RESUMEN
Prions are infectious proteins that possess multiple self-propagating structures. The information for strains and structural specific barriers appears to be contained exclusively in the folding of the pathological isoform, PrP(Sc). Many recent studies determined that de novo prion strains could be generated in vitro from the structural conversion of recombinant (rec) prion protein (PrP) into amyloidal structures. Our aim was to elucidate the conformational diversity of pathological recPrP amyloids and their biological activities, as well as to gain novel insights in characterizing molecular events involved in mammalian prion conversion and propagation. To this end we generated infectious materials that possess different conformational structures. Our methodology for the prion conversion of recPrP required only purified rec full-length mouse (Mo) PrP and common chemicals. Neither infected brain extracts nor amplified PrP(Sc) were used. Following two different in vitro protocols recMoPrP converted to amyloid fibrils without any seeding factor. Mouse hypothalamic GT1 and neuroblastoma N2a cell lines were infected with these amyloid preparations as fast screening methodology to characterize the infectious materials. Remarkably, a large number of amyloid preparations were able to induce the conformational change of endogenous PrPC to harbor several distinctive proteinase-resistant PrP forms. One such preparation was characterized in vivo habouring a synthetic prion with novel strain specified neuropathological and biochemical properties.
Asunto(s)
Enfermedades por Prión/patología , Priones/química , Priones/metabolismo , Secuencia de Aminoácidos , Proteínas Amiloidogénicas/química , Animales , Western Blotting , Línea Celular , Modelos Animales de Enfermedad , Ratones , Microscopía de Fuerza Atómica , Datos de Secuencia Molecular , Proteínas Priónicas , Priones/síntesis química , Conformación Proteica , Pliegue de Proteína , Proteínas Recombinantes/síntesis química , Proteínas Recombinantes/químicaRESUMEN
Prion pathologies are characterized by the conformational conversion of the cellular prion protein (PrPC) into a pathological infectious isoform, known as PrPSc. The latter acquires different abnormal conformations, which are associated with specific pathological phenotypes. Recent evidence suggests that prions adapt their conformation to changes in the context of replication. This phenomenon is known as either prion selection or adaptation, where distinct conformations of PrPSc with higher propensity to propagate in the new environment prevail over the others. Here, we show that a synthetically generated prion isolate, previously subjected to protein misfolding cyclic amplification (PMCA) and then injected in animals, is able to change its biochemical and biophysical properties according to the context of replication. In particular, in second transmission passage in vivo, two different prion isolates were found: one characterized by a predominance of the monoglycosylated band (PrPSc-M) and the other characterized by a predominance of the diglycosylated one (PrPSc-D). Neuropathological, biochemical, and biophysical assays confirmed that these PrPSc possess distinctive characteristics. Finally, PMCA analysis of PrPSc-M and PrPSc-D generated PrPSc (PrPSc-PMCA) whose biophysical properties were different from those of both inocula, suggesting that PMCA selectively amplified a third PrPSc isolate. Taken together, these results indicate that the context of replication plays a pivotal role in either prion selection or adaptation. By exploiting the ability of PMCA to mimic the process of prion replication in vitro, it might be possible to assess how changes in the replication environment influence the phenomenon of prion selection and adaptation.
Asunto(s)
Adaptación Fisiológica , Priones/metabolismo , Animales , Fenómenos Biofísicos , Ratones , Enfermedades por Prión/metabolismo , Enfermedades por Prión/patología , Priones/aislamiento & purificación , Pliegue de ProteínaRESUMEN
Prion diseases are a group of neurodegenerative disorders associated with the conformational conversion of the cellular prion protein (PrPC) into an abnormal misfolded form named PrPSc. Other than accumulating in the brain, PrPSc can bind PrPC and force it to change conformation to PrPSc. The exact mechanism which underlies the process of PrPC/PrPSc conversion still needs to be defined and many molecules or cofactors might be involved. Several studies have documented an important role of PrPC to act as receptor for abnormally folded forms of α-synuclein which are responsible of a group of diseases known as synucleinopathies. The presence of PrPC was required to promote efficient internalization and spreading of abnormal α-synuclein between cells. In this work, we have assessed whether α-synuclein exerts any role in PrPSc conversion and propagation either in vitro or in vivo. Indeed, understanding the mechanism of PrPC/PrPSc conversion and the identification of cofactors involved in this process is crucial for developing new therapeutic strategies. Our results showed that PrPSc was able to efficiently propagate in the brain of animals even in the absence of α-synuclein thus suggesting that this protein did not act as key modulator of prion propagation. Thus, α-synuclein might take part in this process but is not specifically required for sustaining prion conversion and propagation.
Asunto(s)
Priones/metabolismo , alfa-Sinucleína/deficiencia , Animales , Encéfalo/metabolismo , Endopeptidasa K/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Pliegue de Proteína , alfa-Sinucleína/metabolismoRESUMEN
Pin1 is a peptidyl-prolyl isomerase that induces the cis-trans conversion of specific Ser/Thr-Pro peptide bonds in phosphorylated proteins, leading to conformational changes through which Pin1 regulates protein stability and activity. Since down-regulation of Pin1 has been described in several neurodegenerative disorders, including Alzheimer's Disease (AD), Parkinson's Disease (PD) and Huntington's Disease (HD), we investigated its potential role in prion diseases. Animals generated on wild-type (Pin1+/+), hemizygous (Pin1+/-) or knock-out (Pin1-/-) background for Pin1 were experimentally infected with RML prions. The study indicates that, neither the total depletion nor reduced levels of Pin1 significantly altered the clinical and neuropathological features of the disease.
Asunto(s)
Isomerasa de Peptidilprolil/metabolismo , Enfermedades por Prión/metabolismo , Animales , Modelos Animales de Enfermedad , Peptidilprolil Isomerasa de Interacción con NIMA/genética , Peptidilprolil Isomerasa de Interacción con NIMA/metabolismo , Isomerasa de Peptidilprolil/genética , Fosforilación , Enfermedades por Prión/genética , Proteínas Priónicas/genética , Proteínas Priónicas/metabolismoRESUMEN
Protein misfolding and aggregation is a central feature of several neurodegenerative disorders including Alzheimer's disease (AD), in which assemblies of amyloid ß (Aß) peptides accumulate in the brain in the form of parenchymal and/or vascular amyloid. A widely accepted concept is that AD is characterized by distinct clinical and neuropathological phenotypes. Recent studies revealed that Aß assemblies might have structural differences among AD brains and that such pleomorphic assemblies can correlate with distinct disease phenotypes. We found that in both sporadic and inherited forms of AD, amyloid aggregates differ in the biochemical composition of Aß species. These differences affect the physicochemical properties of Aß assemblies including aggregation kinetics, resistance to degradation by proteases and seeding ability. Aß-amyloidosis can be induced and propagated in animal models by inoculation of brain extracts containing aggregated Aß. We found that brain homogenates from AD patients with different molecular profiles of Aß are able to induce distinct patterns of Aß-amyloidosis when injected into mice. Overall these data suggest that the assembly of mixtures of Aß peptides into different Aß seeds leads to the formation of distinct subtypes of amyloid having distinctive physicochemical and biological properties which result in the generation of distinct AD molecular subgroups.
Asunto(s)
Enfermedad de Alzheimer/clasificación , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/análisis , Amiloide/química , Encéfalo/patología , Agregación Patológica de Proteínas , Péptidos beta-Amiloides/química , Animales , Angiopatía Amiloide Cerebral/patología , Fenómenos Químicos , Modelos Animales de Enfermedad , Humanos , RatonesRESUMEN
We developed a novel therapeutic strategy for Alzheimer's disease (AD) exploiting the properties of a natural variant of Amyloid-ß (Aß) carrying the A2V substitution, which protects heterozygous carriers from AD by its ability to interact with wild-type Aß, hindering conformational changes and assembly thereof. As prototypic compound we designed a six-mer mutated peptide (Aß1-6A2V), linked to the HIV-related TAT protein, which is widely used for brain delivery and cell membrane penetration of drugs. The resulting molecule [Aß1-6A2VTAT(D)] revealed strong anti-amyloidogenic effects in vitro and protected human neuroblastoma cells from Aß toxicity. Preclinical studies in AD mouse models showed that short-term treatment with Aß1-6A2VTAT(D) inhibits Aß aggregation and cerebral amyloid deposition, but a long treatment schedule unexpectedly increases amyloid burden, although preventing cognitive deterioration. Our data support the view that the AßA2V-based strategy can be successfully used for the development of treatments for AD, as suggested by the natural protection against the disease in human A2V heterozygous carriers. The undesirable outcome of the prolonged treatment with Aß1-6A2VTAT(D) was likely due to the TAT intrinsic attitude to increase Aß production, avidly bind amyloid and boost its seeding activity, warning against the use of the TAT carrier in the design of AD therapeutics.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos beta-Amiloides/química , Fármacos Neuroprotectores/farmacología , Fragmentos de Péptidos/química , Agregación Patológica de Proteínas/fisiopatología , Proteínas Recombinantes de Fusión/farmacología , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/síntesis química , Enfermedad de Alzheimer/fisiopatología , Sustitución de Aminoácidos , Animales , Encéfalo/efectos de los fármacos , Encéfalo/fisiopatología , Química Encefálica , Cognición/efectos de los fármacos , Modelos Animales de Enfermedad , Conducta Exploratoria/efectos de los fármacos , Femenino , Humanos , Inyecciones Intraperitoneales , Ratones , Ratones Transgénicos , Fármacos Neuroprotectores/síntesis química , Unión Proteica , Proteínas Recombinantes de Fusión/síntesis químicaRESUMEN
Prion diseases are caused by a conformational modification of the cellular prion protein (PrP (C)) into disease-specific forms, termed PrP (Sc), that have the ability to interact with PrP (C) promoting its conversion to PrP (Sc). In vitro studies demonstrated that anti-PrP antibodies inhibit this process. In particular, the single chain variable fragment D18 antibody (scFvD18) showed high efficiency in curing chronically prion-infected cells. This molecule binds the PrP (C) region involved in the interaction with PrP (Sc) thus halting further prion formation. These findings prompted us to test the efficiency of scFvD18 in vivo. A recombinant Adeno-Associated Viral vector serotype 9 was used to deliver scFvD18 to the brain of mice that were subsequently infected by intraperitoneal route with the mouse-adapted scrapie strain RML. We found that the treatment was safe, prolonged the incubation time of scrapie-infected animals and decreased the burden of total proteinase-resistant PrP (Sc) in the brain, suggesting that scFvD18 interferes with prion replication in vivo. This approach is relevant for designing new therapeutic strategies for prion diseases and other disorders characterized by protein misfolding.
Asunto(s)
Encéfalo/patología , Vectores Genéticos/uso terapéutico , Plásmidos/uso terapéutico , Priones/inmunología , Scrapie/patología , Scrapie/terapia , Anticuerpos de Cadena Única/inmunología , Adenoviridae/genética , Animales , Encéfalo/inmunología , Encéfalo/metabolismo , Vectores Genéticos/genética , Células HEK293 , Humanos , Ratones , Plásmidos/genética , Scrapie/genética , Scrapie/inmunología , Anticuerpos de Cadena Única/genéticaRESUMEN
In Creutzfeldt-Jakob disease (CJD), molecular typing based on the size of the protease resistant core of the disease-associated prion protein (PrP(Sc) ) and the M/V polymorphism at codon 129 of the PRNP gene correlates with the clinico-pathologic subtypes. Approximately 95% of the sporadic 129MM CJD patients are characterized by cerebral deposition of type 1 PrP(Sc) and correspond to the classic clinical CJD phenotype. The rare 129MM CJD patients with type 2 PrP(Sc) are further subdivided in a cortical and a thalamic form also indicated as sporadic fatal insomnia. We observed two young patients with MM2-thalamic CJD. Main neuropathological features were diffuse, synaptic PrP immunoreactivity in the cerebral cortex and severe neuronal loss and gliosis in the thalamus and olivary nucleus. Western blot analysis showed the presence of type 2A PrP(Sc) . Challenge of transgenic mice expressing 129MM human PrP showed that MM2-thalamic sporadic CJD (sCJD) was able to transmit the disease, at variance with MM2-cortical sCJD. The affected mice showed deposition of type 2A PrP(Sc) , a scenario that is unprecedented in this mouse line. These data indicate that MM2-thalamic sCJD is caused by a prion strain distinct from the other sCJD subtypes including the MM2-cortical form.
Asunto(s)
Síndrome de Creutzfeldt-Jakob , Polimorfismo Genético/genética , Priones/genética , Priones/metabolismo , Tálamo/metabolismo , Tálamo/patología , Adulto , Animales , Síndrome de Creutzfeldt-Jakob/genética , Síndrome de Creutzfeldt-Jakob/patología , Síndrome de Creutzfeldt-Jakob/transmisión , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Ratones , Ratones Transgénicos , Adulto JovenRESUMEN
The amyloidotic form of bovine spongiform encephalopathy (BSE) termed BASE is caused by a prion strain whose biological properties differ from those of typical BSE, resulting in a clinically and pathologically distinct phenotype. Whether peripheral tissues of BASE-affected cattle contain infectivity is unknown. This is a critical issue since the BASE prion is readily transmissible to a variety of hosts including primates, suggesting that humans may be susceptible. We carried out bioassays in transgenic mice overexpressing bovine PrP (Tgbov XV) and found infectivity in a variety of skeletal muscles from cattle with natural and experimental BASE. Noteworthy, all BASE muscles used for inoculation transmitted disease, although the attack rate differed between experimental and natural cases (â¼70% versus â¼10%, respectively). This difference was likely related to different prion titers, possibly due to different stages of disease in the two conditions, i.e. terminal stage in experimental BASE and pre-symptomatic stage in natural BASE. The neuropathological phenotype and PrP(res) type were consistent in all affected mice and matched those of Tgbov XV mice infected with brain homogenate from natural BASE. The immunohistochemical analysis of skeletal muscles from cattle with natural and experimental BASE showed the presence of abnormal prion protein deposits within muscle fibers. Conversely, Tgbov XV mice challenged with lymphoid tissue and kidney from natural and experimental BASE did not develop disease. The novel information on the neuromuscular tropism of the BASE strain, efficiently overcoming species barriers, underlines the relevance of maintaining an active surveillance.