Axonal transport of the cellular prion protein is increased during axon regeneration

The cellular prion protein, PrPc, is a glycosylphosphatidylinositol-anchored cell surface glycoprotein and a protease-resistant conformer of the protein may be the infectious agent in transmissible spongiform encephalopathies. PrPc is localized on growing axons in vitro and along fibre bundles that contain elongating axons in developing and adult brain. To determine whether the growth state of axons influenced the expression and axonal transport of PrPc, we examined changes in the protein following post-traumatic regeneration in the hamster sciatic nerve. Our results show (1) that PrPc in nerve is significantly increased during nerve regeneration; (2) that this increase involves an increase in axonally transported PrPc; and (3) that the PrPc preferentially targeted for the newly formed portions of the regenerating axons consists of higher molecular weight glycoforms. These results raise the possibility that PrPc may play a role in the growth of axons in vivo, perhaps as an adhesion molecule interacting with the extracellular environment through specialized glycosylation.     

朊蛋白生理功能研究进展

朊蛋白作为一种高度保守的细胞膜糖蛋白,广泛分布于机体各组织器官,参与信号跨膜传导、细胞黏附、铜离子代谢、抗细胞凋亡、抗氧化应激等过程。近年来,随着对朊蛋白结构、生理功能、变构机制等的深入研究,对它的认识已不再局限于一种单纯的致病因子,朊蛋白在遗传进化、生理功能上所表现出的重要作用已成为新的研究热点。我们首先分析了朊蛋白的细胞定位、转运及组织分布,其次对朊蛋白在神经系统、肿瘤发生、胚胎发育过程中发挥的生理功能做简要介绍,最后对该蛋白的研究前景进行了展望。     

Interaction of Doppel with the full-length laminin receptor precursor protein

Doppel (Dpl) is a homolog of normal cellular prion protein (PrPc) with unknown functions. Ectopic expression of Dpl in the central nervous system (CNS) causes neurotoxicity and this effect is rescued by the expression of PrPc. However, the molecular basis for the protective effect of PrPc remains unclear. Using a yeast two-hybrid system, we showed that Dpl binds the full-length 37-kDa laminin receptor precursor protein (LRP), one of the receptors of PrPc. The interaction was also validated by immunoprecipitation and immunoblotting using transfected cell lines and in vivo derived tissues. Further mapping experiments showed that although the middle fragment containing residues 100-220 of LRP was able to interact with Dpl, deletion of the N-terminal domain of the full-length LRP abolished its interaction with Dpl. These results suggest that while both PrPc and Dpl interact with LRP, the domains that are involved in the binding are not the same. Our results may have implications for the molecular mechanisms of Dpl-PrPc antagonism and physiological roles of Dpl.     

Botulinum toxin's axonal transport from periphery to the spinal cord

Axonal transport of enzymatically active botulinum toxin A (BTX-A) from periphery to the CNS has been described in facial and trigeminal nerve, leading to cleavage of synaptosomal-associated protein 25 (SNAP-25) in central nuclei. Aim of present study was to examine the existence of axonal transport of peripherally applied BTX-A to spinal cord via sciatic nerve. We employed BTX-A-cleaved SNAP-25 immunohistochemistry of lumbar spinal cord after intramuscular and subcutaneous hind limb injections, and intraneural BTX-A sciatic nerve injections. Truncated SNAP-25 in ipsilateral spinal cord ventral horns and dorsal horns appeared after single peripheral BTX-A administrations, even at low intramuscular dose applied (5 U/kg). Cleaved SNAP-25 appearance in the spinal cord after BTX-A injection into the sciatic nerve was prevented by proximal intrasciatic injection of colchicine (5 mM, 2 μl). Cleaved SNAP-25 in ventral horn, using choline-acetyltransferase (ChAT) double labeling, was localized within cholinergic neurons. These results extend the recent findings on BTX-A retrograde axonal transport in facial and trigeminal nerve. Appearance of truncated SNAP-25 in spinal cord following low-dose peripheral BTX-A suggest that the axonal transport of BTX-A occurs commonly following peripheral application.     

PrPc on the road: trafficking of the cellular prion protein

The glycosylphosphatidylinositol (GPI)-anchored cellular prion protein (PrPc) has a fundamental role in prion diseases. Intracellular trafficking of PrPc is important in the generation of protease resistant PrP species but little is known of how endocytosis affects PrPc function. Here, we discuss recent experiments that have illuminated how PrPc is internalized and what are the possible destinations taken by the protein. Contrary to what would be expected for a GPI-anchored protein there is increasing evidence that clathrin-mediated endocytosis and classical endocytic organelles participate in PrPc trafficking. Moreover, the N-terminal domain of PrPc may be involved in sorting events that can direct the protein during its intracellular journey. Indeed, the concept that the GPI-anchor determines PrPc trafficking has been challenged. Cellular signaling can be triggered or be regulated by PrPc and we suggest that endocytosis of PrPc may influence signaling in several ways. Definition of the processes that participate in PrPc endocytosis and intracellular trafficking can have a major impact on our understanding of the mechanisms involved in PrPc function and conversion to protease resistant conformations.     

Cell-based immunotherapy of prion diseases by adoptive transfer of antigen-loaded dendritic cells or antigen-primed CD4+ T lymphocytes

Prion diseases are neurodegenerative conditions caused by the transconformation of a normal host glycoprotein, the cellular prion protein (PrPc) into a neurotoxic, self-aggregating conformer (PrPSc). TSEs are ineluctably fatal and no treatment is yet available. In principle, prion diseases could be attacked from different angles including: blocking conversion of PrPc into PrPSc, accelerating the clearance of amyloid deposits in peripheral tissues and brain, stopping prion progression in secondary lymphoid organs, reducing brain inflammation and promoting neuronal healing. There are many indications that adaptive and innate immunity might mediate those effects but so far, the achievements of immunointervention have not matched all expectations. Difficulties arise from the impossibility to diagnose TSE before substantial brain damage, poor accessibility of the CNS to immunological agents, deep immune tolerance to self-PrP, and short term effects of many immune interventions contrasting with the slow progression of TSEs. Here, we discuss two approaches, inspired from cancer immunotherapy, which might overcome some of those obstacles. One is vaccination with antigen-pulsed or antigen-transduced dendritic cells to bypass self-tolerance. The other one is the adoptive transfer of PrP-sensitized CD4+ T cells which can promote humoral, cell-mediated or regulatory responses, coordinate adaptive and innate immunity and have long lasting effects.     

Cellular prion protein is expressed in a subset of neuroendocrine cells of the rat gastrointestinal tract.

Prion diseases are believed to develop from the conformational change of normal cellular prion protein (PrPc) to a pathogenic isoform (PrPsc). PrPc is present in both the central nervous system and many peripheral tissues, although protein concentration is significantly lower in non-neuronal tissues. PrPc expression is essential for internalization and replication of the infectious agent. Several works have pointed to the gastrointestinal (GI) tract as the principal site of entry of PrPsc, but how passage through the GI mucosa occurs is not yet known. Here we studied PrPc expression using Western blot, RT-PCR, and immunohistochemistry in rat GI tract. PrPc mRNA and protein were detected in corpus, antrum, duodenum, and colon. Immunoreactivity was found in scattered cells of the GI epithelium. With double immunofluorescence, these cells have been identified as neuroendocrine cells. PrPc immunostaining was found in subsets of histamine, somatostatin (Som), ghrelin, gastrin (G), and serotonin (5HT) cells in stomach. In small and large bowel, PrPc cells co-localized with subpopulations of 5HT-, Som-, G-, and peptide YY-immunolabeled cells. Our results provide evidence for a possible and important role of endocrine cells in the internalization of PrPsc from gut lumen.     

Redox-regulation of intrinsic prion expression in multicellular prostate tumor spheroids

The cellular function of the intrinsic prion protein (PrPc) remains largely unknown. In the present study PrPc expression was investigated in multicellular prostate tumor spheroids and was correlated to the intracellular redox state as evaluated using the fluorescent dye 2'7'-dichlorodihydrofluorescein diacetate (H2DCFDA). In small tumor spheroids (diameter 100 +/- 20 microm) reactive oxygen species (ROS) levels were increased as compared with large (diameter 250 +/- 50 microm) spheroids. ROS generation was mediated by the mitochondrial respiratory chain and a NADPH oxidaselike enzyme, because carbonylcyanide-m-chlorophenylhydrazone (CCCP), rotenone, and diphenylene iodonium chloride (DPI) significantly reduced ROS levels. The elevated ROS were correlated to an increased expression of PrPc, Cu/Zn superoxide dismutase (SOD-1), and catalase in small as compared with large spheroids. In large tumor spheroids, PrPc was predominantly expressed in the peripheral cell layers and colocalized with SOD-1 and catalase. Raising intracellular ROS in large tumor spheroids by hydrogen peroxide, menadione, buthionine sulfoximine (BSO), and incubation in glutamine-reduced medium increased PrPc expression. In small spheroids PrPc was downregulated after incubation with the radical scavengers dehydroascorbate (DHA) and vitamin E. Our data indicate that PrPc expression in tumor spheroids is related to the intracellular redox state and may participate in antioxidative defense.     

Selective prion protein binding to synaptic components is modulated by oxidative and nitrosative changes induced by copper(II) and peroxynitrite in cholinergic synaptosomes, unveiling a role for calcineurin B and thioredoxin

Choline acetyltransferase (ChAT) and choline transport are decreased after nitrosative stress. ChAT activity is altered in scrapie-infected neurons, where oxidative stress develops. Cellular prion protein (PrPc) may play a neuroprotective function in participating in the redox control of neuronal environment and regulation of copper metabolism, a role impaired when PrPc is transformed into PrPSc in prion pathologies. The complex cross-talk between PrPc and cholinergic neurons was analyzed in vitro using peroxynitrite and Cu2+ treatments on nerve endings isolated from Torpedo marmorata, a model of the motoneuron pre-synaptic element. Specific interactions between solubilized synaptic components and recombinant ovine prion protein (PrPrec) could be demonstrated by Biacore technology. Peroxynitrite abolished this interaction in a concentration-dependent way and induced significant alterations of neuronal targets. Interaction was restored by prior addition of peroxynitrite trapping agents. Cu2+ (in the form of CuSO4) treatment of synaptosomes triggered a milder oxidative effect leading to a bell-shaped increase of PrPrec binding to synaptosomal components, counteracted by the natural thiol agents, glutathione and thioredoxin. Copper(II)-induced modifications of thiols in several neuronal proteins. A positive correlation was observed between PrPrec binding and immunoreactive changes for calcineurin B and its partners, suggesting a synergy between calcineurin complex and PrP for copper regulation.     

Cell-based immunotherapy of prion diseases by adoptive transfer of antigen-loaded dendritic cells or antigen-primed CD4+ T lymphocytes

Prion diseases are neurodegenerative conditions caused by the transconformation of a normal host glycoprotein, the cellular prion protein (PrPc) into a neurotoxic, self-aggregating conformer (PrPSc). TSEs are ineluctably fatal and no treatment is yet available. In principle, prion diseases could be attacked from different angles including: blocking conversion of PrPc into PrPSc, accelerating the clearance of amyloid deposits in peripheral tissues and brain, stopping prion progression in secondary lymphoid organs, reducing brain inflammation and promoting neuronal healing. There are many indications that adaptive and innate immunity might mediate those effects but, so far, the achievements of immunointervention have not matched all expectations. Difficulties arise from the impossibility to diagnose TSE before substantial brain damage, poor accessibility of the CNS to immunological agents, deep immune tolerance to self-PrP and short term effects of many immune interventions contrasting with the slow progression of TSEs. Here, we discuss two approaches, inspired from cancer immunotherapy, which might overcome some of those obstacles. One is vaccination with antigen-pulsed or antigen-transduced dendritic cells to bypass self-tolerance. The other one is the adoptive transfer of PrP-sensitized CD4⁺ T cells which can promote humoral, cellmediated or regulatory responses, coordinate adaptive and innate immunity and have long lasting effects.Key words: prion, TSE, dendritic cells, CD4⁺ T cells, cellular immunotherapy, vaccination, adoptive cell transfer     

The cellular prion protein: a new partner of the lectin CBP70 in the nucleus of NB4 human promyelocytic leukemia cells.

Prion diseases are characterized by the presence of an abnormal isoform of the cellular prion protein (PrPc) whose physiological role still remains elusive. To better understand the function of PrPc, it is important to identify the different subcellular localization(s) of the protein and the different partners with which it might be associated. In this context, the PrPc-lectins interactions are investigated because PrPc is a sialoglycoprotein which can react with lectins which are carbohydrate-binding proteins. We have previously characterized a nuclear lectin CBP70 able to recognize N-acetyl-beta-D-glucosamine residues in HL60 cells. Using confocal immunofluorescence, flow-cytofluorometry, and Western-blotting, we have found that PrPc is expressed in the nucleus of the NB4 human promyelocytic leukemia cell line. It was also found that the lectin CBP70 is localized in NB4 cell nuclei. Moreover, several approaches revealed that PrPc and CBP70 are colocalized in the nucleus. Immunoprecipitation experiments showed that these proteins are coprecipitated and interact via a sugar-dependent binding moiety. In conclusion, PrPc and CBP70 are colocalized in the nuclear compartment of NB4 cells and this interaction may be important to better understand the biological function and possibly the conversion process of PrPc into its pathological form (PrPsc).     

Isolation of bovine follicular dendritic cells allows the demonstration of a particular cellular prion protein

As interaction of cellular prion protein (PrPc) and the infectious agent (PrPres) appears to be a crucial pathogenic step promoted by homology, variation in PrPc isoforms on bovine immune cells may explain the absence of infectivity in most bovine lymph organs. In this study, we examined PrPc expression in bovine lymph organs (tonsils and lymph nodes) and on isolated follicular dendritic cells (FDCs). We used a panel of different monoclonal antibodies (MoAbs) raised against different epitopes of prion protein. Two MoAbs recognise amino acids 79-92 (SAF 34 and SAF 32 Mo-Abs); the 6H4 antibody reacts with a specific peptide comprising the 144-152 amino acids, and the 12F10 MoAb recognises the sequence 142-160. After immunolabelling of frozen sections of lymph organs with 6H4 or 12F10 MoAbs, we detected cellular prion protein in germinal centres. However, using the SAF 34 or SAF 32 antibodies, PrPc was revealed outside the lymphoid tissues. No PrPc was observed in the germinal centres. Therefore, we adapted the method of FDC isolation, making it suitable for the study of PrPc expression on their surface. Using electron microscopy, the presence of PrPc on the surface of FDCs was demonstrated only with 6H4 MoAb. These results suggest that bovine follicular dendritic cells express a particular form of prion protein. Either the N-terminal part of PrPc is cleaved or the accessibility of the specific epitope (79-92) of SAF 34 MoAb is abolished by interaction with other molecules. This particular isoform of PrPc on bovine FDCs might be related to the apparent absence of infectivity in lymph organs in cattle affected by bovine spongiform encephalopathy.     

Mapping the functional domain of the prion protein.

Prion diseases such as Creutzfeldt-Jakob disease are possibly caused by the conversion of a normal cellular glycoprotein, the prion protein (PrPc) into an abnormal isoform (PrPSc). The process that causes this conversion is unknown, but to understand it requires a detailed insight into the normal activity of PrPc. It has become accepted from results of numerous studies that PrPc is a Cu-binding protein and that its normal function requires Cu. Further work has suggested that PrPc is an antioxidant with an activity like that of a superoxide dismutase. We have shown in this investigation that this activity is optimal for the whole protein and that deletion of parts of the protein reduce or abolish this activity. The protein therefore contains an active domain requiring certain regions such as the Cu-binding octameric repeat region and the hydrophobic core. These regions show high evolutionary conservation fitting with the idea that they are important to the active domain of the protein.     

Metallic prions

Prion diseases, also referred to as transmissible spongiform encephalopathies, are characterized by the deposition of an abnormal isoform of the prion protein in the brain. However, this aggregated, fibrillar, amyloid protein, termed PrPSc, is an altered conformer of a normal brain glycoprotein, PrPc. Understanding the nature of the normal cellular isoform of the prion protein is considered essential to understanding the conversion process that generates PrPSc. To this end much work has focused on elucidation of the normal function and activity of PrPc. Substantial evidence supports the notion that PrPc is a copper-binding protein. In conversion to the abnormal isoform, this Cu-binding activity is lost. Instead, there are some suggestions that the protein might bind other metals such as Mn or Zn. PrPc functions currently under investigation include the possibility that the protein is involved in signal transduction, cell adhesion, Cu transport and resistance to oxidative stress. Of these possibilities, only a role in Cu transport and its action as an antioxidant take into consideration PrPc's Cu-binding capacity. There are also more published data supporting these two functions. There is strong evidence that during the course of prion disease, there is a loss of function of the prion protein. This manifests as a change in metal balance in the brain and other organs and substantial oxidative damage throughout the brain. Thus prions and metals have become tightly linked in the quest to understand the nature of transmissible spongiform encephalopathies.     

Limited Trafficking of a Neurotropic Virus Through Inefficient Retrograde Axonal Transport and the Type I Interferon Response

Poliovirus is an enteric virus that rarely invades the human central nervous system (CNS). To identify barriers limiting poliovirus spread from the periphery to CNS, we monitored trafficking of 10 marked viruses. After oral inoculation of susceptible mice, poliovirus was present in peripheral neurons, including vagus and sciatic nerves. To model viral trafficking in peripheral neurons, we intramuscularly injected mice with poliovirus, which follows a muscle–sciatic nerve–spinal cord–brain route. Only 20% of the poliovirus population successfully moved from muscle to brain, and three barriers limiting viral trafficking were identified. First, using light-sensitive viruses, we found limited viral replication in peripheral neurons. Second, retrograde axonal transport of poliovirus in peripheral neurons was inefficient; however, the efficiency was increased upon muscle damage, which also increased the transport efficiency of a non-viral neural tracer, wheat germ agglutinin. Third, using susceptible interferon (IFN) α/β receptor knockout mice, we demonstrated that the IFN response limited viral movement from the periphery to the brain. Surprisingly, the retrograde axonal transport barrier was equivalent in strength to the IFN barrier. Illustrating the importance of barriers created by the IFN response and inefficient axonal transport, IFN α/β receptor knockout mice with muscle damage permitted 80% of the viral population to access the brain, and succumbed to disease three times faster than mice with intact barriers. These results suggest that multiple separate barriers limit poliovirus trafficking from peripheral neurons to the CNS, possibly explaining the rare incidence of paralytic poliomyelitis. This study identifies inefficient axonal transport as a substantial barrier to poliovirus trafficking in peripheral neurons, which may limit CNS access for other viruses.     

Physiopathologic implications of the structural and functional domains of the prion protein

Prion diseases are invariably fatal neurodegenerative disorders affecting man and various animal species. A large body of evidence supports the notion that the causative agent of these diseases is the prion, which, devoid of nucleic acids, is composed largely, if not entirely, of a conformationally abnormal isoform (PrP(Sc) of the cellular prion protein (PrPc). PrPc is a highly conserved and ubiquitously expressed sialoglycoprotein, the normal function of which is, however, still ill defined. Several modules have been recognised in PrPc structure. Their extensive analysis by different experimental approaches, including transgenic animal models, has allowed to assigning to several modules a putative role in PrPc physiology. Concurrently, it has underscored the possibility that alteration of specific domains may determine the switching from a beneficial role of PrPc into one that becomes detrimental to neurons, and/or promote the conversion of PrPc into the pathogenic PrP(Sc) conformer.     

Mutagenesis identifies a conserved tyrosine residue important for the activity of uroporphyrinogen III synthase from Anacystis nidulans

Cellular prion (PrPc) is a plasma membrane glycosyphosphatidylinositol-anchored protein present in neurons but also in other cell types. Protein conservation among species suggests that PrPc may have important physiological roles. Cellular and molecular approaches have established several novel features of the regulation of PrPc expression, cellular trafficking as well as its participation in copper uptake, protection against oxidative stress, cell adhesion, differentiation, signaling and cell survival. It is therefore likely that PrPc plays pleiotropic roles in neuronal and non-neuronal cells, and as such the loss of function of PrPc may be an important component of various diseases.     

Distribution and submicroscopic immunogold localization of cellular prion protein (PrPc) in extracerebral tissues

In transmissible spongiform encephalopathies (TSE), such as scrapie in animals and Creutzfeldt-Jakob disease in humans, the central event is the conversion of a host-encoded amyloidogenic protein (PrPc) into an abnormal isoform (PrPsc) that accumulates as amyloid in TSE brain. PrPc is a membrane sialoglycoprotein synthesized in the central nervous system and elsewhere. We have examined the ultrastructural localization of PrPc in numerous hamster and some human extracerebral tissues, by means of a post-embedding electron-microscopic method combined with immunogold labeling. In stomach, intestine, lung, and kidney from hamsters, and in stomach, kidney, and spleen from humans, immunogold labeling specific for PrPc is observed on various cellular substructures related to secretory pathways: Golgi apparatus, secretory globules, and plasma membrane. In mucous epithelial cells of stomach and intestine, PrPc appears to be concentrated in secretory globules, suggesting a role for PrPc in the secretory function of the digestive tract. The secretory aspect of PrPc may be a key to understanding the physiopathological mechanisms underlying TSE.