BACKGROUND INFORMATION: Cellular prion protein (PrPC ) is infamous for its role in prion diseases. The physiological function of PrPC remains enigmatic, but several studies point to its involvement in cell differentiation processes. To test this possibility, we monitored PrPC changes during the differentiation of prion-susceptible CAD 5 cells, and then we analysed the effect of PrPC ablation on the differentiation process. RESULTS: Neuronal CAD 5 cells differentiate within 5 days of serum withdrawal, with the majority of the cells developing long neurites. This process is accompanied by an up to sixfold increase in PrPC expression and enhanced N-terminal β-cleavage of the protein, which suggests a role for the PrPC in the differentiation process. Moreover, the majority of PrPC in differentiated cells is inside the cell, and a large proportion of the protein does not associate with membrane lipid rafts. In contrast, PrPC in proliferating cells is found mostly on the cytoplasmic membrane and is predominantly associated with lipid rafts. To determine the importance of PrPC in cell differentiation, a CAD 5 PrP-/- cell line with ablated PrPC expression was created using the CRISPR/Cas9 system. We observed no considerable difference in morphology, proliferation rate or expression of molecular markers between CAD 5 and CAD 5 PrP-/- cells during the differentiation initiated by serum withdrawal. CONCLUSIONS: PrPC characteristics, such as cell localisation, level of expression and posttranslational modifications, change during CAD 5 cell differentiation, but PrPC ablation does not change the course of the differentiation process. SIGNIFICANCE: Ablation of PrPC expression does not affect CAD 5 cell differentiation, although we observed many intriguing changes in PrPC features during the process. Our study does not support the concept that PrPC is important for neuronal cell differentiation, at least in simple in vitro conditions.
In this study we attempted to characterize cellular prion-like protein (PrPC) properties and its possible interactions with metal ions. In the first part of this study we described the structure of prion-like protein, its biochemical properties and its role in human or animal organism. In the next and the most important part we summarized accessible information about the metals and heavy metals which can bind prion-like protein. Especially the heavy metals could have really important role in the process of protein misfolding, but this role is still unknown. In the last section we focused on transmissible spongiform encephalopathies, their incubation period and clinical signs.
- MeSH
- Cadmium MeSH
- Metals MeSH
- Humans MeSH
- Copper MeSH
- Prion Diseases * MeSH
- Prions * MeSH
- PrPC Proteins metabolism MeSH
- PrPSc Proteins metabolism pathogenicity MeSH
- Metals, Heavy * MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
In this study we tried to describe the role of prion proteins in living organisms. PrPC is a metal binding protein and there is a presumption that the metals may play a role in the pathogenic conversion of PrPC to PrPSc. Equally important link between prion diseases and metals is a protein Metallothionein (MT). The brain specific form of Metallothionein is MT–III. MT-III participates in formation of neurodegenerative diseases and maintains the metal balance in a brain. It was revealed that the lower level of MT-III can lead to the formation of neurofibrillary clusters characteristic of Alzheimer‘s disease. Recently, there are hypotheses that prion diseases may have common mechanisms of other neurodegenerative diseases such as Alzheimer‘s, Parkinson‘s or Huntington‘s disease. These hypotheses obviously ignited great scientific interest in the subject and the common features of these diseases are now intensively studied.
- MeSH
- Metals adverse effects MeSH
- Humans MeSH
- Metallothionein * MeSH
- Prion Diseases * etiology MeSH
- PrPC Proteins MeSH
- PrPSc Proteins * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
Prionová onemocnění jsou způsobena proteinovými infekčními částicemi, odolnými vůči běžným sterilizačním postupům. Nejčastější je Creutzfeldtova-Jakobova nemoc, jejíž diagnóza je založena na kombinaci klinického obrazu (rychle progredující demence, poruchy hybnosti pyramidového či extrapyramidového typu, myoklonus, zrakově-prostorové a mozečkové projevy a akinetický mutizmus) a průkazu proteinu 14–3–3 v likvoru a/nebo periodických projevů na EEG. MRI obraz hypersignálů v difuzním vážení a FLAIR sekvencích subkortikálně v putamen a ncl. caudatus a v některých korových zónách byl nedávno zahrnut do upravených diagnostických kritérií WHO. Včasné rozpoznání prionového onemocnění má zásadní význam pro pacienta i jeho rodinu u genetických forem. Při neexistenci kauzální terapie lze ušetřit pacienta zbytečných pomocných vyšetření, mnohdy i invazivních, a přejít včas na účinnou paliativní péči.
Prion diseases are caused by proteinous infectious particles, which are very resistant to usual sterilization procedures. The most current is Creutzfeldt-Jakob disease. Its diagnosis is based on clinical findings (rapidly progressive dementia, pyramidal or extrapyramidal involvement, myoclonus, visuo-spatial impairment and akinetic mutism) and positive 14–3–3 protein in CSF and/or periodic activity on EEG. MRI hyperintensities in FLAIR and diffusion weighted images in subcortical structures (putamen and caudate) and in some cortical areas, are currently being added to new WHO diagnostic criteria. Early recognition of a prion disease is crucial for the patient and his family, mainly in case of genetic forms of diseases. Since causal therapy is inexistent, many futile investigations including invasive procedures can be avoided and adequate palliative care can be introduced.
- Keywords
- prion, 14–3–3 protein,
- MeSH
- Creutzfeldt-Jakob Syndrome diagnosis classification pathology MeSH
- Dementia diagnosis MeSH
- Electroencephalography MeSH
- Financing, Organized MeSH
- Gerstmann-Straussler-Scheinker Disease genetics MeSH
- Immunohistochemistry MeSH
- Insomnia, Fatal Familial genetics MeSH
- Humans MeSH
- Magnetic Resonance Imaging MeSH
- Mutation MeSH
- Polymorphism, Genetic MeSH
- Prion Diseases etiology genetics classification MeSH
- 14-3-3 Proteins MeSH
- PrPC Proteins analysis genetics MeSH
- PrPSc Proteins analysis genetics MeSH
- Check Tag
- Humans MeSH
BACKGROUND: Recent transmissions of variant Creutzfeldt-Jakob disease by blood transfusion emphasize the need for the development of prion screening tests. The detection of prions in blood is complicated by the presence of poorly characterized cellular prion protein (PrP(C) ) in both plasma and blood cells. According to published studies, most of PrP(C) in blood cells resides in platelets (PLTs) and white blood cells. STUDY DESIGN AND METHODS: To clarify conflicting reports about the quantity of PrP(C) associated with human red blood cells (RBCs), quantitative flow cytometry, Western blot (WB), and enzyme-linked immunosorbent assay (ELISA) were used to measure protein levels in healthy donors. RESULTS: RBCs expressed 290 ± 140 molecules of PrP(C) per cell, assuming equimolar binding of monoclonal antibody (MoAb) 6H4 to PrP(C). Binding of alternate PrP(C) MoAbs, FH11 and 3F4, was substantially lower. WB estimated the level of PrP(C) per cell on RBCs to be just four times lower than in PLTs. A similar level of PrP(C) was detected using ELISA. The weak binding of commonly used MoAb 3F4 was not caused by PrP(C) conformation, truncation, or glycosylation, suggesting a covalent modification, likely glycation, of the 3F4 epitope. CONCLUSIONS: Taken together, human RBCs express low but significant amounts of PrP(C) /cell, which makes them, due to high RBC numbers, major contributors to the pool of cell-associated PrP(C) in blood. Previous reports utilizing MoAb 3F4 may have underestimated the amount of PrP(C) in RBCs. Likewise, screening tests for the presence of the abnormal prion protein in blood may be difficult if the abnormal protein is modified similar to RBC PrP(C).
- MeSH
- Creutzfeldt-Jakob Syndrome metabolism prevention & control transmission MeSH
- Enzyme-Linked Immunosorbent Assay MeSH
- Epitopes immunology MeSH
- Erythrocyte Membrane metabolism MeSH
- Erythrocytes metabolism MeSH
- Glycosylation MeSH
- Blood Transfusion MeSH
- Humans MeSH
- Antibodies, Monoclonal immunology MeSH
- Mass Screening methods MeSH
- Transfusion Reaction MeSH
- PrPC Proteins immunology metabolism MeSH
- Flow Cytometry MeSH
- Blotting, Western MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The recently shown transmissibility of variant Creutzfeldt-Jakob disease (vCJD) by blood transfusion emphasises the need for better understanding of the cellular prion protein (PrPc) in blood. A substantial amount of cell-associated PrPc in blood resides in platelets. Platelet activation leads to up-regulation of PrPc on the platelet surface and its release on exosomes and microparticles. The sub-cellular localisation and function of platelet PrPc, however, is poorly understood. In the present study, we investigated the association of PrPc with platelet lipid rafts and the platelet cytoskeleton. Immuno-fluorescence microscopy showed that the signals of PrPc and P-selectin, both of which occupy intracellular alpha granules, were separated on the membrane, suggesting organisation in different membrane domains. A flotation assay of platelet lysates demonstrated that a relatively small portion of platelet PrPc floats with lipid rafts, regardless of platelet activation status. This was reversed by depolymerisation of the platelet cytoskeleton, which led to flotation of most platelet PrPc, suggesting that interactions with the cytoskeleton prevent flotation of PrPc rafts. This association of PrPc with the platelet cytoskeleton was confirmed by its presence in both the isolated membrane skeleton and actin cytoskeleton. Platelet activation significantly increased the amount of PrPc associated with the cytoskeleton. Our results indicate that the localisation of PrPc in platelets is complex, with the majority of PrPc present within platelet lipid rafts linked to the platelet cytoskeleton. This localisation places PrPc in a position where it can interact with proteins involved in platelet signalling and eventually with vCJD prions.
- MeSH
- Platelet Activation MeSH
- Biopolymers MeSH
- Cell Membrane chemistry MeSH
- Cytoplasmic Granules chemistry MeSH
- Cytoskeleton chemistry MeSH
- Microscopy, Fluorescence MeSH
- Humans MeSH
- Membrane Microdomains chemistry MeSH
- P-Selectin MeSH
- PrPC Proteins blood MeSH
- Blood Platelets chemistry ultrastructure MeSH
- Check Tag
- Humans MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
Cellular prion protein (PrPc) participates in the pathogenesis of prion diseases but its normal function remains unclear. PrPc is expressed on hematopoietic cells, including erythroid precursors. We investigated the role of PrPc in erythropoiesis in vivo with phenylhydrazine-induced acute anemia. Induction of equivalent anemia in wild-type (WT) and Prnp-/- mice resulted in a higher number of circulating reticulocytes, hematocrits and spleen weights in WT mice than in Prnp-/- mice on Days 5 and 7. Examination of bone marrow erythroid precursor cells (Ter119+) on Day 5 revealed no significant differences in the number of these cells between the two types of animals. However, a higher percentage of Ter119+ cells were going through apoptosis in Prnp-/- mice than in WT mice. Plasma erythropoietin (Epo) levels and Epo mRNA in kidneys peaked on Day 3 in response to anemia for both types of animals but rose less in Prnp-/- (5500 pg/ml ) than in WT (18,000 pg/ml) animals. Administration of recombinant human Epo to mice produced an equivalent reticulocyte response in both types of animals suggesting that the potential for erythroid generation is intact in Prnp-/- animals. These observations indicate that PrPc may modulate tissue hypoxia-sensing mechanisms or effect hypoxia target gene expression.
- MeSH
- Acute Disease MeSH
- Erythropoietin blood administration & dosage pharmacology MeSH
- Erythroid Precursor Cells physiology MeSH
- Erythropoiesis MeSH
- Phenylhydrazines MeSH
- Financing, Organized MeSH
- Anemia, Hemolytic chemically induced blood metabolism MeSH
- Hypoxia metabolism MeSH
- Mice, Mutant Strains MeSH
- Mice, Knockout MeSH
- Mice MeSH
- Oxidants pharmacology MeSH
- PrPC Proteins physiology blood MeSH
- Reticulocytes physiology drug effects MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
The cellular prion protein (PrPc) is a membrane glycoprotein expressed on many human cells including platelets. We investigated the cellular localization of platelet PrPc. In resting platelets most PrPc was localized inside the cells. The correlation of PrPc and P-selectin surface up-regulation after platelet activation suggested its association with alpha-granules. This was confirmed by normal expression of PrPc on Hermansky-Pudlak syndrome platelets, which lack dense granules, and failure of gray platelet syndrome platelets, which lack alpha-granules, to up-regulate PrPc. Our results warrant further studies on the role of platelet PrPc in the transmission of prion diseases by blood transfusion.
- MeSH
- Creutzfeldt-Jakob Syndrome blood transmission MeSH
- Cytoplasmic Granules chemistry ultrastructure MeSH
- Hermanski-Pudlak Syndrome blood MeSH
- Blood Transfusion MeSH
- Humans MeSH
- Transfusion Reaction MeSH
- Prions blood MeSH
- PrPC Proteins blood MeSH
- Blood Platelets chemistry MeSH
- Check Tag
- Humans MeSH
