Perturbation of endoplasmic reticulum homeostasis facilitates prion replication

Prion diseases are fatal and infectious neurodegenerative disorders characterized by the accumulation of an abnormally folded form of the prion protein (PrP), termed PrP(Sc). Prion replication triggers endoplasmic reticulum (ER) stress, neuronal dysfunction, and apoptosis. In this study we analyze the effect of perturbations in ER homeostasis on PrP biochemical properties and prion replication. ER stress led to the generation of a mis-folded PrP isoform, which is detergent-insoluble and protease-sensitive. To understand the mechanism by which ER stress generates PrP misfolding, we assessed the contribution of different signaling pathways implicated in the unfolded protein response. Expression of a dominant negative form of IRE1 alpha or XBP-1 significantly increased PrP aggregation, whereas overexpression of ATF4 or an active mutant form of XBP-1 and ATF6 had the opposite affect. Analysis of prion replication in vitro revealed that the PrP isoform generated after ER stress is more efficiently converted into PrP(Sc) compared with the protein extracted from untreated cells. These findings indicate that ER-damaged cells might be more susceptible to prion replication. Because PrP(Sc) induces ER stress, our data point to a vicious cycle accelerating prion replication, which may explain the rapid progression of the disease.     

Prion protein polymerisation triggered by manganese-generated prion protein seeds

Prion diseases are neurodegenerative diseases that can be transmitted between individuals. The exact cause of these diseases remains unknown. However, one of the key events associates with the disease is the aggregation of a cellular protein, the prion protein. The mechanism of this is still unclear. However, it is likely that the aggregation is trigged by a seeding mechanism in which an oligomer of the prion protein is able to catalyse polymerisation of further prion protein into larger aggregates. We have developed a model of this process using an oligomeric species generated from recombinant protein by exposure to manganese. On fractionation of the seeding species, we estimated that the smallest size the oligomer would be is an octomer. We analysed the catalytic mechanism of the seeding oligomer and its interaction with substrate. Different domains of the protein are necessary for the seeding ability of the prion protein as opposed to those required for it to form a substrate for the polymerisation reaction. Prion seeds formed from different sheep alleles are able to reproduce the characteristics of scrapie in terms of resistance to disease. However, we were also able to generate prion seed from chicken PrP a species where no prion disease is known. Our findings provide an insight into the aggregation process of the prion protein and its potential relation to disease progress.     

Molecular mechanisms of neurotoxicity of pathological prion protein

Transmissible Spongiform Encephalopathies or prion related disorders are fatal and infectious neurodegenerative diseases characterized by extensive neuronal apoptosis and accumulation of a misfolded form of the cellular prion protein (PrP), denoted PrP(Sc). Although the mechanism of neurodegeneration and the involvement of PrP(Sc) is far from clear, data indicates that neuronal apoptosis might be related to activation of several signaling pathways, including proteasome dysfunction, alterations in prion maturation pathway and endoplasmic reticulum (ER) stress. In this article we describe recent studies investigating the molecular mechanism of PrP(Sc) neurotoxicity. We propose a model in which the key step in the pathogenesis of prion disorders, independent on their etiology, is the alteration of ER-homeostasis due to drastic modifications of the physicochemical properties of PrP, leading to the activation of ER-dependent signaling pathways that controls cellular survival.     

A nonfibrillar form of the fusogenic prion protein fragment [118-135] induces apoptotic cell death in rat cortical neurons

Neuronal loss is a salient feature of prion diseases. However, its cause and mechanism, particularly its relationship with the accumulation and precipitation of the pathogenic, protease-resistant isoform PrP(Sc) of the cellular prion protein PrP(C), are still an enigma. Several studies suggest that neuronal loss could occur through a process of programmed cell death, which is consistent with the lack of inflammation in these conditions. By analogy with the pathological events occurring during the development of Alzheimer's disease, controversies still exist regarding the relationship between amyloidogenesis, prion aggregation, and neuronal loss. We recently demonstrated that a prion protein fragment (118-135) displayed membrane-destabilizing properties and was able to induce, in a nonfibrillar form, the fusion of unilamellar liposomes. To unravel the mechanism of prion protein neurotoxicity, we characterize the effects of the human Pr[118-135] peptide on rat cortical neurons. We demonstrate that low concentrations of the Pr[118-135] peptide, in a nonfibrillar form, induce a time- and dose- dependent apoptotic cell death, including caspase activation, DNA condensation, and fragmentation. This toxicity might involve oxidative stress, because antioxidant molecules, such as probucol and propyl gallate, protect neurons against prion peptide toxicity. By contrast, a nonfusogenic variant Pr[118-135, 0 degrees ] peptide, which displays the same amino acid composition but several amino acid permutations, is not toxic to cortical neurons, which emphasizes the critical role of the fusogenic properties of the prion peptide in its neurotoxicity. Taken together, our results suggest that the interaction between the Pr[118-135] peptide and the plasma membrane of neurons might represent an early event in a cascade leading to neurodegeneration.     

Copper-dependent functions for the prion protein

Prion diseases such as bovine spongiform encephalopathy and Creutzfeldt-Jakob disease are fatal neurodegenerative diseases. These diseases are characterized by the conversion of a normal cellular protein, the prion protein, to an abnormal isoform that is thought to be responsible for both pathogenesis in the disease and the infectious nature of the disease agent. Understanding the biology and metabolism of the normal prion protein is therefore important for understanding the nature of these diseases. This review presents evidence for the normal function of the cellular prion protein, which appears to depend on its ability to bind copper (Cu). There is now considerable evidence that the prion protein is an antioxidant. Once the prion protein binds Cu, it may have an activity like that of a superoxide dismutase. Conversion of the prion protein to an abnormal isoform might lead to a loss of antioxidant protection that could be responsible for neurodegeneration in the disease.     

Stressing out the ER: a role of the unfolded protein response in prion-related disorders

Transmissible Spongiform Encephalopathies are fatal and infectious neurodegenerative diseases characterized by extensive neuronal apoptosis and the accumulation of an abnormally folded form of the cellular prion protein (PrP), denoted PrP(SC). Compelling evidence suggests the involvement of several signaling pathways in prion pathogenesis, including proteasome dysfunction, alterations in the protein maturation pathways and the unfolded protein response. Recent reports indicate that endoplasmic reticulum stress due to the PrP misfolding may be a critical factor mediating neuronal dysfunction in prion diseases. These findings have applications for developing novel strategies for treatment and early diagnosis of transmissible spongiform encephalopathies and other neurodegenerative diseases.     

Small RNA deep sequencing reveals a distinct miRNA signature released in exosomes from prion-infected neuronal cells

Prion diseases are transmissible neurodegenerative disorders affecting both humans and animals. The cellular prion protein, PrP^C, and the abnormal infectious form, PrP^Sc, are found associated with exosomes, which are small 50–130 nm vesicles released from cells. Exosomes also contain microRNAs (miRNAs), a class of non-coding RNA, and have been utilized to identify miRNA signatures for diagnosis of disease. While some miRNAs are deregulated in prion-infected brain tissue, the role of miRNA in circulating exosomes released during prion disease is unknown. Here, we investigated the miRNA profile in exosomes released from prion-infected neuronal cells. We performed the first small RNA deep sequencing study of exosomes and demonstrated that neuronal exosomes contain a diverse range of RNA species including retroviral RNA repeat regions, messenger RNA fragments, transfer RNA fragments, non-coding RNA, small nuclear RNA, small nucleolar RNA, small cytoplasmic RNA, silencing RNA as well as known and novel candidate miRNA. Significantly, we show that exosomes released by prion-infected neuronal cells have increased let-7b, let-7i, miR-128a, miR-21, miR-222, miR-29b, miR-342-3p and miR-424 levels with decreased miR-146 a levels compared to non-infected exosomes. Overall, these results demonstrate that circulating exosomes released during prion infection have a distinct miRNA signature that can be utilized for diagnosis and understanding pathogenic mechanisms in prion disease.     

朊病毒病相关细胞凋亡

朊病毒病是一组蛋白质折叠异常的蛋白质构象病,由正常朊蛋白PrP^C转化为具蛋白酶抗性的异常朊蛋白PrP^Sc并在中枢神经系统聚集引起.其主要的病理变化为神经元的丢失,脑组织的海绵样变及神经胶质细胞增生.目前认为其神经元丢失的主要原因为凋亡及自噬等,而神经元的凋亡涉及多种因素及途径,机制较为复杂.对神经元凋亡的研究将有助于揭示朊病毒病的发病机制.     

Ablation of the metal ion-induced endocytosis of the prion protein by disease-associated mutation of the octarepeat region.

The neurodegenerative spongiform encephalopathies, or prion diseases, are characterized by the conversion of the normal cellular form of the prion protein PrP(C) to a pathogenic form, PrP(Sc) [1]. There are four copies of an octarepeat PHGG(G/S)WGQ that specifically bind Cu(2+) ions within the N-terminal half of PrP(C) [2--4]. This has led to proposals that prion diseases may, in part, be due to abrogation of the normal cellular role of PrP(C) in copper homeostasis [5]. Here, we show that murine PrP(C) is rapidly endocytosed upon exposure of neuronal cells to physiologically relevant concentrations of Cu(2+) or Zn(2+), but not Mn(2+). Deletion of the four octarepeats or mutation of the histidine residues (H68/76 dyad) in the central two repeats abolished endocytosis, indicating that the internalization of PrP(C) is governed by metal binding to the octarepeats. Furthermore, a mutant form of PrP that contains nine additional octarepeats and is associated with familial prion disease [6] failed to undergo Cu(2+)-mediated endocytosis. For the first time, these results provide evidence that metal ions can promote the endocytosis of a mammalian prion protein in neuronal cells and that neurodegeneration associated with some prion diseases may arise from the ablation of this function due to mutation of the octarepeat region.     

Prion protein gene-deficient cell lines: powerful tools for prion biology

Prion diseases are zoonotic infectious diseases commonly transmissible among animals via prion infections with an accompanying deficiency of cellular prion protein (PrP(C)) and accumulation of an abnormal isoform of prion protein (PrP(Sc)), which are observed in neurons in the event of injury and disease. To understand the role of PrP(C) in the neuron in health and diseases, we have established an immortalized neuronal cell line HpL3-4 from primary hippocampal cells of prion protein (PrP) gene-deficient mice by using a retroviral vector encoding Simian Virus 40 Large T antigen (SV40 LTag). The HpL3-4 cells exhibit cell-type-specific proteins for the neuronal precursor lineage. Recently, this group and other groups have established PrP-deficient cell lines from many kinds of cell types including glia, fibroblasts and neuronal cells, which will have a broad range of applications in prion biology. In this review, we focus on recently obtained information about PrP functions and possible studies on prion infections using the PrPdeficient cell lines.     

Prion Pathogenesis is Independent of Caspase-12

The pathogenic mechanism(s) underlying neurodegenerative diseases associated with protein misfolding is unclear. Several studies have implicated ER stress pathways in neurodegenerative conditions, including prion disease, amyotrophic lateral sclerosis, Alzheimer's disease and many others. The ER stress response and up-regulation of ER stress-responsive chaperones is observed in the brains of patients affected with Creutzfeldt-Jacob disease and in mouse models of prion diseases. In particular, the processing of caspase-12, an ER-localized caspase, correlates with neuronal cell death in prion disease. However, the contribution of caspase-12 to neurodegeneration has not been directly addressed in vivo. We confirm that ER stress is induced and that caspase-12 is proteolytically processed in a murine model of infectious prion disease. To address the causality of caspase-12 in mediating infectious prion pathogenesis, we inoculated mice deficient in caspase-12 with prions. The survival, behavior, pathology and accumulation of proteinase K-resistant PrP are indistinguishable between caspase-12 knockout and control mice, suggesting that caspase-12 is not necessary for mediating the neurotoxic effects of prion protein misfolding.     

Genesis of mammalian prions: from non-infectious amyloid fibrils to a transmissible prion disease

The transmissible agent of prion disease consists of a prion protein in its abnormal, β-sheet rich state (PrP(Sc)), which is capable of replicating itself according to the template-assisted mechanism. This mechanism postulates that the folding pattern of a newly recruited polypeptide chain accurately reproduces that of a PrP(Sc) template. Here we report that authentic PrP(Sc) and transmissible prion disease can be generated de novo in wild type animals by recombinant PrP (rPrP) amyloid fibrils, which are structurally different from PrP(Sc) and lack any detectable PrP(Sc) particles. When induced by rPrP fibrils, a long silent stage that involved two serial passages preceded development of the clinical disease. Once emerged, the prion disease was characterized by unique clinical, neuropathological, and biochemical features. The long silent stage to the disease was accompanied by significant transformation in neuropathological properties and biochemical features of the proteinase K-resistant PrP material (PrPres) before authentic PrP(Sc) evolved. The current work illustrates that transmissible prion diseases can be induced by PrP structures different from that of authentic PrP(Sc) and suggests that a new mechanism different from the classical templating exists. This new mechanism designated as "deformed templating" postulates that a change in the PrP folding pattern from the one present in rPrP fibrils to an alternative specific for PrP(Sc) can occur. The current work provides important new insight into the mechanisms underlying genesis of the transmissible protein states and has numerous implications for understanding the etiology of neurodegenerative diseases.     

Mechanism of aggregation and membrane interactions of mammalian prion protein

The cellular prion protein (PrP^C), which is present ubiquitously in all mammalian neurons, is normally found to be linked to the cell membrane through a glycosylphosphatidylinositol (GPI) anchor. The conformational conversion of PrP^C into misfolded and aggregated forms is associated with transmissible neurodegenerative diseases known as prion diseases. The importance of different misfolded conformations in prion diseases, and the mechanism by which prion aggregates induce neurotoxicity remain poorly understood. Multiple studies have been shown that the toxicity of misfolded prion protein is directly correlated with its ability to interact with and perturb membranes. This review describes the current progress toward understanding prion protein misfolding and aggregation, as well as the interaction of prion protein aggregates with lipid membrane.     

Identification of an Intracellular Site of Prion Conversion

Prion diseases are fatal, neurodegenerative disorders in humans and animals and are characterized by the accumulation of an abnormally folded isoform of the cellular prion protein (PrP^C), denoted PrP^Sc, which represents the major component of infectious scrapie prions. Characterization of the mechanism of conversion of PrP^C into PrP^Sc and identification of the intracellular site where it occurs are among the most important questions in prion biology. Despite numerous efforts, both of these questions remain unsolved. We have quantitatively analyzed the distribution of PrP^C and PrP^Sc and measured PrP^Sc levels in different infected neuronal cell lines in which protein trafficking has been selectively impaired. Our data exclude roles for both early and late endosomes and identify the endosomal recycling compartment as the likely site of prion conversion. These findings represent a fundamental step towards understanding the cellular mechanism of prion conversion and will allow the development of new therapeutic approaches for prion diseases.     

Early Increase and Late Decrease of Purkinje Cell Dendritic Spine Density in Prion-Infected Organotypic Mouse Cerebellar Cultures

Prion diseases are infectious neurodegenerative diseases associated with the accumulation of protease-resistant prion protein, neuronal loss, spongiform change and astrogliosis. In the mouse model, the loss of dendritic spines is one of the earliest pathological changes observed in vivo, occurring 4–5 weeks after the first detection of protease-resistant prion protein in the brain. While there are cell culture models of prion infection, most do not recapitulate the neuropathology seen in vivo. Only the recently developed prion organotypic slice culture assay has been reported to undergo neuronal loss and the development of some aspects of prion pathology, namely small vacuolar degeneration and tubulovesicular bodies. Given the rapid replication of prions in this system, with protease-resistant prion protein detectable by 21 days, we investigated whether the dendritic spine loss and altered dendritic morphology seen in prion disease might also develop within the lifetime of this culture system. Indeed, six weeks after first detection of protease-resistant prion protein in tga20 mouse cerebellar slice cultures infected with RML prion strain, we found a statistically significant loss of Purkinje cell dendritic spines and altered dendritic morphology in infected cultures, analogous to that seen in vivo. In addition, we found a transient but statistically significant increase in Purkinje cell dendritic spine density during infection, at the time when protease-resistant prion protein was first detectable in culture. Our findings support the use of this slice culture system as one which recapitulates prion disease pathology and one which may facilitate study of the earliest stages of prion disease pathogenesis.     

Prion Pathogenesis is Independent of Caspase-12

The pathogenic mechanism(s) underlying neurodegenerative diseases associated with protein misfolding is unclear. Several studies have implicated ER stress pathways in neurodegenerative conditions, including prion disease, amyotrophic lateral sclerosis, Alzheimer''s disease and many others. The ER stress response and upregulation of ER stress-responsive chaperones is observed in the brains of patients affected with Creutzfeldt-Jacob disease and in mouse models of prion diseases. In particular, the processing of caspase-12, an ER-localized caspase, correlates with neuronal cell death in prion disease. However, the contribution of caspase-12 to neurodegeneration has not been directly addressed in vivo. We confirm that ER stress is induced and that caspase-12 is proteolytically processed in a murine model of infectious prion disease. To address the causality of caspase-12 in mediating infectious prion pathogenesis, we inoculated mice deficient in caspase-12 with prions. The survival, behavior, pathology and accumulation of proteinase K-resistant PrP are indistinguishable between caspase-12 knockout and control mice, suggesting that caspase-12 is not necessary for mediating the neurotoxic effects of prion protein misfolding.     

Globular and pre-fibrillar prion aggregates are toxic to neuronal cells and perturb their electrophysiology

Prion diseases are characterised at autopsy by neuronal loss and accumulation of amorphous protein aggregates and/or amyloid fibrils in the brains of humans and animals. These protein deposits result from the conversion of the cellular, mainly alpha-helical prion protein (PrP(C)) to the beta-sheet-rich isoform (PrP(Sc)). Although the pathogenic mechanism of prion diseases is not fully understood, it appears that protein aggregation is itself neurotoxic and not the product of cell death. The precise nature of the neurotoxic species and mechanism of cell death are yet to be determined, although recent studies with other amyloidogenic proteins suggest that ordered pre-fibrillar or oligomeric forms may be responsible for cellular dysfunction. In this study we have refolded recombinant prion protein (rPrP) to two distinct forms rich in beta-sheet structure with an intact disulphide bond. Here we report on the structural properties of globular aggregates and pre-fibrils of rPrP and show that both states are toxic to neuronal cells in culture. We show that exogenous rPrP aggregates are internalised by neuronal cells and found in the cytoplasm. We also measured the changes in electrophysiological properties of cultured neuronal cells on exposure to exogenous prion aggregates and discuss the implications of these findings.     

Hydrogen peroxide cleavage of the prion protein generates a fragment able to initiate polymerisation of full length prion protein

The prion protein is central to the disease pathogenesis of a variety of neurodegenerative diseases such as CJD. The protein is only able to initiate the disease process following post-translational modification. The main characteristic of this change is the ability of this altered isoform to polymerise. We wish to determine if altered cleavage of the protein could generate a protein fragment able to initiate polymerisation. During normal metabolic breakdown the protein is initially cleaved at a single site at around amino acid residue 111/112 in the mouse sequence. A second site before amino acid residue 90 has been postulated as an alternative cleavage point. We have provided evidence that hydrogen peroxide as low as 50 microM in the presence of copper, iron or manganese (but not nickel, magnesium or zinc) can cleave the recombinant protein near this site and requires a GXXH motif in the protein sequence. This reaction results in the production of 6 and 19 kDa fragments of the protein. This cleavage pattern occurs in prion proteins from different species (mouse, chicken and turtle) and is enhanced by modification of the octameric repeat region. The 19 kDa fragment produced by this reaction is protease sensitive. This fragment in a pure form caused the polymerisation of wild-type prion protein by a seeding mechanism. Therefore our results provide a possible mechanism by which altered cleavage of the prion protein could result in the kind of protein polymerisation associated with prion diseases.     

Parkin Overexpression Ameliorates PrP106–126-Induced Neurotoxicity via Enhanced Autophagy in N2a Cells

Transmissible spongiform encephalopathies (TSEs) are caused by the accumulation of the abnormal prion protein scrapie (PrP^Sc). Prion protein aggregation, misfolding, and cytotoxicity in the brain are the major causes of neuronal dysfunction and ultimate neurodegeneration in all TSEs. Parkin, an E3 ubiquitin ligase, has been studied extensively in all major protein misfolding aggregating diseases, especially Parkinson’s disease and Alzheimer’s disease, but the role of parkin in TSEs remains unknown. Here we investigated the role of parkin in a prion disease cell model in which neuroblastoma2a (N2a) cells were treated with prion peptide PrP106–126. We observed a gradual decrease in the soluble parkin level upon treatment with PrP106–126 in a time-dependent manner. Furthermore, endogenous parkin colocalized with FITC-tagged prion fragment106–126. Overexpression of parkin in N2a cells via transfection repressed apoptosis by enhancing autophagy. Parkin-overexpressing cells also showed reductions in apoptotic BAX translocation to the mitochondria and cytochrome c release to the cytosol, which ultimately inhibited activation of proapoptotic caspases. Taken together, our findings reveal a parkin-mediated cytoprotective mechanism against PrP106–126 toxicity, which is a novel potential therapeutic target for treating prion diseases.     

Prions and the proteasome

Prion diseases are fatal neurodegenerative disorders that include Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy in animals. They are unique in terms of their biology because they are caused by the conformational re-arrangement of a normal host-encoded prion protein, PrPC, to an abnormal infectious isoform, PrPSc. Currently the precise mechanism behind prion-mediated neurodegeneration remains unclear. It is hypothesised than an unknown toxic gain of function of PrPSc, or an intermediate oligomeric form, underlies neuronal death. Increasing evidence suggests a role for the ubiquitin proteasome system (UPS) in prion disease. Both wild-type PrPC and disease-associated PrP isoforms accumulate in cells after proteasome inhibition leading to increased cell death, and abnormal beta-sheet-rich PrP isoforms have been shown to inhibit the catalytic activity of the proteasome. Here we review potential interactions between prions and the proteasome outlining how the UPS may be implicated in prion-mediated neurodegeneration.