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C. Siberchicot et al.
   Supplementary Figure S1F). In contrast, compared to their 3- month old counterparts, ST- and LT-HSC frequencies respectively increased 2- and 8.3-fold in 11-month old WT mice but did not change (ST-HSC) or was only 3-fold increased (LT-HSC) in 11-month old KO mice (Figure 1I). DNA repair was slightly dependent on PrPC in aged HSC. A 1.4-fold increased activity of Ape1 in WT HSC was found between three months and 11 months without any change in Ape1 mRNA level. In KO HSC, Ape1 activity also increased between three months and 11 months, but to a lesser extent (1.2-fold) than in WT HSC. Interestingly, this increased activity was associated with an increased Ape1 mRNA level (Figure 1J and K).
Altogether, these results show that PrPC deficiency is associated with decreased HSC determination towards the myeloid lineage, and decreased number of HSC and decreased Ape1 activity in old mice.
Prnp expression is up-regulated in myeloid progenitors and hematopoietic stem cell subpopulations after
in vivo radiation exposure
Hematopoietic stem cell aging is associated with increased oxidative stress28 and PrPC has been shown to protect cells from oxidative stress.17-20 To characterize the role of PrPC during the oxidative stress of hematopoiesis, we performed total body irradiation (TBI) on WT and KO mice. Survival curves of WT and KO mice exposed to increasing gamma-radiation doses showed that a higher percentage of irradiated KO mice died earlier than irradi- ated WT mice even if statistical significance between both genotypes was not reached (P=0.0792 at 7 Gy) (Figure 2A). When KO mice were grafted with BM from non-irradiat- ed WT mice 24 hours (h) after a 7 Gy irradiation, they did not die, indicating that the higher sensitivity of KO mice to TBI was not due to the BM microenvironment of KO mice and that they died from hematopoietic syndrome.
One hour after a 7 Gy TBI, a 1.5-fold increase in Prnp mRNA level was found in HSC, CMP and GMP but not in MPP and MEP (Figure 2B). These data are consistent with the observed Prnp upregulation in neuronal tissues after exposure to genotoxic stress,21 and suggest a potential role of PrPC in response to radiation in GMP, CMP, and HSC.
Cellular prion protein-dependent increase in the DNA repair activity of Ape1 is associated with radioprotection of CMP and GMP
Cellular prion protein prevents cell death in response to alkylating agent or H2O2 exposure by directly stimulating the DNA repair activity of Ape1.21 Without irradiation, Ape1 activity was similar in WT and KO progenitors (Figure 2C). One hour after a 7 Gy TBI, Ape1 activity increased in all WT irradiated myeloid progenitors ana- lyzed (from 1.5- to 1.7-fold) but not in their KO counter parts (Figure 2D). In HSC and MPP, Ape1 activity was similar in WT and KO mice (Figure 2E) but increased only in WT HSC after a 7 Gy TBI (Figure 2F). Whatever the subpopulation analyzed, the radiation-induced Ape1 activity was not associated with an increase in Ape1 mRNA level (Online Supplementary Figure S2A and B). These data show that, after irradiation, Ape1 activity in all myeloid progenitor subpopulations and in HSC is stimulated in a PrPC-dependent manner.
As the radiation-induced death of myeloid progenitors is dependent on apoptosis,5 we quantified apoptosis in WT and KO myeloid progenitors 1 and 12 h after TBI at
7 Gy. One hour after irradiation, apoptotic (Annexin V- positive cells) and dead cell (Annexin V-negative and Hoechst-positive cells) fractions increased only in CMP and were higher in KO compared to WT CMP (Figure 2G and Online Supplementary Figure S2C). Twelve hours after irradiation, both apoptotic (Figure 2H) and dead (Online Supplementary Figure S2D) cell fractions increased in all myeloid progenitor subpopulations in both WT and KO mice. However, higher rates of apoptosis and cellular death were observed in irradiated KO compared to WT GMP. In accordance with this, significantly lower frequen- cies of CMP and GMP were found in KO versus WT irra- diated mice 18 h after irradiation (Figure 2I). Myeloid pro- genitors from Prnp ZH3/ZH3 mice exhibited the same radiation sensitivity than those from Prnp-/- mice, shown by a similar reduced number compared to mice in non- irradiated conditions (Online Supplementary Figure S2E). Altogether, these results suggest that PrPC-dependent stimulation of the DNA repair activity of Ape1 is required for the radioprotection of myeloid progenitors.
Discussion
Despite numerous studies, the physiological role of PrPC remains elusive. Recently, we showed that PrPC can stimulate an important DNA repair pathway, the BER, in neuronal tissues through interaction with and stimulation of its key enzyme, APE1.21 Here we show that the same mechanism can be proposed for the radioprotection of myeloid progenitors, HSC determination, and the expan- sion of the HSC compartment during aging.
Previous studies22-24,29 indicated a decreased Prnp expres- sion during differentiation of hematopoietic cells. Here, we performed an extended study of Prnp expression in dif- ferent hematopoietic subpopulations and showed a 3-fold higher Prnp expression level in MEP compared to their progenitors CMP, suggesting that the correlation between Prnp downregulation and cellular differentia- tion24 may not be a general feature in hematopoiesis. Furthermore, and contrary to a previous study,25 we found that KO mice have less myeloid progenitors. This discrep- ancy could be explained by the fact that, in the previous study, younger mice were analyzed (7-10-week old mice compared to the 3-month and 11-month old mice used in the present study) and by the number of backcrosses (4 vs. >10) that might influence the phenotype of Prnp knockout mice.30 Finally, we found a higher frequency of KO ST-HSC and KO MPP in the G0 phase. These populations being the direct precursors of myeloid progenitors, the increased quiescence of these cells might account for the decreased myeloid progenitor subpopulations.31 Strikingly, both KO CMP and GMP exhibited a lower plating effi- ciency despite no significant change in their cell cycle in vivo. Whether the microenvironment of these cells could compensate in vivo for an intrinsic growth deficiency observed in vitro remains to be clarified.
Prnp expression in the HSC compartment increased 8- fold with age. This higher expression was associated with the known elevated frequency of both ST- and LT-HSC.27,32 PrPC deficiency was associated with no increase in ST- HSC and with a diminished increase in LT-HSC with age, suggesting that PrPC plays a role in the age-dependent increase in HSC. Although an aging-associated increase in HSC numbers has been known for a long time and is
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