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).

Two recent UK cases of vCJD transmission by blood transfusion emphasize urgent need of donor screening test for prion diseases. Pathological form of prion protein, PrPsc, is currently the only specific marker of prion diseases, but its detection in blood poses significant challenge. Blood contains substantial amount of normal PrPc which supposedly differs from PrPsc only in its conformation. Majority of cell associated PrPc in blood reside in platelets (PLT). PrPc is also expressed on PBMCs, but their contribution to quantity of blood PrPc is small. The situation is less clear with PrPc on red blood cells (RBC). Others and we have previously shown that PrPc is detectable on RBC by flow cytometry (FACS). However, this was contradicted by another study and by reported minimal content of PrPc in RBC measured by ELISA. To confirm our finding, we used quantitative FACS with fluorescein labeled monoclonal antibodies (MAbs) FH11, 3F4 and 6H4 against different parts of PrPc molecule (PrP23–85, PrP109–112 and PrP144–152, respectively) for evaluation of PrPc expression on RBC of healthy blood donors (n=8). Mean (range) of MAb molecules bound /cell was: FH11 - 36 (13–74), 3F4 - 80 (33–137), 6H4 - 258 (113–557). Interestingly, 3F4 and 6H4 recognized PrPc on PLT in the same samples equally well. Decrease accessibility of 3F4 epitope is one of the characteristics of PrPsc, suggesting that PrPc on RBC may adopt PrPsc like conformation. To test if PrPc on RBC is resistant to proteolysis we treated blood samples with increasing concentrations of proteinase K (1-50 mg/ml, 30 min., 0°C). FACS demonstrated gradual and complete cleavage of PrPc on both PLT and RBC. Lower 3F4 binding to RBC could be explained by expression of N-terminally truncated form of PrPc. Western blot (WB) analysis of RBC ghosts with MAbs 6H4 and AG4 (PrP31–51) confirmed the presence of PrPc in RBC membranes. PrPc seems to be mainly in diglycosylated form, detected as a diffuse band with molecular weight (m.w.) slightly higher (35–38 kDa) than brain PrPc. The difference in PrPc mobility diminished after deglycosylation of PrPc with PNGase. No prominent bands with lower m.w. suggesting presence of truncated PrPc in RBC were detected. If conformation was the cause of 3F4 epitope inaccessibility, this should be reversed by denaturation. Interestingly, 3F4 displayed similar deficiency in PrPc detection also on WB after denaturation of RBC samples with SDS and boiling. At the same time 3F4 and 6H4 exhibited similar sensitivity in detection of PrPc on WB of dilutions of brain or PLT lysate. The remaining explanation of 3F4 reactivity is that its epitope MKHM on RBC PrPc is modified. Recently, modification of Lys residues of PrPsc by advanced glycosylation end products (AGEs) has been reported. We modified Lys residues of brain PrPc on blots by treatment with increasing concentrations of paraformaldehyde and confirmed that Lys modification leads to loss of 3F4 binding. In opposite to situation in peripheral blood, PrPc on erythroid CD71+ cells in cord blood was detected equally well with 3F4 and 6H4, suggesting that modification of PrPc occurs after release of RBC into periphery. Taken together, human RBC express ~ 200 molecules of PrPc/cell. Due to high RBC count even such low level of expression suggests significant contribution to pool of cell associated PrPc in blood (~ 50%). Methods utilizing MAbs FH11 and 3F4 may underestimate quantity of PrPc in RBC. Likewise, screening tests for presence of PrPsc in blood may encounter difficulties if modification similar to one reported here is present.

2032 Cellular prion protein (PrPc) plays a key role in pathogenesis of prion diseases, however, its physiologic function remains unclear. The involvement of PrPc in hematopoiesis was suggested by importance of its expression for self renewal and survival of long term repopulating hematopoietic stem cells. Prion diseases were shown to deregulate transcription of several erythroid genes and we have demonstrated reduction of erythroid cell and erythropoietin production in FVB PrP-/- (Zurich I) mice in response to acute anemia (Zivny J. et al. Blood Cells Mol Dis. 2008;40:302-307). In this study, we exploited different mouse models with manipulated level of PrPc expression to verify the role of PrPc in erythropoiesis. First set of experiments was carried out on PrP-/- (Zurich I) and Tga20 PrP over-expressing mice on a mixed C57Bl6/129Sv genetic background. Inbred C57Bl6 mice served as a wild type control (WT). Induction of acute anemia by phenylhydrazine (PHZ) in PrP-/- and WT mice (n=18) led to drop in the hematocrit (HCT) from 52.5±1.5 and 49.8±2.5% to 37.9± 1.0 and 41.9±3.0% after 24 h, respectively. The course of anemia was significantly deeper in PrP-/- mice at 72 h, 96 h and 120 h (p < 0.01) after PHZ administration. Plasma levels of erythropoietin (Epo) in response to anemia reached higher maximum levels in PrP-/- than WT mice (2250 vs. 1810 pg/mL) although rose more slowly. The level of Epo mRNA in kidneys increased approximately 30-fold in both, WT and PrP-/- mice, however, in WT mice peaked at 24 h whereas in KO mice at 96 h. We repeated the study with smaller groups of PrP-/- and Tga20 mice (n=9) and analysed samples 24 h and 96 h post anemia induction. Random PrP gene re-introduction in Tga20 mice rescued the animals from severe anemia. Decrease in HCT after PHZ administration was significantly lower in Tga20 comparing to PrP-/- mice and was accompanied by less elevated reticulocyte (RTC) count, plasma Epo level and level of Epo mRNA in kidneys. Next we studied the dynamics of unchallenged erythropoiesis in PrP-/-, Tga20 and WT mice by in vivo labelling of blood cells with NHS-biotin and subsequent flow cytometric analysis of relative numbers of newly produced non-labelled RBC. WT mice had significantly enhanced turnover of RBC with higher counts of non-labelled RBC comparing to PrP-/- during 46 days of chase (p < 0.05). Half- life of labeled RBC in WT mice was 20 days, but 32 and 30 days in PrP-/- and Tga20 mice, respectively. Tga20 mice displayed tendency to increased RBC turnover over PrP-/- mice, but the difference was significant only 2 and 33 days after initiation of the experiment. Having in mind possible limitations of experiments conducted in genetically modified inbred mice we have designed second set of experiments in more stringent animal models. We mated C57Bl6/129Sv PrP-/- mice with inbred C57Bl6 and outbred CD-1 mice. Heterozygotes in F1 generation were mated and their PrP -/-, PrP -/+ and PrP +/+ offspring used in the experiments. Anemia was induced by PHZ and blood was sampled from tail vein at defined time points and HCT and RTC count were analysed. In C57Bl6 crossbreeds we observed significantly higher starting HCT level in PrP-/- mice (p < 0.05) compared to PrP-/+ and PrP+/+ mice reaching 53.2±2.3, 50.0±2.1 and 49±2.9%, respectively. Similar decrease in HCT was observed for all PrP groups 24 h after PHZ administration, however, significant differences between PrP-/- and PrP+/+ mice were recorded at 48 h and 72 h. The recovery to normal HCT was again retarded in PrP-/- mice. RTC counts rose more rapidly in PrP+/+ mice after PHZ administration and declined to basal levels faster than in PrP-/- mice, the difference reached significance at 24 h, 48 h and 96 h. Dynamics of unchallenged erythropoiesis in C57Bl6 crossbreeds was similar in all three PrP genotypes with no significant differences in numbers of newly produced RBC during 57 days of the experiment. In CD-1 crossbreed mice no significant differences in HCT and RTC counts were detected after PHZ induced anemia among PrP-/-, PrP-/+ and PrP+/+ siblings. Also the dynamics of unchallenged erythropoiesis was similar in all PrP genotypes. To sum up, our data confirmed the role of PrPc in stress erythropoiesis in studied inbred mouse models. In outbred model the effect of PrP deletion on erythropoiesis seems to be compensated. (GACR310/08/0878, GAUK86408) Disclosures: No relevant conflicts of interest to declare.

No abstract

Cellular prion protein (PrPc) has significant medical importance but its physiological role remains unclear. Some reports indicate that PrPc may play role in the cell survival and/or differentiation. Connection between prion pathogenesis and erythropoiesis was suggested by downregulation of alpha hemoglobin stabilizing protein (AHSP) during prion disease. In addition we recently demonstrated the reduction of erythroid cell and erythropoietin production in PrP-null mice in response to acute anemia (Zivny et al. BCMD 40(2008)302–7). Supporting data for possible involvement of PrPc in hematopoiesis come from the study showing that long-term hematopoietic stem cells of PrP-null bone marrow exhibited impaired self-renewal in serial transplantation of lethally irradiated mouse recipients. Pilot studies in cell culture model, represented by murine erythroleukemic (MEL) cells, indicated upregulation of PrPc expression during erythroid differentiation suggesting its importance in the process. In order to study the role of PrPc during the erythroid differentiation we created 3 stable transfected MEL lines infected with retroviruses coding the short interfering RNA in context of miRNA (1 nonsilencing control - LN and 2 RNAs - LP1 and LP2 targeting murine prion gene (Prnp)). Anti Prnp sequences (HP_285770 and HP_288208) were chosen from publicly available database (RNAi codex). As a vector we used MSCV/LTRmiR30-PIG (Open Biosystems) retroviral plasmid which was transfected to packaging cell line HEK293GP2 and medium containing the viruses was used to infect the MEL cells. Upon selection with 0.5 μg/mL of Puromycin we obtained ~95% of positive cells (estimated by eGFP expression). Downregulation of PrPc was verified by western blot and qRT-PCR. Significant inhibition of PrPc extending from ~70 to more than 90 % compared to LN - MEL cells was observable during the 6 day course of the differentiation (Fig.). We analyzed the expression of 3 genes by qRT-PCR: c-myb, AHSP and hemoglobin alpha (HBA). C-myb is required for early cellular expansion and its downregulation allows the terminal differentiation of cells. Interestingly on the start of differentiation we found ~70% upregulation of c-myb in lines with silenced PrPc. After entering of cells to differentiation, all lines downregulated the c-myb, but the decrease in c-myb expression was more pronounced in LP1 and LP2 cells (15% of initial expression after 24 hours) in comparison with LN cells (30%). Analysis of AHSP and HBA, showed similar upregulation in all cell lines. Since many reports suggest cytoprotective role of PrPc in prevention of apoptosis we analyzed potential influence of PrPc on MEL cell survival. Previous study showed that neither physiological upregulation nor enhanced expression by exogenous delivered Prnp cDNA altered percentage of apoptotic MEL cells during differentiation. We wondered if opposite approach- downregulation of PrPc will affect cell survival. Flow cytometry analysis upon cell staining with 7-AAD and Hoechst 33342 showed that inhibition of translation of PrPc during differentiation probably does not play role in sensitization of cells to apoptosis under physiological conditions. We hypothesized that if under normal conditions cell lines do not differ in level of apoptosis, then they could react differentially after exposure to external stress. We promoted the disturbing conditions by several different treatments: oxidative stress by presence of H2O2 (500 μM), Staurosporin (125 nM) and elevated incubation temperature (40°C). We also treated uninduced cells with increasing concentration of Cu2+ or exposed them to serum deprivation. Contrary to belief that absence of PrPc sensitizes the cells against damage, we found that inhibition of PrPc expression did not altered level of apoptosis/necrosis in MEL cells. Finally, we have introduced the new cell culture model for study of PrPc involvement in erythroid differentiation. Our initial observations suggest that direct cytoprotection is not mode of PrPc action in studied process, but it may play role in cell cycle which is a subject of undergoing research. Figure Figure