prion protein: from physiology to cancer biology · be involved in cancer biology. 2. human prion...

Cancer Letters 290 (2010) 1–23

Contents lists available at ScienceDirect

Cancer Letters

journal homepage: www.elsevier .com/locate /canlet

Mini-review

Prion protein: From physiology to cancer biology

Maryam Mehrpour *, Patrice CodognoINSERM, U 756, Université Paris Sud 11, Faculté de Pharmacie, 92290 Châtenay Malabry, France

a r t i c l e i n f o

Article history:Received 19 May 2009Received in revised form 10 July 2009Accepted 13 July 2009

Keywords:ApoptosisMetastasisDrug resistanceBreastProstate and gastric cancer

0304-3835/$ - see front matter � 2009 Elsevier Ireldoi:10.1016/j.canlet.2009.07.009

Abbreviations: ABC transporter, ATP-Binding CADAM, a disintegrin and metalloprotease; AKT, v-aviral oncogene homolog; ATM, ataxia-telangiectasiaassociated X protein; Bcl-2, B-cell leukemia/lymphomBcl-2-like 1; BH, Bcl-2 Homology; BID, BH3 interagonist; CNS, central nervous system; CDKN2a, CyclInhibitor 2a; CTSD, endolysosomal aspartate proteadendritic cells; DED, Death Effector Domain; DD, Ddeath-inducing signaling complex; DR, Death RecepGrowth Factor; ERK, Extracellular signal-Regulated KMAPK; FADD, Fas-associated DD Kinase; FLICE, FADDconverting enzyme; FLIP, FLICE-like inhibitory Protenoglycans; GPI, Glycosyl-phosphatidylinositol; HSE,JNK, c-Jun N-terminal kinase; LRP1, [LDL (low-receptor-related protein-1]; LRP/LR, laminin receptreceptor; MAPKs, mitogen-activated protein kinasesMEK, mitogen-activated protein kinase kinase;Leukemia sequence 1; MOMP, Mitochondrial Outeabilization; MDR, multidrug resistant; MRP, massociated protein; NF-jB, nuclear factor-jB; NKPIPLC, phosphoinositol phospholipase C; PrPc, CePrPSc, Scrapie Prion Protein; PTP, Permeability TrPhosphoinositide 3-Kinase; PIP, Phosphoinositol Phooxygen species; SOD, superoxide dismutase; STATand Activator of Transcription 3; TNF, Tumour Necroreceptor; TRAIL, Tumor Necrosis Factor-RelatedLigand; TRAIL-R, TRAIL Receptor.

* Corresponding author. Tel.: +33 663431562.E-mail address: [email protected] (M.

a b s t r a c t

Prion protein (PrPc) was originally viewed solely as being involved in prion disease, butnow several intriguing lines of evidence have emerged indicating that it plays a fundamen-tal role not only in the nervous system, but also throughout the human body. PrPc isexpressed most abundantly in the brain, but has also been detected in other non-neuronaltissues as diverse as lymphoid cells, lung, heart, kidney, gastrointestinal tract, muscle, andmammary glands. Recent data indicate that PrPc may be implicated in biology of glioblas-toma, breast cancer, prostate and gastric cancer. Over expression of PrPc is correlated to theacquisition by tumor cells of a phenotype for resistance to cell death induced by TNF alphaand TRAIL or antitumor drugs such as paclitaxel and anthracyclines. PrPc may promotetumorigenesis, proliferation and G1/S transition in gastric cancer cells. This review revisitsthe physiological functions of PrPc, and its possible implications for cancer biology.

� 2009 Elsevier Ireland Ltd. All rights reserved.

and Ltd. All rights reserved.

assette transporter;kt murine thymomamutated; Bax, Bcl-2-a 2; BCL-XL/Bcl-2L1,

acting domain deathin-Dependent Kinasese cathepsin D; DC,eath Domain; DISC,

tor; EGFR, Epidermialinases also known as-like interleukin-1 b-in; GAG, Glycosami-heat shock element;density lipoprotein)or precursor/laminin

also known as ERK;MCL1, Myeloid Cellr Membrane Perme-ultidrug resistance-

, Natural Killer Cell;llular Prion Protein;ansition Pore; PI3K,sphate; ROS, reactive3, Signal Transducersis Factor; TNFR, TNF

Apoptosis-Inducing

Mehrpour).

1. Introduction

‘‘Prion” is an abbreviation devised by Stanley Prusinerfor ‘‘proteinaceous infectious particle”. Prion diseases area group of transmissible neurodegenerative disorders thatincludes Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler syndrome (GSS), kuru, and fatal familialinsomnia (FFI) in humans, as well as scrapie and bovinespongiform encephalopathy in animals [1]. These diseasesare caused by the conversion of the host cellular prion pro-tein (PrPc) into scrapie prion protein (PrPSc), a b-sheet-richconformer that is infectious in the absence of nucleic acid[1,2]. This conformational conversion and subsequent dis-orders necessitate the presence of PrPc, since the absenceof endogenous PrPc totally precludes PrPSc-mediated infec-tivity and neurotoxicity [3]. Prion disease can also arisesporadically, as a result of an as-yet uncharacterized sto-chastic event causing a PrPc to PrPSc conversion, or bydominant mutations in the gene encoding PrPc (PRNP inhumans), which results in the formation of a mutant PrPcthat more readily undergoes spontaneous conversion toPrPSc.

Prions accumulate not only in the central nervous sys-tem (CNS), but also in lymphoid organs, as has been shown

http://dx.doi.org/10.1016/j.canlet.2009.07.009

mailto:[email protected]

http://www.sciencedirect.com/science/journal/03043835

http://www.elsevier.com/locate/canlet

2 M. Mehrpour, P. Codogno / Cancer Letters 290 (2010) 1–23

in new variant and sporadic CJD patients, and in some ani-mals (for review see [4]). A new study finds that tunnellingnanotubes (TNT) are important for the intercellular trans-fer of prions during neuroinvasion [5]. Heikenwalder etal. now report that a lymphotoxin-dependent prion repli-cation can occur in inflammatory stromal cells of granulo-mas [6].

Although a great deal is known about the role of PrPSc inthe disease process, the normal function of PrPc hasremained elusive. A variety of functions have been pro-posed for mammalian PrPc, including involvement in celldeath and survival, oxidative stress, immunomodulation,differentiation, metal ion trafficking [7], cell adhesion [8],and transmembrane signaling (for review see [9]). Severalintriguing lines of evidence have emerged recently indicat-ing that PrPc plays a fundamental role not only in the ner-vous system, but also throughout the human body. Thisimplies that PrPc may be involved in resistance to apopto-sis, proliferation and metastasis of human cancer cells.

Recently several excellent reviews have summarizedthe basic processes: the pathogenic pathways that lead totissue pathology in different forms of prion disease andphenotype variability (see [10–12,4] for review). In thepresent review, after a brief description of the prion locusand protein structure, we look at what is known aboutthe physiological function of PrPc, and see how it couldbe involved in cancer biology.

2. Human prion protein locus and a fourth prion gene

Molecular genetic studies conducted with human sub-jects have revealed that the PRNP gene, together withtwo other genes (PRND and PRNT), is localized in the p12/p13 region of chromosome 20, which has been designatedthe PRNP locus. These three genes are located within a 55-kb region (PRNP-20 kb–PRND-3 kb–PRNT). The PRNP genespans 20 kbp, and is composed of two exons [13]. Muta-tions in the repeat region and elsewhere in the PRNP genehave been linked to prion diseases (see [4] for review). Ele-ven different single-point mutations have been shown tobe associated with CJD, ten different point mutations areassociated with GSS, and the D178N mutation causes FFI[14]. Each mutation is associated with either a methionineor a valine at codon 129, which can severely affect the type,onset and duration of the disease [15]. Multiple transcriptvariants (2479 nucleotides) encoding the same proteinhave been found for this gene (NCBI database, Gene). Anal-ysis of both adult and fetal human tissues has confirmedthe ubiquitous but variable expression profile of PRNP,with the highest levels being observed in the CNS and tes-tis [13]. This gene encodes a 253-amino acid ubiquitousprotein of 32–35 kDa, which is expressed by all knownmammals, predominantly in the brain, breast myoepithe-lial cells, lymphocytes and stromal cells of lymphoidorgans [1,16,17]. PRND, which also harbors two exons,encodes a 179-amino acid protein, Doppel, which bearssome similarity to PRNP in terms of both structure andtopology of the proteins [18]. Doppel shares approximately25% identity of amino acid sequence with the C-terminalglobular domain of PrPc, and is expressed in various tissues

during fetal development. However, in astrocytomas, aparticular kind of glial tumor, PRND is overexpressed, andDoppel is localized in the cytoplasm of the tumor cells [19].

The third gene at this locus, PRNT, has only recentlybeen identified in humans. PRNT encodes three alternativesplicing transcripts, and is expressed exclusively in theadult testis. It is now thought that this gene does not en-code a protein.

The fourth member of the prion gene family was iden-tified during a search of publicly-available databases fornucleotide sequences with similarities to the PRNPsequence. The protein and gene have been designated Sha-doo and SPRN, respectively. SPRN is not part of the Priongenomic locus, but is located on chromosomes 7 and 10in mice and humans, respectively. Like PRNP and PRND,the entire open reading frame of SPRN is contained withina single exon. Analysis of expression patterns implies thatSPRN expression is restricted to the brain, suggesting thatunlike Doppel, the Shadoo protein may be pertinent toprion-associated CNS phenomena (see [20] for review).

3. Structure of prion and prion-like proteins

Human PrPc contains a signal peptide (1–22), five octa-peptide repeats of the sequence PHGGGWGQ (51–91), ahighly-conserved hydrophobic domain (106–126), threepeptide sequences forming an a-helix structure (a1–a2–a3), two peptide sequences forming a b-helix structure,and a signal sequence for a glycosylphosphatidylinositol(GPI) anchor (231–253) (Fig. 1a). Like many GPI-anchoredproteins, PrPc is found in cholesterol-rich lipid raft do-mains within the membrane (lipid rafts are regions ofmembranes resistant to cold detergent extraction). PrPccontains two consensus sequences for N-linked glycosyla-tion (T181 and T197) and un-, mono-, and di-glycosylatedversions of PrPc are simultaneously present in the cell. Adisulfide bond between Cys 179 and Cys 214 is essentialfor proper folding of the protein [21].

The octarepeat region is notable for several reasons.Firstly, the histidine residues are capable of binding copperat four copper-binding sites located within the octarepeatregion, and copper is known to induce the endocytosis ofPrPc. Secondly, expansions of the octarepeat domain (withup to thirteen total repeats) are known to cause geneticprion disease.

The structures of human and mouse recombinant Dop-pel and Shadoo have been reviewed in depth in [20]. Thetertiary structure of Doppel is very similar to that of theC-terminal domain of PrPc, but it lacks the N-terminalhydrophobic and octarepeat regions. Shadoo is expressedin the CNS and, like full length PrPc, protects the brainagainst the neurotoxic effects of Doppel and N-terminallydeleted forms of PrPc (DPrP).

The structures of PrPc from various species have beendetermined, and are remarkably similar (Fig. 1b).

4. Cellular localization and trafficking of PrPc

The biogenesis of PrPc is characterized by a series of co-and post-translational modifications. It involves importing

Fig. 1. (a) Schematic representation of the domain architecture of the prion protein family members. The human PrPc molecule contains a signal peptide(1–22), five octapeptide repeats (51–91), a highly-conserved hydrophobic domain (106–126), three peptide sequences implicated in the a-helix structure(a1–a2–a3), two peptide sequences implicated in the b-helix structure, and a signal sequence for the GPI anchor (231–254). Both Doppel and PrPc havestructured C-terminal domains consisting of three a-helices and two short b-strands, as well as basically-charged N-terminal regions. Disulfide bridges areindicated above the proteins (–S–S–) and N-glycosylation sites (Gly) are denoted. Repetitive regions are found in both PrPc and Shadoo; the formerpossessing octarepeats capable of binding copper, and the latter possessing tetrarepeats rich in arginine and glycine residues. A well-conservedhydrophobic tract is also illustrated in PrPc and Shadoo. (b) Color-coded alignment of the PrPc sequence in various species. The peptide sequence of humanPrPc (top) was aligned with sequences found in the mouse (middle), and hamster using the bioinformatics Espript (Easy Sequencing in Postscript). Thissequence alignment shows a high degree of homology between the different PrPC species (about 90% – amino acids shown in red on a white backgroundhave the same physicochemical properties, and those in white on red background are identical). Structures of the a helices and b sheets are indicated. Twored stars indicate sites of N-glycosylation. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of thisarticle.)

M. Mehrpour, P. Codogno / Cancer Letters 290 (2010) 1–23 3


the nascent chain into the endoplasmic reticulum, and theattachment of two N-linked core glycans, disulfide bondingand a GPI anchor. After processing the glycans to formcomplex structures in the Golgi compartment, PrPc is tar-geted to the outer leaflet of the plasma membrane(Fig. 2). PrPc can adopt multiple topologies, including afully translocated form (SecPrP), two transmembrane formswith opposite topologies (NtmPrP and CtmPrP), and a cyto-solic soluble form (CytPrP) [22]. Chakrabarti and Hegdenow report that both CytPrP and CtmPrP inactivatingMahogunine, an E3 ubiquitin ligase, provide a plausibleexplanation for neuronal dysfunction and diseases [23].In cerebellar granule neurons cultured from transgenicmice, CtmPrP was found to be concentrated in the Golgiapparatus, rather than in the endoplasmic reticulum as in

Fig. 2. Diagram showing the mechanism of endocytosis of PrPc. PrPc is located init dissociates from the rafts and moves laterally into non-raft regions of the ptransmembrane adaptor, which facilitates the endocytosis of PrPc through clathrworks on the plasma membrane to internalize cargo in clathrin-mediated enmacroglobulin receptor), also known as LRP1 or CD91, is a protein forming a recemediated endocytosis. AP180 is also a protein that plays an important rolesimultaneously binding both membrane lipids (via an ANTH domain) and clathrinvaginating vesicles. Furthermore, recent findings in neuronal and non-neuroExosomes are membrane vesicles that are released into the extracellular environsurface. Exosome secretion can be used by cells to eject molecules into targeted inalso use exosomes as intercellular communication devices for transferring prote

transfected cell lines [24]. Confocal scanning fluorescenceanalysis showed that PrPc was highly expressed in theGolgi apparatus of the TNF-resistant MCF7 cell line. Inaddition, TNF-resistant MCF7 cells are sensitive to phos-phoinositol phospholipase C (PIPLC) treatment, confirmingthat the PrPc form is overexpressed at the surface of thesetumor cells [25].

Like many cell surface proteins PrPc, can undergo twodistinct endoproteolytic cleavages [26]. The ‘normal’ con-stitutive cleavage of PrPc, which occurs in the brain andin cultured cells, between residues 110 and 111, leadingto the formation of a 9-kDa, soluble, N-terminal fragment(N1; residues 23–110), and a 17 kDa C-terminal fragment(C1 or PrP-II) that is still attached to the membrane viathe GPI anchor [27–30]. This ‘normal’ cleavage of PrPc

cholesterol-rich lipid rafts in the plasma membrane. After binding to Cu2+,

lasma membrane. The N-terminal polybasic region then interacts with ain-coated pits [58]. The AP2 adaptor complex is a multimeric protein thatdocytosis. Low-density lipoprotein receptor-related protein 1 (alpha-2-ptor found in the plasma membrane of human cells involved in receptor-in clathrin-mediated endocytosis of synaptic vesicles. It is capable ofin and is therefore thought to recruit clathrin to the membrane of newly

nal cell models indicate the association of PrPc with secreted exosomes.ment following exocytic fusion of multivesicular endosomes with the celltra-luminal vesicles of multivesicular bodies, but particular cell types mayins and lipids from one cell to another [107].


has been designated a-cleavage [31], and it is stimulatedby agonists of the protein kinase C pathway [32]. a-Cleav-age may be mediated by two members of the ADAM family(ADMA: a disintegrin and metalloprotease) ADAM 10 andADAM 17 [33].

PrPc can also be cleaved within or adjacent to theoctapeptide repeats to generate a 19-kDa, GPI-anchoredC-terminal fragment C2, and the corresponding 7-kDa, N-terminal fragment N2 (residues 23–90) [27,28,34]). Thiscleavage event is mediated by reactive oxygen species(ROS), and is termed b-cleavage [31,35].

4.1. Nucleocytoplasmic isoforms of PrPc

Using high-resolution cryoimmunogold electronmicroscopy in the mouse hippocampus, PrPc has also beenfound in the cytosol of a subpopulation of neurons in thehippocampus, neocortex and thalamus, but not of thosein the cerebellum. In normal and cancerous breast and gas-tric tissues, PrPc is detected in the cytoplasm and on theplasma membrane. These data highlight the widely docu-mented but still controversial question of whether cyto-solic PrP protein is toxic/non-toxic. To the best of ourknowledge, overexpression of normal cytosolic PrPc hasnever been shown to be toxic in any tumor sample ortransfected cells. Wild-type PrPc has been reported to beprocessed by the endoplasmic reticulum associated withthe degradation proteasome pathway in the course ofwhich they can be misdirected to the cytosol. In addition,exposing cultured cells with proteasome inhibitors inducesthe accumulation of an aggregated and non-glycosylatedform of PrPc in the cytoplasm.

Increased synthesis of CtmPrP has been shown to coin-cide with progressive neurodegeneration in GSS syndromepatients with an A117V mutation, and in transgenic micecarrying a triple mutation within the hydrophobic domain[36,37].

Recent data indicate that CytPrP is translated from adownstream AUG (coding for M8 in HuPrP). The authorssuggest that shortening of the signal sequence dictateswhether this isoform spills into the cytosol or not, whenceit can either penetrate into the nucleus or form insolublecytosolic aggregates if the proteasome is inhibited [38].The PrPc construct lacking both the N-terminal signal se-quence and the GPI anchor (23–230) is found mainly inthe nuclei, independently of the presence of nuclear local-ization signals. Overexpression of 20–230 PrPc in the nu-cleus induces the formation of multinucleated cells [39].There were no significant differences in the viability ofthe cells expressing 23–230 PrPc, raising questions aboutthe neurodegenerative phenomena observed in transgenicmice overexpressing 23–230 PrPc [40]. Recombinant 23–230 Prpc binds to chromatin [41], and this DNA-bindingcapacity could account for the nuclear accumulation of23–230 PrPc. This is reminiscent of a truncated PrPc mu-tant, Prpc Q 160stop, which is almost exclusively locatedin the nucleus of transfected cells along the proteasomalpathway [42]. Conflicting results have recently been re-ported for Doppel protein biosynthetic trafficking in astro-cytic tumor cells, probably resulting from abnormal post-translational processes: these modifications make it

impossible for Doppel to locate relative to the plasmamembrane, and lead to its intracellular accumulation inthe lysosomes [43].

4.2. Endocytosis and internalization of PrPc

PrPc can be constitutively internalized at the plasmamembrane (Fig. 2). The route and mechanism of by whichPrPc is internalized are still debated, as caveolae/raft andclathrin-dependent processes have all been reported tobe involved [44–55]. Particularly interesting was the dem-onstration that PrPc interacts functionally with Fyn, in acaveolin-1 like manner in 1C11 cells, leading to activationof this tyrosine kinase activity [56]. More recently, Sunyachand collaborators have shown that experimental condi-tions intended to block endocytosis also prevent PrPcinternalization, and concomitantly abolish PrPc-mediated,p53-dependent, caspase-3 activation in human cells. Wecan therefore conclude that the p53-dependent caspase-3activation triggered by PrPc is directly dependent uponits endocytosis in human cell lines [57]. For PrPc to under-go endocytosis by clathrin-coated pits, it needs to leave thelipid rafts before being internalized, because the rigidstructure of raft lipids is unlikely to be able to accommo-date the tight curvature of coated pits. This phenomenonoccurs after the binding of copper to the OR [49,58], butits physiological significance is unknown (Fig. 2). Zinc alsoinduces endocytosis of PrPc via clathrin-coated pits[48,45]. PrPc binds to several proteins present at the cellsurface including STI1, N-CAM, 37-kDa/67-kDa lamininreceptor (see Table 1 supplementary data). Of these pro-teins, only the 37-kDa/67-kDa laminin receptor has beenimplicated directly in the internalization of PrPc [59]; how-ever, it was shown to be responsible for the internalizationof only 25–50% of membrane-bound recombinant PrPc[60]. Low-density lipoprotein receptor-related protein 1(LRP1) was subsequently shown to control both biosyn-thetic and endocytic, trafficking of PrPc [61,62].

It is still unclear whether caveolae-mediated endocyto-sis is involved in PrPc trafficking in epithelial or/and tumorcells. However, the data suggest that transit through thisunusual pathway might be involved in the implication ofPrPc in tumor resistance to cell death induced by a deathreceptor ligand.

5. Interaction partners of PrPc

The commonly held opinion is that PrPc exerts a part ofits function via interaction with other cell-surfacecomponents.

5.1. Prion protein ligands

To influence intracellular signal-transduction pathways,GPI-anchored proteins need to interact with a transmem-brane adaptor, thereby enabling the transduction of anextracellular signal to occur. An example of one suchprotein is uPAR, which is involved in cellular adhesion,differentiation, proliferation, and migration mediated bythe interaction with transmembrane adaptors such as


integrins, G protein-coupled receptors, and caveolins [63].Analogously, PrPc may bind to a transmembrane protein orto a protein complex that mediates functional associationwith intracellular pathways. Knowledge about how PrPcinteracts with cellular protein is essential if we are to pro-pose a realistic possible function for PrPc. Several methods,such as yeast-two hybrid, coimmunoprecipitation, andcross-linking experiments, have identified several part-ners. Indeed, PrPc has been shown to interact with variousmacromolecules at the cell membrane, in endocytic com-partments and in the secretory pathway. Moreover, somecytosolic candidates have also been identified. Several ofthe candidates are listed and reviewed in [4,11,64]. Allthe PrPc interaction partners so far identified are summa-rized in supplementary data Table 1. They include mem-brane proteins (receptors, enzymes, Caveolin-1, Na–K–ATPase, and a potassium channel), cytoplasmic proteins(components of the cytoskeleton, heat-shock proteins,adaptor proteins involved in signaling), and even the nu-clear protein CBP70. Several of these interaction partnersare known to play a role in synaptic vesicle function.Unfortunately, the physiological relevance of most of theproposed interaction partners remains unconfirmed.

In tumor cells, the crucial challenge is to identify theproteins that interact with PrPc. Although most PrPc is lo-cated on the cell surface, significant amounts are also pres-ent within the cytoplasm of the tumor cells, and in asubpopulation of neurons found in the cortex, hippocam-pus and thalamus. The 37-kDa/67-kDa laminin receptorprecursor/laminin receptor (LRP/LR), which acts as a recep-tor for PrPc and viruses, has been identified [65]. Recently,LRP/LR has been shown to be overexpressed in various can-cer cell lines. The metastatic potential of cancer cells corre-lates with LRP/LR levels [66].

The toxic deletion mutants of PrPc may destroy such acomplex by competing for the binding of some complexcomponents yet failing to interact with the signal-trans-ducing components. Indeed, several models for the toxicityof PrPc deletion mutants have proposed that PrPc binds toa transmembrane receptor, and that deletion mutantseither induce a toxic signal [67] or prevent a survival signal[68,69] (see Section 8.1.3).

5.2. Copper and PrPc

Copper is believed to be the switch that turns on theangiogenesis process in tumor cells. It has been observedthat abnormally high serum copper levels are found in pa-tients with many types of progressive tumors. Early workshowed that PrPc can bind Cu2+ ions in vivo (see [70,71]for review). The co-ordination geometry, stoichiometryand affinity of Cu2+ for PrPc are the subject of much debate.Binding of Cu2+ to PrPc may depend on the concentrationof metal and on the pH. Soon after the initial publication,it was shown that Cu2+ binding causes structural changesin PrPc, suggesting that the misfolding and fibrillizationof PrPc may be profoundly influenced by the presence ofCu2+ ions. In addition, Cu2+ binding has been shown to in-duce endocytosis of PrPc, as described in the previous sec-tion. However, a major role for PrPc in Cu2+ transport intothe cell was ruled out, as PrPc expression levels do not

seem to affect Cu2+ delivery. The affinity and number ofCu2+-binding sites support the suggestion that PrPc couldact as an anti-oxidant by binding potentially harmfulCu2+ ions and sacrificially quenching the free radicals gen-erated as a result of copper redox cycling. The octapeptide-repeating region constitutes a novel glycosaminoglycan(GAG)-binding sequence and His-bound Cu2+ may act asa cofactor for intermolecular recognition reactions, allow-ing the formation of PrPc–Cu2+ GAG assemblies that maybe crucial entities in PrPc metabolism [72].

It has recently been reported that endogenous PrPc rap-idly reacts to Cu2+ in murine neuro-2a and human HeLacells. Specifically, the Cu2+-induced elevation of PrPc ismodulated through transcriptional up-regulation medi-ated via the ataxia-telangiectasia mutated (ATM). The ele-vated PrPc protects against copper-induced oxidativestresses and cell death and plays an active role in modula-tion of intracellular copper concentration [73].

6. Physiological functions of PrPc

The striking lack of a phenotype reported for the firstPrnp knockout mouse [74] is surprising in view of the factthat PrPc is found in a wide range of species [see [4] forreview]. The exact biological functions of PrPc proteinbeyond the nervous system are still unknown, but manyfindings from transgenic mice or the expression of PrPcsuggest that this protein is involved in many biologicalprocesses. Functional roles detected in the nervous and im-mune systems, as well as in other organs, are reviewed indepth in [10–12,75,76]. We briefly examine several of thecellular processes influenced by PrPc (Table 1), and thengo onto focus on the role of PrPc in programmed cell death.

6.1. Function of the PrPc in the nervous system

PrPc is expressed throughout the entire CNS, and occursat particularly high levels in the hippocampus, striatum,and frontal cortex, with an apparently wide subcellulardistribution, including synaptic sites. Several processes inthe nervous system have been shown to be influenced byPrPc. Neurite outgrowth, including the growth of axonsand dendrites, was observed to be reduced in neurons lack-ing PrPc. A possible role of PrPc in synaptic function is sup-ported by the suggestion that PrP-null mice seem todisplay impaired spatial learning and altered excitatoryand inhibitory neurotransmission [77,78].

Levels of PrPc in adult life are associated with possiblechanges in motility, anxiety, and equilibrium [79]. RecentlyKhosravani et al. [80] have reported that PrPc attenuatesglutamate excitotoxicity both in vitro and in vivo by inhib-iting N-methyl-D-aspartate receptors (NMDARs) contain-ing NR2D subunits. These data are also consistent withan important role of PrPc in synaptic function. In addition,in the transgenic mice, maintenance of myelinated axonsin the white matter is impaired. The authors therefore sug-gest that, native PrPc mediates an important neuroprotec-tive role in virtue of its ability to inhibit NR2D subunits.Several studies have also suggested that PrPc may provideneuroprotection (see below).

Table 1Physiological function of PrPc.

PrPc is involved in many cellular and physiological processes in the central nervous system, lymphoid organs, cancer cells, and embryonic development. Atthe cellular level, PrPc is involved in adhesion, differentiation, proliferation, immunomodulation, intracellular communication, Cu2+ and redox homeostasis,oxidative stress and cell death and survival.


6.2. Function of the PrPc in olfactory behavior and physiology

Recently using the Prnp-deficient mouse and PrPc neu-ronal-specific expression transgenic mice Le Pichon et al.have reported that PrPc is important in the normal pro-cessing of sensory information by the olfactory system[81]. The findings suggest that PrPc may be important inlocal circuit function in the olfactory system, and so may,in turn, influence odor perception (see also [82]).

In summary, it is thought that PrPc is involved in CNSdevelopment and differentiation, and in neurite out-growth. PrPc also mediates early synaptic transmission inneurons. Other physiological functions affected by PrPcare the regulation of circadian rhythms, memory forma-tion, and cognitive function (see [83] for review).

6.3. Function of the PrPc in the immune system

Functional roles detected in the immune systems are re-viewed in depth in [84]. Although PrP(�/�) mice have beenreported to display only minor alterations in immune func-tion, recent work has suggested that PrP is required for theself-renewal of hematopoietic stem cells [85]. The animalshave not been reported to display spontaneous tumors orgreater susceptibility to infections than PrP+/+ mice.

The role of PrPc in innate and adaptive immunity iscomplex. PrPc is expressed widely in the immune system,and in mature lymphoid and myeloid compartments(human T- and B-lymphocytes, natural killer (NK) cells,platelets, monocytes, dendritic cells (DCs) and folliculardendritic cells). It is upregulated in T-cell activation, and

may be expressed at higher levels during human NK celldifferentiation, with particularly high levels on CD56+

CD3+ NK T cells. Furthermore, antibody cross-linking ofsurface PrP modulates T-cell activation, and leads to rear-rangements of lipid raft constituents and increased phos-phorylation of signaling proteins. Although PrPc residesin membrane microclusters of the immunological synapseduring lymphocyte activation, the role of PrPc in the for-mation of immunological synapses is controversial [86].Recently, the contribution of PrPc in alloantigen and majorhistocompatibility complex (MHC)-driven DC-T cell inter-action was reported. An absence of PrPc on DCs results ina reduced allogenic T-cell response. Moreover, PrPc isresponsible for the phagocytic capacity of macrophagesby activating ERK1/2 and Akt kinase.

In human T-lymphocytes, PrPc interacts with zeta-chain associated protein (ZAP)-70, a transduction signalresponsible for T-cell activation and proliferation. Theexpression of interleukin-2 by T cells is enhanced in thepresence of PrPc.

In summary, these observations suggest that PrPc playsa functional role in T-cell development, activation and pro-liferation (see [83] for review).

6.4. Function of PrPc in cell adhesion

In association with cell adhesion, distinct functions ofPrPc were observed, depending on the cell type: neuronaldifferentiation, epithelial and endothelial barrier integrity,trans-endothelial migration of monocytes, T-cell activation(see [87]).


6.4.1. Cell adhesion and extracellular matrix neuritogenesisLehmann’s lab has reported that neuroblastoma (N2a)

cells in culture expressing the PrPc at high levels aggre-gated to a much greater extent than control cells [8]. Thissuggests that PrPc promotes cell adhesion interactions thatare copper-independent, and that this might involve inter-actions with the N-terminal part of the protein. Some ofthe interaction partners of PrPc identified so far play a rolein adhesion, including Laminin, a structural component ofbasement membrane [88], LRP/LR a non-integrin lamininreceptor [89], heparan sulfate proteoglycans [65,90],neuropilin [184] stress-inducible protein-1 (STI1) a heat-shock-related protein [91] and neural cell adhesion mole-cule (N-CAMs) [92]. Both cis and trans interactionsbetween N-CAM at the neuronal surface and PrPc promotethe recruitment of N-CAM to lipid rafts, thereby regulatingthe activation of fyn kinase, an enzyme involved in N-CAM-mediated signaling. Cis and trans interactions betweenN-CAM and PrPc promote neurite outgrowth [93].

STI-1, which, as noted above, is a partner of PrPc withneuroprotective activity through activation of PKA [94];could induce the growth of neurites in hippocampal neu-rons via activation of ERK [95]. Recently the same labwas reported that PrPc endocytosis is a necessary step tomodulate STI1-dependent ERK1/2 signaling involved inneuritogenesis [76]. All these data reveal the involvementof PrPc in neuritogenesis, in particular its contribution tocell adhesion on a matrix rich in laminin.

6.4.2. Cell–cell adhesion and junctional proteinA preferential localization of PrPc at the level of inter-

cellular junctions has recently been observed in brainendothelial cells that form the blood–brain barrier [87].Mixed cultures of wild-type and PrPc-deficient mousebrain endothelial cells showed that PrPc accumulation atcell–cell contacts was probably dependent on homophilicinteractions between adjacent cells. Interaction PrPc/PrPcbetween two adjacent cells could involve a head–tail con-formation similar to that described for the oligomerizationof PrPc in an in vitro model [96]. Moreover, anti-PrPc anti-bodies unexpectedly inhibited the transmigration of U937human monocytic cells as well as that of freshly isolatedmonocytes through human brain endothelial cells. Theseobservations suggest that PrPc, like PECAM-1 (plateletendothelial cell adhesion molecule), is expressed by brainendothelium as a junctional protein that is involved inthe trans-endothelial migration of monocytes.

6.4.3. PrPc mediates Ca2+-independent and -dependent celladhesion and signaling in zebra fish embryos

A severe PrPc loss-of-function phenotype was reportedrecently in zebra fish embryos, which is characterized bythe loss of embryonic cell adhesion and arrested gastrula-tion [97]. Using zebra fish, mouse, and Drosophila cells,they showed that PrPc: (1) mediates Ca2+-independenthomophilic cell adhesion and signaling; and (2) modulatesCa2+-dependent cell adhesion by regulating the delivery ofE-cadherin to the plasma membrane. In vivo, time-lapseanalyses reveal that the arrested gastrulation in PrPcknock-down embryos is due to deficient morphogeneticcell movements, which rely on E-cadherin-based adhesion.

Moreover, the local accumulation of PrPc at cell contactsites was concomitant with the activation of Src-related ki-nases, the recruitment of reggie/flotillin microdomains,and the reorganization of the actin cytoskeleton, which isconsistent with a role of PrPc in the modulation of celladhesion via signaling. Taken together, these data revealevolutionarily-conserved roles of PrP in cell communica-tion, which ultimately impinge on the stability of adherentcell junctions during embryo development.

7. Signal transduction of PrPc

Recent studies have advanced the hypothesis that sev-eral signaling pathways or signaling components, such asPI3-kinase/Akt, Cyclic AMP/protein kinase A (PKA), PKC,Fyn, and Erk1/2, are modulated by PrPc expression, itscross-linking, or its interaction with another protein(Fig. 3).

Taken as a whole, these studies indicate that endoge-nously-expressed PrPc can modify several intracellular sig-naling pathways involved in proliferation, differentiation,cell adhesion, cell trafficking, and transmembrane signal-ing, in order to determine cell survival.

8. PrPc and programmed cell death

Some intriguing evidence has emerged recently indicat-ing that PrPc may exert a cytoprotective activity againstvarious kinds of internal or environmental stress (see[9,11,98] for review). The original suggestion that PrPccould possibly be involved in programmed cell death arosefrom the identification of a significant similarity with theBcl-2 homology domain 2 (BH2) and its ability to bind toBcl-2 in a yeast double-hybrid approach [99,100]. Anti-apoptotic activity of PrPc was then demonstrated in a vari-ety of experimental systems, including mice, culturedmammalian cells, and yeast.

8.1. PrPc promotes neuroprotection in vivo

8.1.1. PrP-null miceSeveral lines of PrP knockout mice have been generated

by homologous recombination in embryonic stem cells inmany laboratories (for details see Table 2 supplementarydata). These mice can be divided in two groups. Group 1includes Prnp0/0 [Zürich I] [74] and Prnp�/� [Edinburgh][101]. These mice display discrete neurophysiologicalchanges and demyelination of their peripheral nerves withage. In contrast with these earliest lines, three lines gener-ated subsequently: Prnp�/� [Nagasaki], Rcm0, and Prnp�/�

[Zürich II] [102–104] develop ataxia and Purkinje cell losslater in life. Neurodegeneration in Nagasaki, Zurich II, andRcm0 PrP-null mice, and their rescue by the reintroductionof Prnp as a transgene have suggested that PrPc is a neuro-protective protein. However, it was later shown that thestrategy used to generate the second group of knockoutmice led to overexpression of the Prnd gene. As describedin previous section, Prnd gene encodes Doppel a prion-likeprotein. Doppel protein is specifically expressed in Pur-kinje cells, where it induces cell death and results in the

Fig. 3. Signaling through PrPc. Antibody-mediated cross-linking of PrPc-induced activation of the Fyn kinase, a downstream event, in neuronaldifferentiated cells in a caveolin-1-dependent manner [53]. The same group also claims to have identified ERK1/2 phosphorylation [97]. They argued thatsince ERK1/2 are fully controlled through NADPH oxidase-dependent ROS production; the basic function of PrPc is to maintain cellular redox homeostasis[53,97,98]. Fyn kinase activation and downstream activation of ERK1/2 have also been reported in a hypothalamic cell line [99]. Furthermore, PrPc cross-linking also modulates the serotonergic receptor that is coupled to G-protein activity in differentiated neuronal cells [100]. PrPc binding peptide activatedboth the ERK- and the PKA-pathway in retinal explants from neonatal mice [92]. Although Fyn kinase activity occurred mainly in neurite outgrowths, boththe PI3-kinase/Akt pathway and the regulation of Bcl-2 and Bax expression contributed to the survival effect elicited by PrPc. The PKA and Erk signalingpathways contributed to both neurite outgrowth and neuronal survival [91,101]. Interactions between N-CAM at the neuronal surface and PrPc regulate theactivation of Fyn kinase, and promote neurite outgrowth [91]. Two other studies have reported a link between PrPc expression and the induction of PI 3-kinase activity, and have shown that this mechanism probably involved the octarepeat region of PrPc. Phosphorylation of Akt was lower in the brains of PrP-null mice than in wild type both under control condition and soon after an ischemic insult [102,103]. Recently, the same lab reported controversial data. Theauthors suggest that overexpression of PrPc alters postischemic Erk1/2 phosphorylation, but not Akt phosphorylation, and protects against focal cerebralischemia. The engagement of PrPc with STI1 induces neuroprotective signals that rescue cells from apoptosis via cAMP/protein kinase A and the Erksignaling pathways. Interaction with STI1 induced different signaling pathways, promoting neuroprotection by PKA activation and neuritogenesis byactivation of the MAPK pathway (see [7] for review). PrPc also binds to laminin in a manner that could involve the mitogen-activated protein (MAP) kinase/ERK pathway [104]. Intracellular endocytosed PrPc may interact with proteins involved in signaling pathways, including growth factor receptor bindingprotein (Grb2), an adaptor protein that is involved in neuronal survival by activating ERK1/2 and MAP kinases [105]. As with other GPI-anchor proteins, PrPccan transfer from cell to cell, and may transduce signals to a large number of cells [106]. PrPc may play several differential roles in the cell death response,such as a proapoptotic role during ER stress, and an anti-apoptotic role during oxidative stress-induced cell death. ER stress-induced activation of caspases,PKCdelta, and apoptosis were all significantly exacerbated in PrPc cells, whereas H2O2-induced proapoptotic changes were all but suppressed in PrPc-positive cells, in contrast to PrPc-negative cells. Overexpression of the kinase-inactive form of PKCdelta suppressed both ER and oxidative stress-inducedapoptosis. These results suggest that PrPc enhances the susceptibility of neural cells to impairment of protein processing and trafficking, but decreases theirvulnerability to oxidative insults. The authors suggest that PKCdelta is a key downstream mediator of cellular stress-induced neuronal apoptosis [146].Taken as a whole, these studies indicate that endogenously-expressed PrPc can modify several intracellular signaling pathways involved in proliferation,differentiation, cell adhesion, cell trafficking, and transmembrane signaling, in order to determine cell survival. Arrows indicate activation, hammersindicate inhibition. Dotted lines indicate the hypothetical part of the pathway.


ataxic phenotype of these mice. This implies that overex-pression of Doppel in the absence of PrP, rather than theabsence of PrPc itself, was responsible for the neurotoxicityobserved in Nagasaki, Zurich II, and Rmc0 mice. Preciselywhy the overexpression of Doppel is deleterious is still un-clear. On the basis of the observation that Doppel expres-sion induced heme oxygenase 1 (HO-1) and neuronal andinducible nitric oxide synthases (nNOS and iNOS), suggest-ing an increase in oxidative stress in the brains of theDoppel-expressing Prnp0/0 mice [105], proposed thatDoppel expression exacerbates oxidative damage by antag-onizing the anti-oxidant function if wild-type PrPc.

Other studies in knockout mice provide further evi-dence in support of the neuroprotective role of PrPin vivo. Interestingly, after traumatic brain injury, PrP isone of the two most highly up-regulated proteins, suggest-ing that it contributes to post-trauma recovery [106]. Thepromoter of PRNP contains a heat shock element that

may respond to stress, and result in the overexpressionof PrPc [107]. Zurich I Prnp0/0 mice are more susceptibleto seizures [108]. Increased susceptibility of these mice isunlikely to be due to Doppel expression, since Zurich IPrnp0/0 mice do not over-express Doppel.

PrPc-null mice are more susceptible to neuronal lossafter experimental brain injury [109]. Examination thephenotype of PrPc-null mice reveals higher levels of nucle-ar factor NF-jB and Mn superoxide dismutase, COX-IV,lower levels of Cu/Zn superoxide dismutase activity,decreased p53, and altered melatonin levels.

8.1.2. PrP transgenic miceDespite all the uncertainties surrounding the neuropro-

tective functions of PrPc at the molecular level, someknowledge was obtained by expressing a series of par-tially-deleted Prnp variants in transgenic mice (see Table3 supplementary data and [4]).


This showed that deletions encompassing residues 32through 106 did not cause any abnormalities [69]. Incontrast, two larger deletions (D32–121 or D32–134,collectively referred to PrPDN) caused progressive neuro-degenerative illness in the mice lacking both copies ofthe endogenous Prnp gene. Strikingly, a single Prnp allelewas sufficient to completely abrogate the phenotype ofthese animals. This phenomenon is similar to that involv-ing the phenotype observed in Nagasaki, Zurich II, andRmc0 mice. Doppel is structurally similar to PrPDN, sinceit lacks regions homologous to the flexible, N-terminal tailof PrP (see Fig. 1A).

Full-length secreted PrPc was released when its car-boxy-terminal hydrophobic domain (PrP231#), which isnormally replaced by a GPI anchor, was deleted. This failedto induce any pathological phenotype [110]. In contrast,targeting PrPc to the cytosol (cyPrP = PrPD1–22 231#), bydeleting its amino-terminal leader peptide (which targetsPrPc to the endoplasmic reticulum and to the secretorypathway) induced ataxia with cerebellar degenerationand gliosis [40]. Co-expression of wild-type PrPc did notinfluence the phenotype of these mice. In contrast toin vivo data, in cultured cells, a range of in vitro data hasshown that cyPrP is not toxic [111,112] and, moreover, ap-pears to be the predominant PrPc form with an anti-apop-totic function against Bax [113]. Whether cytoplasmicexpression of PrPc and its cytotoxicity represent realisticmodels of the events occurring during prion disease re-mains very hotly debated.

The function of PrPc may depend on the region contain-ing three a-helices in concert with charged amino acidclusters (aa 95–110). With the exception of a small dele-tion (PrPD104–114) [36], ablation of charged amino acid clus-ters (aa 95–110) in combination with a partial or completedeletion of the a-helices region elicits severe disorders inmice. PrPD32–121 and PrPD32–134 transgenic mice sufferfrom ataxia and cerebellar granule cell loss, in addition towidespread white matter disease [114,69]. The lattercondition is also seen in mice expressing deletionsencompassing all (PrPD94–134) or part (PrPD105–125) of thecentral domain (CD) [67,68]. These disorders are radicallydifferent from those seen in prion infections, and none ofthem is accompanied by pathological aggregation of PrP.However, this phenotype is reversed by the co-expressionof wild-type PrPc.

Insertion mutations affecting the octapeptide repeat(OR) are associated with hereditary human prion disease.However, transgenic studies indicate that the OR is notrequired for PrPc to function or to be convertible into PrPSc.It has been shown that mice expressing OR-deficient PrPcmutants do not develop pathologies (see [4] for review).This was unexpected, because a range of in vitro data hadidentified the OR as being responsible for binding copper(reviewed in [115]), and for conferring protection againstoxidative stress (see below). On the other hand, transgenicmice expressing nine supernumerary octapeptide repeats,out of a total of 14 proline and glycine-rich repeats [116],which models a human familial CJD-linked mutation, dodevelop ataxia and cerebellar atrophy, granule cell loss, gli-osis, progressive myopathy, and PrP deposition. The latterphenotype resembles its human counterpart in some ways

[117], although no transmission to wild-type miceoccurred [118]. However, co-expression of wild-type PrPcdid not influence the phenotypes of these mice.

8.1.3. A molecular model for Prnp/Prnd interactionsThe fact that PrPc suppresses the phenotypes induced

by PrPDN, PrPD105–125 and Doppel suggests that a commonmechanism facilitates the protective effect of PrPc againsteach of these toxic proteins. One model that has been pro-posed attempts to explain the interactions between Doppeland PrPc in terms of an unidentified prion protein receptor,termed LPrP (Fig. 4).

One facet of this model is that it invokes a hypotheticalprion-like protein termed ‘p’ to explain the lack of a pheno-type in Prnp0/0 mice. p also binds to LPrP, and can initiatethe positive signaling event that promotes cell survival. Ithas recently been shown that like PrPc, Shadoo is capableof blocking Doppel-induced apoptosis in Prnp0/0 CGNs[20]. When the hydrophobic domain is eliminated, Shadooloses its neuroprotective capacity in a manner similar toPrPc. Together with the biochemical properties shared byShadoo and PrPc mentioned, above these observationsunderscore similarities between Shadoo and the hypothet-ical p protein.

Another facet is the hypothetical ligand suggested inthis model. Among the various PrPc ligands identified todate (see supplementary Table 1), at least STI1, the interac-tion of which with PrPc mediates neuroprotection [91,94],has been shown to bind to the PrPc113–128 domain, which isdeleted in the truncated molecules with neurodegenera-tive activity. However, there is no evidence that STI1 is infact the putative PrPc ligand/receptor proposed in the dif-ferent models [67,68].

In summary, PrP-null mice are more susceptible toischemia, Doppel and N-terminally truncated PrP-inducedcytotoxicity. Expression of wild-type PrPc completelyabrogates the neurodegenerative phenotype of miceexpressing Doppel, or N-terminally truncated forms ofPrP [69,102,119]. Clearly, further progress in elucidatingthe mechanism underlying PrPc cytoprotection will re-quire identification of other proteins that interact physi-cally and functionally with PrPc and connect it to the celldeath and survival pathway.

8.2. PrPc promotes neuroprotection in vitro

The first experimental data was obtained by theKuwahara laboratory using cell lines established fromhippocampal neurons (HpL3-4 cells) derived from NgskPrP-null or wild-type mice. PrP-null cells died quicklywhen deprived of serum, whereas their wild-type counter-parts survived [120]. Similarly, in HpL3-4 cells serumdeprivation led to p53 and Bax upregulation, cleavage ofboth caspase-3 and poly(ADP-ribose) polymerase (PARP),decreased Bcl-2, increased levels of mitochondrial calcium,and lower mitochondrial membrane potential. In contrast,transfection of knockout cells with a Prnp expression vec-tor prevented all these typical apoptotic changes [121].However, the neuronal HpL3-4 cells had been derived frommice over-expressing Doppel, and so their increased sensi-tivity to serum deprivation might be a consequence of

Fig. 4. PrPc-mediated cell survival or cell death in transgenic mice. (Top left) In wild-type mice, Prpc binds to a hypothetical ligand (LPrP) and this initiatesan as-yet-unidentified signaling event that promotes cell survival. This binding event can occur in both the cis and trans configurations. This implies thatthere are two binding sites for LPrP on PrPc: a C-terminal anchoring site, and an N-terminal effector site which enables signaling. (Top right) When Doppelor DPrP are expressed in Prnp+/+ mice, PrPc displays higher affinity for LPrP, and this prevents or reduces Doppel/DPrP binding. (Bottom left) in Prnp-nullmice, a hypothetical Prpc-like protein, p, binds to LPrP and initiates the same cell survival signaling. p has the same N-terminal effector domain as PrPc, andcan therefore elicit signaling activity through LPrP. (Bottom right) when delta PrP or Doppel bind to LPrP in Prnp-null mice, cell death signaling is initiatedsince both proteins lack the N-terminal effector domain required for prosurvival signaling [183].


Doppel overexpression rather than of the absence of PrPc.To investigate whether PrPc inhibits apoptotic neuronalcell death without Doppel, an immortalized cell line wasestablished (NpL2) from the brain of Zürich I Prnp0/0 mice,which do not display any ectopic expression of Doppel. Theresults showed that PrPc potently inhibited serum-inducedapoptotic cell death (see supplementary Table 2). Further-more, PrPc expression enhanced SOD activity in NpL2 cells.These findings indicate that Doppel production did not af-fect the anti-apoptotic and anti-oxidative functions of PrPc.The authors suggested that PrPc may be directly implicatedin protection against oxidative stress [122].

One of the clearest examples of a cytoprotective activityof PrPc is the protein’s ability to protect human fetal neu-rons in culture against the apoptosis induced by Bax. Whenhuman fetal neurons in culture were microinjected with aplasmid encoding Bax, �90% of the neurons underwentapoptosis; but when the neurons were co-injected withboth Bax- and PrP-encoding plasmids, the percentage ofapoptotic cells fell to �10% [123].

The octapeptide repeat domain that displays some sim-ilarity with the BH2 domain of Bcl-2 is essential for PrP’sneuroprotective function against Bax. Furthermore, famil-ial PrPc mutations D178N and T183A, which are associatedwith the human prion diseases FFI and familial atypicalspongiform encephalopathy, partially or completely abol-ish PrPc anti-Bax function (Table 2 supplementary data[124]). The authors therefore propose that the normalstructure of PrPc is probably important for the anti-Baxfunction. While PrPc needs to achieve some aspects of

maturity (glycosylation and transport) in order to performits anti-Bax function, this does not require the GPI anchor-ing of PrPc [123]. Furthermore, PrPc expressed uniquely inthe cytosol is also able to inhibit Bax [112,124]. Theauthors also showed that the GSS-associated A117V PrPmutant with a valine at codon 129 retained the anti-Baxfunction, while all of the eleven CJD-associated PrP muta-tions carrying a methionine at codon 129 and the FFI asso-ciated D178N PrP mutant lost their ability to prevent Bax-mediated formation of condensed chromatin in humanneurons [125]. Recently the same laboratory, using variousSyrian hamster PrP mutants, has confirmed that the cyto-solic form of PrPc is the predominant form carrying outthe anti-Bax function [113]. The physiological significanceof cytoplasmic PrP is uncertain at this point, since very lit-tle of this form appears to be generated in vivo from thewild-type molecule [22].

Roucou et al. have reported that the cytoprotective ef-fect of PrPc against Bax appeared to be specific, since PrPcdid not prevent the apoptosis induced by caspase, Bak,t-Bid, staurosporine, or thapsigargin [124]. PrPc does notcolocalize with Bax in normal or apoptotic primaryneurons, and did not prevent Bax-mediated cytochrome crelease in a mitochondrial cell-free system. PrPc could pro-tect against Bax-mediated cell death by preventing the Baxproapoptotic conformational change that is the first step inBax activation. The authors conclude that, unlike Bcl-2, butlike BAR and BI-1, PrPc probably does not prevent Bax-mediated cell death by directly interacting with Bax. Theauthors propose that in mammalian cells, both Bcl-2 and


PrPc maintain Bax in an inactive state, thus conferring neu-roprotection. They suggest that the conversion of PrPc intoPrPSc inactivates antiapoptotic PrPc, leaving Bcl-2 as theonly major Bax inhibitor. At this stage Bax and Bcl-2 arestill in balance, but neurons are more sensitive to apoptoticinsults. Any subsequent events leading to a further de-crease in the expression of Bcl-2, such as those observedin the aging CNS and loss of PrPc neuroprotective function,would result in increase neurotoxicity (see [98] forreview).

Li et al. used mouse PrPc to analyze the involvement ofPrPc in Bax-induced cell death in yeast ([126]; see [11] forreview). The authors found that PrPc potently suppressedthe death of yeast cells expressing mammalian Bax. In con-trast, cytosolic PrP-(23–231) failed to rescue the growth ofBax-expressing yeast, indicating that protective activity re-quires PrPc to be targeted to the secretory pathway. In con-trast to the data reported by Leblanc’s group in neuroncells, Li et al. found that the octapeptide repeat domain isnot essential for PrPc’s neuroprotective function againstBax in a yeast system, whereas a charged region encom-passing residues 23–31 is. Although yeast does not containendogenous Bcl-2 family members or caspases, the initialevents underlying Bax activity in yeast and mammaliancells are similar, including translocation of the protein tomitochondria, the release of cytochrome c, and alterationsin mitochondrial function [127]. Only a mutant containingnine extra octapeptide repeats failed to suppress Bax-induced cell death in several mutants associated with hu-man familial prion diseases (Table 3 supplementary data).In yeast, the protective effect of PrPc seems to be related toits interactions with endogenous yeast proteins that occurdownstream of Bax in a context of cellular stress or a tox-icity pathway.

Other studies have shown that the PrPc binds to STI1,and that the interaction between these two proteins atthe cell surface can rescue cultured retinal cells from theapoptosis induced by treatment with anisomycin. A func-tional role for this interaction is supported by the fact thatboth proteins are found on neuronal cell surfaces in theCNS. Neuroprotection depended on the binding of specificdomains in both proteins: STI1 230–245 and PrPc 113–128. This constitutes a further demonstration that this pro-tection depends on an increase in the levels of cyclic AMPthat activates protein kinase A [91,94,95]. Other studiesalso suggest that the interaction between STI1 and PrPc in-duces cytoprotective signals in a PrPc-transfected neuronalcell line, by regulating SOD activity involving the NH2-ter-minal domain of PrPc [128,129]. The first question is howthe binding of PrPc at the outer membrane activates aden-ylyl cyclase, which is normally regulated by G proteins onthe inner membrane. We can find the beginnings of anexplanation in the data reported by Mouillet-Richard etal. [130]. These authors propose that the antibody-medi-ated ligation of PrPc may affect the potency or dynamicsof G-protein activation by agonist-bound serotonergicreceptors. The PrPc-dependent modulation of 5-HT recep-tor coupling is restricted to 1C115-HT cells expressing acomplete serotonergic phenotype. Critically, it involves aPrPc-caveolin platform implemented on the neurites of1C115-HT cells during differentiation.

8.3. Prion protein and oxidative stress

Two papers from same laboratory have suggested thatPrPc may act as a free radical scavenger and/or sensor mol-ecule for oxidative stress in tumor cells. The first hints ofthis came from in vitro studies of prostate tumor spheroids.ROS levels were correlated to an increased expression ofPrPc, Cu/Zn SOD-1, and catalase in small (diameter100 ± 20 lm) versus large spheroids (diameter250 ± 50 lm). These data suggest that PrPc expression intumor spheroids is related to the intracellular redox state,and may contribute to the anti-oxidative defense [131].The authors suggest that since PrPc expression is corre-lated with the intrinsic intracellular redox state, it is alsolinked to tumor size. A subsequent study has demonstratedthat treatment with growth factors and TNF-a results in in-creased PrPc in glioma tumor spheroids as a result of anelevation in intracellular ROS levels. Elevation of PrPcmay occur as a stress response in order to enhance theanti-oxidant capacity of cells, and compete for oxidativeinsults. Overexpression of either wild-type PrPc or mutantV210I PrP in neuroblastoma tumor spheroids resulted in asignificant reduction in intracellular ROS levels, and de-creased growth factor-induced ROS generation. Theauthors suggest that PrP may play a role in the cellularanti-oxidant system [132].

In CNS, several lines of evidence suggest that PrPc mayplay a role in protecting cells against oxidative stress (see[133] for a review).

The sensitivity of human neuroblastoma cells to oxida-tive stress is increased by the expression of two disease-associated mutants of PrP, namely PG14 and A116 V, whichare incapable of undergoing ROS-mediated b-cleavage,implying that this cleavage event plays a critical role inthe cellular resistance to oxidative stress [134]. These find-ings suggest that b-cleavage of PrPc is an initial conse-quence following exposure to ROS in the extracellularenvironment and contributes to a pathway involved inthe anti-oxidant protection of neuronal cells. This is consis-tent with recent data from N2 a mouse neuroblastomashowing that the protective effect of PrPc against oxidativestress involves the octarepeat region, but not the residues110–135 domain nor the high-affinity copper-binding sitedescribed for human residues His96/His111 [135].

How PrPc protects cells against oxidative stress remainsunclear. One possibility is that PrPc could itself have SODactivity, and thereby mediate the anti-oxidant function[136]. However, there is significant controversy about thereality of this alleged SOD activity in vitro [137] andin vivo [138]. A second hypothesis is that PrPc may act indi-rectly to protect cells against oxidative stress by upregulat-ing the activities of other proteins, such as Cu–Zn SOD orglutathione reductase [139,140], that detoxify ROS. Theactivities of other anti-oxidant enzymes, such as catalaseand glutathione reductase, have been reported to be de-creased in Prnp0/0 mice [141,142], but whether PrPc playsa direct role in regulating these molecules remains to bedetermined.

It is also possible that PrPc acts either upstream ordownstream of ROS to protect cells against oxidativestress. In some situations, such as oxidative stress, for


example, may activate apoptotic pathways [143]. In suchcases, the anti-apoptotic effects of PrPc may account forthe protein’s ability to protect cells against oxidative stress.Consistent with this, Choi et al., using neural cells frommice expressing PrPc and PrPc-knockout, showed that PrPcinterferes with divalent metal Mn uptake, and protects thecells against H2O2- and Mn-induced oxidative stress andapoptotic cell death [144].

Another example is the data reported by Jörg Tatzelt’slaboratory showing that green tea extracts interfere withthe stress-protective activity of PrPc and the formation ofPrPsc [145]. The major polyphenols in green tea induce ra-pid transition of PrPc into a detergent-insoluble conforma-tion. As a consequence, the mature PrPc in treated cells isdepleted and is localized at the cell surface and degradedin lysosomal compartments. These cells are protectedagainst PrPSc propagation; however, they are more vulner-able to stress-induced cell death.

Using mouse neural cells expressing PrPc and PrPc-knockout, Anantharam et al. have recently reported thatPrPc plays a proapoptotic role during ER stress, and anantiapoptotic role during oxidative stress-induced celldeath. These results suggest that PrPc enhances the sus-ceptibility of neural cells to impairment of protein process-ing and trafficking, but decreases their vulnerability tooxidative insults. The authors suggest that PKCdelta is akey downstream mediator of cellular stress-induced neu-ronal apoptosis. This study suggests that PrPc may playsseveral differential roles in the cell death response, suchas a proapoptotic role during ER stress, and an anti-apopto-tic role during oxidative stress-induced cell death [146].

8.4. The domain involved in PrP cytoprotective activity indifferent cell types

The first region (residues 23–31 in mouse) is known toplay a role in endocytic trafficking of PrP [49,58], and inlocalization the protein to lipid rafts [147]. This regionhas been found to be essential for PrPc protection againstBax in yeast [126], and against Doppel in cerebellar granuleneurons [148]. In transgenic mice, deletions beginning atresidue 32 (PrPD32–80 and PrPD32–93), do not affect the abil-ity of PrP to suppress neurodegeneration induced byPrPDN (Table 3 supplementary data). In contrast, deletionof residues 23–88 obliterates the ability of PrP to rescuemice from Doppel-induced toxicity [149]. These two re-sults together suggest that residues 23–31 are implicatedin protecting against PrPDN and Doppel, although it willbe necessary to generate Tg(PrPD23–31) mice to confirm thisinference. The requirement for residues 23–31 suggeststhat PrPc cytoprotective activity may depend on itsrelocation to the cell surface, lipid rafts, or endosomalcompartments.

Another domain that has been tested is the C-terminal,GPI addition signal. In both yeast [126] and human neu-rons [123], deletion of this region does not impair the abil-ity of PrPc to suppress Bax-mediated apoptosis, implyingthat the cytoprotective activity of PrP does not requiretethering to the plasma membrane. Similarly, the rescueof granule neurons from Doppel-induced apoptosis byPrP does not require the GPI anchor [150]. A related issue

concerns the necessity for the expression of PrP in thesecretory pathway. Deletion of the N- and C-terminal sig-nal peptides (PrP23–231) completely abolishes the abilityof PrP to rescue yeast from Bax-induced cell death. Simi-larly, deleting its amino-terminal leader peptide provokedataxia, with cerebellar degeneration and gliosis [40]. Coex-pression of wild-type PrPc did not influence the pheno-types of these mice. In contrast, PrP23–231 retains fullrescue activity against Bax in cultured human neurons[111,112] and, moreover, appears to be the predominantPrPc form displaying an anti-apoptotic function againstBax [113].

Whether this discrepancy implies a fundamental differ-ence in the cytoprotective pathways operating in varioussystems remains to be determined.

The role of the PrP octapeptide repeats has also beeninvestigated in several other systems. The repeats areknown to bind copper ions, which have been postulatedto play a role in the function of PrPc (see Section 5.2). Dele-tion of the octapeptide repeats abolishes the ability of PrPcto protect cortical neurons against Bax [123], granule neu-rons against Doppel [148], and immortalized hippocampalneurons against serum deprivation [129]. In contrast, elim-inating the repeats does not affect the ability of PrPc toprotect yeast against Bax [126], or transgenic mice againstPrPDN [69]. These observations are difficult to reconcile,and suggest that the octapeptide repeats may perform dif-ferent functions in different cell types, or in the context ofdifferent toxic insults.

Another interesting point of comparison concerns theeffect of disease-causing mutations on the rescuing activityof PrP. D178N and E199K mutants retain the ability to res-cue yeast from Bax-mediated cell death [126]. In contrast,PG14, a mutant harboring an insertion of nine additionaloctapeptide repeats, displays no rescue activity [126]. Bio-chemical analysis of these mutant proteins suggests thatthe loss of protective activity by PG14 may be due to itsaggregated state, whereas D178N and E199K are com-pletely soluble in yeast. This observation is consistent withstudies in transgenic mice, since PG14 PrP is aggregatedwhen expressed in the brain [116], and also displays par-tially impaired ability to suppress the neurotoxicity ofPrPD32–134 [151]. PrP carrying the E199K mutation, whichhas been shown to be completely soluble in mouse models[152], maintains the ability to suppress Doppel-inducedneurodegeneration [149]. Although soluble in yeast,D178N PrPc is partially detergent resistant in cultured neu-rons [111], which may explain the inability of this mutantto rescue neurons from Bax [123]. Westergard et al. sug-gested that the cytoprotective activity of PrPc may requirethe presence of a soluble form of the protein, and so muta-tions inducing aggregation impair activity [11].

8.5. The protective function of PrPc: controversy story

As far as we are aware, normal PrPc has never beenshown to be toxic when overexpressed in a tumor sampleor transfected cells. Therefore, PrPc is not toxic by itself,and tumor cells that endogenously, transiently or stablyover-express PrPc can be routinely obtained in the labora-tory. However, in the human embryonic kidney 293 cell


line, the rabbit epithelial Rov9 cell line, and the murinecortical TSM1 cell line, PrPc over-expression increases thesusceptibility of these cells to the apoptotic inducer,staurosporine [153,154]. The authors show that this cellu-lar response was due to activation of caspase-3 via tran-scriptional and post-transcriptional control of theproapoptotic oncogene p53 [153,154].

Another demonstrative example is the study of theapoptotic events induced by exposure to HuPrP106–126peptide in primary culture of murine cortical neuronsand in transgenic mice 338 cortical neurons. The aggre-gated peptide did indeed trigger massive neuronal deathwithin 24 h. The authors showed that this cellular responsewas due to the activation of c-Jun-N-terminal kinase (see[39] for review).

Finally, in vivo cross-linking of PrPc with specific mono-clonal antibodies that target the PrPc epitope within therange of 95–105 induced rapid and extensive apoptosisin hippocampal and cerebellar neurons [155]. The authorssuggest that dimerization of PrPc initiates an apoptoticcascade, possibly via an as-yet-unidentified secondarymolecule. Interruption of the PrPc-mediated survival sig-naling produced by association of PrPc with another mole-cule was dismissed on the basis that the Fab fragments didnot produce any effect. However, Linden et al. found thelatter argument far from compelling, since we cannot ex-clude the possibility that sustained ligand-induced signal-ing is disrupted amidst the widespread effects of antibodycross-linking, and particularly of the extensive lateral reor-ganization of cell surface molecules [9].

These findings conflict with reports indicating that theabsence of PrPc sensitizes cells to apoptotic stimuli, andalso with the fact that activation of ERKs in response todeath stimuli is believed to have an antiapoptotic effect.The most likely explanation for this discrepancy is thatthe effect of PrPc may depend on the cell type and deathstimuli involved. We can find the beginnings of an expla-nation in the data reported by Wu et al. [156]. Theseauthors compared the anti-apoptotic activities of mouse,hamster, and bovine PrP by expressing mouse PrP, hamsterPrP, and bovine PrP in HpL3-4 cells. Cells expressing Ham-ster PrP or Bovine PrP displayed the typical features ofapoptosis, whereas HpL3-4 cells expressing mouse PrP dis-played lower levels of apoptosis. These results indicate thatPrP has a species-specific anti-apoptotic function, suggest-ing that interaction between murine PrP and murine hostfactors is required for its anti-apoptotic activity.

Another study (see Section 8.3) has suggested that PrPcmay play differential roles in the cell death response, i.e., aproapoptotic role during ER stress, and an anti-apoptoticrole during oxidative stress-induced cell death [146].

Recent reports show that a conformational change inBcl-2 occurs during the onset of apoptosis, although it issuggested that this conformational change may be an inte-gral part of its antiapoptotic function [157]. Interaction ofBcl-2 with nuclear orphan receptor may convert the Bcl-2from a protector to a killer molecule, via conformationalchange [158]. We suggest that in the cell with Bcl-2 andPrPc protectors, both PrPc and Bcl-2 maintain Bax in aninactive state and confer cell protection. The interactionof Bcl-2 with another factor converts Bcl-2 from a protector

to a killer molecule via conformational change. Amongstother substances, Bcl-2 and Bcl-XL can be cleaved byendogenous caspases to yield a potent proapoptotic car-boxy-terminal fragment. As a consequence, overexpressionof these proteins would accelerate cell death after caspaseactivation [159]. This hypothesis is consistent with otherreports, indicating that transfection of the Bcl-2 gene inMCF-7 did not reduce TNF sensitivity [160]. PrPc, likeBcl-2, may be converted into a lethal protein. PrPc has acaspase-like site at amino acid 145, and this may generatea potent proapoptotic PrPc fragment, like that seen withthe 145 STOP codon mutation that is associated with a vas-cular form of prion disease [161]. Lastly, it is possible thatunknown factors convert PrPc from a protector to a killermolecule via a conformational change that induces celldeath.

8.6. PrPc and autophagy

The functional significance of autophagy and its rela-tionship to cell death in the nervous system is poorlyunderstood. Liberski et al. have suggested that autophagyplays a role in transmissible spongiform encephalopathies[162]. the scrapie-responsive gene 1 (Scrg1) is induced inTSE and brain injuries, and associated with autophagy[163]. As previously described in Ngsk deficient mice, ecto-pic expression of Doppel in central neurons induces signif-icant Purkinje cell death (see Section 8.1.1). Both beforeand during significant Purkinje cell loss, Scrg1 and theautophagic markers (LC3 and p62) were modulated inthe Ngsk deficient mice Purkinje cell. The present and pre-vious data indicate that Doppel triggers autophagy andapoptosis in Ngsk deficient mice Purkinje cell [164]. Serumdeprivation can induce both cell death pathways in varioustypes of cells. A recent report has shown that autophagy ishigher in Zürich I Prnp0/0 hippocampal neuronal cells thanin wild-type mice cells. This up-regulated autophagicactivity was retarded by the introduction of PrPc intoPrnp0/0 cells, but not by that of PrPc lacking the octapeptiderepeat region. Thus, the octapeptide repeat region of PrPcmay play a pivotal role in controlling the autophagy exhib-ited by PrPc in neuronal cells [165].

9. PrPc and tumor resistance

New genetic and biochemical approaches have led toremarkable progress in our understanding of cancer biol-ogy during the past decade [166]. One of the most impor-tant advances has been the recognition that resistance tocell death, particularly to apoptotic cell death, is an impor-tant aspect of both tumorigenesis and the development ofresistance to drugs used to treat cancer [166–168]. Muchrecent research on new cancer therapies has therefore fo-cused on devising ways to overcome this resistance andto trigger the death of tumor cells. Although the detailedmechanisms underlying tumor-cell resistance to apoptosisremain to be characterized, some important componentsand steps in this process have already been elucidated.For decades, clinicians and scientists have been puzzledby the fact that tumor cells simultaneously acquire the


capability to avoid immune surveillance mechanisms andevade the cytotoxic action of diverse cytotoxic insults, forexample, DNA damage, microtubule destabilization ortopoisomerase inhibition. The concept of cancer stem cells,and new genetic and biochemical approaches used inbreast cancer also suggest that drug resistance is probablynot primarily a consequence of acquired genetic altera-tions selected during or after therapy, but rather an inher-ent aspect of the malignant behavior of cancer cells fromthe outset. Various mechanisms, including genetic instabil-ity, oncogene overexpression, tumor suppressor downreg-ulation, epigenetic modifications, loss of cell cycle controland the impact of the tumor microenvironment lead tothe development of tumor resistance to cell death. Under-standing these mechanisms at the molecular level providesdeeper insights into carcinogenesis, influences therapeuticstrategy and might, ultimately, lead to new therapeutic ap-proaches based on modulating susceptibility to apoptosis.

9.1. Breast cancer cells

9.1.1. TNF-family mediated cell deathIn order to identify the genetic determinants of tumor-

cell resistance to the cytotoxic action of TNF, we have ap-plied cDNA microarrays to a human breast carcinomaTNF-sensitive MCF7 cell line and its established TNF-resis-tant clone. A great number of genes involved in the PI3K/Akt signaling pathway were differentially expressed. Unex-pectedly, endogenous PrPc was found to be overexpressedat both the mRNA (17-fold) and protein levels (10-fold) inTNF-resistant derivative cells as compared to the TNF-sen-sitive MCF7 cell line. Examining a panel of human breastcarcinoma cell lines for their sensitivity to TNF and theirPrPc expression revealed a close correlation between sus-ceptibility to the cytotoxic action of TNF and expressionof PrPc. Furthermore, the ectopic expression of human PrPcconferred TNF resistance on previously TNF-sensitiveMCF7 cells, by a mechanism involving alteration of cyto-chrome c release from mitochondria and nuclear conden-sation. These data showed for the first time that theectopic expression of PrPc can protect a breast cancer cellline from TNF-mediated cell death by interfering with themitochondrial pathway. These data demonstrate that Prpcis involved in tumor resistance [169]. In our follow-onstudy, we used the human breast adenocarcinoma TRAIL-sensitive MCF7 cell line and two resistant counterparts:the multidrug resistant (MDR) human breast cancer cellline, MCF-7/AdR and a TRAIL-resistant clone. We showedthat silencing PrPc facilitated the activation of proapoptot-ic Bax by downregulation of Bcl-2 expression, therebyabolishing the resistance of breast cancer cells to TRAIL-induced apoptosis. Our data showed that the cytoprotec-tive effect of PrPc in estrogen-receptor positive cells ap-peared to be specific to the TNF family, since PrPc did notprevent apoptosis induced by epirubicin/adriamycin in thismodel. We also showed that TRAIL-mediated apoptosis inPrPc knocked-down cells was associated with caspase pro-cessing, Bid cleavage and Mcl-1 degradation. However, thisincreased susceptibility to apoptosis was not associatedwith any increase in the recruitment of TRAIL receptorsor intracellular signaling molecule to DISC. It has been

established that Bcl-2 and Mcl-1 antagonize cell death bysequestering members of the proapoptotic family, includ-ing the BH3-only family and multidomain proapoptotics.We report that in the presence of PrPc and under TRAIL-apoptotic conditions (Fig. 5), small amounts of tBid wereproduced. tBid has a much higher affinity for Mcl-1 thanBcl-2, and consequently tBid interferes only weakly withthe anti-apoptotic function of Bcl-2 [170]. Mcl-1, whichhas high affinity for tBid, interacts with and blocks tBid-mediated cell death. In the absence of PrPc and underTRAIL-apoptotic conditions, Bcl-2 expression was abol-ished. We suggest that as a result of this, more tBid wasavailable to interact with Mcl-1, and thus neutralized theanti-apoptotic function of Mcl-1 to a greater extent. Inaddition, degradation of Mcl-1 by TRAIL-activated cas-pase-3 may produce a proapoptotic form of Mcl-1 thatmediates a Bax-dependent apoptotic cascade. In addition,the apoptotic signal leads to caspase-3 activation, whichactivates caspase 8, which in turn further increases tBidformation independently of DISC formation. This acceler-ates the cross-talk between the extrinsic and intrinsicpathways via an amplification loop, thereby abolishingthe resistance of breast cancer cells to TRAIL-inducedapoptosis.

9.1.2. Bax-mediated cell deathAt the molecular level, the anti-Bax activity of PrPc de-

scribed in the previous section in human primary neurons,human differentiated neuronal NT2 teratocarcinoma cells,and Saccharomyces cerevisiae is also observed in MCF7 cellline. In human neurons and MCF-7 cells, PrPc prevents theinitial conformational change of Bax, resulting in the trans-location of Bax to the mitochondrial membrane, the releaseof cytochrome c, and ultimately cell death. PrPc is thereforeconsidered to be a bona fide Bax inhibitor; it does not pre-vent active caspase-mediated cell death, and its anti-apop-totic effect is specific to Bax as it does not inhibit Bak orBid. However, despite being Bax-specific, PrPc does notinteract directly with Bax itself. This suggests that PrPc re-quires an intermediate to carry out its anti-Bax function.The authors suggest that this intermediate is probablynot Bcl-2 protein, because PrPc can protect againstBax-mediated cell death in yeasts, which are geneticallydeficient for Bcl-2 gene family members ([11,98,113].However, a double-immunoprecipitation approach is nec-essary to confirm this in MCF-7. The predominant anti-Bax form of PrPc is cytosolic PrP (CyPrP) rather than themore abundant, cell-surface glycosylphosphatidylinositol-anchored PrP [113,125]. Recently, using structure–functionanalyses, the same team has reported that deletions of 1, 2,or 3 OR (but not of all four ORs) abolish the anti-Bax func-tion of CyPrPs. Deletion of alpha-helix 3 (PrP23–199) orfurther C-terminal deletions of alpha-helices 1 and 2, andbeta-strands 1 and 2 (PrP23–172, PrP23–160, PrP23–143,and PrP23–127) abolish CyPrPs protection against Bax-mediated cell death. The replacement of helix-3 amino acidresidues K204, V210, and E219 by proline inhibits the anti-Bax function of CyPrP. The substitution of K204, but not ofV210 and E219, by alanine residues also prevents CyPrPsanti-Bax function. Expression of PrPs helix 3 producesanti-Bax activity in MCF-7 cells and in human neurons.

Fig. 5. PrPc-mediated TRAIL cell death in breast cancer. In the presence of PrPc and under TRAIL-apoptotic conditions, small amounts of tBid were produced.tBid has a much higher affinity for Mcl-1 than Bcl-2, and consequently tBid interferes only weakly with the anti-apoptotic function of Bcl-2. Mcl-1, whichhas high affinity for tBid, interacts with and blocks tBid-mediated cell death. In the absence of PrPc, and under TRAIL-apoptotic conditions, Bcl-2 expressionwas abolished. We propose that as a result, more tBid was available to interact with Mcl-1, and thus neutralized the anti-apoptotic function of Mcl-1 to agreater extent. In addition, degradation of Mcl-1 by TRAIL-activated caspase-3 may produce a proapoptotic form of Mcl-1 that mediates a Bax-dependentapoptotic cascade. Furthermore, the apoptotic signal leads to caspase-3 activation, which activates caspase 8 and this in turn further increases tBidformation independently of DISC formation. This accelerates the cross-talk between the extrinsic and intrinsic pathways via an amplification loop, therebyabolishing the resistance of breast cancer cells to TRAIL-induced apoptosis [25].


Taken as a whole, these results indicate that although theOR domain has an influence on PrPs anti-Bax function, he-lix 3 is both necessary and sufficient for the anti-Bax func-tion of CyPrP [171].

9.1.3. Paclitaxel-mediated cell deathPaclitaxel, a member of the taxanes family, is a microtu-

bule-stabilizing agent that acts primarily by interferingwith spindle microtubule dynamics, causing cell cycle ar-rest and apoptosis. Moreover, altered expression of the ef-flux transporters MDR1/P-glycoprotein (P-gp or ABC1) andmultiresistance-associated proteins (MRP) has been shownboth in vitro and in vivo to cause intrinsic or acquired resis-tance to commonly-used cytotoxic drugs, such as the anth-racyclines, taxanes, anti-metabolites and platinum agents,all of which are substrates of either the MDR1/P-gp orMRP transporters (see [172] for review). P-gp substrates(anthracyclines, taxanes, anti-metabolites and platinumagents) significantly enhanced the in vitro invasion capac-ity of MCF-7/Adr cells.

Physical interaction between P-gp and PrPc has recentlybeen reported [173]. Blocking P-gp activity or depletingPrPc in vitro inhibited the paclitaxel-induced invasion po-tential of MCF-7/Adr. Paclitaxel also facilitated the forma-tion of P-gp/PrPc clusters located in caveolar domains,and promoted the association of P-gp with caveolin-1. Bothcaveolin-1 and caveolar integrity were required for the

drug-induced invasion potential of MCF-7/Adr to be ex-pressed. In addition, silencing PrPc abolished the resistanceof breast cancer cells to paclitaxel-induced apoptosis. Pac-litaxel-mediated apoptosis in PrPc knocked-down cellswas associated with caspase processing, up-regulation ofBax, and downregulation of Bcl-2 expression. It was shownthat at high concentrations, paclitaxel is able to kill cancercells that express Bcl-2; it inhibits the antiapoptotic activ-ity of Bcl-2 by inducing its phosphorylation. JNK, but notERK or p38 MAPK, appears to be involved in the phosphor-ylation of Bcl-2 induced by paclitaxel [174]. It would beinteresting to assess the phosphorylation of Bcl-2 in PrPcknocked-down cells treated by paclitaxel.

9.2. Gastric cancer cells

Du et al., using the adriamycin-sensitive gastric carci-noma cell line SGC7901/ADR and its derivative resistanceclone reported that PrPc is involved in multidrug resis-tance. Overexpression of PrPc conferred resistance to bothP-gp-related and P-gp-unrelated drugs on SGC7901. PrPcknock-down expression partially reverses the multidrug-resistant phenotype of SGC7901/ADR. PrPc significantlyupregulated the expression of the P-gp, but not that ofMRP or glutathione S-transferase pi. PrPc can also suppressadriamycin-induced apoptosis, and alter the expression ofBcl-2 and Bax [175]. These data are not consistent with our


data showing that PrPc knock-down did not influence thesensitivity of adriamycin-resistant breast cell lines to adri-amycin-induced apoptosis. The discrepancy could beattributable to the different in vitro models used.

A subsequent study from the same laboratory hasshown that PrPc is upregulated in several gastric cancercell lines at both the mRNA and protein levels during hy-poxia. Human gastric cancer MKN28 cells, transfected withhuman PrPc promoter (�2253 � +289), which containedthe heat shock element (HSE), expressed higher luciferaseactivities. The authors suggest that some transcriptionalfactors phosphorylated by ERK1/2, could in turn interactwith the HSE in the promoter of PrPc, resulting in theupregulation of PrPc in the MKN28 gastric cancer cell lineduring hypoxia. Downregulation of PrPc makes gastric can-cer cells more sensitive to hypoxia-induced drug sensitiv-ity [176]. More recently, the same team has suggestedthat PrPc-induced MDR in gastric cancer is associated withactivation of the PI3K/Akt pathway. Inhibition of PI3K/Aktby LY2940002 or Akt siRNA leads to the inhibition ofPrPc-induced drug resistance and P-gp upregulation in gas-tric cancer cells, suggesting a possible novel mechanism bywhich PrPc regulates gastric cancer cell survival [177].

9.3. Glioblastoma cells

Gliomas are tumors derived from the glia or their pre-cursors within the CNS. Clinically, gliomas are divided intofour grades, and the glioblastoma multiforme (GBM), alsoreferred to as grade IV astrocytoma, is the most aggressiveand most common glioma in humans. The prognosis forpatients with GBM remains dismal, with a median survivalof 9–12 months.

STI-1, which, as we noted in previous section, acts inpartnership with PrPc to produce neuroprotective activity[94,95]. In the human glioblastoma-derived cell lineA172, thymidine incorporation assays have shown thatSTI1 is both secreted as a result of proliferation of thesecells and also induces further proliferation. The authorssuggest that the STI1 may also promote glioma prolifera-tion via the MAPK and PI3K pathways [178].

10. PrPc in tumor progression and response to therapy

10.1. Breast cancer

Breast cancer is a major cause of morbidity and mortal-ity in women world wide [1]. Anthracyclines-based che-motherapy administered either before surgery (neo-adjuvant) or after surgery (adjuvant) in patients with stageI–III breast cancers improves survival rates [2] Neverthe-less, the 10-year absolute benefit of adjuvant anthracy-clines-based chemotherapy is limited, ranging between2% and 11%, indicating that most patients do not in factbenefit from this treatment [3]. There is therefore a needto be able to predict which patients could benefit fromadjuvant chemotherapy in order to be able to tailor adju-vant treatments to the patient, and deliver the right drugat the right time, and for the optimum length of time.

Immunohistochemistry analysis of PrPc expression innormal breast has shown that PrPc is expressed in thecytoplasm and plasma membrane of normal myoepithelialcells, but not of the luminal cells. Moreover, PrPc wasfound in lymphocytes and fibroblasts in breast tissue.The expression of PrPc by primary tumors was assessedby immunohistochemistry in a series of 756 patients in-cluded in two randomized trials comparing anthracy-cline-based chemotherapy to no chemotherapy. Thepositive rate of PrPc expression in breast cancer tissueswas 15% (113/756). Tissue microarray analysis alsoshowed that 33% of estrogen-receptor (ER)-negative pa-tients expressed PrPc, versus only 5% of ER-positive pa-tients. This indicates that the expression of PrPc isassociated with the ER-negative subclass in breast cancer(P < 0.001). Comparison of the 10-year overall survival rateand multivariate analysis showed the ER-negative patientswhose tumors expressed PrPc did not seem to benefit fromchemotherapy [16].

10.2. Gastric cancer

Fan’s team has shown that PrPc is predominantly lo-cated in the cytoplasm and plasma membrane of gastriccancer cells. PrPc expression was higher in gastric cancertissues than in adjacent non-tumorous tissues or normalgastric mucosa. Comparison of PrPc expression at primarysites with that at corresponding metastatic sites in 22 pa-tients suggested that PrPc was prone to be highly ex-pressed in metastatic gastric cancer, but did notdistinguish between primary sites and metastatic sitesfrom the same metastatic gastric cancer [179]. Theseobservations are consistent with our data suggesting thatin patients with breast cancer, 65% of PrPc- positive tumorswere grade III versus 26% of PrPc-negative tumors.

PrPc significantly promoted the adhesive, invasive, andin vivo metastatic capacities of the gastric cancer cell linesSGC7901 and MKN45. PrPc also increased promoter activ-ity and the expression of MMP11 by activating phosphor-ylated ErK1/2 in gastric cancer cells. The N-terminalregion of PrPc may promote the invasive and metastaticactivities of gastric cancer cells at least to some extent,by activating the MEK/ERK pathway and the consequenttransactivation of MMP11 [179]. Subsequent studies haveindicated that overexpression of PrPc might also promotethe proliferation of gastric cancer cells at least partially byactivating the PI3K/Akt pathway and the subsequent tran-scriptional activation of CyclinD1 to accelerate the G1/S-phase transition. The octapeptide repeat region may playan indispensable role in this (see Table 2) [180]. One octa-peptide repeat deletion was found in several gastric can-cer cell lines, and its mutation frequency was higher ingastric cancer [181]. Overexpression of PrPc containingone octapeptide repeat deletion might therefore promotethe proliferation of gastric cancer cells, at least partiallythrough transcriptional activation of CyclinD3 in additionto CyclinD1 to accelerate G1/S-phase transition. The effectof PrPc with one octapeptide repeat deletion in promotingproliferation was greater than that of wild-type PrPc[182].

Table 2Structure–activity relationships for PrP in cancer cells.

Breast carcinoma cell line MCF-7 Gastric carcinoma cell line SGC7901

Bax-induced cell death TNF family-inducedcell death

Paclitaxel-inducedcell death

Adriamycin-induced celldeath

P-glycoprotein (P-gp) relatedand P-gp nonrelated drugsinduce cell death

Adriamycin-induced celldeath

Invasion andmetastasis ofgastric cancer

Proliferation andG1/S transition ofgastric cancer

PrPc molecule Rescue PrPc molecule Rescue PrPcmolecule

Rescue PrPcmolecule

Rescue Promotes

Human wild type +[a,b,cand o]

Syrian hamsterwild-type PrP

+[c] Humanwildtype

+[u] +[w] �[v] Humanwild type

+[q] +[q,s] +[r] +[t]

A117V +[b] (DSTE)D104–112

+[c] D22–47 +[t]

A117V/M129V +[b] D24–90 ND ND �[r] +[t]Called MH2M(mouse andhamster) G123P

+[c]

129M/V +[b] (KH/II)K110I/H111I

�[c]

D178N �[b]D178N/M129V �[b] (AV3) A113V/

A115V/A118/V�[c]

V180I ±[b] A120L +[c]V180I/M129V +[b] (N4 A120L) A2R/

N3R/A120L�[c]

T188A �[b] (N7aDSTE) L4D/W7G/D104–112

+[c]

T188A/M129V +[b]D51–90 octarepeat ND D51–90

octarepeatND D51–90

octarepeatND ND +[r] �[t]

E196K �[b] (N7a A120L)L4D/W7G A120L

�[c] PrPDc(96–230)

ND ND +[r] +[t]

E196KM129V +[b] Op n-PrP +[c]E200K ±[b] Prl–PrP +[c]E200K/M129V +[b]V203I +[b]V203I/M129V +[b]R208H �[b]R208H/M129V +[b]V210I/M129V �[b]E211Q ±[b]E211Q M129V ±[b]M232R/M129V �[b]M232R ±[b]P238S/M129V �[b]D232–254 (GPI) +[a] D232–254 (GPI) +[c]23–231 (Cytosolic

PrPc)+[z] 23–231

(Cytosolic PrPc)+[c] 23–231

(CytosolicPrPc)

+[t]

198–231 Helix 3 +[z]

[a] Ref. [123]; [b] Ref. [125]; [c] Ref. [113]; [o] Ref. [124]; [q] Ref. [175]; [r] Ref. [179]; [s] Ref. [177]; [t] Ref. [180]; [u] Ref. [169]; [v] Ref. [25]; [w] Ref. [173]; [z] Ref. [171]; ND: not determined.

18M

.Mehrpour,P.Codogno

/CancerLetters

290(2010)

1–23


11. Concluding remarks

PrPc was originally viewed solely as being involved inprion disease, but now several intriguing lines of evidencehave emerged indicating that it plays a fundamental rolenot only in the nervous system, but also throughout thehuman body. This means that it could well be involved inresistance to apoptosis, and the proliferation and metasta-sis of human cancer cells. PrPc therefore has both vital andlethal functions. It has a proapoptotic role during ER stress,but an anti-apoptotic role during oxidative stress-inducedcell death which makes it difficult to obtain reproducibledata that can be extrapolated to the wider context. Manyquestions concerning the role of PrPc in cancer biology re-mains to be answered. The situation is complex as manyinterconnected pathways may be involved and interferewith each other.

Conflict of interest

None declared.

Acknowledgments

We apologize to those investigators whose work wasnot cited or discussed because of space limitations. Wethank the members of our laboratories, past and present,for their support and advice. This work was supported byGrants from INSERM. We thank Franck Meslin for his helpfor figures design.

Appendix A. Supplementary material

Supplementary data associated with this article can befound, in the online version, at doi:10.1016/j.canlet.2009.07.009.

References

[1] A. Aguzzi, M. Polymenidou, Mammalian prion biology: one centuryof evolving concepts, Cell 116 (2004) 313–327.

[2] S.B. Prusiner, The prion diseases, Brain Pathol. 8 (1998) 499–513.[3] H. Bueler, Mice devoid of PrP are resistant to scrapie, Cell 73 (1993)

1339–1347.[4] A. Aguzzi, F. Baumann, J. Bremer, The prion’s elusive reason for

being, Annu. Rev. Neurosci. 31 (2008) 439–477.[5] K. Gousset, C. Zurzolo, Tunnelling nanotubes: a highway for prion

spreading?, Prion 3 (2009)[6] M. Heikenwalder, M.O. Kurrer, I. Margalith, J. Kranich, N. Zeller, J.

Haybaeck, M. Polymenidou, M. Matter, J. Bremer, W.S. Jackson, S.Lindquist, C.J. Sigurdson, A. Aguzzi, Lymphotoxin-dependent prionreplication in inflammatory stromal cells of granulomas, Immunity29 (2008) 998–1008.

[7] P.C. Pauly, D.A. Harris, Copper stimulates endocytosis of the prionprotein, J. Biol. Chem. 273 (1998) 33107–33110.

[8] A. Mange, O. Milhavet, D. Umlauf, D. Harris, S. Lehmann, PrP-dependent cell adhesion in N2a neuroblastoma cells, FEBS Lett. 514(2002) 159–162.

[9] R. Linden, V.R. Martins, M.A. Prado, M. Cammarota, I. Izquierdo, R.R.Brentani, Physiology of the prion protein, Physiol. Rev. 88 (2008)673–728.

[10] B. Caughey, G.S. Baron, Prions and their partners in crime, Nature443 (2006) 803–810.

[11] L. Westergard, H.M. Christensen, D.A. Harris, The cellular prionprotein (PrP(C)): its physiological function and role in disease,Biochim. Biophys. Acta 1772 (2007) 629–644.

[12] G.G. Kovacs, H. Budka, Prion diseases: from protein to cellpathology, Am. J. Pathol. 172 (2008) 555–565.

[13] E. Makrinou, J. Collinge, M. Antoniou, Genomic characterization ofthe human prion protein (PrP) gene locus, Mamm. Genome 13(2002) 696–703.

[14] R. Medori, H.J. Tritschler, A. LeBlanc, F. Villare, V. Manetto, H.Y.Chen, R. Xue, S. Leal, P. Montagna, P. Cortelli, et al., Fatal familialinsomnia, a prion disease with a mutation at codon 178 of the prionprotein gene, N. Engl. J. Med. 326 (1992) 444–449.

[15] P. Gambetti, Q. Kong, W. Zou, P. Parchi, S.G. Chen, Sporadic andfamilial CJD: classification and characterisation, Br. Med. Bull. 66(2003) 213–239.

[16] F. Meslin, R. Conforti, C. Mazouni, N. Morel, G. Tomasic, F. Drusch,M. Yacoub, J.C. Sabourin, J. Grassi, S. Delaloge, M.C. Mathieu, S.Chouaib, F. Andre, M. Mehrpour, Efficacy of adjuvant chemotherapyaccording to prion protein expression in patients with estrogenreceptor-negative breast cancer, Ann. Oncol. 18 (2007) 1793–1798.

[17] A. Didier, R. Dietrich, M. Steffl, M. Gareis, M.H. Groschup, S. Muller-Hellwig, E. Martlbauer, W.M. Amselgruber, Cellular prion protein inthe bovine mammary gland is selectively expressed in activelactocytes, J. Histochem. Cytochem. 54 (2006) 1255–1261.

[18] S. Sakaguchi, Physiological functions of prion protein and its rolesin the pathogenesis of prion diseases, Seikagaku 79 (2007) 843–852.

[19] S. Comincini, V. Ferrara, A. Arias, A. Malovini, A. Azzalin, L. Ferretti,E. Benericetti, M. Cardarelli, M. Gerosa, M.G. Passarin, S. Turazzi, R.Bellazzi, Diagnostic value of PRND gene expression profiles inastrocytomas: relationship to tumor grades of malignancy, Oncol.Rep. 17 (2007) 989–996.

[20] J.C. Watts, B. Drisaldi, V. Ng, J. Yang, B. Strome, P. Horne, M.S. Sy, L.Yoong, R. Young, P. Mastrangelo, C. Bergeron, P.E. Fraser, G.A.Carlson, H.T. Mount, G. Schmitt-Ulms, D. Westaway, The CNSglycoprotein Shadoo has PrP(C)-like protective properties anddisplays reduced levels in prion infections, EMBO J. 26 (2007)4038–4050.

[21] N. Stahl, S.B. Prusiner, Prions and prion proteins, FASEB J. 5 (1991)2799–2807.

[22] R.S. Stewart, D.A. Harris, Mutational analysis of topologicaldeterminants in prion protein (PrP) and measurement oftransmembrane and cytosolic PrP during prion infection, J. Biol.Chem. 278 (2003) 45960–45968.

[23] O. Chakrabarti, R.S. Hegde, Functional depletion of mahogunin bycytosolically exposed prion protein contributes toneurodegeneration, Cell 137 (2009) 1136–1147.

[24] R.S. Stewart, D.A. Harris, A transmembrane form of the prionprotein is localized in the Golgi apparatus of neurons, J. Biol. Chem.280 (2005) 15855–15864.

[25] F. Meslin, A. Hamai, P. Gao, A. Jalil, N. Cahuzac, S. Chouaib, M.Mehrpour, Silencing of prion protein sensitizes breast adriamycin-resistant carcinoma cells to TRAIL-mediated cell death, Cancer Res.67 (2007) 10910–10919.

[26] N.M. Hooper, Roles of proteolysis and lipid rafts in the processing ofthe amyloid precursor protein and prion protein, Biochem. Soc.Trans. 33 (2005) 335–338.

[27] K.M. Pan, N. Stahl, S.B. Prusiner, Purification and properties of thecellular prion protein from Syrian hamster brain, Protein Sci. 1(1992) 1343–1352.

[28] A. Jimenez-Huete, P.M. Lievens, R. Vidal, P. Piccardo, B. Ghetti, F.Tagliavini, B. Frangione, F. Prelli, Endogenous proteolytic cleavageof normal and disease-associated isoforms of the human prionprotein in neural and non-neural tissues, Am. J. Pathol. 153 (1998)1561–1572.

[29] S.-L. Shyng, M.T. Huber, D.A. Harris, A prion protein cyclesbetween the cell surface and an endocytic compartment incultured neuroblastoma cells, J. Biol. Chem. 268 (1993) 15922–15928.

[30] K. Nieznanski, H. Nieznanska, K.J. Skowronek, K.M. Osiecka, D.Stepkowski, Direct interaction between prion protein and tubulin,Biochem. Biophys. Res. Commun. 334 (2005) 403–411.

[31] A. Mange, F. Beranger, K. Peoc’h, T. Onodera, Y. Frobert, S. Lehmann,Alpha- and beta-cleavages of the amino-terminus of the cellularprion protein, Biol. Cell 96 (2004) 125–132.

[32] B. Vincent, E. Paitel, Y. Frobert, S. Lehmann, J. Grassi, F. Checler,Phorbol ester-regulated cleavage of normal prion protein inHEK293 human cells and murine neurons, J. Biol. Chem. 275(2000) 35612–35616.

[33] B. Vincent, E. Paitel, P. Saftig, Y. Frobert, D. Hartmann, B. DeStrooper, J. Grassi, E. Lopez-Perez, F. Checler, The disintegrinsADAM10 and TACE contribute to the constitutive and phorbol




ester-regulated normal cleavage of the cellular prion protein, J. Biol.Chem. 276 (2001) 37743–37746.

[34] A. Taraboulos, A. Raeber, D.R. Borchelt, D. Serban, S.B. Prusiner,Synthesis and trafficking of prion proteins in cultured cells, Mol.Biol. Cell 3 (1992) 851–863.

[35] N.T. Watt, D.R. Taylor, A. Gillott, D.A. Thomas, W.S. Perera, N.M.Hooper, Reactive oxygen species-mediated beta-cleavage of theprion protein in the cellular response to oxidative stress, J. Biol.Chem. 280 (2005) 35914–35921.

[36] R.S. Hegde, J.A. Mastrianni, M.R. Scott, K.A. DeFea, P. Tremblay, M.Torchia, S.J. DeArmond, S.B. Prusiner, V.R. Lingappa, Atransmembrane form of the prion protein in neurodegenerativedisease, Science 279 (1998) 827–834.

[37] R.S. Stewart, P. Piccardo, B. Ghetti, D.A. Harris, Neurodegenerativeillness in transgenic mice expressing a transmembrane form of theprion protein, J. Neurosci. 25 (2005) 3469–3477.

[38] M.E. Juanes, G. Elvira, A. Garcia-Grande, M. Calero, M. Gasset,Biosynthesis of prion protein nucleocytoplasmic isoforms byalternative initiation of translation, J. Biol. Chem. 284 (2009)2787–2794.

[39] C. Crozet, F. Beranger, S. Lehmann, Cellular pathogenesis in priondiseases, Vet. Res. 39 (2008) 44.

[40] J. Ma, R. Wollmann, S. Lindquist, Neurotoxicity andneurodegeneration when PrP accumulates in the cytosol, Science298 (2002) 1781–1785.

[41] A. Mange, C. Crozet, S. Lehmann, F. Beranger, Scrapie-like prionprotein is translocated to the nuclei of infected cells independentlyof proteasome inhibition and interacts with chromatin, J. Cell Sci.117 (2004) 2411–2416.

[42] H. Lorenz, O. Windl, H.A. Kretzschmar, Cellular phenotyping ofsecretory and nuclear prion proteins associated with inheritedprion diseases, J. Biol. Chem. 277 (2002) 8508–8516.

[43] E. Sbalchiero, A. Azzalin, S. Palumbo, G. Barbieri, A. Arias, L.Simonelli, L. Ferretti, S. Comincini, Altered cellular distributionand sub-cellular sorting of doppel (Dpl) protein in humanastrocytoma cell lines, Cell Oncol. 30 (2008) 337–347.

[44] M.A. Prado, J. Alves-Silva, A.C. Magalhaes, V.F. Prado, R. Linden, V.R.Martins, R.R. Brentani, PrPc on the road: trafficking of the cellularprion protein, J. Neurochem. 88 (2004) 769–781.

[45] N.M. Hooper, D.R. Taylor, N.T. Watt, Mechanism of the metal-mediated endocytosis of the prion protein, Biochem. Soc. Trans. 36(2008) 1272–1276.

[46] D.A. Harris, Trafficking, turnover and membrane topology of PrP, Br.Med. Bull. 66 (2003) 71–85.

[47] M. Nunziante, S. Gilch, H.M. Schatzl, Essential role of the prionprotein N terminus in subcellular trafficking and half-life of cellularprion protein, J. Biol. Chem. 278 (2003) 3726–3734.

[48] W.S. Perera, N.M. Hooper, Ablation of the metal ion-inducedendocytosis of the prion protein by disease-associated mutationof the octarepeat region, Curr. Biol. 11 (2001) 519–523.

[49] C. Sunyach, A. Jen, J. Deng, K.T. Fitzgerald, Y. Frobert, J. Grassi, M.W.McCaffrey, R. Morris, The mechanism of internalization ofglycosylphosphatidylinositol-anchored prion protein, EMBO J. 22(2003) 3591–3601.

[50] M. Marella, S. Lehmann, J. Grassi, J. Chabry, Filipin preventspathological prion protein accumulation by reducing endocytosisand inducing cellular PrP release, J. Biol. Chem. 277 (2002) 25457–25464.

[51] K. Kaneko, M. Vey, M. Scott, S. Pilkuhn, F.E. Cohen, S.B. Prusiner,COOH-terminal sequence of the cellular prion protein directssubcellular trafficking and controls conversion into the scrapieisoform, Proc. Natl. Acad. Sci. USA 94 (1997) 2333–2338.

[52] S. Sabharanjak, P. Sharma, R.G. Parton, S. Mayor, GPI-anchoredproteins are delivered to recycling endosomes via a distinct cdc42-regulated, clathrin-independent pinocytic pathway, Dev. Cell 2(2002) 411–423.

[53] L. Johannes, C. Lamaze, Clathrin-dependent or not: is it still thequestion?, Traffic 3 (2002) 443–451

[54] P.J. Peters, A. Mironov Jr., D. Peretz, E. van Donselaar, E. Leclerc, S.Erpel, S.J. DeArmond, D.R. Burton, R.A. Williamson, M. Vey, S.B.Prusiner, Trafficking of prion proteins through a caveolae-mediatedendosomal pathway, J. Cell Biol. 162 (2003) 703–717.

[55] D. Sarnataro, A. Caputo, P. Casanova, C. Puri, S. Paladino, S.S.Tivodar, V. Campana, C. Tacchetti, C. Zurzolo, Lipid rafts andclathrin cooperate in the internalization of PrP in epithelial FRTcells, PloS one 4 (2009) e5829.

[56] S. Mouillet-Richard, M. Ermonval, C. Chebassier, J.L. Laplanche, S.Lehmann, J.M. Launay, O. Kellermann, Signal transduction throughprion protein, Science 289 (2000) 1925–1928.

[57] C. Sunyach, F. Checler, Combined pharmacological, mutationaland cell biology approaches indicate that p53-dependentcaspase 3 activation triggered by cellular prion is dependenton its endocytosis, J. Neurochem. 92 (2005) 1399–1407.

[58] D.R. Taylor, N.T. Watt, W.S. Perera, N.M. Hooper, Assigningfunctions to distinct regions of the N-terminus of the prionprotein that are involved in its copper-stimulated, clathrin-dependent endocytosis, J. Cell Sci. 118 (2005) 5141–5153.

[59] S. Gauczynski, J.M. Peyrin, S. Haik, C. Leucht, C. Hundt, R. Rieger, S.Krasemann, J.P. Deslys, D. Dormont, C.I. Lasmezas, S. Weiss, The 37-kDa/67-kDa laminin receptor acts as the cell-surface receptor forthe cellular prion protein, EMBO J. 20 (2001) 5863–5875.

[60] S. Gauczynski, C. Hundt, C. Leucht, S. Weiss, Interaction of prionproteins with cell surface receptors, molecular chaperones, andother molecules, Adv. Protein Chem. 57 (2001) 229–272.

[61] D.R. Taylor, N.M. Hooper, The low-density lipoprotein receptor-related protein 1 (LRP1) mediates the endocytosis of the cellularprion protein, Biochem. J. 402 (2007) 17–23.

[62] C.J. Parkyn, E.G. Vermeulen, R.C. Mootoosamy, C. Sunyach, C.Jacobsen, C. Oxvig, S. Moestrup, Q. Liu, G. Bu, A. Jen, R.J. Morris,LRP1 controls biosynthetic and endocytic trafficking of neuronalprion protein, J. Cell Sci. 121 (2008) 773–783.

[63] F. Blasi, P. Carmeliet, uPAR: a versatile signalling orchestrator, Nat.Rev. Mol. Cell Biol. 3 (2002) 932–943.

[64] K.S. Lee, R. Linden, M.A. Prado, R.R. Brentani, V.R. Martins, Towardscellular receptors for prions, Rev. Med. Virol. 13 (2003) 399–408.

[65] C. Hundt, J.M. Peyrin, S. Haik, S. Gauczynski, C. Leucht, R. Rieger,M.L. Riley, J.P. Deslys, D. Dormont, C.I. Lasmezas, S. Weiss,Identification of interaction domains of the prion protein with its37-kDa/67-kDa laminin receptor, EMBO J. 20 (2001) 5876–5886.

[66] C. Zuber, S. Knackmuss, G. Zemora, U. Reusch, E. Vlasova, D. Diehl,V. Mick, K. Hoffmann, D. Nikles, T. Frohlich, G.J. Arnold, B. Brenig, E.Wolf, H. Lahm, M. Little, S. Weiss, Invasion of tumorigenic HT1080cells is impeded by blocking or downregulating the 37-kDa/67-kDalaminin receptor, J. Mol. Biol. 378 (2008) 530–539.

[67] A. Li, S.J. Barmada, K.A. Roth, D.A. Harris, N-terminally deletedforms of the prion protein activate both Bax-dependent and Bax-independent neurotoxic pathways, J. Neurosci. 27 (2007) 852–859.

[68] F. Baumann, M. Tolnay, C. Brabeck, J. Pahnke, U. Kloz, H.H. Niemann,M. Heikenwalder, T. Rulicke, A. Burkle, A. Aguzzi, Lethal recessivemyelin toxicity of prion protein lacking its central domain, EMBO J.26 (2007) 538–547.

[69] D. Shmerling, I. Hegyi, M. Fischer, T. Blattler, S. Brandner, J. Gotz, T.Rulicke, E. Flechsig, A. Cozzio, C. von Mering, C. Hangartner, A.Aguzzi, C. Weissmann, Expression of amino-terminally truncatedPrP in the mouse leading to ataxia and specific cerebellar lesions,Cell 93 (1998) 203–214.

[70] G.L. Millhauser, Copper and the prion protein: methods, structures,function, and disease, Ann. Rev. Phys. Chem. 58 (2007) 299–320.

[71] J.H. Viles, M. Klewpatinond, R.C. Nadal, Copper and the structuralbiology of the prion protein, Biochem. Soc. Trans. 36 (2008) 1288–1292.

[72] R. Gonzalez-Iglesias, M.A. Pajares, C. Ocal, J.C. Espinosa, B. Oesch, M.Gasset, Prion protein interaction with glycosaminoglycan occurswith the formation of oligomeric complexes stabilized by Cu(II)bridges, J. Mol. Biol. 319 (2002) 527–540.

[73] K. Qin, L. Zhao, R.D. Ash, W.F. McDonough, R.Y. Zhao, ATM-mediated transcriptional elevation of prion in response to copper-induced oxidative stress, J. Biol. Chem. 284 (2009) 4582–4593.

[74] H. Bueler, Normal development and behaviour of mice lacking theneuronal cell-surface PrP protein, Nature 356 (1992) 577–582.

[75] J.D. Isaacs, R.J. Ingram, J. Collinge, D.M. Altmann, G.S. Jackson, Thehuman prion protein residue 129 polymorphism lies within acluster of epitopes for T cell recognition, J. Neuropathol. Exp.Neurol. 65 (2006) 1059–1068.

[76] F.A. Caetano, M.H. Lopes, G.N. Hajj, C.F. Machado, C. Pinto Arantes,A.C. Magalhaes, P. Vieira Mde, T.A. Americo, A.R. Massensini, S.A.Priola, I. Vorberg, M.V. Gomez, R. Linden, V.F. Prado, V.R. Martins,M.A. Prado, Endocytosis of prion protein is required for ERK1/2signaling induced by stress-inducible protein 1, J. Neurosci. 28(2008) 6691–6702.

[77] J. Collinge, M.A. Whittington, K.C. Sidle, C.J. Smith, M.S. Palmer, A.R.Clarke, J.G. Jefferys, Prion protein is necessary for normal synapticfunction, Nature 370 (1994) 295–297.

[78] J.R. Criado, M. Sanchez-Alavez, B. Conti, J.L. Giacchino, D.N. Wills,S.J. Henriksen, R. Race, J.C. Manson, B. Chesebro, M.B. Oldstone,Mice devoid of prion protein have cognitive deficits that arerescued by reconstitution of PrP in neurons, Neurobiol. Dis. 19(2005) 255–265.


[79] B. Lobao-Soares, R. Walz, C.G. Carlotti Jr., A.C. Sakamoto, F. Calvo,A.L. Terzian, J.A. da Silva, L. Wichert-Ana, N.C. Coimbra, M.M.Bianchin, Cellular prion protein regulates the motor behaviourperformance and anxiety-induced responses in geneticallymodified mice, Behav. Brain Res. 183 (2007) 87–94.

[80] H. Khosravani, Y. Zhang, S. Tsutsui, S. Hameed, C. Altier, J. Hamid, L.Chen, M. Villemaire, Z. Ali, F.R. Jirik, G.W. Zamponi, Prion proteinattenuates excitotoxicity by inhibiting NMDA receptors, J. Cell Biol.181 (2008) 551–565.

[81] C.E. Le Pichon, M.T. Valley, M. Polymenidou, A.T. Chesler, B.T.Sagdullaev, A. Aguzzi, S. Firestein, Olfactory behavior andphysiology are disrupted in prion protein knockout mice, Nat.Neurosci. 12 (2009) 60–69.

[82] D.A. Wilson, R.A. Nixon, Sniffing out a function for prion proteins,Nat. Neurosci. 12 (2009) 7–8.

[83] W. Hu, B. Kieseier, E. Frohman, T.N. Eagar, R.N. Rosenberg, H.P.Hartung, O. Stuve, Prion proteins: physiological functions and rolein neurological disorders, J. Neurol. Sci. 264 (2008) 1–8.

[84] J.D. Isaacs, G.S. Jackson, D.M. Altmann, The role of the cellular prionprotein in the immune system, Clin. Exp. Immunol. 146 (2006) 1–8.

[85] C.C. Zhang, A.D. Steele, S. Lindquist, H.F. Lodish, Prion protein isexpressed on long-term repopulating hematopoietic stem cells andis important for their self-renewal, Proc. Natl. Acad. Sci. USA 103(2006) 2184–2189.

[86] C. Paar, S. Wurm, W. Pfarr, A. Sonnleitner, C. Wechselberger, Prionprotein resides in membrane microclusters of the immunologicalsynapse during lymphocyte activation, Eur. J. Cell Biol. 86 (2007)253–264.

[87] P. Viegas, N. Chaverot, H. Enslen, N. Perriere, P.O. Couraud, S.Cazaubon, Junctional expression of the prion protein PrPC by brainendothelial cells: a role in trans-endothelial migration of humanmonocytes, J. Cell Sci. 119 (2006) 4634–4643.

[88] E. Graner, Cellular prion protein binds laminin and mediatesneuritogenesis, Mol. Brain Res. 76 (2000) 85–92.

[89] R. Rieger, F. Edenhofer, C.I. Lasmezas, S. Weiss, The human 37-kDalaminin receptor precursor interacts with the prion protein ineukaryotic cells, Nat. Med. 3 (1997) 1383–1388.

[90] A.D. Snow, T.N. Wight, D. Nochlin, Y. Koike, K. Kimata, S.J.DeArmond, S.B. Prusiner, Immunolocalization of heparan sulfateproteoglycans to the prion protein amyloid plaques of Gerstmann-Straussler syndrome, Creutzfeldt-Jakob disease and scrapie, Lab.Invest. 63 (1990) 601–611.

[91] S.M. Zanata, Stress-inducible protein 1 is a cell surface ligand forcellular prion that triggers neuroprotection, EMBO J. 21 (2002)3307–3316.

[92] G. Schmitt-Ulms, G. Legname, M.A. Baldwin, H.L. Ball, N. Bradon, P.J.Bosque, K.L. Crossin, G.M. Edelman, S.J. DeArmond, F.E. Cohen, S.B.Prusiner, Binding of neural cell adhesion molecules (N-CAMs) to thecellular prion protein, J. Mol. Biol. 314 (2001) 1209–1225.

[93] A. Santuccione, V. Sytnyk, I. Leshchyns’ka, M. Schachner, Prionprotein recruits its neuronal receptor NCAM to lipid rafts to activatep59fyn and to enhance neurite outgrowth, J. Cell Biol. 169 (2005)341–354.

[94] L.B. Chiarini, A.R. Freitas, S.M. Zanata, R.R. Brentani, V.R. Martins, R.Linden, Cellular prion protein transduces neuroprotective signals,EMBO J. 21 (2002) 3317–3326.

[95] M.H. Lopes, G.N. Hajj, A.G. Muras, G.L. Mancini, R.M. Castro, K.C.Ribeiro, R.R. Brentani, R. Linden, V.R. Martins, Interaction of cellularprion and stress-inducible protein 1 promotes neuritogenesis andneuroprotection by distinct signaling pathways, J. Neurosci. 25(2005) 11330–11339.

[96] K.J. Knaus, M. Morillas, W. Swietnicki, M. Malone, W.K. Surewicz,V.C. Yee, Crystal structure of the human prion protein reveals amechanism for oligomerization, Nat. Struct. Biol. 8 (2001) 770–774.

[97] E. Malaga-Trillo, G.P. Solis, Y. Schrock, C. Geiss, L. Luncz, V.Thomanetz, C.A. Stuermer, Regulation of embryonic cell adhesionby the prion protein, PLoS Biol. 7 (2009) e55.

[98] X. Roucou, A.C. LeBlanc, Cellular prion protein neuroprotectivefunction: implications in prion diseases, J. Mol. Med. 83 (2005) 3–11.

[99] C. Kurschner, J.I. Morgan, Analysis of interaction sites in homo- andheteromeric complexes containing Bcl-2 family members and thecellular prion protein, Brain Res. Mol. Brain Res. 37 (1996) 249–258.

[100] C. Kurschner, J.I. Morgan, The cellular prion protein (PrP) selectivelybinds to Bcl-2 in the yeast two-hybrid system, Brain Res. Mol. BrainRes. 30 (1995) 165–168.

[101] J.C. Manson, A.R. Clarke, P.A. McBride, I. McConnell, J. Hope, PrPgene dosage determines the timing but not the final intensity or

distribution of lesions in scrapie pathology, Neurodegeneration 3(1994) 331–340.

[102] R.C. Moore, I.Y. Lee, G.L. Silverman, P.M. Harrison, R. Strome, C.Heinrich, A. Karunaratne, S.H. Pasternak, M.A. Chishti, Y. Liang, P.Mastrangelo, K. Wang, A.F. Smit, S. Katamine, G.A. Carlson, F.E.Cohen, S.B. Prusiner, D.W. Melton, P. Tremblay, L.E. Hood, D.Westaway, Ataxia in prion protein (PrP)-deficient mice isassociated with upregulation of the novel PrP-like protein doppel,J. Mol. Biol. 292 (1999) 797–817.

[103] D. Rossi, A. Cozzio, E. Flechsig, M.A. Klein, T. Rulicke, A. Aguzzi, C.Weissmann, Onset of ataxia and Purkinje cell loss in PrP null miceinversely correlated with Dpl level in brain, EMBO J. 20 (2001) 694–702.

[104] S. Sakaguchi, S. Katamine, N. Nishida, R. Moriuchi, K. Shigematsu, T.Sugimoto, A. Nakatani, Y. Kataoka, T. Houtani, S. Shirabe, H. Okada,S. Hasegawa, T. Miyamoto, T. Noda, Loss of cerebellar Purkinje cellsin aged mice homozygous for a disrupted PrP gene, Nature 380(1996) 528–531.

[105] B.S. Wong, T. Liu, R. Li, T. Pan, R.B. Petersen, M.A. Smith, P. Gambetti,G. Perry, J.C. Manson, D.R. Brown, M.S. Sy, Increased levels ofoxidative stress markers detected in the brains of mice devoid ofprion protein, J. Neurochem. 76 (2001) 565–572.

[106] P.G. Marciano, J. Brettschneider, E. Manduchi, J.E. Davis, S. Eastman,R. Raghupathi, K.E. Saatman, T.P. Speed, C.J. Stoeckert Jr., J.H.Eberwine, T.K. McIntosh, Neuron-specific mRNA complexityresponses during hippocampal apoptosis after traumatic braininjury, J. Neurosci. 24 (2004) 2866–2876.

[107] W.C. Shyu, M.C. Kao, W.Y. Chou, Y.D. Hsu, B.W. Soong, Heat shockmodulates prion protein expression in human NT-2 cells,Neuroreport 11 (2000) 771–774.

[108] R. Walz, O.B. Amaral, I.C. Rockenbach, R. Roesler, I. Izquierdo, E.A.Cavalheiro, V.R. Martins, R.R. Brentani, Increased sensitivity toseizures in mice lacking cellular prion protein, Epilepsia 40 (1999)1679–1682.

[109] S. Hoshino, K. Inoue, T. Yokoyama, S. Kobayashi, T. Asakura, A.Teramoto, S. Itohara, Prions prevent brain damage afterexperimental brain injury: a preliminary report, Acta Neurochir.Suppl. 86 (2003) 297–299.

[110] B. Chesebro, M. Trifilo, R. Race, K. Meade-White, C. Teng, R.LaCasse, L. Raymond, C. Favara, G. Baron, S. Priola, B. Caughey, E.Masliah, M. Oldstone, Anchorless prion protein results ininfectious amyloid disease without clinical scrapie, Science 308(2005) 1435–1439.

[111] L. Fioriti, S. Dossena, L.R. Stewart, R.S. Stewart, D.A. Harris, G.Forloni, R. Chiesa, Cytosolic prion protein (PrP) is not toxic in N2acells and primary neurons expressing pathogenic PrP mutations, J.Biol. Chem. 280 (2005) 11320–11328.

[112] X. Roucou, Q. Guo, Y. Zhang, C.G. Goodyer, A.C. LeBlanc, Cytosolicprion protein is not toxic and protects against Bax-mediated celldeath in human primary neurons, J. Biol. Chem. 278 (2003) 40877–40881.

[113] D.T. Lin, J. Jodoin, M. Baril, C.G. Goodyer, A.C. Leblanc, Cytosolicprion protein is the predominant anti-Bax prion protein form:exclusion of transmembrane and secreted prion protein forms inthe anti-Bax function, Biochim. Biophys. Acta 1783 (2008) 2001–2012.

[114] I. Radovanovic, N. Braun, O.T. Giger, K. Mertz, G. Miele, M. Prinz, B.Navarro, A. Aguzzi, Truncated prion protein and Doppel aremyelinotoxic in the absence of oligodendrocytic PrPC, J. Neurosci.25 (2005) 4879–4888.

[115] N. Vassallo, J. Herms, Cellular prion protein function in copperhomeostasis and redox signalling at the synapse, J. Neurochem. 86(2003) 538–544.

[116] R. Chiesa, P. Piccardo, B. Ghetti, D.A. Harris, Neurological illness intransgenic mice expressing a prion protein with an insertionalmutation, Neuron 21 (1998) 1339–1351.

[117] R. Chiesa, B. Drisaldi, E. Quaglio, A. Migheli, P. Piccardo, B. Ghetti,D.A. Harris, Accumulation of protease-resistant prion protein (PrP)and apoptosis of cerebellar granule cells in transgenic miceexpressing a PrP insertional mutation, Proc. Natl. Acad. Sci. USA97 (2000) 5574–5579.

[118] R. Chiesa, P. Piccardo, E. Quaglio, B. Drisaldi, S.L. Si-Hoe, M. Takao, B.Ghetti, D.A. Harris, Molecular distinction between pathogenic andinfectious properties of the prion protein, J. Virol. 77 (2003) 7611–7622.

[119] G. Mitteregger, M. Vosko, B. Krebs, W. Xiang, V. Kohlmannsperger,S. Nolting, G.F. Hamann, H.A. Kretzschmar, The role of theoctarepeat region in neuroprotective function of the cellularprion protein, Brain Pathol. 17 (2007) 174–183.


[120] C. Kuwahara, A.M. Takeuchi, T. Nishimura, K. Haraguchi, A.Kubosaki, Y. Matsumoto, K. Saeki, Y. Matsumoto, T. Yokoyama, S.Itohara, T. Onodera, Prions prevent neuronal cell-line death, Nature400 (1999) 225–226.

[121] B. Kim, H. Lee, J. Choi, J. Kim, E. Choi, R. Carp, K. YS, The cellularprion protein (PrP (C)) prevents apoptotic neuronal cell death andmitochondrial dysfunction induced by serum deprivation, BrainRes. Mol. Brain Res. 124 (2004) 40–50.

[122] T. Nishimura, A. Sakudo, Y. Hashiyama, A. Yachi, K. Saeki, Y.Matsumoto, M. Ogawa, S. Sakaguchi, S. Itohara, T. Onodera, Serumwithdrawal-induced apoptosis in ZrchI prion protein (PrP) gene-deficient neuronal cell line is suppressed by PrP, independent ofDoppel, Microbiol. Immunol. 51 (2007) 457–466.

[123] Y. Bounhar, Y. Zhang, C.G. Goodyer, A. LeBlanc, Prion proteinprotects human neurons against Bax-mediated apoptosis, J. Biol.Chem. 276 (2001) 39145–39149.

[124] X. Roucou, P.N. Giannopoulos, Y. Zhang, J. Jodoin, C.G. Goodyer, A.LeBlanc, Cellular prion protein inhibits proapoptotic Baxconformational change in human neurons and in breastcarcinoma MCF-7 cells, Cell Death Differ. 12 (2005) 783–795.

[125] J. Jodoin, S. Laroche-Pierre, C.G. Goodyer, A.C. LeBlanc, Defectiveretrotranslocation causes loss of anti-Bax function in humanfamilial prion protein mutants, J. Neurosci. 27 (2007) 5081–5091.

[126] A. Li, D.A. Harris, Mammalian prion protein suppresses Bax-induced cell death in yeast, J. Biol. Chem. 280 (2005) 17430–17434.

[127] H. Zha, H.A. Fisk, M.P. Yaffe, N. Mahajan, B. Herman, J.C. Reed,Structure–function comparisons of the proapoptotic protein Bax inyeast and mammalian cells, Mol. Cell Biol. 16 (1996) 6494–6508.

[128] A. Sakudo, M. Hamaishi, T. Hosokawa-Kanai, K. Tuchiya, T.Nishimura, K. Saeki, Y. Matsumoto, S. Ueda, T. Onodera, Absenceof superoxide dismutase activity in a soluble cellular isoform ofprion protein produced by baculovirus expression system,Biochem. Biophys. Res. Commun. 307 (2003) 678–683.

[129] A. Sakudo, D.C. Lee, T. Nishimura, S. Li, S. Tsuji, T. Nakamura, Y.Matsumoto, K. Saeki, S. Itohara, K. Ikuta, T. Onodera, Octapeptiderepeat region and N-terminal half of hydrophobic region of prionprotein (PrP) mediate PrP-dependent activation of superoxidedismutase, Biochem. Biophys. Res. Commun. 326 (2005) 600–606.

[130] S. Mouillet-Richard, M. Pietri, B. Schneider, C. Vidal, V. Mutel, J.M.Launay, O. Kellermann, Modulation of serotonergic receptorsignaling and cross-talk by prion protein, J. Biol. Chem. 280(2005) 4592–4601.

[131] H. Sauer, A. Dagdanova, J. Hescheler, M. Wartenberg, Redox-regulation of intrinsic prion expression in multicellular prostatetumor spheroids, Free Radic. Biol. Med. 27 (1999) 1276–1283.

[132] H. Sauer, K. Wefer, V. Vetrugno, M. Pocchiari, C. Gissel, A.Sachinidis, J. Hescheler, M. Wartenberg, Regulation of intrinsicprion protein by growth factors and TNF-alpha: the role ofintracellular reactive oxygen species, Free Radic. Biol. Med. 35(2003) 586–594.

[133] O. Milhavet, S. Lehmann, Oxidative stress and the prion protein intransmissible spongiform encephalopathies, Brain Res. Brain Res.Rev. 38 (2002) 328–339.

[134] N.T. Watt, N.M. Hooper, Reactive oxygen species (ROS)-mediatedbeta-cleavage of the prion protein in the mechanism of the cellularresponse to oxidative stress, Biochem. Soc. Trans. 33 (2005) 1123–1125.

[135] M. Malaise, H.M. Schatzl, A. Burkle, The octarepeat region of prionprotein, but not the TM1 domain, is important for the antioxidanteffect of prion protein, Free Radic. Biol. Med. 45 (2008) 1622–1630.

[136] K.L. Brown, Scrapie replication in lymphoid tissues depends onprion protein-expressing follicular dendritic cells, Nat. Med. 5(1999) 1308–1312.

[137] S. Jones, M. Batchelor, D. Bhelt, A.R. Clarke, J. Collinge, G.S. Jackson,Recombinant prion protein does not possess SOD-1 activity,Biochem. J. 392 (2005) 309–312.

[138] G. Hutter, F.L. Heppner, A. Aguzzi, No superoxide dismutase activityof cellular prion protein in vivo, Biol. Chem. 384 (2003) 1279–1285.

[139] D.R. Brown, W.J. Schulz-Schaeffer, B. Schmidt, H.A. Kretzschmar,Prion protein-deficient cells show altered response to oxidativestress due to decreased SOD-1 activity, Exp. Neurol. 146 (1997)104–112.

[140] D.R. Brown, R.S. Nicholas, L. Canevari, Lack of prion proteinexpression results in a neuronal phenotype sensitive to stress, J.Neurosci. Res. 67 (2002) 211–224.

[141] F. Klamt, F. Dal-Pizzol, M.J. Conte da Frota, R. Walz, M.E. Andrades,E.G. da Silva, R.R. Brentani, I. Izquierdo, J.C. Fonseca Moreira,Imbalance of antioxidant defense in mice lacking cellular prionprotein, Free Radic. Biol. Med. 30 (2001) 1137–1144.

[142] A.R. White, S.J. Collins, F. Maher, M.F. Jobling, L.R. Stewart, J.M.Thyer, K. Beyreuther, C.L. Masters, R. Cappai, Prion protein-deficient neurons reveal lower glutathione reductase activity andincreased susceptibility to hydrogen peroxide toxicity, Am. J.Pathol. 155 (1999) 1723–1730.

[143] B. Halliwell, Oxidative stress and neurodegeneration: where are wenow?, J Neurochem. 97 (2006) 1634–1658.

[144] C.J. Choi, V. Anantharam, N.J. Saetveit, R.S. Houk, A. Kanthasamy,A.G. Kanthasamy, Normal cellular prion protein protects againstmanganese-induced oxidative stress and apoptotic cell death,Toxicol. Sci. 98 (2007) 495–509.

[145] A.S. Rambold, M. Miesbauer, D. Olschewski, R. Seidel, C. Riemer, L.Smale, L. Brumm, M. Levy, E. Gazit, D. Oesterhelt, M. Baier, C.F.Becker, M. Engelhard, K.F. Winklhofer, J. Tatzelt, Green tea extractsinterfere with the stress-protective activity of PrP and theformation of PrP, J. Neurochem. 107 (2008) 218–229.

[146] V. Anantharam, A. Kanthasamy, C.J. Choi, D.P. Martin, C.Latchoumycandane, J.A. Richt, A.G. Kanthasamy, Opposing roles ofprion protein in oxidative stress- and ER stress-induced apoptoticsignaling, Free Radic. Biol. Med. 45 (2008) 1530–1541.

[147] A.R. Walmsley, F. Zeng, N.M. Hooper, The N-terminal region of theprion protein ectodomain contains a lipid raft targetingdeterminant, J. Biol. Chem. 278 (2003) 37241–37248.

[148] B. Drisaldi, J. Coomaraswamy, P. Mastrangelo, B. Strome, J. Yang, J.C.Watts, M.A. Chishti, M. Marvi, O. Windl, R. Ahrens, F. Major, M.S. Sy,H. Kretzschmar, P.E. Fraser, H.T. Mount, D. Westaway, Geneticmapping of activity determinants within cellular prion proteins: N-terminal modules in PrPC offset pro-apoptotic activity of theDoppel helix B/B0 region, J. Biol. Chem. 279 (2004) 55443–55454.

[149] R. Atarashi, N. Nishida, K. Shigematsu, S. Goto, T. Kondo, S.Sakaguchi, S. Katamine, Deletion of N-terminal residues 23–88from prion protein (PrP) abrogates the potential to rescue PrP-deficient mice from PrP-like protein/doppel-inducedneurodegeneration, J. Biol. Chem. 278 (2003) 28944–28949.

[150] K. Qin, L. Zhao, Y. Tang, S. Bhatta, J.M. Simard, R.Y. Zhao, Doppel-induced apoptosis and counteraction by cellular prion protein inneuroblastoma and astrocytes, Neuroscience 141 (2006) 1375–1388.

[151] A. Li, P. Piccardo, S.J. Barmada, B. Ghetti, D.A. Harris, Prion proteinwith an octapeptide insertion has impaired neuroprotectiveactivity in transgenic mice, EMBO J. 26 (2007) 2777–2785.

[152] H. Rosenmann, G. Talmor, M. Halimi, A. Yanai, R. Gabizon, Z.Meiner, Prion protein with an E200K mutation displays propertiessimilar to those of the cellular isoform PrP(C), J. Neurochem. 76(2001) 1654–1662.

[153] E. Paitel, C. Sunyach, C. Alves da Costa, J.C. Bourdon, B. Vincent, F.Checler, Primary cultured neurons devoid of cellular prion displaylower responsiveness to staurosporine through the control of p53at both transcriptional and post-transcriptional levels, J. Biol.Chem. 279 (2004) 612–618.

[154] E. Paitel, R. Fahraeus, F. Checler, Cellular prion protein sensitizesneurons to apoptotic stimuli through Mdm2-regulated and p53-dependent caspase 3-like activation, J. Biol. Chem. 278 (2003)10061–10066.

[155] L. Solforosi, J.R. Criado, D.B. McGavern, S. Wirz, M. Sanchez-Alavez,S. Sugama, L.A. DeGiorgio, B.T. Volpe, E. Wiseman, G. Abalos, E.Masliah, D. Gilden, M.B. Oldstone, B. Conti, R.A. Williamson, Cross-linking cellular prion protein triggers neuronal apoptosis in vivo,Science 303 (2004) 1514–1516.

[156] G. Wu, K. Nakajima, N. Takeyama, M. Yukawa, Y. Taniuchi, A.Sakudo, T. Onodera, Species-specific anti-apoptotic activity ofcellular prion protein in a mouse PrP-deficient neuronal cell linetransfected with mouse, hamster, and bovine Prnp, Neurosci. Lett.446 (2008) 11–15.

[157] P.K. Kim, M.G. Annis, P.J. Dlugosz, L.B.D.W. Andrews, Duringapoptosis bcl-2 changes membrane topology at both theendoplasmic reticulum and mitochondria, Mol. Cell 14 (2004)523–529.

[158] B. Lin et al., Conversion of Bcl-2 from protector to killer byinteraction with nuclear orphan receptor Nur77/TR3, Cell 116(2004) 527–540.

[159] R. Clem, E.H. Cheng, C.L. Karp, D.G. Kirsch, K. Ueno, A. Takahashi,M.B. Kastan, D.E. Griffin, W.C. Earnshaw, M.A. Veliuona, H. JM,Modulation of cell death by Bcl-XL through caspase interaction,Proc. Natl. Acad. Sci. USA 95 (1998) 554–559.

[160] B. Vanhaesebroeck, J.C. Reed, D. De Valck, J. Grooten, T. Miyashita, S.Tanaka, R. Beyaert, F. Van Roy, W. Fiers, Effect of bcl-2 proto-oncogene expression on cellular sensitivity to tumor necrosisfactor-mediated cytotoxicity, Oncogene 8 (1993) 1075–1081.


[161] B. Ghetti, P. Piccardo, M.G. Spillantini, Y. Ichimiya, M. Porro, F.Perini, T. Kitamoto, J. Tateishi, C. Seiler, B. Frangione, O. Bugiani, G.Giaccone, F. Prelli, M. Goedert, S.R. Dlouhy, T. F, Vascular variant ofprion protein cerebral amyloidosis with tau-positiveneurofibrillary tangles: the phenotype of the stop codon 145mutation in PRNP, Proc. Natl. Acad. Sci. USA 93 (1996) 744–748.

[162] P.P. Liberski, D.R. Brown, B. Sikorska, B. Caughey, P. Brown, Celldeath and autophagy in prion diseases (transmissible spongiformencephalopathies), Folia Neuropatholo. 46 (2008) 1–25.

[163] M. Dron, Y. Bailly, V. Beringue, A.M. Haeberle, B. Griffond, P.Y.Risold, M.G. Tovey, H. Laude, F. Dandoy-Dron, SCRG1, a potentialmarker of autophagy in transmissible spongiformencephalopathies, Autophagy 2 (2006) 58–60.

[164] S. Heitz, N.J. Grant, R. Leschiera, A.M. Haeberle, V. Demais, G.Bombarde, Y. Bailly, Autophagy and cell death of Purkinje cellsoverexpressing doppel in Ngsk Prnp-deficient mice, Brain Pathol.(2008).

[165] J.M. Oh, H.Y. Shin, S.J. Park, B.H. Kim, J.K. Choi, E.K. Choi, R.I. Carp,Y.S. Kim, The involvement of cellular prion protein in theautophagy pathway in neuronal cells, Mol. Cell. Neurosci. 39(2008) 238–247.

[166] D. Hanahan, R.A. Weinberg, The hallmarks of cancer, Cell 100(2000) 57–70.

[167] D.R. Green, G.I. Evan, A matter of life and death, Cancer Cell 1(2002) 19–30.

[168] R.W. Johnstone, A.A. Ruefli, S.W. Lowe, Apoptosis: a link betweencancer genetics and chemotherapy, Cell 108 (2002) 153–164.

[169] M. Diarra-Mehrpour, S. Arrabal, A. Jalil, X. Pinson, C. Gaudin, G.Pietu, A. Pitaval, H. Ripoche, M. Eloit, D. Dormont, S. Chouaib, Prionprotein prevents human breast carcinoma cell line from tumornecrosis factor alpha-induced cell death, Cancer Res. 64 (2004)719–727.

[170] J.G. Clohessy, J. Zhuang, J. de Boer, G. Gil-Gomez, H.J. Brady, Mcl-1interacts with truncated Bid and inhibits its induction ofcytochrome c release and its role in receptor-mediated apoptosis,J. Biol. Chem. 281 (2006) 5750–5759.

[171] S. Laroche-Pierre, J. Jodoin, A.C. LeBlanc, Helix 3 is necessary andsufficient for prion protein’s anti-Bax function, J. Neurochem. 108(2009) 1019–1031.

[172] B.T. McGrogan, B. Gilmartin, D.N. Carney, A. McCann, Taxanes,microtubules and chemoresistant breast cancer, Biochim. Biophys.Acta 1785 (2008) 96–132.

[173] Q.Q. Li, X.X. Cao, J.D. Xu, Q. Chen, W.J. Wang, F. Tang, Z.Q. Chen, X.P.Liu, Z.D. Xu, The role of P-glycoprotein/cellular prion protein

interaction in multidrug-resistant breast cancer cells treated withpaclitaxel, Cell Mol. Life Sci. 66 (2009) 504–515.

[174] R.K. Srivastava, Q.S. Mi, J.M. Hardwick, D.L. Longo, Deletion of theloop region of Bcl-2 completely blocks paclitaxel-inducedapoptosis, Proc. Natl. Acad. Sci. USA 96 (1999) 3775–3780.

[175] J. Du, Y. Pan, Y. Shi, C. Guo, X. Jin, L. Sun, N. Liu, T. Qiao, D. Fan,Overexpression and significance of prion protein in gastric cancerand multidrug-resistant gastric carcinoma cell line SGC7901/ADR,Int. J. Cancer 113 (2005) 213–220.

[176] J. Liang, F. Bai, G. Luo, J. Wang, J. Liu, F. Ge, Y. Pan, L. Yao, R. Du, X. Li,R. Fan, H. Zhang, X. Guo, K. Wu, D. Fan, Hypoxia inducedoverexpression of PrP(C) in gastric cancer cell lines, Cancer Biol.Ther. 6 (2007) 769–774.

[177] J. Liang, F. Ge, C. Guo, G. Luo, X. Wang, G. Han, D. Zhang, J. Wang, K.Li, Y. Pan, L. Yao, Z. Yin, X. Guo, K. Wu, J. Ding, D. Fan, Inhibition ofPI3K/Akt partially leads to the inhibition of PrP(C)-induced drugresistance in gastric cancer cells, FEBS J. 276 (2009) 685–694.

[178] R.B. Erlich, S.A. Kahn, F.R. Lima, A.G. Muras, R.A. Martins, R. Linden,L.B. Chiarini, V.R. Martins, V. Moura Neto, STI1 promotes gliomaproliferation through MAPK and PI3K pathways, Glia 55 (2007)1690–1698.

[179] Y. Pan, L. Zhao, J. Liang, J. Liu, Y. Shi, N. Liu, G. Zhang, H. Jin, J. Gao, H.Xie, J. Wang, Z. Liu, D. Fan, Cellular prion protein promotes invasionand metastasis of gastric cancer, FASEB J. 20 (2006) 1886–1888.

[180] J. Liang, Y. Pan, D. Zhang, C. Guo, Y. Shi, J. Wang, Y. Chen, X. Wang, J.Liu, X. Guo, Z. Chen, T. Qiao, D. Fan, Cellular prion protein promotesproliferation and G1/S transition of human gastric cancer cellsSGC7901 and AGS, FASEB J. 21 (2007) 2247–2256.

[181] J. Liang, J.B. Wang, Y.L. Pan, J. Wang, L.L. Liu, X.Y. Guo, L. Sun, T. Lin,S. Han, H.H. Xie, F. Yin, X.G. Guo, D. Fan, High frequency occurrenceof 1-OPRD variant of PRNP gene in gastric cancer cell lines andChinese population with gastric cancer, Cell Biol. Int. 30 (2006)920–923.

[182] J. Liang, J. Wang, G. Luo, Y. Pan, G. Han, X. Wang, C. Guo, D. Zhang, F.Yin, X. Zhang, J. Liu, J. Wang, X. Guo, K. Wu, D. Fan, Function ofPrP(C)(1-OPRD) in biological activities of gastric cancer cell lines, J.Cell. Mol. Med. (2009).

[183] J.C. Watts, D. Westaway, The prion protein family: diversity, rivalry,and dysfunction, Biochim. Biophys. Acta 1772 (2007) 654–672.

[184] D.C. West, C.G. Rees, L. Duchesne, S.J. Patey, C.J. Terry, J.E. Turnbull,M. Delehedde, C.W. Heegaard, F. Allain, C. Vanpouille, D. Ron, D.G.Fernig, Interactions of multiple heparin binding growth factorswith neuropilin-1 and potentiation of the activity of fibroblastgrowth factor-2, J. Biol. Chem. 280 (2005) 13457–13464.

prion protein: from physiology to cancer biology · be involved in cancer biology. 2. human prion...

Documents