Desarrollo de un nuevo sistema de cribado masivo basado en la propagación in vitro de priones recombinantes infecciosos para la detección de compuestos con actividad anti-priónica.

136 p.Las Encefalopatías Espongiformes Transmisibles (EET) son un grupo de enfermedades neurodegenerativas fatales que se caracterizan por el acúmulo de fibras amiloides de la proteína PrPSc en el sistema nervioso central de los pacientes. Debido a que no existe ningún tratamiento disponible para estas enfermedades, uno de los principales objetivos del campo de estudio de las EET es la búsqueda de fármacos que inhiban o retrasen el proceso de propagación de las fibras amiloides. Para ello, el uso de sistemas de cribado masivo de compuestos con posible actividad anti-priónica es uno de los métodos más prometedores. En esta tesis doctoral se ha desarrollado con éxito un sistema de propagación in vitro depriones altamente infecciosos y aplicable a un método de cribado masivo. Utilizando este novedoso método, se ha cribado una biblioteca de 2500 compuestos, detectándose 3 compuestos con actividad inhibidora no descritos hasta la actualidad. A continuación, se ha confirmado su actividad inhibidora de la propagación utilizando otros métodos de propagación in vitro e in cellula, demostrando la utilidad el nuevo sistema de propagación in vitro para el descubrimiento de nuevos compuestos con actividad antipriónica y abriendo una puerta a un posible tratamiento de estas enfermedades fatales mediante los compuestos descritos en esta tesis doctoral.CICbioGun

Desarrollo de un nuevo sistema de cribado masivo basado en la propagación in vitro de priones recombinantes infecciosos para la detección de compuestos con actividad anti-priónica.

136 p.Las Encefalopatías Espongiformes Transmisibles (EET) son un grupo de enfermedades neurodegenerativas fatales que se caracterizan por el acúmulo de fibras amiloides de la proteína PrPSc en el sistema nervioso central de los pacientes. Debido a que no existe ningún tratamiento disponible para estas enfermedades, uno de los principales objetivos del campo de estudio de las EET es la búsqueda de fármacos que inhiban o retrasen el proceso de propagación de las fibras amiloides. Para ello, el uso de sistemas de cribado masivo de compuestos con posible actividad anti-priónica es uno de los métodos más prometedores. En esta tesis doctoral se ha desarrollado con éxito un sistema de propagación in vitro depriones altamente infecciosos y aplicable a un método de cribado masivo. Utilizando este novedoso método, se ha cribado una biblioteca de 2500 compuestos, detectándose 3 compuestos con actividad inhibidora no descritos hasta la actualidad. A continuación, se ha confirmado su actividad inhibidora de la propagación utilizando otros métodos de propagación in vitro e in cellula, demostrando la utilidad el nuevo sistema de propagación in vitro para el descubrimiento de nuevos compuestos con actividad antipriónica y abriendo una puerta a un posible tratamiento de estas enfermedades fatales mediante los compuestos descritos en esta tesis doctoral.CICbioGun

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

Enfermedades priónicas: historia, diversidad e importancia socioeconómica como paradigma de las Enfermedades Raras

Rare 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: prions.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 priones

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

A Novel, Reliable and Highly Versatile Method to Evaluate Different Prion Decontamination Procedures

International audienc

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

Understanding the key features of the spontaneous formation of bona fide prions through a novel methodology that enables their swift and consistent generation

Abstract Among transmissible spongiform encephalopathies or prion diseases affecting humans, sporadic forms such as sporadic Creutzfeldt–Jakob disease are the vast majority. Unlike genetic or acquired forms of the disease, these idiopathic forms occur seemingly due to a random event of spontaneous misfolding of the cellular PrP (PrPC) into the pathogenic isoform (PrPSc). Currently, the molecular mechanisms that trigger and drive this event, which occurs in approximately one individual per million each year, remain completely unknown. Modelling this phenomenon in experimental settings is highly challenging due to its sporadic and rare occurrence. Previous attempts to model spontaneous prion misfolding in vitro have not been fully successful, as the spontaneous formation of prions is infrequent and stochastic, hindering the systematic study of the phenomenon. In this study, we present the first method that consistently induces spontaneous misfolding of recombinant PrP into bona fide prions within hours, providing unprecedented possibilities to investigate the mechanisms underlying sporadic prionopathies. By fine-tuning the Protein Misfolding Shaking Amplification method, which was initially developed to propagate recombinant prions, we have created a methodology that consistently produces spontaneously misfolded recombinant prions in 100% of the cases. Furthermore, this method gives rise to distinct strains and reveals the critical influence of charged surfaces in this process