Fatal Familial Insomnia (FFI) is a genetic prion disorder caused by the conformational conversion of the cellular form of the prion protein (PrP^C) into an abnormally folded conformer, named prion or PrP^Sc, which acquires toxic properties and accumulates in the brain (Lugaresi et al., 1986). In 1992, it was shown that the cause of the disease is an autosomal-dominant mutation at codon 178 of the PrP^C encoding gene (PRNP), resulting in aspartic acid (D) to asparagine (N) substitution (D178N) (Medori et al., 1992). Since a main clinical feature of the disease was the presence of a severe and untreatable insomnia, it was named FFI. The pathology primarily affects the thalamus that shows neuronal loss, spongiform changes, astrogliosis, and moderate PrP^Sc deposition (Montagna et al., 2003; Macchi et al., 1997). In cases of long disease duration, such alterations involve also the cerebral cortex. The polymorphism at codon 129 of the PRNP (methionine [M] or valine [V]) plays a significant role in disease heterogeneity (Monari et al., 1994). For instance, the presence of MM (FFI^D178N-129MM) causes FFI with rapid progression (7–18 months), sleep disturbances, and dysautonomia. The presence of MV (FFI^D178N-129MV) is associated with longer disease duration (20–35 months) with equilibrium and gait dysfunctions, while the presence of VV causes a distinct genetic disease, referred to as familial Creutzfeldt–Jakob disease (fCJD^D178N-129VV) (Gambetti et al., 1995). Several lines of evidence suggest that codon 129 polymorphism in PRNP does not influence the age at disease onset (Montagna et al., 2006). PrP^C is synthesized in the rough endoplasmic reticulum where it undergoes several post-translation modifications, including the addition of a GPI anchor to its C-terminal part, the formation of a disulfide bridge between two cysteine residues (Cys179-Cys214), and the N-linked glycosylation at two asparagine residues (Asn181 and Asn197) (Turk et al., 1988). Finally, in the Golgi apparatus, the oligosaccharides are modified to produce complex-type chains rich in sialic acid (Caughey et al., 1989), which have an important role in targeting PrP^C to neuronal synapses (Bate et al., 2016). The pattern of glycosylation gives rise to different PrP^C forms: the di-glycosylated (70%), the mono-glycosylated (25%), and the un-glycosylated (5%) species (Caughey et al., 1989; Rudd et al., 1999). All PrP^C species are soluble in detergent and sensitive to proteinase K (PK) digestion. The D178N mutation reduces the structural stability of the PrP^C, which is more prone to convert into PrP^Sc and aggregate (Liemann and Glockshuber, 1999). This latter is less soluble in detergent and is partially resistant to PK digestion. After PK treatment, the resistant un-glycosylated band of PrP^Sc associated with FFI^D178N-129MM or FFI^D178N-129MV shows a molecular weight of 19 kDa, while that associated with fCJD^D178N-129VV has a molecular weight of 21 kDa (Monari et al., 1994). These differences suggest that the mutation at position 178 and the polymorphism at 129 of the PRNP affect the tertiary structure of the protein, thus influencing the cleavage of PK at the N-terminal region. Such alterations in PrP^Sc structure might be the factor responsible for the phenotypic heterogeneity of FFI. The ability of the D178N mutation to promote conversion of PrP^C into PrP^Sc that results in the generation of spontaneous prion pathology was shown in mice genetically modified to express murine PrP with the D177N mutation (the equivalent to human D178N) where PrP^Sc is barely detectable (Bouybayoune et al., 2015; Jackson et al., 2009). The infectious properties of FFI-PrP^Sc were extensively investigated by transmission studies in wild-type animals (Tateishi et al., 1995; Sasaki et al., 2005) and in several lines of mice genetically modified to express human PrP (Telling et al., 1994; Telling et al., 1995) as well as primates (Tateishi et al., 1995; Brown et al., 1994; Collinge et al., 1995; Telling et al., 1996; Takeuchi et al., 2019). Results of these studies showed that several factors, including the polymorphisms at codon 129 of the incoming FFI-PrP^Sc inoculum, the prion titer of the FFI brain areas used to challenge the animals, and the PrP^C amino acid sequence of the recipient animals, could significantly influence the transmission efficiency. Notably, when inoculated in mice expressing a chimeric human-mouse PrP, FFI-PrP^Sc is able to promote the generation of prions characterized by similar biochemical features of the original inoculum (Telling et al., 1996). In a recent report, FFI was efficiently transmitted to knock-in mice expressing the bank vole PrP (BvPrP) with methionine at codon 109 of the PrP^C (Takeuchi et al., 2019). Also in this case, the PrP^Sc generated in mice showed the typical biochemical features of FFI-PrP^Sc (predominance of the di-glycosylated band and migration of the un-glycosylated PrP species at 19 kDa). By exploiting the recently developed Protein Misfolding Cyclic Amplification (PMCA) (Saborio et al., 2001) and Real-Time Quaking-Induced Conversion (RT-QuIC) assays (Atarashi et al., 2011), it was shown that FFI-PrP^Sc can promote PrP^C misfolding and aggregation in vitro (Orrú et al., 2015). In this context, we have previously shown that the PMCA is able to detect traces of PrP^Sc present in the olfactory mucosa (OM) of a patient with FFI (at a late stage of the disease) using the brain of bank voles (Bv109M) as a reaction substrate (Redaelli et al., 2017).

In the present study, we verified whether PMCA-generated products possess infectious properties when challenged in mice genetically modified to express the BvPrP with methionine at codon 109 (BvPrP-Tg407) (Espinosa et al., 2016). In particular, animals were inoculated with (1) raw FFI or Alzheimer’s disease (AD) brain homogenates (FFI-BH or AD-BH), (2) their PMCA products (FFI-BH_PMCA or AD-BH_PMCA), and (3) PMCA products obtained from OM of FFI or AD patients (FFI-OM_PMCA or AD-OM_PMCA). Our results indicate that BvPrP-Tg407 mice are highly susceptible to infection by FFI-BH but also by the PMCA products (FFI-BH_PMCA and FFI-OM_PMCA). Particularly, animals inoculated with PMCA products developed prion isolates and neuropathological changes significantly different from those observed in mice challenged with FFI-BH. It is likely that, through PMCA, we have generated bank vole-adapted FFI prion isolates that acquired infectious properties distinct from that of the original FFI, and this resulted in the appearance of different pathological phenotypes of the disease. As expected, none of the animals inoculated with AD-related samples developed prion pathology.

This is the second study showing that PrP^Sc amplified by PMCA from peripheral tissues of patients with prion diseases is infectious when challenged in susceptible mice. In particular, our findings show that the PMCA-amplified products either generated from the brain or the OM of FFI patients are infectious when inoculated in transgenic mice expressing the BvPrP (BvPrP-Tg407). Remarkably, BvPrP-Tg407 mice do not develop spontaneous pathology (Espinosa et al., 2016). Regardless of the inoculum, all mice injected with PMCA products developed very similar neuropathological alterations characterized by focal and plaque-like deposits of PrP^res mainly affecting the thalamus and the striatum. In contrast, animals inoculated with FFI brain homogenate (not subjected to PMCA) showed different histopathological features, for example, a stronger synaptic and diffuse PrP^res immunoreactivity with focal and plaque-like deposits consistently affecting the thalamus, the striatum, and the deep layer of the cerebral cortex. Notably, the intensity of prion deposition observed in all mice was paralleled by a proportional glial activation. Unfortunately, we did not have the possibility to inject the raw FFI-OM because the original sample underwent several analyses (including extensive PMCA and RT-QuIC investigations, as reported in the work of Redaelli et al., 2017) and we did not have enough material for the inoculation. An additional OM sampling (finalized to animals’ injection) was not possible because the patient died soon after the first OM collection. However, a very recent study (Raymond et al., 2020) showed that OM of patients with sporadic CJD are able to induce prion disease when challenged in transgenic mice overexpressing the human PrP with methionine at codon 129 (Tg66). In this study, only a small percentage of animals developed the disease, thus suggesting that the infectivity in OM is very low.

The use of BvPrP-Tg407 mice was dictated by the fact that the PrP^Sc present in OM of the FFI patient was efficiently amplified by using the brain of bank vole (Bv109M) as a PMCA reaction substrate (Redaelli et al., 2017). Therefore, to avoid additional passages of species, we decided to inject BvPrP-Tg407 mice while being conscious that the original properties of the FFI prions could have been altered. However, after injection, this model has generated a bank vole-adapted FFI prion isolate that, to some extent, retained few features of the original human FFI strain.

We have attempted to amplify the FFI-PrP^Sc from OM by using the brain of mice expressing the human PrP with methionine at codon 129 of PRNP (HuPrP129MM) as a PMCA substrate, but we were not successful. Using this model, we have shown that the PrP^Sc amplified from the urine of patients with the new variant CJD (vCJD) was able to maintain the same biochemical and neuropathological features of the vCJD-PrP^Sc present in the brain (Cali et al., 2019). However, in the present work, we were required to use the bank vole PrP^C as an efficient acceptor of the FFI prions and our results need to take into consideration these experimental constraints.

The first important finding of this study was that BvPrP-Tg407 mice were highly susceptible to human FFI prions (100% attack rate). It is known that the transmission of prions between species is an inefficient process because of the species barrier that is mainly dictated by the amino acid differences between the PrP^Sc of the donor and the PrP^C of the acceptor species, and the conformational properties of PrP^Sc aggregates (Hagiwara et al., 2013). It is known that the BvPrP can be converted into PrP^Sc by a number of human and animal prion strains with high efficiency (Watts et al., 2014; Nonno et al., 2006; Agrimi et al., 2008). There is only one work showing that FFI prions are efficiently transmissible to knock-in mice expressing BvPrP with methionine at codon 109 of Prnp (Takeuchi et al., 2019). Our work confirmed that also our model is susceptible to FFI. In particular, animals inoculated with FFI-BH showed mild spongiform changes and glial activation mainly localized in the thalamus, striatum, and deep layer of the frontal cortex. They accumulated PrP^res that was characterized by the typical FFI glycoform profile: predominance of the di-glycosylated PrP band and migration of the un-glycosylated band at 19 kDa. However, several lines of evidence indicate that the BvPrP is prone to convert into PrP^Sc while retaining these biochemical features, regardless of the inocula (Espinosa et al., 2016). For instance, upon inoculation with different human and animal prion strains, transgenic mice expressing the BvPrP with methionine or isoleucine at codon 109 (TgM109 or TgI109, respectively) developed prion pathology and all generated PrP^res were characterized by a predominance of the di-glycosylated band, even in those challenged with human or animal prion strains with the mono-glycosylated predominant PrP band (e.g., sporadic CJD or Rocky Mountain Laboratory prion strain (RML)) (Watts et al., 2014). This has been observed also in transgenic mice expressing BvPrP with isoleucine at codon 109 and the D178 mutation that spontaneously developed prion diseases with the presence of a PrP^res with a predominant di-glycosylated band (Watts et al., 2016). Many other studies showed that this phenomenon occurred also in other transgenic mouse models inoculated with FFI, thus suggesting that, regardless of the recipient mice, some biochemical features typical of the FFI are reproduced after inoculation (Telling et al., 1996; Takeuchi et al., 2019).

For this reason, although the FFI glycoform profile was maintained upon transmission in BvPrP-Tg407 mice, we need to consider that this finding might be related to some intrinsically features of the animal model used. Another alteration that we have noticed in the brain of many FFI-BH-inoculated mice, as previously published (Jackson et al., 2009), was a significant enlargement of the ventricular spaces. This change was not found in the brain of mice inoculated with FFI-BH_PMCA and FFI-OM_PMCA. Surprisingly, in one animal of the FFI-BH-inoculated group we have identified a PrP^res form characterized by the predominance of the mono-glycosylated band with the un-glycosylated one always migrating at 19 kDa. In addition, compared to all the other prions of this group, it differentially behaved to PK and Gdn-HCl treatments (Figure 4—figure supplement 1) and PrP^Sc was not detectable by immunohistochemistry, even by treating the samples with lower concentrations of PK. Finally, this animal did not show the typical ventricles enlargement observed in the brains of the other mice of this group. Therefore, we decided to exclude this animal from the rest of the group in consideration of the fact that it might have generated a distinct prion isolate. There is a compelling evidence that some human prion strains originally believed to be ‘pure’ are instead composed by a mixture of PrP^Sc variants (Cassard et al., 2020; Kobayashi et al., 2011; Bishop et al., 2010; Morales et al., 2016). Interestingly, a very recent publication of the group of Kitamoto Tetsuyuki showed that even the FFI^D178N-129MM strain might not be pure and that at least two distinct prion strains can be responsible for the disease, one associated with the typical clinical presentation of the disease and the other associated with an atypical phenotype (Takeuchi et al., 2019). It is therefore conceivable that the mono-glycosylated PrP^res observed in one of the animals inoculated with FFI-BH might have emerged either because the FFI propagates as a mixture of prions (as also shown for other sporadic forms of prion diseases) or as a random event due to specific limitations imposed by the animal model used. Indeed, these mice have never been challenged with FFI before and seem to be potentially permissive to underrepresented prion strains eventually coexisting in the same isolate.

Prior to injection in BvPrP-Tg407 mice, we wanted to assess whether and to what extent the PMCA analysis could have modified the original features of PrP^Sc present in FFI-BH, eventually generating a different infectious prion isolate. To this aim, we have performed PK and Gdn-HCl characterization of PrP^Sc present in FFI-BH and FFI-BH_PMCA and found that after PMCA the biochemical properties of PrP^Sc have slightly changed. Unfortunately, due to lack (raw OM) or insufficient material (FFI-OM_PMCA that was thoroughly inoculated in mice), we could not evaluate whether the amplification of FFI-OM might have generated a PrP^Sc with different biochemical properties. We have then evaluated the biochemical and neuropathological alterations developed in the brain of all injected mice, with a special focus on those challenged with FFI-BH, FFI-BH_PMCA, and FFI-OM_PMCA. Notably, mice inoculated with FFI-BH and FFI-BH_PMCA developed distinct pathologies, while those inoculated with FFI-OM_PMCA showed alterations analogous to those found in FFI-BH_PMCA-challenged animals, although less pronounced. For this reason, the PMCA might have produced prion isolates with similar infectious properties, regardless of the tissue used for the amplification (BH vs. OM). This evidence prompted us to speculate that prions present in the brain and OM could be similar, thus explaining the pathological analogies observed in mice challenged with PMCA-generated products. However, it should be considered that other factors (e.g., genetic or environmental) of the BvPrP-Tg407 recipient mice might have further controlled the formation of these prions. What is certain, in any case, is that these PMCA-produced prions are not the results of a de novo generation since we did not observe any PrP^res in amplified brain or OM samples collected from the AD patient that were subjected to the same amplification procedures. Overall, these products did not induce pathology when challenged in BvPrP-Tg407 mice. To confirm the absence of prions (even trace amounts) in the brain of these animals, we have performed RT-QuIC experiments that showed the lack of seeding activity in all AD-related samples. As expected, strong RT-QuIC responses were obtained from the analyses of mice inoculated with FFI-related material.

Taken together these results showed that the FFI brain homogenate was able to induce pathology in BvPrP-Tg407 mouse model and promoted the generation of two distinct prion isolates, one more represented that was characterized by the prevalence of the di-glycosylated band (83%) and another less represented characterized by the prevalence of the mono-glycosylated band (17%). Both isolates showed distinct features and support the fact that FFI might not be a pure strain, even among FFI^D178N-129MM subjects. As previously mentioned, all PMCA products were infectious and elicited analogous biochemical and neuropathological changes when challenged in BvPrP-Tg407 mice, probably due to prior selective effects of the PMCA on prions present in the brain or OM of the FFI patient. To better investigate this aspect, we have compared the biochemical properties of PrP^Sc present in FFI-BH_PMCA (prior to passaging in mice) with those of the PrP^Sc generated in the brain of FFI-BH_PMCA-injected mice. We have observed that the inoculation generated a PrP^Sc significantly more resistant to PK digestion and less stable against Gdn-HCl compared to that of the inoculum. Thus, it might be hypothesized that other than a possible PMCA selection of prions present in the brain or OM of the FFI patients, additional processes of adaptation might have occurred in the brain of inoculated mice.

In contrast, animals inoculated with AD-BH, AD-BH_PMCA, and AD-OM_PMCA did not show any prion-related pathology.

From this study, we can conclude that BvPrP-Tg407 mice are susceptible to both the FFI prions and their bank vole-adapted PMCA-generated products. We were intrigued by the fact that mice inoculated with FFI-OM_PMCA developed less PrP^res deposits compared to those inoculated with FFI-BH_PMCA. However, by using the methods generally employed to study prion strains (e.g., PK, Gdn-HCl, PNGase F assays), we did not detect other differences supporting that this group of mice might have developed a distinct prion isolate from that observed in FFI-BH_PMCA-inoculated animals. Additional and more advanced studies are needed to further investigate this aspect. Moreover, we have noticed that animal number #1 of the FFI-OM_PMCA group showed two distinct un-glycosylated PrP^res fragments, thus further supporting the possibility that PMCA might have generated a multitude of unstable prion isolates capable of inducing slightly distinct alterations even within the same group of animals. This finding was detectable only with the use of the N-terminal 6D11 antibody and was not observed with the use of the C-terminal Sha31 antibody.

In summary, our injected mice developed prion pathologies that recapitulate some of the key clinical hallmarks of FFI (e.g., vacuolation, glial activation, and PrP^res deposition mainly affecting the thalamus) and, although with some limitations, this model can be used to (1) deepen some of the pathological events associated with FFI, (2) uncover new factors eventually involved in its phenotypic heterogeneity, and (3) evaluate the effects of therapeutic compounds to interfere with the progression of the disease in a more phenotypic-specific way. The recent discovery that FFI patients with the same genotype might have distinct clinic-pathological presentations of the disease suggests that still unidentified factors (including the presence of distinct PrP^Sc strains), other than the genetic background, may be responsible for the pathogenesis of the disease. This evidence clearly indicates that the mutation can trigger the onset of the disease but many other factors modulate the pathological process, which results in a heterogeneous presentation of the disease. The possibility of detecting PrP^Sc in the OM can be exploited for basic research experiments, for therapeutic studies but also for monitoring disease progression, especially in patients eventually enrolled in clinical trials. This study shows that OM contains traces of FFI-PrP^Sc that can be efficiently amplified by PMCA using the brain of bank vole as substrate and that the amplified products are infectious when challenged in mice that express the bank vole PrP^C (BvPrP-Tg407).

The fact that the PMCA products are as infectious as the FFI brain homogenate raises major biosafety concerns that require the adoption of specific precautions when manipulating amplified material (Bistaffa et al., 2017). For diagnostic purpose, a safer alternative is represented by the RT-QuIC assay that, according to the latest evidence, was found not to replicate biohazardous prions in vitro (Raymond et al., 2020).

Although with some experimental constraints, we showed that the OM of FFI patients contains potentially infectious prions. All inoculated animals developed pathology in the thalamus, which is also the area typically affected in the brain of patients with FFI. Moreover, the glycoform ratio of the PrP^res was similar to that found in the brain of diseased patients. Therefore, some of the typical features of the FFI-PrP^Sc have been carefully reproduced in this animal model, even in samples that underwent PMCA treatment before the injection. We are aware of the fact that the experiments have been performed using the bank vole PrP^C as acceptor of FFI prions, either in vitro (PMCA with Bv109M substrate) or in vivo (BvPrP-Tg407). Hence, some important FFI-PrP^Sc features might have been lost due to the passage of the species barrier, as already shown by several experimental transmission studies described in the literature (Bartz et al., 2000; Peretz et al., 2002; Bruce et al., 1994). Additional studies with more suitable in vitro and in vivo models are still needed to investigate whether the properties of the FFI-PrP^Sc present in the OM and brain are similar or whether they are influenced by the environment of the tissues. Having the possibility to collect (with non-invasive procedures) and amplify PrP^Sc from the OM of FFI patients and test its infectious properties in mice will consent to deepen the pathological mechanisms associated with individual disease phenotype, identify the factors (other than the mutation and the polymorphism at codon 129) eventually involved in the phenotypic heterogeneity of the disease, and uncover new potential targets for the development of individual therapies capable of blocking the progression of this devastating condition. An increasing number of studies are being published to support the important role that the OM might have for research and diagnosis purposes in the field of prion and other neurodegenerative diseases (Redaelli et al., 2017; Raymond et al., 2020; Orrú et al., 2014; De Luca et al., 2019).

All data generated or analyzed during this study are included in the manuscript.

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Author details

1. Edoardo Bistaffa

   Fondazione IRCCS Istituto Neurologico Carlo Besta, Division of Neurology 5 and Neuropathology, Milan, Italy

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Writing - original draft, Writing - review and editing

   Contributed equally with

   Alba Marín-Moreno

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7441-153X

2. Alba Marín-Moreno

   Centro de Investigación en Sanidad Animal (CISA-INIA), Valdeolmos, Madrid, Spain

   Contribution

   Data curation, Investigation, Methodology, Writing - original draft

   Contributed equally with

   Edoardo Bistaffa

   Competing interests

   No competing interests declared

3. Juan Carlos Espinosa

   Centro de Investigación en Sanidad Animal (CISA-INIA), Valdeolmos, Madrid, Spain

   Contribution

   Data curation, Investigation, Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-6719-9902

4. Chiara Maria Giulia De Luca

   1. Fondazione IRCCS Istituto Neurologico Carlo Besta, Division of Neurology 5 and Neuropathology, Milan, Italy
   2. Scuola Internazionale Superiore di Studi Avanzati (SISSA), Department of Neuroscience, Laboratory of Prion Biology, Trieste, Italy

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

5. Federico Angelo Cazzaniga

   Fondazione IRCCS Istituto Neurologico Carlo Besta, Division of Neurology 5 and Neuropathology, Milan, Italy

   Contribution

   Data curation, Investigation, Methodology

   Competing interests

   No competing interests declared

6. Sara Maria Portaleone

   ASST Santi Paolo e Carlo, Department of Health Sciences, Otolaryngology Unit, Università Degli Studi di Milano, Milan, Italy

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

7. Luigi Celauro

   Scuola Internazionale Superiore di Studi Avanzati (SISSA), Department of Neuroscience, Laboratory of Prion Biology, Trieste, Italy

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

8. Giuseppe Legname

   Scuola Internazionale Superiore di Studi Avanzati (SISSA), Department of Neuroscience, Laboratory of Prion Biology, Trieste, Italy

   Contribution

   Supervision, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

9. Giorgio Giaccone

   Fondazione IRCCS Istituto Neurologico Carlo Besta, Division of Neurology 5 and Neuropathology, Milan, Italy

   Contribution

   Supervision, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

10. Juan Maria Torres

    Centro de Investigación en Sanidad Animal (CISA-INIA), Valdeolmos, Madrid, Spain

    Contribution

    Conceptualization, Supervision, Funding acquisition, Writing - original draft, Writing - review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-0443-9232

11. Fabio Moda

    Fondazione IRCCS Istituto Neurologico Carlo Besta, Division of Neurology 5 and Neuropathology, Milan, Italy

    Contribution

    Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review and editing

    For correspondence

    Fabio.Moda@istituto-besta.it

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-2820-9880

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