Prion protein deficiency impairs hematopoietic stem cell determination and sensitizes myeloid progenitors to irradiation

Abstract

Highly conserved among species and expressed in various types of cells, numerous roles have been attributed to the cellular prion protein (PrPC). In hematopoiesis, PrPC regulates hematopoietic stem cell self-renewal but the mechanisms involved in this regulation are unknown. Here we show that PrPC regulates hematopoietic stem cell number during aging and their determination towards myeloid progenitors. Furthermore, PrPC protects myeloid progenitors against the cytotoxic effects of total body irradiation. This radioprotective effect was associated with increased cellular prion mRNA level and with stimulation of the DNA repair activity of the Apurinic/pyrimidinic endonuclease 1, a key enzyme of the base excision repair pathway. Altogether, these results show a previously unappreciated role of PrPC in adult hematopoiesis, and indicate that PrPC-mediated stimulation of BER activity might protect hematopoietic progenitors from the cytotoxic effects of total body irradiation.

Figure 1

PrPC contributes to mouse hematopoietic homeostasis. (A) quanta-tive real-time polymerase chain reaction (qRT-PCR) analysis of Prnp expression, normalized to Rplp0 in the indicated bone marrow (BM) subpopulations: CMP: common myeloid progenitor; GMP: granulocyte-macrophage progenitor; MEP: megakaryocyte-erythrocyte progenitor purified by flow cytometry from BM of 3-month old mice (n=7-9). Data are presented as mean±standard error of mean (SEM). Means with different letters are significantly different (P<0.05). (B) Flow cytometry analysis of the PrPC protein expression in the indicated BM supopulations. Graph depicts ratio of median fluorescence intensity (MFI) in wild-type (WT) and knock-out (KO) control cells (n=5-6). Data are presented as mean±SEM. Means with different letters are significantly different (P<0.05) (CMP vs. MEP, P=0.06). (C) qRT-PCR analysis of Prnp expression, normalized to Actb in hematopoietic stem cell (HSC) (LSK CD135⁻) and in multipotent progenitor (MPP) (LSK CD135⁺) purified by flow cytometry from BM of 3-month old mice (n=9); Data are presented as mean±SEM. ***P<0.001. (D) Frequencies of myeloid progenitors in WT (black bars) and KO (white bars) BM from 3-month old mice (n=6-10). Data are presented as mean±SEM. *P<0.05; **P<0.01. (E) Frequencies of lymphoid progenitors (CLP), MPP, ST- and LT-HSC in the BM from WT (black bars) and KO (white bars) mice (n=6). Data are presented as mean±SEM. (F) Distribution of WT (black bars) and KO (white bars) HSC and MPP in each phase of the cell cycle. Data are presented as mean±SEM. *P<0.05; **P<0.01. ns: not significant. (G) In vitro plating efficiency of CMP and GMP purified by flow cytometry from BM of WT (black bars) and KO (white bars) mice (n=6-9). Data are presented as mean±SEM. **P<0.01. (H) qRT-PCR analysis of Prnp expression, normalized to Actb in WT and KO HSC and MPP purified by flow cytometry from BM of 3-month and 11-month old mice (n=6-9). Data are presented as the mean±SEM. ***P<0.001 or ^###P<0.001. (I) Frequencies of MPP, ST-and LT-HSC in BM from 3-month and 11-month old WT (black bars) and KO (white bars) mice. Data are presented as the mean±SEM fold change of the frequencies in 11-month relative to 3-month old mice (n=6-10). **P<0.01 or ^##P<0.01. (J) Ape1 endonuclease activity in 3-month (opened) and 11-month (hatched) old WT (dark) and KO (light) HSC. Data are presented as the mean±SEM. (n=4-5). *P<0.05 or ^#P<0.05. (K) qRT-PCR analysis of Ape1 expression, normalized to Actb in WT (dark) and KO (light) HSC purified by flow cytometry from BM of 3-month (opened bars) and 11-month (hatched bars) old mice. Data are presented as mean±SEM. (n=7-9). *P<0.05 or ^#P<0.05.

Figure 2

PrPC favors survival of mice exposed to moderate doses of γ-rays and protects common myeloid progenitors (CMP) from radiation-induced death. (A) Kaplan-Meier survival plots of wild-type (WT) (solid lines) and knock-out (KO) (dashed lines) mouse overall survival after total body irradiation (TBI) at indicated doses: n=5, 10 Gy; n=13, 6.5 Gy; n=28, 7 Gy; for each genotype. Arrow points to the 100% survival of both KO and WT irradiated mice (7 Gy) after transplantation of WT bone marrow (BM) cells (n=5). (B) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of Prnp expression in hematopoietic stem cell (HSC), multi-potent progenitor (MPP), and myeloid progenitors 1 hour (h) after irradiation (7 Gy) (n=6). Prnp RNA levels were normalized to Actb (HSC and MPP) or Rplp0 (myeloid progenitors). Data are presented as mean±standard error of mean (SEM) fold change of normalized Prnp RNA levels in irradiated relative (IR) to control cells (Ctrl). *P<0.05; **P<0.01. (C) Ape1 endonuclease activity in myeloid progenitor subpopulations from WT (black bars) and KO (white bars) mice (n=5-8). Data are presented as mean±SEM. Means with different letters are significantly different (P<0.05). (D) Ape1 endonuclease activity in myeloid progenitors from WT (black bars) and KO (white bars) mice (n=5-8) 1 h after irradiation (7 Gy). Data are presented as mean±SEM fold change of Ape1 endonuclease in irradiated relative to non-irradiated control cells. *P<0.05; **P<0.01. (E) Ape1 endonuclease activity in HSC and MPP from WT (black bars) and KO (white bars) mice (n=5-8). Data are presented as mean±SEM. (F) Ape1 endonuclease activity in HSC and MPP from WT (black bars) and KO (white bars) mice (n=4-5) 1 h after irradiation (7 Gy). Data are presented as mean±SEM fold change of Ape1 endonuclease in irradiated relative to non-irradiated control cells. *P<0.05 or ^#P<0.05. (G) Percentage of apoptotic myeloid progenitors (AnnexineV-positive cells) in BM from WT (black bars) and KO (white bars) mice (n=6-8) 1 h after irradiation (7 Gy). Data are presented as mean±SEM fold change of percentage of apoptotic cells in irradiated relative to non-irradiated control myeloid progenitors. **P<0.01; ***P<0.001; ^#P<0.05. (H) Percentage of apoptotic myeloid progenitors (AnnexineV-positive cells) in BM from WT (dark gray bars, WTIR) and KO (light gray bars, KOIR) mice (n=6-8) 1 h after irradiation (7 Gy). Data are presented as mean±SEM. Non-irradiated control WT (black bars) and KO (white bars) myeloid progenitors are shown. **P<0.01; ^#P<0.05. (I) Percentage of myeloid progenitors in BM from WT (black bars) and KO (white bars) mice (n=6-7) 18 h after irradiation (7 Gy). Data are presented as mean±SEM of the percentage of cells remaining in BM 18 h after irradiation compared to non-irradiated control. **P<0.01.