Packaging of prions into exosomes is associated with a novel pathway of PrP processing

Prion diseases are fatal, transmissible neurodegenerative disorders associated with conversion of the host-encoded prion protein (PrP(C)) into an abnormal pathogenic isoform (PrP(Sc)). Following exposure to the infectious agent (PrP(Sc)) in acquired disease, infection is propagated in lymphoid tissues prior to neuroinvasion and spread within the central nervous system. The mechanism of prion dissemination is perplexing due to the lack of plausible PrP(Sc)-containing mobile cells that could account for prion spread between infected and uninfected tissues. Evidence exists to demonstrate that the culture media of prion-infected neuronal cells contain PrP(Sc) and infectivity but the nature of the infectivity remains unknown. In this study we have identified PrP(C) and PrP(Sc) in association with endogenously expressing PrP neuronal cell-derived exosomes. The exosomes from our prion-infected neuronal cell line were efficient initiators of prion propagation in uninfected recipient cells and to non-neuronal cells. Moreover, our neuronal cell line was susceptible to infection by non-neuronal cell-derived exosome PrP(Sc). Importantly, these exosomes produced prion disease when inoculated into mice. Exosome-associated PrP is packaged via a novel processing pathway that involves the N-terminal modification of PrP and selection of distinct PrP glycoforms for incorporation into these vesicles. These data extend our understanding of the relationship between PrP and exosomes by showing that exosomes can establish infection in both neighbouring and distant cell types and highlight the potential contribution of differentially processed forms of PrP in disease distribution. These data suggest that exosomes represent a potent pool of prion infectivity and provide a mechanism for studying prion spread and PrP processing in cells endogenously expressing PrP.     

Prion transmission in blood and urine: what are the implications for recombinant and urinary-derived gonadotrophins?

Evidence is emerging that suggests that the protease-resistant isoform (PrP(sc)) of the normal cellular prion protein (PrP(c)) can be detected in the blood and urine of animals and humans with transmissible spongiform encephalopathies (TSEs). The production of the human menopausal and recombinant gonadotrophin preparations for use in ovarian stimulation protocols in fertility treatment is one area where the pharmaceutical industry needs to be vigilant and take appropriate steps to ensure that the safety of such drugs remains as high as ever. The recombinant preparations utilize fetal calf serum or other animal sera or proteins as part of a culture medium during production. Human urinary-derived menotrophin preparations are exposed to the theoretical risk of infection from menopausal donors of urine. Nevertheless, the failure to demonstrate irrefutably infectivity following intracerebral inoculation with urine from TSE-infected hosts suggests that the risk associated with products derived from urine is merely theoretical. Despite the paucity of evidence to date and its relevance to the infectious spread of TSEs, it is important that robust measures are implemented to either remove or inactivate PrP(sc) in order to minimize contamination. Validation of each production process is required to assess the likelihood of contamination.     

Hypothesis of interference to superinfection between bovine spastic paresis and bovine spongiform encephalopathy; suggestions for experimentation, theoretical and practical interest

Sub-acute transmissible spongiform encephalopathies (TSEs) or prion diseases are diseases of little known etiology. The origin of these diseases would appear to be an abnormal protease-resistant prion protein (PrP(res)) which would be infectious by directly inducing its defective conformation to the normal native protein (PrP(C)). This hypothesis does not account for certain aspects of TSEs, such as interference to superinfection: in laboratory animals, inoculation by means of an attenuated strain with a long incubation period protects against later infection by a very virulent strain with a short incubation period. The hypothesis is put forward that there exists a possibility of interference to superinfection between neurodegenerative diseases of unknown origin, thought to be similar to TSEs, and a later infection by a TSE. The study of this interference between bovine spastic paresis (BSP) and bovine spongiform encephalopathy (BSE) could be used as a model for this hypothesis. BSP is a very rare disease among cattle, of unknown etiology; it is curable, in the very early stages, by using tryptophan and especially lithium, potentiated by copper and manganese. An etiology close to that of TSEs has been suggested on several occasions. If interference could be demonstrated between BSP and BSE, interesting data would be provided concerning the etiology, the pathogenesis and possibly the treatment and prevention of these diseases. Notably, such data could lead to the development of a treatment and a prevention with lithium and amino acids precursors of neuromediators (tryptophan, tyrosine, glutamic acid, etc.), as well as the developing of a vaccine to combat TSEs, especially BSE and scrapie.     

Validation of a luminescence immunoassay for the detection of PrP(Sc) in brain homogenate

A luminescence immunoassay (LIA) was developed for the diagnosis of bovine spongiform encephalopathy (BSE) in brain tissue using two different monoclonal antibodies for capture and detection of the protease-resistant fragment of the pathological prion protein (PrP27-30). PrP27-30 currently represents the most reliable marker for the infectious particle (denominated prion) causing transmissible spongiform encephalopathies (TSEs). Internal and official validation studies of this assay are described using brain homogenates from ascertained BSE positive and negative cows. Using more than 300 positive and 1400 negative bovine or ovine samples, an excellent sensitivity and specificity of 100% were demonstrated. More than 1000-fold dilutions of a BSE positive homogenate still resulted in a clear positive signal. In combination with a simple homogenisation procedure for the preparation of the samples, this assay lends itself for large scale screening of cattle and sheep for TSEs using complete automation of the process.     

Neuroinvasion of prions: insights from mouse models

The prion was defined by Stanley B. Prusiner as the infectious agent that causes transmissible spongiform encephalopathies. A pathological protein accumulating in the brain of scrapie-infected hamsters was isolated in 1982 and termed prion protein (PrPSc). Its cognate gene Prnp was identified more than a decade ago by Charles Weissmann, and shown to encode the host protein PrP(C). Since the latter discovery, transgenic mice have contributed many important insights into the field of prion biology, including the understanding of the molecular basis of the species barrier for prions. By disrupting the Prnp gene, it was shown that an organism that lacks PrP(C) is resistant to infection by prions. Introduction of mutant PrP genes into PrP-deficient mice was used to investigate the structure-activity relationship of the PrP gene with regard to scrapie susceptibility. Ectopic expression of PrP in PrP knockout mice proved a useful tool for the identification of host cells competent for prion replication. Finally, the availability of PrP knockout mice and transgenic mice overexpressing PrP allows selective reconstitution experiments aimed at expressing PrP in neurografts or in specific populations of haemato- and lymphopoietic cells. The latter studies have allowed us to clarify some of the mechanisms of prion spread and disease pathogenesis.     

Effects on the serotoninergic system in sub-acute transmissible spongiform encephalopathies: current data, hypotheses, suggestions for experimentation

Sub-acute transmissible spongiform encephalopathies (TSEs), or prion diseases, are affections in which little is known of their etiology. The predominant theory stipulates that an abnormal protease-resistant prion protein (PrPres) would be infectious by directly inducing its defective conformation to the normal native protein (PrPC). The function of PrPC remains unknown. The preferred localization of PrPC at the level of the synapses supposes a function in neuronal transmission. Several neurotransmitter systems (acetylcholine, GABA, dopamine, etc.) are damaged in TSEs, mainly the serotonin (5-HT) system. At a hypothetical level, PrPC would play a trophic and functional role by regulating the capture of amino acid precursors of neurotransmitters and the functions of neuroreceptors, in particular regarding tryptophan and 5-HT receptors. By comparison with the modes of action of Ras proteins and of the envelope glycoprotein of jaagsiekte sheep retrovirus, the adaptation of an oncogenic model is suggested for the mode of action of PrPres. The sequence of events could be the following: capture of PrPres and forming of an abnormal receptor, chronic disturbance of transduction pathways, more particularly of the phosphatidylinositol-3 kinase (PI-3K)/protein kinase B (Akt)/glycogen synthetase kinase 3 (GSK 3)/Wnt-beta catenin pathway, deregulation of the PrP gene and infrequent and transitory forming of abnormal RNA messengers and, finally, the forming of abnormal proteins and the deterioration of the serotoninergic system. The involvement of endogenous nucleic acids is supposed. The infectious agent of TSEs could be an ancestral form of retrovirus, such as a retrotransposon using the prion protein as an envelope glycoprotein. Pharmacological tests, by comparison with a rare disease of unknown etiology in cattle, bovine spastic paresis, are suggested with the amino acid precursors of neuromediators (tryptophan, tyrosine, glutamic acid, etc.) and with lithium, neuroprotector and regulator of the serotonergic system.     

Chimeric porcine reproductive and respiratory syndrome viruses reveal full function of genotype 1 envelope proteins in the backbone of genotype 2

Earlier studies indicated that transgenic (tg) mice engineered to express prion protein (PrP) lacking the glycophosphatidylinositol (GPI^−/−) membrane anchor formed abnormal proteinase-resistant prion (PrPsc) amyloid deposits in their brains and hearts when infected with the RML strain of murine scrapie. In contrast, RML scrapie infection of normal mice with a GPI-anchored PrP did not deposit amyloid with PrPsc in the brain or the heart. Here we report that scrapie-infected GPI^−/− PrP tg mice also deposit PrP and transmissible infectious material in the gut, kidneys, and islets of Langerhans. Similar to previously reported amyloid deposits in the brain and heart, amyloid deposits were found in the gut; however, no amyloid deposited in the islets. By high-resolution electron microscopy, we show PrP is located primarily in α cells and also β cells. Islets contain abundant insulin and there is no abnormality in glucose metabolism in infected GPI^−/− PrP tg mice.     

An autoimmune response causes transmissible spongiform encephalopathies

Misfolded prion protein (PrP) is generally accepted as causing transmissible spongiform encephalopathies (TSEs) by aligning alongside normal host prion protein and inducing it to change to the misfolded configuration. This paper disputes this theory, and proposes that, rather than causing TSEs, misfolded PrP is the result of an autoimmune response to the host PrP, a component both of nerve cells and of lymphocytes. Autoimmunity is initiated by detachment of the phosphotidylinositol glycolipid anchor as a result of exposure to organophosphate pesticides. Once PrP is detached, antibodies are mobilized against it. In some individuals, point mutations, like the codon 129 met-val substitution, have evolved as a self-defence mechanism, causing a change in PrP to the misfolded, protease-resistant form seen in TSEs. Increased PrP production, both in response to nerve damage, and as a component of lymphocytes stimulated to proliferate in response to PrP, produces a positive feedback mechanism, resulting in symptoms of brain destruction.     

Disease-associated prion protein is not detectable in human systemic amyloid deposits

Cerebral and cardiac amyloid deposits have been reported after scrapie infection in transgenic mice expressing variant prion protein (PrP(C)) lacking the glycophosphatidylinositol anchor. The amyloid fibril protein in the systemic amyloid deposits was not characterized, and there is no clinical or pathological association between prion diseases and systemic amyloidosis in humans. Nevertheless, in view of the potential clinical significance of these murine observations, we tested both human amyloidotic tissues and isolated amyloid fibrils for the presence of PrP(Sc), the prion protein conformation associated with transmissible spongiform encephalopathy (TSE). We also sequenced the complete prion protein gene, PRNP, in amyloidosis patients. No specific immunohistochemical staining for PrP(Sc) was obtained in the amyloidotic cardiac and other visceral tissues of patients with different types of systemic amyloidosis. No protease-resistant prion protein, PrP(res), was detectable by Western blotting of amyloid fibrils isolated from cardiac and other systemic amyloid deposits. Only the complete normal wild-type PRNP gene sequence was identified, including the usual distribution of codon 129 polymorphisms. These reassuringly negative results do not support the idea that there is any relationship of prions or TSE with human systemic amyloidosis, including cardiac amyloid deposition.     

Fast, reversible interaction of prion protein with RNA aptamers containing specific sequence patterns

Summary. One of the unsolved problems in prion diseases relates to the physiological function of cellular prion protein (PrP), of which a misfolded isoform is the major component of the transmissible spongiform encephalopathies agent. Knowledge of the PrP-binding molecules may help in elucidating its role and understanding the pathological events underlying prion diseases. Because nucleic acids are known to bind PrP, we attempted to identify the preferred RNA sequences that bind to the ovine recombinant PrP. An in vitro selection approach (SELEX) was applied to a pool of 80-nucleotide(nt)-long RNAs containing a randomised 40-nt central region. The most frequently isolated aptamer, RM312, was also the best ligand (20 nM K_D value), according to both surface plasmon resonance and filter binding assays. The fast rates of association and dissociation of RM312 with immobilized PrP, which are reminiscent of biologically relevant interactions, could point to a physiological function of PrP towards cellular nucleic acids. The minimal sequence that we found necessary for binding of RM312 to PrP presents a striking similarity with one previously described PrP aptamer of comparable affinity. In addition, we here identify the two lysine clusters contained in the N-terminal part of PrP as its main nucleic-acid binding sites.     

朊蛋白相关蛋白Shadoo与朊病毒病

朊病毒病,即传染性海绵状脑病(transmissible spongiform encephalopathies,TSEs),是一类传染性、致死性神经退行性疾病.在朊病毒病的病理过程中,细胞正常朊蛋白PrPC转化为异常构象的PrPSc是至关重要的,但是PrPC的正常生理功能仍不清楚.国外学者利用比较基因组学发现了一个新的朊蛋白相关蛋白-Shadoo(Sho).Sho与PrPC在氨基酸序列和细胞定位的相似性及主要在脑组织表达,使它成为一个非常值得研究的PrP相关蛋白.对Sho可能存在的与PrPC重叠的功能甚至直接相互作用的研究工作,将对今后揭示PrPC正常生理功能以及揭示Prion病发病机制具有重要现实意义.     

Prion diseases: from protein to cell pathology

Prion diseases or transmissible spongiform encephalopathies are fatal neurodegenerative conditions in humans and animals that originate spontaneously, genetically or by infection. Conformational change of the normal (cellular) form of prion protein (PrP c) to a pathological, disease-associated form (PrP TSE) is considered central to pathogenesis and formation of the infectious agent or prion. Neuronal damage is central to clinical manifestation of prion diseases but poorly understood. In this review, we analyze the major pathogenetic pathways that lead to tissue pathology in different forms of disease. Neuropathogenesis of prion diseases evolves in complex ways on several front lines, most but not all of which exist also in other neurodegenerative as well as infectious diseases. Whereas intracellular accumulation of PrP forms might significantly impair cell function and lead to cytopathology, mere extracellular deposition of PrP TSE is questionable as a direct cytotoxic factor. Tissue damage may result from several parallel, interacting, or subsequent pathways. Future studies should clarify the trigger(s) and sequence of these processes and whether, and which, one is dominating or decisive.     

What can we learn from the oral intake of prions by sheep?

The central nervous system is the ultimate target of prions, the agents responsible for fatal neurodegenerative diseases known as transmissible spongiform encephalopathies (TSEs). The neuro-invasive phase and its related clinical signs take place after a long incubation period. During this asymptomatic phase, however, active transport and replication of the infectious agent take place in peripheral sites. The oral infection route has been extensively studied because of its implication in the recent epidemic of bovine spongiform encephalopathy (BSE) in cattle and of the resulting human cases of variant Creutzfeldt-Jakob disease (vCJD). Rodent models have been useful in studying some aspects of this pathogenesis. Now, new data on the initial steps of oral infection have been obtained in sheep. This species is naturally infected with scrapie by horizontal transmission and there is strong evidence implicating the oral route. Furthermore, the existence of resistant and susceptible genotypes offers the possibility of comparative studies. The data were obtained using surgical and biochemical procedures to modulate the efficiency of oral infection and show that, in sheep, the abnormal prion protein (PrP) associated with the infectious agent crosses the intact intestinal barrier at the level of the enterocytes and then passes rapidly into lymph. These steps are identical in susceptible and resistant sheep. Thereafter, replication takes place in lymphoid structures. Other results in the same study indicate that alimentary fluids almost completely degrade the PrP of the inoculum. Though not directly transposable to human diseases, in which it is not possible to study these early stages, these data allow the elaboration of a simplified concept for the pathogenesis of TSEs. They also suggest that human contamination at the level of the oral cavity might be more important than previously suspected.     

The Priori Diseases

The human prion diseases are fatal neurodegenerative maladies that may present as sporadic, genetic, or infectious illnesses. The sporadic form is called Creutzfeldt-Jakob disease (CJD) while the inherited disorders are called familial (f) CJD, Gerstmann-Sträussler-Scheinker (GSS) disease and fatal familial insomnia (FFI). Prions are transmissible particles that are devoid of nucleic acid and seem to be composed exclusively of a modified protein (PrPSc).The normal, cellular PrP (PrPC) is converted into PrPSc through a posttranslational process during which it acquires a high β-sheet content. In fCJD, GSS, and FFI, mutations in the PrP gene located on the short arm of chromosome 20 are the cause of disease. Considerable evidence argues that the prion diseases are disorders of protein conformation.     

The pathogenic mechanisms of prion diseases

Transmissible spongiform encephalopathies (TSEs) or prion diseases are a group of fatal neurodegenerative diseases of humans and animals, including bovine spongiform encephalopathy (BSE) of cattle, scrapie of sheep, and Creutzfeldt-Jakob disease (CJD) of humans. Prion diseases have become an important issue in public health and in the scientific world not only due to the possible relationship between BSE and new variant CJD (nvCJD) but also due to the unique biological features of the infectious agent. Although the nature of the infectious agent and the pathogenic mechanisms of prion diseases are not fully understood, considerable evidence suggests that an abnormal form (PrP(Sc)) of a host prion protein (PrP(C)) may compose substantial parts of the infectious agent and that various factors such as oxidative stress and calcium cytotoxicity are associated with the pathogenesis of prion diseases. Here, we briefly review and discuss the pathogenic mechanisms of prion diseases. These advances in understandings of fundamental biology of prion diseases may open the possibilities for the prevention and treatment of these unusual diseases and also suggest applications in more common neurodegenerative disorders such as Alzheimer's disease (AD) and Parkinson's disease (PD).     

Prion and anti-codon usage: Does infectious PrP alter tRNA abundance to induce misfolding of PrP?

The "protein-only" hypothesis of prion diseases views the infectious agent as devoid of nucleic acids and consisting of misfolded prion proteins (PrP(Sc)) which, upon infiltration into host cells, act as a template that induces transformation of wild-type protein (PrP(C)) to the pathological form by unknown mechanisms. The two isoforms are identical in amino-acid composition. By analogy to reported "silent" mutations in which utilization of relatively rare tRNAs alter protein folding pattern, we postulate that misfolded PrP(Sc) alters tRNAs abundance in prion-infected cells and results in different rates of co-translational folding of PrP, leading to the formation of additional misfolded PrP(Sc). We analyze experiments that might link tRNAs to prions. This concept of "PrP-seed and tRNA-soil" envisages a vicious cycle in which PrP(Sc) levels govern specific tRNA usage, whose alteration subsequently transforms resident PrP(C) to PrP(Sc), causing the cycle to repeat itself ad infinitum.     

Molecular interaction between prion protein and GFAP both in native and recombinant forms in vitro

Gliosis of glial fibrillary acidic protein (GFAP) associated astrocytes is considered to be one of the hallmarks of transmissible spongiform encephalopathies (TSEs). In the present study, remarkable GFAP-PrP(Sc) or GFAP-PrP(C) complexes were separately detected in the brain homogenates of 263 K (Scrapie)-infected or normal hamsters by co-immunoprecipitation assay. To get more exact molecular evidences for interaction between prion protein (PrP) and GFAP, various recombinant PrP or GFAP proteins were expressed using prokaryotic-expressing and in vitro translation system. Using pull down and co-immunoprecipitation assays, reliable molecular interaction between PrP and GFAP was observed, and proteinase K (PK)-digested PrP(Sc) molecules were confirmed to be able to bind the recombinant GFAP specifically as well. The region within PrP that was responsible for interaction with GFAP was narrowed to PK-resistant core of PrP (i.e. aa 91-230). The study of the association of PrP with GFAP supplies the molecular evidence for the observation of co-localization of PrP(Sc) and GFAP in the brains of TSEs and may further provide insight into a potential role of GFAP in the biological function of PrP and the pathogenesis of prion diseases.     

Possible cell-free prion replication.

Spongiform encephalopathies, such as scrapie or bovine spongiform encephalopathy in animals, or kuru, Creutzfeld-Jakob disease (CJD) and Gerstmann-Str?ussler-Scheinker disease (GSS) in man, seem to be caused by a transmissible agent whose nature is still a matter of debate. The properties of this agent which has been designated as prion, differ from those of any other known infectious agents, including viruses and viroids. Several lines of evidence suggest that the prion is devoid of nucleic acid and is identical with a modified form of a normal host protein. This lack of nucleic acid poses the question of how a protein moiety can propagate itself as a transmissible agent. Here it is proposed that prion replication is a process similar to crystallization and as such, the propagation of prions can take place as a sort of chain-reaction in an in vitro cell-free system.     

Transgenic analysis of prion diseases

Prion diseases are fatal transmissible neurological disorders afflicting a range of mammalian species. Although still controversial, a large body of data suggests that the causative agent may be composed entirely of a small glycoprotein. The brains of infected animals have accumulations of a pathogenic protease-resistant isoform (PrPsc) of a normal host-encoded glycoprotein, PrPc or prion protein. A number of lines of biochemical evidence implicate the disease-specific isoform, PrPsc, as the transmissible agent and genetic analysis has shown tight linkage between PrP gene mutations and polymorphisms and differential susceptibility to prion diseases, Perhaps the strongest evidence for a protein-only model of the agent is that PrP gene-ablated mice are resistant to scrapie and that mice with PrP mutation, corresponding to those found in a human familial prion disease, spontaneously develop a transmissible prion disease. This review describes the critical role that transgenic technology has played in the study of the biology of prion diseases and considers the issues raised by this work.