RESUMO
Exposure of cultured primary neurons to preformed α-synuclein fibrils (PFFs) leads to the recruitment of endogenous α-synuclein and its templated conversion into fibrillar phosphorylated α-synuclein (pα-synF) aggregates resembling those involved in Parkinson's disease (PD) pathogenesis. Pα-synF was described previously as inclusions morphologically similar to Lewy bodies and Lewy neurites in PD patients. We discovered the existence of a conformationally distinct, nonfibrillar, phosphorylated α-syn species that we named "pα-syn*." We uniquely describe the existence of pα-syn* in PFF-seeded primary neurons, mice brains, and PD patients' brains. Through immunofluorescence and pharmacological manipulation we showed that pα-syn* results from incomplete autophagic degradation of pα-synF. Pα-synF was decorated with autophagic markers, but pα-syn* was not. Western blots revealed that pα-syn* was N- and C-terminally trimmed, resulting in a 12.5-kDa fragment and a SDS-resistant dimer. After lysosomal release, pα-syn* aggregates associated with mitochondria, inducing mitochondrial membrane depolarization, cytochrome C release, and mitochondrial fragmentation visualized by confocal and stimulated emission depletion nanoscopy. Pα-syn* recruited phosphorylated acetyl-CoA carboxylase 1 (ACC1) with which it remarkably colocalized. ACC1 phosphorylation indicates low ATP levels, AMPK activation, and oxidative stress and induces mitochondrial fragmentation via reduced lipoylation. Pα-syn* also colocalized with BiP, a master regulator of the unfolded protein response and a resident protein of mitochondria-associated endoplasmic reticulum membranes that are sites of mitochondrial fission and mitophagy. Pα-syn* aggregates were found in Parkin-positive mitophagic vacuoles and imaged by electron microscopy. Collectively, we showed that pα-syn* induces mitochondrial toxicity and fission, energetic stress, and mitophagy, implicating pα-syn* as a key neurotoxic α-syn species and a therapeutic target.
Assuntos
Autofagia/efeitos dos fármacos , Mitofagia/efeitos dos fármacos , Neurotoxinas , Doença de Parkinson/metabolismo , alfa-Sinucleína , Acetil-CoA Carboxilase/química , Acetil-CoA Carboxilase/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Química Encefálica , Técnicas de Cultura de Células , Células Cultivadas , Humanos , Lisossomos/metabolismo , Camundongos , Mitocôndrias , Neurotoxinas/química , Neurotoxinas/metabolismo , Neurotoxinas/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Fosforilação , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , alfa-Sinucleína/toxicidadeRESUMO
We recently identified a truncated and phosphorylated form of α-synuclein, pα-syn*, as a key neurotoxic α-synuclein species found in cultured neurons, as well as in mouse and Parkinson's disease patients' brains. Small pα-syn* aggregates localize to mitochondria and induce mitochondrial damage and fragmentation. Herein, we investigated the molecular basis of pα-syn*-induced toxicity. By immunofluorescence, we found phosphorylated MKK4, JNK, ERK5 and p38 MAPKs in pα-syn* inclusions. pJNK colocalized with pα-syn* at mitochondria and mitochondria-associated ER membranes where it was associated with BiP and pACC1, markers for the ER and energy deprivation, respectively. We also found that pα-syn* aggregates are tightly associated with small ptau aggregates of similar size. Pα-syn*/ptau inclusions localized to areas of mitochondrial damage and to mitophagic vesicles, showing their role in mitochondrial toxicity, mitophagy induction and their removal along with damaged mitochondrial fragments. Several MAPKs may act cooperatively to phosphorylate tau, notably JNK, p38 and GSK3ß, a non-MAPK that was also found phosphorylated in the vicinity of pα-syn*/ptau aggregates. These results add insight into the mechanisms by which pα-syn* exerts its toxic effects that include the phosphorylation of several kinases of the MAPK pathway, as well as the formation of ptau at the mitochondrial membrane, likely contributing to mitotoxicity. Thus pα-syn* appears to be the trigger of a series of kinase mediated pathogenic events and a link between α-syn pathology and tau, another protein known to aggregate in Parkinson's disease and other synucleinopathies.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Mitocôndrias/metabolismo , Doenças Neurodegenerativas/metabolismo , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Ativação Enzimática/fisiologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/patologia , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/fisiopatologia , FosforilaçãoRESUMO
Prion diseases such as Creutzfeldt-Jakob disease (CJD) are incurable and rapidly fatal neurodegenerative diseases. Because prion protein (PrP) is necessary for prion replication but dispensable for the host, we developed the PrP-FRET-enabled high throughput assay (PrP-FEHTA) to screen for compounds that decrease PrP expression. We screened a collection of drugs approved for human use and identified astemizole and tacrolimus, which reduced cell-surface PrP and inhibited prion replication in neuroblastoma cells. Tacrolimus reduced total cellular PrP levels by a nontranscriptional mechanism. Astemizole stimulated autophagy, a hitherto unreported mode of action for this pharmacophore. Astemizole, but not tacrolimus, prolonged the survival time of prion-infected mice. Astemizole is used in humans to treat seasonal allergic rhinitis in a chronic setting. Given the absence of any treatment option for CJD patients and the favorable drug characteristics of astemizole, including its ability to cross the blood-brain barrier, it may be considered as therapy for CJD patients and for prophylactic use in familial prion diseases. Importantly, our results validate PrP-FEHTA as a method to identify antiprion compounds and, more generally, FEHTA as a unique drug discovery platform.
Assuntos
Astemizol/farmacologia , Autofagia/efeitos dos fármacos , Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Doenças Priônicas/tratamento farmacológico , Príons/metabolismo , Tacrolimo/farmacologia , Animais , Astemizol/uso terapêutico , Western Blotting , Linhagem Celular Tumoral , Transferência Ressonante de Energia de Fluorescência/métodos , Ensaios de Triagem em Larga Escala/métodos , Humanos , Estimativa de Kaplan-Meier , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Prion diseases are infectious and belong to the group of protein misfolding neurodegenerative diseases. In these diseases, neuronal dysfunction and death are caused by the neuronal toxicity of a particular misfolded form of their cognate protein. The ability to specifically target the toxic protein conformer or the neuronal death pathway would provide powerful therapeutic approaches to these diseases. The neurotoxic forms of the prion protein (PrP) have yet to be defined but there is evidence suggesting that at least some of them differ from infectious PrP (PrP(Sc)). Herein, without making an assumption about size or conformation, we searched for toxic forms of recombinant PrP after dilution refolding, size fractionation, and systematic biological testing of all fractions. We found that the PrP species most neurotoxic in vitro and in vivo (toxic PrP, TPrP) is a monomeric, highly α-helical form of PrP. TPrP caused autophagy, apoptosis, and a molecular signature remarkably similar to that observed in the brains of prion-infected animals. Interestingly, highly α-helical intermediates have been described for other amyloidogenic proteins but their biological significance remains to be established. We provide unique experimental evidence that a monomeric α-helical form of an amyloidogenic protein represents a cytotoxic species. Although toxic PrP has yet to be purified from prion-infected brains, TPrP might be the equivalent of one highly neurotoxic PrP species generated during prion replication. Because TPrP is a misfolded, highly neurotoxic form of PrP reproducing several features of prion-induced neuronal death, it constitutes a useful model to study PrP-induced neurodegenerative mechanisms.
Assuntos
Neurotoxinas/química , Neurotoxinas/toxicidade , Príons/química , Príons/toxicidade , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/patologia , Neurotoxinas/metabolismo , Doenças Priônicas/patologia , Príons/metabolismo , Estrutura Secundária de ProteínaRESUMO
The mechanisms underlying prion-linked neurodegeneration remain to be elucidated, despite several recent advances in this field. Herein, we show that soluble, low molecular weight oligomers of the full-length prion protein (PrP), which possess characteristics of PrP to PrPsc conversion intermediates such as partial protease resistance, are neurotoxic in vitro on primary cultures of neurons and in vivo after subcortical stereotaxic injection. Monomeric PrP was not toxic. Insoluble, fibrillar forms of PrP exhibited no toxicity in vitro and were less toxic than their oligomeric counterparts in vivo. The toxicity was independent of PrP expression in the neurons both in vitro and in vivo for the PrP oligomers and in vivo for the PrP fibrils. Rescue experiments with antibodies showed that the exposure of the hydrophobic stretch of PrP at the oligomeric surface was necessary for toxicity. This study identifies toxic PrP species in vivo. It shows that PrP-induced neurodegeneration shares common mechanisms with other brain amyloidoses like Alzheimer disease and opens new avenues for neuroprotective intervention strategies of prion diseases targeting PrP oligomers.
Assuntos
Encéfalo/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Doenças Priônicas/induzido quimicamente , Príons/toxicidade , Animais , Anticorpos Bloqueadores/farmacologia , Anticorpos Monoclonais/farmacologia , Apoptose/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Células Cultivadas , Feminino , Membranas Intracelulares/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Neurônios/patologia , Doenças Priônicas/metabolismo , Doenças Priônicas/patologia , Príons/química , Príons/imunologia , Príons/metabolismo , Isoformas de Proteínas , Estrutura Secundária de ProteínaRESUMO
The uncertain extent of human exposure to bovine spongiform encephalopathy (BSE)--which can lead to variant Creutzfeldt-Jakob disease (vCJD)--is compounded by incomplete knowledge about the efficiency of oral infection and the magnitude of any bovine-to-human biological barrier to transmission. We therefore investigated oral transmission of BSE to non-human primates. We gave two macaques a 5 g oral dose of brain homogenate from a BSE-infected cow. One macaque developed vCJD-like neurological disease 60 months after exposure, whereas the other remained free of disease at 76 months. On the basis of these findings and data from other studies, we made a preliminary estimate of the food exposure risk for man, which provides additional assurance that existing public health measures can prevent transmission of BSE to man.
Assuntos
Encefalopatia Espongiforme Bovina/transmissão , Contaminação de Alimentos , Doenças dos Primatas/transmissão , Animais , Encéfalo/patologia , Química Encefálica , Bovinos , Síndrome de Creutzfeldt-Jakob/transmissão , Ingestão de Alimentos , Encefalopatia Espongiforme Bovina/patologia , Macaca fascicularis , Proteínas PrPSc/análiseRESUMO
BACKGROUND: The unique resistance of prions to classic methods of decontamination, and evidence that prion diseases can be transmitted iatrogenically by medical devices pose a serious infection control challenge to health-care facilities. In view of the widespread tissue distribution of the variant Creutzfeldt-Jakob disease agent in human beings, new practicable decontamination procedures are urgently needed. METHODS: We adapted an in-vivo method using stainless steel wires contaminated with prions to the hamster-adapted scrapie strain 263K. A new in-vitro protocol of surface contamination compatible with subsequent biochemical detection of PrP(res) (protease-resistant form of the prion protein) from the treated surface was developed to explore the mechanisms of action of methods of decontamination under test. These models were used to investigate the effectiveness of innovative physical and chemical methods of prion inactivation. FINDINGS: Standard chemical decontamination methods (NaOH 1N, NaOCl 20000 ppm) and autoclaving in water at 134 degrees C reduced infectivity by >5.6 log10 lethal doses; autoclaving without immersion was somewhat less effective (4-4.5 log reduction). Three milder treatments, including a phenolic disinfectant, an alkaline cleaner, and the combination of an enzymatic cleaner and vaporised hydrogen peroxide (VHP) were also effective. VHP alone, which can be compatible with electronic components, achieved an approximately 4.5 log reduction in infectivity (equivalent to autoclaving without water immersion). INTERPRETATION: New decontamination procedures are proposed to ensure the safety of medical and surgical instruments as well as surfaces that cannot withstand the currently recommended prion inactivation procedures.
Assuntos
Encéfalo/microbiologia , Desinfecção/métodos , Contaminação de Equipamentos , Implantes Experimentais/microbiologia , Doenças Priônicas/prevenção & controle , Animais , Transfusão de Sangue , Cricetinae , Feminino , Mesocricetus , Doenças Priônicas/microbiologia , Scrapie/prevenção & controle , Scrapie/transmissão , Instrumentos CirúrgicosRESUMO
While there is a growing consensus on the understanding of the propagation pathways after oral infection of transmissible spongiform encephalopathy (TSE) agents and even if the central role of follicular dendritic cells is identified, little is known about the key players in the first steps of the infection and about the site of the disease development. We investigated the role of gut macrophages, which are capable of capturing aggregates of the prion protein. PLGA particles containing clodronate were designed in order to be orally administered and to target Peyer's patches for inducing gut-associated macrophages suicide in mice. Mice were subsequently infected with scrapie or BSE by the oral route. It was found that the efficacy of macrophage suppression in the Peyer's patches correlated well with an earlier appearance of PrPres in these formations and with a higher amount of PrPres at a later stage of the infection. Thus, the capture of infectious particles that have crossed the epithelial gut barrier and their elimination by macrophages seems to be a key event to restrict the amount of agent initiating the infection.
Assuntos
Sistema Digestório/patologia , Encefalopatia Espongiforme Bovina/patologia , Macrófagos/patologia , Scrapie/patologia , Animais , Bovinos , Fenômenos Químicos , Físico-Química , Ácido Clodrônico , Composição de Medicamentos , Corantes Fluorescentes , Imuno-Histoquímica , Ácido Láctico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Varredura , Microesferas , Tamanho da Partícula , Nódulos Linfáticos Agregados/patologia , Ácido Poliglicólico , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Polímeros , RodaminasRESUMO
Prion strain identification has been hitherto achieved using time-consuming incubation time determinations in one or more mouse lines and elaborate neuropathological assessment. In the present work, we make a detailed study of the properties of PrP-overproducing Tga20 mice. We show that in these mice the four prion strains examined are rapidly and faithfully amplified and can subsequently be discriminated by a cell-based procedure, the Cell Panel Assay.
Assuntos
Bioensaio/métodos , Príons/classificação , Príons/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas PrPC/metabolismo , Proteínas PrPSc/metabolismo , Príons/patogenicidade , Especificidade da EspécieRESUMO
The absence of specific immune response is a hallmark of prion diseases. However, in vitro and in vivo experiments have provided evidence that an anti-PrP humoral response could have beneficial effects. Prophylactic passive immunization performed at the time of infection delayed or prevented disease. Nonetheless, the potential therapeutic effect of PrP antibodies administered shortly before the clinical signs has never been tested in vivo. Moreover, a recent study showed the potential toxicity of PrP antibodies administered intracerebrally. We aimed at evaluating the effect of a prolonged intracerebral anti-PrP antibody administration at the time of neuroinvasion in BSE infected Tg20 mice. Unexpectedly, despite a good penetration of the antibodies in the brain parenchyma, the treatment was not protective against the development of BSE. Instead, it led to an extensive neuronal loss, strong astrogliosis and microglial activation. Since this effect was observed after injection of anti-PrP antibodies as whole IgGs, F(ab')(2) or Fab fragments, the toxicity was directly related to the ability of the antibodies to recognize native PrP and to the intracerebral concentration achieved, and not to the Fc portion or the divalence of the antibodies. This experiment shows that a prolonged treatment with anti-PrP antibodies by the intracerebral route can induce severe side-effects and calls for caution with regard to the use of similar approaches for late therapeutic interventions in humans.
Assuntos
Anticorpos/farmacologia , Encefalopatia Espongiforme Bovina/tratamento farmacológico , Fragmentos Fab das Imunoglobulinas/farmacologia , Imunoglobulina G/farmacologia , Príons/antagonistas & inibidores , Animais , Anticorpos/imunologia , Anticorpos/uso terapêutico , Formação de Anticorpos/imunologia , Encéfalo/imunologia , Bovinos , Encefalopatia Espongiforme Bovina/genética , Encefalopatia Espongiforme Bovina/imunologia , Imunização Passiva/métodos , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/uso terapêutico , Imunoglobulina G/imunologia , Imunoglobulina G/uso terapêutico , Camundongos , Camundongos Transgênicos , Príons/imunologiaRESUMO
BACKGROUND: Recently, we showed that the 37-kDa/67-kDa laminin receptor (LRP/LR) acts as the receptor of the cellular prion protein. METHODS: For the present study, we investigated the binding of the murine scrapie prion protein (moPrP27-30) to baby hamster kidney (BHK) cells, using the Semliki Forest virus system. RESULTS: The enhanced binding of moPrP27-30 to BHK cells expressing moLRP::FLAG was inhibited by the LRP/LR-specific antibody W3, which suggests that LRP/LR acts as a receptor for the scrapie form of the prion protein, PrP(Sc). This finding was confirmed by a parallel study that showed that bovine prions are internalized by human enterocytes via LRP/LR. The heparan sulfate mimetics HM5004 and HM2602 reduced PrP27-30 binding to moLRP-expressing cells to approximately 30% and approximately 20%, respectively, at a concentration of 10 microg/mL, whereas pentosan polysulfate (SP54) and phycarin sulfate (PS3) both reduced the binding to approximately 40% at a concentration of 100 microg/mL. CONCLUSIONS: We suggest that the inhibition reported elsewhere of PrP(Sc) synthesis and the incubation times prolonged in rodent models by these sulfated glycans are due to the inhibition of the LRP/LR-dependent binding of prions to the target cells.
Assuntos
Heparitina Sulfato/farmacologia , Proteínas PrPSc/metabolismo , Príons/fisiologia , Receptores de Laminina/fisiologia , Animais , Linhagem Celular , Cricetinae , Rim , Camundongos , Plasmídeos , Príons/efeitos dos fármacos , RNA Viral/genética , Vírus da Floresta de Semliki/efeitos dos fármacos , Vírus da Floresta de Semliki/genética , Vírus da Floresta de Semliki/fisiologiaRESUMO
Human prion diseases, such as Creutzfeldt-Jakob disease (CJD), are neurodegenerative and fatal. Sporadic CJD (sCJD) can be transmitted between humans through medical procedures involving highly infected organs, such as the central nervous system. However, in variant CJD (vCJD), which is due to human contamination with the bovine spongiform encephalopathy (BSE) agent, lymphoreticular tissue also harbors the transmissible spongiform encephalopathy-associated prion protein (PrP(TSE)), which poses a particularly acute risk for iatrogenic transmission. Two blood transfusion-related cases are already documented. In addition, the recent observation of PrP(TSE) in spleen and muscle in sCJD raised the possibility that peripheral PrP(TSE) is not limited to vCJD cases. We aimed to clarify the peripheral pathogenesis of human TSEs by using a nonhuman primate model which mimics human diseases. A highly sensitive enzyme-linked immunosorbent assay was adapted to the detection of extraneural PrP(TSE). We show that affected organs can be divided into two groups. The first is peripheral organs accumulating large amounts of PrP(TSE), which represent a high risk of iatrogenic transmission. This category comprises only lymphoreticular organs in the vCJD/BSE model. The second is organs with small amounts of PrP(TSE) associated with nervous structures. These are the muscles, adrenal glands, and enteric nervous system in the sporadic, iatrogenic, and variant CJD models. In contrast to the first set of organs, this low level of tissue contamination is not strain restricted and seems to be linked to secondary centrifugal spread of the agent through nerves. It might represent a risk for iatrogenic transmission, formerly underestimated despite previous reports of low rates of transmission from peripheral organs of humans to nonhuman primates (5, 10). This study provides an additional experimental basis for the classification of human organs into different risk categories and a rational re-evaluation of current risk management measures.
Assuntos
Síndrome de Creutzfeldt-Jakob/patologia , Príons/isolamento & purificação , Animais , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Variação Genética , Humanos , Imuno-Histoquímica , Macaca fascicularisRESUMO
While the exact function of the cellular prion protein (PrP(C)) remains unknown, there are several leads due to increasing knowledge on the localisation and interaction of PrP(C) with other molecules. This chapter will concentrate on these aspects. Identified ligands of PrP(C) mainly belong to the categories of heat-shock proteins, membrane-bound receptors, or heparan sulphates. The possible synaptic role of PrP(C) has been exemplified by electrophysiological findings in PrP(o/o) mice and the studies of PrP(C) as a copper-binding molecule that could regulate the copper content of the synaptic cleft. The latter property of PrP(C) may also endow PrP(C) with the activity of a copper-dependent superoxide dismutase. Binding of PrP(C) to signalling molecules suggests a role as a transmitter of information from the extracellular milieu to the cell and a trigger for a molecular cascade. This agrees with new data on PrP(C) receptors and the role of PrP(C) in cell survival.
Assuntos
Membrana Celular/metabolismo , Neurônios Aferentes/metabolismo , Proteínas PrPC/fisiologia , Animais , Diferenciação Celular , Sobrevivência Celular , Cobre/metabolismo , Glicosaminoglicanos/metabolismo , Proteínas de Choque Térmico/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Proteínas PrPC/metabolismo , Ligação Proteica , Receptores de Superfície Celular/metabolismo , Receptores de Laminina/metabolismo , Transdução de Sinais/fisiologiaRESUMO
The 37-kDa/67-kDa laminin receptor (LRP/LR) plays a major role in the propagation of PrPSc, the abnormal form of the prion protein. In order to ablate the expression of LRP/LR in mouse brain we generated transgenic mice ectopically expressing antisense LRP RNA in the brain under control of the neuron-specific enolase (NSE) promoter. Hemizygous transgenic mice TgN(NSEasLRP)2 showed a significant reduction of LRP/LR protein levels in hippocampal and cerebellar brain regions. These mice might act as powerful tools to investigate the role of the laminin receptor in scrapie pathogenesis.
Assuntos
Encéfalo/metabolismo , Expressão Gênica , Camundongos/genética , RNA Antissenso/metabolismo , Receptores de Laminina/metabolismo , Animais , Linhagem Celular , Camundongos Transgênicos , Peso Molecular , Fosfopiruvato Hidratase/genética , Regiões Promotoras Genéticas , Ratos , Receptores de Laminina/genéticaRESUMO
The accumulation of PrP(Sc) in scrapie-infected neuronal cells has been prevented by three approaches: (i) transfection of ScMNB cells with an antisense laminin receptor precursor (LRP) RNA-expression plasmid, (ii) transfection of ScN2a cells and ScGT1 cells with small interfering RNAs (siRNAs) specific for the LRP mRNA, and (iii) incubation of ScN2a cells with an anti-LRP/LR antibody. LRP antisense RNA and LRP siRNAs reduced LRP/LR expression and inhibited the accumulation of PrP(Sc) in these cells. The treatments also reduced PrP(c) levels. The anti-LRP/LR antibody, W3, abolished PrP(Sc) accumulation and reduced PrP(c) levels after seven days of incubation. Cells remained free of PrP(Sc) after being cultured for 14 additional days without the antibody, whereas the PrP(c) level was restored. Our results demonstrate the necessity of the laminin receptor (LRP/LR) for PrP(Sc) propagation in cultured cells and suggest that LRP/LR-specific antibodies could be used as powerful therapeutic tools in the treatment of transmissible spongiform encephalopathies.
Assuntos
Neurônios/metabolismo , Proteínas PrPSc/metabolismo , Receptores de Laminina/metabolismo , Scrapie/metabolismo , Animais , Transporte Biológico , Peso Molecular , Neuroblastoma , Proteínas PrPSc/genética , RNA Antissenso/farmacologia , RNA Interferente Pequeno/farmacologia , Receptores de Laminina/química , Receptores de Laminina/genética , Receptores de Laminina/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonucleases , Scrapie/genética , Células Tumorais CultivadasRESUMO
The accumulation of PrP(res), the protease-resistant abnormal form of the host-encoded cellular prion protein, PrP(C), plays a central role in transmissible spongiform encephalopathies. Human contamination by bovine spongiform encephalopathy (BSE) has propelled many scientific teams on a highway for anti-prion drug development. This study reports that heparan sulfate mimetics (HMs), developed originally for their effect on tissue regeneration, abolish prion propagation in scrapie-infected GT1 cells. PrP(res) does not reappear for up to 50 days post-treatment. When tested in vivo, one of these compounds, HM2602, hampered PrP(res) accumulation in scrapie- and BSE-infected mice and prolonged significantly the survival time of 263K scrapie-infected hamsters. Interestingly, HM2602 is an apparently less toxic and more potent inhibitor of PrP(res) accumulation than dextran sulfate 500, a molecule known to exhibit anti-prion properties in vivo. Kinetics of PrP(res) disappearance in vitro and unaffected PrP(C) levels during treatment suggest that HMs are able to block the conversion of PrP(C) into PrP(res). It is speculated that HMs act as competitors of endogenous heparan sulfates known to act as co-receptors for the prion protein. Since these molecules are particularly amenable to drug design, their anti-prion potential could be developed further and optimized for the treatment of prion diseases.