prion 
 diseases , also known as transmissible spongiform encephalopathies 
 or tses , are a group of rare neurodegenerative disorders that affect 
 humans and other mammals .
animal forms 
 of prion disease include sheep scrapie , chronic wasting disease in 
 deer and bovine spongiform encephalopathy in cattle .
human prion disorders include familial , sporadic and variant 
 creutzfeldt  jakob disease ( cjd ) , as well as kuru and gerstmann  strussler 
scheinker 
 ( gss ) syndrome . a critical event in prion pathogenesis is the conversion 
 of host - encoded prion protein ( prp ) into a misfolded isoform , 
 termed prp , which is widely believed to be the primary 
 component of the infectious prion agent .
pathogenic prp has the same amino acid sequence as prp but has significantly 
 more -sheet structure and is no longer monomeric .
variable disease phenotypes induced by different prion strains are often attributed to specific prp conformations , perhaps defined by specific interactions between 
 amino acid side - chains within individual  strands .
although no direct link between a specific 
 structural motif in 
 prp and a specific in vivo prion phenotype has been made , 
 prp anchoring to the plasma membrane has been shown to 
 profoundly influence prion disease phenotypes .
prp is normally attached to the plasma membrane by a glycosylphosphatidylinositol 
 ( gpi ) anchor .
when wild - type c57bl/10 
 mice are infected intracranially with the rml mouse prion strain , 
 prp accumulates in diffuse nonamyloid deposits and brain 
 pathology is distinguished by the hallmark gray matter spongiosis 
 for which tses are named .
conversely , when transgenic mice homozygous 
 for prp lacking the gpi anchor ( tg44 or 
mice ) are infected with the same rml strain , the incubation time is 
 longer and prp accumulates in dense perivascular amyloid 
 plaques with little or no gray matter spongiosis . similar amyloid 
 prp deposition and disease resulting in death has been 
 observed in humans with an aberrant stop codon in the c - terminal portion 
 of the prp sequence , resulting in the generation of prp lacking the 
 gpi anchoring group as well as some amino acid residues .
thus , gpi - anchoring of prp to the plasma membrane appears 
 to be a primary factor determining the type of prp deposition 
 and the development of brain pathology . despite the neuropathological 
 differences between these two prion 
 disease phenotypes , the mechanisms underlying these differences remain 
 unclear .
both anchorless and wild - type mice displayed a similar cytokine 
 release profile when infected with the 22l prion agent , suggesting 
 a similar inflammatory response .
when 
 enriched prp samples from rml - infected wild - type or anchorless 
 tg44 mouse brains were compared by infrared spectroscopy , gross differences 
 in secondary structure were minimal .
furthermore , 
 infectivity replicated to high levels in both types of mice and , when 
 passaged back into wild - type mice , both led to prion disease typical 
 of the original rml strain .
taken together , 
 the data suggest that the dramatic phenotypic differences between 
 these two prion diseases is determined more by the presence or absence 
 of the gpi anchor on prp rather than by any changes in 
 strain - specific -sheet conformation within prp .
multiple studies have used microarray analysis to determine 
 whether 
 different pathways underlie differences in disease pathogenesis between 
 mouse prion strains or various types of human cjd .
a postgenomic , 
 proteomics approach has been utilized in some studies , several of which have focused on the identification of potential 
 prion disease biomarkers ( refs ( 26 , 31 ) , and see ref ( 32 ) for review ) . however , there are no genomic or proteomic studies 
 directly comparing two different prion diseases induced by the same 
 prion strain . in the absence of any strain - dependent phenotypic changes
, 
 such an analysis could provide useful insights into the general pathways 
 which underlie different prion disease phenotypes . in the present 
 study ,
wild - type c57bl/10 and anchorless tg44 mice 
 were infected intracranially with the rml mouse prion strain .
brain 
 homogenates from clinically positive mice were profiled for gross 
 differences between the nonamyloid ( c57bl/10 mice ) and amyloid ( tg44 
 mice ) disease phenotypes by utilizing mass spectrometry to identify 
 changes in protein abundance .
proteomic comparison of these two disease 
 phenotypes allowed us to distinguish the biochemical aspects of prion 
 pathogenesis that were likely related to a generalized host inflammatory 
 response versus those that may be more specific to each disease model .
dithiothreitol , iodoacetamide , 
 ammonium bicarbonate , trifluoroethanol ( tfe ) , tributylphosphine , and 
 membrane solubilization buffer were purchased from sigma ( st . louis , 
 mo , u. s. a. ) .
porcine trypsin ( # v5111 ) was purchased from promega 
 ( madison , wi , u. s. a. ) .
burdick & jackson water and acetonitrile 
 were purchased from vwr ( pittsburgh , pa , u. s. a. ) .
formic acid ( fa ) 
 ampules and imperial coomassie blue stain were purchased from thermo - fisher 
 scientific ( pittsburgh , pa , u. s. a. ) .
sds - page was run using reagents 
 and gels from life technologies - invitrogen corporation ( carlsbad , 
 ca , u. s. a. ) , with nupage bis  tris gels .
briefly , 
 prp staining was done using an avidin  biotin complex immunoperoxidase 
 protocol using anti - prp antibody d13 ( in - pro biotechnology , south 
 san francisco , ca , u. s. a. ) . staining with a 1% thioflavin s solution 

the mice used 
 in this study were housed at the rocky mountain laboratories ( rml ) 
 in a facility accredited by the association for assessment and accreditation 
 of laboratory animal care , and the research protocols used were approved 
 by the nih rml animal care and use committee .
uninfected wild - type 
 mice ( c57bl/10snj ) were obtained from jackson laboratories ( bar harbor , 
 me , u. s. a. ) .
transgenic mice homozygous for prp without 
 the gpi anchor ( tg44 ) were described previously .
six to eight week old mice were inoculated intracerebrally 
 as described with 50 l of a 1% 
 brain homogenate of rml containing 0.71.0  10 id50 .
mice were euthanized at the time of advanced 
 clinical disease as described previously and this was defined as the incubation period .
wild - type c57bl/10 
 mice developed clinical signs with a mean incubation time of 148  
 9 days postinfection ( dpi ) , whereas tg44 mice developed clinical disease 
 with a mean incubation time of 318  16 dpi .
for age - matched 
 controls , uninoculated 188 day old c57bl/10 mice and 401 day old tg44 
 mice were used . for all mice ,
brain samples were collected and flash 
 frozen in liquid nitrogen then kept at 80 c until processed . 

brain tissue was homogenized as a 20% ( w / v ) brain homogenate in 0.01 
 m tris , ph 7.2 , using a mini bead beater ( biospec , inc . ) , then vortexed 
 for 1 min followed by 1 min sonication in a misonix cuphorn sonicator 
 and kept at 80 c until needed .
brain homogenates analyzed without 
 proteinase k ( pk ) treatment were diluted from 20% to 2.5% and boiled 
 in tris - buffered saline , ph 8 , and sample buffer ( life technologies ) 
 with 0.5% -mercaptoethanol .
brain homogenates treated with 
 pk were diluted to 6% in the presence of 0.1 m tris , ph 8.3 , 1% triton 
 x-100 , 1% sodium deoxycholate , then digested with 125 g / ml 
 pk for 1 h at 37 c and then boiled in an equal volume of sample 
 buffer . portions of each sample ( 4 l ) were separated by sds - page 
 on a 10% bis  tris gel with mes running buffer ( life technologies ) 
 and then transferred to an immobilon pvdf - fl membrane ( millipore ) 
 by wet transfer using towbin s buffer .
samples were probed 
 for prp with mouse monoclonal antibody 6d11 ( covance inc . ) at a dilution 
 of 1:15  000 in tbst buffer for 1 h. 6d11 recognizes residues 
 93109 of mouse prp .
anti - apoe antibody ( calbiochem # 178479 ) was used 
 at 1:1000 and antiactin ( abcam # ab1801 ) was used at 1:1000 .
secondary 
 antibodies ( li - cor biosciences ) were irdye 800cw or irdye 680cw anti - mouse , 
 anti - rabbit or anti - goat , used at a 1:15  000 dilution in tbst 
 buffer for 40 min .
imaging was performed with an odyssey imaging system 
 ( li - cor ) using the 800 and 680 channels .
the in - gel tryptic digest 
 procedure was conducted using 16 individual brains with 4 from each 
 biological group : ( 1 ) rml - infected tg44 mice , ( 2 ) age - matched uninoculated 
 tg44 mice , ( 3 ) rml - infected wild - type c57bl/10 mice , and ( 4 ) age - matched 
 c57bl/10 uninoculated mice .
the 20% ( w / v ) brain homogenates were diluted 
 with denaturant to a final concentration of 5.3 m urea , 1.5 m thiourea , 
 0.8% detergent ( 3-(4-heptyl)phenyl-3hydroxypropyl)dimethylammoniopropanesulfonate ) , 
 2 mm tributylphosphine and 14 mm dtt , and then warmed at 37 c 
 for 20 min .
iodoacetamide was then added to a concentration of 50 
 mm and incubated at room temperature with gentle agitation in the 
 dark for 30 min .
the alkylation reaction was quenched with dtt to 
 a final concentration of 80 mm .
the mixture was then diluted with 
 4x sample buffer ( life technologies ) , vortexed briefly , incubated 
 at 37 c for 20 min , and then subjected to centrifugation for 
 30 min at 22  000 g .
a 20 l portion of 
 the reduced and alkylated mixture corresponding to 0.5 mg brain equivalents 
 or approximately 50 g of total protein was loaded for sds - page 
 onto a 10% bis  tris 1.5 mm thick nupage gel with mes running 
 buffer and run for 90 min at 150 v constant voltage .
this was repeated 
 for each of the 4 biological replicates from each of the 4 sample 
 groups for a total of 16 lanes ( 16 brains ) .
gels were subsequently 
 destained with water , cut with a razor blade into 24 slices and then 
 stored in individual tubes at 20 c until needed . in - gel 
 tryptic digestion of each gel slice was conducted overnight in a mixture 
 of 10% trifluoroethanol in 50 mm ammonium bicarbonate , ph 8 at 37 
 c for 16 h and then extracted with 70% acetonitrile as described 
 previously .
each digest was then subjected 
 to centrifugation at 22  000 g ( 10 min ) and 
 transferred to an autosampler vial for liquid chromatography tandem 
 mass spectrometry ( lc  ms / ms ) analysis .
ms / ms using an agilent 1200 
 interfaced to an xct ultra ion trap via a microfluidic hplc chip - cube 
 and a nanospray source .
the ion trap was calibrated externally using 
 a tuning mix provided by agilent specifically for this instrument . 

data - dependent ms acquisition was performed with dry gas ( nitrogen / air ) 
 set to 4 l / min at 350 c , ms capillary voltage 1800 v , and a 
 maximum accumulation time of 150 ms .
the ms scan range was set to 
 3001400 m / z in the ultrascan 
 mode .
four parent ions were selected for each ms / ms cycle with a fragmentation 
 amplitude of 1.0 v and the smartfrag setting on .
active exclusion 
 for fragmentation was set after 2 spectra in order to facilitate detection 
 of less abundant ions .
the tryptic digests were loaded onto the chip 
 ( agilent # g4240 - 62006 , zorbax 300sb - c18 , 5 m , 75 m  
 150 mm ) with an autosampler and washed with buffer a ( 3% acetonitrile / h2o and 0.1% formic acid ) prior to elution at 300 nl / min by 
 reversed - phase chromatography .
the gradient was 610% buffer 
 b ( 90% acetonitrile and 0.1% formic acid ) over 12 min , to 20% b by 
 65 min , to 30% b by 120 min , to 40% b by 140 min , and to 80% b by 
 147 min .
the column was washed with 95% b until 155 min , followed 
 by a 4 min postrun re - equilibration at 3% b with a total run time 
 of approximately 160 min .
separate chips were used for each biological 
 grouping of samples in order to minimize the possibility of contamination .
the mgf files were searched 
 using mascot daemon against a target database ( www.uniprot.org ) filtered for mouse taxonomy consisting of 16  315 entries . 

a separate reverse - sequence decoy database with the same number of 
 entries was created from the target database using a perl script provided 
 by nusep inc .
( athens , ga , u. s. a. ) . the trypsin / p search parameters 
 for mascot protein identification consisted of one missed tryptic 
 cleavage allowed with a fixed carbamidomethylation ( + 57 , cys ) and 
 a variable oxidation ( + 16 , met ) .
mass tolerances of 2.4 and 1.0 da 
 were used for parent and monoisotopic fragment ions , respectively . 

the resulting dat files from mascot were used as input files for further 
 analysis using the proteoiq software bioinformatics platform ( premier 
 biosoft inc . ) .
protein identifications with a protein false discovery 
 rate ( fdr ) of 1% were calculated using proteoiq s implementation 
 of the provalt algorithm with the constraints 
 that only mascot ion scores of 30 and only peptides of 7 
 amino acids in length were considered in these initial calculations . 

the resulting 2026 protein identifications were further filtered to 
 require at least 2 unique peptides and a protein group probability 
 of 0.9 for a positive identification . 
 additionally , each peptide was limited to only one protein identification 
 such that the number of protein groups equaled the number of protein 
 identifications .
the list of 1567 proteins identified according to 
 these criteria is provided as supporting information table 1 .
two data sets 
 were derived from the 
 original 1567 protein identifications , one from the c57bl/10 mice 
 and the other from the tg44 mice . for label - free quantitation ,
the 1567 protein identifications ( supporting 
 information table 1 ) were normalized by dividing the spectral 
 count ( spc ) for each protein from each mouse by the mean of all proteins 
 from that individual mouse .
once normalized , a sum was calculated 
 for each protein for each group of mice ( n = 4 ) , 
 and the fold change for each protein was calculated by dividing the 
 sum for the four scrapie - infected mice by the sum from the four uninfected 
 mice .
a set 
 of  values was obtained by running an analysis of variance 
 ( anova ; partek genomics suite v6.6 ) for the comparisons of interest , 
 and multiple test corrected using the false discovery rate ( fdr ) method 
 of benjamini and hochberg . any proteins 
 with a spc average below 5 in both
the infected and uninfected groups 
 were flagged as below detection because it has been reported that 
 mean counts of less than 5 spectra are often unreliable for quantitation . in order to identify the proteins that were most affected 
 by prion infection in each mouse line ,
individual rankings were derived based upon the combination 
 of greatest fold change , lowest  value , lowest coefficient 
 of variation , and spectral counts from the two conditions compared 
 ( i.e. , uninfected versus infected brain homogenate ) .
all four criteria 
 were thus given equal weight in the analysis and were used to estimate 
 changes in relative protein abundance between rml - infected brain homogenates 
 and their respective uninfected controls for each mouse strain .
the 
 resulting four ranks for any given protein were summed and divided 
 by the square of their sum to produce one number from all ranks .
this 
 final number was then ranked in descending order to produce the overall 
 reported rank .
thus , the protein with the best result across all criteria 
 produced a rank of one , followed by the rank of two for second best 
 and continuing for each of the 1567 proteins identified . based on 
 the final protein rankings ,
the top 100 proteins were used for subsequent 
 ipa analysis ( supporting information table 
 2 ) .
functional analyses and 
 pathways were examined with ingenuity pathway analysis ( ipa , ingenuity 
 systems , www.ingenuity.com ) .
the top 100 ranked proteins 
 from the tg44 and the c57bl/10 lists , most of which had  values 
 of 0.05 , were evaluated against the ingenuity knowledge base . 

the ipa database organizes lists of genes into graphical representations 
 based upon text - mining of published interactions between the genes 
 of interest and various predefined networks , disease classifications , 
 canonical pathways or biofunctions .
the scores assigned to the data 
 sets by ipa , based upon their degree of overlap with the experimental 
 data , are the negative exponent of the right - tailed fisher s 
 exact test result .
the  values reported estimate the probability 
 that each disease classification or pathway assigned to the data could 
 have been determined by random chance .
for example , a  value 
 of 0.01 would indicate a 1% false discovery rate for a set of protein 
 associations with a particular classification .
a random set of 100 
 proteins from the swiss - prot murine database generated ipa  
 values of 7.1  101.5  10 ( data not shown ) .
therefore , we considered the association 
 of proteins with an ipa  value of 5  10 with a particular molecular functional group to be significant and 
 outside of the realm of random chance .
after intracranial inoculation of rml mouse scrapie , 
 significant gray matter spongiosis was observed only in rml infected 
 c57bl/10 mice ( figure 1a inset ) .
c57bl/10 mice 
 expressing gpi - anchored prp also exhibited diffuse prp deposits in the brain ( figure 1a ) 
 and had a disease incubation time of approximately 150 days .
by contrast , 
 the same inoculum into tg44 mice , which express anchorless prp , led to the deposition of prp in large plaques 
 ( figure 1b ) with little or no gray matter spongiosis 
 ( figure 1b inset ) and an incubation time of 
 approximately 320 days . when brain sections were stained with the 
 amyloid - binding dye thioflavin s ,
the diffuse prp deposits 
 in c57bl/10 mice were negative ( data not shown ) , whereas the large 
 plaque - like prp deposits in tg44 mice were positive ( figure 1c ) .
the disease phenotype of rml infected c57bl/10 
 mice will therefore be referred to as nonamyloid , whereas that of 
 rml infected tg44 mice will be referred to as amyloid .
consistent 
 with previous results , these data show that the presence 
 or absence of a gpi anchor on prp can induce two markedly 
 distinct prion diseases . moreover , the neuropathological differences 
 observed between the two mouse models suggest that the biological 
 pathways underlying prion pathogenesis in these mice fundamentally 
 differ .
infection of c57bl/10 and tg44 mice with the rml prion strain yields 
 two distinct disease phenotypes .
( a ) immunostaining of rml infected 
 c57bl/10 brain tissue with d13 prion antibody shows diffuse prp deposition in most regions of the brain in addition to spongiform 
 degeneration .
( b ) immunostaining of rml infected tg44 brain tissue 
 with d13 prion antibody shows prp deposition in the absence 
 of spongiform degeneration .
( c ) staining of the brain hippocampal 
 region with the amyloid - specific fluorescent dye thioflavin s confirms 
 the presence of amyloid plaque in rml infected tg44 brain .
thioflavin 
 s staining is negative in uninfected tg44 mice ( inset ) . in order to gain more insight into these potential differences
brain homogenates from infected 
 versus uninfected tg44 and c57bl/10 mice were examined , each with 
 four biological replicates , for a total of 16 individual brains . a 
 representative coomassie blue stained gel loaded with
approximately 
 50 g of protein for each replicate demonstrated a similar protein 
 staining profile for each sample ( figure 2a ) . 

as outlined in figure 2b , the initial list 
 of proteins identified was set to a false discovery rate ( fdr ) threshold 
 of 1% , resulting in 2026 nonredundant 
 protein identifications .
these data were additionally filtered so 
 that only proteins identified by 2 unique peptides and a protein 
 group probability of 0.9 based upon the protein prophet algorithm were selected for further analysis .
the resulting 
 protein list contained 1567 high - confidence protein identifications 
 ( supporting information table 1 ) , derived 
 from a total of 12  834 unique peptides across all of the biological 
 groups .
the number of protein identifications was similar between 
 groups ( figure 2c , left panel ) with the vast 
 majority of proteins common to all four groups ( figure 2c , right panel ) , indicating good consistency in protein sampling . identification 
 of proteins from prion - infected and uninfected brain 
 homogenates .
( a ) coomassie blue staining of brain homogenate from 
 each of the 16 individual brains used for lc  ms / ms analysis . 

ms / ms data collection , processing and 
 searching with mascot , and spectral counting with proteoiq resulted 
 in 2026 proteins with a calculated fdr of 1% .
further filtering 
 of the data resulted in 1567 high - confidence protein identifications . 

( c ) the number of protein identifications delineated by biological 
 group in the pie chart shows approximately 1500 identifications per 
 group .
the venn diagram shows that 1470 proteins ( 94% ) of all the 
 proteins identified were common to all of the biological groups studied 
 prior to further filtering of the data for label - free quantitation . 

tg - u = uninfected tg44 mice ; tg - sc = rml infected tg44 mice ; c57-u 
 = uninfected c57bl/10 mice ; c57-sc = rml infected c57bl/10 mice .
the relative apparent abundance 
 of the proteins between sample 
 groups was examined with the goal of comparing the global effects 
 of prion infection in the amyloid and nonamyloid disease phenotypes 
 at the end - stage of clinical disease . in order to semiquantitatively 
 compare the relative abundance of proteins between the groups , spectral 
 counting was utilized .
spectral counting is often used as a practical , 
 effective approach for estimating the relative abundance of proteins 
 between biological samples and is the preferred method for quantifying profiling data from 
 ion trap mass spectrometers such as the one used in this study . as expected , prp was 
 identified as a protein with an altered relative abundance when compared 
 to uninfected controls .
based upon spectral counts , there was an approximate 
 5-fold increase in prp in uninfected c57bl/10 mice when 
 compared to uninfected tg44 mice ( figure 3a ) . 

our semiquantitative analysis using spectral counts was therefore 
 consistent with previous findings suggesting that uninfected tg44 
 mice express approximately 8-fold less prp relative to 
 uninfected c57bl/10 mice .
the amount of 
 prp in brain homogenates from the two prion - infected mouse lines was 
 also significantly different . in the absence of pk treatment ,
total 
 prp levels ( i.e. , prp and prp ) were significantly 
 higher in mice with the amyloid disease phenotype than in any other 
 sample group , demonstrating an apparent 91-fold higher concentration 
 of total prp ( table 1 ) when compared to age - matched 
 controls ( figure 3a ) .
by contrast , a 2-fold 
 increase in total prp was observed with the nonamyloid disease phenotype 
 ( table 1 ) when compared to their uninfected 
 controls ( figure 3a ) .
these differences in 
 the total amount of prp were confirmed by immunoblot analysis ( supporting information figure 1a ) .
after pk digestion , 
 protease - resistant prp was found only in the infected 
 samples ( figure 3b ) .
the increased levels of 
 total prp in both strains of infected mice therefore appears to represent 
 the generation and accumulation of prp .
( a ) a spectral count comparison 
 of prp between sample groups shows that total prp concentrations are 
 lowest in the uninfected tg44 brains ( tg - u ) and highest in the rml 
 infected tg44 brains ( tg - sc ) .
data shown are the mean and sem of four 
 individual mouse brains per group .
( b ) brain homogenates were subjected 
 to treatment with proteinase k and then probed with 6d11 , showing 
 that prp is present only in the prion - infected samples . 

rank 
 out of 1567 identified proteins 
 based upon lowest p value , highest fold change , best 
 count combination , and lowest coefficient of variation .
each of the 1567 identified proteins 
 was ranked for both the amyloid and nonamyloid phenotypes and the 
 top 100 ranked proteins ( supporting information table 2 ) from each data set were compared .
these proteins had the 
 strongest association with each disease phenotype and were thus the 
 primary focus of our subsequent data analysis
. only 17 of the top 
 100 proteins were common to both disease phenotypes . in every instance , 
 proteins that showed a significant fold increase or decrease in the 
 amyloid disease phenotype mice showed similar fold increases or decreases 
 in the nonamyloid disease phenotype ( table 1 ) .
consistent with the gliosis observed in both prion disease models , the astrocyte protein glial fibrillary acidic protein ( gfap ) was 
 increased 5.8 and 8.9 fold in the amyloid and nonamyloid disease phenotypes , 
 respectively ( table 1 ) .
proteins such as vimentin , -2-macroglobulin , and 
 -2-hs - glycoprotein , all of which have been linked to neuroinflammation 
 in prion as well as other neurodegenerative diseases , were increased anywhere from 2.826.7 fold in both disease 
 phenotypes ( table 1 )
. the increased levels 
 of vimentin in prion infected mice was further confirmed by immunoblot 
 analysis ( supporting information figure 
 1b ) . finally , multiple complement factors which have been linked to 
 neuroinflammatory responses also had 
 elevated spectral counts in both prion disease models with increases 
 in relative abundance ranging from 7.827.6 fold ( table 1 ) .
it was unlikely that the increased relative abundance 
 of these proteins was due to age - related differences between c57bl/10 
 and tg44 mice because they were present at comparable levels in uninfected 
 controls from both strains of mice ( figure 4a  f ) .
thus , the similar expression levels of these proteins 
 in mice with both the amyloid and nonamyloid disease phenotypes may 
 reflect a similar response and activation of astroglia and microglia 
 to the two different types of brain damage that result from rml prion 
 infection in tg44 and c57bl/10 mice .
no significant age - related differences 
 in protein abundance in 
 c57bl/10 and tg44 mice .
average spectral counts ( spc ) for proteins 
 associated with neuroinflammation ( a  f ) and lipid metabolism 
 ( g  i ) .
in general , the relative abundance of these proteins 
 was the same between uninfected c57bl/10 ( c57-u ) and tg44 mice ( tg - u ) , 
 but greater in abundance in rml infected tg44 mice ( tg - sc ) when compared 
 to rml infected c57bl/10 ( c57-u ) mice .
data shown are the mean and 
 sem of four individual mouse brains per group . in mice with the amyloid 
 disease phenotype , expression levels of several key apolipoproteins 
 ( apod , apoe , apoj ) as well as two other proteins involved in lipid 
 transport ,
peroxisomal multifunctional enzyme type 2 and low density 
 lipoprotein receptor related protein 1 ( lrp1 ) , were elevated when 
 compared to uninfected age - matched controls ( table 2 ) .
these proteins were generally much higher ranked in the 
 amyloid - associated data set than they were in the nonamyloid data 
 set . the greater abundance of these proteins in mice with the amyloid 
 phenotype was further confirmed by immunoblot for one of them , apoe 
 ( supporting information figure 1c ) .
furthermore , 
 the differences in relative abundance did not appear to be due to 
 age - related differences between tg44 and c57bl/10 mice because these 
 proteins were present at comparable levels in uninfected controls 
 from both strains of mice ( figure 4g 
these data suggest that , although lipid metabolism and transport 
 are altered in both the amyloid and nonamyloid phenotypes , this disruption 
 is more strongly linked to the amyloid form of disease .
rank 
 out of 1567 identified proteins 
 based upon lowest p value , highest fold change , best 
 count combination , and lowest coefficient of variation .
information derived from the ingenuity 
 knowledge database . in order to further differentiate the two prion disease 
 phenotypes , the top 100 ranked proteins in each data set were analyzed 
 using ingenuity pathway analysis ( ipa ) which maps the molecular and 
 cellular pathways associated with those proteins .
our initial analysis 
 showed that individual proteins were often associated with multiple 
 functional processes , making it difficult to determine which proteins 
 and pathways were most specifically relevant to prion disease .
therefore , 
 to identify the potential molecular mechanisms involved in pathogenesis , 
 we focused our initial analysis on the molecular and cellular function 
 categories , particularly on those subcategories which contained the 
 highest percentage of unique proteins ( table 3 ) . using these criteria ,
cell death and survival was the top scoring 
 group in the nonamyloid phenotype , with a higher percentage of unique 
 proteins when compared to the other groups ( 29% versus 38% , 
 see table 3 ) .
by contrast , in the amyloid phenotype , 
 the highest percentage of unique proteins was associated with the 
 molecular transport category ( 23% ) followed by cell death and survival 
 at 14% ( table 3 ) .
thus , whereas cellular pathways 
 related to cell death and survival were associated with both the amyloid 
 and nonamyloid disease phenotypes , pathways involved in molecular 
 transport were most strongly implicated in the amyloid disease phenotype .
the 100 top - ranked 
 proteins associated 
 with the amyloid and nonamyloid disease phenotypes were subjected 
 to ipa analysis and the top molecular and functional categories determined .
number of proteins in the top 
 100 
 from each data set that ipa associated with that functional category .
percentage of proteins which 
 are 
 uniquely associated with the specific molecular and cellular function 
 category listed and thus do not overlap with the other three . in mice with the amyloid disease phenotype
, the 39 proteins implicated 
 in molecular transport ( table 4 ) were involved 
 in the binding and transport of multiple molecules including amino 
 acids , metals , and adp / atp .
interestingly , many of these proteins 
 were associated with metal - dependent processes , many of which involved 
 calcium ( figure 5a ) .
further analysis revealed 
 another protein known to bind calcium , calcineurin subunit type b1 , 
 that had not been identified by the ipa analysis .
finally , multiple 
 proteins involved in calcium transport , regulation , and homeostasis 
 were also linked to molecular transport in the amyloid phenotype ( supporting information table 3 ) .
thus , our data 
 suggest that metal ion transport , and in particular calcium homeostasis , 
 is disrupted in the amyloid form of prion infection .
the majority of molecular 
 transport proteins disrupted in prion - infected 
 tg44 mice were associated with metal - dependent processes .
( a ) relative 
 distribution of the metal - associated molecular transport proteins 
 from table 4 . ( b ) venn diagram illustrating 
 the overlap between molecular transport ( table 4 ) and cell death and survival ( table 6 ) in 
 the amyloid phenotype and their overlap with known calcium binding 
 proteins from each category .
proteins shown in bold are unique 
 to molecular transport and were not found associated with any other 
 ipa - derived molecular and cellular functions in the nonamyloid disease 
 phenotype .
rank 
 out of 1567 identified proteins 
 based upon lowest p value , highest fold change , best 
 count combination , and lowest coefficient of variation .
the dashed 
 line ( --- ) indicates the protein was not among the top 100 ranked 
 proteins .
proteins involved 
 in calcium binding 
 or calcium - mediated transport , regulation , and signaling .
interestingly , many of the proteins 
 in the molecular transport 
 category were also involved in synaptic vesicle transport , a process 
 known to be regulated by calcium .
they 
 included synaptosomal - associated protein 25 ( snap25 ) and syntaxin 
 1a , which are two of the three proteins present in a complex necessary 
 for synaptic fusion and neurotransmitter release , as well as several 
 proteins involved in synaptic vesicle exocytosis including syntaxin 1b , rabphilin 3a , and members of the ras - related 
 proteins ( table 4 ) .
all of these synaptic vesicle 
 proteins were present in lower abundance when compared to noninfected 
 controls , suggesting significant disruption to synaptic vesicle transport 
 in prion - infected mice with the amyloid disease phenotype .
the highest scoring molecular function category 
 associated with the top 100 proteins in mice with the nonamyloid disease 
 phenotype was cell death and survival , with 48% of the 100 input proteins 
 linked to this category ( table 5 ) . of those , 
 29% ( 14 out of 48 ) were uniquely associated with cell death and survival 
 and were not found in any of the other categories ( table 3 ) . the cell death and survival category proteins 
 had diverse functions including protein folding , cytoskeletal organization , 
 rna processing , apoptosis , and intracellular calcium regulation .
however , 
 there was an increase in the relative levels of three proteins associated 
 with the mitochondrial inner membrane .
two of these , the mitochondrial 
 inner membrane protein ( mitofilin ) and heat shock protein hspa9 , are 
 part of the mitochondrial inner membrane .
the third is apoptosis - inducing 
 factor 1 ( aif1 ) , which is anchored to the mitochondrial inner membrane .
finally , the fold expression of several proteins 
 associated with the ubiquitin / proteasome pathway was also increased . 
 these included heat shock protein hspa5 ( or bip ) , which interacts 
 with proteins involved in endoplasmic reticulum associated degeneration 
 ( erad ) , as well as the protein chaperonin tcp1 ( table 5 ) .
thus , the data are consistent with disruptions to mitochondria , 
 er and the ubiquitin / proteasome pathway as part of the process of 
 cell death in the nonamyloid prion disease phenotype .
proteins shown in bold are unique 
 to cell death and were not found associated with any other molecular 
 and cellular functions identified by ipa in the nonamyloid disease 
 phenotype .
rank 
 out of 1567 identified proteins 
 based upon lowest p value , highest fold change , best 
 count combination , and lowest coefficient of variation .
the dashed 
 line ( --- ) indicates the protein was not among the top 100 ranked 
 proteins .
proteins involved 
 in calcium binding 
 or calcium - mediated transport , regulation , and signaling . cell death and survival
was also 
 the second highest scoring ipa 
 functional category in prion - infected mice with the amyloid phenotype 
 and contained 42 of the top 100 input proteins , 14% of which uniquely 
 segregated into this category ( table 6 ) .
many of these proteins , including annexin a2 , 
 gelsolin , syntaxin 1a and snap25 , overlapped with those proteins associated 
 with calcium binding and molecular transport ( figure 5b ) .
this overlap suggested a strong link between perturbations 
 in calcium homeostasis and cell death in the disease with the amyloid 
 phenotype .
interestingly , however , none of the mitochondrial or ubiquitin / proteasome 
 pathway proteins identified in the mice with the nonamyloid phenotype 
 were found in the top 100 ranked proteins in mice with the amyloid 
 disease phenotype .
in fact , there was minimal overlap with the proteins 
 associated with cell death and survival in the nonamyloid versus the 
 amyloid disease phenotypes ( 9 out of 90 , or 10% ) .
moreover , several 
 of these 9 proteins , including gfap , vimentin , complement , and -2-macroglobulin 
 have previously been associated with a strong inflammatory response 
 in prion diseases and are considered global neuroinflammatory markers , indicating 
 that they are not functionally restricted to pathways mediating cell 
 death .
thus , our data suggest that different cell death pathways may 
 predominate in the amyloid and nonamyloid disease phenotypes .
proteins shown in bold are unique 
 to cell death and were not found associated with any other molecular 
 and cellular functions identified by ipa in the nonamyloid disease 
 phenotype . fold change in 
 abundance relative 
 to uninfected control .
rank 
 out of 1567 identified proteins 
 based upon lowest p value , highest fold change , best 
 count combination , and lowest coefficient of variation .
the dashed 
 line ( --- ) indicates the protein was not among the top 100 ranked 
 proteins .
proteins involved 
 in calcium binding 
 or calcium - mediated transport , regulation , and signaling .
previous 
 genomic studies in prion - infected mice have suggested 
 that disruptions to metal binding , calcium homeostasis , synaptic transmission , 
 lipid metabolism , and protein folding / degradation are involved in 
 the pathogenesis of prion diseases .
similarly , genomic 
 and proteomic analysis of tissue from human cjd patients has suggested 
 that disruptions to molecular transport , calcium homeostasis , mitochondria , 
 synapses and glucose metabolism may be involved in the pathogenesis 
 of cjd .
our proteomics analysis of two different models of 
 mouse prion disease also implicated imbalances in metal binding , calcium 
 homeostasis , mitochondria , synaptic transmission , lipid metabolism , 
 and molecular transport as factors in the late stages of prion disease . 

however , unlike previous studies , our comparison of the two distinctive 
 prion disease phenotypes induced by a single prion strain has also 
 allowed us to distinguish which pathways were more strongly linked 
 to amyloid or nonamyloid forms of prion disease as well as which likely 
 represented general responses to prion infection .
our analysis 
 primarily focused on the 100 proteins that were linked 
 most strongly to either the amyloid or nonamyloid disease phenotype . 

of these , only 17 were common to both disease phenotypes and most 
 had some link to neuroinflammation ( table 1 ) .
this is consistent with previous studies showing that multiple 
 genes and proteins associated with the inflammatory response are upregulated 
 in different types of mouse and human prion disease .
astrocytes which have been activated in response 
 to damage or infection produce complement factors and increase the 
 expression levels of both vimentin and gfap , all of which are present at increased levels in our two mouse models 
 ( table 1 ) . the increased abundance of -2-macroglobulin 
 may be related to its ability to act as a carrier protein for interleukin-6 or to remove proteinases at the site of inflammation .
likewise , -2-hs - glycoprotein , an acute 
 phase protein which binds calcium and has anti - inflammatory properties , 
 was also increased in abundance in both disease models . although the magnitude of the changes in fold 
 expression was not always identical between the two phenotypes , proteins 
 which were increased or decreased in mice with the amyloid phenotype 
 mice were also increased or decreased in mice with the nonamyloid 
 phenotype ( table 1 ) . interestingly , many 
 of these same proteins are also increased or 
 decreased in abundance in other human neurodegenerative diseases such 
 as alzheimer s disease ( ad ) and parkinson s disease . 

for example , upregulation of vimentin has recently been described 
 in neuronal cells of ad patients as part of a damage response mechanism , 
 whereby the neurons attempt to repair synapses and re - establish dendritic 
 branches lost during the disease process .
-2-macroglobulin has been found in senile ad plaques , whereas -2-hs - glycoprotein has been found , 
 albeit at decreased levels , in the cerebrospinal fluid of alzheimer s 
 patients .
complement factors ( table 1 ) have 
 also been proposed to play a role in both parkinson s and alzheimer s 
 diseases when taken together , the data 
 suggest that the changes in the relative abundance of these proteins 
 during disease may represent similar , general responses related to 
 the damage in the brain which occurs during neurodegeneration rather 
 than a direct response to the deposition of prp as amyloid 
 versus nonamyloid .
an increased relative abundance of several 
 lipoproteins was also 
 observed in both disease phenotypes ( table 2 ) .
notably , the lipoproteins apod , apoe and clusterin ( apoj ) as well 
 as the low density lipoprotein receptor - related protein ( lrp1 ) , were 
 elevated in both data sets but present at higher levels in the amyloid 
 disease phenotype ( table 2 ) .
apoe consistently  copurifies 
 with prp isolated from infected animal tissue and elevated levels of clusterin have been observed 
 in the hippocampus of prion infected mice , raising the possibility that these molecules have a functional role 
 in prion pathogenesis .
the ability of clusterin to inhibit the formation 
 of amyloid as well as the link between 
 lrp1 and the apoe and -2-macroglobulin - mediated clearance of 
 -amyloid may explain their elevated levels 
 in the amyloid disease phenotype . it is possible that other clusterin 
 functions , such as its ability to prevent apoptosis induced by the 
 mitochondrial release of cytochrome c , may also be relevant in the nonamyloid disease phenotype where proteins 
 necessary for the integrity of the mitochondrial crista ( table 5 ) are also elevated .
overall , our data suggest that 
 disruption of cholesterol and lipid metabolism and alterations in 
 the mechanisms for the clearance of misfolded proteins occur in both 
 amyloid and nonamyloid forms of prion disease . however , these 
 changes are more pronounced in the mice with the 
 amyloid disease phenotype .
thus , the presence of amyloid may exacerbate 
 disruptions in lipid metabolism and protein clearance .
consistent 
 with this idea , apod and apoe have been reported as candidate biomarkers 
 for ad and prion disease and are known 
 to colocalize with amyloid plaque in both alzheimer s disease and cerebral amyloid angiopathy . moreover
, 
 the ability of clusterin to inhibit the formation of amyloid as well as the link between lrp1 and the apoe 
 and -2-macroglobulin - mediated clearance of alzheimer s 
 disease -amyloid may explain their elevated 
 levels in the disease phenotypes where amyloid is a prominent feature .
in addition to the perturbation of the lipid transport proteins 
 that we observed in both phenotypes , other molecular transport proteins 
 were also found to have significantly changed in abundance in mice 
 with the amyloid disease phenotype ( table 4 ) .
approximately one - third of these are known to bind metals such 
 as iron , zinc , manganese , copper and magnesium ( figure 5a ) indicating a metal imbalance that could contribute to the 
 buildup of reactive oxygen species and reduction of superoxide dismutase 
 activity that has been observed during prion infection .
interestingly , multiple proteins were involved 
 with calcium transport , regulation , and binding ( supporting information table 3 ) , suggesting a significant 
 disruption to calcium homeostasis in mice with the amyloid disease 
 phenotype .
divalent calcium ions are universal signaling molecules 
 with cells maintaining an approximately 20  000-fold lower concentration 
 of divalent calcium ions in the intracellular compartment compared 
 to the extracellular compartment .
imbalances 
 in this tightly controlled gradient can trigger a wide variety of 
 cellular processes and dysregulation 
 of calcium has been implicated in the pathogenesis of prion disease .
our data suggest that in addition to dysregulation 
 of calcium homeostasis , 
 synaptic vesicle transport may also be impaired in the mice with the 
 amyloid phenotype .
synaptic vesicle transport and exocytosis is triggered 
 by an influx of calcium at the synapse leading to fusion of the soluble 
 nsf attachment protein receptor ( or snare ) complex to the membrane 
 and the subsequent release of neurotransmitters from the synaptic 
 vesicle .
snare is essential for synaptic 
 vesicle fusion to the membrane and syntaxin-1 and snap25 , two critical 
 proteins which comprise snare , are decreased in abundance in mice 
 with the amyloid phenotype .
similarly , the levels of syntaxin 1b , 
 rab3a , rab3b and rabphilin-3a , all of which participate in the fusion 
 of synaptic vesicles , are also decreased ( table 4 ) .
interestingly , altered cycling of rab3a has recently 
 been reported in cjd and alterations 
 in synaptic vesicle fusion and recycling can lead to disruptions in 
 neurotransmitter release and deficits in learning and memory , a phenotype 
 which has been described in the prion - infected mice with amyloid disease .
thus , our data suggest that impairment of synaptic 
 vesicle fusion and neurotransmitter release triggered by changes in 
 calcium homeostasis may underlie the learning and memory deficits 
 seen in mice with the amyloid disease phenotype . snare proteins 
 have also been associated with neurodegeneration 
 in cjd , and synaptic degeneration precedes neurodegeneration 
 in prion infected mice .
consistent with 
 this hypothesis , ipa analysis linked syntaxin-1 and snap25 to cell 
 death and survival pathways only in mice with the amyloid disease 
 phenotype ( table 6 ) . however
, synaptic degeneration 
 has been observed in multiple mouse prion disease models , including the rml strain used for the current studies .
furthermore , a decrease in snap25 and syntaxin 
 has been reported in rml - infected wild - type mice .
thus , contrary to the present data , synaptic 
 transport can also be impaired in nonamyloid forms of prion disease . 

the reason for this discrepancy is not clear but it is possible that 
 decreased expression of snare proteins in the amyloid phenotype is 
 the result of changes in synaptic vesicle fusion not linked to snare - mediated 
 neurodegeneration , perhaps because of the lack of membrane anchored 
 prp in these transgenic mice .
this hypothesis is consistent 
 with the lack of significant gray matter spongiosis in the amyloid 
 disease phenotype as well as studies showing no correlation between 
 decreased snap25 and apoptosis in rml infected mice .
in the proteins associated with cell death and survival 
 ( tables 5 and 6 ) , there 
 was very little 
 overlap between the amyloid and nonamyloid disease phenotypes even 
 though multiple proteins linked to calcium homeostasis were identified 
 for both disease forms .
this suggested that the dominant mechanisms 
 underlying calcium - associated cell death may differ between the two 
 mouse models .
for example , only in the nonamyloid phenotype was there 
 an increased abundance of both chaperonin containing tcp1 and the 
 er protein bip , both of which are part of a large chaperonin complex 
 that interacts with multiple proteins linked to erad ( table 5 ) .
these data are consistent with the possibility 
 that the ubiquitin - proteasome system is involved in prion pathogenesis 
 but that it may be more disrupted in the nonamyloid disease phenotype .
mitochondrial dysfunction has been proposed as a mechanism 
 of neurodegeneration 
 in prion disease .
our data also implicated mitochondrial 
 pathways of apoptosis in neurodegeneration but only in the nonamyloid 
 disease phenotype .
mitochondria have been linked to apoptosis via 
 the release of pro - apoptotic factors such as cytochrome c and aif1 
 from the inner mitochondrial membrane .
disruptions in this membrane have been observed in 
 prion infected mice in regions of synaptic degeneration .
mitofilin and hspa9 , two proteins associated 
 with the mitochondrial inner membrane organizing system ( minos ) responsible 
 for maintaining cristae morphology , were present at higher levels 
 only in the nonamyloid phenotype .
the level of aif1 , an apoptosis 
 inducing factor anchored to the mitochondrial inner membrane , was 
 also elevated . in response to
increased cytoplasmic calcium , mitochondrial 
 calcium also increases leading to permeabilization of the mitochondrial 
 outer membrane , changes in cristae morphology , and the production 
 of reactive oxygen species .
the resultant cleavage of aif1 from the 
 inner mitochondrial membrane allows it to translocate to the nucleus 
 where it triggers dna fragmentation and apoptosis , primarily in neurons .
the altered levels of mitofilin , hspa9 , aif1 
 and multiple proteins involved in calcium homeostasis are all consistent 
 with this mechanism of mitochondria - associated apoptosis in mice with 
 the nonamyloid disease phenotype .
overall , our data suggest 
 that different mechanisms of cell death 
 may predominate in amyloid versus nonamyloid forms of prion disease , 
 with erad and mitochondria induced apoptosis implicated primarily 
 in the nonamyloid disease phenotype when compared to the amyloid disease 
 phenotype .
this difference may be driven more by the presence of the 
 gpi anchor on prp , either because of the possible function 
 of prp in cell signaling and apoptosis or because of the cell - surface conversion of prp by prp .
thus , rather than being a phenotype necessarily 
 encoded by prp , our data suggest that the spongiform 
 change associated with prion diseases is linked to membrane - anchored 
 prp and to complex calcium - regulated changes in mitochondria 
 and the erad / ubiquitin - proteasome system .
the lack of gray matter 
 spongiosis in the nonamyloid phenotype mice , despite very high levels 
 of abnormal prp , is consistent with this hypothesis and 
 further suggests that spongiform change is not necessarily driven 
 by a loss of function of membrane anchored prp or by the 
 amount of prp deposited .
in fact , the deposition of anchorless 
 prp as amyloid may be somewhat protective , possibly resulting 
 in slower disease progression
alternatively , it may simply take longer for 
 prp amyloid to alter brain homeostasis sufficiently to 
 trigger the mechanisms that eventually lead to neurodegeneration .
 prion 
 diseases are a heterogeneous group of neurodegenerative disorders 
 affecting various mammals including humans . 
 prion diseases are characterized 
 by a misfolding of the host - encoded prion protein ( prpc ) into a pathological isoform termed prpsc . in wild - type 
 mice 
 , prpc is attached to the plasma membrane by a glycosylphosphatidylinositol 
 ( gpi ) anchor and prpsc typically accumulates in diffuse 
 nonamyloid deposits with gray matter spongiosis . 
 by contrast , when 
 mice lacking the gpi anchor are infected with the same prion inoculum , 
 prpsc accumulates in dense perivascular amyloid plaques 
 with little or no gray matter spongiosis . in order to evaluate whether 
 different host biochemical pathways were implicated in these two phenotypically 
 distinct prion disease models 
 , we utilized a proteomics approach . 
 in both models , infected mice displayed evidence of a neuroinflammatory 
 response and 
 complement activation . 
 proteins involved in cell death 
 and calcium homeostasis were also identified in both phenotypes . 
 however , 
 mitochondrial pathways of apoptosis were implicated only in the nonamyloid 
 form , whereas metal binding and synaptic vesicle transport were more 
 disrupted in the amyloid phenotype . 
 thus , following infection with 
 a single prion strain , prpc anchoring to the plasma membrane 
 correlated not only with the type of prpsc deposition but 
 also with unique biochemical pathways associated with pathogenesis . 
Introduction
Experimental
Procedures
Results
Discussion