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Glycans are not necessary to maintain the pathobiological features of bovine spongiform encephalopathy [1]

['Alicia Otero', 'Centro De Encefalopatías Y Enfermedades Transmisibles Emergentes', 'Universidad De Zaragoza', 'Iss Aragón', 'Zaragoza', 'Tomás Barrio', 'Umr Inrae-Envt Interactions Hôtes-Agents Pathogènes', 'Ihap', 'Institute Nationale De Recherche Pour L Alimentation', 'L Agriculture Et L Environnement']

Date: 2022-12 

The role of the glycosylation status of PrP C in the conversion to its pathological counterpart and on cross-species transmission of prion strains has been widely discussed. Here, we assessed the effect on strain characteristics of bovine spongiform encephalopathy (BSE) isolates with different transmission histories upon propagation on a model expressing a non-glycosylated human PrP C . Bovine, ovine and porcine-passaged BSE, and variant Creutzfeldt-Jakob disease (vCJD) isolates were used as seeds/inocula in both in vitro and in vivo propagation assays using the non-glycosylated human PrP C -expressing mouse model (TgNN6h). After protein misfolding cyclic amplification (PMCA), all isolates maintained the biochemical characteristics of BSE. On bioassay, all PMCA-propagated BSE prions were readily transmitted to TgNN6h mice, in agreement with our previous in vitro results. TgNN6h mice reproduced the characteristic neuropathological and biochemical hallmarks of BSE, suggesting that the absence of glycans did not alter the pathobiological features of BSE prions. Moreover, back-passage of TgNN6h-adapted BSE prions to BoTg110 mice recovered the full BSE phenotype, confirming that the glycosylation of human PrP C is not essential for the preservation of the human transmission barrier for BSE prions or for the maintenance of BSE strain properties.

Bovine spongiform encephalopathy (BSE), publicly known as “mad cow disease”, is a neurodegenerative disorder affecting cattle, caused by unconventional agents called prions. BSE can naturally transmit to human beings, producing the variant form of Creutzfeldt-Jakob disease (vCJD), which caused an unprecedented health and economic crisis in the UE. Prions are composed of PrP Sc , a misfolded form of the cellular protein PrP C , which can be variably glycosylated by conjugation with sugar molecules at two positions of its sequence. Several studies reported the role of PrP C -attached sugars on important aspects of prion biology, such as the existence of different prion strains. Here, we demonstrate that it is possible to propagate BSE prions (from different animal and human sources) in a non-glycosylated human PrP C environment without loss of their strain properties. Different BSE isolates were successfully transmitted to a transgenic mouse model expressing non-glycosylated human PrP C , and these animals manifested neuropathological and biochemical signs compatible with BSE. To definitely prove the maintenance of the strain, non-glycosylated BSE prions were transmitted to their original host: transgenic mice expressing cattle PrP C . These animals recovered the full BSE phenotype, confirming that the glycosylation of human PrP C is not relevant for the propagation of this particular prion strain.

In the present study, we assessed the behavior of BSE isolates with different transmission histories when propagated in a mouse model expressing non-glycosylated human PrP C (TgNN6h mice) [ 34 ], following both in vitro and in vivo approaches. Using protein misfolding cycling amplification (PMCA), all BSE prions propagated in the non-glycosylated human substrate. However, they showed different propagation efficiencies, in a way that was consistent with the existence of a transmission barrier. PMCA-propagated prions were readily transmitted to TgNN6h mice on bioassay, which developed biochemical and neuropathological hallmarks strongly indicative of BSE. Moreover, when TgNN6h-adapted BSE prions were back-passaged to a host expressing bovine PrP (BoTg110), the full BSE phenotype was recovered. Taken together, our results suggest that the absence of glycans alter neither the strength of the human transmission barrier for BSE nor the BSE strain pathobiological features.

A common feature of prion diseases is the accumulation of the pathological prion protein (PrP Sc ) in the central nervous system (CNS) of affected individuals. PrP Sc is a self-propagating, misfolded isoform of the host-encoded cellular prion protein (PrP C ), a membrane-anchored glycoprotein that is abundantly expressed in the CNS [ 10 – 12 ]. PrP C sequence contains two consensus sites for N-glycosylation, involving asparagine residues at positions 181 and 197 in the human PrP sequence (or corresponding positions in other species) which can be variably occupied [ 13 ], generating di-, mono-, and unglycosylated mature forms of PrP [ 14 ]. Several studies have demonstrated the key role of N-linked glycans in the intracellular trafficking and membrane location of PrP C [ 15 – 19 ]. This protein has a still-elusive function [ 20 , 21 ]. Although it was thought that the expression of PrP C in the cellular membrane was fundamental for the development of prion disease [ 22 – 25 ], it has been shown that mice expressing an anchorless PrP C (lacking the GPI attachment to the cell membrane) can develop a fatal transmissible amyloid encephalopathy [ 26 ]. Prion protein glycosylation can significantly modulate the interactions between heterologous PrP C and PrP Sc molecules, suggesting that glycans could be determining not only in the conversion efficiency of PrP C into its pathological counterpart, but also in the cross-species transmission of prions [ 27 ]. Other works using transgenic mice expressing an anchorless PrP C , which due to altered post-translational processing is poorly glycosylated, obtained similar results [ 28 ]. In addition, in the context of a defined host, PrP C can be misfolded into a great variety of prion strains, which are characterized by unique clinical features, neuropathological patterns and biological properties, including distinctive ratios of PrP Sc glycoforms. In general, strain properties are faithfully recapitulated upon serial passages in the same animal species [ 29 – 33 ]. Thus, the role of PrP C glycosylation in the transmission barrier phenomenon and in the encoding of strain-specific properties has been widely investigated in vivo.

Prion diseases are fatal neurodegenerative disorders that include scrapie in sheep and goats, bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease (CWD) in cervids and Creutzfeldt-Jakob disease (CJD) in humans. Following its association with the variant form of Creutzfeldt-Jakob disease (vCJD) in humans [ 1 ], BSE represented one of the major public health crises in Europe in the last decades. Several studies strongly suggest that the BSE epidemic was caused by a single strain, which can be transmitted to a wide range of hosts without apparent alteration of its pathobiological features [ 2 – 6 ]. However, its pathogenicity towards humans can be enhanced through passages in other species, such as sheep and goats [ 7 , 8 ] or macaques [ 9 ].

A Spongiosis and PrP Sc deposition profiles. Spongiform lesions and PrP Sc deposition were evaluated semiquantitatively on a scale of 0 (absence of lesions/deposits) to 5 (high intensity lesion/deposition) in the following brain areas: frontal cortex (Fc), parietal cortex (Pc), septal area (Sa), corpus callosum (Cc), hippocampus (Hc), thalamus (T), hypothalamus (Ht), mesencephalon (Mes), pons (Po), cerebellum (Cbl), and medulla oblongata (Mo). Non-glycosylated isolates generated very similar neuropathological features to those of cattle BSE when transmitted to BoTg110 mice. B Immunohistochemical analysis of the brains of BoTg110 mice inoculated with the different isolates (pons). All inocula produced intense vacuolation and PrP Sc plaques especially intense at the pons level. Immunohistochemistry was performed using the 6H4 antibody (1:1,000).

In addition, these mice showed widespread spongiform degeneration, especially prominent in septal area, thalamus, medulla oblongata and pons, with lower scores in hippocampus, mesencephalon, and cerebellar cortex. This distribution of neuropathological lesions is very similar to that observed by other authors in BoTg110 mice inoculated with BSE [ 43 , 44 ]. All BoTg110 mice that succumbed to disease developed fine-punctate, granular and plaque-like deposits, following a similar brain distribution ( Fig 6 ). This morphological pattern has been described before in the same model inoculated with BSE isolates [ 4 , 43 ].

Brain homogenates at 1% from challenged mice were digested with 85 μg/mL (for original isolates) or 170 μg/mL (for TgNN6h and BoTg110 brains) of proteinase K and analyzed by Western blot using 6H4 antibody (1:10,000). A transition from a classical three-banded BSE pattern in the original isolates to a single 19-kDa band-containing pattern in TgNN6h mice was observed, followed by a complete recovery of the three-banded pattern in BoTg110 mice. Note that the banding pattern of the pBSE original isolate differs from the classical BSE pattern in being predominantly monoglycosylated (a hallmark imposed by porcine PrP C ), while after passage to BoTg110 the pattern is identical to those of other BSE sources. Red arrows indicate passage history.

Two additional factors may have contributed to this reduced transmission efficiency: 1) the fact that TgNN6h mouse brains express ~40% less PrP C than a human brain, thus accumulating lower amounts of PrP Sc and infectivity, and 2) the use of 1% (instead of the standard 10%) brain homogenates as inocula, which further reduces the infective dose administered to each animal. In order to prove this hypothesis, we performed an in vitro titration experiment. Both the original cattle BSE isolate and the BSE-TgNN6h, sBSE-TgNN6h, and pBSE-TgNN6h inocula that were used to infect BoTg110 mice were serially diluted and submitted to two serial PMCA rounds in BoTg110 substrate. While cattle BSE was able to propagate up to dilution 10 −5 , all of the TgNN6h-passaged isolates propagated only up to dilution 10 −1 ( S4 Fig ). These results allowed us to conclude that the propagative dose that accumulates in the brain of TgNN6h mice is significantly lower than that of BSE-infected cattle, thus explaining the low transmission efficiency of these isolates in first-passage BoTg110 mice.

While cattle BSE readily transmitted to BoTg110 mice (100% attack rate) and incubation periods of around 300 dpi, as previously reported [ 4 , 7 ], all three non-glycosylated isolates propagated with incomplete attack rates and longer and heterogeneous incubation periods ( Table 2 ). These discrepant results are unlikely to reflect a change in the strain properties of BSE prions upon passage in the TgNN6h model (since, as stated below, the biochemical and neuropathological hallmarks were identical to those obtained with the original cattle BSE isolate). Rather, they evidence the transmission barrier naturally occurring between cattle and human, likely increased by the two additional amino acid changes introduced in the TgNN6h PrP C sequence to impede glycosylation.

Immunohistochemical analyses revealed that the plaques that were observed with hematoxylin and eosin staining were composed of prion protein, presenting a PrP amyloid core (confirmed by Congo red staining, as showed in S2 Fig ) generally surrounded by a halo of spongiform degeneration ( Fig 4B ). These neuropathological changes are termed florid plaques and are characteristic of vCJD in humans [ 41 , 42 ]. Large florid plaques were detected in the cerebral cortex, thalamus, hypothalamus, and white matter structures such as the corpus callosum. In certain brain areas, especially in the hippocampus, plaques were usually confluent and very disruptive. In other areas, such as cerebellum and medulla oblongata, amyloid plaques were more discretely distributed. In addition to plaques, granular deposits of prion protein were detected throughout the brain. No differences in neuropathological characteristics were observed among the different in vitro-adapted BSE sources or between the first and second passage of TgNN6h mice.

A Spongiosis and PrP Sc deposition profiles. Spongiform lesions and PrP Sc deposition were evaluated semiquantitatively on a scale of 0 (absence of lesions/deposits) to 5 (high intensity lesion/deposition) in the following brain areas: frontal cortex (Fc), parietal cortex (Pc), septal area (Sa), corpus callosum (Cc), hippocampus (Hc), thalamus (T), hypothalamus (Ht), mesencephalon (Mes), pons (Po), cerebellum (Cbl), and medulla oblongata (Mo). All BSE inocula showed almost identical neuropathological profiles when transmitted to TgNN6h mice. B Hematoxylin and eosin staining and immunohistochemical analysis of the brains of BSE-PMCA, sBSE-PMCA, pBSE-PMCA or vCJD-PMCA affected mice showed the presence of conspicuous plaque-like deposits, surrounded by areas of severe spongiform change (florid plaques). Scale: x20, insert pictures: x60. Immunohistochemistry was performed using the 3F4 antibody (1:1,000). Brain areas: Parietal cortex (BSE-PMCA), mesencephalon (sBSE-PMCA), and thalamus (pBSE-PMCA and vCJD-PMCA).

All TgNN6h animals that accumulated PrP Sc in the brain presented a banding pattern characterized by a clear band with a molecular weight of 19 kDa, likely corresponding to non-glycosylated BSE PrP Sc . A lower band (around 15 kDa) was consistently observed only in these animals ( Fig 3 ), but not in Tg340 mice. Deglycosylation of BSE-infected TgNN6h samples with PNGase F did not provoke a shift of this band ( S1 Fig ). In addition, Tg340 samples treated with PNGase F showed a reduction of the three-bands pattern to a single 19–20 kDa band, but it did not show the 15 kDa band ( S1 Fig ). Therefore, we believe that this band corresponds to an endoproteolytic fragment specific to TgNN6h mice, although the reason why it only arises from unglycosylated PrP Sc remains to be investigated.

At first passage, none of the Tg340 inoculated mice developed disease after the inoculation of BSE, sBSE, or pBSE isolates. However, one pBSE-inoculated Tg340 mice culled at 735 dpi was positive for PrP Sc in the brain by Western blot. In contrast, vCJD transmitted to Tg340 mice, leading to incomplete attack rates and long survival periods ( Table 1 ), indicative of a considerable transmission barrier. These results, although contrasting with those published elsewhere [ 7 ], were expected since the inoculations were performed with 1% brain homogenates, i.e. 10 times less infectious material than in most other studies, that use 10% brain homogenates. In addition, the BSE and vCJD isolates used were different from those inoculated in the present study, and therefore it is possible that these isolates contained different levels of infectivity. This, together with the prolonged incubation times close to the lifespan of the animals observed also in previous studies, could explain the absence of transmission in our case.

Notably, no TgNN6h mouse inoculated with the direct (i.e. not PMCA-propagated) BSE, sBSE, or pBSE isolates developed disease or accumulated detectable levels of PrP Sc in the brain for up to 600 dpi ( Table 1 ); this was expected given the low PrP C expression level of these animals (0.6x) and agrees with studies in which BSE was unable to infect knock-in models (thus expressing physiological levels of the protein) [ 40 ]. However, after 584 dpi, one animal from the TgNN6h group challenged with the direct vCJD inoculum developed neurological signs, particularly tremor and ataxic gait. This animal and five other vCJD-inoculated TgNN6h mice that were culled at ∼700 dpi with no signs of clinical disease presented PrP Sc accumulation in the brain ( Table 1 ).

TgNN6h mice inoculated with the in vitro-propagated BSE-PMCA, sBSE-PMCA, pBSE-PMCA and vCJD-PMCA isolates succumbed to prion disease on first passage with a 100% attack rate. TgNN6h animals that developed clinical disease showed hyperesthesia, kyphosis and ataxic gait in a first stage of the disease, followed by weight loss, lethargy and ruffled coat in a later phase. These transmissions to TgNN6h mice occurred with similar survival periods. Second passage of BSE-PMCA, sBSE-PMCA and pBSE-PMCA isolates resulted also in 100% attack rates in TgNN6h mice in all cases, with survival periods similar to those seen in the first passage ( Table 1 ).

The original inocula (consisting of 1% brain homogenates) and the in vitro-adapted inocula (following sufficient serial PMCA passages to eliminate any traces of the original seed) were inoculated in TgNN6h mice. This transgenic line expresses non-glycosylated human 129M PrP C at approximately 60% of the physiological levels [ 34 ]. The original, non-adapted isolates were also bioassayed in Tg340 mice, which overexpresses a fully glycosylated human 129M PrP C (at levels 4-fold higher than those detected in human brain), as controls [ 7 ]. At least 6 animals of either transgenic line were inoculated with each of the isolates ( Table 1 ).

The products obtained from PMCA propagation on either cattle or human substrate showed the PrP Sc signature characteristic of BSE, i.e. the non-glycosylated band at 19 kDa and a predominance of the diglycosylated species ( Fig 2 ). This was true also for pBSE, whose original isolate presented, at variance with the rest of BSE sources, a banding pattern characterized by a predominance of the monoglycosylated band; this is likely a particularity of porcine PrP [ 35 , 39 ] and does not entail a modification of the BSE strain contained in this inoculum. In contrast, cattle BSE, sBSE and vCJD original isolates presented the prototypic BSE banding pattern from the beginning.

As predicted from previous studies demonstrating the absence of transmission barrier of BSE prions towards its natural host, all isolates propagated in just one 24-h PMCA round in wild-type cattle brain substrate ( Fig 1B ). In contrast, the distinct BSE isolates showed different propagation efficiencies when using normal human brain homogenate as substrate. The vCJD inoculum propagated in all replicates in the first round, whereas BSE, sBSE and pBSE isolates did not overcame the barrier until round 3 ( Fig 1C ). Again, this result agreed with the presence of a transmission barrier towards humans and suggests that the difficulties encountered with the TgNN6h substrate for propagating BSE prions is likely unrelated to its glycosylation status, but rather linked to the human PrP C sequence.

Original inocula: brain homogenates from BSE-infected cattle, BSE-infected sheep (sBSE), BSE-infected pig (pBSE), or a vCJD patient; PMCA in TgNN6h substrate: isolates generated in vitro after 19 rounds of PMCA in TgNN6h substrate; PMCA in cattle substrate: isolates generated in vitro after 13 rounds of PMCA in wild-type bovine substrate; PMCA in human substrate: isolates generated in vitro after 16 rounds of PMCA in wild-type human substrate. Original inocula and samples propagated in cattle and human substrate were digested with 85 μg/mL of proteinase K (PK), while TgNN6h-propagated samples were digested with 170 μg/mL PK, and analyzed by Western blot using monoclonal antibody 6H4 (1:10,000); bands corresponding to incomplete digestion are marked with asterisks. Undigested TgNN6h, cattle, and human substrates were loaded as controls.

As expected, given the absence of different glycoforms in TgNN6h PrP C , PMCA products derived from the propagation of all four BSE sources on TgNN6h substrate showed a PrP Sc signature characterized by a single, non-glycosylated band at 19 kDa ( Fig 2 ). Given that PMCA is a highly stochastic technique (the efficiency among rounds can differ significantly), and that these samples were submitted for SDS-PAGE and Western blot without prior adjustment of quantities, the differences in signal intensity observed among different TgNN6h-propagated isolates are likely due to dissimilarities in PrP Sc amount rather than to different resistances to PK.

Each BSE isolate was subjected, in quadruplicate, to 15 serial PMCA rounds in TgNN6h substrate to compare their abilities to induce the misfolding of non-glycosylated human PrP C . The vCJD isolate readily propagated in TgNN6h substrate with a 100% efficacy (4/4 positive replicates) after a single 24 h round. In contrast, cattle BSE, sheep-passaged BSE, and pig-passaged BSE required more than one PMCA rounds to propagate, and they did so with different efficiencies ( Fig 1A ), in a way consistent with the existence of a transmission barrier. In particular, BSE required 15 rounds for a 25% amplification, and sBSE needed 7 rounds to show 75% propagation, whereas pBSE reached 100% propagation efficiency within 9 rounds. The fact that BSE propagated significantly worse than sBSE (and pBSE) in human substrate likely correlates with its more difficult transmission capability in vivo when compared to sheep-passaged BSE, reported elsewhere [ 8 ].

In order to assess the in vitro misfolding ability of non-glycosylated human PrP and how the lack of glycans could affect the human transmission barrier for BSE prions, TgNN6h mice brain homogenates were seeded in vitro with BSE isolates of different origins: classical cattle BSE (BSE), sheep (sBSE) and pig-passaged BSE (pBSE) [ 35 ] and human vCJD. As mentioned before, classical BSE prions present particular abilities to spread to distinct animal species [ 36 – 38 ], including humans [ 1 ], and, moreover, they show singular stable pathobiological features [ 2 , 4 , 5 ].

Discussion

In the present study, we aimed at assessing whether the absence of glycans in the human PrPC could impact the transmission barrier and the strain properties of BSE prions. BSE is the only animal prion strain for which a natural transmission to humans has been described, leading to its human counterpart variant Creutzfeldt-Jakob disease (vCJD). Prions accumulating in the brain of vCJD patients maintain BSE pathobiological features [1,45,46]. Aside from classical BSE, the potential cross-species transmission of prions to humans has been demonstrated for classical scrapie [47], CWD [48], and L-BSE [49–51] by experimental challenge of transgenic mice overexpressing human PrPC and/or in vitro propagation techniques. However, these successful transmissions are the exception rather than the rule since many other similar studies have reported opposite results [48,52–54]. Indeed, in parallel experiments (not shown in this publication) we seeded the TgNN6h substrate with SSBP/1, atypical scrapie, CWD, L-BSE and H-BSE isolates and subjected them to 15 rounds of serial PMCA. None of these prion strains was able to propagate in this non-glycosylated human substrate. TgNN6h mice intracerebrally challenged with the same strains did not succumb to disease or accumulate PrPSc in their brains.

In our study, all BSE isolates propagated in the TgNN6h substrate. The vCJD isolate showed the highest efficiency on in vitro propagation (Fig 1A) and, in correlation with this, it was the only one able to transmit in vivo after the inoculation of the original isolate (Table 1). This behavior parallels the results obtained when the same BSE isolates were propagated in wild-type human substrate (Fig 1B), suggesting that the absence of glycans in TgNN6h PrPC does not alter the human transmission barrier for BSE prions. The higher efficiency of vCJD with respect to other BSE prions for propagating in wild-type human substrate has been previously reported [48]. Since PMCA was designed as a methodology to accelerate the misfolding process [55], it is conceivable that the compatibility of amino acid sequences between seed and substrate and other slight adaptations of vCJD to the human brain environment accounts for the high propagation efficiency in human substrate observed for this particular isolate [45]. In addition, transmission studies in humanized transgenic mice and primates have demonstrated that BSE, although showing very stable pathobiological features upon transmission, can readily adapt to the new host [9,40], and once transmitted to human beings in the form of vCJD, the barrier for human-to-human transmission is substantially reduced [40]. This fact could explain the results obtained in the TgNN6h mouse bioassay, since vCJD was the only isolate that transmitted to mice at first passage after the direct inoculation of the original source (Table 1).

The propagation efficiency of all BSE sources was absolute in the bovine substrate (Fig 1C). BSE-derived prions, irrespective of their original host (cattle, sheep, pig or human) transmit to transgenic mice expressing bovine PrPC with no differences in regard to survival times or pathobiological characteristics, indicating a lack of a transmission barrier for the propagation of BSE prions in a bovine PrP substrate [38]. However, they transmit poorly to transgenic human models [38,40,50,56], pointing to the existence of a strong transmission barrier between cattle and humans, even though the transmission of BSE to humans naturally occurs producing vCJD [1].

Among BSE isolates sourced from animal species, we observed that both sBSE and pBSE isolates propagated in TgNN6h substrate more efficiently than cattle BSE (Fig 1A). These results are in accordance with previous studies showing that experimental sBSE prions propagate more efficiently than cattle BSE in transgenic mice expressing human PrPC [7,57], which was attributed to a better structural compatibility between sheep PrPSc and human PrPC [7]. However, we did not observe the same behavior with the wild-type human substrate. The reason for this could be related to the higher PrPC expression levels of human brain in comparison with TgNN6h, which, in combination with the extraordinary sensitivity of PMCA [55,58] make these differences undetectable in vitro.

The implication of the glycosylation status of PrPC on intra- and inter-species transmission of prion strains has been discussed at length, and several studies have suggested that glycosylation of PrPC could be a key factor influencing transmission barrier [15,27,59,60]. Wiseman and collaborators showed that transgenic mice expressing non-glycosylated murine PrPC (G3 mice) were completely resistant to prion disease, and that this alteration was associated to the absence of the first glycosylation site since the elimination of the second site resulted in the efficient transmission of human prions. [60]. As aforementioned, we did not observe any significant alterations of the cross-species transmission barrier for BSE prions in the TgNN6h non-glycosylated human model. Differences in the isolates and/or the transgenic models used could explain the discrepancies between our results and those obtained with the G3 transgenic line [60]. Nevertheless, it is also possible that glycans affect prion propagation in a strain or species-dependent manner. BSE is a particular strain, with the ability of propagating in PrPC from different species without significantly altering its features [6].

All TgNN6h mice inoculated with the in vitro-propagated BSE-PMCA, sBSE-PMCA, or pBSE-PMCA isolates succumbed to prion disease in ∼200 dpi and proved positive for PrPSc accumulation by immunohistochemical techniques and Western blot (Figs 3 and 4). Animals inoculated with direct brain homogenates BSE, sBSE and pBSE did not develop disease in more than 700 dpi. These results corroborate that the direct inoculation requires incubation times longer than the normal lifespan of these mice (especially considering that inoculation were performed with 1% brain homogenates and that this model expresses human PrPC at levels of only 0.6x), while PMCA serial propagation in TgNN6h substrate greatly facilitates overcoming the transmission barrier by the progressive stabilization and adaptation of the in vitro generated prions [61].

Mice inoculated with BSE-PMCA, sBSE-PMCA, pBSE-PMCA and vCJD-PMCA presented very severe neuropathological changes, and showed abundant deposits with the characteristics of florid plaques described in vCJD-affected patients and humanized models (Fig 4) [7,42]. Unglycosylated prions have been reported to favor extracellular plaque formation in murine models of prion disease, in which these prions colocalize with plaque deposits [62]. These results, together with the results obtained in the present study, suggest that florid plaques observed in humanized mice and humans are preferentially formed from unglycosylated PrPSc. Our PMCA-propagated inocula were very similar with respect to the distribution of neuropathological changes (Fig 3), which also coincided with those previously described for Tg340 [7] and Tg650 [63] mice (expressing fully glycosylated human PrPC) inoculated with BSE and BSE-related prions. Overall, our results indicate that the neuropathological hallmarks of BSE were maintained after transmission to a humanized non-glycosylated host.

When analyzed by Western blot, TgNN6h mice that developed disease displayed a single unglycosylated band with a molecular weight of 19 kDa (Fig 3), in most cases accompanied by a low molecular weight fragment, likely a consequence of endoprotease activity on the samples. This lower fragment can be also observed in undigested TgNN6h substrate (Fig 2), but not in Tg340 mice inoculated with vCJD, not even after deglycosylation (S1 Fig). Although we did not further characterized this 15 kDa band seemingly characteristic of TgNN6h mice, we believe that it may correspond to an endoproteolytic fragment similar to others previously described [64], and that apparently occur preferentially on unglycosylated substrate. This glycoprofile was almost identical to that displayed by in vitro-propagated seeds in TgNN6h substrate (Fig 2). These biochemical features are suggestive of BSE [65], indicating that glycans are likely not necessary to maintain the pathobiological features of classical BSE prions. This was later unequivocally proved by the observation that BoTg110 inoculated with TgNN6h-passaged BSE prions developed a prion disease whose neuropathological hallmarks and biochemical features fully coincided with those of BoTg110 animals infected with reference BSE isolates in this study and others [4,7]. These TgNN6h-passaged BSE prions, however, transmitted with incomplete attack rates to BoTg110 mice, in contrast with the results obtained by Padilla and colleagues with normally glycosylated BSE prions. These discrepant results could be explained by the transmission barrier existing between human and bovine PrP sequences, likely increased due to the presence of two additional asparagine-to-glutamine substitutions in the TgNN6h model to avoid glycosylation. Also, the fact that TgNN6h mouse brains contain ~40% less PrPC than human brain could lead to a lower accumulation of PrPSc and infectivity in this model, and the use of 1% brain homogenates for inoculation, instead of the 10% used in previous studies, could contribute to a reduction in the infective dose administered to each BoTg110 animal. A second passage in the same animal model would definitely demonstrate that the incomplete attack rates observed in first-passage BoTg110 animals are due to the existence of such transmission barrier and/or to reduced infectivity accumulating in TgNN6h brains, rather than to an alteration in the strain features of BSE upon passage in the unglycosylated model. In fact, neuropathological features in BoTg110 mice inoculated with the TgNN6h-passaged BSE prions were very similar from those reported by us and other authors in BoTg110 mice inoculated with fully glycosylated BSE prions, regarding distribution [43,44] and morphology of PrPSc deposits [4,43]. Therefore, a second passage in BoTg110 was deemed unnecessary to prove that propagation upon an unglycosylated PrPC does not alter BSE strain features. Nonetheless, in order to confirm that TgNN6h mouse brains accumulate less infectivity with respect to the classical BSE isolate from cattle, an in vitro titration experiment was performed by submitting serial dilutions of all the inocula to two serial PMCA rounds in BoTg110 substrate. As shown in S4 Fig, original cattle BSE isolate was detectable up to dilution 10−5 in the second PMCA round, whereas the three inocula derived from TgNN6h brain homogenates were able to propagate only up to dilution 10−1, i.e. a 10,000-fold lower propagation capability. This apparently lower titer, together with a transmission barrier and the long incubation times characteristic of this strain, which are close to the lifespan of the animals, can explain the incomplete attack rates observed in vivo.

Several studies have suggested that the glycosylation status of host PrPC could strongly determine the phenotypic characteristics of the infecting strain [66] and the transmission efficiency of prions between different species [60]. However, these effects have been observed with some strains but not with others, which indicates that glycans may not be essential for the retention of strain-specific properties [66], or that the strains present dramatically different requirements with respect to the glycosylation status of host PrPC. Piro et al., 2009 showed that unglycosylated PrPSc molecules, generated in vitro using an enzymatically deglycosylated mouse PrPC as a substrate, maintained their strain-dependent neuropathological and biochemical features when inoculated in wild-type mice. These results led to the hypothesis that unglycosylated PrPSc molecules can encode strain-specific patterns of PrPSc accumulation [67]. This conclusion was further supported by Moudjou et al., 2016 by the propagation of 127S scrapie prions in ovine PrP glycosylation mutants and their subsequent transmission to Tg338 ovinized mice. Non-glycosylated, PMCA-propagated 127S prions reproduced the neuropathological and biochemical features of normally glycosylated 127S prions when inoculated in Tg338 mice. In addition, these unglycosylated prions recovered the three-band pattern when propagated in wild-type PrPC by PMCA. Thus, they concluded that glycans do not play a major role in determining strain-specific properties, and that these are encoded in the structural backbone of PrPSc [68].

In line with this, we observed that TgNN6h mice that developed the disease after transmission of BSE isolates showed similar pathobiological features to those described in natural cases of human transmissions [42,65] and in BSE/vCJD-challenged transgenic mice expressing fully glycosylated human PrPC [7,63]. In addition to this, the back-passage of these unglycosylated prions in their original PrPC environment, i.e. BoTg110 mice expressing wild-type cattle PrPC, resulted in the recovery of the BSE full phenotype. BoTg110 mice were chosen for this study as they transmit the BSE agent in absence of any transmission barrier, and are thus the most suitable model to assess whether unglycosylated BSE prions recover their prototypal characteristics. Alternatively, a back-passage of the TgNN6h-propagated BSE isolates into humanized, instead of bovinized, transgenic mice would also provide interesting clues on the properties of these unglycosylated BSE prions. Overall, thus, our results suggest that glycans are not vital on the determination of this transmission barrier or for the conservation of the pathobiological features of BSE prions.

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