Degradation Of PrP Research Articles

Carbon Microspheres Composite PbO2 Anode to Enhance Electrode Activity for Degradation of Isopropylantipyrine: Kinetics and Mechanism

An innovative CMS-PbO2 electrode was fabricated by combining hydrothermally synthesized carbon microspheres (CMS) on a PbO2 electrode by electrodeposition. Using the CMS-PbO2 electrode, the main factors affecting Isopropylantipyrine (PRP) degradation were studied. Under optimum process conditions, the concentration of PRP was 50 mg l−1, the applied current density was 30 mA cm−2, the electrolyte (Na2SO4) concentration was 0.1 mol l−1, and pH was 7. The PRP degradation rate reached 100%, and chemical oxygen demand (COD) removal rate reached 43.42% after 120 min of electrochemical oxidation. Using field-emission scanning electron microscopy, X-ray diffraction, X-ray photoelectron spectroscopy, cyclic voltammetry, linear sweep voltammetry, and electrochemical impedance spectroscopy, the electrochemical performance of the two electrodes was discussed. The composite of carbon microspheres successfully improved the electrochemical activity of the electrode and its conductivity. Furthermore, the ability of the two electrodes to generate hydroxyl radicals was compared and the possible degradation pathway of PRP was considered. In addition, electrode stability and safety were evaluated by accelerated lifetime experiments and detection of lead ions in solution after electrochemical oxidation. The CMS-electrode was more stable and safer than the PbO2 electrode. The CMS-PbO2 electrode provides a new strategy for the treatment of pharmaceutical wastewater.

Degradation behaviors of Isopropylphenazone and Aminopyrine and their genetic toxicity variations during UV/chloramine treatment

Combination of ultraviolet and chloramine (i.e., UV/chloramine) treatment has been attracting increasingly attention in recent years due to its high efficiency in removing trace organic contaminants. This study investigated the degradation behaviors of two pyrazolone pharmaceuticals (i.e., Isopropyl phenazone (PRP) and Aminopyrine (AMP)) and their genetic toxicity variations during UV/chloramine treatment. The results showed that chloramine could hardly degrade PRP and AMP, while UV/chloramine greatly increased the observed first-order rate constant (kobs) of PRP and AMP degradation. The quenching and probe experiments illustrated that the reactive chlorine species (RCS) contributed dominantly to PRP removal, and hydroxyl radical (HO•) was the major contributor to the degradation of AMP, while the reactive amine radicals (RNS) could hardly degrade them. The overall degradation rates of PRP and AMP decreased as pH increased from 6.5 to 10. The kobs of PRP and AMP increased along with NH2Cl dosage increasing and reached a plateau at higher concentrations (0.2–0.5 mM). The present background carbonate (HCO3−, 1–10 mM), chloride (Cl−, 1–10 mM) and natural organic matter (NOM, 5-10 mg-C L−1) exhibited inhibition impacts on PRP and AMP degradation. In addition, the intermediates/products of PRP and AMP were identified and their general degradation pathways were proposed to be hydroxylation, deacetylation, and dephenylization. Specifically, Cl-substitution was inferred during PRP degradation, while demethylation in tertiary amine group was only observed in AMP degradation. These mechanisms including the main reactive sites of PRP and AMP were further confirmed by the frontier orbitals calculation. Moreover, the results of the genetic toxicity according to the micronucleus test of Viciafaba root tip indicated that UV/chloramine treatment could partially reduce the genetic toxicity of PRP and AMP.

Ubiquitin-specific protease 14 modulates degradation of cellular prion protein.

Prion diseases are fatal neurodegenerative disorders characterized by the accumulation of prion protein (PrPC). To date, there is no effective treatment for the disease. The accumulated PrP, termed PrPSc, forms amyloid fibrils and could be infectious. It has been suggested that PrPSc is abnormally folded and resistant to proteolytic degradation, and also inhibits proteasomal functions in infected cells, thereby inducing neuronal death. Recent work indicates that the ubiquitin-proteasome system is involved in quality control of PrPC. To reveal the significance of prion protein ubiqitination, we focused on ubiquitin-specific protease 14 (USP14), a deubiqutinating enzyme that catalyzes trimming of polyubiquitin chains and plays a role in regulation of proteasomal processes. Results from the present study showed that treatment with a selective inhibitor of USP14 reduced PrPC, as well as PrPSc, levels in prion-infected neuronal cells. Overexpression of the dominant negative mutant form of USP14 reduced PrPSc, whereas wildtype USP14 increased PrPSc in prion-infected cells. These results suggest that USP14 prevents degradation of both normal and abnormal PrP. Collectively, a better understanding about the regulation of PrPSc clearance caused by USP14 might contribute greatly to the development of therapeutic strategies for prion diseases.

Pyroglutamyl-N-terminal prion protein fragments in sheep brain following the development of transmissible spongiform encephalopathies

Protein misfolding, protein aggregation and disruption to cellular proteostasis are key processes in the propagation of disease and, in some progressive neurodegenerative diseases of the central nervous system, the misfolded protein can act as a self-replicating template or prion converting its normal isoform into a misfolded copy of itself. We have investigated the sheep transmissible spongiform encephalopathy, scrapie, and developed a multiple selected reaction monitoring (mSRM) mass spectrometry assay to quantify brain peptides representing the “ragged” N-terminus and the core of ovine prion protein (PrPSc) by using Q-Tof mass spectrometry. This allowed us to identify pyroglutamylated N-terminal fragments of PrPSc at residues 86, 95 and 101, and establish that these fragments were likely to be the result of in vivo processes. We found that the ratios of pyroglutamylated PrPSc fragments were different in sheep of different breeds and geographical origin, and our expanded ovine PrPSc assay was able to determine the ratio and allotypes of PrP accumulating in diseased brain of PrP heterozygous sheep; it also revealed significant differences between N-terminal amino acid profiles (N-TAAPs) in other types of ovine prion disease, CH1641 scrapie and ovine BSE. Variable rates of PrP misfolding, aggregation and degradation are the likely basis for phenotypic (or strain) differences in prion-affected animals and our mass spectrometry-based approach allows the simultaneous investigation of factors such as post-translational modification (pyroglutamyl formation), conformation (by N-TAAP analysis) and amino-acid polymorphisms (allotype ratio) which affect the kinetics of these proteostatic processes.

Prion degradation pathways: Potential for therapeutic intervention.

Prion diseases are fatal neurodegenerative disorders. Pathology is closely linked to the misfolding of native cellular PrPC into the disease-associated form PrPSc that accumulates in the brain as disease progresses. Although treatments have yet to be developed, strategies aimed at stimulating the degradation of PrPSc have shown efficacy in experimental models of prion disease. Here, we describe the cellular pathways that mediate PrPSc degradation and review possible targets for therapeutic intervention. This article is part of a Special Issue entitled ‘Neuronal Protein’.

Comparison of the Anti-Prion Mechanism of Four Different Anti-Prion Compounds, Anti-PrP Monoclonal Antibody 44B1, Pentosan Polysulfate, Chlorpromazine, and U18666A, in Prion-Infected Mouse Neuroblastoma Cells

Molecules that inhibit the formation of an abnormal isoform of prion protein (PrPSc) in prion-infected cells are candidate therapeutic agents for prion diseases. Understanding how these molecules inhibit PrPSc formation provides logical basis for proper evaluation of their therapeutic potential. In this study, we extensively analyzed the effects of the anti-PrP monoclonal antibody (mAb) 44B1, pentosan polysulfate (PPS), chlorpromazine (CPZ) and U18666A on the intracellular dynamics of a cellular isoform of prion protein (PrPC) and PrPSc in prion-infected mouse neuroblastoma cells to re-evaluate the effects of those agents. MAb 44B1 and PPS rapidly reduced PrPSc levels without altering intracellular distribution of PrPSc. PPS did not change the distribution and levels of PrPC, whereas mAb 44B1 appeared to inhibit the trafficking of cell surface PrPC to organelles in the endocytic-recycling pathway that are thought to be one of the sites for PrPSc formation. In contrast, CPZ and U18666A initiated the redistribution of PrPSc from organelles in the endocytic-recycling pathway to late endosomes/lysosomes without apparent changes in the distribution of PrPC. The inhibition of lysosomal function by monensin or bafilomycin A1 after the occurrence of PrPSc redistribution by CPZ or U18666A partly antagonized PrPSc degradation, suggesting that the transfer of PrPSc to late endosomes/lysosomes, possibly via alteration of the membrane trafficking machinery of cells, leads to PrPSc degradation. This study revealed that precise analysis of the intracellular dynamics of PrPC and PrPSc provides important information for understanding the mechanism of anti-prion agents.

Biodegradation of prions in compost.

Composting may serve as a practical and economical means of disposing of specified risk materials (SRM) or animal mortalities potentially infected with prion diseases (transmissible spongiform encephalopathies, TSE). Our study investigated the degradation of prions associated with scrapie (PrP(263K)), chronic waste disease (PrP(CWD)), and bovine spongiform encephalopathy (PrP(BSE)) in lab-scale composters and PrP(263K) in field-scale compost piles. Western blotting (WB) indicated that PrP(263K), PrP(CWD), and PrP(BSE) were reduced by at least 2 log10, 1-2 log10, and 1 log10 after 28 days of lab-scale composting, respectively. Further analysis using protein misfolding cyclic amplification (PMCA) confirmed a reduction of 2 log10 in PrP(263K) and 3 log10 in PrP(CWD). Enrichment for proteolytic microorganisms through the addition of feather keratin to compost enhanced degradation of PrP(263K) and PrP(CWD). For field-scale composting, stainless steel beads coated with PrP(263K) were exposed to compost conditions and removed periodically for bioassays in Syrian hamsters. After 230 days of composting, only one in five hamsters succumbed to TSE disease, suggesting at least a 4.8 log10 reduction in PrP(263K) infectivity. Our findings show that composting reduces PrP(TSE), resulting in one 50% infectious dose (ID50) remaining in every 5600 kg of final compost for land application. With these considerations, composting may be a viable method for SRM disposal.

Ubiquitin Ligase gp78 Targets Unglycosylated Prion Protein PrP for Ubiquitylation and Degradation

Prion protein PrP is a central player in several devastating neurodegenerative disorders, including mad cow disease and Creutzfeltd-Jacob disease. Conformational alteration of PrP into an aggregation-prone infectious form PrPSc can trigger pathogenic events. How levels of PrP are regulated is poorly understood. Human PrP is known to be degraded by the proteasome, but the specific proteolytic pathway responsible for PrP destruction remains elusive. Here, we demonstrate that the ubiquitin ligase gp78, known for its role in protein quality control, is critical for unglycosylated PrP ubiquitylation and degradation. Furthermore, C-terminal sequences of PrP protein are crucial for its ubiquitylation and degradation. Our study reveals the first ubiquitin ligase specifically involved in prion protein PrP degradation and PrP sequences crucial for its turnover. Our data may lead to a new avenue to control PrP level and pathogenesis.

Human Tonsil-Derived Follicular Dendritic-Like Cells are Refractory to Human Prion Infection in Vitro and Traffic Disease-Associated Prion Protein to Lysosomes

The molecular mechanisms involved in human cellular susceptibility to prion infection remain poorly defined. This is due, in part, to the absence of any well characterized and relevant cultured human cells susceptible to infection with human prions, such as those involved in Creutzfeldt-Jakob disease. In variant Creutzfeldt-Jakob disease, prion replication is thought to occur first in the lymphoreticular system and then spread into the brain. We have, therefore, examined the susceptibility of a human tonsil-derived follicular dendritic cell-like cell line (HK) to prion infection. HK cells were found to display a readily detectable, time-dependent increase in cell-associated abnormal prion protein (PrP(TSE)) when exposed to medium spiked with Creutzfeldt-Jakob disease brain homogenate, resulting in a coarse granular perinuclear PrP(TSE) staining pattern. Despite their high level of cellular prion protein expression, HK cells failed to support infection, as judged by longer term maintenance of PrP(TSE) accumulation. Colocalization studies revealed that exposure of HK cells to brain homogenate resulted in increased numbers of detectable lysosomes and that these structures immunostained intensely for PrP(TSE) after exposure to Creutzfeldt-Jakob disease brain homogenate. Our data suggest that human follicular dendritic-like cells and perhaps other human cell types are able to avoid prion infection by efficient lysosomal degradation of PrP(TSE).

Proteolysis of abnormal prion protein with a thermostable protease from Thermococcus kodakarensis KOD1

The abnormal prion protein (scrapie-associated prion protein, PrP(Sc)) is considered to be included in the group of infectious agents of transmissible spongiform encephalopathies. Since PrP(Sc) is highly resistant to normal sterilization procedures, the decontamination of PrP(Sc) is a significant public health issue. In the present study, a hyperthermostable protease, Tk-subtilisin, was used to degrade PrP(Sc). Although PrP(Sc) is known to be resistant toward proteolytic enzymes, Tk-subtilisin was able to degrade PrP(Sc) under extreme conditions. The level of PrP(Sc) in brain homogenates was found to decrease significantly in vitro following Tk-subtilisin treatment at 100°C, whereas some protease-resistant fractions remain after proteinase K treatment. Rather small amounts of Tk-subtilisin (0.3U) were required to degrade PrP(Sc) at 100°C and pH8.0. In addition, Tk-subtilisin was observed to degrade PrP(Sc) in the presence of sodium dodecyl sulfate or other industrial surfactants. Although several proteases degrading PrP(Sc) have been reported, practical decontamination procedures using enzymes are not available. This report aims to provide basic information for the practical use of a proteolytic enzyme for PrP(Sc) degradation.

The effects of lysosomal and proteasomal inhibitors on abnormal forms of prion protein degradation in murine macrophages

It has been reported that macrophages degrade infectious forms of prion protein (PrP(Sc) ). In order to investigate the mechanisms underlying PrP(Sc) degradation in macrophages, the effects of lysosomal and proteasomal inhibitors on macrophage cell lines which were incubated with scrapie-affected brain homogenate were studied. PrP(Sc) degradation was inhibited in the presence of both proteasomal and lysosomal inhibitors. Indirect fluorescence assays to determine the cellular localization of PrP(Sc) were undertaken. PrP(Sc) colocalized with the lysosomal membrane protein Lamp-1 and ubiquitin, a protein that is related to the proteasome. The present data indicate that macrophages might degrade PrP(Sc) via the lysosomal and proteasomal pathways.

Manganese Upregulates Cellular Prion Protein and Contributes to Altered Stabilization and Proteolysis: Relevance to Role of Metals in Pathogenesis of Prion Disease

Prion diseases are fatal neurodegenerative diseases resulting from misfolding of normal cellular prion (PrP(C)) into an abnormal form of scrapie prion (PrP(Sc)). The cellular mechanisms underlying the misfolding of PrP(C) are not well understood. Since cellular prion proteins harbor divalent metal-binding sites in the N-terminal region, we examined the effect of manganese on PrP(C) processing in in vitro models of prion disease. Exposure to manganese significantly increased PrP(C) levels both in cytosolic and in membrane-rich fractions in a time-dependent manner. Manganese-induced PrP(C) upregulation was independent of messenger RNA transcription or stability. Additionally, manganese treatment did not alter the PrP(C) degradation by either proteasomal or lysosomal pathways. Interestingly, pulse-chase analysis showed that the PrP(C) turnover rate was significantly altered with manganese treatment, indicating increased stability of PrP(C) with the metal exposure. Limited proteolysis studies with proteinase-K further supported that manganese increases the stability of PrP(C). Incubation of mouse brain slice cultures with manganese also resulted in increased prion protein levels and higher intracellular manganese accumulation. Furthermore, exposure of manganese to an infectious prion cell model, mouse Rocky Mountain Laboratory-infected CAD5 cells, significantly increased prion protein levels. Collectively, our results demonstrate for the first time that divalent metal manganese can alter the stability of prion proteins and suggest that manganese-induced stabilization of prion protein may play a role in prion protein misfolding and prion disease pathogenesis.

Lithium induces clearance of protease resistant prion protein in prion‐infected cells by induction of autophagy

Lithium is used for several decades to treat manic-depressive illness (bipolar affective disorder). Recently, it was found that lithium induces autophagy, thereby promoting the clearance of mutant huntingtin and alpha-synucleins in experimental systems. We show here for the first time that lithium significantly reduces the amount of pathological prion protein (PrP(Sc)) in prion-infected neuronal and non-neuronal cultured cells by inducing autophagy. Treatment of prion-infected cells with 3-methyladenine, a potent inhibitor of autophagy, counteracted the anti-prion effect of lithium, demonstrating that induction of autophagy mediates degradation of PrP(Sc). Co-treatment with lithium and rapamycin, a drug widely used to induce autophagy, had an additive effect on PrP(Sc) clearance compared to treatment with either drug alone. In addition, we provide evidence that the ability to reduce PrP(Sc) and to induce autophagy is common for diverse lithium compounds, not only for the drug lithium chloride, usually administered in clinical therapy. Furthermore, we show here that besides reduction of PrP(Sc)-aggregates, lithium-induced autophagy also slightly reduces the levels of cellular prion protein. Limiting the substrate available for conversion of cellular prion protein into PrP(Sc) may provide an additional mechanism for reduction of PrP(Sc) by lithium-induced autophagy.

The effect of Fenton reaction on protease-resistant prion protein (PrP Sc) degradation and scrapie infectivity

In prion diseases, metal imbalances in brain and/or metal substitutions for copper in prion protein suggest that metal-catalyzed oxidation (MCO) and oxidative stress may affect cellular function and accumulation of protease-resistant prion protein (PrP Sc). We examined the effect of metal-induced oxidative stress by Fenton reaction on prion protein with regard to its degradation, insolubility, and infectivity. Precipitation and insolubility of prion protein were induced by Fenton reaction in scrapie-infected brain homogenate. Results showed an increase in hydroxylation products (thiobarbituric acid reactive substances; TBARS) and a decrease of ferrous ion (Fe 2+) levels after Fenton reaction. Efficiency of metal-induced oxidation was higher for Fe 2+ than Mn 2+. Compared to untreated samples, there was increased susceptibility to proteolytic degradation of PrP Sc after treatment with 3.12–12.5 mM Fe 2+–Mn 2+/H 2O 2. Interestingly, we observed that Fenton reaction could extend incubation periods, indicating a decrease in scrapie infectivity. These results suggest that PrP Sc hydroxylation and degradation may affect PrP conversion and the pathogenesis of prion diseases.

Synthetic fibril peptide promotes clearance of scrapie prion protein by lysosomal degradation

Transmissible spongiform encephalopathies are infectious and neurodegenerative disorders that cause neural deposition of aggregates of the disease-associated form of PrP(Sc). PrP(Sc) reproduces by recruiting and converting the cellular PrP(C), and ScN2a cells support PrP(Sc) propagation. We found that incubation of ScN2a cells with a fibril peptide named P9, which comprises an intrinsic sequence of residues 167-184 of mouse PrP(C), significantly reduced the amount of PrP(Sc) in 24 hr. P9 did not affect the rates of synthesis and degradation of PrP(C). Interestingly, immunofluorescence analysis showed that the incubation of ScN2a cells with P9 induced colocalization of the accumulation of PrP with cathepsin D-positive compartments, whereas the accumulation of PrP in the cells without P9 colocalized mainly with lysosomal associated membrane proteins (LAMP)-1-positive compartments but rarely with cathepsin D-positive compartments in perinuclear regions. Lysosomal enzyme inhibitors attenuated the anti-PrP(Sc) activity; however, a proteasome inhibitor did not impair P9 activity. In addition, P9 neither promoted the ubiquitination of cellular proteins nor caused the accumulation of LC3-II, a biochemical marker of autophagy. These results indicate that P9 promotes PrP(Sc) redistribution from late endosomes to lysosomes, thereby attaining PrP(Sc) degradation.

CpG and LPS can interfere negatively with prion clearance in macrophage and microglial cells

Cells of the innate immune system play important roles in the progression of prion disease after peripheral infection. It has been found in vivo and in vitro that the expression of the cellular prion protein (PrP(c)) is up-regulated on stimulation of immune cells, also indicating the functional importance of PrP(c) in the immune system. The aim of our study was to investigate the impact of cytosine-phosphate-guanosine- and lipopolysaccharide-induced PrP(c) up-regulation on the uptake and processing of the pathological prion protein (PrP(Sc)) in phagocytic innate immune cells. For this purpose, we challenged the macrophage cell line J774, the microglial cell line BV-2 and primary bone marrow-derived macrophages in a resting or stimulated state with various prion strains, and monitored the uptake and clearance of PrP(Sc). Interestingly, stimulation led either to a transient increase in the level of PrP(Sc) relative to unstimulated cells or to a decelerated degradation of PrP(Sc). These features were dependent on cell type and prion strain. Our data indicate that the stimulation of innate immune cells may be able to support transient prion propagation, possibly explained by an increased PrP(c) cell surface expression in stimulated cells. We suggest that stimulation of innate immune cells can lead to an imbalance between the propagation and degradation of PrP(Sc).

Mechanistic insights into the cure of prion disease by novel antiprion compounds.

Prion diseases are fatal neurodegenerative disorders. Identification of possible therapeutic tools is important in the search for a potential treatment for these diseases. Congo red is an azo dye that has been used for many years to detect abnormal prion protein in the brains of diseased patients or animals. Congo red has little therapeutic potential for the treatment of these diseases due to toxicity and poor permeation of the blood-brain barrier. We have prepared two Congo red derivatives, designed without these liabilities, with potent activity in cellular models of prion disease. One of these compounds cured cells of the transmissible agent. The mechanism of action of these compounds is possibly multifactorial. The high affinity of Congo red derivatives, including compounds that are ineffective and are effective at the cure of prion disease, for abnormally folded prion protein suggests that the amyloidophylic property of these derivatives is not as critical to the mechanism of action as other effects. Congo red derivatives that are effective at the cure of prion disease increased the degradation of abnormal PrP by the proteasome. Therefore, the principal mechanism of action of the Congo red analogues was to prevent inhibition of proteasomal activity by PrPSc.

Prions and retroviruses: an endosomal rendezvous?

The past two decades have seen the emergence of endocytic pathways as highly regulated systems for the sorting, selective degradation and recycling of nearly all cell‐surface membrane proteins. At the centre of these pathways are complex endosomal structures known as multivesicular bodies (MVBs) that house intraluminal vesicles (ILVs) formed by invagination and budding from the limiting membrane (Fig 1A). The ILVs contain membrane proteins from the Golgi complex and cell surface that are destined typically for degradation after the fusion of MVBs with lysosomes. This constitutive fate of ILVs is universal to eukaryotes and represents one of the main pathways for membrane protein degradation (Katzmann et al , 2002). Figure 1. Protein transport in the endo‐lysosomal pathways. ( A ) Topological relationships of the multivesicular body (MVB). Different types of cell‐surface proteins (indicated by blue and red molecules) can be internalized and transported by various routes (arrows) to endosomal compartments. The endosomal compartment is known as an MVB when its lumen contains intraluminal vesicles (ILVs) that are formed by budding from the limiting membrane. If the MVB fuses with the plasma membrane, ILVs are released into the extracellular space and become known as exosomes. ( B ) Transport into and out of the multivesicular body (MVB). Pathways linking the MVB with other compartments, such as the early endosome (EE), Golgi, lysosome and plasma membrane, are all regulated in a substrate‐specific and cell‐type‐specific manner to control the localization and fate of various membrane proteins (blue, red and green molecules). Alterations of these transport pathways is emerging as a key facet of viral pathogenesis and might also influence the intracellular accumulation and cell‐to‐cell transmission of prions. For example, adjacent cells, through interactions among cell surface signalling or adhesion molecules, might stimulate localized MVB fusion with the plasma membrane to facilitate exosome release and intercellular transfer of their cargo. …

Cellular tolerance of prion protein PrP in yeast involves proteolysis and the unfolded protein response

Secretory proteins undergo a stringent quality control process in the endoplasmic reticulum (ER). Misfolded ER proteins are returned to the cytosol and destroyed by the proteasome. Prion protein PrP is degraded by the proteasome in mammalian cells. However, the significance of proteolysis on PrP-induced cell death is controversial. Moreover, the specific pathway involved in PrP degradation remains unknown. Here, we demonstrate that the unglycosylated form of human PrP is subjected to the ER-associated protein degradation (ERAD) process in the yeast Saccharomyces cerevisiae. We also show that unglycosylated PrP is degraded by the Hrd1–Hrd3 pathway. Accumulation of misfolded proteins triggers the unfolded protein response (UPR), which promotes substrate refolding. Interestingly, we find that the expression of PrP leads to growth impairment in cells deficient in UPR and ERAD. These findings raise the possibility that decreased UPR activity and proteolysis may contribute to the pathogenesis of some prion-related diseases.

Anterograde axonal transport of the exogenous cellular isoform of prion protein in the chick visual system

The cellular isoform of endogenous, newly synthesized prion protein (PrP c) can be transported by axons in the anterograde direction. To determine whether a mechanism exists for secreted PrP c to be internalized and then axonally transported, we analyzed internalization and anterograde axonal transport of radiolabeled recombinant PrP c after its intraocular injection in chick embryos. Internalization and axonal transport of exogenous PrP c to the midbrain by retinal ganglion cells (RGCs) is efficient, saturable and likely receptor-mediated. Ultrastructural quantitative localization of radiolabeled PrP c within RGC soma showed significant labeling of vesicular/endosomal compartments and much less labeling present over the Golgi apparatus and lysosomes, which indicates slow degradation of exogenous PrP c in this system. These data show that a mechanism exists to internalize a secreted form of PrP c and then to axonally transport such PrP c in an anterograde direction. This may provide an additional, novel mechanism for prion protein to spread among neurons.