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1.
Acta Neuropathol Commun ; 8(1): 83, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32517816

RESUMO

In the human prion disease Creutzfeldt-Jakob disease (CJD), different CJD neuropathological subtypes are defined by the presence in normal prion protein (PrPC) of a methionine or valine at residue 129, by the molecular mass of the infectious prion protein PrPSc, by the pattern of PrPSc deposition, and by the distribution of spongiform change in the brain. Heterozygous cases of CJD potentially add another layer of complexity to defining CJD subtypes since PrPSc can have either a methionine (PrPSc-M129) or valine (PrPSc-V129) at residue 129. We have recently demonstrated that the relative amount of PrPSc-M129 versus PrPSc-V129, i.e. the PrPSc allotype ratio, varies between heterozygous CJD cases. In order to determine if differences in PrPSc allotype correlated with different disease phenotypes, we have inoculated 10 cases of heterozygous CJD (7 sporadic and 3 iatrogenic) into two transgenic mouse lines overexpressing PrPC with a methionine at codon 129. In one case, brain-region specific differences in PrPSc allotype appeared to correlate with differences in prion disease transmission and phenotype. In the other 9 cases inoculated, the presence of PrPSc-V129 was associated with plaque formation but differences in PrPSc allotype did not consistently correlate with disease incubation time or neuropathology. Thus, while the PrPSc allotype ratio may contribute to diverse prion phenotypes within a single brain, it does not appear to be a primary determinative factor of disease phenotype.


Assuntos
Encéfalo/patologia , Síndrome de Creutzfeldt-Jakob/patologia , Síndrome de Creutzfeldt-Jakob/transmissão , Proteínas PrPC/patogenicidade , Proteínas PrPSc/patogenicidade , Animais , Encéfalo/metabolismo , Síndrome de Creutzfeldt-Jakob/metabolismo , Heterozigoto , Humanos , Camundongos Transgênicos , Proteínas PrPC/metabolismo , Proteínas PrPSc/metabolismo
2.
Int J Mol Sci ; 21(11)2020 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-32545192

RESUMO

The prion protein (PrP) is an enigmatic molecule with a pleiotropic effect on different cell types; it is localized stably in lipid raft microdomains and it is able to recruit downstream signal transduction pathways by its interaction with various biochemical partners. Since its discovery, this lipid raft component has been involved in several functions, although most of the publications focused on the pathological role of the protein. Recent studies report a key role of cellular prion protein (PrPC) in physiological processes, including cellular differentiation. Indeed, the PrPC, whose expression is modulated according to the cell differentiation degree, appears to be part of the multimolecular signaling pathways of the neuronal differentiation process. In this review, we aim to summarize the main findings that report the link between PrPC and stem cells.


Assuntos
Microdomínios da Membrana/metabolismo , Proteínas PrPC/metabolismo , Células-Tronco/metabolismo , Animais , Diferenciação Celular/fisiologia , Humanos , Neurônios/metabolismo , Neurônios/patologia , Proteínas PrPC/genética , Proteínas PrPC/patogenicidade , Células-Tronco/patologia
3.
PLoS Pathog ; 14(8): e1007214, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30089152

RESUMO

Although misfolding of normal prion protein (PrPC) into abnormal conformers (PrPSc) is critical for prion disease pathogenesis our current understanding of the underlying molecular pathophysiology is rudimentary. Exploiting an electrophysiology paradigm, herein we report that at least modestly proteinase K (PK)-resistant PrPSc (PrPres) species are acutely synaptotoxic. Brief exposure to ex vivo PrPSc from two mouse-adapted prion strains (M1000 and MU02) prepared as crude brain homogenates (cM1000 and cMU02) and cell lysates from chronically M1000-infected RK13 cells (MoRK13-Inf) caused significant impairment of hippocampal CA1 region long-term potentiation (LTP), with the LTP disruption approximating that reported during the evolution of murine prion disease. Proof of PrPSc (especially PrPres) species as the synaptotoxic agent was demonstrated by: significant rescue of LTP following selective immuno-depletion of total PrP from cM1000 (dM1000); modestly PK-treated cM1000 (PK+M1000) retaining full synaptotoxicity; and restoration of the LTP impairment when employing reconstituted, PK-eluted, immuno-precipitated M1000 preparations (PK+IP-M1000). Additional detailed electrophysiological analyses exemplified by impairment of post-tetanic potentiation (PTP) suggest possible heightened pre-synaptic vulnerability to the acute synaptotoxicity. This dysfunction correlated with cumulative insufficiency of replenishment of the readily releasable pool (RRP) of vesicles during repeated high-frequency stimulation utilised for induction of LTP. Broadly comparable results with LTP and PTP impairment were obtained utilizing hippocampal slices from PrPC knockout (PrPo/o) mice, with cM1000 serial dilution assessments revealing similar sensitivity of PrPo/o and wild type (WT) slices. Size fractionation chromatography demonstrated that synaptotoxic PrP correlated with PK-resistant species >100kDa, consistent with multimeric PrPSc, with levels of these species >6 ng/ml appearing sufficient to induce synaptic dysfunction. Biochemical analyses of hippocampal slices manifesting acute synaptotoxicity demonstrated reduced levels of multiple key synaptic proteins, albeit with noteworthy differences in PrPo/o slices, while such changes were absent in hippocampi demonstrating rescued LTP through treatment with dM1000. Our findings offer important new mechanistic insights into the synaptic impairment underlying prion disease, enhancing prospects for development of targeted effective therapies.


Assuntos
Endopeptidase K/metabolismo , Proteínas PrPC/patogenicidade , Doenças Priônicas/etiologia , Príons/patogenicidade , Sinapses/patologia , Doença Aguda , Animais , Encefalopatias/etiologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas PrPC/metabolismo , Proteólise , Sinapses/efeitos dos fármacos
4.
Proc Natl Acad Sci U S A ; 114(36): 9617-9622, 2017 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-28827331

RESUMO

In transmissible spongiform encephalopathies (TSEs), which are lethal neurodegenerative diseases that affect humans and a wide range of other mammalian species, the normal "cellular" prion protein ([Formula: see text]) is transformed into amyloid aggregates representing the "scrapie form" of the protein ([Formula: see text]). Continued research on this system is of keen interest, since new information on the physiological function of [Formula: see text] in healthy organisms is emerging, as well as new data on the mechanism of the transformation of [Formula: see text] to [Formula: see text] In this paper we used two different approaches: a combination of the well-tempered ensemble (WTE) and parallel tempering (PT) schemes and metadynamics (MetaD) to characterize the conformational free-energy surface of [Formula: see text] The focus of the data analysis was on an 11-residue polypeptide segment in mouse [Formula: see text](121-231) that includes the [Formula: see text]2-[Formula: see text]2 loop of residues 167-170, for which a correlation between structure and susceptibility to prion disease has previously been described. This study includes wild-type mouse [Formula: see text] and a variant with the single-residue replacement Y169A. The resulting detailed conformational landscapes complement in an integrative manner the available experimental data on [Formula: see text], providing quantitative insights into the nature of the structural transition-related function of the [Formula: see text]2-[Formula: see text]2 loop.


Assuntos
Proteínas PrPC/química , Substituição de Aminoácidos , Animais , Humanos , Camundongos , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Proteínas PrPC/genética , Proteínas PrPC/patogenicidade , Doenças Priônicas/etiologia , Doenças Priônicas/genética , Doenças Priônicas/metabolismo , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta
5.
Emerg Infect Dis ; 23(6): 946-956, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28518033

RESUMO

In the United-Kingdom, ≈1 of 2,000 persons could be infected with variant Creutzfeldt-Jakob disease (vCJD). Therefore, risk of transmission of vCJD by medical procedures remains a major concern for public health authorities. In this study, we used in vitro amplification of prions by protein misfolding cyclic amplification (PMCA) to estimate distribution and level of the vCJD agent in 21 tissues from 4 patients who died of clinical vCJD and from 1 asymptomatic person with vCJD. PMCA identified major levels of vCJD prions in a range of tissues, including liver, salivary gland, kidney, lung, and bone marrow. Bioassays confirmed that the quantitative estimate of levels of vCJD prion accumulation provided by PMCA are indicative of vCJD infectivity levels in tissues. Findings provide critical data for the design of measures to minimize risk for iatrogenic transmission of vCJD.


Assuntos
Bioensaio , Síndrome de Creutzfeldt-Jakob/diagnóstico , Proteínas PrPC/química , Animais , Doenças Assintomáticas , Medula Óssea/metabolismo , Medula Óssea/patologia , Síndrome de Creutzfeldt-Jakob/metabolismo , Síndrome de Creutzfeldt-Jakob/patologia , Feminino , Humanos , Rim/metabolismo , Rim/patologia , Fígado/metabolismo , Fígado/patologia , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos , Proteínas PrPC/metabolismo , Proteínas PrPC/patogenicidade , Dobramento de Proteína , Glândulas Salivares/metabolismo , Glândulas Salivares/patologia , Reino Unido
6.
J Virol ; 90(23): 10660-10669, 2016 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-27654300

RESUMO

Bank vole is a rodent species that shows differential susceptibility to the experimental transmission of different prion strains. In this work, the transmission features of a panel of diverse prions with distinct origins were assayed both in bank vole expressing methionine at codon 109 (Bv109M) and in transgenic mice expressing physiological levels of bank vole PrPC (the BvPrP-Tg407 mouse line). This work is the first systematic comparison of the transmission features of a collection of prion isolates, representing a panel of diverse prion strains, in a transgenic-mouse model and in its natural counterpart. The results showed very similar transmission properties in both the natural species and the transgenic-mouse model, demonstrating the key role of the PrP amino acid sequence in prion transmission susceptibility. However, differences in the PrPSc types propagated by Bv109M and BvPrP-Tg407 suggest that host factors other than PrPC modulate prion strain features. IMPORTANCE: The differential susceptibility of bank voles to prion strains can be modeled in transgenic mice, suggesting that this selective susceptibility is controlled by the vole PrP sequence alone rather than by other species-specific factors. Differences in the phenotypes observed after prion transmissions in bank voles and in the transgenic mice suggest that host factors other than the PrPC sequence may affect the selection of the substrain replicating in the animal model.


Assuntos
Arvicolinae/genética , Arvicolinae/fisiologia , Proteínas PrPC/patogenicidade , Doenças Priônicas/etiologia , Príons/patogenicidade , Animais , Encéfalo/fisiopatologia , Bovinos , Síndrome de Creutzfeldt-Jakob/etiologia , Síndrome de Creutzfeldt-Jakob/genética , Síndrome de Creutzfeldt-Jakob/transmissão , Modelos Animais de Doenças , Suscetibilidade a Doenças , Interações Hospedeiro-Patógeno , Humanos , Camundongos , Camundongos Transgênicos , Proteínas PrPC/genética , Proteínas PrPC/fisiologia , Doenças Priônicas/genética , Doenças Priônicas/transmissão , Príons/genética , Príons/fisiologia , Ovinos , Especificidade da Espécie
7.
J Virol ; 90(23): 10752-10761, 2016 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-27654299

RESUMO

Chronic wasting disease (CWD) in cervids and bovine spongiform encephalopathy (BSE) in cattle are prion diseases that are caused by the same protein-misfolding mechanism, but they appear to pose different risks to humans. We are interested in understanding the differences between the species barriers of CWD and BSE. We used real-time, quaking-induced conversion (RT-QuIC) to model the central molecular event in prion disease, the templated misfolding of the normal prion protein, PrPc, to a pathogenic, amyloid isoform, scrapie prion protein, PrPSc We examined the role of the PrPc amino-terminal domain (N-terminal domain [NTD], amino acids [aa] 23 to 90) in cross-species conversion by comparing the conversion efficiency of various prion seeds in either full-length (aa 23 to 231) or truncated (aa 90 to 231) PrPc We demonstrate that the presence of white-tailed deer and bovine NTDs hindered seeded conversion of PrPc, but human and bank vole NTDs did the opposite. Additionally, full-length human and bank vole PrPcs were more likely to be converted to amyloid by CWD prions than were their truncated forms. A chimera with replacement of the human NTD by the bovine NTD resembled human PrPc The requirement for an NTD, but not for the specific human sequence, suggests that the NTD interacts with other regions of the human PrPc to increase promiscuity. These data contribute to the evidence that, in addition to primary sequence, prion species barriers are controlled by interactions of the substrate NTD with the rest of the substrate PrPc molecule. IMPORTANCE: We demonstrate that the amino-terminal domain of the normal prion protein, PrPc, hinders seeded conversion of bovine and white-tailed deer PrPcs to the prion forms, but it facilitates conversion of the human and bank vole PrPcs to the prion forms. Additionally, we demonstrate that the amino-terminal domain of human and bank vole PrPcs requires interaction with the rest of the molecule to facilitate conversion by CWD prions. These data suggest that interactions of the amino-terminal domain with the rest of the PrPc molecule play an important role in the susceptibility of humans to CWD prions.


Assuntos
Proteínas PrPC/genética , Proteínas PrPC/patogenicidade , Doenças Priônicas/etiologia , Sequência de Aminoácidos , Animais , Arvicolinae , Encéfalo/metabolismo , Bovinos , Cervos , Suscetibilidade a Doenças , Encefalopatia Espongiforme Bovina/etiologia , Encefalopatia Espongiforme Bovina/genética , Encefalopatia Espongiforme Bovina/metabolismo , Especificidade de Hospedeiro/genética , Humanos , Proteínas PrPC/química , Doenças Priônicas/genética , Doenças Priônicas/metabolismo , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Homologia de Sequência de Aminoácidos , Doença de Emaciação Crônica/etiologia , Doença de Emaciação Crônica/genética , Doença de Emaciação Crônica/metabolismo
8.
J Virol ; 90(23): 10867-10874, 2016 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-27681129

RESUMO

Prions are proteinaceous pathogens responsible for subacute spongiform encephalopathies in animals and humans. The prions responsible for bovine spongiform encephalopathy (BSE) are zoonotic agents, causing variant Creutzfeldt-Jakob disease (CJD) in humans. The transfer of prions between species is limited by a species barrier, which is thought to reflect structural incompatibilities between the host cellular prion protein (PrPC) and the infecting pathological PrP assemblies (PrPSc) constituting the prion. A BSE strain variant, designated L-BSE and responsible for atypical, supposedly spontaneous forms of prion diseases in aged cattle, demonstrates zoonotic potential, as evidenced by its capacity to propagate more easily than classical BSE in transgenic mice expressing human PrPC and in nonhuman primates. In humanized mice, L-BSE propagates without any apparent species barrier and shares similar biochemical PrPSc signatures with the CJD subtype designated MM2-cortical, thus opening the possibility that certain CJD cases classified as sporadic may actually originate from L-type BSE cross-transmission. To address this issue, we compared the biological properties of L-BSE and those of a panel of CJD subtypes representative of the human prion strain diversity using standard strain-typing criteria in human PrP transgenic mice. We found no evidence that L-BSE causes a known form of sporadic CJD. IMPORTANCE: Since the quasi-extinction of classical BSE, atypical BSE forms are the sole BSE variants circulating in cattle worldwide. They are observed in rare cases of old cattle, making them difficult to detect. Extrapolation of our results suggests that L-BSE may propagate in humans as an unrecognized form of CJD, and we urge both the continued utilization of precautionary measures to eliminate these agents from the human food chain and active surveillance for CJD phenotypes in the general population.


Assuntos
Síndrome de Creutzfeldt-Jakob/genética , Encefalopatia Espongiforme Bovina/genética , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Bovinos , Síndrome de Creutzfeldt-Jakob/etiologia , Síndrome de Creutzfeldt-Jakob/transmissão , Modelos Animais de Doenças , Encefalopatia Espongiforme Bovina/etiologia , Encefalopatia Espongiforme Bovina/transmissão , Variação Genética , Especificidade de Hospedeiro , Humanos , Camundongos , Camundongos Transgênicos , Proteínas PrPC/genética , Proteínas PrPC/patogenicidade , Proteínas PrPSc/genética , Proteínas PrPSc/patogenicidade
9.
J Virol ; 89(18): 9524-31, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26157118

RESUMO

UNLABELLED: The propensity for transspecies prion transmission is related to the structural characteristics of the enciphering and new host PrP, although the exact mechanism remains incompletely understood. The effects of variability in prion protein on cross-species prion transmission have been studied with animal bioassays, but the influence of prion protein structure versus that of host cofactors (e.g., cellular constituents, trafficking, and innate immune interactions) remains difficult to dissect. To isolate the effects of protein-protein interactions on transspecies conversion, we used recombinant PrP(C) and real-time quaking-induced conversion (RT-QuIC) and compared chronic wasting disease (CWD) and classical bovine spongiform encephalopathy (cBSE) prions. To assess the impact of transmission to a new species, we studied feline CWD (fCWD) and feline BSE (i.e., feline spongiform encephalopathy [FSE]). We cross-seeded fCWD and FSE into each species' full-length, recombinant PrP(C) and measured the time required for conversion to the amyloid (PrP(Res)) form, which we describe here as the rate of amyloid conversion. These studies revealed the following: (i) CWD and BSE seeded their homologous species' PrP best; (ii) fCWD was a more efficient seed for feline rPrP than for white-tailed deer rPrP; (iii) conversely, FSE more efficiently converted bovine than feline rPrP; (iv) and CWD, fCWD, BSE, and FSE all converted human rPrP, although not as efficiently as homologous sCJD prions. These results suggest that (i) at the level of protein-protein interactions, CWD adapts to a new species more readily than does BSE and (ii) the barrier preventing transmission of CWD to humans may be less robust than estimated. IMPORTANCE: We demonstrate that bovine spongiform encephalopathy prions maintain their transspecies conversion characteristics upon passage to cats but that chronic wasting disease prions adapt to the cat and are distinguishable from the original prion. Additionally, we showed that chronic wasting disease prions are effective at seeding the conversion of normal human prion protein to an amyloid conformation, perhaps the first step in crossing the species barrier.


Assuntos
Síndrome de Creutzfeldt-Jakob , Encefalopatia Espongiforme Bovina , Proteínas PrPC , Doença de Emaciação Crônica , Animais , Arvicolinae , Gatos , Bovinos , Síndrome de Creutzfeldt-Jakob/metabolismo , Síndrome de Creutzfeldt-Jakob/transmissão , Encefalopatia Espongiforme Bovina/metabolismo , Encefalopatia Espongiforme Bovina/transmissão , Humanos , Proteínas PrPC/química , Proteínas PrPC/metabolismo , Proteínas PrPC/patogenicidade , Especificidade da Espécie , Doença de Emaciação Crônica/metabolismo , Doença de Emaciação Crônica/transmissão
10.
PLoS Pathog ; 10(9): e1004366, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25211026

RESUMO

The central event underlying prion diseases involves conformational change of the cellular form of the prion protein (PrP(C)) into the disease-associated, transmissible form (PrP(Sc)). Pr(PC) is a sialoglycoprotein that contains two conserved N-glycosylation sites. Among the key parameters that control prion replication identified over the years are amino acid sequence of host PrP(C) and the strain-specific structure of PrPSc. The current work highlights the previously unappreciated role of sialylation of PrP(C) glycans in prion pathogenesis, including its role in controlling prion replication rate, infectivity, cross-species barrier and PrP(Sc) glycoform ratio. The current study demonstrates that undersialylated PrP(C) is selected during prion amplification in Protein Misfolding Cyclic Amplification (PMCAb) at the expense of oversialylated PrP(C). As a result, PMCAb-derived PrP(Sc) was less sialylated than brain-derived PrP(Sc). A decrease in PrPSc sialylation correlated with a drop in infectivity of PMCAb-derived material. Nevertheless, enzymatic de-sialylation of PrP(C) using sialidase was found to increase the rate of PrP(Sc) amplification in PMCAb from 10- to 10,000-fold in a strain-dependent manner. Moreover, de-sialylation of PrP(C) reduced or eliminated a species barrier of for prion amplification in PMCAb. These results suggest that the negative charge of sialic acid controls the energy barrier of homologous and heterologous prion replication. Surprisingly, the sialylation status of PrP(C) was also found to control PrP(Sc) glycoform ratio. A decrease in Pr(PC) sialylation levels resulted in a higher percentage of the diglycosylated glycoform in PrP(Sc). 2D analysis of charge distribution revealed that the sialylation status of brain-derived PrP(C) differed from that of spleen-derived PrP(C). Knocking out lysosomal sialidase Neu1 did not change the sialylation status of brain-derived PrP(C), suggesting that Neu1 is not responsible for desialylation of PrP(C). The current work highlights previously unappreciated role of PrP(C) sialylation in prion diseases and opens multiple new research directions, including development of new therapeutic approaches.


Assuntos
Ácido N-Acetilneuramínico/metabolismo , Proteínas PrPC/metabolismo , Proteínas PrPSc/metabolismo , Doenças Priônicas/transmissão , Dobramento de Proteína , Animais , Western Blotting , Encéfalo/metabolismo , Encéfalo/patologia , Glicosilação , Masculino , Mesocricetus , Camundongos , Camundongos Knockout , Neuraminidase/metabolismo , Neuraminidase/fisiologia , Proteínas PrPC/química , Proteínas PrPC/patogenicidade , Proteínas PrPSc/química , Proteínas PrPSc/patogenicidade , Doenças Priônicas/metabolismo , Doenças Priônicas/patologia , Baço/metabolismo , Baço/patologia
12.
Viruses ; 4(11): 3109-31, 2012 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-23202518

RESUMO

The human cellular prion protein (PrP(C)) is a glycosylphosphatidylinositol (GPI) anchored membrane glycoprotein with two N-glycosylation sites at residues 181 and 197. This protein migrates in several bands by Western blot analysis (WB). Interestingly, PNGase F treatment of human brain homogenates prior to the WB, which is known to remove the N-glycosylations, unexpectedly gives rise to two dominant bands, which are now known as C-terminal (C1) and N-terminal (N1) fragments. This resembles the ß-amyloid precursor protein (APP) in Alzheimer disease (AD), which can be physiologically processed by α-, ß-, and γ-secretases. The processing of APP has been extensively studied, while the identity of the cellular proteases involved in the proteolysis of PrP(C) and their possible role in prion biology has remained limited and controversial. Nevertheless, there is a strong correlation between the neurotoxicity caused by prion proteins and the blockade of their normal proteolysis. For example, expression of non-cleavable PrP(C) mutants in transgenic mice generates neurotoxicity, even in the absence of infectious prions, suggesting that PrP(C) proteolysis is physiologically and pathologically important. As many mouse models of prion diseases have recently been developed and the knowledge about the proteases responsible for the PrP(C) proteolysis is accumulating, we examine the historical experimental evidence and highlight recent studies that shed new light on this issue.


Assuntos
Proteínas PrPC/fisiologia , Animais , Modelos Animais de Doenças , Proteínas Ligadas por GPI/fisiologia , Humanos , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/fisiologia , Proteínas PrPC/patogenicidade , Doenças Priônicas/etiologia
13.
PLoS One ; 7(8): e44051, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22937149

RESUMO

Before prion uptake and infection can occur in the lower gastrointestinal system, ingested prions are subjected to anaerobic digestion in the rumen of cervids and bovids. The susceptibility of soil-bound prions to rumen digestion has not been evaluated previously. In this study, prions from infectious brain homogenates as well as prions bound to a range of soils and soil minerals were subjected to in vitro rumen digestion, and changes in PrP levels were measured via western blot. Binding to clay appeared to protect noninfectious hamster PrP(c) from complete digestion, while both unbound and soil-bound infectious PrP(Sc) proved highly resistant to rumen digestion. In addition, no change in intracerebral incubation period was observed following active rumen digestion of unbound hamster HY TME prions and HY TME prions bound to a silty clay loam soil. These results demonstrate that both unbound and soil-bound prions readily survive rumen digestion without a reduction in infectivity, further supporting the potential for soil-mediated transmission of chronic wasting disease (CWD) and scrapie in the environment.


Assuntos
Digestão/fisiologia , Proteínas PrPC/metabolismo , Doenças Priônicas/metabolismo , Rúmen/metabolismo , Animais , Cricetinae , Masculino , Proteínas PrPC/patogenicidade , Doenças Priônicas/transmissão , Solo
14.
PLoS Pathog ; 7(12): e1002402, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22144895

RESUMO

Prion diseases are characterised by the accumulation of PrP(Sc), an abnormally folded isoform of the cellular prion protein (PrP(C)), in affected tissues. Following peripheral exposure high levels of prion-specific PrP(Sc) accumulate first upon follicular dendritic cells (FDC) in lymphoid tissues before spreading to the CNS. Expression of PrP(C) is mandatory for cells to sustain prion infection and FDC appear to express high levels. However, whether FDC actively replicate prions or simply acquire them from other infected cells is uncertain. In the attempts to-date to establish the role of FDC in prion pathogenesis it was not possible to dissociate the Prnp expression of FDC from that of the nervous system and all other non-haematopoietic lineages. This is important as FDC may simply acquire prions after synthesis by other infected cells. To establish the role of FDC in prion pathogenesis transgenic mice were created in which PrP(C) expression was specifically "switched on" or "off" only on FDC. We show that PrP(C)-expression only on FDC is sufficient to sustain prion replication in the spleen. Furthermore, prion replication is blocked in the spleen when PrP(C)-expression is specifically ablated only on FDC. These data definitively demonstrate that FDC are the essential sites of prion replication in lymphoid tissues. The demonstration that Prnp-ablation only on FDC blocked splenic prion accumulation without apparent consequences for FDC status represents a novel opportunity to prevent neuroinvasion by modulation of PrP(C) expression on FDC.


Assuntos
Células Dendríticas Foliculares/metabolismo , Regulação da Expressão Gênica , Proteínas PrPC/biossíntese , Proteínas PrPC/patogenicidade , Doenças Priônicas/metabolismo , Baço/metabolismo , Animais , Células Dendríticas Foliculares/patologia , Camundongos , Camundongos Knockout , Proteínas PrPC/genética , Doenças Priônicas/genética , Doenças Priônicas/patologia , Baço/patologia
15.
Biochem Biophys Res Commun ; 410(2): 159-63, 2011 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-21679685

RESUMO

As mutations in the PRNP gene account for human hereditary prion diseases (PrDs), it is crucial to elucidating how these mutations affect the central pathogenic conformational transition of normal cellular prion protein (PrP(C)) to abnormal scrapie isoform (PrP(Sc)). Many studies proposed that these pathogenic mutations may make PrP more susceptible to conformational change through altering its structure stability. By evaluating the most recent observations regarding pathogenic mutations, it was found that the pathogenic mutations do not exert a uniform effect on the thermodynamic stability of the human PrP's structure. Through analyzing the reported PrDs-related mutations, we found that 25 out of 27 mutations possess strong directional selection, i.e., enhancing hydrophobicity or decreasing negative and increasing positive charge. Based on the triggering role reported by previous studies of facilitating factors in PrP(C) conversion, e.g., lipid and polyanion, we proposed that the mutation-induced changes may strengthen the interaction between PrP and facilitating factors, which will accelerate PrP conversion and cause PrDs.


Assuntos
Proteínas PrPC/genética , Proteínas PrPC/patogenicidade , Proteínas PrPSc/genética , Proteínas PrPSc/patogenicidade , Seleção Genética , Análise Mutacional de DNA , Glicosilfosfatidilinositóis/química , Humanos , Mutação , Proteínas PrPC/química , Proteínas PrPSc/química , Sinais Direcionadores de Proteínas/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína/genética
16.
Prion ; 4(4): 292-301, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20930564

RESUMO

Neurodegenerative diseases are often associated with misfolding and deposition of specific proteins in the nervous system. The prion protein, which is associated with transmissible spongiform encephalopathies (TSEs), is one of them. The normal function of the cellular form of the prion protein (PrP(C)) is mediated through specific signal transduction pathways and is linked to resistance to oxidative stress, neuronal outgrowth and cell survival. In TSEs, PrP(C) is converted into an abnormally folded isoform, called PrP(Sc), that may impair the normal function of the protein and/or generate toxic aggregates. To investigate these molecular events we performed a two-dimensional gel electrophoresis comparison of neuroblastoma N2a cells expressing different amounts of PrP(C) and eventually infected with the 22L prion strain. Mass spectrometry and peptide mass fingerprint analysis identified a series of proteins with modified expression. They included the chaperones Grp78/BiP, protein disulfide-isomerase A6, Grp75 and Hsp60 which had an opposite expression upon PrPC expression and PrP(Sc) production. The detection of these proteins was coherent with the idea that protein misfolding plays an important role in TSEs. Other proteins, such as calreticulin, tubulin, vimentin or the laminin receptor had their expression modified in infected cells, which was reminiscent of previous results. Altogether our data provide molecular information linking PrP expression and misfolding, which could be the basis of further therapeutic and pathophysiological research in this field.


Assuntos
Neuroblastoma/metabolismo , Proteínas PrPC/metabolismo , Proteômica/métodos , Animais , Western Blotting , Extratos Celulares , Linhagem Celular Tumoral , Eletroforese em Gel Bidimensional , Chaperona BiP do Retículo Endoplasmático , Camundongos , Modelos Biológicos , Proteínas PrPC/patogenicidade , Proteínas PrPSc/metabolismo
17.
Science ; 327(5969): 1132-5, 2010 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-20110469

RESUMO

The prion hypothesis posits that a misfolded form of prion protein (PrP) is responsible for the infectivity of prion disease. Using recombinant murine PrP purified from Escherichia coli, we created a recombinant prion with the attributes of the pathogenic PrP isoform: aggregated, protease-resistant, and self-perpetuating. After intracerebral injection of the recombinant prion, wild-type mice developed neurological signs in approximately 130 days and reached the terminal stage of disease in approximately 150 days. Characterization of diseased mice revealed classic neuropathology of prion disease, the presence of protease-resistant PrP, and the capability of serially transmitting the disease; these findings confirmed that the mice succumbed to prion disease. Thus, as postulated by the prion hypothesis, the infectivity in mammalian prion disease results from an altered conformation of PrP.


Assuntos
Fosfatidilgliceróis/química , Proteínas PrPSc/química , Proteínas PrPSc/patogenicidade , Doenças Priônicas/etiologia , Príons/química , Príons/patogenicidade , RNA/química , Animais , Encéfalo/patologia , Química Encefálica , Linhagem Celular , Endopeptidase K/metabolismo , Escherichia coli/genética , Feminino , Glicosilação , Fígado/química , Camundongos , Neurônios/química , Proteínas PrPC/química , Proteínas PrPC/patogenicidade , Proteínas PrPSc/análise , Doenças Priônicas/patologia , Dobramento de Proteína , Proteínas Recombinantes/química
18.
J Neuropathol Exp Neurol ; 68(8): 870-9, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19606066

RESUMO

Previous studies indicate that RNA may be required for proteinase-resistant prion protein (PrP) amplification and for infectious prion formation in vitro, suggesting that RNA molecules may function as cellular cofactors for abnormal PrP (PrPSc) formation and become part of the structure of the infectious agent. To address this question, we used chemicals that can cleave phosphodiester bonds of RNA and assessed their effects on the infectious agent. Lithium aluminum hydride, a reducing agent that can induce reductive cleavage of oxidized molecules such as carbonyls, carboxyl acids, esters, and phosphodiester bonds, did not affect cellular PrP degradation; however, it destroyed PrPSc, extended the scrapie incubation period, and markedly reduced total RNA concentrations. These results prompted us to investigate whether RNA molecules are cofactors for PrPSc propagation. RNase A treatment of partially purified PrP and of 263K scrapie brain homogenates was sufficient to increase the sensitivity of PrPSc to proteinase K degradation. This is the first evidence that suggests that RNA molecules are a component of PrPSc. Treatment with RNase A alone and PrP degradation by RNase A plus proteinase K in vitro, however, did not result in loss of scrapie infectivity compared with the effects of lithium aluminum hydride. Together, these data suggest that RNA molecules may be important for maintaining the structure of PrPSc and that oxidized molecules can be important in scrapie agent replication and prion infectivity.


Assuntos
Compostos de Alumínio/farmacologia , Compostos de Lítio/farmacologia , Príons/sangue , Príons/patogenicidade , RNA/metabolismo , Substâncias Redutoras/farmacologia , Scrapie/metabolismo , Compostos de Alumínio/uso terapêutico , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Cricetinae , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Compostos de Lítio/uso terapêutico , Masculino , Mesocricetus , Proteínas PrPC/sangue , Proteínas PrPC/metabolismo , Proteínas PrPC/patogenicidade , Príons/metabolismo , Proteínas Quinases/farmacologia , Substâncias Redutoras/uso terapêutico , Ribonucleases/farmacologia , Scrapie/tratamento farmacológico , Scrapie/mortalidade , Scrapie/patologia
19.
Biochim Biophys Acta ; 1792(6): 555-63, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19281844

RESUMO

The prion protein (PrP) is essential for the pathogenesis of prion disease. PrP has been detected in the cytosol of neurons and transgenic mice expressing PrP in the cytosol (cyPrP) under a pan-neuronal promoter developed rapid cerebellar granule neuron degeneration. Yet, it remains unclear whether cyPrP is capable to cause toxicity in other neuronal populations. Here, we report that transgenic mice expressing cyPrP in the forebrain neurons developed behavioral abnormalities including clasping and hyperactivity. These mice had reduced thickness in cortex and developed astrogliosis in hippocampal and cortical regions. Moreover, cyPrP in these mice was recognized by the A11 anti-oligomer antibody and was associated with the hydrophobic lipid core of membranes, indicating that cyPrP oligomer caused membrane perturbation contributes to cyPrP neurotoxicity. Together, our results clearly revealed that cyPrP is able to cause toxicity in different neuronal populations, supporting a role of cyPrP in PrP-mediated neurodegenerative disorders.


Assuntos
Síndromes Neurotóxicas/metabolismo , Proteínas PrPC/patogenicidade , Doenças Priônicas/patologia , Prosencéfalo/patologia , Animais , Comportamento Animal , Córtex Cerebral/patologia , Citoplasma/metabolismo , Hipocampo/patologia , Homozigoto , Bicamadas Lipídicas/metabolismo , Camundongos , Camundongos Transgênicos , Neurônios/metabolismo , Síndromes Neurotóxicas/patologia , Proteínas PrPC/metabolismo
20.
Methods Mol Biol ; 459: 265-73, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18576161

RESUMO

The unconventional nature of the infectious agent of prion diseases poses a challenge to conventional infection control methodologies. The extra neural tissue distribution of variant and sporadic Creutzfeldt-Jakob disease has increased concern regarding the risk of prion disease transmission via general surgical procedures and highlighted the need for decontamination procedures that can be incorporated into routine processing. This chapter describe a quantitative method for assessing the prionocidal activity of chemical and physical decontamination methods against surface-bound prion infectivity.


Assuntos
Bioensaio/métodos , Proteínas PrPC/patogenicidade , Animais , Encéfalo/patologia , Descontaminação , Camundongos , Propriedades de Superfície
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