RESUMEN
Neurodegenerative diseases are often characterized by the codeposition of different amyloidogenic proteins, normally defining distinct proteinopathies. An example is represented by prion diseases, where the classical deposition of the aberrant conformational isoform of the prion protein (PrPSc) can be associated with tau insoluble species, which are usually involved in another class of diseases called tauopathies. How this copresence of amyloidogenic proteins can influence the progression of prion diseases is still a matter of debate. Recently, the cellular form of the prion protein, PrPC, has been investigated as a possible receptor of amyloidogenic proteins, since its binding activity with Aß, tau, and α-synuclein has been reported, and it has been linked to several neurotoxic behaviors exerted by these proteins. We have previously shown that the treatment of chronically prion-infected cells with tau K18 fibrils reduced PrPSc levels. In this work, we further explored this mechanism by using another tau construct that includes the sequence that forms the core of Alzheimer's disease tau filaments in vivo to obtain a distinct fibril type. Despite a difference of six amino acids, these two constructs form fibrils characterized by distinct biochemical and biological features. However, their effects on PrPSc reduction were comparable and probably based on the binding to PrPC at the plasma membrane, inhibiting the pathological conversion event. Our results suggest PrPC as receptor for different types of tau fibrils and point out a role of tau amyloid fibrils in preventing the pathological PrPC to PrPSc conformational change.
Asunto(s)
Enfermedades Neurodegenerativas , Enfermedades por Prión , Priones , Proteínas tau , Humanos , Proteínas Amiloidogénicas , Enfermedades por Prión/metabolismo , Proteínas Priónicas , Priones/metabolismo , Proteínas tau/metabolismoRESUMEN
Tauopathies are prevalent, invariably fatal brain diseases for which no cure is available. Tauopathies progressively affect the brain through cell-to-cell transfer of tau protein amyloids, yet the spreading mechanisms remain unknown. Here we show that the cellular prion protein (PrPC ) facilitates the uptake of tau aggregates by cultured cells, possibly by acting as an endocytic receptor. In mouse neuroblastoma cells, pull-down experiments revealed that tau amyloids bind to PrPC . Confocal images of both wild-type and PrPC -knockout N2a cells treated with fluorescently labeled synthetic tau fibrils showed that the internalization was reduced in isogenic cells devoid of the gene encoding PrPC . Pre-treatment of the same cells with antibodies against N-proximal epitopes of PrPC impaired the binding of tau amyloids and decreased their uptake. Surprisingly, exposure of chronically prion-infected cells to tau amyloids reduced the accumulation of aggregated prion protein and this effect lasted for more than 72 hr after amyloid removal. These results point to bidirectional interactions between the two proteins: while PrPC mediates the entrance of tau fibrils in cells, PrPSc buildup is greatly reduced in their presence, possibly because of an impairment in the prion conversion process.
Asunto(s)
Amiloide/metabolismo , Proteínas PrPC/metabolismo , Proteínas tau/metabolismo , Animales , Línea Celular Tumoral , Células Cultivadas , Ratones , Proteínas Priónicas/metabolismo , Unión Proteica/fisiologíaRESUMEN
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
BACKGROUND AIMS: Adipose-derived mesenchymal stromal cells (ASC) are known to promote neuroprotection and neuroregeneration in vitro and in vivo. These biological effects are probably mediated by paracrine mechanisms. In recent years, nanovesicles (NV) and microvesicles (MV) have been shown to play a major role in cell-to-cell communication. We tested the efficacy of NV and MV obtained from ASC in mediating neuroprotection and neuroregeneration in vitro. METHODS: We exposed neuronal cells (both cell line and primary cultures) to oxidative stress in the presence or not of NV or MV. RESULTS: In this experimental setting, we found that low doses of NV or MV protected neurons from apoptotic cell death. We then assessed the neuroregenerative effect of NV/MV in cerebellar slice cultures demyelinated with lysophosphatidylcholine. We observed that low but not higher doses of NV and MV increased the process of remyelination and activated nestin-positive oligodendroglial precursors. CONCLUSIONS: Taken together, our data in vitro support the relevance of ASC vesicles as a source of protecting and regenerating factors that might modulate the microenvironment in neuro-inflammatory as well as in neurodegenerative disorders. The present findings may suggest that stromal cell-derived vesicles might represent a potential therapeutic tool, enabling the safe administration of stromal cell effector factors, avoiding the cellular counterpart.
Asunto(s)
Tejido Adiposo/citología , Micropartículas Derivadas de Células/clasificación , Células Madre Mesenquimatosas/citología , Regeneración Nerviosa/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Animales , Línea Celular Tumoral , Micropartículas Derivadas de Células/metabolismo , Micropartículas Derivadas de Células/ultraestructura , Electroforesis en Gel de Poliacrilamida , Humanos , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Neuronas/metabolismoRESUMEN
BACKGROUND: The current diagnosis of Alzheimer's disease (AD) is based on a series of analyses which involve clinical, instrumental and laboratory findings. However, signs, symptoms and biomarker alterations observed in AD might overlap with other dementias, resulting in misdiagnosis. METHODS: Here we describe a new diagnostic approach for AD which takes advantage of the boosted sensitivity in biomolecular detection, as allowed by seed amplification assay (SAA), combined with the unique specificity in biomolecular recognition, as provided by surface-enhanced Raman spectroscopy (SERS). RESULTS: The SAA-SERS approach supported by machine learning data analysis allowed efficient identification of pathological Aß oligomers in the cerebrospinal fluid of patients with a clinical diagnosis of AD or mild cognitive impairment due to AD. CONCLUSIONS: Such analytical approach can be used to recognize disease features, thus allowing early stratification and selection of patients, which is fundamental in clinical treatments and pharmacological trials.
Asunto(s)
Enfermedad de Alzheimer , Disfunción Cognitiva , Humanos , Espectrometría Raman , Enfermedad de Alzheimer/diagnóstico , Aprendizaje Automático , SemillasRESUMEN
Prion diseases are characterized by the self-assembly of pathogenic misfolded scrapie isoforms (PrPSc) of the cellular prion protein (PrPC). In an effort to achieve a theranostic profile, symmetrical bifunctional carbazole derivatives were designed as fluorescent rigid analogues of GN8, a pharmacological chaperone that stabilizes the native PrPC conformation and prevents its pathogenic conversion. A focused library was synthesized via a four-step route, and a representative member was confirmed to have native fluorescence, including a band in the near-infrared region. After a cytotoxicity study, compounds were tested on the RML-infected ScGT1 neuronal cell line, by monitoring the levels of protease-resistant PrPSc. Small dialkylamino groups at the ends of the molecule were found to be optimal in terms of therapeutic index, and the bis-(dimethylaminoacetamido)carbazole derivative 2b was selected for further characterization. It showed activity in two cell lines infected with the mouse-adapted RML strain (ScGT1 and ScN2a). Unlike GN8, 2b did not affect PrPC levels, which represents a potential advantage in terms of toxicity. Amyloid Seeding Assay (ASA) experiments showed the capacity of 2b to delay the aggregation of recombinant mouse PrP. Its ability to interfere with the amplification of the scrapie RML strain by Protein Misfolding Cyclic Amplification (PMCA) was shown to be higher than that of GN8, although 2b did not inhibit the amplification of human vCJD prion. Fluorescent staining of PrPSc aggregates by 2b was confirmed in living cells. 2b emerges as an initial hit compound for further medicinal chemistry optimization towards strain-independent anti-prion compounds.
Asunto(s)
Carbazoles , Proteínas PrPC , Enfermedades por Prión , Agregado de Proteínas , Animales , Ratones , Carbazoles/química , Carbazoles/farmacología , Carbazoles/uso terapéutico , Línea Celular , Imagen Óptica , Enfermedades por Prión/diagnóstico , Enfermedades por Prión/tratamiento farmacológico , Proteínas PrPC/antagonistas & inhibidores , Proteínas PrPC/química , Agregado de Proteínas/efectos de los fármacosRESUMEN
[This corrects the article DOI: 10.3389/fnagi.2022.848991.].
RESUMEN
The conformational conversion of the cellular prion protein (PrPC) into a misfolded, aggregated and infectious scrapie isoform is associated with prion disease pathology and neurodegeneration. Despite the significant number of experimental and theoretical studies the molecular mechanism regulating this structural transition is still poorly understood. Here, via Nuclear Magnetic Resonance (NMR) methodologies we investigate at the atomic level the mechanism of the human HuPrP(90-231) thermal unfolding and characterize the conformational equilibrium between its native structure and a ß-enriched intermediate state, named ß-PrPI. By comparing the folding mechanisms of metal-free and Cu2+-bound HuPrP(23-231) and HuPrP(90-231) we show that the coupling between the N- and C-terminal domains, through transient electrostatic interactions, is the key molecular process in tuning long-range correlated µs-ms dynamics that in turn modulate the folding process. Moreover, via thioflavin T (ThT)-fluorescence fibrillization assays we show that ß-PrPI is involved in the initial stages of PrP fibrillation, overall providing a clear molecular description of the initial phases of prion misfolding. Finally, we show by using Real-Time Quaking-Induced Conversion (RT-QuIC) that the ß-PrPI acts as a seed for the formation of amyloid aggregates with a seeding activity comparable to that of human infectious prions.
RESUMEN
Sporadic Creutzfeldt-Jakob disease (sCJD) is a rare neurodegenerative disorder caused by the conformational conversion of the prion protein (PrPC) into an abnormally folded form, named prion (or PrPSc). The combination of the polymorphism at codon 129 of the PrP gene (coding either methionine or valine) with the biochemical feature of the proteinase-K resistant PrP (generating either PrPSc type 1 or 2) gives rise to different PrPSc strains, which cause variable phenotypes of sCJD. The definitive diagnosis of sCJD and its classification can be achieved only post-mortem after PrPSc identification and characterization in the brain. By exploiting the Real-Time Quaking-Induced Conversion (RT-QuIC) assay, traces of PrPSc were found in the olfactory mucosa (OM) of sCJD patients, thus demonstrating that PrPSc is not confined to the brain. Here, we have optimized another technique, named protein misfolding cyclic amplification (PMCA) for detecting PrPSc in OM samples of sCJD patients. OM samples were collected from 27 sCJD and 2 genetic CJD patients (E200K). Samples from 34 patients with other neurodegenerative disorders were included as controls. Brains were collected from 26 sCJD patients and 16 of them underwent OM collection. Brain and OM samples were subjected to PMCA using the brains of transgenic mice expressing human PrPC with methionine at codon 129 as reaction substrates. The amplified products were analyzed by Western blot after proteinase K digestion. Quantitative PMCA was performed to estimate PrPSc concentration in OM. PMCA enabled the detection of prions in OM samples with 79.3% sensitivity and 100% specificity. Except for a few cases, a predominant type 1 PrPSc was generated, regardless of the tissues analyzed. Notably, all amplified PrPSc were less resistant to PK compared to the original strain. In conclusion, although the optimized PMCA did not consent to recognize sCJD subtypes from the analysis of OM collected from living patients, it enabled us to estimate for the first time the amount of prions accumulating in this biological tissue. Further assay optimizations are needed to faithfully amplify peripheral prions whose recognition could lead to a better diagnosis and selection of patients for future clinical trials.
RESUMEN
Cytoplasmic aggregation of the primarily nuclear TAR DNA-binding protein 43 (TDP-43) affects neurons in most amyotrophic lateral sclerosis (ALS) and approximately half of frontotemporal lobar degeneration (FTLD) cases. The cellular prion protein, PrPC, has been recognized as a common receptor and downstream effector of circulating neurotoxic species of several proteins involved in neurodegeneration. Here, capitalizing on our recently adapted TDP-43 real time quaking induced reaction, we set reproducible protocols to obtain standardized preparations of recombinant TDP-43 fibrils. We then exploited two different cellular systems (human SH-SY5Y and mouse N2a neuroblastoma cells) engineered to express low or high PrPC levels to investigate the link between PrPC expression on the cell surface and the internalization of TDP-43 fibrils. Fibril uptake was increased in cells overexpressing either human or mouse prion protein. Increased internalization was associated with detrimental consequences in all PrP-overexpressing cell lines but was milder in cells expressing the human form of the prion protein. As described for other amyloids, treatment with TDP-43 fibrils induced a reduction in the accumulation of the misfolded form of PrPC, PrPSc, in cells chronically infected with prions. Our results expand the list of misfolded proteins whose uptake and detrimental effects are mediated by PrPC, which encompass almost all pathological amyloids involved in neurodegeneration.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas PrPC/genética , Proteínas PrPC/metabolismo , Proteínas Priónicas/metabolismo , Animales , Transporte Biológico , Línea Celular , Supervivencia Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/farmacología , Humanos , Ratones , Proteínas PrPC/clasificación , Proteínas Priónicas/genéticaRESUMEN
Parkinson's disease (PD) is the most common neurodegenerative movement disorder. The neuropathological hallmark of the disease is the loss of dopamine neurons of the substantia nigra pars compacta. The clinical manifestations of PD are bradykinesia, rigidity, resting tremors and postural instability. PD patients often display non-motor symptoms such as depression, anxiety, weakness, sleep disturbances and cognitive disorders. Although, in 90% of cases, PD has a sporadic onset of unknown etiology, highly penetrant rare genetic mutations in many genes have been linked with typical familial PD. Understanding the mechanisms behind the DA neuron death in these Mendelian forms may help to illuminate the pathogenesis of DA neuron degeneration in the more common forms of PD. A key step in the identification of the molecular pathways underlying DA neuron death, and in the development of therapeutic strategies, is the creation and characterization of animal models that faithfully recapitulate the human disease. In this review, we outline the current status of PD modeling using mouse, rat and non-mammalian models, focusing on animal models for autosomal recessive PD.
RESUMEN
Sporadic Creutzfeldt-Jakob disease (sCJD) is a rare and fatal neurodegenerative disorder with an incidence of 1.5 to 2 cases per million population/year. The disease is caused by a proteinaceous infectious agent, named prion (or PrPSc), which arises from the conformational conversion of the cellular prion protein (PrPC). Once formed, PrPSc interacts with the normally folded PrPC coercing it to undergo similar structural rearrangement. The disease is highly heterogeneous from a clinical and neuropathological point of view. The origin of this variability lies in the aberrant structures acquired by PrPSc. At least six different sCJD phenotypes have been described and each of them is thought to be caused by a peculiar PrPSc strain. Definitive sCJD diagnosis requires brain analysis with the aim of identifying intracerebral accumulation of PrPSc which currently represents the only reliable biomarker of the disease. Clinical diagnosis of sCJD is very challenging and is based on the combination of several clinical, instrumental and laboratory tests representing surrogate disease biomarkers. Thanks to the advent of the ultrasensitive Real-Time Quaking-Induced Conversion (RT-QuIC) assay, PrPSc was found in several peripheral tissues of sCJD patients, sometimes even before the clinical onset of the disease. This discovery represents an important step forward for the clinical diagnosis of sCJD. In this manuscript, we present an overview of the current applications and future perspectives of RT-QuIC in the field of sCJD diagnosis.
Asunto(s)
Biomarcadores/análisis , Síndrome de Creutzfeldt-Jakob/clasificación , Síndrome de Creutzfeldt-Jakob/diagnóstico , Pruebas Diagnósticas de Rutina/métodos , Proteínas PrPSc/metabolismo , Bioensayo , Síndrome de Creutzfeldt-Jakob/genética , Síndrome de Creutzfeldt-Jakob/metabolismo , Humanos , Proteínas PrPSc/químicaRESUMEN
Background: Fatal Familial Insomnia (FFI) is a genetic prion disease caused by the D178N mutation in the prion protein gene (PRNP) in coupling phase with methionine at PRNP 129. In 2017, we have shown that the olfactory mucosa (OM) collected from FFI patients contained traces of PrPSc detectable by Protein Misfolding Cyclic Amplification (PMCA). Methods: In this work, we have challenged PMCA-generated products obtained from OM and brain homogenate of FFI patients in BvPrP-Tg407 transgenic mice expressing the bank vole prion protein to test their ability to induce prion pathology. Results: All inoculated mice developed mild spongiform changes, astroglial activation, and PrPSc deposition mainly affecting the thalamus. However, their neuropathological alterations were different from those found in the brain of BvPrP-Tg407 mice injected with raw FFI brain homogenate. Conclusions: Although with some experimental constraints, we show that PrPSc present in OM of FFI patients is potentially infectious. Funding: This work was supported in part by the Italian Ministry of Health (GR-2013-02355724 and Ricerca Corrente), MJFF, ALZ, Alzheimer's Research UK and the Weston Brain Institute (BAND2015), and Euronanomed III (SPEEDY) to FM; by the Spanish Ministerio de Economía y Competitividad (grant AGL2016-78054-R [AEI/FEDER, UE]) to JMT and JCE; AM-M was supported by a fellowship from the INIA (FPI-SGIT-2015-02).
Asunto(s)
Insomnio Familiar Fatal/etiología , Mucosa Olfatoria/química , Proteínas PrPSc/administración & dosificación , Animales , Humanos , Ratones , Ratones TransgénicosRESUMEN
Parkinson's disease (PD) and multiple system atrophy (MSA) are caused by two distinct strains of disease-associated α-synuclein (αSynD). Recently, we have shown that olfactory mucosa (OM) samples of patients with PD and MSA can seed the aggregation of recombinant α-synuclein by means of Real-Time Quaking-Induced Conversion (αSyn_RT-QuIC). Remarkably, the biochemical and morphological properties of the final α-synuclein aggregates significantly differed between PD and MSA seeded samples. Here, these aggregates were given to neuron-like differentiated SH-SY5Y cells and distinct inflammatory responses were observed. To deepen whether the morphological features of α-synuclein aggregates were responsible for this variable SH-SY5Y inflammatory response, we generated three biochemically and morphologically distinct α-synuclein aggregates starting from recombinant α-synuclein that were used to seed αSyn_RT-QuIC reaction; the final reaction products were used to stimulate SH-SY5Y cells. Our study showed that, in contrast to OM samples of PD and MSA patients, the artificial aggregates did not transfer their distinctive features to the αSyn_RT-QuIC products and the latter induced analogous inflammatory responses in cells. Thus, the natural composition of the αSynD strains but also other specific factors in OM tissue can substantially modulate the biochemical, morphological and inflammatory features of the αSyn_RT-QuIC products.
Asunto(s)
Inflamación/patología , Atrofia de Múltiples Sistemas/metabolismo , Atrofia de Múltiples Sistemas/patología , Mucosa Olfatoria/metabolismo , Mucosa Olfatoria/patología , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , alfa-Sinucleína/metabolismo , Diferenciación Celular , Línea Celular Tumoral , Humanos , Neuroblastoma/patología , Agregado de Proteínas , Proteínas Recombinantes/metabolismo , alfa-Sinucleína/ultraestructuraRESUMEN
BACKGROUND: Detection of the pathological and disease-associated alpha-synuclein (αSynD) in the brain is required to formulate the definitive diagnosis of multiple system atrophy (MSA) and Parkinson's disease (PD). We recently showed that αSynD can be detected in the olfactory mucosa (OM) of MSA and PD patients. For this reason, we have performed the first interlaboratory study based on α-synuclein Real-Time Quaking-Induced Conversion (αSyn_RT-QuIC) analysis of OM samples collected from PD and MSA patients with the parkinsonian (MSA-P) and cerebellar (MSA-C) phenotypes. METHODS: OM samples were prospectively collected from patients with a probable diagnosis of MSA-P (n = 20, mean disease duration 4.4 years), MSA-C (n = 10, mean disease duration 4 years), PD (n = 13, mean disease duration 8 years), and healthy control subjects (HS) (n = 11). Each sample was analyzed by αSyn_RT-QuIC in two independent specialized laboratories, one located in Italy (ITA-lab) and one located in the USA (USA-lab). Both laboratories have developed and used harmonized αSyn_RT-QuIC analytical procedures. Results were correlated with demographic and clinical data. RESULTS: The αSyn_RT-QuIC analysis reached a 96% interrater agreement of results (IAR) between laboratories (Kappa = 0.93, 95% CI 0.83-1.00). In particular, αSyn_RT-QuIC seeding activity was found in the OM of 9/13 patients with PD (sensitivity 69%, IAR 100%) and 18/20 patients with MSA-P (sensitivity 90%, IAR 100%). Interestingly, samples collected from patients with MSA-C did not induce αSyn_RT-QuIC seeding activity, except for one subject in USA-lab. Therefore, we found that MSA-P and MSA-C induced opposite effects. Regardless of disease diagnosis, the αSyn_RT-QuIC seeding activity correlated with some clinical parameters, including the rigidity and postural instability. CONCLUSIONS: Our study provides evidence that OM-αSynD may serve as a novel biomarker for accurate clinical diagnoses of PD, MSA-P, and MSA-C. Moreover, αSyn_RT-QuIC represents a reliable assay that can distinguish patients with MSA-P from those with MSA-C, and may lead to significant advancements in patients stratification and selection for emerging pharmacological treatments and clinical trials.
Asunto(s)
Atrofia de Múltiples Sistemas , Enfermedad de Parkinson , Humanos , Laboratorios , Atrofia de Múltiples Sistemas/patología , Mucosa Olfatoria/química , Mucosa Olfatoria/patología , Enfermedad de Parkinson/diagnóstico , Enfermedad de Parkinson/patología , Reproducibilidad de los Resultados , alfa-SinucleínaRESUMEN
Alzheimer's disease (AD) is the most common neurodegenerative disorder worldwide. The distinctive neuropathological feature of AD is the intracerebral accumulation of two abnormally folded proteins: ß-amyloid (Aß) in the form of extracellular plaques, and tau in the form of intracellular neurofibrillary tangles. These proteins are considered disease-specific biomarkers, and the definite diagnosis of AD relies on their post-mortem identification in the brain. The clinical diagnosis of AD is challenging, especially in the early stages. The disease is highly heterogeneous in terms of clinical presentation and neuropathological features. This phenotypic variability seems to be partially due to the presence of distinct Aß conformers, referred to as strains. With the development of an innovative technique named Real-Time Quaking-Induced Conversion (RT-QuIC), traces of Aß strains were found in the cerebrospinal fluid of AD patients. Emerging evidence suggests that different conformers may transmit their strain signature to the RT-QuIC reaction products. In this review, we describe the current challenges for the clinical diagnosis of AD and describe how the RT-QuIC products could be analyzed by a surface-enhanced Raman spectroscopy (SERS)-based systems to reveal the presence of strain signatures, eventually leading to early diagnosis of AD with the recognition of individual disease phenotype.
RESUMEN
Neurodegenerative diseases (NDs) such as Alzheimer's disease (AD), Parkinson's disease (PD), atypical parkinsonisms, frontotemporal dementia (FTLD) and prion diseases are characterized by the accumulation of misfolded proteins in the central nervous system (CNS). Although the cause for the initiation of protein aggregation is not well understood, these aggregates are disease-specific. For instance, AD is characterized by the intraneuronal accumulation of tau and extracellular deposition of amyloid-ß (Aß), PD is marked by the intraneuronal accumulation of α-synuclein, many FTLD are associated with the accumulation of TDP-43 while prion diseases show aggregates of misfolded prion protein. Hence, misfolded proteins are considered disease-specific biomarkers and their identification and localization in the CNS, collected postmortem, is required for a definitive diagnosis. With the development of two innovative cell-free amplification techniques named Protein Misfolding Cyclic Amplification (PMCA) and Real-Time Quaking-Induced Conversion (RT-QuIC), traces of disease-specific biomarkers were found in CSF and other peripheral tissues (e.g., urine, blood, and olfactory mucosa) of patients with different NDs. These techniques exploit an important feature shared by many misfolded proteins, that is their ability to interact with their normally folded counterparts and force them to undergo similar structural rearrangements. Essentially, RT-QuIC and PMCA mimic in vitro the same pathological processes of protein misfolding which occur in vivo in a very rapid manner. For this reason, they have been employed for studying different aspects of protein misfolding but, overall, they seem to be very promising for the premortem diagnosis of NDs.
Asunto(s)
Priones/metabolismo , Animales , Sistema Libre de Células , Humanos , Proteínas PrPSc/química , Proteínas PrPSc/metabolismo , Enfermedades por Prión/diagnóstico , Enfermedades por Prión/metabolismo , Enfermedades por Prión/patología , Priones/química , Pliegue de ProteínaRESUMEN
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.
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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.
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Priones/metabolismo , alfa-Sinucleína/deficiencia , Animales , Encéfalo/metabolismo , Endopeptidasa K/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Pliegue de Proteína , alfa-Sinucleína/metabolismoRESUMEN
Chronic wasting disease (CWD) is a highly contagious prion disease affecting captive and free-ranging cervid populations. CWD has been detected in United States, Canada, South Korea and, most recently, in Europe (Norway, Finland and Sweden). Animals with CWD release infectious prions in the environment through saliva, urine and feces sustaining disease spreading between cervids but also potentially to other non-cervids ruminants (e.g. sheep, goats and cattle). In the light of these considerations and due to CWD unknown zoonotic potential, it is of utmost importance to follow specific surveillance programs useful to minimize disease spreading and transmission. The European community has already in place specific surveillance measures, but the traditional diagnostic tests performed on nervous or lymphoid tissues lack sensitivity. We have optimized a Real-Time Quaking-Induced Conversion (RT-QuIC) assay for detecting CWD prions with high sensitivity and specificity to try to overcome this problem. In this work, we show that bank vole prion protein (PrP) is an excellent substrate for RT-QuIC reactions, enabling the detection of trace-amounts of CWD prions, regardless of prion strain and cervid species. Beside supporting the traditional diagnostic tests, this technology could be exploited for detecting prions in peripheral tissues from live animals, possibly even at preclinical stages of the disease.