Impairment of Erythropoiesis In Inbred Cellular Prion Protein Deficient Mice.

Abstract 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)