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1.
Int J Mol Sci ; 25(11)2024 May 30.
Artículo en Inglés | MEDLINE | ID: mdl-38892177

RESUMEN

Alpha-synuclein seed amplification assays (αSyn-SAAs) have emerged as promising diagnostic tools for Parkinson's disease (PD) by detecting misfolded αSyn and amplifying the signal through cyclic shaking and resting in vitro. Recently, our group and others have shown that multiple biospecimens, including CSF, skin, and submandibular glands (SMGs), can be used to seed the aggregation reaction and robustly distinguish between patients with PD and non-disease controls. The ultrasensitivity of the assay affords the ability to detect minute quantities of αSyn in peripheral tissues, but it also produces various technical challenges of variability. To address the problem of variability, we present a high-yield αSyn protein purification protocol for the efficient production of monomers with a low propensity for self-aggregation. We expressed wild-type αSyn in BL21 Escherichia coli, lysed the cells using osmotic shock, and isolated αSyn using acid precipitation and fast protein liquid chromatography (FPLC). Following purification, we optimized the ionic strength of the reaction buffer to distinguish the fluorescence maximum (Fmax) separation between disease and healthy control tissues for enhanced assay performance. Our protein purification protocol yielded high quantities of αSyn (average: 68.7 mg/mL per 1 L of culture) and showed highly precise and robust αSyn-SAA results using brain, skin, and SMGs with inter-lab validation.


Asunto(s)
Enfermedad de Parkinson , alfa-Sinucleína , alfa-Sinucleína/genética , alfa-Sinucleína/química , alfa-Sinucleína/aislamiento & purificación , alfa-Sinucleína/metabolismo , Humanos , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/genética , Concentración Osmolar , Reproducibilidad de los Resultados , Escherichia coli/genética , Escherichia coli/metabolismo
2.
PLoS Pathog ; 17(7): e1009765, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-34260664

RESUMEN

Prions are comprised solely of PrPSc, the misfolded self-propagating conformation of the cellular protein, PrPC. Synthetic prions are generated in vitro from minimal components and cause bona fide prion disease in animals. It is unknown, however, if synthetic prions can cross the species barrier following interspecies transmission. To investigate this, we inoculated Syrian hamsters with murine synthetic prions. We found that all the animals inoculated with murine synthetic prions developed prion disease characterized by a striking uniformity of clinical onset and signs of disease. Serial intraspecies transmission resulted in a rapid adaptation to hamsters. During the adaptation process, PrPSc electrophoretic migration, glycoform ratios, conformational stability and biological activity as measured by protein misfolding cyclic amplification remained constant. Interestingly, the strain that emerged shares a strikingly similar transmission history, incubation period, clinical course of disease, pathology and biochemical and biological features of PrPSc with 139H, a hamster adapted form of the murine strain 139A. Combined, these data suggest that murine synthetic prions are comprised of bona fide PrPSc with 139A-like strain properties that efficiently crosses the species barrier and rapidly adapts to hamsters resulting in the emergence of a single strain. The efficiency and specificity of interspecies transmission of murine synthetic prions to hamsters, with relevance to brain derived prions, could be a useful model for identification of structure function relationships between PrPSc and PrPC from different species.


Asunto(s)
Proteínas PrPC/metabolismo , Proteínas PrPSc/metabolismo , Enfermedades por Prión/metabolismo , Enfermedades por Prión/transmisión , Animales , Cricetinae , Ratones , Especificidad de la Especie
3.
J Biol Chem ; 297(5): 101344, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-34710372

RESUMEN

Conversion of normal prion protein (PrPC) to the pathogenic PrPSc conformer is central to prion diseases such as Creutzfeldt-Jakob disease and scrapie; however, the detailed mechanism of this conversion remains obscure. To investigate how the N-terminal polybasic region of PrP (NPR) influences the PrPC-to-PrPSc conversion, we analyzed two PrP mutants: ΔN6 (deletion of all six amino acids in NPR) and Met4-1 (replacement of four positively charged amino acids in NPR with methionine). We found that ΔN6 and Met4-1 differentially impacted the binding of recombinant PrP (recPrP) to the negatively charged phospholipid 1-palmitoyl-2-oleoylphosphatidylglycerol, a nonprotein cofactor that facilitates PrP conversion. Both mutant recPrPs were able to form recombinant prion (recPrPSc) in vitro, but the convertibility was greatly reduced, with ΔN6 displaying the lowest convertibility. Prion infection assays in mammalian RK13 cells expressing WT or NPR-mutant PrPs confirmed these differences in convertibility, indicating that the NPR affects the conversion of both bacterially expressed recPrP and post-translationally modified PrP in eukaryotic cells. We also found that both WT and mutant recPrPSc conformers caused prion disease in WT mice with a 100% attack rate, but the incubation times and neuropathological changes caused by two recPrPSc mutants were significantly different from each other and from that of WT recPrPSc. Together, our results support that the NPR greatly influences PrPC-to-PrPSc conversion, but it is not essential for the generation of PrPSc. Moreover, the significant differences between ΔN6 and Met4-1 suggest that not only charge but also the identity of amino acids in NPR is important to PrP conversion.


Asunto(s)
Encéfalo/metabolismo , Mutación , Proteínas PrPC/metabolismo , Proteínas PrPSc/metabolismo , Enfermedades por Prión/metabolismo , Animales , Línea Celular , Ratones , Proteínas PrPC/genética , Proteínas PrPSc/genética , Enfermedades por Prión/genética , Conejos
4.
Nucleic Acids Res ; 48(19): 10615-10631, 2020 11 04.
Artículo en Inglés | MEDLINE | ID: mdl-32776089

RESUMEN

Lowering of prion protein (PrP) expression in the brain is a genetically validated therapeutic hypothesis in prion disease. We recently showed that antisense oligonucleotide (ASO)-mediated PrP suppression extends survival and delays disease onset in intracerebrally prion-infected mice in both prophylactic and delayed dosing paradigms. Here, we examine the efficacy of this therapeutic approach across diverse paradigms, varying the dose and dosing regimen, prion strain, treatment timepoint, and examining symptomatic, survival, and biomarker readouts. We recapitulate our previous findings with additional PrP-targeting ASOs, and demonstrate therapeutic benefit against four additional prion strains. We demonstrate that <25% PrP suppression is sufficient to extend survival and delay symptoms in a prophylactic paradigm. Rise in both neuroinflammation and neuronal injury markers can be reversed by a single dose of PrP-lowering ASO administered after the detection of pathological change. Chronic ASO-mediated suppression of PrP beginning at any time up to early signs of neuropathology confers benefit similar to constitutive heterozygous PrP knockout. Remarkably, even after emergence of frank symptoms including weight loss, a single treatment prolongs survival by months in a subset of animals. These results support ASO-mediated PrP lowering, and PrP-lowering therapeutics in general, as a promising path forward against prion disease.


Asunto(s)
Oligonucleótidos Antisentido/uso terapéutico , Enfermedades por Prión/terapia , Proteínas Priónicas/genética , Tratamiento con ARN de Interferencia/métodos , Animales , Encéfalo/metabolismo , Encéfalo/patología , Línea Celular , Ratones , Ratones Endogámicos C57BL , Oligonucleótidos Antisentido/química , Proteínas Priónicas/metabolismo
5.
Proc Natl Acad Sci U S A ; 116(12): 5765-5774, 2019 03 19.
Artículo en Inglés | MEDLINE | ID: mdl-30842285

RESUMEN

Mutations in the vacuolar protein sorting 35 ortholog (VPS35) gene represent a cause of late-onset, autosomal dominant familial Parkinson's disease (PD). A single missense mutation, D620N, is considered pathogenic based upon its segregation with disease in multiple families with PD. At present, the mechanism(s) by which familial VPS35 mutations precipitate neurodegeneration in PD are poorly understood. Here, we employ a germline D620N VPS35 knockin (KI) mouse model of PD to formally establish the age-related pathogenic effects of the D620N mutation at physiological expression levels. Our data demonstrate that a heterozygous or homozygous D620N mutation is sufficient to reproduce key neuropathological hallmarks of PD as indicated by the progressive degeneration of nigrostriatal pathway dopaminergic neurons and widespread axonal pathology. Unexpectedly, endogenous D620N VPS35 expression induces robust tau-positive somatodendritic pathology throughout the brain as indicated by abnormal hyperphosphorylated and conformation-specific tau, which may represent an important and early feature of mutant VPS35-induced neurodegeneration in PD. In contrast, we find no evidence for α-synuclein-positive neuropathology in aged VPS35 KI mice, a hallmark of Lewy body pathology in PD. D620N VPS35 expression also fails to modify the lethal neurodegenerative phenotype of human A53T-α-synuclein transgenic mice. Finally, by crossing VPS35 KI and null mice, our data demonstrate that a single D620N VPS35 allele is sufficient for survival and early maintenance of dopaminergic neurons, indicating that the D620N VPS35 protein is fully functional. Our data raise the tantalizing possibility of a pathogenic interplay between mutant VPS35 and tau for inducing neurodegeneration in PD.


Asunto(s)
Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/fisiología , Proteínas tau/metabolismo , Animales , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/fisiología , Técnicas de Sustitución del Gen , Masculino , Ratones , Mutación , Enfermedades del Sistema Nervioso/patología , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/fisiopatología , Neuropatología , Enfermedad de Parkinson/genética , Transporte de Proteínas , alfa-Sinucleína/metabolismo , Proteínas tau/fisiología
6.
PLoS Pathog ; 15(12): e1008139, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31815959

RESUMEN

Prion or PrPSc is the proteinaceous infectious agent causing prion diseases in various mammalian species. Despite decades of research, the structural basis for PrPSc formation and prion infectivity remains elusive. To understand the role of the hydrophobic region in forming infectious prion at the molecular level, we report X-ray crystal structures of mouse (Mo) prion protein (PrP) (residues 89-230) in complex with a nanobody (Nb484). Using the recombinant prion propagation system, we show that the binding of Nb484 to the hydrophobic region of MoPrP efficiently inhibits the propagation of proteinase K resistant PrPSc and prion infectivity. In addition, when added to cultured mouse brain slices in high concentrations, Nb484 exhibits no neurotoxicity, which is drastically different from other neurotoxic anti-PrP antibodies, suggesting that the Nb484 can be a potential therapeutic agent against prion disease. In summary, our data provides the first structure-function evidence supporting a crucial role of the hydrophobic region of PrP in forming an infectious prion.


Asunto(s)
Proteínas PrPSc/química , Proteínas PrPSc/efectos de los fármacos , Proteínas Priónicas/química , Proteínas Priónicas/efectos de los fármacos , Anticuerpos de Dominio Único/farmacología , Animales , Ratones , Conformación Proteica , Dominios Proteicos/efectos de los fármacos , Relación Estructura-Actividad
7.
PLoS Pathog ; 14(1): e1006797, 2018 01.
Artículo en Inglés | MEDLINE | ID: mdl-29385212

RESUMEN

Very solid evidence suggests that the core of full length PrPSc is a 4-rung ß-solenoid, and that individual PrPSc subunits stack to form amyloid fibers. We recently used limited proteolysis to map the ß-strands and connecting loops that make up the PrPSc solenoid. Using high resolution SDS-PAGE followed by epitope analysis, and mass spectrometry, we identified positions ~116/118, 133-134, 141, 152-153, 162, 169 and 179 (murine numbering) as Proteinase K (PK) cleavage sites in PrPSc. Such sites likely define loops and/or borders of ß-strands, helping us to predict the threading of the ß-solenoid. We have now extended this approach to recombinant PrPSc (recPrPSc). The term recPrPSc refers to bona fide recombinant prions prepared by PMCA, exhibiting infectivity with attack rates of ~100%. Limited proteolysis of mouse and bank vole recPrPSc species yielded N-terminally truncated PK-resistant fragments similar to those seen in brain-derived PrPSc, albeit with varying relative yields. Along with these fragments, doubly N- and C-terminally truncated fragments, in particular ~89/97-152, were detected in some recPrPSc preparations; similar fragments are characteristic of atypical strains of brain-derived PrPSc. Our results suggest a shared architecture of recPrPSc and brain PrPSc prions. The observed differences, in particular the distinct yields of specific PK-resistant fragments, are likely due to differences in threading which result in the specific biochemical characteristics of recPrPSc. Furthermore, recombinant PrPSc offers exciting opportunities for structural studies unachievable with brain-derived PrPSc.


Asunto(s)
Encéfalo/metabolismo , Proteínas PrPSc/química , Priones/química , Proteolisis , Proteínas Recombinantes/química , Animales , Arvicolinae , Femenino , Ratones , Ratones Transgénicos , Proteínas PrPSc/metabolismo , Priones/metabolismo , Estructura Secundaria de Proteína
8.
J Neurosci ; 38(38): 8211-8232, 2018 09 19.
Artículo en Inglés | MEDLINE | ID: mdl-30093536

RESUMEN

α-Synuclein (αsyn) is the key protein that forms neuronal aggregates in the neurodegenerative disorders Parkinson's disease (PD) and dementia with Lewy bodies. Recent evidence points to the prion-like spread of αsyn from one brain region to another. Propagation of αsyn is likely dependent on release, uptake, and misfolding. Under normal circumstances, this highly expressed brain protein functions normally without promoting pathology, yet the underlying endogenous mechanisms that prevent αsyn spread are not understood. 14-3-3 proteins are highly expressed brain proteins that have chaperone function and regulate protein trafficking. In this study, we investigated the potential role of the 14-3-3 proteins in the regulation of αsyn spread using two models of αsyn spread. In a paracrine αsyn model, 14-3-3θ promoted release of αsyn complexed with 14-3-3θ. Despite higher amounts of released αsyn, extracellular αsyn showed reduced oligomerization and seeding capability, reduced internalization, and reduced toxicity in primary mixed-gender mouse neurons. 14-3-3 inhibition reduced the amount of αsyn released, yet released αsyn was more toxic and demonstrated increased oligomerization, seeding capability, and internalization. In the preformed fibril model, 14-3-3 θ reduced αsyn aggregation and neuronal death, whereas 14-3-3 inhibition enhanced αsyn aggregation and neuronal death in primary mouse neurons. 14-3-3s blocked αsyn spread to distal chamber neurons not exposed directly to fibrils in multichamber, microfluidic devices. These findings point to 14-3-3s as a direct regulator of αsyn propagation, and suggest that dysfunction of 14-3-3 function may promote αsyn pathology in PD and related synucleinopathies.SIGNIFICANCE STATEMENT Transfer of misfolded aggregates of α-synuclein from one brain region to another is implicated in the pathogenesis of Parkinson's disease and other synucleinopathies. This process is dependent on active release, internalization, and misfolding of α-synuclein. 14-3-3 proteins are highly expressed chaperone proteins that interact with α-synuclein and regulate protein trafficking. We used two different models in which toxicity is associated with cell-to-cell transfer of α-synuclein to test whether 14-3-3s impact α-synuclein toxicity. We demonstrate that 14-3-3θ reduces α-synuclein transfer and toxicity by inhibiting oligomerization, seeding capability, and internalization of α-synuclein, whereas 14-3-3 inhibition accelerates the transfer and toxicity of α-synuclein in these models. Dysfunction of 14-3-3 function may be a critical mechanism by which α-synuclein propagation occurs in disease.


Asunto(s)
Proteínas 14-3-3/metabolismo , Encéfalo/metabolismo , Neuronas/metabolismo , alfa-Sinucleína/metabolismo , Animales , Encéfalo/patología , Cuerpos de Lewy/metabolismo , Cuerpos de Lewy/patología , Enfermedad por Cuerpos de Lewy/metabolismo , Enfermedad por Cuerpos de Lewy/patología , Ratones , Neuronas/patología , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Transporte de Proteínas/fisiología
9.
J Biol Chem ; 293(48): 18494-18503, 2018 11 30.
Artículo en Inglés | MEDLINE | ID: mdl-30275016

RESUMEN

Prion diseases are neurodegenerative disorders that affect many mammalian species. Mammalian prion proteins (PrPs) can misfold into many different aggregates. However, only a small subpopulation of these structures is infectious. One of the major unresolved questions in prion research is identifying which specific structural features of these misfolded protein aggregates are important for prion infectivity in vivo Previously, two types of proteinase K-resistant, self-propagating aggregates were generated from the recombinant mouse prion protein in the presence of identical cofactors. Although these two aggregates appear biochemically very similar, they have dramatically different biological properties, with one of them being highly infectious and the other one lacking any infectivity. Here, we used several MS-based structural methods, including hydrogen-deuterium exchange and hydroxyl radical footprinting, to gain insight into the nature of structural differences between these two PrP aggregate types. Our experiments revealed a number of specific differences in the structure of infectious and noninfectious aggregates, both at the level of the polypeptide backbone and quaternary packing arrangement. In particular, we observed that a high degree of order and stability of ß-sheet structure within the entire region between residues ∼89 and 227 is a primary attribute of infectious PrP aggregates examined in this study. By contrast, noninfectious PrP aggregates are characterized by markedly less ordered structure up to residue ∼167. The structural constraints reported here should facilitate development of experimentally based high-resolution structural models of infectiosus mammalian prions.


Asunto(s)
Priones/química , Priones/metabolismo , Animales , Proteínas de la Membrana Bacteriana Externa/química , Biocatálisis , Espectrometría de Masas , Ratones , Oxidación-Reducción , Priones/síntesis química , Priones/genética , Agregación Patológica de Proteínas/genética , Agregación Patológica de Proteínas/metabolismo , Estructura Secundaria de Proteína
10.
PLoS Pathog ; 13(7): e1006491, 2017 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-28704563

RESUMEN

Prions, characterized by self-propagating protease-resistant prion protein (PrP) conformations, are agents causing prion disease. Recent studies generated several such self-propagating protease-resistant recombinant PrP (rPrP-res) conformers. While some cause prion disease, others fail to induce any pathology. Here we showed that although distinctly different, the pathogenic and non-pathogenic rPrP-res conformers were similarly recognized by a group of conformational antibodies against prions and shared a similar guanidine hydrochloride denaturation profile, suggesting a similar overall architecture. Interestingly, two independently generated non-pathogenic rPrP-res were almost identical, indicating that the particular rPrP-res resulted from cofactor-guided PrP misfolding, rather than stochastic PrP aggregation. Consistent with the notion that cofactors influence rPrP-res conformation, the propagation of all rPrP-res formed with phosphatidylglycerol/RNA was cofactor-dependent, which is different from rPrP-res generated with a single cofactor, phosphatidylethanolamine. Unexpectedly, despite the dramatic difference in disease-causing capability, RT-QuIC assays detected large increases in seeding activity in both pathogenic and non-pathogenic rPrP-res inoculated mice, indicating that the non-pathogenic rPrP-res is not completely inert in vivo. Together, our study supported a role of cofactors in guiding PrP misfolding, indicated that relatively small structural features determine rPrP-res' pathogenicity, and revealed that the in vivo seeding ability of rPrP-res does not necessarily result in pathogenicity.


Asunto(s)
Endopeptidasas/química , Enfermedades por Prión/metabolismo , Proteínas Priónicas/química , Animales , Biocatálisis , Dimerización , Endopeptidasas/metabolismo , Ratones , Fosfatidilgliceroles/metabolismo , Enfermedades por Prión/genética , Proteínas Priónicas/genética , Proteínas Priónicas/metabolismo , Unión Proteica , Conformación Proteica , ARN/química , ARN/genética , ARN/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
11.
J Proteome Res ; 17(7): 2460-2469, 2018 07 06.
Artículo en Inglés | MEDLINE | ID: mdl-29762036

RESUMEN

Parkinson's disease is the second most common neurodegenerative disease. In the vast majority of cases the origin is not genetic and the cause is not well understood, although progressive accumulation of α-synuclein aggregates appears central to the pathogenesis. Currently, treatments that slow disease progression are lacking, and there are no robust biomarkers that can facilitate the development of such treatments or act as aids in early diagnosis. Therefore, we have defined metabolomic changes in the brain and serum in an animal model of prodromal Parkinson's disease. We biochemically profiled the brain tissue and serum in a mouse model with progressive synucleinopathy propagation in the brain triggered by unilateral injection of preformed α-synuclein fibrils in the olfactory bulb. In total, we accurately identified and quantified 71 metabolites in the brain and 182 in serum using 1H NMR and targeted mass spectrometry, respectively. Using multivariate analysis, we accurately identified which metabolites explain the most variation between cases and controls. Using pathway enrichment analysis, we highlight significantly perturbed biochemical pathways in the brain and correlate these with the progression of the disease. Furthermore, we identified the top six discriminatory metabolites and were able to develop a model capable of identifying animals with the pathology from healthy controls with high accuracy (AUC (95% CI) = 0.861 (0.755-0.968)). Our study highlights the utility of metabolomics in identifying elements of Parkinson's disease pathogenesis and for the development of early diagnostic biomarkers of the disease.


Asunto(s)
Sangre/metabolismo , Encéfalo/metabolismo , Enfermedad de Parkinson/metabolismo , Síntomas Prodrómicos , Animales , Biomarcadores/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Metaboloma , Ratones , Enfermedad de Parkinson/diagnóstico
12.
PLoS Pathog ; 11(7): e1004958, 2015 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-26136122

RESUMEN

The prion hypothesis postulates that the infectious agent in transmissible spongiform encephalopathies (TSEs) is an unorthodox protein conformation based agent. Recent successes in generating mammalian prions in vitro with bacterially expressed recombinant prion protein provide strong support for the hypothesis. However, whether the pathogenic properties of synthetically generated prion (rec-Prion) recapitulate those of naturally occurring prions remains unresolved. Using end-point titration assay, we showed that the in vitro prepared rec-Prions have infectious titers of around 104 LD50/µg. In addition, intraperitoneal (i.p.) inoculation of wild-type mice with rec-Prion caused prion disease with an average survival time of 210-220 days post inoculation. Detailed pathological analyses revealed that the nature of rec-Prion induced lesions, including spongiform change, disease specific prion protein accumulation (PrP-d) and the PrP-d dissemination amongst lymphoid and peripheral nervous system tissues, the route and mechanisms of neuroinvasion were all typical of classical rodent prions. Our results revealed that, similar to naturally occurring prions, the rec-Prion has a titratable infectivity and is capable of causing prion disease via routes other than direct intra-cerebral challenge. More importantly, our results established that the rec-Prion caused disease is pathogenically and pathologically identical to naturally occurring contagious TSEs, supporting the concept that a conformationally altered protein agent is responsible for the infectivity in TSEs.


Asunto(s)
Modelos Animales de Enfermedad , Enfermedades por Prión/patología , Priones/patogenicidad , Proteínas Recombinantes/administración & dosificación , Animales , Inmunohistoquímica , Inyecciones Intraperitoneales , Ratones , Priones/administración & dosificación , Proteínas Recombinantes/síntesis química
13.
Curr Opin Neurol ; 29(4): 459-66, 2016 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-27257944

RESUMEN

PURPOSE OF REVIEW: We describe evidence supporting the hypothesis that α-synuclein has a prion-like role in Parkinson's disease and related α-synucleinopathies, and discuss how this novel thinking impacts the development of diagnostics and disease-modifying therapies. RECENT FINDINGS: Observations that immature dopamine neurons grafted to Parkinson's disease patients can develop Lewy bodies triggered a surge of interest in the putative prion-like properties of α-synuclein. We recount results from experiments which confirm that misfolded α-synuclein can exhibit disease-propagating properties, and describe how they relate to the spreading of α-synuclein aggregates in α-synucleinopathies. We share insights into the underlying molecular mechanisms and their relevance to novel therapeutic targets. Finally, we discuss what the initial triggers of α-synuclein misfolding might be, where in the body the misfolding events might take place, and how this can instruct development of novel diagnostic tools. We speculate that differences in anatomical trigger sites and variability in α-synuclein fibril structure can contribute to clinical differences between α-synucleinopathies. SUMMARY: The realization that α-synuclein pathology can propagate between brain regions in neurodegenerative diseases has deepened and expanded our understanding of potential pathogenic processes which can lead to the development of novel diagnostic tools as well as the identification of new therapeutic targets.


Asunto(s)
Enfermedad de Parkinson/etiología , Enfermedad de Parkinson/patología , Enfermedades por Prión/etiología , Priones/metabolismo , alfa-Sinucleína/fisiología , Humanos , Enfermedad de Parkinson/fisiopatología , Enfermedades por Prión/metabolismo , Enfermedades por Prión/fisiopatología , Pliegue de Proteína
14.
Proc Natl Acad Sci U S A ; 109(22): 8546-51, 2012 May 29.
Artículo en Inglés | MEDLINE | ID: mdl-22586108

RESUMEN

Infectious prions containing the pathogenic conformer of the mammalian prion protein (PrP(Sc)) can be produced de novo from a mixture of the normal conformer (PrP(C)) with RNA and lipid molecules. Recent reconstitution studies indicate that nucleic acids are not required for the propagation of mouse prions in vitro, suggesting the existence of an alternative prion propagation cofactor in brain tissue. However, the identity and functional properties of this unique cofactor are unknown. Here, we show by purification and reconstitution that the molecule responsible for the nuclease-resistant cofactor activity in brain is endogenous phosphatidylethanolamine (PE). Synthetic PE alone facilitates conversion of purified recombinant (rec)PrP substrate into infectious recPrP(Sc) molecules. Other phospholipids, including phosphatidylcholine, phosphatidylserine, phosphatidylinositol, and phosphatidylglycerol, were unable to facilitate recPrP(Sc) formation in the absence of RNA. PE facilitated the propagation of PrP(Sc) molecules derived from all four different animal species tested including mouse, suggesting that unlike RNA, PE is a promiscuous cofactor for PrP(Sc) formation in vitro. Phospholipase treatment abolished the ability of brain homogenate to reconstitute the propagation of both mouse and hamster PrP(Sc) molecules. Our results identify a single endogenous cofactor able to facilitate the formation of prions from multiple species in the absence of nucleic acids or other polyanions.


Asunto(s)
Encéfalo/metabolismo , Ácidos Nucleicos/metabolismo , Fosfatidiletanolaminas/metabolismo , Priones/metabolismo , Animales , Western Blotting , Encéfalo/patología , Cricetinae , Inmunohistoquímica , Ratones , Fosfatidilcolinas/metabolismo , Fosfatidilgliceroles/metabolismo , Fosfatidilinositoles/metabolismo , Fosfatidilserinas/metabolismo , Priones/química , Priones/genética , Pliegue de Proteína , ARN/metabolismo , Proteínas Recombinantes/metabolismo
15.
Proc Natl Acad Sci U S A ; 109(28): E1938-46, 2012 Jul 10.
Artículo en Inglés | MEDLINE | ID: mdl-22711839

RESUMEN

Prions containing misfolded prion protein (PrP(Sc)) can be formed with cofactor molecules using the technique of serial protein misfolding cyclic amplification. However, it remains unknown whether cofactors materially participate in maintaining prion conformation and infectious properties. Here we show that withdrawal of cofactor molecules during serial propagation of purified recombinant prions caused adaptation of PrP(Sc) structure accompanied by a reduction in specific infectivity of >10(5)-fold, to undetectable levels, despite the ability of adapted "protein-only" PrP(Sc) molecules to self-propagate in vitro. We also report that changing only the cofactor component of a minimal reaction substrate mixture during serial propagation induced major changes in the strain properties of an infectious recombinant prion. Moreover, propagation with only one functional cofactor (phosphatidylethanolamine) induced the conversion of three distinct strains into a single strain with unique infectious properties and PrP(Sc) structure. Taken together, these results indicate that cofactor molecules can regulate the defining features of mammalian prions: PrP(Sc) conformation, infectivity, and strain properties. These findings suggest that cofactor molecules likely are integral components of infectious prions.


Asunto(s)
Priones/química , Animales , Catálisis , Femenino , Glicosilación , Ratones , Ratones Endogámicos C57BL , Microscopía de Fuerza Atómica/métodos , Fosfatidiletanolaminas/química , Priones/metabolismo , Conformación Proteica , Desnaturalización Proteica , Pliegue de Proteína , Proteínas Recombinantes/química , Urea/química
16.
FASEB J ; 27(12): 4768-75, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-23970796

RESUMEN

The prion hypothesis is strongly supported by the fact that prion infectivity and the pathogenic conformer of prion protein (PrP) are simultaneously propagated in vitro by the serial protein misfolding cyclic amplification (sPMCA). However, due to sPMCA's enormous amplification power, whether an infectious prion can be formed de novo with bacterially expressed recombinant PrP (rPrP) remains to be satisfactorily resolved. To address this question, we performed unseeded sPMCA with rPrP in a laboratory that has never been exposed to any native prions. Two types of proteinase K (PK)-resistant and self-perpetuating recombinant PrP conformers (rPrP-res) with PK-resistant cores of 17 or 14 kDa were generated. A bioassay revealed that rPrP-res(17kDa) was highly infectious, causing prion disease in wild-type mice with an average survival time of about 172 d. In contrast, rPrP-res(14kDa) completely failed to induce any disease. Our findings reveal that sPMCA is sufficient to initiate various self-perpetuating PK-resistant rPrP conformers, but not all of them possess in vivo infectivity. Moreover, generating an infectious prion in a prion-free environment establishes that an infectious prion can be formed de novo with bacterially expressed rPrP.


Asunto(s)
Priones/química , Animales , Encéfalo/patología , Ratones , Priones/patogenicidad , Pliegue de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química
17.
Mol Neurobiol ; 2024 May 14.
Artículo en Inglés | MEDLINE | ID: mdl-38743210

RESUMEN

PRNP Q160X is one of the five dominantly inheritable nonsense mutations causing familial prion diseases. Till now, it remains unclear how this type of nonsense mutations causes familial prion diseases with unique clinical and pathological characteristics. Human prion protein (PrP) Q160X mutation is equivalent to Q159X in mouse PrP, which produces the mutant fragment PrP1-158. Through intracerebroventricular injection of recombinant adeno-associated virus in newborn mice, we successfully overexpressed mouse PrP1-158-FLAG in the central nervous system. Interestingly, high level PrP1-158-FLAG expression in the brain caused death in these mice with an average survival time of 60 ± 9.1 days. Toxicity correlated with levels of PrP1-158-FLAG but was independent of endogenous PrP. Histopathological analyses showed microgliosis and astrogliosis in mouse brains expressing PrP1-158-FLAG and most of PrP1-158-FLAG staining appeared intracellular. Biochemical characterization revealed that the majority of PrP1-158-FLAG were insoluble and a significant part of PrP1-158-FLAG appeared to contain an un-cleaved signal peptide that may contribute to its cytoplasmic localization. Importantly, an ~10-kDa proteinase K-resistant PrP fragment was detected, which was the same as those observed in patients suffering from this type of prion diseases. To our knowledge, this is the first animal study of familial prion disease caused by Q159X that recapitulates key features of human disease. It will be a valuable tool for investigating the pathogenic mechanisms underlying familial prion diseases caused by nonsense mutations.

18.
Asian J Psychiatr ; 92: 103900, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-38218004

RESUMEN

Major depressive disorder (MDD) seriously threatens human health. BRICS, known as an acronym for "Brazil, Russia, India, China, and South Africa," were also actively carrying out researches on MDD. This study aimed to conduct a bibliometric study of research on MDD conducted by the BRICS. By searching in the Web of Science and using the software Vosviewer and Citespace as analysis tools, this study analyzed the cooperation network at the country, institution, author-specific levels, the research hotspots and trends from BRICS between 2003-2022. A total of 10,911 articles were finally included. Our findings showed that researches on MDD from BRICS rapidly increased during the past two decades. China and India have shown explosive growth, while South Africa has the largest average "Usage Count" and "Time Cited". The current cooperation partners of the BRICS were mainly high-income countries and other developing countries with similar cultures, languages, and geographical locations. Institutions in high-income countries served as the main bridges for BRICS cooperation, while at the author level, some core authors in the BRICS countries serve as centers. China showed a flexible model in domestic partnership, but institutions and authors in the other four countries have gathered to cooperate within the group. BRICS research on MDD mainly focused on cognitive science, brain science, epidemiology, and disease mechanisms. The keywords"gut microbiota", "network analysis," "machine learning" and "sleep quality" showed explosive growth and might become research hotspots in the near future. This bibliometric analysis provided a science knowledge graph and references for other researchers.


Asunto(s)
Trastorno Depresivo Mayor , Humanos , Encéfalo , Bibliometría , Brasil , China
19.
J Infect Public Health ; 17(10): 102538, 2024 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-39270469

RESUMEN

BACKGROUND: With double pressures of endemic and imported emerging infectious diseases (EIDs), China's ability to detect, prevent and control the unknown virus is of regional and global interest. This study aimed to establish an R&D Blueprint for EIDs in China by identifying the list of prioritized diseases and medical countermeasures (MCMs) that need proactive actions for the next pandemic. METHODS: The process mainly referred to the World Health Organization's prioritization methodology, supplemented by pipeline landscape, rapid risk assessment and multi-dimensional analysis. The study included five steps: 1) identifying potential pathogens, 2) screening into the long list, 3) prioritizing the long list, 4) identifying the final list and 5) generating an R&D Blueprint. RESULTS: China's R&D Blueprint identified 14 viral pathogens and two virus groups (i.e., Influenza HxNy and Coronavirus X) for proactive and representative MCM development. At least one diagnostic candidate in preclinical study, and one therapeutic and one vaccine candidate in Phase I/II clinical trials for each prioritized pathogen were suggested to be developed as strategic national stockpiles. Various generalized and innovative platform technologies were also highlighted for enhancing overall capacities of EID preparedness and response, covering basic research, experiment, detection, prevention and control, surveillance and information sharing. CONCLUSIONS: This is the first study in developing countries that established an R&D Blueprint of prioritized diseases, countermeasures and technologies. Our findings could help to drive pre-emptive scientific and technological actions toward emerging pathogens that may cause the next epidemic and could provide evidence-based strategies for developing countries to establish their national health research agenda tailored to health and research context under resource-limited settings.


Asunto(s)
Enfermedades Transmisibles Emergentes , Países en Desarrollo , Pandemias , Humanos , China/epidemiología , Enfermedades Transmisibles Emergentes/prevención & control , Enfermedades Transmisibles Emergentes/epidemiología , Pandemias/prevención & control , COVID-19/epidemiología , COVID-19/prevención & control , Investigación , Investigación Biomédica
20.
J Clin Oncol ; : JCO2400099, 2024 Sep 06.
Artículo en Inglés | MEDLINE | ID: mdl-39241208

RESUMEN

PURPOSE: We aim to determine the effectiveness of adding electroacupuncture to standard triple antiemetic therapy for treating chemotherapy-induced nausea and vomiting (CINV). METHODS: From March 2022 to December 2023, a randomized, blind, sham-controlled trial conducted across six Chinese hospitals investigated patients with breast cancer undergoing highly emetogenic chemotherapy (HEC). Patients were randomly assigned to either true electroacupuncture (n = 120) or sham electroacupuncture (n = 119) groups, with both groups receiving standard triple antiemetic therapy. The primary end point was the proportion of complete protection (no vomiting, no need for rescue treatment, and no significant nausea, as evaluated using the visual analog scale [VAS]) within 120 hours after receiving HEC. RESULTS: Among 239 randomly assigned patients, 235 (98.3%) completed the trial. In the full analysis set, compared with the sham electroacupuncture group, the true electroacupuncture group demonstrated a significant increase in the complete protection rate from 34.5% to 52.9% (P = .004). Additionally, true electroacupuncture also showed enhanced total control (4.3% v 13.4%; P = .014), no significant nausea (37.9% v 58.8%; P = .001), no nausea (4.3% v 13.4%; P = .014), and nausea VAS score = 0 mm (4.3% v 12.6%; P = .023). However, the occurrence of no vomiting in the overall stage was similar (76.7% v 73.9%; P = .622) in both groups. Post hoc exploratory analysis showed a significantly higher rate of complete protection during the delayed stage in the true electroacupuncture group compared with the sham electroacupuncture group, with no significant difference observed during the acute stage. CONCLUSION: Adding true electroacupuncture to standard triple antiemetic therapy significantly enhances the efficacy of CINV treatment in patients with breast cancer receiving HEC.

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