prion diseases are a family of progressive fatal neurodegenerative diseases of infectious , sporadic or inherited origin , which affect humans and other animals . central to the pathogenesis is the conversion of a host - encoded prion protein , prp , into a misfolded , protease resistant isoform , prp , which accumulates in the brain and constitutes the infectious agent responsible for the disease . prion diseases are typically diagnosed by manifestation of dementia and locomotor symptoms , which correlate with extensive neuronal loss and prp deposition . at the histological level , neuronal loss is preceded by synaptic pathology and early synaptic failure is a well - documented component of prion diseases . however , little is known about the underlying mechanisms of synaptic degeneration , and the sequence of events involved in the neurodegeneration is not yet completely understood . it has been proposed that impairment in mitochondrial function could be responsible for the synaptic pathology observed in prion diseases . by investigating a wide range of mitochondrial parameters , it was shown that mitochondrial function was impaired in prion diseases and that mitochondrial damage could potentially be the initial cause of synaptic pathology . additionally , several neurodegenerative diseases marked by the accumulation of misfolded proteins , such as alzheimer and parkinson diseases , are associated with synaptic dysfunction and mitochondrial abnormalities . evidence that mitochondria could have a role in prion pathology comes from the finding that prp can interact with neuronal mitochondria and impair their function . in transgenic mice overexpressing prp , redistribution of prp to the mitochondria induces the loss of mitochondrial membrane potential ( mmp ) and cytochrome c release , resulting in caspase-3-dependent apoptosis , predominantly in hippocampal neurons . moreover , the expression of prp as a cytosolic protein induces the formation of cytosolic aggregates , mitochondrial clustering and promotes cell death by a process that involves depolarization of the mitochondrial membrane , release of cytochrome c and caspase activation . finally , the synthetic peptide prp-(106126 ) , which has been shown to be neurotoxic , can induce the rapid depolarization of mitochondrial membranes , cytochrome c release and apoptosis in a panel of different neuronal cells . while the mechanism of prion disease development remains unclear , mitochondrial damage and dysfunction has emerged as the possible initial cause of synaptic degeneration , which eventually results in apoptotic neuronal cell death . the possibility that the mitochondria constitute the primary site of the proapoptotic pathway opens up new avenues for early diagnosis and particularly for early treatment . prp is a glycoprotein that localizes to the plasma membrane via a c - terminally linked glycosylphosphatidylinositol ( gpi ) anchor . this posttranslational modification concerns over 100 different proteins in humans , including adhesion molecules , surface receptors and enzymes . gpi anchor addition occurs in the lumen of the endoplasmic reticulum ( er ) , where the pre - assembled gpi anchor is transferred to the acceptor amino acid ( called the omega site ) by the action of a multimeric gpi transamidase . the gpi transamidase recognizes a c - terminal gpi - anchor signal peptide ( gpi - sp ) , which is cleaved concomitantly to the attachment of the gpi - anchor . the gpi - sp consists of a hydrophilic region ( 510 amino acids ) followed by a hydrophobic sequence ( 1520 amino acids ) , but overall lacks a well - defined consensus sequence . as such , they are not usually conserved across species , with the significant exception of prp , whose gpi - sp shows a remarkable degree of conservation . additionally , prp gpi - sp harbors 2 autosomal dominant pathogenic mutations responsible for genetic forms of prion diseases ( m232r and p238s ) ; these mutations are particularly perplexing since the gpi - sp is replaced by a gpi anchor and is absent in the mature prion protein . because of its unusual level of conservation , and because of the presence of unexplained pathogenic mutations in its sequence , prp gpi - sp represents an ideal model to address the fate of the gpi - anchor signal peptides . to investigate the metabolism of prp gpi - sp we recently created a hybrid construct where the c - terminus of gfp had been fused to the gpi - sp of prp , which had then been linked to the double tag myc - his via a flexible gly - ser linker . as a control , we fused gfp to the gpi - sp of folate receptor ( fr ) ; the gpi - sps of fr and prp have the same length ( 24 amino acids ) and an identical size of the hydrophilic ( 9 amino acids ) and the hydrophobic ( 14 amino acids ) regions . we were able to show that , while fr gpi - sp was retained within the lumen of the er , prp gpi - sp was selectively transported to the cytoplasm , where it was targeted for degradation by the proteasome . interestingly , when the pathogenic point mutation p238s was introduced into our construct , prp gpi - sp was spared from degradation . linking the p238s point mutation to the accumulation of prp gpi - sp potentially establishes a link between the accumulation of prp gpi - sp and prp induced neurodegeneration , opening up the possibility that prp gpi - sp could play a role in prion diseases . we show that , differently form the control gpi - sp of folate receptor , the gpi - sp of prp is targeted to the mitochondria , and causes their fragmentation and loss of mmp . to further explore the function of prp gpi - sp , we decided to investigate the fate of prp gpi - sp following its retrotranslocation into the cytoplasm . because the retrotranslocation of proteins into the cytosol is ubiquitination - dependent , we reasoned that the expression of a cytosolic form of prp gpi - sp ( cprpgpi - sp ) would allow prp gpi - sp to escape ubiquitination . indeed , it has been shown that er proteins that are targeted for proteasomal degradation accumulate in the cytosol when degradation is prevented by proteasome inhibitors ( as is the case for prp gpi - sp ) , but remain restricted to the lumen of the er under ubiquitination - deficient conditions , confirming that the release of the protein into the cytosol is linked to ubiquitination . the cytosolic versions of prp 's and fr 's gpi - sps ( cprpgpi - sp and cfrgpi - sp respectively ) were cloned starting from gfp - fr - mh and gfp - prp - mh ( described in ref . constructs the gpi - sp was linked to a myc - his ( mh ) tag via a gly - ser flexible linker , as described in ref . the scheme in figure 1a represents the starting construct ( gfp - gpi - tag ) and circled in red is the part expressed as cytosolic protein . the constructs were expressed in hela cells and analyzed by western blot ( wb ) and immunofluorescence ( if ) . wb showed that both constructs produced a single band at the expected molecular weight ( fig . 1b ) ; by if analysis , the 2 constructs showed significantly different localization patterns . cfrgpi - sp appeared to localize to a reticular network that extended across the cytoplasm and around the nucleus ( fig . 1c ) , most likely representing the er . this localization is not surprising , as the hydrophobic region of gpi - sps can potentially function as an er - signal sequence , explaining why cfrgpi - sp localizes to the er . specifically , prp gpi - sp has been shown to work as an er - signal sequence when linked to the n - term of gfp . however , when expressed in hela cells , cprp - gpi - sp localized to discrete tubular elements ( fig . 1d ) , reminiscent of the mitochondrial network . to confirm the localization of our constructs , cells expressing cfrgpi - sp were co - stained with the er marker calnexin ( fig . g ) and cells expressing cprpgpi - sp were labeled with the mitochondrial marker tom20 ( fig . 1h j ) . as shown in figure 1e  j , complete colocalization confirmed that cprpgpi - sp localizes to the mitochondria , whereas cfrgpi - sp localizes to the er . 
 figure 1.expression and localization of cprpgpi - sp and cfrgpi - sp . wb ( a ) and if ( b , c ) of hela cells expressing cfrgpi - sp and cprpgpi - sp . ( d  f ) colocalization between cfrgpi - sp ( green ) and the er marker calnexin(red ) . ( g  i ) colocalization between cprpgpi - sp ( green ) and the mitochondrial marker tom20 ( red ) . nuclei are in blue . 
  expression and localization of cprpgpi - sp and cfrgpi - sp . wb ( a ) and if ( b , c ) of hela cells expressing cfrgpi - sp and cprpgpi - sp . ( d  f ) colocalization between cfrgpi - sp ( green ) and the er marker calnexin(red ) . ( g  i ) colocalization between cprpgpi - sp ( green ) and the mitochondrial marker tom20 ( red ) . while cfrgpi - sp er localization had no effect on the structure of the compartment or on the viability of the transfected cells ( data not shown ) , we noticed that in cells expressing cprpgpi - sp the mitochondria were affected in a time - dependent fashion : within the first hours after transfection , cprpgpi - sp localized to intact active mitochondria , as shown by costaining with mitotracker red cmxros . mitotracker red cmxros was used to indicate the alteration of mitochondrial membrane potential ( mmp ) : cells were transfected with cprpgpi - sp and incubated with mitotracker red cmxros ( 100 nm , 30  min ) 12h , 24h and 36h after transfection ( fig . after fixation , the degree of mmp was visualized qualitatively as red fluorescence while the morphology of nuclei could be observed as blue fluorescence ( dapi staining ) . because the uptake of mitotracker red depends on mmp , loss in mmp causes a dramatic decrease of red fluorescence . as shown in figure 2 , the shape of mitochondria dramatically changed , appearing fragmented at 24h and extensively vacuolarized at 36h after transfection ; at the same time the nuclei appeared condensed , a characteristic morphology of cells undergoing apoptosis ( fig . moreover , the degree of red fluorescence in mitochondria containing cprpgpi - sp decreased from 12h to 24h and almost completely disappeared 36h after transfection . the degree of colocalization between mitochondria and cprpgpi - sp was quantified by calculating manders overlap coefficient ( fig . manders m1 and m2 coefficients measure the portion of the pixels in each channel ( here red and green ) that coincides with a signal in the other channel . the m coefficient varies from 0 to 1 , where 1 indicates complete overlap . after each time - point ( 12h , 24h , 36h ) , the value of m1 ( amount of mitotracker colocalizing with cprpgpi - sp ) was 0.9  0.05 , indicating that virtually all mitotracker signal overlaps with cprpgpi - sp 's signal . however , the value of m2 ( amount of cprpgpi - sp colocalizing with mitotracker ) decreased from 0.9 ( at 12h after transfection ) to 0.4 and 0.1 ( 24h and 36h after transfection respectively ) , indicating that the majority of cprpgpi - sp had lost mitotracker staining , which is indicative of loss in mmp . hela cells transfected with cprpgpi - sp for 12h ( a  c ) , 24h ( d  f ) and 36h ( g  i ) were stained to visualize cprpgpi - sp ( green ) and the status of mmp via mitotracker ( red ) . colocalzation efficiency was measured using imagej software and shown by mander 's coefficient ( j ) . m1 = amount of mitotracker colocalizing with cprpgpi - sp ; m2 = amount of cprpgpi - sp colocalizing with mitotracker . hela cells transfected with cprpgpi - sp for 12h ( a  c ) , 24h ( d  f ) and 36h ( g  i ) were stained to visualize cprpgpi - sp ( green ) and the status of mmp via mitotracker ( red ) . colocalzation efficiency was measured using imagej software and shown by mander 's coefficient ( j ) . m1 = amount of mitotracker colocalizing with cprpgpi - sp ; m2 = amount of cprpgpi - sp colocalizing with mitotracker . the average and standard deviation were obtained by the analysis of 5 images . in conclusion , we were able to show that prp gpi - sp , when expressed as a cytosolic peptide , is able to localize to the mitochondria and to induce mitochondria fragmentation and vacuolarization , which are accompanied by loss of mmp and ultimately apoptosis . this phenotype of mitochondrial damage and mitochondrial - dependent apoptosis strengthens the evidence that mitochondria play a role in prion pathology . our results , for the first time , identify the gpi - sp of prp as a novel candidate responsible for the impairment in mitochondrial function involved in the synaptic pathology observed in prion diseases . it is conceivable that during prion infection prp gpi - sp may accumulate in the cytoplasm , bind to mitochondria and induce depolarization of the mitochondrial membrane , release of cytochrome c and caspase activation , resulting in neuronal cell death . p. cossart , institut pasteur , paris , france ) were maintained in dulbecco 's modified eagle 's medium ( dmem ) ( invitrogen ) supplemented with 10% fetal calf serum ( fcs ) in a 5% co2 incubator at 37c . cells were transfected at 70% confluence using fugene6 ( roche diagnostic ) according to manufacturer 's protocol . for immunofluorescence and western blot the following antibodies were used : primary antibodies : rabbit anti - his ( a gift from dr . dautry , institut pasteur ) ; tom20 ( bd transduction laboratories , # 612278 ) ; calreticulin ( stressgen # spa-600 ) ; myc9e10 ( sigma # m4439 ) . secondary antibodies for if : alexa fluor 488 goat anti mouse ( 1:500 ) and alexa fluor 594 goat anti rabbit ( 1:500 ) from molecular probes . mitotracker red cmx ros ( invitrogen , carlsbad , ca ) was used at a final concentration of 50  nm for 15  min . dna constructs : gpi - sps were inserted into pcdna3.1mychis expression vector ( invitrogen ) using nhei / bamhi restriction sites . ssgfp - fr - mh and ssgfp - prp - mh described in were used as templates . primers : 5-gga gac cca agc tgg cta gcc acc atg aag gaa ttc-3 and bghrv ; the use of a common fw primer resulted in the addition of 8aa upstream of the omega site . immunofluorescence : hela cells plated on 12 mm glass coverslips were fixed with 4% formaldehyde , permeabilized with 0.1% tritonx100 in pbs for 5  min , then incubated in blocking buffer ( pbs containing 10% fcs ) for 30  min at room temperature . coverslips were then mounted onto glass slides with aqua / poly mount ( polysciences ) and visualized using a zeiss observer z1 156 inverted microscope with 63 objective controlled by axiovision software ( zeiss , thornwood , ny ) . the degree of colocalization between mitochondria ( red ) and ga - bodipy ( green ) was quantified using the colocalization finder and jacop plug - in of imagej software ( http://rsb.info.nih.gov/ij/ ) . western blot : cells were lysed with page loading buffer ( 60  mm tris , ph 6.8 , 5% 2-mercaptoethanol , 2% sds , 0.01% bromophenol blue , and 10% glycerol ) . page using a 420% gradient tris - tricine gel ( mini - protean tris - tricine precast gels , biorad ) . proteins were transferred on whatman protran nitrocellulose membranes ( 0.1um pore size ) ( sigma ) . the membrane was kept in blocking buffer ( 50 mn , tris , ph 7.5 , 150  mm nacl , 0.05% tween 20 , and 3% bsa ) and then incubated with the primary antibody diluted in blocking buffer . hrp - conjugated secondary antibodies and ecl reagents from amersham ( ge healthcare ) were used for detection on kodak biomax films . the work in c.z . lab was supported by fellowships from the federation of european biochemical societies ( febs ) , the fondation pour la recherche mdicale ( frm ) , the european union [ grant number fp7-kbbe-20072a-22287 ] , the anr ( agence nationale de la recherche ) ( grant numbers anr-09-blan-0122 ) and the pasteur  weizmann foundation ( 20102012 )