Role of Prion protein in premature senescence of human fibroblasts

Accepted Manuscript Title: Role of Prion protein in premature senescence of human fibroblasts Authors: Emmanuelle Boilan, Virginie Winant, Elise Dumortier, Benaissa ElMoualij, Pascale Quatresooz, Heinz D. Osiewacz, Florence Debacq-Chainiaux, Olivier Toussaint PII: DOI: Reference:

S0047-6374(17)30087-8 http://dx.doi.org/doi:10.1016/j.mad.2017.08.002 MAD 10976

To appear in:

Mechanisms of Ageing and Development

Received date: Revised date: Accepted date:

31-3-2017 29-6-2017 3-8-2017

Please cite this article as: Boilan, Emmanuelle, Winant, Virginie, Dumortier, Elise, ElMoualij, Benaissa, Quatresooz, Pascale, Osiewacz, Heinz D., DebacqChainiaux, Florence, Toussaint, Olivier, Role of Prion protein in premature senescence of human fibroblasts.Mechanisms of Ageing and Development http://dx.doi.org/10.1016/j.mad.2017.08.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Role of Prion protein in premature senescence of human fibroblasts Emmanuelle Boilana1, Virginie Winanta, Elise Dumortiera, Benaissa ElMoualijb, Pascale Quatresoozb, Heinz D. Osiewaczc, Florence Debacq-Chainiauxa,* and Olivier Toussainta a

Unité de Recherche en Biologie Cellulaire (URBC) - Namur Research Institute for Life Sciences

(Narilis), University of Namur, Belgium b c

Human Histology-CRPP, University of Liège, Liège, Belgium Institute of Molecular Biosciences, Johann Wolfgang Goethe University, Frankfurt am Main,

Germany *

Corresponding author: URBC-Narilis, University of Namur, 61, rue de Bruxelles, B-5000 Namur,

Belgium Phone: +32 81 72 44 57, Fax: + 32 81 72 41 35, [email protected]

Highlights 

Prion protein expression is overexpressed in copper-induced premature senescence

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Prion silencing induces appearance of several biomarkers of senescence

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Prion protein has a protective effect on stress and copper-induced senescence

Abstract

Prion protein (PrP) is essentially known for its capacity to induce neurodegenerative prion diseases in mammals caused by a conformational change in its normal cellular isoform (PrPC) into an infectious and disease-associated misfolded form, called scrapie isoform (PrPSc). Although its sequence is highly conserved, less information is available on its physiological role under normal conditions. However, increasing evidence supports a role for PrPC in the cellular response to oxidative stress. In the present study, a new link between PrP and senescence is highlighted. The role of PrP in premature senescence induced by copper was investigated. WI-38 human fibroblasts were incubated with copper sulfate (CuSO4) to trigger premature senescence. This induced an increase of PrP mRNA level, an increase of protein abundance of the normal form of PrP and a nuclear localization of the protein. Knockdown of PrP expression using specific small interfering RNA (siRNA) gave rise to appearance of several biomarkers of senescence as a senescent

1

Present address : CESI, Chaussée de Louvain 290, 5004 Bouge, Belgium ; email : [email protected] 1

morphology, an increase of senescence associated β-galactosidase activity and a decrease of the cellular proliferative potential. Overall these data suggest that PrP protects cells against premature senescence induced by copper. Keywords

Senescence, Copper, Prion protein, Oxidative stress, Fibroblasts 1. Introduction

Prion protein (PrP) is essentially known for its capacity to induce neurodegenerative prion diseases in mammals, such as bovine spongiform encephalopathy, ovine scrapie and human CreutzfeldtJakob disease (Colby and Prusiner, 2011; Das and Zou, 2016). This infectious state is caused by a conformational change in its normal cellular isoform (PrPC) into an infectious and diseaseassociated misfolded form, called scrapie (PrPSc) (Colby and Prusiner, 2011). PrPC is a Nglycosylated glycosyl-phosphatidyl-inositol-anchored protein that has a predominant α-helical structure, degradable by proteinase-K (a protease that exhibits broad substrate specificity). In contrast, PrPSc is predominantly a β-sheet structure following a conformational change of the hydrophobic region, which makes this form resistant to proteinase-K digestion (Pan et al., 1993). This is a perfect example of the well-established “protein-only hypothesis” (Mays and Soto, 2016; Soto, 2011), when a cellular protein undergoes a conformational change, its normal function is compromised or the protein acquires a new activity. However, despite PrP sequence is highly conserved during evolution, suggesting an important role of this protein, less information is known on its function in normal conditions. PrPC can form a dynamic platform on the cell surface for signaling molecules triggering transmembrane signaling (Petrakis and Sklaviadis, 2006) and is thought to be involved in several functions including cell proliferation and differentiation (Lee and Baskakov, 2010), cell adhesion (Schmitt-Ulms et al., 2001), and protection against oxidative stress (Cazaubon et al., 2007). PrPC binds copper through its N-terminal domain that contains 4 to 5 repeats of 8 residues (PHGGGWGQ) (Brown et al., 1997; Rachidi et al., 2005). Since free copper ions are highly toxic because they contribute to the generation of ROS via Fenton chemistry, the binding of copper ions through the octarepeat region of PrPC is thought to provide an antioxidant activity in mouse neural crest-derived cells and in human HeLa cells (Qin et al., 2009). Copper is an essential trace metal for the development and maintenance of cells. It plays a key role in the catalytic site of several enzymes (e.g. cytochrome oxidase, Cu/Zn superoxide dismutase) through its property to change their oxidation state (Balamurugan and Schaffner, 2006). On the other side, an excess of copper may generate reactive oxygen species (ROS) (Jomova and 2

Valko, 2011). To keep cellular copper levels well balanced, organisms have developed several protective mechanisms to control the uptake, distribution, detoxification and elimination of copper. These mechanisms are not fully efficient and ROS can still damage various molecules such as lipids, proteins and nucleic acids. Cellular senescence, first described in vitro by Hayflick in the 1960’s, is defined as an irreversible arrest of the cellular divisions, associated with a « critical » shortening of the telomeres [for a review, (Campisi and d'Adda di Fagagna, 2007)], and for this reason also called “replicative senescence”. Senescent fibroblasts are characterized by several biomarkers such as typical morphology, senescence-associated beta-galactosidase activity (SA-gal) (Dimri et al., 1995), altered gene expression (Dumont et al., 2000), senescence-associated heterochromatin foci (SAHFs), senescence-associated DNA-damage foci (SDFs) (d'Adda di Fagagna et al., 2003) and lamin B1 loss (Freund et al., 2012). The combined search of p16INK-4A and SA-βgal has allowed to detect cells with characteristics of senescence in vivo, and it was shown that they accumulate with age in multiple tissues from both humans and rodents. Moreover, these cells are present at sites of certain age-related pathologies, including atherosclerotic lesions, skin ulcers and arthritic joints, as well as benign and preneoplastic hyperproliferative lesions in the prostate and liver (Krtolica and Campisi, 2002). Recently, Baker et al. showed that the presence of senescent cells in vivo are involved in age-related disorders as sarcopenia and cataract in mice (Baker et al., 2016; Baker et al., 2011). Therefore, cellular senescence is now recognized as one of the nine hallmarks of aging (Lopez-Otin et al., 2013), but the mechanisms responsible for its occurrence are still largely unknown (Toutfaire et al., 2017). A variety of stresses including exposure to oxidative stress and generation of DNA damage induce a senescent-like phenotype, called stress-induced premature senescence or SIPS (Debacq-Chainiaux et al., 2016; Toussaint et al., 2002). Since Hayflick’s experiments, fibroblast is the most studied cell type in senescence, and has the advantage to exhibit most of the established, ubiquitous hallmarks of ageing (Tigges et al., 2014). A link between cellular copper homeostasis and aging was previously shown in the fungal aging model Podospora anserina and in human diploid fibroblasts (HDFs) (Scheckhuber et al., 2009). In old cultures of P. anserina, several phenotypic changes occur which are collectively referred to as the “senescence syndrome” (Scheckhuber and Osiewacz, 2008). It has been demonstrated that a decrease of copper concentration in culture medium of P. anserina results in lifespan extension (Gredilla et al., 2006). In particular, impairments the activity of mitochondrial cytochrome c oxidase, a copper containing protein complex of the respiratory chain, induces the expression of an alternative oxidase, especially efficient in reducing the generation of mitochondrial ROS generation and in increasing lifespan (Gredilla et al., 2006; Stumpferl et al., 2004). One of the corresponding mutants (grisea) that is affected in copper-uptake is characterized by a changed gene expression profile (Servos et al., 2012). In addition, an intracellular change in copper distribution occurs in P. anserina during senescence leading to age-related changes in the expression of copper-regulated 3

genes (Philipp et al., 2013; Scheckhuber et al., 2009). As the proteins and mechanisms implicated in copper metabolism are highly conserved throughout evolution (Bleackley and Macgillivray, 2011), the potential role of cellular copper homeostasis was investigated in cellular senescence. Overexpression of copper-related genes was found in senescent HDFs suggesting that cellular copper levels also increase during senescence in mammalian cells, which was confirmed by several methods. Moreover, it was shown that the incubation of WI-38 HDFs with a sublethal concentration of copper sulfate (CuSO4) leads to the premature appearance of some senescence biomarkers (Boilan et al., 2013). In this work, we show that PrPC is a putative biomarker of aging and is implicated in cellular senescence. The potential role of PrP was subsequently investigated in senescence induced by copper by using siRNA to knockdown PrP gene expression.

2. Materials and methods

2.1. WI-38 HDFs culture and exposure to CuSO4 WI-38 fetal lung HDFs (American Type Culture Collection, #CCL-75) were cultivated as described in (Dumont et al., 2000). Sub-confluent young WI-38 HDFs at about 60 % of in vitro proliferative lifespan (± 27 population doublings) were plated at 14,000 cells/cm2 in Basal Medium Eagle (BME, Gibco) + 10 % fetal bovine serum (FBS, Gibco, USA). At 24 hours after seeding, the cells were incubated in the culture medium with 500 µM of CuSO4 (CuSO4.5H2O, UCB) for 16 hours in a cell incubator (HERAcell 240, Qlab) (Boilan et al., 2013). Controls were incubated in BME + 10 % FBS. After treatment, cells were washed once with phosphate buffer saline (10 mM phosphate, 155 mM NaCl, pH 7.4 (PBS), Lonza) and incubated with BME + 10 % FBS.

2.2. RT-PCR At different times after the end of the incubation period with CuSO4, total RNA from WI-38 HDFs was isolated using “TRI Reagent Solution” method (AB AM9738, Ambion). Total RNA (2 µg) was reversed transcribed using First Strand cDNA synthesis kit for RT-PCR (Roche). PCR amplification primers (IDT and Applied Biosystems) used are: 23 kDa (GCC TAC AAG AAA GTT TGC CTA TCT G-TGA GCT GTT TCT TCT TCC GGT AGT) and PrP (ATG ATG GAG CGC GTG GTT-CAT GCT CGA TCC TCT CTG GTA A). PCR mixture contained SYBR Green PCR Mastermix (Applied Biosystem) and primers at optimal concentrations. A hot start at 95°C for 5 minutes was performed, then 40 cycles at 95°C for 15 seconds and at 65°C for 1 minute were repeated using the ABI PRISM 7000 SDS Thermal Cycler (Applied Biosystem). The abundance of 23 kDa mRNA was used as housekeeping gene. Relative abundances were determined based on Ct difference considering individual amplification efficiencies (Schefe et al., 2006). Three biological replicates 4

were performed.

2.3. Extraction of proteins Extraction of total proteins was performed at different times after the end of the incubation period with CuSO4. WI-38 HDFs were washed with ice-cold PBS and scrapped in lysis buffer DLA (7M Urea, 2M Thiourea, 4 % Chaps, 30 mM Tris, 60 mM DTT and 0,16 % PIC). The lysates were agitated on a shaking plate for 25 minutes at room temperature (RT), sonicated for 5 minutes and centrifuged at 13,000 rpm for 15 minutes. The supernatants were collected and frozen at -80°C. Extraction and separation of nuclear and cytoplasmic proteins were performed immediately after the incubation period with CuSO4 in 75 cm2 flasks. Cells were washed with PBS containing 1 mM Na2MoO4 and 5 mM NaF, incubated on ice for 8 minutes with 10 ml cold Hypotonic Buffer (HB, 20 mM HEPES, 5 mM NaF, 1 mM Na2MoO4 0.1 mM EDTA) and scrapped in 100 μl Hypotonic Buffer containing 0.2% NP-40 (Sigma), a protease inhibitor cocktail (1:25) and phosphatase inhibitors (1:25). Lysates were centrifuged for 30 seconds at 13,000 rpm. Sedimented nuclei were resuspended in 15 μl HB containing 20% glycerol and protease/phosphatase inhibitors. Extraction was performed for 30 minutes at 4°C by the addition of 15 μl HB containing 20% glycerol, 0.8 M NaCl and protease/phosphatase inhibitors.

2.4. Proteinase K treatment In order to differentiate PrPC and PrPSc forms of PrP, protein extracts were incubated with increasing concentrations of Proteinase K (Sigma) (0, 25, 50 and 100 µg/ml) for 30 minutes at 37°C.

2.5. Western blot analysis Proteins were assessed (Pierce method, Sigma) before electrophoresis. 10 µg of proteins were separated on standard NU-PAGE 4-12% Bis-Tris (MES) gels with SeeBlue® Plus2 Pre-Stained Standard (Invitrogen #LC5925). After migration, proteins were transferred to pure nitrocellulose membranes (BioRad) for Licor revelation. After a 2 hours blocking in PBS and Licor blocking buffer (v:v) (Odyssey Infrared Imaging System Licor Biosciences), the membrane was incubated with the primary antibody for 2 hours, in Licor blocking buffer-Tween 0.1 %. The antibodies used are: Antiα-tubulin antibody (Sigma #T5168, 1/7500) and Anti-Prion protein antibody (AjRoboscreen #0102000701, 1/1000). After incubation, the membranes were washed with PBS-Tween 0.1 % for 5 minutes, 4 times. Membranes were then incubated with the secondary antibody for 1 hour, in Licor blocking buffer-Tween 0.1 %. The binding of antibodies was visualized using fluorescent probe-coupled secondary antibodies: anti-mouse Odyssey Infrared Imaging System Licor Biosciences #926-32210, (1/7500). After incubation, the membrane was washed with PBS-Tween 0.1 % for 5 minutes, 4 times and PBS alone twice for 5 min. Revelation of dried membrane was 5

performed by Odyssey Infrared Imaging System Licor Biosciences software (Odyssey Infrared Imaging System Licor Biosciences). Three biological replicates were performed.

2.6. Confocal microscopy WI-38 HDFs were seeded at a density of 20,000 cells/cm2 in BME + 10 % FBS on cover glasses in a 24-well culture plate. At 24 hours, the cells were incubated with CuSO4. After 16 hours of incubation with CuSO4, the cells were washed with ice-cold PBS and fixed for 10 minutes using 4 % paraformaldehyde (Merck, Germany) in PBS. Cells were then washed 3 times with freshly made PBS and permeabilized with Triton X-100 1 % (Merck) in PBS for 5 minutes at RT. After 3 washings in PBS containing 2 % of Bovine Serum Albumin (BSA) (Sigma), cells were incubated with the primary Anti-Prion protein antibody (AjRoboscreen #0102000701, 1/200) in PBS-BSA 2 %. Cells were then washed 3 times with PBS-BSA 2 %. The binding of antibodies was visualized using secondary antibodies coupled with Alexa Fluorescent dye 488 nm (anti-mouse Alexa 488-Ab Molecular probes #A-11001, 1/500). After 3 washings with PBS-BSA 2 % and 1 with PBS, nuclei were marked with TOPRO-3 (Molecular probes) and cells were washed 3 times with PBS. Cover glasses were mounted in Mowiol (Molecular probes) on microscope slides and were incubated overnight at 4°C. The cells were analyzed using a TCD confocal laser microscope (Leica) equipped with appropriate filters. Three biological replicates were performed.

2.7. PrP siRNA transfection Cells were seeded at 12,000 cells/cm2 and then incubated with siRNA corresponding to the human gene coding for PrP (SMARTpool siRNA M-011101-01-0010, Dharmacon) or with siRNA negative control Non-targeting smart pool (NTS) for 24 hours (Dharmacon, cat. #D-001206-13-20). siRNAs were mixed with Opti-MEM medium (Gibco) and BME + 10 % FBS to obtain a final concentration of 50 nM. Dharmafect was used as a transfection reagent, following the manufacturer’s instructions (Dharmacon). At 24 hours after siRNA incubation, the cells were washed with PBS and the medium was changed for BME + 10 % FBS for 8 hours before CuSO4 incubation (Fig. 3a).

2.8. Senescence biomarkers 2.8.1. Senescence Associated β-galactosidase activity (SA-βgal) Cells were seeded in 6-well culture plates at 20,000 cells/well at 24 hours after the end of the CuSO4 incubation period. SA-βgal was determined 48 hours later, as described in (DebacqChainiaux et al., 2009). The population of SA-βgal positive cells was determined by counting 400 cells per well. The proportion of positive cells was given as the percentage of total number of cells counted in each well. Three biological replicates were performed. 2.8.2. [3H]-thymidine incorporation Cells were seeded in a 24-well culture plate at 10,000 cells/well 24 hours after the end of the 6

CuSO4 incubation period. 24 hours after seeding, cells were grown in 1 ml of BME + 10 % FBS supplemented with 1 µl [3H]-thymidine (specific activity: 2 Ci/mmol, Du Pont NEN) for 24 hours in 5 % CO2. After incubation, cells were treated as described in (Dumont et al., 2000). Data were normalized to the cellular protein content by Pierce method assay. Three biological replicates were performed.

2.9. Statistical analysis All statistical analyzes were performed using the SigmaPlot 12.5 software. Data are reported as mean ± 1 standard deviation (± 1 SD). Depending on the data to compare (two or many groups), statistical differences between means were evaluated by either two-tailed paired t-test or by twoway ANOVA with Holm-Sidak test as post hoc test. Normality test and equal variance test passed. A P < 0.05 was considered statistically significant (*, #, 0.01 < P < 0.05; **, ##, 0.001 < P < 0.01; ***, ###, P < 0.001). Corresponding statistical tests are outlined in figure captions. 3. Results

3.1. PrP overexpression in copper-induced premature senescence We previously showed that PrP is overexpressed in replicative senescence and in UVB-SIPS in HDFs (Scheckhuber et al., 2009). In order to confirm the link between PrP and copper, we studied the expression of PrP in copper-induced premature senescence of human fibroblasts. WI-38 HDFs were incubated for 16 hours with CuSO4 at 500 µM, conditions that induce premature senescence (Boilan et al., 2013; Scheckhuber et al., 2009). We detected an increase of PrP mRNA abundance of 3.7 ± 0.3 just after the end of the incubation with CuSO4 in comparison with control (CTL) cells (Fig. 1a). By western blot analysis we found an increase of PrP total protein abundance just after the end of the incubation with CuSO4 (Fig. 1b). In this analysis, we detected several bands corresponding to PrP. These bands probably correspond to glycosylated or phosphorylatedforms of PrP (Giannopoulos et al., 2009; Otvos and Cudic, 2002). The PrP forms were characterized in the following section.

3.2. Cellular localization and characterization of PrP in WI-38 HDFs PrP is often described as a plasma membrane protein (Linden et al., 2008). We analyzed the cellular localization of PrP after 16 hours of incubation with CuSO4. Surprisingly, the analysis of immuno-labeled PrP by confocal microscopy showed a cytoplasmic and a (peri)nuclear localization in WI-38 HDFs (Fig. 2a). An increase in the protein abundance was also observed, confirming the western blot analysis (Fig. 1b). To confirm the localization of PrP, cytoplasmic and nuclear extracts were investigated by western blot analysis. PrP was found in both extracts (Fig. 2b), with an increased abundance after CuSO4 incubation. Again, several bands were detected for PrP in both

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extracts. Next we tested whether CuSO4 incubation induces an increase of PrPC form (constitutive expression in the cells) or of PrPSc form (misfolded form of PrPC) using Proteinase K (PK) digestion (Fig. 2c). We showed the bands corresponding to PrP protein disappeared after PK treatment at 50 or 100 µg/ml. This confirms that it is the constitutive form of PrP (PrPC) which is expressed in WI38 HDFs with or without CuSO4 incubation. 3.3. PrP silencing induces premature senescence of WI-38 HDFs with or without CuSO4 treatment In order to study the potential involvement of PrP in CuSO4-induced premature senescence, we did a knockdown of its expression by using specific siRNA. The experimental procedure, described in Materials and Methods, is shown in Fig. 3a. PrP mRNA level decreased by 96.8 ± 5.5% or by 90.6 ± 5.9% in siRNA transfected cells (CTL or CuSO4 respectively) compared to non-transfected CTL cells (Fig. 3b). This decrease in PrP expression following siRNA transfection was confirmed at the protein level by western blot analysis in total extracts (Fig. 3c). PrP expression completely disappeared in siRNA-transfected cells with or without CuSO4 treatment. WI-38 HDFs incubated for 16 hours with CuSO4 enter premature senescence as revealed by an increase of SA-βgal positive cells and growth arrest (Boilan et al., 2013; Scheckhuber et al., 2009). In a next series of experiments we first confirmed that CuSO4 treatment induces a senescent morphology, an increase in the proportion of SA-βgal positive cells and a reduced proliferation in non-transfected CTL cells (Fig. 4a, 4b, 4c, 4d) as previously detected (Boilan et al., 2013). Larger cells with a larger flattened cytoplasm containing many vacuoles and cytoplasmic filaments characterize senescent morphology (Bayreuther et al., 1988; Cristofalo and Kritchevsky, 1969). Surprisingly, we detected that PrP silencing by itself induces the expression of several biomarkers of senescence, even in control cells not exposed to CuSO4 (Fig. 4a, 4b, 4c, 4d). First, a clear senescent morphology is detected in PrP siRNA transfected control cells (Fig. 4a). Secondly, the percentage of positive cells for SA-βgal increased from 12.7 ± 2.9 % in non-transfected control cells to 28.6 ± 7.1 % in PrP siRNA transfected control cells (Fig. 4b, 4c), level comparable after the CuSO4 treatment. Thirdly, a significant decrease in proliferative potential was detected in PrP siRNA transfected control cells (Fig. 4d). These different biomarkers of senescence were even more induced after CuSO4 incubation in PrP siRNA transfected cells. These results suggest that knockdown of PrP expression induces by itself premature senescence.

4. Discussion

Metal binding proteins are important regulators of cellular homeostasis. Among these, PrP has been identified as a copper binding protein. PrPC binds copper via an octarepeat region within the N-terminal part of the protein. The octarepeat region of PrPC is able to reduce Cu(II) to Cu(I) in vitro (Opazo et al., 2003). PrPC can be considered as a transporter of copper, a sink for excess copper, 8

a copper-dependent receptor, or a scavenger of Cu(II) generated free radicals (Viles et al., 2008). PrP and the amyloid precursor protein (APP), another copper binding protein, are both related to neurodegenerative diseases (Creutzfeldt-Jakob disease and Alzheimer’s disease respectively). Their function as copper binding protein must certainly play a role in the neurodegenerative process, probably by influencing the oxidant and anti-oxidant balance. Neurodegenerative diseases and among them Prion diseases are age-related diseases (Prusiner, 1998). However, few studies have examined the potential role of PrP in cellular senescence. PrP is overexpressed in replicative senescence and in SIPS (Scheckhuber et al., 2009). An incubation of WI-38 HDFs with a sublethal concentration of CuSO4 induces premature senescence and an overexpression of copper-related genes as PrP in HDFs (Boilan et al., 2013; Matos et al., 2012; Scheckhuber et al., 2009). Nothing had been published so far concerning the role of PrP in premature senescence of normal HDFs. We showed here that the abundance of PrP is increased in normal HDFs after incubation with CuSO4 at mRNA and protein levels. We detected many bands representing probably the unglycosylated, mono- and di-glycosylated forms of PrP. The induction of PrP gene expression by copper was shown in different cell types (Canello et al., 2012; Varela-Nallar et al., 2006). VarelaNallar et al. reports that copper induces PrPC expression in primary neurons (Varela-Nallar et al., 2006). The characterization of PrPC induced by copper presents a normal glycosylation pattern, sensitivity to proteinase K confirming that it is the PrPC form, and localization of PrPC at the cell surface and in an intracellular compartment identified as the Golgi complex (Varela-Nallar et al., 2006). In the current study, we characterized the cellular localization of PrPC induced by CuSO4 and observed the presence of PrP in the nuclear and cytoplasmic compartments of HDFs. The presence of PrP in nuclei was confirmed by western blot analysis. In the literature, the cellular localization of PrPC remains a subject of debate. PrPC is described in different cellular compartments in different cell types. In endocrine and neuronal cells, Strom et al. discovered that PrP is localized in the nucleus and interacts with histones and lamin B1 and these interactions suggest that PrP would be involved in transcriptional regulation in the nucleus (Strom et al., 2011). It has been shown that lamin B1 depletion in senescent cells triggers large-scale changes in gene expression and the chromatin landscape (Shah et al., 2013) and that the redistribution of the lamin B1 genomic binding profile affects rearrangement of heterochromatic domains and SAHF formation during senescence (Sadaie et al., 2013). All this information suggests a potential link between PrP and senescence. Mange et al. showed that PrPSc accumulates in the nuclei of Prion-infected cells independently of proteasome activity and interacts with chromatin in vivo. The presence of PrPSc in the nucleus required an intact microtubule network (Mange et al., 2004). We characterized the conformational form of PrP after incubation with CuSO4 with sensitivity to 9

proteinase K digestion, which means PrP induced by copper in our model is the PrPC form. Leclerc et al. showed that copper induced conformational changes in the N-terminal part of the cell-surface PrP (Leclerc et al., 2006). However, the consequences of copper binding on PrP function are unclear. Copper seems to be able to modify aggregation properties of PrP but the mechanisms are not completely understood (Thakur et al., 2011). To investigate the potential role of PrP in senescence of WI-38 HDFs, we did a knockdown of the PrP gene expression using a specific siRNA and subsequently incubated cells with CuSO4. We observed that incubation with PrP siRNA induced the appearance of senescence biomarkers like senescent morphology, an increase in the proportion of SA-βgal positive cells and a decrease of the proliferative potential independently of CuSO4 presence. This appears to be in contradiction with the fact that PrP was previously described to play a protective role against oxidative stress. PrP seems to have a superoxide dismutase (SOD)-like function and this activity was supported by the binding of a single Cu(II) ion to the PrP octapeptide repeat region (Mange et al., 2004). Watt et al. showed that the expression of PrPC protects human neuroblastoma cells against DNA damage under basal and oxidative stress conditions (Watt et al., 2007). This property is influenced by the presence and the integrity of the octapeptide repeats of PrPC and is lost in disease-associated mutants of the protein or upon conversion to PrPSc. Recent studies revealed that PrP plays multiple roles, notably in proliferation, cell-cell adhesion, signal transduction and nucleic acid folding. The in vitro studies indicated that PrP is able to bind nucleic acid and possesses annealing activity, similarly to nucleic acid chaperone proteins playing essential roles in DNA and RNA metabolism (Guichard et al., 2011). We hypothesize that the protective role of PrPC against oxidative damage becomes progressively defective during aging. This dysfunction can result from conformational modifications, posttranslational modifications, a link with chelating-proteins or with PrP, etc. It could be possible that a migration of PrP into a particular cell compartment occurs during aging and the consequences of this phenomenon would be a loss of PrP functions in basal conditions and an increase of sensitivity to stress conditions. Interestingly, some of the glycosylation modifications of PrPC during aging resemble to those found on PrPSc (Lawson et al., 2005; Rudd et al., 2001). Goh et al. (Goh et al., 2007) proposed that glycosylation patterns of PrPC are related to aging, PrPSc and the loss of PrPC functions. For example, it was shown in transgenic mice overexpressing PrPC that aged mice exhibited an aberrant mitochondrial localization of PrPC concomitant with cytochrome c release into the cytosol, caspase-3 activation, DNA fragmentation, and decreased proteasomal activity, while younger mice did not (Hachiya et al., 2005). In conclusion, our study shows a novel indirect link between the PrPC and senescence mechanisms. PrPC seems to play a protective role against premature senescence. The characterization of PrPC in senescent cells could help to understand the potential loss of function of 10

PrPC during aging and the apparition of other neurodegenerative disorders as PrPC seems to regulate the production of the neurotoxic amyloid-beta peptide in Alzheimer’s disease (Parkin et al., 2007). Acknowledgements E. Boilan is a recipient of “Fonds de la Recherche dans l'industrie et l'agriculture (FRIA)”, Belgium. O. Toussaint and F. Debacq-Chainiaux are respectively Senior Research Associate and Research Associate of the FNRS, Belgium. We thank the European Commission for large-scale collaborative projects “MyoAge”, contract HEALTH-F2-2009-223576, and “Markage”, contract HEALTH-20072.2.2-3 from the Seventh FP. The authors declare no conflict of interest.

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Legends of the figures

Figure 1: Overexpression of PrP in copper-induced premature senescence in WI-38 HDFs (a) Abundance of PrP mRNA in WI-38 HDFs after 16 hours of incubation with 0 (CTL) or 500 μM of CuSO4 using Real Time RT-PCR. The abundance of 23 kDa mRNA was used as reference. Results (means of biological triplicates ± 1 SD) are expressed by comparison with the mRNA abundance of the respective mRNA species in CTL cells. Statistical analysis was performed by two-tailed paired t-test. **, 0.001 < P < 0.01. (b) Western blot analysis of PrP after 16 hours of incubation with 0 (CTL) or 500 μM of CuSO 4. Protein abundance of α-tubulin was used to assess protein loading.

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Figure 2: Cellular localization and characterization of PrP in WI-38 HDFs (a) Immunofluorescence detection of PrP (green) after 16 hours of incubation with 0 (CTL) or 500 μM of CuSO4. TOPRO-3 dye was used to detect nuclei (blue). (b) Western blot analysis of PrP after 16 hours of incubation with 0 (CTL) or 500 μM of CuSO4. Recombinant Prion protein (PrPrec, AjRoboscreen #0101000301) was added in the last lane as control. Protein abundance of α-tubulin was used to assess protein loading. (c) Western blot analysis of PrP after 16 hours of incubation with 0 (CTL) or 500 μM of CuSO4. The same protein extracts were treated with different concentrations (0, 25, 50 and 100 μg/ml) of Proteinase K (PK) to differentiate PrPC (constitutive) from PrPSc (scrapie) form of Prion protein.

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Figure 3: Silencing of PrP expression by specific siRNA in WI-38 HDFs (a) Experimental design and time frame of CuSO4 incubation. (b) mRNA abundance of PrP in cells after 16 hours of incubation with 0 (CTL) or 500 μM of CuSO4 in non-transfected cells, in transfected cells with specific PrP siRNA or with non-targeting siRNA by Real Time RT-PCR. mRNA abundance of 23 kDa was used as reference. Results (means of biological triplicates ± 1 SD) are expressed by comparison with the mRNA abundance of the respective mRNA species in non-transfected CTL cells. Statistical analysis was performed by twoway ANOVA and Holm-Sidak test as post hoc test. * CuSO4 versus corresponding CTL, # CTL versus other CTL. #, 0.01 < P < 0.05; *** P < 0.001. (c) Western blot analysis of PrP after 16 hours of incubation with 0 (CTL) or 500 μM of CuSO4, in non-transfected CTL cells, transfected cells with specific PrP siRNA or with non-targeting siRNA (transfected CTL cells). Protein abundance of α-tubulin was used to assess protein loading.

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Figure 4: PrP siRNA induces premature senescence in WI-38 HDFs with or without CuSO4 incubation (a) Morphology of cells at 72 hours after the end of incubation with 0 (CTL) or 500 μM of CuSO4 for 16 hours, in non-transfected cells, in transfected cells with specific PrP siRNA or with non-targeting siRNA, using optical microscope with phase contrast (scale bar = 100 µm). (b) Histochemical detection of SA-ßgal at 72 hours after the end of incubation with 0 (CTL) or 500 μM of CuSO4 for 16 hours, in non-transfected cells, in transfected cells with specific PrP siRNA or with non-targeting siRNA, using optical microscope with phase contrast (scale bar = 100 µm). (c) Percentage of positive cells for SA-βgal at 72 hours after the end of the incubation with 0 (CTL) or 500 μM of CuSO4 for 16 hours, in non-transfected cells, in transfected cells with specific PrP siRNA or non-targeting siRNA. Results (means of biological triplicates ± 1 SD) are expressed in percentage of blue cells. Statistical analysis was performed by two-way ANOVA and Holm-Sidak test as post hoc test. * CuSO4 versus corresponding CTL, # CTL versus other CTL. *, #, 0.01 < P < 0.05; ** 0.001 < P < 0.01 (d) Estimation of proliferative potential by incorporation of [ 3H]-thymidine at 72 hours after the end of incubation with 0 (CTL) or 500 μM of CuSO4 for 16 hours, in non-transfected cells, in transfected cells with specific PrP siRNA or non-targeting siRNA. Results (means of biological triplicates ± 1 SD) are expressed in percentage compared to values for non-transfected CTL cells (100%). Statistical analysis was performed by two-way ANOVA and Holm-Sidak test as post hoc test. * CuSO4 versus corresponding CTL, # CTL versus other CTL. ***, ###, P < 0.001.

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