Page 57 - Haematologica May 2020

PrPC-dependent hematopoietic homeostasis radioprotection of hematopoietic progenitors
disorders called prion diseases.16 The normal prion protein was shown to protect cells from oxidative stress.17-20 It also gives protection from DNA damage by promoting Ape1 DNA repair activity and cell survival through an interac- tion with Ape1.21 During hematopoiesis, PrPC is highly expressed in HSC and hematopoietic progenitors22-24 and PrPC deficiency is associated with decreased HSC self- renewal.25 As oxidative stress and DNA damage help determine HSC cell fate,26 PrPC might participate in the maintenance of the hematopoietic system and its response to cytotoxic stresses. To address these points, we used Prnp knockout mice to study the consequences of PrPC deficiency on hematopoiesis of young and old adult mice, and on the response of hematopoietic stem cells (HSC) and hematopoietic progenitors to gamma-irradia- tion.
Methods
Mice
Mice experiments were carried out in compliance with the European Community Council Directive (EC/2010/63) and were approved by our institutional ethics committee (CetEA-CEA- DRF–n. 17-096). The B6.129S7-Prnptm1Cwe/Orl mice were from the European Mutant Mouse Archive and bred in our animal facil- ity. We also used Prnp ZH3/ZH3 mice provided by A. Aguzzi (Zurich, Switzerland) and C57BL/6 mice were purchased from Charles River.
Cell sorting and flow cytometry analysis of bone marrow cells
Murine BM cells were flushed out of femurs, tibiae, hip bone and humeruses using a syringe filled with DPBS and filtered through a 70 μm-cell strainer. After red blood cell lysis using NH4Cl solution (STEMCELL Technologies), mononuclear cells were phenotyped using different antibody cocktails from Biolegend, e-Bioscience or Beckton Dickinson. Flow cytometry analysis was performed with a BD FACS LSRIITM flow cytometer (BD Biosciences) and cell sorting with a FACS Influx cell sorter (Becton Dickinson). Data were analyzed with FlowJo software. Antibodies and gating strategies for hematopoietic subset analysis and sorting are described in the Online Supplementary Methods. For RT-PCR and Ape1 endonuclease activity experiments, aliquots of 50,000 myeloid progenitor cells were sorted in PBS whereas aliquots of 10,000 HSC and multi-potent progenitors (MPP) were sorted in PBS/1%BSA.
Ape1 endonuclease activity
Cell extracts were obtained by sonication of pelleted BM sorted cells in 20mM Tris-HCl, pH 7.5, 250mM NaCl, 1mM EDTA, 20mM sucrose, and protease inhibitor cocktail 0.1% (Sigma- Aldrich P2714). For progenitor analysis, 50,000 cells were suspend- ed in 125 mL extraction buffer. For HSC or MEP analysis, 10,000 sorted cells were suspended in 30μL extraction buffer. After soni- cation, the homogenate was centrifuged at 20,000 x g for 30 min- utes (min) at 4°C and aliquots of the supernatant were stored at -80°C. Ape1 endonuclease activity was measured using a 5’-end labeled 34-mer oligonucleotide containing a single tetrahydrofu- ranyl artificial AP site at position 16 hybridized to its complemen- tary oligonucleotide containing a cytosine opposite the lesion (Eurogentec). To measure Ape1 activity in BM progenitors, 1-4 mL of cell extract were incubated for 30 min at 30°C in 10 mL of reac- tion buffer containing 25mM Tris-HCl pH 7.5, 5mM MgCl2, 5% glycerol, 52mM NaCl, BSA 1 mg/mL and 150 fmoles of the
hybridized oligonucleotide. To determine Ape1 activity in BM HSC and MPP, 1-4 mL of cell extract were incubated for 10 min at 30°C in 10 mL reaction buffer containing 25mM Tris-HCl pH 7.5, 5mM MgCl2, 5% glycerol, 52mM NaCl, and 150 fmoles of the hybridized oligonucleotide. The reaction was stopped by adding 4μL of denaturating buffer (80% formamide, 0.1% bromophenol blue, 10mM EDTA) followed by heating for 5 min at 95°C. The products of the reaction were resolved by denaturating 7M urea- 20% polyacrylamide gel electrophoresis. Gels were scanned using a Typhoon 5 (GE Healthcare Life Sciences) and band intensifies were quantified with ImageQuant TL 8.1 (GE Healthcare Life Sciences).
Statistical analyses
Quantitative data are presented as the mean±standard error of mean (SEM). Statistical significance was assayed using the non- parametric Mann Whitney U-test (GraphPad Prism software).
Additional information concerning the materials and methods used are to be found in the Online Supplementary Methods.
Results
Cellular prion protein is differentially expressed in hematopoietic stem cells and myeloid progenitors and is involved in hematopoietic stem cell expansion during aging
To address the potential functions of PrPC in hematopoietic stem and progenitor cells (HSPC), we first characterized the expression pattern of Prnp in different purified hematopoietic subpopulations, i.e. common myeloid progenitors (CMP), granulocyte-monocyte pro- genitors (GMP), megakaryocyte-erythrocyte progenitors (MEP), MPP, and hematopoietic stem cells (HSC). The highest level of Prnp mRNA was found in MEP while they were 2.7-fold and 4.3-fold lower in CMP and GMP, respectively (Figure 1A). These differences in mRNA expression were also found at the protein level (Figure 1B and Online Supplementary Figure S1A). The Prnp mRNA level in purified HSC was 2.5-fold higher than in MPP (Figure 1C).
To determine if PrPC has a role in hematopoiesis, we first compared BM from 3-month old Prnp+/+ (WT) and Prnp-/- (KO) mice. WT and KO mice showed similar peripheral blood counts (data not shown) and BM cellularity (Online Supplementary Figure S1B). However, the frequency of CMP, GMP and MEP was significantly reduced in KO mice compared to WT mice (Figure 1D), whereas CLP, MPP, ST-HSC and LT-HSC frequencies were similar (Figure 1E). The differences between KO and WT myeloid progenitor frequencies were not associated with increased apoptosis (Online Supplementary Figure S1C) or cell cycle alteration (Online Supplementary Figure S1D) but with a higher percentage of quiescent MPP and ST-HSC (Figure 1F). These results suggest a defect of determination of HSPC (MPP and ST-HSC) towards the myeloid lineage in Prnp-/- mice. Finally, clonogenic assay using purified CMP and GMP showed a significantly decreased plating effi- ciency of Prnp-/- CMP and GMP (Figure 1G). Taken togeth- er, these results show intrinsic myeloid differentiation deficiencies in Prnp-/- HSC and progenitors and suggest that the reduction of cycling MPP and ST-HSC contributes to the lower myeloid progenitor content in the BM from KO compared to WT mice.
Aging of HSC is associated with an increased percent-
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