Prion Diseases: History, Diversity, and Socioeconomic Importance as a Paradigm of Rare Diseases

Las enfermedades raras son aquellas patologías que afectan a una proporción muy reducida de la población (menos de 50 casos por cada 100 000 personas). Por esta razón, la investigación sobre sus causas y mecanismos, algo imprescindible para dar con una forma de tratarlas o prevenirlas, es insuficiente. Por ello, los pacientes denuncien la falta de cobertura del sistema sanitario y la discriminación social que supone padecer una de estas patologías. Entre las enfermedades raras, se encuentran las denominadas enfermedades priónicas o encefalopatías espongiformes transmisibles. A pesar de ser relativamente conocidas gracias a la crisis sanitaria que supuso el “mal de las vacas locas” a finales del siglo pasado, se conoce todavía relativamente poco sobre estas patologías que afectan tanto a animales como a humanos. En este monográfico se pretende dar a conocer la fascinante historia y la diversidad de las enfermedades priónicas, que sacudieron los cimientos de la biología conocida hasta los años 80 al poner en escena a un nuevo y desconcertante tipo de agente infeccioso: los prionesRare disease are those pathologies that affect a reduced proportion of the population (less than 50 cases per 100 000 people). For this reason, the research on their causes and mechanisms, which is essential to find a way to treat or prevent them, is insufficient. This causes that the patients report a lack of coverage by the health system and the social discrimination that suffering one of these pathologies entails. Among rare diseases, we find the so-called prion diseases or transmissible spongiform encephalopathies. Although they are relatively well-known due to the health crisis provoked by the “mad cow disease” at the end of the last century, there are still many uncertainties about these disorders that affect both animals and humans. This monograph aims at bringing to light the fascinating history and the diversity of prion diseases, which shook the foundations of the biology known before the 1980s by bringing out a new and puzzling type of infectious agent: prion

An Augmented Reality App to Learn to Interpret the Nutritional Information on Labels of Real Packaged Foods

[EN] Healthy eating habits involve controlling your diet. It is important to know how to interpret the nutritional information of the packaged foods that you consume. These packaged foods are usually processed and contain carbohydrates and fats. Monitoring carbohydrates intake is particularly important for weight-loss diets and for some pathologies such as diabetes. In this paper, we present an augmented reality app for helping interpret the nutritional information about carbohydrates in real packaged foods with the shape of boxes or cans. The app tracks the full object and guides the user in finding the surface or area of the real package where the information about carbohydrates is located using augmented reality and helps the user to interpret this information. The portions of carbohydrates (also called carb choices or carb servings) that correspond to the visualized food are shown. We carried out a study to check the effectiveness of our app regarding learning outcomes, usability, and perceived satisfaction. A total of 40 people participated in the study (20 men and 20 women). The participants were between 14 and 55 years old. The results reported that their initial knowledge about carb choices was very low. This indicates that education about nutritional information in packaged foods is needed. An analysis of the pre-knowledge and post-knowledge questionnaires showed that the users had a statistically significant increase in knowledge about carb choices using our app. Gender and age did not influence the knowledge acquired. The participants were highly satisfied with our app. In conclusion, our app and similar apps could be used to effectively learn how to interpret the nutritional information on the labels of real packaged foods and thus help users acquire healthy life habits.Juan, M.; Charco, JL.; García García, I.; Mollá Vayá, RP. (2019). 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Glycans are not necessary to maintain the pathobiological features of bovine spongiform encephalopathy

The role of the glycosylation status of PrPC 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 PrPC. 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 PrPC-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 PrPC is not essential for the preservation of the human transmission barrier for BSE prions or for the maintenance of BSE strain properties

Dogs are resistant to prion infection, due to the presence of aspartic or glutamic acid at position 163 of their prion protein

Unlike other species, prion disease has never been described in dogs even though they were similarly exposed to the bovine spongiform encephalopathy (BSE) agent. This resistance prompted a thorough analysis of the canine PRNP gene and the presence of a negatively charged amino acid residue in position 163 was readily identified as potentially fundamental as it differed from all known susceptible species. In the present study, the first transgenic mouse model expressing dog prion protein (PrP) was generated and challenged intracerebrally with a panel of prion isolates, none of which could infect them. The brains of these mice were subjected to in vitro prion amplification and failed to find even minimal amounts of misfolded prions providing definitive experimental evidence that dogs are resistant to prion disease. Subsequently, a second transgenic model was generated in which aspartic acid in position 163 was substituted for asparagine (the most common in prion susceptible species) resulting in susceptibility to BSE‐derived isolates. These findings strongly support the hypothesis that the amino acid residue at position 163 of canine cellular prion protein (PrPC) is a major determinant of the exceptional resistance of the canidae family to prion infection and establish this as a promising therapeutic target for prion diseases.MINECO/FEDER. Grant Numbers: AGL2015‐65046‐C2‐1‐R, AGL2008‐05296‐C02 Interreg. Grant Number: POCTEFA EFA148/1

Bona fide atypical scrapie faithfully reproduced for the first time in a rodent model

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 a ladder-like PrPres pattern with a predominant 7–10 kDa band. Brain homogenates from spontaneously diseased 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 PrPC), Tg361 (human V129 PrPC), TgVole (bank vole I109 PrPC), bank vole (I109I PrPC), 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.info:eu-repo/semantics/publishedVersio

Bona fide atypical scrapie faithfully reproduced for the first time in a rodent model

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 a ladder-like PrPres pattern with a predominant 7–10 kDa band. Brain homogenates from spontaneously diseased 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 PrPC), Tg361 (human V129 PrPC), TgVole (bank vole I109 PrPC), bank vole (I109I PrPC), 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.info:eu-repo/semantics/publishedVersio

Development of a New largely scalable in vitro prion propagation method for the production of infectious recombinant prions for high resolution structural studies.

The resolution of the three-dimensional structure of infectious prions at the atomic level is pivotal to understand the pathobiology of Transmissible Spongiform Encephalopathies (TSE), but has been long hindered due to certain particularities of these proteinaceous pathogens. Difficulties related to their purification from brain homogenates of disease-affected animals were resolved almost a decade ago by the development of in vitro recombinant prion propagation systems giving rise to highly infectious recombinant prions. However, lack of knowledge about the molecular mechanisms of the misfolding event and the complexity of systems such as the Protein Misfolding Cyclic Amplification (PMCA), have limited generating the large amounts of homogeneous recombinant prion preparations required for high-resolution techniques such as solid state Nuclear Magnetic Resonance (ssNMR) imaging. Herein, we present a novel recombinant prion propagation system based on PMCA that substitutes sonication with shaking thereby allowing the production of unprecedented amounts of multi-labeled, infectious recombinant prions. The use of specific cofactors, such as dextran sulfate, limit the structural heterogeneity of the in vitro propagated prions and makes possible, for the first time, the generation of infectious and likely homogeneous samples in sufficient quantities for studies with high-resolution structural techniques as demonstrated by the preliminary ssNMR spectrum presented here. Overall, we consider that this new method named Protein Misfolding Shaking Amplification (PMSA), opens new avenues to finally elucidate the three-dimensional structure of infectious prions

Recombinant PrP and Its Contribution to Research on Transmissible Spongiform Encephalopathies

The misfolding of the cellular prion protein (PrPC) into the disease-associated isoform (PrPSc) and its accumulation as amyloid fibrils in the central nervous system is one of the central events in transmissible spongiform encephalopathies (TSEs). Due to the proteinaceous nature of the causal agent the molecular mechanisms of misfolding, interspecies transmission, neurotoxicity and strain phenomenon remain mostly ill-defined or unknown. Significant advances were made using in vivo and in cellula models, but the limitations of these, primarily due to their inherent complexity and the small amounts of PrPSc that can be obtained, gave rise to the necessity of new model systems. The production of recombinant PrP using E. coli and subsequent induction of misfolding to the aberrant isoform using different techniques paved the way for the development of cell-free systems that complement the previous models. The generation of the first infectious recombinant prion proteins with identical properties of brain-derived PrPSc increased the value of cell-free systems for research on TSEs. The versatility and ease of implementation of these models have made them invaluable for the study of the molecular mechanisms of prion formation and propagation, and have enabled improvements in diagnosis, high-throughput screening of putative anti-prion compounds and the design of novel therapeutic strategies. Here, we provide an overview of the resultant advances in the prion field due to the development of recombinant PrP and its use in cell-free systems

Generation of a new infectious recombinant prion: a model to understand Gerstmann–Sträussler–Scheinker syndrome

Abstract Human transmissible spongiform encephalopathies (TSEs) or prion diseases are a group of fatal neurodegenerative disorders that include Kuru, Creutzfeldt-Jakob disease, Gerstmann-Sträussler-Scheinker syndrome (GSS), and fatal familial insomnia. GSS is a genetically determined TSE caused by a range of mutations within the prion protein (PrP) gene. Several animal models, based on the expression of PrPs carrying mutations analogous to human heritable prion diseases, support that mutations might predispose PrP to spontaneously misfold. An adapted Protein Misfolding Cyclic Amplification methodology based on the use of human recombinant PrP (recPMCA) generated different self-propagating misfolded proteins spontaneously. These were characterized biochemically and structurally, and the one partially sharing some of the GSS PrPSc molecular features was inoculated into different animal models showing high infectivity. This constitutes an infectious recombinant prion which could be an invaluable model for understanding GSS. Moreover, this study proves the possibility to generate recombinant versions of other human prion diseases that could provide a further understanding on the molecular features of these devastating disorders

A Protein Misfolding Shaking Amplificationbased method for the spontaneous generation of hundreds of bona fide prions

Prion diseases are a group of rapidly progressing neurodegenerative disorders caused by the misfolding of the endogenous prion protein (PrPC) into a pathogenic form (PrPSc). This process, despite being the central event underlying these disorders, remains largely unknown at a molecular level, precluding the prediction of new potential outbreaks or interspecies transmission incidents. In this work, we present a method to generate bona fide recombinant prions de novo, allowing a comprehensive analysis of protein misfolding across a wide range of prion proteins from mammalian species. We study more than 380 different prion proteins from mammals and classify them according to their spontaneous misfolding propensity and their conformational variability. This study aims to address fundamental questions in the prion research field such as defining infectivity determinants, interspecies transmission barriers or the structural influence of specific amino acids and provide invaluable information for future diagnosis and therapy applications.The authors would like to thank the following for their support: IKERBasque foundation, personnel from vivarium, IT service (in particular to Sara Gómez Ramos for her assistance with the PrPdex webpage), maintenance departments of CIC bioGUNE, Neiker and IRTA-CReSA. The authors would also like to acknowledge the work from past laboratory members of the Prion Research Lab from CIC bioGUNE, that despite not directly involved in the manuscript have contributed along the years to the development of all the methods and techniques currently used in the laboratory (specially to Tomás Barrio and Leire Hervá for their efforts at the initial and end stages of the work, respectively). Finally, we would like to thank Jesús R. Requena for always useful scientific discussions and advice. The present work was partially funded by different grants awarded by “Ministerio de Economía y Competitividad” (Spanish Government), grant numbers PID2021-122201OB-C21, PID2021-1222010BC22, PID2021-125946OB-I00 and IJC2020-045506-I, funded by MCIN/ AEI /10.13039/501100011033 and co-financed by the European Regional Development Fund (ERDF), and by the Instituto de Salud Carlos III (ISCIII), grant number AC21_2/00024, to J.C. Additionally, CIC bioGUNE currently holds a Severo Ochoa Excellence accreditation, CEX2021- 001136-S, also funded by MCIN/AEI /10.13039/501100011033. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.info:eu-repo/semantics/publishedVersio