brains after digestion with proteinase K (PK). Electrophoretic polymorphic ­PrPC variant and with expression levels migration profiles of prions from TgVole (1 × and 4x, with respect to mouse PrP expression levels) inoculated intracerebrally with close to physiological. Additionally, TgShI112 mice were ShTgSPON are similar and characterized by the presence of a readily susceptible to inoculation with an atypical scrapie 7–10 kDa band, also indistinguishable from ovine atypical scrapie isolate, demonstrating their usefulness to model acquired isolate inoculated in the same models. Both models challenged atypical prion disease in addition to the spontaneous one. with ShTgSPON show the characteristic 7–10 kDa fragment In fact, the resulting lesional pattern was indistinguish- indistinguishable from atypical scrapie, indicating the spontaneous formation of an atypical ovine prion in TgSh112I model. Anti PrP able from that obtained when this model was inoculated antibody 12B2, 1:5000. MW: Molecular weight with the ShTgSPON inoculum (obtained from pooling five brains from TgShI112 with confirmed prion dis- ease). The lesional and P ­ rPres deposition profile observed inoculation not only to the same model, but also to other in TgShI112 mice inoculated with both ShTgSPON and models carrying wild type ovine ­ PrPC (Tg338, Tg501 atypical scrapie distinctly involved the cerebellum while and most interestingly to the original host: AHQ/AHQ sparing the remaining brain areas. This differed consid- sheep) and to models expressing bank vole P ­ rPC (TgVole erably from the profile observed in the spontaneously 1x & 4x, and bank vole). In addition, bioassays on human- sick ShTgI112 mice in which other areas such as the ized transgenic mice have not shown so far, at least on hippocampus, thalamus, striatum and neocortex were first passage, a zoonotic potential. consistently involved. An explanation for this difference Upon biochemical characterization (Fig. 2) the elec- could be the length of the incubation period, which in trophoretic migration profile of the ­PrPres deposited in both cases (152 dpi and 177 dpi, respectively) was way TgShI112 mice brains after proteinase K (PK) digestion shorter than the time required for the spontaneous phe- strongly resembled the partially PK-susceptible multi- notype to occur; the cerebellum in this model is likely a banded patterns with a 7–10 kDa band described in atyp- brain area particularly (and interestingly likewise) sus- ical scrapie cases [10]. Some variability was observed in ceptible to prions from both isolates upon inoculation. the banding pattern form individual animals that devel- These animals developed a marked ataxia that led to their oped disease spontaneously and also in those inoculated euthanasia, had the disease been allowed to progress with the ShTgSPON inoculum, mainly regarding the further, it is likely that the prosencephalic brain regions presence or absence of a band of approximately 15 kDa would have been involved and, ultimately would have (detectable in the sample from Fig. 2, but absent in sam- overlapped with the spontaneous phenotype. ple from Fig. 4), whereas high molecular weight bands Altogether, these results suggest that this novel ani- and the characteristic 7–10 kDa band remain unaltered, mal model gives rise to a prion isolate, the ShTgSPON, Vidal et al. Acta Neuropathologica Communications (2022) 10:179 Page 18 of 22 Fig. 10 Imunohistochemical and biochemical analysis of sheep inoculated with ShTgSPON compared to atypical scrapie. a ­PrPres Immunohistochemistry showed a fine granular deposition pattern, particularly abundant in the outer molecular layer of the cerebellar cortex that ­ rPres antibody 8G8, 1:1000). b Western blotting is identical to that observed in sheep inoculated intrecerebrally with atypical scrapie isolate (Anti P comparison of a healthy sheep brain homogenate (Ov NBH), a classical scrapie isolate (SSBP/1), two different isolates of atypical (Nor98-like) ovine scrapie (01: Cerebellum O-1233 AHQ/AHQ; 02: Cerebellum O-1238 AHQ/AHQ) and the prions extracted from ShTgSPON inoculated AHQ/AHQ sheep after digestion with proteinase K (PK). Anti PrP antibody 12B2, 1:5000. Electrophoretic migration profile of P ­ rPSc detected in different brain areas of one of the sheep inoculated with diseased-TgShI112 brain homogenate (ShTgSPON), shows a 7–10 kDa band characterisitc of atypical prion disease of sheep, similar to atypical scrapie isolates from sheep used here to compare. Some variability can be observed among the different brain areas regarding the exact size of this low molecular weight band, matching with subtle differences among the two different atypical scrapie isolates used for comparison, likely due to the presence of fragments with ragged ends after PK digestion more evident in low molecular weight products. MW: Molecular weight with strain features similar to those of Nor98-like atypi- sheep to compare the ShTgSPON isolate with an ovine cal scrapie isolates and that it could be the most suit- atypical scrapie isolate. The results obtained in all the able animal model available not only to investigate the challenged models (Tg338, Tg501, TgVole, bank vole spontaneous misfolding event but also, for any other and AHQ/AHQ sheep) showed strikingly similar neuro- aspect related to transmission and pathogenesis of atypi- pathological and biochemical features, thus confirming cal prions. Despite the similarities between ShTgSPON our initial suspicion. Along this line, both inocula showed and Nor98 regarding biochemical and histopathological little transmission barrier when inoculated in the Tg338 features upon inoculation in TgShI112, differences were model as shown by the slight shortening of the incuba- detected in terms of incubation periods. Such differ- tion period on the second passage (Table 2). Most inter- ences may likely respond to distinct titres from the two estingly, the preliminary results of the sheep bioassay inocula, although the possibility of being slightly different (Fig. 10) definitely show that the phenotype encoded in strains cannot be completely ruled out. The emergence of ShTgSPON inoculum is undistinguishable from that gen- potentially distinct atypical prion strains in this model, if erated with an intracerebral inoculation of atypical scra- confirmed, would also be of great interest. Given the low pie isolate. number of atypical prion disease in nature [considering Remarkably, transmission studies of atypical prions to atypical scrapie, some GSS cases and Variably Protease- either wild type animals, or models bearing isoleucine sensitive Prionopathy (VPSPr) within this category due in the position equivalent to that of bank voles, showed to the biochemical signature of their P­ rPres], but the great an enhanced susceptibility to ShTgSPON and natural phenotypical heterogeneity among them [26, 50, 60, 63, atypical scrapie isolates. TgShI112 models, despite their 71], a largely uncharacterized strain variability could exist lower levels of expression when compared to Tg338 (8x) which could be assessed through this new model. and Tg501 (2x), showed generally much shorter incuba- To further ascertain this finding, multiple bioassays tion periods upon inoculation of ShTgSPON and atypi- were carried out in transgenic mouse models and in cal scrapie isolates. Similarly, bank vole-based models Vidal et al. Acta Neuropathologica Communications (2022) 10:179 Page 19 of 22 (bank voles with I109 polymorphism, TgVole 1 × and 4x), In summary, we have herein presented experimen- show 100% attack rates and strikingly short incubation tal evidence to claim that expression of the sheep ­PrPC periods. While bank voles with M109 polymorphism do with an isoleucine in position 112 in a transgenic mouse not succumb to infection with atypical scrapie isolates in model, results in the spontaneous generation of a bona more than 600 days [61]. This indicates that isoleucine at fide transmissible prion disease with strain features position 109 from bank vole or equivalents could favour undistinguishable from small ruminants atypical/Nor98 propagation of atypical forms of prion disease in a gener- scrapie. This model will be of great interest on the one alized manner, independently from transmission barriers, hand, to study the pathogenesis of atypical sporadic prion what may help gain insight on the differences between phenotypes in small ruminants, such as where does the classical and atypical prion strains. initial misfolding event take place or how long before dis- Even though the I112 polymorphism, upon overex- ease onset is ­PrPres detectable and in which tissues. On pression, favours misfolding of ovine ­PrPC with atypi- the other hand, it may also be an invaluable model to test cal scrapie strain features, it is obviously not the sole anti-prion therapeutic agents as a model of human pri- determinant of the spontaneous appearance of Nor98 onopathies with atypical P ­ rPres features such as GSS and strain in sheep, since no atypical scrapie cases have been VPSPr. described among animals bearing this polymorphism. Although the low number of sheep with such variant and Supplementary Information the absence of active scrapie surveillance in Asia may The online version contains supplementary material available at https://​doi.​ have precluded detection. Atypical scrapie has not been org/​10.​1186/​s40478-​022-​01477-7. linked to a particular PRNP mutation, but the presence Additional file 1. Supplementary figures 1 to 4. Fig. S1 ­PrPC expression of phenylalanine in codon 141 [53] and the presence of levels in TgShI112 mouse lines L456 and L460 compared to a normal a histidine in codon 154 [21] have been associated with sheep brain ­PrPC expression by Western blotting. Fig. S2 Anatomopatho‑ this phenotype, however, these polymorphisms neither logical analysis of sheepBSE-inoculated TgShI112 mice. Fig. S3: Compara‑ tive anatomopathological analysis of Tg338 (Ovine VRQ ­PrPC) and Tg501 do account for all cases. (ovine ARQ ­PrPC) mice inoculated with TgShSPON, atypical scrapie and While TgShI112 mice were apparently more suscep- SSBP/1. Fig. S4: Analysis of ShTgSPON-inoculated compared to atypical tible to atypical scrapie than the wild type counterparts scrapie-inoculated TgVole mice. were, they showed very low susceptibility to one strain causing classical scrapie (SSBP/1). Nonetheless, the fact Acknowledgements The authors would like to thank the following for their support: IKERBasque that one of the inoculated animals developed disease, foundation, vivarium and maintenance from CIC bioGUNE and Patricia Piñeiro and that inoculation with sheepBSE resulted in a 100% for technical support. Maria de la Sierra Espinar, Samanta Giler and Marta Valle attack rate with a distinguishable lesion profile, undoubt- (IRTA-CReSA) for technical support, Dr. Bernat Pérez de Val for his assistance in data analysis, IRTA-CReSA’s biocontainment unit staff for excellent care and edly demonstrate that classical prion disease can also maintenance of the animals, and also Dr. Olivier Andréoletti from UMR INRAE- be modelled in this transgenic mouse line. A possible ENVT, Toulouse for providing the sheepBSE inoculum. explanation to the low susceptibility of these models to Author contributions the classical scrapie strain used herein may come from JC, EV, MASM, HE, JRR and RB conceived the project, acquired funding. MASM, other polymorphic positions within ovine PrP. The gen- EV, HE, SPL, MPC, AO, JMC, BM, RLM, CMD, MG, MO, GC, MdB, NLL, LP and CdA, otype of the TgShI112 animals is ARQ (A136, R154 and performed experiments and analysed data, JMT, VB, EV, HE, GT, JJB, JRR, JC, RN, RB and MP interpreted results and oversaw the study. EV, MASM, HE and JC Q171) regarding the three most studied polymorphic wrote the manuscript with contributions from all authors. sites in sheep P­ rPC influencing susceptibility to scrapie, which has been already reported to show lower suscep- Funding The present work was partially funded by “Ministerio de Economía y tibility than other genotypes to SSBP/1 inoculation [39]. Competitividad” (Spanish Government), Grant Nos. AGL 2013–46756-P, Along this line, analysing the effect of alternative geno- PID2021-122201OB-C21, and PID2020-117465 GB-I00 which are partially sup‑ types together with I112 polymorphism on the propaga- ported by EU FEDER funds, by the Infectious diseases edition of the Fundació La Marató de TV3 Grant No. ATYPRION 201821–30-31–32 and by the Project tion of classical and atypical scrapie strains could be of No. EFA148/16 REDPRION. The project EFA148/16 REDPRION was 65% co- great interest to determine whether some other amino financed by the European Regional Development Fund (ERDF) through the acid variant could hinder formation and propagation of Interreg V-A Spain-France-Andorra program (POCTEFA 2014–2020). POCTEFA aims to reinforce the economic and social integration of the French–Span‑ one strain type versus the other. However, as it is proba- ish–Andorran border. Its support is focused on developing economic, social bly the case for 141F and 154H, the P ­ rPC bearing the I112 and environmental cross-border activities through joint strategies favouring polymorphism seems to be more prone to misfold into a sustainable territorial development. IRTA is supported by CERCA Programme/ Generalitat de Catalunya. structure encoding the atypical scrapie strain rather than other polymorphisms that favour the classical counter- Availability of data and materials parts. Accordingly, atypical cases are frequently found in The datasets during and/or analysed during the current study available from the corresponding author on reasonable request. classical scrapie-resistant genotypes [24, 28, 45, 65]. Vidal et al. Acta Neuropathologica Communications (2022) 10:179 Page 20 of 22 Declarations Collinge J (2020) Spontaneous generation of prions and transmissible PrP amyloid in a humanised transgenic mouse model of A117V GSS. 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