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Keywords:

  • cell signaling;
  • cellular prion protein;
  • filopodia;
  • gene expression;
  • microarray;
  • proliferation

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Funding
  8. References
  9. Supporting Information
Thumbnail image of graphical abstract

The prion protein (PrP) plays a key role in prion disease pathogenesis. Although the misfolded and pathologic variant of this protein (PrPSC) has been studied in depth, the physiological role of PrPC remains elusive and controversial. PrPC is a cell-surface glycoprotein involved in multiple cellular functions at the plasma membrane, where it interacts with a myriad of partners and regulates several intracellular signal transduction cascades. However, little is known about the gene expression changes modulated by PrPC in animals and in cellular models. In this article, we present PrPC-dependent gene expression signature in N2a cells and its implication in the most overrepresented functions: cell cycle, cell growth and proliferation, and maintenance of cell shape. PrPC over-expression enhances cell proliferation and cell cycle re-entrance after serum stimulation, while PrPC silencing slows down cell cycle progression. In addition, MAP kinase and protein kinase B (AKT) pathway activation are under the regulation of PrPC in asynchronous cells and following mitogenic stimulation. These effects are due in part to the modulation of epidermal growth factor receptor (EGFR) by PrPC in the plasma membrane, where the two proteins interact in a multimeric complex. We also describe how PrPC over-expression modulates filopodia formation by Rho GTPase regulation mainly in an AKT-Cdc42-N-WASP-dependent pathway.

In this study, we analyzed the PrPC-dependent gene expression signature of neuroblastoma (N2a) cells after transient acute up-regulation and down-regulation of PrPC. We demonstrate that PrPC plays roles in proliferation and neuritogenesis through modulation of EGFR activity. This approach will give new insights into the molecular mechanisms by which PrPC regulates key cellular functions in cell physiology.

Abbreviations used
AKT/PKB

protein kinase B

cdc42

cell division cycle 42

EGF

epidermal growth factor

EGFR

epidermal growth factor receptor

ERK1/2

extracellular signal-regulated kinases

FBS

fetal bovine serum

N2a

Neuro2a

N-WASP

Neural Wiskott–Aldrich syndrome protein

PrPC

cellular prion protein

PrPSC

scrapie prion protein

SDS-PAGE

sodium dodecyl sulfate–polyacrylamide gel electrophoresis

The normal, cellular isoform of the prion protein, PrPC, is a highly conserved glycoprotein tethered to the cell membrane by a glycosyl phosphatidyl inositol anchor (GPI) (see Linden et al. 2008; Aguzzi et al. 2008 for review). Expression of PrPC is required for prion propagation, since deletion of the Prnp gene in mice, specifically in neurons, entails complete protection against scrapie infection (Bueler et al. 1993). Many efforts have been made to increase knowledge of PrPSC, the misfolded and pathogenic form of PrPC, and to determine the molecular mechanisms by which the prion may promote the misfolding of more protein into PrPSC. However, understanding the physiological role of PrPC remains a challenging issue, because of the many roles described for it (Linden et al. 2008). It has been reported that PrPC plays a role in several cellular processes, including proliferation and cell–cell adhesion (Aguzzi et al. 2008; Linden et al. 2008). Moreover, PrPC has been found to be a copper-binding protein (Brown et al. 1997), which reveals new physiological functions as an antioxidant and in cell homeostasis (Viles et al. 2008). Several lines of research point to a role in neuroprotection against a wide range of insults, such as excitotoxicity and serum starvation (Llorens and Del Rio 2012).

Studies using various mouse models have demonstrated the pleiotropic phenotype of the Prnp gene that extends from changes in gene expression profiles to particular cellular functions (Steele et al. 2007; Rangel et al. 2009; Benvegnu et al. 2011). In fact, Prnp−/− mice present cognitive deficits, depressive-like behavior, anxiety-related disorders and alterations in circadian activity (Steele et al. 2007). In contrast, Prnp-over-expressing mice present some deficiencies, such as cell degeneration in the PNS and CNS at more advanced stages (Westaway et al. 1994) but without the age-related behavioral impairments observed in knockout mice, some immunological defects (Jouvin-Marche et al. 2006), and enhanced synaptic facilitation (Rangel et al. 2009). In addition, data obtained from mice expressing eGFP under the Prnp promoter (Bailly et al. 2004; Barmada et al. 2004) as well as Prnp-over-expressing mice (Westaway et al. 1994) showed some differences in PrPC expression. This reinforces the idea that Prnp expression can be modulated in specific cells by a highly regulated process during development and adult life (Benvegnu et al. 2011). To gain insight into the molecular mechanisms under the control of PrPC expression, multiple gene expression approaches have been carried out in transgenic Prnp mice (Rangel et al. 2009; Chadi et al. 2010; Benvegnu et al. 2011) and in neuronal and non-neuronal stably transfected cell lines (Satoh et al. 2000; Satoh and Yamamura 2004). In these studies, there was no coincidence in gene regulation data, and there was a lack of shared signaling pathways over-represented by gene expression changes. This reinforces the notion that the role of PrPC may depend on the cell type and its environment.

In parallel, a lack of knowledge about the events triggered by PrPC in baseline conditions and its expression changes in physiological conditions hinders understanding of the molecular mechanisms under its regulation. Several signaling pathways have been implicated in the transmission of the effects downstream from PrPC, mainly through its interaction with extracellular partners (Santuccione et al. 2005; Santos et al. 2011) and neurotransmitter receptors (Khosravani et al. 2008; Carulla et al. 2011). Indeed, it is suggested that PrPC may be located in specialized membrane environments such as lipid rafts (Harmey et al. 1995; Taylor and Hooper 2006; Lewis and Hooper 2011) and in post-synaptic density (Carulla et al. 2011) forming multimeric complexes with several interacting partners involved in multiple cellular functions. In this regard, PrPC clustering, as a model of PrPC-mediated signaling, modulates the downstream activation of Fyn-ERK1/2 and protein kinase B (AKT) pathways (Mouillet-Richard et al. 2000; Santuccione et al. 2005; Toni et al. 2006).

In this study, we analyzed the PrPC-dependent gene expression signature of neuroblastoma (N2a) cells after transient acute up-regulation and down-regulation of PrPC. To functionally validate the transcriptomic approach, we analyzed the implication of PrPC expression in two of the molecular functions over-represented in our gene expression analysis: cell growth and proliferation, and cell shape maintenance. Finally, to evaluate the molecular mechanisms regulated by PrPC, we examined the activation of ERK1/2 and AKT signaling pathways and the role of PrPC in epidermal growth factor/epidermal growth factor receptor (EGF/EGFR)-dependent mitogenic stimulation and neurite formation in an AKT-Cdc42-N-WASP-dependent pathway.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Funding
  8. References
  9. Supporting Information

Reagents and antibodies

EGF, cytochalasin D, and wortmannin were from Sigma (Poole, UK). Cycloheximide, anti-cyclin D1, anti-SNAP25, anti-p-EGFR(Tyr1173), total anti-ERK, anti-p-p44/42 MAPK(Erk1/2)(Thr202/Tyr204), anti-p-AKT-1(Ser473), anti-PARP, anti-Grb2, and anti-p-Src(Tyr416) were from Cell Signalling (Beverly, MA, USA). Anti-cyclin E, anti-Cdk2, anti-Egr1, anti-p-Elk1(Ser383),and anti-AKT were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), anti-Gap43, anti-actin, and anti-tubulin were from Millipore (Billerica, MA, USA) and anti-c-fos was from Calbiochem (Darmstadt, Germany). Anti-PrP-SAF61 (immunoprecipitation/immunofluorescence/PrPC clustering) was from Spi-Bio&Cayman Chemical (Massy Cedex, France) and anti-PrP-6H4 (western blot) was from Prionics (Schlieren, Switzerland). Anti-Ki67-MM1 was from Novocastra (Newcastle-upon-Tyne, UK). Anti-Flotilin-1 was from BD Bioscience (San Diego, CA, USA).

Cell culture and treatments

PrPC siRNA used for prion silencing and the Scramble siRNA sequence used as a control were obtained from (Daude et al. 2003) and were synthesized by Dharmacon (Lafayette, LA, USA). pcDNA3.1-PrPC and pcDNA3.1-PrP-ΔF35 were kind gifts from Prof. D. Harris (Boston University) and Prof. A. Aguzzi (University Hospital of Zurich). N2a (N2a) and SK-N-SH (SK) neuroblastoma cell lines with low PrPC expression levels were grown at 37°C, 5.5% CO2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 4.5 g/L glucose, 10% fetal bovine serum (FBS), and antibiotics (all from Invitrogen-Life Technologies, Barcelona, Spain).

For siRNA transfection, cells were plated at 0.25 × 106 cells/mL and transfected the next day using Lipofectamine 2000 reagent in Optimem™ medium, following the manufacturer's instructions (Invitrogen-Life Technologies). Four hours after transfection, cells were washed and the medium was replaced with DMEM containing 10% FBS. PrPC up-regulation and down-regulation was checked by western blot analysis and RT-qPCR. For serum restimulation experiments, the day after transfections cells were serum-starved for 24 h and restimulated with medium containing 10% FBS or EGF (150 ng/μL) for the indicated times.

For N2a differentiation analysis, transfected cells were serum-starved for 24 h and fixed in 4% paraformaldehyde for 10 min at 20°C. Coverslips were processed for immunofluorescence assays, as indicated.

Cell proliferation analysis

The proliferation of N2a cells was analyzed using the colorimetric WST-1 assay (Roche, Mannheim, Germany) and Ki67 labeling. WST-1 ((4-[3-(4-Iodophenyl)-2H-5-tetrazolio)]-1,3-benzene-disulfonate) is cleaved to formazan salts, which are soluble in the culture medium and analyzable in a PowerWave™ XS Microplate spectrophotometer (Biotek® Instruments, Inc., Winooski, CA, USA). Ki67 analysis was carried out using the Ki67-MM1 monoclonal antibody, which recognizes the Ki67 antigen that is only present during active phases of the cell cycle and absent in G0.

Microarray analysis

Total RNA for the microarray analysis was quantified with a NanoDrop ND-1000 spectrophotometer, followed by quality assessment with the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), according to the manufacturer's instructions. Acceptable quality values were in the 1.8–2.2 range for A260/A280 ratios, > 0.9 for the rRNA ratio (28S/18S) and > 8.0 for RIN (RNA Integrity Number). Biotinylated cRNA was prepared using the Illumina RNA Amplification Kit (Ambion, Austin, TX, USA), according to the manufacturer's instructions, starting with 200 ng total RNA from each sample. cRNA was purified and each sample was hybridized on Illumina mouse-6 v1.1 expression bead chips, following the manufacturer's instructions. After 16 h of hybridization, the arrays were washed, dried, stained with Cy3-Streptavidin, and scanned using Illumina BeadScan software on the Illumina BeadArray scanning system (Illumina, Inc., San Diego, CA, USA). The raw data were summarized per probe using BeadStudio Gene Expression software, and the summary data file was processed using the Lumi package (Du et al. 2008), developed within the Bioconductor project in the R statistical programming environment (Gentleman et al. 2004). Data were normalized using robust spline normalization (RSN) and variance stabilizing transformation (VST) methods. The log2 intensities were media-centered and log ratios were computed as differences in log2 intensities for each probe. Whole genome expression data results were filtered, with criteria selection of a 1.2- or 1.5-fold change, as indicated, and a Q-value of less than 5%.

Network and pathway analysis

Ingenuity pathway analysis 3.1 software (IPA; Ingenuity Systems, Redwood City, CA, USA) was used to evaluate the functional significance of PrPC-induced gene profiles. Specified lists of genes identified by RankProd as being affected by PrPC expression were used for the network generation and pathway analyses implemented in IPA tools. HUGO official gene symbols for the selected gene lists were uploaded into the IPA suite, and were then mapped to the Ingenuity Pathway Knowledge Base. The so-called focus genes were then used to generate biological networks. A score was calculated for each network, according to the fit of the original set of significant genes. This score reflects the negative logarithm of the p-value, which indicates the likelihood of the focus genes in a network being found together by random chance. The significance for biological functions was then assigned to each network by determining a p-value for the enrichment of the genes in the network for such functions, compared with the whole Ingenuity Pathway Knowledge Base as a reference set.

RT-qPCR

RT-qPCR was performed on total RNA extracted from N2a cells with the miRVana isolation kit (Ambion). Purified RNAs were used to generate the corresponding cDNAs, which served as PCR templates for the mRNA quantification. The primers used in this study for RT-qPCR validation are given in Figure S7. PCR amplification and detection were performed with the Roche LightCycler 480 detector, using 2x SYBR Green Master Mix (Roche, Barcelona, Spain) as a reagent, following the manufacturer's instructions. The reaction profile was as follows: denaturation–activation cycle (95°C for 10 min) followed by 40 cycles of denaturation–annealing–extension (95°C, 10 min; 72°C, 1 min; 98°C, continuous). mRNA levels were calculated using the LightCycler 480 software (Roche). The data were analyzed using the ΔΔCt method, which provides the target gene expression values as fold changes in the problem sample compared with a calibrator sample. Both problem and calibrator samples were normalized with the relative expression of a housekeeping gene [glyceraldehyde-3-phosphate dehydrogenase (GAPDH)].

Western blotting

N2a cells were lysed with ice-cold lysis buffer – 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% (wt/vol) Triton X-100, 0.5% (wt/vol) Nonidet P-40 (IGEPAL; Sigma), glycerol 10%, 1 mM EDTA, 1 mM EGTA, and protease and phosphatase inhibitors – and centrifuged at 15 000 g for 20 min. After protein quantification, samples were boiled in Laemmli sample buffer at 100°C for 5 min, followed by 8–15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis. They were then electrotransferred to polyvinylidene fluoride (PVDF) membranes (Millipore) and processed for immunoblotting with the ECL Plus kit (Amersham-Pharmacia Biotech, GE Healthcare Bio-Sciences, Piscataway, NJ, USA).

Immunoprecipitation

N2a cells were lysed with ice-cold lysis buffer – 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% (wt/vol) Triton X-100, 0.5% (wt/vol) Nonidet P-40 (IGEPAL; Sigma), glycerol 10%, 1 mM EDTA, 1 mM EGTA, and protease and phosphatase inhibitors – and centrifuged at 15 000 g for 20 min. A total of 1 mg of protein extract was mixed with 10 μL of protein A/G-Sepharose (Sigma) previously equilibrated with lysis buffer for 2 h (pre-clearing). Samples were centrifuged, and the supernatant was mixed with the indicated immunoprecipitation antibodies plus 10 μL of protein A/G–Sepharose overnight at 4°C. The immunocomplexes were washed in lysis buffer three times and once in lysis buffer plus 0.25 M NaCl. Proteins attached to Sepharose beads were eluted with SDS-PAGE sample buffer, subjected to SDS-PAGE, transferred to PVDF membranes and probed with the indicated antibodies.

FACS analysis

Subconfluent N2a cells were harvested 24 or 48 h after transfection. Briefly, cells were washed with ice-cold phosphate-buffered saline (PBS), trypsinized, centrifuged, and pellet washed in PBS. Cells were re-suspended in 0.5 mL of PBS, added to 4.5 mL of 70% cold-ethanol and kept at −20°C until analysis. Cells were centrifuged for 5 min at 200 g, washed once in PBS and centrifuged again. The pellet was re-suspended in 1 mL of propidium iodide/Triton X-100/RNAsa solution [Triton 0.1% (Sigma), 0.2 mg/mL RNAsa A DNAsa free (Sigma) and 0.02 mg/mL (Molecular Probes, Life Technologies) in PBS]. Samples were processed using a Beckman Coulter Epics XL flow cytometer and analyzed with Summitv4.3 software (Brea, CA, USA). The proliferative index (PI) was determined as the [(S + G2)/(S + G2 + G1)] value.

Immunofluorescence

For the immunocytochemistry assays on cell cultures, N2a cells were grown on glass coverslips, and transfected as described above. At the indicated times, cells were rinsed twice with cold PBS and fixed with paraformaldehyde 4% for 10 min at 20°C. After 1 h of blocking with 10% FBS-PBS-0.2% gelatin, coverslips were incubated overnight at 4°C with indicated antibodies in 10% FBS-PBS-0.2% gelatin. Successive washes with 1% FBS-PBS-0.2% gelatin were carried out to reduce non-specific reactivity and were followed by 45 min incubation with Alexa Fluor-488 secondary antibodies (Molecular Probes) in 10% FBS-PBS-0.2% gelatin. Cells were washed in 10% FBS-PBS-0.2% gelatin. They were then mounted on coverslips with Vectashield containing 150 ng/mL Hoechst and analyzed on a LEICA DMR microscope (LEICA, Wetzlar, Germany).

Lipid raft isolation

Lipid raft isolation was performed as described in (Macdonald and Pike 2005). N2a cells were scraped in PBS, centrifuged at 250 g for 5 min at 4°C and re-suspended in 500 mM sodium carbonate, pH 11.0 containing protease and phosphatase inhibitors. Cells were lysed using a Dounce homogenizer (20 strokes) followed by 10 passages through a 27-gauge needle. The sample was mixed with an equal volume of 90% sucrose in MES buffer (25 mM MES, pH 6.5, 150 mM NaCl). It was placed at the bottom of a centrifuge tube and followed by 35% and 5% sucrose layers. The gradient was centrifuged at 175 000 g for 20 h. It was then divided into twelve fractions and processed for western blot.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Funding
  8. References
  9. Supporting Information

PrPC-dependent gene expression signature in N2a cells

N2a cells were selected for their endogenous expression of PrPC and their potential prion infectivity (e.g., Chesebro et al. 1993) compared to other cell lines. Global transcriptional profiling was used to obtain a snapshot of the state of N2a cells with endogenous low levels of PrPC 24 h after ectopic silencing and over-expression of PrPC. The modulation of PrPC expression levels was confirmed by RT-qPCR (siRNA-PrPC/Scramble = −3.66-fold decrease and pcDNA-PrPC/pcDNA = 34.91-fold increase) (Figure S1). PrPC levels returned to the initial endogenous expression around 84–96 h post-transfection (data not shown). To evaluate the genes whose transcription was regulated, three sets of experiments were analyzed. RNA was extracted and hybridized on Illumina Sentrix 6 mouse v2 bead arrays. Normalized and raw data from these experiments are accessible in the GEO database http://www.ncbi.nlm.nih.gov/geo/ under accession number GSE38887. We used significance analysis of microarray software (SAM) to identify genes differentially expressed in the two treatments (siRNA-PrPC/Scramble and pcDNA-PrPC/pcDNA). Using a 1.5-fold change threshold and a false discovery rate at 5%, we found 259 and 357 mapped IDs, regulated as a consequence of PrPC silencing and over-expression, respectively (Figure S2). IPA identified the set of molecular functions that were overrepresented in our experiments by integrating gene expression profiles using gene ontology (GO). The top molecular function overrepresented in both treatments was cell cycle, although cell growth and proliferation also appeared among the top five (Table 1). A set of 27 genes was analyzed that represent key molecules in terms of the respective molecular functions overrepresented in the IPA analysis. Data on these genes were validated by RT-qPCR on the same samples used in the microarray experiments. Results for the 27 analyzed genes were concordant with the microarray results, which included genes involved in cell cycle and proliferation, differentiation, neurite outgrowth, and intracellular trafficking, among other functions. Most of these genes showed an inverse correlation between their expression and PrPC levels (Table 2). Cyclin D1, Cyclin E, Cdk2, Egr1, c-fos, Gap43, and SNAP25 were also validated at protein level by western blot. Although proteins had lower fold changes than those observed for mRNA, they were also regulated in a PrPC-expression-dependent manner that resembled the gene expression pattern (Figure S3).

Table 1. Functional analysis of differentially expressed PrPC-dependent genes in N2a cellsThumbnail image of
Table 2. Experimental validation of microarray data by RT-qPCR
GeneGene nameConditionArrayRT-qPCRFunction
ccndl Cyclin D1

siRNA-PrP/Scramble

pcDNA-PrP/peDNA

−1.30

 1.23

−1.61

 1.51

Regulation of cell cycle: G1 transition
ccne2 Cyclin E2

siRNA-PrP/Scramble

pcDNA-PrP/pcDNA

 1.74

−2.23

 2.24

−2.13

Regulation of cell cycle: G1 transition
Cdkn2a Cyclin-dependent kinase inhibitor 2A

siRNA-PrP/Scramble

pcDNA-PrP/pcDNA

−1.30

 1.70

−1.22

 5.17

Regulation of cell cycle: G1 progression
Cdk2 Cyclin-dependent kinase 2

siRNA-PrP/Scramble

pcDNA-PrP/pcDNA

 1.50

−1.50

 1.60

−3.10

Regulation of cell cycle: G1/S transition
Egr1 Early growth response 1

siRNA-PrP/Scramble

pcDNA-PrP/pcDNA

−1.91

 1.86

−1.36

 1.93

Regulation of mitogenesis & differentiation
Insigl Insulin-induced gene 1

siRNA-PrP/Scramble

pcDNA-PrP/pcDNA

 1.68

−1.64

 2.54

−2.52

Regulation of cell cycle: G0/G1 transition & cholesterol biosynthesis
Txnip Thioredoxin interacting protein

siRNA-PrP/Scramble

pcDNA-PrP/pcDNA

−2.06

 2.10

−2.47

 1.79

Regulation of cell cycle: G0/G1 transition & oxidative stress mediator
Appbpl Amyloid beta precursor protein binding 1

siRNA-PrP/Scramble

pcDNA-PrP/pcDNA

 1.02

−1.45

 1.09

−2.52

Regulation of cell cycle: S/M progression & apoptosis
ATF3 Activating transcription factor 3

siRNA-PrP/Scramble

pcDNA-PrP/pcDNA

−1.10

 1.61

−1.18

 1.47

Regulation of cell cycle & cell death
AurKB Aurora kinase B

siRNA-PrP/Scramble

pcDNA-PrP/pcDNA

 1.87

−1.72

 3.19

−2.38

Regulation of cell cycle: M progression and completion
Akap12 A kinase (PRKA) anchor protein 12

siRNA-PrP/Scramble

pcDNA-PrP/pcDNA

 1.21

−1.30

 2.04

−1.24

Cell growth
c-fos FBJ murine osteosarcoma viral oncogene homotog

siRNA-PrP/Scramble

pcDNA-PrP/pcDNA

−1.10

 1.71

 1.20

 1.30

Cell proliferation, differentiation & apoptosis
Idb2 Inhibitor of DNA binding 2

siRNA-PrP/Scramble

pcDNA-PrP/pcDNA

 1.00

 2.00

 1.18

 2.16

Negatively regulator of cell differentiation
Gap43 Growth-associated protein 43

siRNA-PrP/Scramble

pcDNA-PrP/pcDNA

 3.96

−1.29

 8.09

−1.21

Regulation of neuronal growth cones during development and axonal regeneration
Nm1 Neuritin 1

siRNA-PrP/Scramble

pcDNA-PrP/pcDNA

 1.40

−1.60

 2.00

−1.20

Regulation of neurite outgrowth and arborization
Nsdhl NAD(P) dependent steroid dehydrogenase-like

siRNA-PrP/Scramble

pcDNA-PrP/pcDNA

 1.60

−1.30

 1.91

−1.97

Cholesterol biosynthesis
Aacs Acetoacetyl-CoC synthetase

siRNA-PrP/Scramble

pcDMA-PrP/pcDNA

 2.00

 1.57

 4.27

−2.32

Fatty acid synthesis
Cyp51 Cytochrome P450, family 51, subfamifyA

stRNA-PrP/Scramble

pcDNA-PrP/pcDNA

 1.68

−1.92

 1.54

−2.36

Drug metabolism and cholesterol biosynthesis
Rab7 RAB7A, member RAS oncogene family

siRNA-PrP/Scramble

pcDNA-PrP/pcDNA

 2.12

 1.00

 4.68

 1.15

Vesicular transport
Snx2 Sorting Nexin 2

siRNA-PrP/Scramble

pcDNA-PrP/pcDNA

 1.00

−1.60

 1.02

−2.71

Intracellular traffiking
Stx7 Syntaxin 7

siRNA-PrP/Scramble

pcDNA-PrP/pcDNA

 1.67

−1.50

 1.38

−1.60

Intracellular traffiking
Stx8 Syntaxin 8

siRNA-PrP/Scramble

pcDNA-PrP/pcDNA

−1.56

−1.20

−1.43

−1.12

Intracellular traffiking
Tcp1 t-complex 1

siRNA-PrP/Scramble

pcDNA-PrP/pcDNA

 1.11

−2.06

−1.08

−2.69

Intracellular traffiking & Chaperone activity
Sgol2 Shugoshiun 2

siRNA-PrP/Scramble

pcDNA-PrP/pcDNA

 1.14

−2.32

  1.09

−1.61

Gametogenesis
SNAP25 Synaptosomal-associated protein 25

siRNA-PrP/Scramble

pcDNA-PrP/pcDNA

 1.56

−1.53

 2.99

−3.08

Synaptic Function
Lbr Lamin B receplor

siRNA-PrP/Scramble

pcDNA-PrP/pcDNA

 1.84

−1.34

 2.85

−2.38

Anchorining oflaminin and heterochromatin to Hie inner nuclear membrane
Idhl Isocilrate dehydrogenase 1 {NADP+), soluble

siRNA-PrP/Scramble

pcDNA-PrP/pcDNA

 2.31

−2.46

 2.09

−2.44

NADPH production

PrPC regulates cell cycle and proliferation in N2a cells

WST-1 assay was used to evaluate the direct effects of PrPC expression on N2a proliferation. Down-regulation of PrPC inhibited cell proliferation in asynchronous and serum-restimulated cells. In contrast, PrPC-transfected cells increased proliferation in both experimental conditions (Fig. 1a). Next, we examined the cell cycle at 24 and 48 h after serum restimulation in serum-starved cells, using FACS analysis. The PI indicated that PrPC silencing inhibited cell cycle progression (Scramble PI = 0.39 ± 0.04 vs. siRNA-PrPC PI = 0.27 ± 0.02, mean ± SEM), while PrPC over-expression slightly enhanced G1 entrance and progress throughout the cell cycle (pcDNA PI = 0.40 ± 0.03 vs. pcDNA-PrPC PI = 0.46 ± 0.04, mean ± SEM) (Fig. 1b). In parallel, we used Ki67 immunostaining to study the role of PrPC in cell cycle re-entrance after serum stimulation. Serum-starved N2a cells (0% FBS-Control) lacked Ki67 labeling (Fig. 1c). After 6 h of serum stimulation (10% FBS) in Scramble and pcDNA transfected cells, numerous Ki67+ cells were observed. PrPC silencing decreased the number of Ki67+ cells, while PrPC-transfected cells had higher Ki67 expression (Fig. 1c). Since regulation of PrPC expression has been implicated in some apoptotic mechanisms, we next analyzed whether the decrease in cell number observed after a PrPC decrease was related with increased cell death. This hypothesis was ruled out because of a lack of poly (ADP-ribose) polymerase (PARP) cleavage, which is a marker for apoptotic processes, by western blot (Fig. 1d). In addition, the octapeptide region and the central domain of PrPC play a key role in PrPC-dependent proliferation, since their deletion using over-expression of the F35 truncated form (Δ33-134) (Shmerling et al. 1998) abolished the enhanced proliferation caused by Prnp overexpression (Fig. 1e). Thus, all proliferative analysis methods supported the conclusion that PrPC down-regulation reduces cell growth and proliferation, while increased PrPC expression promotes proliferation and cell cycle progression in N2a cells.

image

Figure 1. Cellular prion protein (PrPC) expression regulates cell proliferation and cell cycle progression in N2a cells. (a) Growth curves of asynchronous (left graph) and serum-restimulated (right graph) N2a cells measured using the WST-1 assay. N2a cells were transfected with Scramble, siRNA-PrPC, pcDNA, and pcDNA-PrPC, as described in the Material and methods section, and cell proliferation was measured for the next 72 h. Error bars indicate ± SEM. (b) Cell cycle distribution of serum-restimulated N2a transfected with Scramble, siRNA-PrPC, pcDNA, and pcDNA-PrPC analyzed by propidium iodide staining and FACS. The mean proliferative index (PI) for each condition is shown. (c) Ki67 immunostaining (green fluorescent signal) and Hoescht staining (blue fluorescent signal) of serum starved (Control) and 6 h serum-restimulated N2a cells transfected with Scramble, siRNA-PrPC, pcDNA, and pcDNA-PrPC. The percentage of Ki67+ cells is expressed relative to its respective controls. Asterisks indicate statistical significance (**< 0.01, anova test). (d) Western blot analysis of N2a cells transfected for 48 h with Scramble, siRNA-PrPC, pcDNA, and pcDNA-PrPC developed with PARP and tubulin (loading control) antibodies. Cycloheximide (CHX) (0.01 mg/mL) was used as a positive control of PARP cleavage, as evidence of apoptosis induction. (e) N2a cells were transfected with pcDNA, pcDNA-PrPC, and pcDNA-∆F35 and analyzed for proliferation and apoptosis as described above. A total of 400 nuclei were analyzed. Asterisks indicate statistical significance (*< 0.05, **< 0.01, anova test). Scale bar in d: 100 μm.

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ERK1/2 and AKT signal pathways are regulated by PrPC expression

Changes in PrPC expression may mediate changes in the activation state of AKT and ERK1/2 signal pathways in specific tissues and cell lines and under specific effectors (Schmalzbauer et al. 2008; Carulla et al. 2011). Since both pathways are critical for proliferative and differentiation processes in neuroblastoma cell lines, and both cellular functions are altered in our model after modulation of PrPC expression, we examined whether these kinase pathways are regulated in non-synchronous N2a cells and after serum restimulation conditions. Densitometry values of p-antibody/total antibody indicated that in non-synchronous PrPC-silenced cells, ERK1/2 and AKT activity was markedly decreased. In contrast, p-ERK1/2 remained unaltered while p-AKT was significantly increased after PrPC over-expression (Fig. 2a). Downstream effectors of ERK1/2, p-Elk-1 levels, resembled ERK1/2 activation, indicating that the effect was transmitted downstream (Fig. 2a). In agreement with these results, we also observed a marked decrease in ERK1/2 phosphorylation after PrPC silencing in the SK-N-SH neuroblastoma cell line and a marginal effect when PrPC was over-expressed (Figure S4). This suggests a common PrPC-dependent regulating mechanism in neuroblastoma cell lines. Since some of the physiological effects after acute regulation of PrPC expression were also observed after serum restimulation of synchronized cells, we analyzed the activation kinetics of ERK1/2 and AKT under these conditions. ERK1/2 presented activation peaks at 5 min, 1 h, and 24 h after serum stimulation, while AKT presented a smooth activation peak starting at 30-min post-serum stimulation and decreasing at 24 h. PrPC silencing induced a reduction in ERK1/2 activation at 6 h and 24 h after serum stimulation, without affecting earlier times. PrPC silencing decreased AKT phosphorylation. However, in contrast to ERK1/2 activation, this inhibition appeared shortly after 30 min following serum stimulation (Fig. 2b). In these conditions, Egr1 expression and p-Elk-1 levels were also down-regulated after serum restimulation, which resembles the effects on ERK and AKT activation (Fig. 2c). When PrPC was over-expressed in the same cellular conditions, we observed marginal effects on ERK1/2 phosphorylation, while an increase in p-AKT was observed at 6 h and 24 h after serum restimulation (Fig. 2d).

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Figure 2. Regulation of extracellular signal-regulated kinases (ERK1/2) and protein kinase B (AKT) activity by cellular prion protein (PrPC) expression in N2a cells. (a) Western blot analysis of asynchronous N2a cells transfected for 24 h with Scramble, siRNA-PrPC, pcDNA, and pcDNA-PrPC, developed with p-ERK1/2, ERK, p-AKT, AKT, and p-Elk-1 antibodies. Densitometric analysis of five independent experiments is shown as the fold change between siRNA-PrPC/Scramble and pcDNA-PrPC/pcDNA. Tubulin was used as a loading control. (b) Western blot analysis of N2a cells transfected for 24 h with Scramble and siRNA-PrPC, serum-restimulated with 10% fetal bovine serum (FBS) for the indicated times and developed with p-ERK1/2, ERK, p-AKT, AKT, and PrP antibodies. Densitometric analysis of five independent experiments is shown as the fold change relative to control samples (serum-starved cells). (c) Western blot analysis of N2a cells transfected for 24 h with Scramble and siRNA-PrPC, serum-restimulated with 10% FBS for the indicated times and developed with Egr1 and p-Elk-1. Tubulin was used as a loading control. Densitometric analysis of five independent experiments is shown as the fold change relative to control samples (serum-starved cells). (d) Western blot analysis of N2a cells transfected for 24 h with pcDNA and pcDNA-PrPC, serum-restimulated with 10% FBS for the indicated times and developed with p-ERK1/2, ERK, p-AKT, AKT and PrP antibodies. Asterisks indicate statistical significance (***< 0.001, anova test).

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Plasma membrane PrPC is required for ERK1/2 regulation

PrPC is an N-linked glycoprotein attached to the cell surface by a GPI anchor. Besides its plasma membrane location, PrPC has been described in endosomes via a high turnover, clathrin-dependent mechanism. Moreover, PrPC has been observed in rough endoplasmic reticulum and Golgi, probably by internalization from non-raft domains (Harris 2003). Indeed, in N2a cells, PrPC labeling can be observed both in plasma membranes and in perinuclear/reticular compartments (data not shown). PrPC silencing causes a decrease in both cellular fractions, indicating a constant PrPC trafficking between reticule and membrane. To establish which PrPC fraction is responsible for the effects over the ERK1/2 pathway, we used the ability of some monoclonal anti-PrPC antibodies to cluster PrPC into the cell membrane (Mouillet-Richard et al. 2000). PrPC clustering and aggregation provokes a well-described increase in ERK1/2 phosphorylation and Fyn kinase activation (Mouillet-Richard et al. 2000; Toni et al. 2006). We treated control and PrPC-silenced cells for 10 min with SAF61 antibody. Western blot analysis of p-ERK1/2 indicated that SAF61 can induce ERK1/2 phosphorylation, which is significantly reduced after PrPC silencing (Scramble 1.9 ± 0.29 vs. siRNA-PrPC 1.3 ± 0.21) (Fig. 3). These results show that the PrPC located at the cell membrane was responsible for the downstream effects on ERK1/2 pathway.

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Figure 3. Cell surface cellular prion protein (PrPC) mediates extracellular signal-regulated kinases (ERK1/2) regulation. Western blot analysis of N2a cells transfected for 24 h with Scramble and siRNA-PrPC and treated with phosphate-buffered saline (PBS) (−) or SAF61 (+). Membranes were developed with p-ERK1/2 and ERK antibodies. Densitometric analysis of three independent experiments is shown as the fold change between SAF61-treated and untreated cells. Asterisks indicate statistical significance (**< 0.01, anova test).

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PrPC regulates EGF-dependent ERK1/2 and AKT activation

Given the ability of PrPC to regulate ERK1/2 and AKT pathways, we searched for putative mitogenic components present in the serum and their respective receptors responsible for these effects. PrPC has been proposed as a potential functional partner of EGFR (Monnet et al. 2004), and a direct interaction has been described between PrPC and the adaptor protein Grb2 (Spielhaupter and Schatzl 2001), suggesting a link between EGFR and downstream MAPK pathway activation. In our experiments, restimulation of PrPC-silenced cells with EGF inhibited p-ERK1/2 and p-AKT activity. Moreover, EGF stimulation increased AKT activation in PrPC over-expressed cells, with marginal effects on p-ERK1/2 (Fig. 4a). This indicates that EGF/EGFR may be one of the main components of the PrPC-dependent ERK1/2 and AKT pathway regulation after serum stimulation.

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Figure 4. Regulation of the epidermal growth factor receptor(EGFR)-extracellular signal-regulated kinases (ERK1/2) pathway by cellular prion protein (PrPC) expression in EGF restimulated N2a cells. (a) Western blot analysis of N2a cells transfected for 24 h with Scramble, siRNA-PrPC, pcDNA, and pcDNA-PrPC, restimulated with EGF for the indicated times and developed with EGFR, p-ERK1/2, ERK, p-AKT, AKT, and PrP antibodies. (b) Cell proliferation analysis using WST-1 in N2a cells transfected with pcDNA and pcDNA-PrPC in the absence and presence of wortmannin and western blot analysis of cell extracts from N2a cells in the absence and presence of wortmannin for 6 h and developed with p-AKT and AKT antibodies. Asterisks indicate statistical significance (**< 0.01, anova test).

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In addition, inhibition of p-AKT activity with the phosphatidylinositide 3-kinase (PI3K) inhibitor wortmannin in EGF-stimulated cells decreased the PrPC-mediated enhancement of cell proliferation, indicating that there is a direct link between PrPC-mediated proliferation induced by EGF and the AKT pathway (Fig. 4b). In contrast, wortmannin treatment of serum-stimulated cells did not significantly affect cell proliferation in PrPC over-expressed cells (data not shown).

PrPC binds to the EFGR macromolecular complex

These results suggest a role for PrPC in regulating EGFR activity, either by modulating its phosphorylation state or through direct interaction between the two proteins. At the time points when PrPC affected ERK1/2 and AKT signaling, we could not observe EGFR phosphorylation (Fig. 5a). Consequently, we explored the possibility of regulation through potential interaction. Endogenous co-immunoprecipitation experiments in N2a cell extracts detected PrPC and EGFR in EGFR and PrPC immunocomplexes, respectively (Fig. 5b). Co-localization of the two proteins was observed in the lipid raft fraction in a sucrose gradient fractionation (Fig. 5c) and with double immunolabeling (Fig. 5d). Furthermore, confocal serial sections in Z-plane of A431 cells (with elevated levels of endogenous EGFR) transfected with PrPC also demonstrated the presence of EGFR and PrPC in intracellular vesicles after immunocytochemical detection of both proteins (Figure S5). This supports the specificity of EGFR and PrPC interaction in cellular compartments. We also observed interaction between PrPC and two components of the EGFR macromolecular complex, Grb2 and p-Src. This indicates that PrPC may be part of the cell membrane complexes that regulate EGF/EGFR signaling (Fig. 5e). To assess whether PrPC interaction with EGFR was altered in the conditions in which ERK and AKT activity was modulated by PrPC in response to EGF (Fig. 4a), we co-immunoprecipitated PrPC and EGFR at 0 h and 24 h post-EGF stimulation in double transfected cells (Fig. 5f). Interestingly, EGFR-PrPC interaction was increased at 24 h post-EGF stimulation, which correlates with changes in high AKT activation and maintenance of ERK1/2 activation when compared to serum-starved cells (Fig. 5f). Thus, it is reasonable to consider that in absence of PrPC, EGF-treated cells showed decreased ERK1/2 and AKT activation (Fig. 4a). Taken together, the present data support the notion that PrPC-EGFR interaction modulates EGFR receptor activity and dynamics, reinforcing previous data (Monnet et al. 2004; Solis et al. 2012).

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Figure 5. Epidermal growth factor receptor (EGFR) binds and co-localizes with cellular prion protein (PrPC). (a) Western blot analysis of p-EGFR in N2a cells after EGF stimulation at indicated times. (b) Co-immunoprecipitation (IP) assay and western blot analysis of endogenous EGFR and PrPC in N2a cells. N2a cell extracts were immunoprecipitated with SAF61 (PrP) antibody and immunoblotted with anti-EGFR antibody (upper panel), and immunoprecipitated with EGFR antibody and immunoblotted with 6H4 (PrP) antibody. An aliquot of the cell extract was used as the assay input. (c) Western blot analysis of N2a detergent-free raft fractions transfected with PrPC with the sucrose gradient method in PrPC transfected N2a cells. Protein distribution in the carbonate step gradient procedure. Membranes were developed with EGFR, PrPC, and Flotilin-1 (lipid raft marker) antibodies. (d) Confocal immunofluorescence images of N2a cells transfected for 24 h with EGFR and PrPC and labeled with SAF61 (PrP) antibody and EGFR (e) Co-immunoprecipitation (IP) assay and western blot analysis of Grb2 and p-Src in N2a cells. N2a cell extracts were immunoprecipitated with SAF61 (PrP) and Grb2 antibodies and immunoblotted with Grb2 and p-Src antibodies. (f) Co-immunoprecipitation (IP) assay and western blot analysis of N2a cells transfected with EGFR and PrPC and restimulated with EGF at the indicated times. N2a cell extracts were immunoprecipitated with SAF61 (PrP) antibody, immunoblotted with anti-EGFR antibody (upper panel), immunoprecipitated with EGFR antibody and immunoblotted with 6H4 (PrP) antibody. An aliquot of the different cell extracts was used as the assay inputs. Scale bar in d: 5 μm.

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PrPC-dependent filopodia formation in N2a cells signals through PI3K, Rho-subfamily GTPases, and N-WASP

PrPC has been shown to regulate neurite outgrowth in several cell lines and hippocampal neurons (e.g., Bodrikov et al. 2011; Loubet et al. 2012). Moreover, in this study, we found several genes involved in neuritogenesis and cellular morphology, regulated by both PrPC silencing and over-expression in our microarray analysis (Table 2). To evaluate the effects of PrPC expression on the morphology of N2a transfected cells, we carried out actin immunofluorescence labeling of cell cultures. In asynchronous cells, PrPC over-expression significantly enhanced filopodia formation (Fig. 6a), in agreement with previous observations (Schrock et al. 2009). These filopodia are also modulated in N2a cells by several proteins such as Delta 1 (Sugiyama et al. 2010), the G protein-coupled receptor kinase 5 (GRK5), and the actin cross-linking family protein 7 (ACF7) (Sanchez-Soriano et al. 2009). In contrast, PrPC silencing does not generate significant phenotypical changes in N2a cells compared to control. This is in contrast to previously published data on other cell types: 1C11 cells (Loubet et al. 2012) and primary hippocampal neurons (Bodrikov et al. 2011; Loubet et al. 2012).

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Figure 6. Cellular prion protein (PrPC) enhances filopodia formation in N2a cells in an protein kinase B (AKT)-Cdc42-N-WASP-dependent manner (a) N2a cells were transfected with Scramble, siRNA-PrPC, pcDNA, and pcDNA-PrPC as described in the Material and methods section. Twenty-four hours post-transfection, cells were fixed and immunostained for anti-actin and stained with Hoescht. A total of 400 cells were analyzed for each condition. (b) N2a cells were transfected with pcDNA-PrPC and 24 h post-transfection treated with cythochalasin D (CCD) at 10 μg/mL for 2 h, fixed and immunostained for anti-actin and stained with Hoescht. (c) N2a cells were transfected with pcDNA-PrPC, pcDNA-PrPC/Cdc42 N17, pcDNA-PrPC/RhoA N19, pcDNA-PrPC/Cdc42 N17, pQBI25-GFP-N-WASP, and pQE-GFP-N-WASP-ΔGBD and 24 h post-transfection were treated or untreated with wortmannin (10 ng/μL) (**< 0.01, ***< 0.001, anova test). A total of 400 cells were analyzed for each condition. (d) N2a cells were transfected with pcDNA-PrPC and pQBI25-GFP-N-WASP or pQE-GFP-N-WASP-ΔGBD. Twenty-four hours post-transfection, cells were fixed and immunostained for anti-actin. Scale bars a and c: 20 μm, b: 10 μm.

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To define the cellular protrusions induced by PrPC over-expression, we treated cells with cytochalasin D, a potent inhibitor of actin polymerization. Cytochalasin D inhibited PrPC-induced protrusions (Fig. 6b), indicating that these are mainly composed of actin filaments. Actin dynamics depends on Rho-subfamily GTPases and downstream effectors (e.g., N-WASP). Therefore, we analyzed filopodia formation in PrPC-transfected N2a co-transfected with the wild type and the dominant-negative forms of Cdc42 (Cdc42 N17), RhoA (RhoA N19), and Rac1 (Rac1 N17) (Fig. 6c). In all cases, a reduction in the total number of N2a-displaying actin bundles in filopodia was observed (Fig. 6c). However, dominant-negative forms of Cdc42 and RhoA significantly reduced PrPC-dependent filopodia formation. In addition, N-WASP (N-WASP-ΔGBD) transfection also reduced PrPC-dependent filopodia formation. Moreover, inhibition of PI3K with wortmannin also decreased the amount of filopodia. This suggests that both the Rho/ROCK and AKT-Cdc42-N-WASP pathways play a role in PrPC-dependent enhanced filopodia formation (Fig. 6c and d). It has been described how N2a cells exhibit enhanced neurite outgrowth after serum deprivation, mainly dependent on EGFR transactivation independently of EGF (e.g., Evangelopoulos et al. 2005). Thus, we aimed to determine whether changes in PrPC expression per se might affect this well-described process (Figure S6). Results indicate that neither PrPC silencing nor over-expression-impaired neuritogenesis compared to control and Scramble treated cells; reinforcing the notion of the putative role of PrPC expression levels modulating neuritogenesis in EGF-stimulated cells.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Funding
  8. References
  9. Supporting Information

Opposing effects of PrPC expression on cell proliferation and variable expression levels in different types of proliferating cells have been reported (see below). Although highly associated with epitope determination and technical resolution, some proliferating cells can be labeled using PrPC antibodies (Linden et al. 2008) while others cannot (Steele et al. 2006). However, neural cell lines (Kim et al. 2005) and oligodendrocyte precursors in vitro (Bribian et al. 2012) from Prnp−/− mice showed high rates of proliferation. Moreover, PrPC levels may change from elevated to lower expression in proliferating cell types (e.g., Steele et al. 2006; Liang et al. 2007). Thus, it is reasonable to consider that PrPC expression may modulate cell cycle control dynamics and differentiation by acting along with additional factors in a cell-specific way (e.g., in the nervous system Steele et al. 2006; Bribian et al. 2012). In fact, data obtained from mutant mice and in vitro cellular models have failed to reveal a clear genomic signature modulated by PrPC (see Introduction and Steele et al. 2007 for details).

In this study, we demonstrate that increased PrPC expression enhances cell proliferation in N2a cells, while PrPC-silenced cells decrease cell cycle and proliferation without inducing cell death. Our microarray data support these observations, since key genes in cell growth and proliferation are regulated after acute PrPC up-regulation. Interestingly, Cyclin D1, required for cell cycle G1/S transition, is up-regulated in PrPC over-expressed cells and down-regulated in PrPC-silenced cells. This result is one of the few coincidences so far between several gene expression studies and proteomic analyses. Indeed, Cyclin D1 has been observed to be up-regulated after stable over-expression of PrPC and down-regulated after PrPC depletion in gastric cancer cell lines (Liang et al. 2007), and down-regulated in the adult brain of Prnp−/− mice (Brown et al. 2002) and Prnp−/−-derived fibroblasts (Benvegnu et al. 2011). More relevantly, Liang and coworkers demonstrated that PrPC over-expression stimulates promoter activity of Cyclin D1 (Liang et al. 2007). Together, these observations demonstrate that regulation of Cyclin D1 by PrPC is one of the common mechanisms in different cell types and tissues by which PrPC may regulate cell cycle progression. Increased presence of PrPC in the cytosol has been considered neurotoxic (e.g., Ma et al. 2002; Fioriti et al. 2005), although there is some controversy on this point. In this study, we have demonstrated that kinase activation (e.g., ERK1/2) is mediated by GPI-anchored plasma membrane PrPC, thereby ruling out unspecific effects because of cytosolic protein accumulation. In our experiments, transfections clearly increased the amount of plasma membrane PrPC, but only slightly increase cytosolic PrPC (data not shown).

PrPC has been associated with several effectors and proteins in plasma membrane complexes, such as neurotransmitter receptors (e.g., Carulla et al. 2011), adhesion molecules (Schmitt-Ulms et al. 2001; Santuccione et al. 2005), and copper. These interactions can modulate a diverse array of cellular functions and signaling pathways such as PrPC-Laminin-mediated neuritogenesis (Beraldo et al. 2011), PrPC-STI1-mediated neural progenitor/stem cells self-renewal (Santos et al. 2011) and GluR6/PSD-95 interaction, as well as further JNK3 activation in response to kainate (see Llorens and Del Rio 2012 for details).

Here, we demonstrate that PrPC is present in the macromolecular complexes associated with EGFR, and also that it is associated with proteins implicated in the downstream transmission of the mitogenic stimuli (Grb2, Src). In addition, PrPC modulates the activity of two EGFR-related pathways, MAPK and AKT, whose activity is critical for G1 cell cycle progression and Cyclin D1 expression (Humtsoe and Kramer 2010). PrPC regulates the second wave of sustained activity that persists throughout the G1 phase for MAPK and the AKT activity peak after serum and EGF restimulation in serum-starved and asynchronous cells. Whether these effects on the EGFR pathway and cell proliferation involve other unknown components or specific cellular compartments warrants further research.

In this regard, we observed that PrPC co-localizes with EGFR in lipid raft fractions and modulates the expression of lipid raft proteins (such as Gap43) and proteins involved in vesicular transport and intracellular trafficking (e.g., syntaxins, nexin-2, t-complex1, and Rab7) (Table 2). Interestingly, Zafar and co-workers demonstrated reciprocal regulation of Rab7 on PrPC expression levels (Zafar et al. 2011). Taken together, these observations reinforce an active role of PrPC in lipid raft dynamics and vesicular trafficking. Indeed, Bodrikov and co-workers demonstrated reduced exocytic vesicle transport to growth cones in cultured hippocampal neurons lacking PrPC, reinforcing the notion that PrPC is necessary for proper neurite extension and growth cone dynamics (Bodrikov et al. 2011). In our experiments using N2a, PrPC deficiency did not lead to significant changes in neurite extension as in other studies (Schrock et al. 2009; Bodrikov et al. 2011; Miranda et al. 2011; Loubet et al. 2012). This could be explained by cellular type specificity, because of the non-sustained effect of transient transfection, or because siRNA inhibition reduced but did not eliminate PrPC expression in our cellular system.

Among other regulatory mechanisms, filopodia dynamics involves AKT, which activates the small GTPase of the Rho-subfamily Cdc42. Cdc42 then interacts with N-WASP, an activator of the Arp2/3 complex, to form actin bundles that enhance filopodia formation (Carlier et al. 1999). Under the cellular conditions in which PrPC enhances filopodia formation and neuritogenesis, AKT activity is enhanced in contrast to ERK1/2. In addition, we observed that both the pharmacological inactivation of AKT and the transfection of mutated forms of RhoA, Rac1, Cdc42, and N-WASP reduced filopodia protrusion induced by PrPC over-expression (especially Cdc42, RhoA, and N-WASP). Loubet and co-workers demonstrated increased membrane clustering of β1 integrin, RhoA over-activation, and impaired neuritogenesis in 1C11 neuroectodermal cells as well as in PC12 cells lacking PrPC (Loubet et al. 2012). It has been generally accepted that N-WASP, as an effector of Cdc42, modulates filopodia formation (Takenawa and Miki 2001). Thus, our results expand these previous observations indicating that the AKT-Cdc42-N-WASP pathway is also regulated by PrPC and EGFR signaling and that it promotes neuritogenesis. Neuritogenesis, axon guidance and cell migration share to some extent many features during development, from the use of substrates to the specific cues regulating chemotaxis and cytoskeleton dynamics. In this scenario, the present data support the notion that PrPC plays crucial roles in proliferation and neuritogenesis. However, we also believe that future cell-specific studies combining gene expression data and functional analysis will reveal new mechanisms involving PrPC.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Funding
  8. References
  9. Supporting Information

The authors thank Prof. Antoni Camins (Universitat de Barcelona) for Cdk2 and Cyclin E antibodies, Prof. Xavier Navarro (Universitat Autònoma de Barcelona) for Gap43 antibody, Prof. Carles Enrich (Universitat de Barcelona) for pQBI25-GFP-N-WASP-WT, pQE-GFP-N-WASP-∆GBD constructions, Prof. David Harris (University of Boston) for pcDNA-PrPC plasmid and Prof. Adriano Aguzzi (University Hospital of Zurich) for pcDNA-ΔF35 plasmid. The authors declare no conflict of interest.

Funding

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Funding
  8. References
  9. Supporting Information

This study was supported by the Seventh Framework Programme of the European Commission [grant number 222887] FP7-PRIORITY to JADR and JMT and [grant number 278486] DEVELAGE to IF. The Spanish Ministry of Science and Innovation [grant numbers BFU2009-10848 and BFU2012-32617], the Generalitat de Catalunya [grant number SGR2009-366], ‘La Caixa’ Obra Social Foundation and the Instituto Salud Carlos III [grant number PI11/03028] to JADR. PC is supported by the FPI program of MICINN.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Funding
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Funding
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
jnc12283-sup-0001-FigureS1-S7.pdfapplication/PDF2507K

Figure S1. Regulation of PrPC expression levels after transient silencing and over-expression in N2a cells.

Figure S2. List of regulated genes in the microarray analysis.

Figure S3. Validation of microarray and RT-qPCR data by western blot.

Figure S4. Regulation of ERK1/2 activity by PrPC expression in SK-N-SH cells.

Figure S5. PrPC and EGFR co-localization in vesicles of A431 cells.

Figure S6. Neuritogenesis in serum-starved N2a.

Figure S7. List of primers used in the study.

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